Enzymatic browning reactionsi appleand

91,84143 Montfavet Cedex, France ABSTRACT: This review examines the parameters of enzymatic browning in apple and apple products that is, phenolic compounds, polyphenoloxidases, and othe

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Enzymatic browning reactions in apple and apple products

Jacques J Nicolas a , Florence C Richard‐Forget b

, Pascale M Goupy b , Marie‐JosèpheAmiot b & Serge Y Aubert b

Version of record first published: 29 Sep 2009

To cite this article: Jacques J Nicolas , Florence C Richard‐Forget , Pascale M Goupy , Marie‐Josèphe Amiot & Serge Y.Aubert (1994): Enzymatic browning reactions in apple and apple products, Critical Reviews in Food Science and Nutrition,34:2, 109-157

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Critical Reviews in Food Science and Nutrition, 34(2): 109-157 (1994)

Enzymatic Browning Reactions in Apple and

Apple Products

Jacques J Nicolas

Chaire de Biochimie Industrielle et Agro-Alimentaire, Conservatoire National des Arts et Metiers,

292 Rue Saint-Martin, 75141 PARIS Cedex 03, France

Florence C Richard-Forget, Pascale M Goupy, Marie-Josèphe Amiot,

and Serge Y Aubert

Laboratoire de Biochimie des Degradations, Station de Technologie des Produits V6getaux, Institut

National de la Recherche Agronomique, Domaine Saint-Paul, B.P 91,84143 Montfavet Cedex, France

ABSTRACT: This review examines the parameters of enzymatic browning in apple and apple products that is,

phenolic compounds, polyphenoloxidases, and other factors (ascorbic acid and peroxidases), both qualitatively and quantitatively Then the relationships between intensity of browning and the browning parameters are discussed, including a paragraph on the methods used for browning evaluation Finally, the different methods for the control of browning are presented.

KEY WORDS: apple, browning, polyphenols, enzymes, polyphenoloxidases

I INTRODUCTION

Based on size of production, the apple is one

of the main fruit crops in the world In 1980, the

world apple production exceeded 35 million t,

corresponding to fourth place after grapes, citrus,

and banana.356 The four major producers are the

U.S., France, Italy, and China More than half of

the apple crop is sold in the fresh produce market

Depending on the year, 40 to 60% of apple

pro-duction is used by the industry Juice, either as

single-strength sweet juice or as fermented juice

(cider), apple sauce, and slices, are the main

prod-ucts of processed apples According to Salunkhe

et al.,357 one fourth of the fruit and vegetables

harvested is never consumed because of spoilage

during postharvest manipulations or processing

Concerning apples, the postharvest losses are

considerably less, because it has been estimated

at less than 10% for the fresh market by Sparks408

and about 14% in developing countries by

Steppe.410 The main causes of losses are physical

injuries, physiological disorders or diseases

dur-ing storage,96 and improper conditions for

pro-cessing In most, if not all cases, the observedsymptoms correspond to a discoloration of theapple or the apple products Thus, bruising, which

is the most common defect of apples186 seen onthe market, results in a flattened area on the side

of the fruit with the flesh browned beneath.352-356Similarly, bitter pit,161 superficial171 and senes-cent scalds, internal (senescent or low-tempera-ture) breakdown,15-166 watercore,239 and coreflush223 all result in browning of either the skin orthe internal tissue Finally, juice, puree, and appleslices that are badly processed brown intenselyand give the final products a bad appearance which

is rejected by the consumer.74287-317 These generaldiscoloration phenomena are mainly related toenzymatic browning However, browning can alsooriginate from nonenzymatic reactions such asthe Maillard reaction,16-106217 which occurs mainly

in heat-processed apple products.228-244328-462Basically, enzymatic browning can be de-fined as an initial enzymatic oxidation of phenolsinto slightly colored quinones.13-47-101-185-251-350-470These quinones are then subjected to further reac-tions, enzymically catalyzed or not, leading to the

