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

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process is impeded by the high cost of the enzyme (S-

adenosylmethionine: catechol O-methyltransferase)

and one of its substrate (S-adenosylmethionine). The

second modification is oxidative ring opening

by protocatechuate 3,4-dioxygenase. However, in

addition to its cost, this enzyme has poor catalytic

activities on caffeic acid and its derivatives.

Action on Reaction Products

0031 Acting on reaction products is the third way of con-

trolling enzymatic browning. o-Quinones are very

reactive primary products (Figures 2 and 3)and,

using chemical means, they can either be reduced

back to o-diphenols or trapped as colorless addition

compounds. However, secondary products resulting

mainly from the oxidative polymerization of o-quin-

ones often give highly colored compounds which

become less reactive as the browning reaction pro-

ceeds. Therefore, almost all the compounds listed

in the section on reaction products which act on

o-quinones, and more especially ascorbic acid and

its derivatives, thiol compounds and sulfite deriva-

tives, can be used for the control of enzymatic

browning. However, their effectiveness in preventing

browning is greatly reduced if their use is delayed

until after the reaction has started.

0032 Besides chemical means, brown pigments can be

removed more or less completely by physical treat-

ments such as ultrafiltration or use of resins in order

to trap polymers of o-quinones. This can be an effi-

cient method for the clarification of liquids such as

fruit juices and wine.

0033 Lastly, several studies have been devoted to the

isolation of ‘natural’ inhibitors of enzymatic brown-

ing. Thus, low-molecular-weight compounds exhibit-

ing inhibitory properties have been detected in

cultures of Penicillium expansum and Dactylium den-

droides. Inhibitors have also been found in anise oil,

cumin, and several medicinal plants. Similarly, it has

been shown that Maillard reaction products and

honey extracts contain substances which are able to

inhibit the PPO activity. Nevertheless, in most cases,

these inhibitory compounds have been only partially

characterized and their exact mechanism of inhibition

is not fully understood. (See Antioxidants: Natural

Antioxidants; Synthetic Antioxidants.)

0034 In recent years, considerable progress has been

made in understanding PPOs and polyphenols of

fruits and vegetables. However, many studies were

restricted to model systems with an enzyme acting

on one phenolic substrate only. There is a great

need for studies on enzymatic action on mixtures of

phenols on the one hand, and on nonenzymatic reac-

tions that follow o-quinone formation on the other.

This research would give valuable information on

how phenols affect both the enzymatic activity and

the kind of brown pigments formed, and it would

help to give a better understanding of the relation

between the extent of browning and the phenolic

composition of fruits and vegetables.

See also: Amines; Antioxidants: Natural Antioxidants;

Synthetic Antioxidants; Ascorbic Acid: Properties and

Determination; Browning: Toxicology of Nonenzymatic

Browning; Colorants (Colourants): Properties and

Determination of Natural Pigments; Enzymes: Functions

and Characteristics; Oxidation of Food Components;

Phenolic Compounds; Tannins and Polyphenols

Further Reading

Amiot MJ, Fleuriet A, Cheynier V and Nicolas J (1997)

Phenolic compounds and oxidative mechanisms in fruits

and vegetables. In: Tomas-Barberan FA, Robins RJ (eds)

Phytochemistry of Fruits and Vegetables, pp. 52–85.

Oxford: Clarendon Press.

Billaud C, Regaudie E, Fayad N, Richard-Forget F and

Nicolas J (1995) Effect of cyclodextrins on polyphenol

oxidation catalyzed by apple polyphenol oxidase. In: Lee

CY, and Whitaker JR (eds) Enzymatic Browning and its

Prevention, pp. 295–312. Washington: American Chem-

ical Society.

Cheynier V, Fulcrand H, Guyot S, Oszmianski J and Mou-

tounet M (1995) Reactions of enzymically generated

quinones in relation to browning in grape musts and

wines. In: Lee CY and Whitaker JR (eds) Enzymatic

Browning and its Prevention, pp. 130–143. Washington:

American Chemical Society.

Flurkey WH, Ratcliff B, Lopez L, Kuglin J and Dawley RM

(1995) Differentiation of fungal tyrosinases and laccases

using selective inhibitors. In: Lee CY and Whitaker JR

(eds) Enzymatic Browning and its Prevention, pp. 81–

89. Washington: American Chemical Society.

Lerch K (1995) Tyrosinase: molecular and active-site struc-

ture. In: Lee CY and Whitaker JR (eds) Enzymatic

Browning and its Prevention, pp. 64–80. Washington:

American Chemical Society.

Macheix JJ, Fleuriet A and Billot J (1990) Fruit Phenolics.

Boca Raton: CRC Press.

Marques L, Fleuriet A and Macheix JJ (1995) Fruit poly-

phenol oxidases. New data on an old problem. In: Lee

CY and Whitaker JR (eds) Enzymatic Browning and its

Prevention, pp. 90–102. Washington: American Chem-

ical Society.

Mayer AM (1987) Polyphenol oxidases in plants. Recent

progress. Phytochemistry 26: 11–20.

McEvily AJ and Iyengar R (1992) Inhibition of enzymatic

browning in foods and beverages. Critical Reviews in

Food Science and Nutrition 32: 253–273.

Nicolas J, Cheynier V, Fleuriet A and Rouet-Mayer MA

(1993) Polyphenols and enzymatic browning. In: Scal-

bert A (ed.) Polyphenolic Phenomena, pp. 165–175.

Paris: INRA Editions.

BROWNING/Enzymatic – Biochemical Aspects 685

Nicolas JJ, Richard-Forget FC, Goupy PM, Amiot MJ and

Aubert SY (1994) Enzymatic browning reactions in

apple and apple products. Critical Reviews in Food

Science and Nutrition 34: 109–157.

Vamos-Vigyazo L (1995) Prevention of enzymatic brown-

ing in fruits and vegetables. A review of principles and

practice. In: Lee CY and Whitaker JR (eds) Enzymatic

Browning and its Prevention, pp. 49–62. Washington:

American Chemical Society.