formation of pigments The colors of the latter

differ widely in hue and intensity, following the

phenols from which they originate and the

environmental factors of the oxidation

reac-tion _50.113,184,243,298,308-311,325,351,395,463 E n z y m a t i c

browning is mainly associated with polyphenol

oxidases, which are able to act on phenols in

the presence of oxygen.253"256-338-350-489-510 Two

kinds of enzymes are classified under this trivial

name

The first class, catechol oxidases (E.C 1.10.3.1),catalyze two distinct reactions (Figure 1): the

hydroxylation of monophenols in o-diphenols

(reaction 1) and the oxidation of o-diphenols in

o-quinones (reaction 2) These two enzymatic

reactions consume oxygen and are referred to

as monophenolase (or cresolase) activity and

o-diphenolase (or catecholase) activity, tively The former activity is not always present,and when both activities are present, the ratio ofcresolase/catecholase activities varies widely from

respec-1 to respec-10 or even 40.34-443The second class, laccases (E.C.I 10.3.2),oxidizes o-diphenols as well as p-diphenols (Fig-ure 2), forming their corresponding quinones.Besides other differences in properties,256-475 theunique ability to oxidize p-diphenols can be used

to distinguish laccase activity from that of thefirst class

The nomenclature of these enzymes176 is what confusing because besides the two numbersE.C.I.10.3.1 and E.C.I 10.3.2, a third one exists,E.C.I.14.18.1 It is referred to as monophenolmonooxygenase (tyrosinase) and corresponds to

some-OH CRESOLASE 1/2 0,

OH CATECHOLASE ,0H V2 0 2

H 2 0

(2)

FIGURE 1 Reactions catalyzed by polyphenoloxidases (E.C 1.14.18.1 and

E.C 1.10.3.1) (1) Hydroxylation of monophenol to o-diphenol; (2) tion of o-diphenol to o-quinone.

dehydrogena-FIGURE 2 Reactions catalyzed by laccases (E.C 1.10.3.2).

the same enzymes as E.C.I.10.3.1, which always

catalyze the hydroxylation of monophenols

The peroxidases (E.C.I 11.1.7) can also be

considered as participating in enzymatic

brown-ing These enzymes, whose primary function is to

oxidize hydrogen donors at the expense of

perox-ides, are highly specific for hydrogen peroxide

On the other hand, they accept a wide range of

hydrogen donors, including polyphenols.36-342-343-443

The primary products of oxidized phenols are

probably quinones similar to those obtained with

polyphenol oxidases Although peroxidases are

distributed widely, especially in plants, they

gen-erally appear to be little involved in enzymatic

browning of fruits and vegetables following a

mechanical stress The explanation could be that

the peroxidase activity is limited by the internal

level of hydrogen peroxide However, their

in-volvement in a slow process such as internal

browning is possible.423424 Nevertheless, the

di-rect involvement of peroxidase in enzymatic

browning of apple and apple products remains

questionable, as does that of laccases The latter

enzymes are mainly present in fungi and in

cer-tain higher plants256 and are almost always absent

in sound fruits and vegetables, with the exception

of peaches146 and apricots.87 Therefore, the bulk

of the discussion below concentrates on the two

main factors of apple enzymatic browning, that

is, phenolic substrates and polyphenoloxidase

(E.C.I.10.3.1) activity, with a small part devoted

to other factors such as peroxidase and ascorbic

acid The different ways to estimate browning,

the correlations among the intensity of browning

and its causal factors, and, finally, the control of

browning are then examined successively

II PARAMETERS OF ENZYMATIC

BROWNING IN APPLE AND APPLE

PRODUCTS

A Phenolic Compounds of Apple and

Apple Products

1 Total and Individual Phenolics in Ripe

Apple Fruit and Apple Juice

A great number of works have been devoted

to the study of phenolics in apple and apple

prod-lics, six classes are present in apple fruit:hydroxycinnamic derivatives, flavonols, antho-cyanins, dihydrochalcones, monomeric flavan-3-ols, and tannins.236 Since the first modernmonograph published by Harborne142-144 in 1964,considerable information has been compiled onthe classes of these phenolics,135-169-238-321-411-454 aswell as on methodologies for their purification,isolation, quantitation, and structure elucida-tion i27.143.162^™^.455