Walker JRL (1995) Enzymatic browning in fruits. Its bio-

chemistry and control. In: Lee CY and Whitaker JR (eds)

Enzymatic Browning and its Prevention, pp. 8–22.

Washington: American Chemical Society.

Zawistowski J, Biliaderis CG and Eskin NAM (1991) Poly-

phenol oxidases. In: Robinson DS and Eskin NAM (eds)

Oxidative Enzymes in Foods, pp. 217–273. London:

Elsevier Applied Sciences.

Enzymatic – Technical Aspects

and Assays

J Nicolas and C Billaud, Conservatoire National des

Arts et Me

tiers, Paris, France

M-A Rouet-Mayer, Centre National de la Recherche

Scientifique, Gif sur Yvette, France

J Philippon (retired), Centre National du Machinisme

Agricole du Ge

nie Rural, des Eaux et des Fore

ts,

France

Background

0001 Enzymatic browning affects numerous plant organs

and seafoods which are rich in oxidizable phenols

such as fruits, vegetables, aromatic plants, fungi, and

shrimps. It occurs during various procedures used in

the food industry: deep-freezing, dehydrofreezing,

freeze-drying, hot-air drying, etc. In practice, there

are three approaches to prevention of this browning:

varietal selection, physical methods, and chemical

inhibitors.

0002 The various methods currently used or likely to be

used in the future for prevention of browning are

reviewed in this article. Deep-frozen fruits and

vegetables are considered in detail, since enzymatic

browning is most difficult to resolve in these prod-

ucts. Also, the preventive methods used during indus-

trial drying and freeze-drying, and even canning and

bottling, are in fact very similar.

0003 A brief review is then given of the methods of

measurement of browning and of the questions raised

by objective assessment.

Practical Methods of Preventing

Browning

Choice of Raw Material

0004Some plant tissues, such as fruits or vegetables, are

less sensitive to browning than others. Most berries –

raspberry, blackcurrant, redcurrant, bilberry – are

virtually unaffected. Other species, however, quickly

brown on freezing and thawing unless previously

subjected to antioxidant treatment. They are gener-

ally rich in total phenols and poor in ascorbic acid in

cases of fruits with pips (apple, pear, quince), and

stone fruits (apricots, peaches, plums).

0005The phenol content of fruits and vegetables varies

naturally according to the degree of ripeness, the soil

and climate, but also depends substantially on intra-

specific genetic characteristics. The Sunbeam peach

variety, for instance, which does not brown, contains

20 times less total phenols than the Elberta variety,

which is sensitive to browning. This plainly illustrates

the value of the development by genetic selection

of new, low-phenol varieties. (See Ascorbic Acid:

Properties and Determination; Phenolic Compounds.)

Physical Methods of Prevention

Mechanical Factors

0006Browning is enhanced by wounding of plant tissue,

notably during industrial processing. Upkeep of

machine blades is important to insure that fruits

and vegetables are cut cleanly.

Effect of Temperature

0007Cold A marked slowing in enzymatic browning is

induced by a reduction in temperature. None the less,

color change is still rapid at 0



C. This means that

the most sensitive products are frozen by methods

allowing the crystallization temperature of water to

be reached as rapidly as possible. This is the case with

mushrooms, for which cryogenic techniques are

obligatory when they are frozen without prior anti-

oxidant treatment. The same is true for peeled slices

of white peach which brown during freezing if the

cooling exceeds 10 min.

0008Once frozen, color change is practically blocked

at the temperature of commercial storage (18



C).

When the temperature rises, browning starts again,

and will be more marked if the cellular structures of

the plant organ have been severely damaged by freez-

ing, chemical peeling, and slicing.

0009Heat Heat treatment, or blanching, without doubt

constitutes the simplest and most direct method

686 BROWNING/Enzymatic – Technical Aspects and Assays

of enzyme inactivation. It consists of brief immer-

sion (from 1 to 6 min depending on size) of the

product in water, boiling syrup, or steam close

to 100



0010 Catechol oxidases are inhibited above approxi-

mately 70



C. Their heat stability is closely pH-

dependent; it is maximal at about pH 6 and decreases

very rapidly below and above this value.

0011 In the deep-freezing of foodstuffs, blanching is

principally applied to vegetables that are eaten

cooked (potatoes, salsify, asparagus, etc.), but it is

also selected to stabilize frozen fruit pure

es, particu-

larly of apricots. Blanching is little used with whole or

sliced frozen fruits that are eaten raw, since it results

in loss of firmness and in flavor changes which are

unacceptable to the consumer. This is why blanching

is only used for fruit with tissue sufficiently imperme-

able to oxygen, to escape deep browning. Examples

are apricot and peach, for which the brief surface

treatment scarcely alters the organoleptic qualities.

When blanching of pears and, particularly, apples

(20–25% lacunae) is incomplete, marked deep

browning occurs on thawing.

Other Physical Methods for Prevention

0012 Electrodialysis, ultrafiltration Electrodialysis, based

on dissociation of the water molecule using a bipolar

membrane, is applicable for the control of enzymatic

browning in food liquids. This technique, applied to

cloudy juice extracted from MacIntosh apples, results

in the reduction of juice pH to 2 with a complete

inhibition of polyphenol oxidase (PPO). After

readjustment at pH 3.5 (the initial juice pH), the

electrodialysis treatment leads to an improvement in

the juice color during storage, a large decrease in

mineral concentration, and a slight loss in malic acid

without noticeable change in the composition of

sugars. The latter modifications result in a less acidic

and somewhat milder apple juice.

0013 Ultrafiltration can be used as a clarification process

in fruit juice processing. Use of polyethersulfone/

polyvinylpyrrolidone membranes permits reduction

of the amounts of polyphenols in apple juice, leading

to a large improvement of the stability of color and

clarity during storage.

0014 High-pressure, ultrasonic waves High-pressure pro-

cessing appeared in the last decade as an alternative to

heat treatments for inactivation of microorganisms

and food-quality enzymes (including PPO). However,

at room temperature, complete inactivation of PPO

requires pressures higher than 400–500 MPa which

are not industrially relevant for the moment. There-

fore, several processes using high pressures in com-

bination with other treatments, such as acidification,

moderate heat treatment, CO

, and ultrasonic waves,

have been proposed to inhibit enzymatic browning in

fruits, vegetables, and crustaceans.