The quantitation of total phenolic compounds

is not accurate because of the extraction method,which often cannot guarantee a total solubiliza-tion of all phenolics, and to the assay method,which is unavoidably a compromise on the reac-tivity or absorption characteristics of the differentclasses of phenols.27-28-81-234-396-398-413

Nevertheless, these measurements still areused for rough comparisons among samples.Indeed, a wide variability is observed in thetotal phenolic contents of ripe fruits and applejuices (Table 1) Obviously, this variation can bepartly attributed to the different methods used bythe authors for the quantitation of phenols How-ever, even when the same method is used, consid-erable variation in results is still evident.69150-448

As in other fruits, the levels of phenolic pounds in apple are highly dependent on manyfactors, such as variety, stage of maturity, andenvironmental factors.236-491 Besides quantitativevariations among the classes of phenolic com-pounds, large variations are also apparent in thequalitative distribution, which are caused by ge-netic and external factors Modern methods, par-ticularly high-performance liquid chromatography,

com-considerable progress in both the tion and quantitation of individual phenolics inapple fruit The main phenolics identified in applefruit and apple products are given in Table 2, andthe contents of the different classes are given inTable 3 for the ripe fruits The tables show that:

separa-1 In the cortex, three phenolics accounted formore than 90% of the total phenolic con-tent for most cultivars: one caffeoyl quinicacid (chlorogenic acid) and two flavan-3-ols ((-)-epicatechin and procyanidin B2)

2 In the peel, the hydroxycinnamic acid

TABLE 1 Total Phenolic Compounds in Ripe Apple Fruit and Apple Juice

0.9-2.10.6-1.20.2-1.90.5-0.7

Apple Juice

Total

0.7-2.5 0.6-2.0 0.5-17 0.15-0.21 0.6-0.8 1.0-1.9 0.49-0.84 0.5-11

0.16-0.44

0.15-0.580.24-0.620.14-0.370.21-15.20.37-0.620.02-0.140.3-2.7

1.49

Ref.

69448157516449381288450323193150172

46430240740763647412614

Note: Values are given in grams per kilogram (FW ripe fruit) or in grams per liter

(juice).

a Chlorogenic acid equivalent.

b o-Diphenols (chlorogenic acid equivalent).

c Gatechin equivalent.

d Tannins.

8 Not given.

' Gallic acid equivalent.

a o-Diphenols (catechol equivalent).

h Tannic acid equivalent.

whereas the flavan-3-ol and flavonolderivatives constitute the major part of thephenolic compounds

3 In the hydroxycinnamic derivatives, quinic

acid is the only hydroxyacid that formsesters with caffeic403 and /7-coumaric41-492acids Although not detected in the originalfruit, 5'-feruloyl quinic acid has beenreported in cell suspensions from applefruit.195 In the latter case, it accumulates inthe latter stage of cell culture growth after

the exponential growth phase Similarbehavior was shown for the sinapoylglucose ester, which was observed only in

in vitro cell suspension cultures from apple

parenchyma.195-307 In apple fruit, besidesquinic acid, hydroxycinnamic acids formesters with sugars and, more precisely, withglucose Thus, Macheix231-232 reported that1-0-p-coumaroyl glucose was one of the majorp-coumaric derivatives in apple (var CalviUeblanc) together with several p-coumaroyl

TABLE 2

Main Phenolics Identified in Apple Fruit and Apple Products (According to Reference 236, with Modifications)