Protection from Oxygen

0015When plant organs cannot be blanched, protection

from air constitutes another way of preventing

browning. Prevention fails, however, as soon as the

anoxic conditions no longer hold, and color changes

are even accelerated by the destruction of cell struc-

tures caused by freezing and thawing.

0016In the food industry, products are protected from

oxygen using a variety of methods, either singly or

combined.

0017Airtight packages These are indispensable for pre-

venting the browning of sensitive fruits with active

oxidase systems. In hermetically sealed metal cans,

pitted cherries in syrup do not brown, even at

7



C, whereas spoilage occurs very rapidly if the

container is not airtight. The differences between air-

tight and nonairtight packages increase as the tem-

perature rises. Browning is also a function of the

degree of can filling, and hence the quantity of

residual air in the headspace.

0018Partial vacuum A partial vacuum of 380 mmHg is

enough to block the browning of frozen peaches in

syrup stored at 7



C. However, stronger vacuums

should be avoided since, on thawing, raw fruit will

have an unpleasant spongy consistency, and fruit

packaged in syrup will tend to acquire an unwanted

translucent appearance. (See Chilled Storage: Pack-

aging Under Vacuum.)

0019Oxygen-poor atmospheres Browning can also be

avoided by keeping the foodstuff in oxygen-poor

atmospheres. This can be achieved chemically, or by

use of an inert gas. The chemical approach involves

the enzymatic oxidation of the sugar present in the

medium (enzymatic deoxygenation), thus leading to

the consumption of oxygen trapped within an airtight

package. The sugar combines with oxygen under the

effect of added glucose oxidase, giving gluconic acid.

This process has been shown to be effective in labora-

tory tests with peaches, but to our knowledge has not

undergone industrial development.

0020The use of carbon dioxide or pure nitrogen atmos-

pheres also effectively protects frozen fruits against

enzymatic browning. Nitrogen, furthermore, affords

better protection of the original flavor and aroma.

0021Minimally processed products (i.e., ready-to-use,

cut fruits and vegetables) are very susceptible to

enzymatic browning since they are wounded, living

BROWNING/Enzymatic – Technical Aspects and Assays 687

tissues. In this case, complete removal of oxygen is

inapplicable due to the risk of metabolic deviations

caused by anaerobic conditions. According to the

type of plant material, the packaging film has to

be chosen by taking into consideration both the

film-selective permeability towards oxygen, carbon

dioxide and water on the one hand, and the plant

respiratory metabolism on the other, in order to

obtain the optimum atmosphere composition

allowing an increased shelf-life upon storage at

refrigerated temperatures.

0022 Sugaring of fruits In this process, fruit is immersed

in a covering liquid, either of a chosen concentration

of sugar syrup, or of the fruit’s own juice drawn out

by contact with dry sugar.

0023 Sugar (generally sucrose) has several effects. It

increases the osmotic pressure of the surrounding

liquid, which contributes to the reduction in size of

ice crystals on freezing, thereby protecting cellular

structures. It also increases the viscosity of the

medium, thus slowing the rate of enzymatic spoilage

reactions. It may also have a slight inhibitory effect on

the enzymes of browning when the syrup concentra-

tion exceeds 20% (w/w). (See Sucrose: Properties and

Determination.)

0024 Sugar is also a well-known and valued enhancer of

taste and aroma. However, it does result in some

dilution of aroma when used in the form of syrup.

This defect is attenuated when sugar is added to fruit

in the dry form, but severe changes in consistency

result, thus harming the fruit’s appearance. In add-

ition, the exuded juice collects in the bottom of

the package and only effectively protects part of the

package contents.

0025 In commercial practice, sugaring is only used with

frozen fruits intended for the manufacture of juice,

syrup, liqueur, or pure

e; the concentrations used

range from 1:9 to, more commonly, 1:4 (dry sugar:

fruit).

0026 Frozen fruits intended for direct consumption are

frequently packaged under a covering syrup. The

syrup concentrations are about 30–40



Brix for cher-

ries, pears, and plums, 40–50



Brix for bananas,

apples, and nectarines, and 40–60



Brix for straw-

berries, apricots, and peaches. None the less, syrup

alone inadequately protects the most sensitive fruits,

and an antioxidant is therefore incorporated. The

most used antioxidant is ascorbic acid, and its deriva-

tives, although other substances such as citric acid are

employed, notably in the case of peeled, frozen

bananas. The covering liquid comprises either a

syrup of pure sucrose, or a mixture of sucrose and

maize glucose. (See Antioxidants: Natural Antioxi-

dants; Synthetic Antioxidants.)

Chemical Methods of Prevention

0027Numerous chemical compounds inhibit enzymatic

browning. Of these, few combine effectiveness with

recognized innocuousness, unaltered product flavor,

and reasonable cost. The use of most of these com-

pounds is banned by legislation.

0028In the case of plant foodstuffs, the legislation of

most countries only permits ascorbic acid and its

derivatives, sodium chloride and, within stricter

limits, citric acid and sulfur dioxide (as well as its

derivatives, sodium sulfite and sodium metabisulfite).

Recently, the GRAS (generally recognized as safe)

status has been attributed to the 4-hexylresorcinol

for use in the prevention of shrimp melanosis (black

spot). (See Legislation: Additives.)

Ascorbic Acid

0029Ascorbic acid, which, with its first oxidation product

dehydroascorbic acid, constitutes vitamin C, reduces

o-quinones progressively as they are formed. Cate-

chol oxidase, which indirectly controls the oxidation

of ascorbic acid, is virtually unaffected during the

reaction, so that browning begins as soon as the

reductant has been consumed. (See Ascorbic Acid:

Properties and Determination.)

0030In practice, concentrations of 0.1–0.3% prove

adequate when, as is often the case, the external

conditions are unfavorable to browning, i.e., low

temperature, acidic and poorly oxygenated medium.