P h e n o l i c A c i d s 4 ' 9 5 ' 1 0 7 - 1 1 2 - 1 6 5 ' 2 2 9 ' 2 3 2 ' 2 3 5 ' 2 6 6 ' 2 7 9 ' 3 0 6 ' 3 3 6 ' 4 0 3 ' 4 8 7 ' 4 9 0 ' 4 9 2 ' 4 9 7

5'-caffeoylquinic, a 4'-caffeoylquinic, 3'-caffeoylquinic, 5'-p-coumaroylquinic, 4'-p-coumaroylquinic,

3'-p-coumaroylquinic, p-coumaroylglucose, caffeoylglucose, feruloylglucose

Note: The major compounds in each class are written in boldfaced type.

a The IUPAC recommendations 177 were applied for the nomenclature of the hydroxycinnamic quinic esters Thus, 5'-caffeoylquinic is chlorogenic acid, 4'-caffeoylquinic acid is cryptochlorogenic acid, and 3'-caffeoylquinic acid is neochlorogenic acid.

b A structure of quercetin-3-O-a-galactoside was proposed by Teuber and Herrmann 425

0 Present in apple cultivars with red-colored skins.

4.

quinic esters Similarly, in their compilation,

Risch and Herrmann336 indicated that the

glucose esters in apple were mainly

p-coumaroyl and feruloyl glucose esters

Glucoside derivatives in which the phenolic

group is engaged in bonding with glucose

are not present in apple fruit, although they

are often encountered in plants.159 Finally,

according to Macheix et al.,237 the overall

balance of the three hydroxycinnamic acids

(either as quinic esters or as glucose esters)

ranges between 75 and 94% for caffeic acid,

5 and 20% forp-coumaric acid, and 1 to 5%

for ferulic acid

In the flavan-3-ol derivatives, procyanidin

B2 (dimer of two (—)-epicatechin units

linked in a bond between C4, the "upper

unit", and C8, the "lower unit") and(-)-epicatechin are the most abundant.35-306The (+)-catechin isomer is also alwayspresent in apple fruit, although sometimesonly as traces.336 According to Mosel andHerrmann,2 8 3 the mean content of(-)-epicatechin in apples is five timeshigher than that of (+)-catechin The latterauthors indicated that (+)-gallocatechin and(—)-epigallocatechin, which are normallyabsent in apples, have been found in somecultivars283-284 in relation to certain climaticfactors and growing conditions The dataconcerning the polymeric forms of flavan-3-ols ("tannins") are scarce, probably owing

to the difficulties encountered in their totalextraction and precise quantitation Almost

TABLE 3 Content in Chlorogenic Acid (CG), Other Hydroxycinnamic Derivatives (HCd), Flavan-3-Ols (F3OI), Flavonols (FVOd), and Dihydrochalcones (DHC) of Ripe Apple Fruit and Apple Juice

6-51 4.3-19 a

18-173 19-55 4.2-32

9-^34-6

18-171 18-171 0.1-20 2-15 3-6 5-150 3-89

0.25-0.91 6.6-23.2

0.73-250.3-5.9

11-510-5.50.35-^.1

15-5012-56

0.96-4.9

4-33

0.2-12 3.5-16 90-261 33-124 3-21.5 13-30 5-29 98-214 98-214 230-650 19.4-81.5 45.5-143 25-127 100-960 50-450 120-575

Apple Juice

0.23-0.45 tr-0.16 3.2-20

0.4-3.3

0.3-46.5 0.1-2.5

FVOd

0-2.9 6-26

4-15 tr-3.8 0.02-O.24

16-52 15.8-142 154-285 78-107 120-550 81-353

0.6-65 8.7-33 18-33 8-220 7-211

Ref.