0031Ascorbic acid is effective above all when thor-

oughly mixed in liquid or paste products (apple

juice, pure

e). It is also used at concentrations of 0.2–

0.25% in syrups covering frozen fruit to enhance the

protective effect against enzymatic browning.

0032One of the drawbacks of ascorbic acid (apart from

its relatively high cost) is that it is readily oxidized

and rapidly disappears from the medium. To counter

this rapid disappearance various derivatives have

been proposed as substitutes, such as ascorbic acid-

2-phosphate (AAP), or ascorbic acid-triphosphate

(AATP). These compounds are not reducing agents,

and therefore remain stable in the presence of oxygen,

but they do hydrolyze progressively, releasing ascor-

bic acid under the action of the phosphatases present

in the plant tissue. Such compounds are not, however,

effective if the medium is too acid (e.g., apple juice,

pH 3.7), since the activity of phosphatases is then too

low. They are effective, though, in solutions of soaked,

sliced apples (0.8%) in cold storage.

0033Ascorbyl palmitate is a fat-soluble substance which

over a long period is more effective than ascorbic

acid in fruit juice when used at a concentration of

200 p.p.m. Its poor aqueous solubility, however,

makes it ineffective in soaking solutions.

688 BROWNING/Enzymatic – Technical Aspects and Assays

Sodium Chloride

0034 Salt is frequently used in the food industry to slow

microbial growth (bacteria, fungi), but is inad-

equately protective at low concentrations and imparts

an unwanted salty flavor at high concentrations. At a

pH below 5.5, however, salt is a good inhibitor of

enzymatic browning, hence the value of using it in

conjunction with citric acid for neutral plant prod-

ucts, such as cultivated mushrooms. (See Preservation

of Food.)

0035 Sodium chloride is sometimes used at low concen-

trations (1–2%) to afford temporary protection from

surface browning of sliced, peeled apples intended for

freezing, or to be sold as ready-to-use fresh foods. The

soaking time is short, usually less than 1 min.

0036 Used in combination with ascorbic acid, sodium

chloride prolongs the duration of action of the acid.

Citric Acid

0037 Oxidative enzyme activity is very pH-dependent (See

previous article Browning: Toxicology of Nonenzy-

matic Browning). Highly acidic (pH < 2.5) or basic

(pH > 10) media which result in protein denaturation

destroy its activity. Although alkalization of the

medium in which fruits are immersed is undesirable

(spontaneous browning following nonenzymatic oxi-

dation of o-diphenols), acidification can prove advan-

tageous in certain circumstances. Apart from acetic

acid used in vinegar production, citric acid is virtually

the only substance authorized for this use. It is some-

times used in deep-freezing and in fruit conserves in

syrup; thus, it is used to neutralize the rinsing water of

halved, chemically peeled peaches, and at the same

time to protect the fruit temporarily against enzym-

atic browning. The dose used is about 1% of the

volume of water. (See Acids: Natural Acids and Acid-

ulants; Enzymes: Functions and Characteristics.)

Sulfur Dioxide and its Derivatives

0038 Sulfur dioxide, which is an antiseptic widely used in

foodstuffs, is by far the most effective of currently

authorized chemical inhibitors of browning. It even

acts at very low concentrations and is inexpensive.

It can also be used with certain fruits to eliminate

unwanted red pigmentation, as in the case of

Bigarreau Napole

on cherries, used in confectionery,

which must first be decolorized using sulfite to insure

that the pigment does not turn brown during heat

treatment.

0039 As an inhibitor of browning, sulfur dioxide

has three actions: formation of colorless addition

products with (colored) o-quinones; reduction of

o-quinones to their original o-diphenols (colorless);

denaturation of catechol oxidase (See Browning:

Toxicology of Nonenzymatic Browning). Sulfur diox-

ide also reacts with many other chemical constituents

of plant tissues (notably ketones and aldehydes). The

strength of the chemical bonds formed varies with the

pH and the type of molecule involved. It would

appear that such products are the cause of the abnor-

mal flavors which sometimes develop when sulfur

dioxide is used at too high a concentration and is

not subsequently eliminated.

0040Sulfur dioxide can be used in the form of a gas or in

aqueous solution as sodium sulfite or disulfite. As a

disulfite, it acts less rapidly, but is more easily con-

trolled and results in a few flavor changes.

0041Of deep-frozen products, apples and apricots are

most suited to sulfur dioxide treatment. Fruit slices or

halved fruits are soaked for 3–4 min in solutions

containing 0.4–0.5% sulfur dioxide.

0042High doses are sometimes authorized and used to

stabilize fruit pulp intended for the making of jams of

standard quality (antiseptic action) and to clear fruits

before drying (notably apricots). In this case, how-

ever, these semiprocessed products will lose most of

the treatment compound during subsequent oper-

ations. Nevertheless, the tendency of national and

international regulations is progressively to restrict

the use of sulfur dioxide, or even to ban it altogether,

since it has been shown on occasion to be implicated

in severe disorders in asthmatics.

4-Hexylresorcinol

00434-Hexylresorcinol is probably the most promising

among the recently identified browning inhibitors.

This compound has obtained the GRAS status for

use in the prevention of shrimp melanosis. In this

case, a low concentration of 4-hexylresorcinol, up to

25-fold lower than sulfites, is required to obtain a

comparable browning inhibition. Moreover, several

studies have shown that this compound used either

alone or in combination with ascorbic acid could

adequately replace sulfites in different plant food-

stuffs such as apple slices, potatoes, avocadoes, or

apple and grape juices.

Assays for Evaluation of Browning

0044Accurate methods are required for the measurement

of browning in tissue slices and extracts. This need is

obvious when different cultivars are to be compared

for susceptibility to browning or for evaluation

of experimental treatments designed to control

enzymatic browning.

0045Basically, two kinds of techniques are available.

The first uses absorbance measurements, usually in

the 400-nm region, on solutions after extraction and

purification of the brown pigments. The second uses

BROWNING/Enzymatic – Technical Aspects and Assays 689

tristimulus reflectance colorimetry and can be applied

directly to cut surfaces or fruit pure

e. Although both

methods are easy and rapid, they do have serious

disadvantages. (See Spectroscopy: Visible Spectros-

copy and Colorimetry.)

0046 Absorbance measurements evaluate only the sol-

uble pigments. It is well known that, as the reaction

proceeds, polymerization occurs (See preceding article

Browning: Toxicology of Nonenzymatic Browning)

and the solubility of a large part of the brown pig-

ments decreases. The insoluble entities are eliminated

during the filtration and centrifugation steps in the

purification process. Moreover, according to the

kind of pigments, which in turn depend on the ori-

ginal phenols and on their relative proportions, the

wavelength of maximum absorbance ranges between

360 and 500 nm in the visible region. Therefore,

absorbance measurements at a single wavelength

are poorly correlated with the visual evaluation of

browning.

0047 Most of the data obtained in tristimulus reflectance

colorimetry are now given as L (lightness), a (green-

ness or redness), and b (blueness or yellowness)

values, or a combination of these factors. However,

the values obtained are highly dependent on the

method of measurement and on the state of the sur-

face of the examined object. Thus, during browning,

variations in tristimulus data are the result of both

chemical and physical changes, the relative import-

ance of the two processes being difficult to assess.

Most authors use the decrease in lightness dL (i.e.,

the difference in L values before and after browning)

to evaluate the extent of browning. Some authors

have proposed a more sophisticated parameter, the

color difference (before and after browning) dE,

which can be calculated as:

dE ¼ H½ðdLÞ

þðdaÞ

þðbÞ



0048 This value gives the distance in color space between

two colors but not the direction in which they differ.

Moreover, neither dL nor dE gives an idea of the

resulting perceived color.

0049 In intervarietal comparisons, numerous authors

have tried to correlate the extent of browning, meas-

ured by one of the above methods with the phenolic

content and/or the PPO activity (Table 1). Clearly,

there is no clear-cut relationship between browning

and any of these chemical parameters for all fruits.

Thus, depending on the fruit species, a correlation can

be obtained with both the parameters, one of them,

or neither. There are two potential explanations for

these difficulties. First, methods for evaluation of

browning are only approximate and improved speci-

ficity is required. Second, the chemical parameters,

and especially, the total phenolic content, are prob-

ably not sufficient to explain the extent of browning.

In the preceding article Browning: Toxicology of

Nonenzymatic Browning, it has been stressed that

PPO has a wide specificity towards phenolic com-

pounds, which are enzyme substrates to varying

degrees. Moreover, the pigments resulting from

these phenolics vary widely in color intensity and

hue. Therefore, the relative balance among the differ-

ent classes of phenols as well as the total phenol

content are probably fundamental in explaining the

degree of browning. Studies carried out both on apple

cultivars and on model solutions of phenolic com-

pounds oxidized under standardized conditions by

purified apple PPO illustrate these remarks. Thus,

the browning susceptibility of apple cultivars can be

adequately determined by the simultaneous measure-

ments of soluble (absorbance at 400 nm) and insol-

uble (lightness) brown pigments. Soluble pigments

are correlated to the amount of hydroxycinnamic

(mainly chlorogenic acid) esters degraded, whereas

insoluble pigments are correlated to the flavan-3-ols

(()-epicatechin and procyanidin B

) degraded. Simi-

larly, in model solutions containing both chlorogenic

acid and ()epicatechin oxidized by a constant

amount of purified apple PPO, the tristimulus color

values and the absorbance values at 400 nm are

strongly correlated to the initial and degraded

amounts of chlorogenic acid and ()-epicatechin

tbl0001Table 1 Relations between the extent of browning, phenolic

content, and polyphenol oxidase (PPO) activity in different fruit

species

Correlationwith

Species Number of cultivars Phenolics PPO Method used

Apple 7 0 0 S (A ¼ 440 nm)

3 þ 0S(A ¼ 440 nm)

40þ S(A ¼ 440 nm)

6–8 þ R (tristimulus)

Avocado 3 0 þ Visual

60þ Visual

Banana 3–5 0 þ Visual

Eggplant 3 þ Visual

Grape 9 (red) 0 þ S(A ¼ 430 nm)

19 (white) 0 0 S (A ¼ 430 nm)

Olive 6 þþR (545 nm)

5 þ S(A ¼ 410 nm)

5 þþS(A ¼ 410 nm)

9 þ S(A ¼ 400 nm)

Peach 6 þ S(A ¼ 395 nm)

Pear 6 þ R (540 nm)

Adapted from Macheix JJ, Fleuriet A and Billot J (1990) Fruit Phenolics,

Boca Raton: CRC Press.

0, no correlation; þ, positive correlation.

S, Absorbance data; R, reflectance data (either at a single wavelength or

tristimulus).

690 BROWNING/Enzymatic – Technical Aspects and Assays

(Table 2). Multilinear regression analysis indicates

that both phenolics play a role in the darkening (de-

crease in L) and yellowness (increase in b). Chloro-

genic acid contributes to redness (increase in a)

whereas ()-epicatechin is almost without effect.

The same kind of experiments carried out on phenolic

compounds extracted from nine different apple culti-

vars shows that the browning extent of the phenolic

solutions after oxidation by purified apple PPO is

strongly correlated to the initial and degraded

amounts of hydroxycinnamic derivatives and flavan-

3-ols (Table 3). However, besides these two classes,

the flavonols and the dihydrochalcones appear to

have a significant influence on the color development

(mainly on the b value). Lastly, additional factors

(other than phenols) may also be influential: some

are chemical, such as the acidity and the concentra-

tion of reductants (e.g., as ascorbic acid), while others

are physical, such as texture. The relative importance

of each of these factors probably varies greatly from

one species to another.

0050 In conclusion, enzymatic browning can be con-

trolled by physical treatments, which are not applic-

able in all circumstances, and by the use of chemical

compounds. Very few of the latter are presently avail-

able to prevent enzymatic browning in foodstuffs,

and none is ideal.