23669172

158 336 283 5,7 322

305, 306 266 35 4 12 322

305, 306

85, 266 35 4 436

12

1.4-2.7 108

— 33 0.48-3.5 80 0.1-1.6 407

Note: All results are given in milligrams per 100 g FW basis or in milligrams per 100 ml (juice)

except those marked by an asterisk (*), which are given in milligrams per 100 g DM basis.

a expressed as caffeic acid equivalent (contains all the caffeoyl esters).

b expressed as p-coumaric and ferulic acids (contains all the coumaroyl and feruloyl esters).

all the data are concerned with apple juicesand ciders, owing to the interest in thesensory and technological properties ofthese products.76-203-208-209-212-280-282-302-407-451-452

A considerable range existed in the totaltannin content of apple juices.80-207-210 Thus,large differences were found in thephenolics of juices obtained from a dessertapple (var Bramley) and a cider apple (var

Dabinett).208 However, the largest differencewas associated with the tannin fraction asthe Dabinett juice contained twofold more

phenolic acids, fivefold more phloridzin, andtenfold more epicatechin, but 20-fold moreprocyanidin B2 than the Bramley juice.208

5 In the flavonol derivatives, the onlycharacteristic feature is the presence ofquercetin as aglycone Methods have beenproposed for the certification of apple juicesbased on their flavonol contents.92-93Although kaempferol derivatives have beenreported by Van Buren450 and Herrmann,158their presence has never been confirmedsince then Similarly, the presence of a

diglucoside quercetin found only by Fisher1 •'

seems unlikely In apple fruit, the

diversity is at the sugar level, because six

different quercetin-3-glycosides have

been characterized fully They are all of

a pyranose form with the exception of

arabinose, which has a furanose form The

amount of each giycoside derivative

(galactose, xylose, glucose, arabinose,

rhamnose, and rutinose) and their relative

balance are highly dependent on variety

(Table 4) According to Teuber and

Herrmann425 and Oleszek et al.,294 hyperin

(the galactoside derivative) is the most

abundant

In the dihydrochalcone derivatives, two

compounds have been characterized in apple

and apple products For a long time, it was

stated that phloridzin

(phloretin-2'-0-glucoside), a characteristic compound of the

genusMalus (Rosaceae),42-468-494 was present

in leaves, stems, and seeds but absent in

fruits.493 However, a first description of

phloridzin was reported in the ethyl acetate

7.

extract of the core tissue of Mclntosh apples95and the methanol extract of peel of Democratapples.111 Then, using HPLC, this compoundwas fully characterized in the different parts

of apple fruit85-305 and in apple juice.497 Inthe same extracts, another phloretin giycosidewas also characterized corresponding to thexyloglucoside294 as well as in GoldenDelicious apple juices and jams.431-432 Thephloridzin content varied from 87 to 331 fig/g

of fresh peel in eight cultivars grown inCanada266 and from 21 to 105 |Xg/g for fivecultivars grown in Spain.306 In the cortex,levels were not as high as in the peel, as theyranged from 0.1 to 0.25 |Llg/g for the lattercultivars306 on a fresh-weight basis and from

10 to 290 (ig/g for 11 French cultivars on adry-weight basis.7

In the anthocyanin derivatives, cyanidin wasthe only aglycone found in apple cultivarswith red-colored skin There is a generalagreement to indicate that ideain (thecyanidin-3-galactoside) is the major pig-ment,412-468 because it represented more than

TABLE 4

Quercetin Giycoside Concentration in Apple Peel (tng.kg- 1 FW Basis) for Three

Cultivars Grown in U.S., Eight Cultivars Grown in Canada, 266 Five Cultivars Grown in Spain, 306 and One in Germany 425

71

80 128 361 369 440 661 69.7 57.9 124 131 347 73

Rutinoside

—

—

117 57 159 185 117 110 139 169 0.5 1.1

3.3

15 19.2

3

Xyloside

100 110 150 210 244 192 119 254 195 145 297

Galactoside + glucoside

360 330

500

604 783 735 554 696 869 594 924 91 78 428 613 954

220 a

Arabinosfde

130 140 200 546 662 589 556 777 539 508 801 21.1 21.3 29.1 47.7 105 75

Ref

35 35 35 266 266 266 266 266 266 266 266 306 306 306 306 306 425

88% of the anthocyanins in 11 cultivarsgrown in England427 and approximately 40%

in the cultivar Scugog grown in Canada.260The presence of a cyanidin-7-arabinosidewas postulated by Sun and Francis.412 How-ever, later on, Timberlake and Bridle427 raisedsome doubt as to the presence of derivativeswith sugars linked in the 7 position ofcyanidin They indicated that all the deriva-tives are esterified by sugars in the 3 posi-tion Moreover, the same authors427 havefound minor pigments corresponding toacylated forms of cyanidin-3-monoglyco-sides, but the nature of the acids involved inacylation was not determined

As already stated, several factors can inducelarge variations in both the quantitative and quali-

tative content of phenolic compounds in apple

2 Phenolic Variations at the Subcellular

Level

At the subcellular level, the phenolics arelocated mainly in the vacuoles Yamaki505 indi-

cated that 97% of the total phenolics present in

apple cells accumulate in vacuoles, while 3% are

in free space and none in cytoplasm According to

the same author,505 the calculated concentration

of phenols is higher than 0.1 M in vacuoles of

immature fruit flesh compared with the 1 to 10 mAf

usually found in mature apples.450

3 Phenolic Variations at the Tissue

Level

Although the subcellular distribution ofsoluble phenolics appears to be homogenous, the

situation is different at the tissue level Thus, it

can be seen in Tables 1 and 3 that the epidermal

and subepidermal layers (peel) have a higher

con-tent of phenolics than the internal tissue (cortex)

In different cultivars, the peel/cortex phenolic

content ratio ranged from 3 to lO.4.35.167-266.305.306-322

In a more precise study on the Calville Blanc

variety, Macheix232-234 divided apple fruit into four

zones, from the outer to the inner parts of the

fruit, corresponding to (1) the peel, (2) the outerpart of the cortex (the major edible portion ofapple), (3) the circular zone surrounding the car-pels, and (4) the central core, respectively Zone

1 was the richest in chlorogenic acid, catechins,and flavonols The flavonol content was less than

40 mg/kg (FW basis) in zones 2 to 4 comparedwith the 178 mg/kg in zone 1 The chlorogenicacid content was the lowest in zone 2 and in-creased slightly in zones 3 and 4 Although lessimportant than in the peel, the catechin contentwas higher in zone 3 than in zones 2 and 4.Concerning chlorogenic acid content in the peel,this last result was slightly contradictory to thegeneral comments given in Section 2.1.1 forTables 2 and 3 This probably comes from differ-ences in the repartitions between peel and cortexused by the authors Risch and Herrmann336 alsoreported a greater amount of chlorogenic acid inthe core than in the outer part of the cortex for theJonathan cultivar Similarly, Harel et al.150 found

a nonuniform concentration of o-diphenols in theflesh of the Grand Alexander cultivar They re-ported that the amount of phenolics was highest

in the peel, lowest in the outer part of cortex, andgradually increased toward the core This unevenspatial distribution of phenolic compounds in applefruit flesh is important, as it can induce differenttissue sensitivities to enzymatic browning