0051 Ascorbic acid is a good inhibitor of browning, but

cannot always be used and is, moreover, relatively

expensive. Citric acid and sodium chloride have

only a limited usefulness and are valuable principally

as a complement to another treatment. Only sulfur

dioxide is effective and inexpensive, but it has un-

desirable side-effects and research is therefore neces-

sary to find a suitable substitute. For certain food

products such as shrimp, 4-hexylresorcinol could be

this substitute.

0052Existing techniques for the measurement of

browning provide useful information on differences

in color and color intensity. However, these tech-

niques give a very imperfect assessment of the degree

of browning as noted by an observer. The main reason

is that human ocular acuity in perception of a differ-

ence in hue or color decreases sharply as the color of

the object darkens or tends towards saturation. Much

more work will be necessary to establish for each

product a strict correlation between instrumental

measurements and consumer perception.

See also: Acids: Natural Acids and Acidulants;

Antioxidants: Natural Antioxidants; Synthetic

Antioxidants; Ascorbic Acid: Properties and

Determination; Browning: Toxicology of Nonenzymatic

Browning; Chilled Storage: Packaging Under Vacuum;

Enzymes: Functions and Characteristics; Legislation:

Additives; Phenolic Compounds; Preservation of

Food; Spectroscopy: Visible Spectroscopy and

Colorimetry; Sucrose: Properties and Determination

tbl0002 Table 2 Equations relating tristimulus color parameters L (lightness), a (redness), and b (yellowness) to initial (in) and oxidized (ox)

chlorogenic acid (CA) and ()-epicatechin (EP) in binary mixtures obtained by multilinear regression analysis

Tristimulus color parameters Initial amount Oxidized amount

L 99–21.0 CA

–25.2 EP

r ¼ 0.96 98.5–19.6 CA

–30.4 EP

r ¼ 0.97

a 4.21 þ 27.9 CA

þ 0.48 EP

r ¼ 0.95 4.57 þ 31.8 CA

–2.0 EP

r ¼ 0.95

b 2.58 þ 51.5 CA

þ 98.2 EP

r ¼ 0.98 4.62 þ 41.3 CA

þ 118.3 EP

r ¼ 0.99

Adapted from Goupy P, Amiot MJ, Richard-Forget F, Duprat F, Aubert S and Nicolas J (1995) Enzymatic browning of model solutions and apple phenolic

extracts by apple polyphenoxidase. Journal of Food Science 60: 497–501, 505.

tbl0003 Table 3 Equations relating tristimulus color parameters L (lightness), a (redness), and b (yellowness) to initial (in) and oxidized (ox)

amount in each phenolic class in apples obtained by multilinear regression analysis

Tr i s t i m u l u s

color

parameters

Initialamount Oxidized amount

L 97.6–5.61 HD

–3.23 FO

þ2.1 FA

–2.39 DC

r ¼ 0.87 98.1–9.29 HD

–0.04 FO

þ 0.53 FA

þ 0.02 DC

r ¼ 0.99

a 0.02 þ 2.75 HD

þ 0.49 FO

–0.74 FA

–0.9 DC

r ¼ 0.92 0.47 þ 2.99 HD

þ 0.72 FO

0.93 FA

– 1.14 DC

r ¼ 0.92

b 5.74 þ 18.3 HD

þ 16.2 FO

–5.46 FA

þ 14.9 DC

r ¼ 0.84 3.92 þ 38 HD

– 1.42 FO

þ 2.77 FA

þ 4.88 DC

r ¼ 0.99

HD, hydroxycinnamic acid derivatives; FO, flavonols; FA, flavanols; DC, dihydrochalcones.

Adapted from Goupy P, Amiot MJ, Richard-Forget F, Duprat F, Aubert S and Nicolas J (1995) Enzymatic browning of model solutions and apple phenolic

extracts by apple polyphenoxidase. Journal of Food Science 60: 497–501, 505.

BROWNING/Enzymatic – Technical Aspects and Assays 691

Further Reading

Amiot MJ, Tacchini M, Aubert S and Nicolas J (1992)

Phenolic composition and browning susceptibility of

various apple cultivars at maturity. Journal of Food

Science 57: 958–962.

Amiot MJ, Fleuriet A, Cheynier V and Nicolas J (1997)

Phenolic compounds and oxidative mechanisms in fruits

and vegetables. In: Tomas-Barberan FA, Robins RJ (eds)

Phytochemistry of Fruits and Vegetables, pp. 52–

85.Oxford: Clarendon Press.

Clydesdale FM (1984) Color measurement. In: Gruenwedel

DW and Whitaker JR (eds) Food Analysis: Principles

and Techniques I: Physical Characterization, pp. 95–

150. New York: Marcel Dekker.

Goupy P, Amiot MJ, Richard-Forget F, Duprat F, Aubert S

and Nicolas J (1995) Enzymatic browning of model

solutions and apple phenolic extracts by apple polyphen-

oxidase. Journal of Food Science 60: 497–501, 505.

Macheix JJ, Fleuriet A and Billot J (1990) Fruit Phenolics.

Boca Raton: CRC Press.

McEvily AJ and Iyengar R (1992) Inhibition of enzymatic

browning in foods and beverages. Critical Reviews in

Food Science and Nutrition 32: 253–273.

Nicolas J, Cheynier V, Fleuriet A and Rouet-Mayer MA

(1993) Polyphenols and enzymatic browning. In: Scal-

bert A (ed.) Polyphenolic Phenomena, pp. 165–

175.Paris: INRA Editions.

Nicolas JJ, Richard-Forget FC, Goupy PM, Amiot MJ and

Aubert SY (1994) Enzymatic browning reactions in

apple and apple products. Critical Reviews in Food

Science and Nutrition 34: 109–157.

Philippon J, Rouet-Mayer MA, Gallet D and Herson A

(1982) Pre

conge

lation des pe

ches entie

res a

l’usage des

conserveries de fruits au sirop. Conse

quences des diffe

entes e

tapes de la nouvelle filie

re technologique sur la

qualite

des fruits de

congele

s et sur celle du produit e

la-

bore

. Refrigeration Science and Technology 4: 302–310.:

Sapers GM, Hicks KB, Phillips JG et al. (1989) Control of

enzymatic browning in apples with ascorbic acid deriva-

tives, polyphenol oxidase inhibitors and complexing

agents. Journal of Food Science 54: 997–1002, 1012.

Vamos-Vigyazo L (1995) Prevention of enzymatic

browning in fruits and vegetables. A review of principles

and practice. In: Lee CY and Whitaker JR (eds) Enzym-

atic Browning and its Prevention, pp. 49–62. Washing-

ton: American Chemical Society.

BUCKWHEAT

G Mazza and B D Oomah, Pacific Agri-Food Research

Centre, Summerland, Canada

Buckwheat

0001 Buckwheat is a crop commonly grown for its black

or gray triangular seeds. It can also be grown as a

green manure crop, a companion crop, a cover crop, a

source of buckwheat honey, and as a pharmaceutical

plant yielding rutin. Buckwheat belongs to the Poly-

gonaceae family and is not a true cereal. Like cereals

such as wheat, corn, and rice, which belong to the

grass family, however, the grain of buckwheat is a dry

fruit.

0002 Buckwheat is believed to have originated in central

and northeastern Asia and was probably cultivated in

China during the fifth and sixth centuries. It was

introduced into Europe through Turkey and Russia

during the fourteenth and fifteenth centuries and into

North America in the seventeenth century. World

production of buckwheat peaked in the early nine-

teenth century, and has declined since then. During

the past 5 years (1995–1999) it has averaged about

2.7 million tonnes, with China and the former Soviet

Union accounting for 58 and 34% of the global pro-

duction, respectively. Other major producing coun-

tries are Brazil, Poland, the USA, Japan, Canada,

France, Bhutan, Republic of Korea, and South Africa.

Types and Cultivars

0003Although the genus Fagopyrum contains at least 15

species of buckwheat, only two are utilized as food

or feed, and wild buckwheat (F. cymosum), mainly

found as tetraploid, is used on a sporadic basis as a

green vegetable or as cattle forage. There are three

major species of buckwheat: common buckwheat,

F. esculentum, tartary buckwheat, F. tataricum, and

perennial buckwheat, F. cymosum. Common buck-

wheat is also known as F. saggitatum, and a form of

tartary buckwheat may be called F. kashmirianum.It

is generally believed, however, that perennial buck-

wheat, particularly the diploid type, is the ancestral

form of both tartary and common buckwheat.

0004Tartary buckwheat is cultivated in the Himalaya

region of India and China, in eastern Canada, and

occasionally in mountain areas of the eastern United

States. It has some frost tolerance and is therefore

692 BUCKWHEAT

grown at higher altitudes where risk of frost damage

is greater. The seeds and products made from

them are greenish in color and somewhat bitter in

taste. It is used primarily as feed or as a mixture of

wheat and buckwheat flour, and often used as a

source of rutin.

0005 Common buckwheat is by far the most economic-

ally important species of buckwheat, accounting for

over 90% of the world production. Many types,

strains, and cultivars of common buckwheat exist.

There are late and early maturing types, Japanese

and European types, summer and fall types. Within

a given type, there may be strains or varieties with tall

or short plants, gray or black seeds, white or pink

flowers. In general, however, common buckwheat

varieties from different parts of the world may be

divided into two major groups. The first group

includes tall, vigorous, late maturing, photoperiod

sensitive varieties found in Japan, Korea, southern

China, Nepal, and India. The second group is rather

insensitive to photoperiod, small and early maturing.

All of the varieties in Europe and northern China

belong to this type.

0006 Prior to 1950, producers of buckwheat planted

unnamed strains of buckwheat harvested from their

own fields or obtained from their neighbors or local

stores. Named varieties developed through plant

breeding were first released in the 1950s. Tokyo, the

oldest of the named cultivars introduced into North

America, was licensed by the Agriculture Canada

Research Station, Ottawa, in 1955. Other cultivars

licensed for production in Canada are: Tempest,

Mancan, and Manor, all developed at the Agriculture

Canada Research Station, Morden, Manitoba since

1965. Mancan, which has large, dark brown seeds,

thick stems and large leaves, is the Canadian cultivar

preferred in the Japanese market because of its large

seed, desirable flavor and color, and high yield of

groats in the milling process. Recently, these older

cultivars have been supplemented by newer cultivars

such as AC Manisoba, Koban, and Koto. These new

cultivars produce larger seeds and have an increased

seed yield.

0007 Cultivars licensed in the USA are Pennquad, re-

leased by the Pennsylvania Agricultural Experimental

Station and the USDA in 1968, and Giant American,

a Japanese-type cultivar apparently selected by a

Minnesota farmer.

0008 Cultivars developed in the former USSR since the

1950s include: Victoria, Galleya, Eneida, Podolyaka,

Diadema, Aelita, and Aestoria. Representative culti-

vars from other areas of the world include: Pulawska,

Emka, and Hruszowska from Poland: Bednja 4n from

Yugoslavia; and Botan-Soba, Shinano No. 1, Kyushu-

Akisoba Shinshu, and Miyazaki Oosoba from Japan.

Plant and Seed Morphology

0009Buckwheat plant is a broad-leaved, erect, herbaceous

plant that grows to a height of 0.70–1.5 m. It has a

main stem and several branches, and can reach full

maturity in less than 90 days. The stem is usually

grooved, succulent, and hollow except for the

nodes. Before maturity, the stems and branches

are green to red in color, and after maturity, they

become brown. The plant has a shallow taproot

from which branched, lateral roots arise. Its root

system is less extensive than that of the cereals and

constitutes only 3–4% of the dry weight of the total

plant.

0010Buckwheat has an indeterminate flowering habit.

The flowers of common buckwheat are perfect but

incomplete. They have no petals, but the calyx is

composed of five petal-like sepals that are usually

white, but may also be pink or red. The flowers are

arranged in dense clusters at the ends of branches or

on short pedicels arising from the axils of the leaves.

Common buckwheat has plants bearing one of two

types of flowers. The pin-type flower has long styles,

or female parts, and short stamens, or male parts,

and the thrum-type has long styles and short pistils.

The pistil consists of a one-celled superior ovary and a

three-part style with knoblike stigmas and is sur-

rounded by eight stamens. Nectar-secreting glands

are located at the base of the ovary. The plants of

common buckwheat are generally self-infertile, as

self-fertilization is prevented by self-incompatibility.

Seed production is usually dependent on cross-

pollination between the pin and thrum flowers.

Honey bees and leaf-cutter bees are effective pollin-

ators. They increase seed set and seed yield.

0011The buckwheat kernel is a triangular, dry fruit

(achene), 4–9 mm in length, consisting of a hull or

pericarp, spermoderm, endosperm, and embryo.

Large seeds tend to be concave-sided, and small

seeds are usually convex-sided. The hull may be

glossy, gray, brown, or black, and it may be solid or

mottled. It may be either smooth or rough with lateral

furrows. The hulls represent 17–26% (in tartary

buckwheat 30–35%) of the kernel weight. Diploid

varieties usually have less hull than tetraploids.

Structure of Kernel

0012Scanning electron microscopy of the buckwheat

kernel has revealed that the hull, spermoderm, endo-

sperm, and embryo are each composed of several

layers. For the hull, these are in order from the outside

toward the inside: epicarp, fiber layers, parenchyma

cells, and endocarp. The spermoderm is composed of

the outer epiderm, the spongy parenchyma, and the

BUCKWHEAT 693

inner epiderm. The endosperm is composed of an

aleurone layer, 10–15 mm thick, and subaleurone

endosperm containing starch granules surrounded

by proteinaceous matrix. The embryo, with its two

cotyledons, extends through the starchy endosperm.

The terminal parts of cotyledons are often parallel

under the kernel surface.

Composition

0013 Proximate composition and selected mineral profiles

of Mancan, Manor, and Tokyo buckwheat groats are

shown in Table 1.

Carbohydrates

0014 Starch is quantitatively the major component of

buckwheat seed, and concentration varies with the

method of extraction and between cultivars. In

whole grain of common buckwheat, the starch con-

tent ranges from 59 to 70% of the dry matter. The

chemical composition of starch isolated from buck-

wheat grains differs from the composition of cereal

starches. The amylose content in buckwheat granules

varies from 15 to 52%, and its degree of polymeriza-

tion varies from 12 to 45 glucose units. Buckwheat

starch granules are irregular, 4–11 mm in size, with

noticeable flat areas due to compact packing in the

endosperm. The starch has a water binding capacity

of 79–104%, a blue value of 0.35, a swelling power

and solubility at 60



C of 2.35 and 0.5%, respectively,

an amylograph viscosity at 92



C of 640 Brabender

Units (BU), an enzyme susceptibility (percentage of

solubilized starch) of 2.63%, and an initial and

final gelatinization temperature of 61.5 and 76.0



respectively.

0015Buckwheat grains also contain 0.65–0.76%

reducing sugars, 0.79–1.16% oligosaccharides, and

0.1–0.2% nonstarchy polysaccharides. Among the

low-molecular-weight sugars, the major component

is sucrose. There is a small amount of arabinose,

xylose, glucose, and probably the disaccharide meli-

biose. (See Sucrose: Properties and Determination.)

Proteins

0016The protein content in buckwheat varies from 7 to

21%, depending on the variety and environmental

factors during growth. Most currently grown culti-

vars yield seeds with 11–15% protein. The major

protein fractions are globulins, which represent

almost half of all proteins. The globulins consist of a

major 13S legumin-like and a minor 8S vicilin-like

fractions. The 13S fraction consists of eight subunits

with molecular weights between 43 000 and 68 000.

The 8S fraction consists mainly of 57 000 polypep-

tides with a few subunits in the 26 000–36 000 range.

The albumin represents about 25% of the total seed

proteins and consists mainly of a low-molecular-mass

single-chain polypeptide of 8–16 kDa. Glutelin repre-

sents only 4% of the total seed protein. Prolamin

has been fractioned into at least two peaks by gel

filtration of Sephacryl S-200 and into three major

and several minor components by sodium dodecyl

sulfate–polyacrylamide gel electrophoresis (SDS–

PAGE). (See Protein: Chemistry.)

0017Buckwheat proteins are particularly rich in lysine.

They contain less glutamic acid and proline, and more

tbl0001 Table 1 Proximate composition, on a dry-weight basis, and selected mineral profile of three dehulled buckwheat cultivars

Assay Cultivar

Mancan (+ SD) Manor (+ SD) Tokyo (+ SD)

Moisture (g per 100 g) 16.2 + 0.9 10.1 + 0.2 10.9 + 0.1

Protein

(g per 100 g) 14.2 + 0.6 14.6 + 0.3 11.9 + 0.4

Crude fiber (g per 100 g) 1.57 + 0.30 1.21 + 0.03 1.57 + 0.10

Ash (g per 100 g) 1.85 + 0.01 1.66 + 0.01 1.39 + 0.01

Lipids

(g per 100 g) 2.6 + 0.3 2.2 + 0.3 2.1 + 0.2

Carbohydrates

(g per 100 g) 79.8 + 1.6 80.3 + 0.8 83.0 + 1.1

K (mg per 100 g) 440 + 0.005 419 + 0.009 407 + 0.005

P (mg per 100 g) 359 + 0.018 347 + 0.003 262 + 0.016

Mg (mg per 100 g) 214 + 0.002 201 + 0.012 195 + 0.010

Ca (mg per 100 g) 18 + 0.74 180.5 + 10.6 220.5 + 6.5

Fe (mg per 100 g) 2.5 + 0.18 21.4 + 0.3 21.2 + 0.9

Zn (mg per 100 g) 2.3 + 0.04 22.0 + 1.6 22.8 + 1.1

Mn (mg per 100 g) 1.0 + 0.02 10.0 + 0.5 10.2 + 0.4

Cu (mg per 100 g) 0.03 3.74 + 0.1 4.3 + 0.2

N  6.25.

Soxhlet, petroleum ether for 8 h.

By difference.

Data from Mazza G (1988) Lipid content and fatty acid composition of buckwheat seed. Cereal Chemistry 65: 122–126, with permission.

694 BUCKWHEAT