4 Phenolic Variations at the Cultivar Level

A considerable variation was observed in thecontent of both total and individual phenolicsamong different cultivars of apple Thus, in theircompilations, Van Buren450 and Herrmann157 in-dicated ranges of 1 to 11 and 1 to 34, respectively,

in the total phenolics content among varieties.Similarly, concerning the main individualphenolics, a variation of 1 to 10 was given forchlorogenic acid by Macheix et al.236 and for(—)-epicatechin by Risch and Herrmann.336 Simi-lar variations (1 to 9) were also found for fla-vonols in apple peel.305 Apple cultivars with green-and yellow-colored skin are pigmented by chloro-phylls, carotenoids,23 and quercetin derivatives503and are obviously devoid of anthocyanins Never-

theless, a large variation (1 to 3) was also

ob-served for ten Delicious apple strains with

red-colored skin grown in the U.S.394 Interestingly

enough, the latter authors394 found a correlation

between the peel luminance L*, measured by a

portable tristimulus colorimeter, and the

antho-cyanin level of deep-red-colored fruits

5 Influence of Maturity and

Postharvest Storage

Although, as already mentioned, the assay

methods for total phenol content are not

accu-rate, there is general agreement that the

con-centrations of phenolic compounds are very high

in young fruits and then rapidly decrease during

fruit development.236 Numerous studies carried

out on the phenolic content of developing apples

reflect this general trend.69-150-157-229-466-488-516 The

phenolic concentrations decline sharply 1 month

after the petal drop, reaching a low level

10 weeks later, and remain approximately

constant thereafter.150-488 On a fruit basis, the

change in total phenols is less pronounced

dur-ing the 4- to 14-week period after the petal

drop Because in that period the number of cells

is fixed, the decrease in the concentration of

total phenols is primarily the result of a dilution

of phenolic compounds in the vacuole.236 After

harvest, the concentration of total phenols

r e m a i n s e s s e n t i a l l y constant or d e c r e a s e s

slightly.35-69-150-172-229-445-448

The changes in content of the different classes

of phenols and of some individual phenols have

also been followed during apple fruit

develop-ment and its subsequent storage after harvest

Thus, during apple development and 1 month

after the petal drop, a general decrease in the

hydroxycinnamic (caffeic, p-coumaric, and

feru-lic) acid derivatives was observed.284 An example

is shown in Figure 3A concerning changes in the

levels of /j-coumaroylquinic acid,

p-coumaroyl-glucose, and chlorogenic acid (by far the most

important).233 For the latter phenolic compound,

similar results were given by several

au-thors.35-69-230-466 During cold storage, the variations

in the content of hydroxycinnamic acid

deriva-tives were less important and, depending on the

cultivars, the authors indicated a decrease,283-284 aconstant level,69 or fluctuations.172-445 On a fruitbasis (Figure 3B), after the initial and rapid rise

in chlorogenic acid (the same was observed for

p c o u m a r o y l q u i n i c acid and p c o u m a r o y l glucose), this phenol steadily accumulated, and itwas only after 10 weeks that its amount decreasedslightly.233 Depending on the cultivars, this lastdecline was not observed283-284 or only during coldstorage.69-172 The variations in catechins were simi-lar to those obtained with hydroxycinnamic de-rivatives The concentrations in catechins rosesharply during the 30 to 40 d after flowering andthen decreased rapidly to stabilize at a low level

-in mature fruit230-283-284 and throughout the storageperiod.35 On a fruit basis, a later peak was ob-served and the decrease was delayed to the end ofthe maturation period.283 Moreover, in two applevarieties, an increase in the (-)-epicatechin-to-(+)-catechin ratio was observed during the pro-gressive growth of the fruit.284 Only a few workshave been devoted to the variations of quercetinand phloretin glycosides The quercetin glyco-sides per gram of fresh weight remained fairlyconstant during the 2 months preceding the com-mercial harvest (var Golden Delicious and GrimesGolden) but, because of fruit growth, increased

on a per fruit basis.503 Moreover, inside the mercial harvest, the same author503 indicated thatthe flavonol content of green fruit was only half

com-of that com-of yellow fruit (var Golden Delicious) Inthat case, no change was observed in the totalflavonol content during storage However, forMclntosh apples, a two- to threefold increase inthe levels of quercetin glycosides was found duringthe first 2 months of cold storage.85 A similartrend was found for phloridzin.85 Finally, althoughthe total quercetin glycosides remained relativelyconstant until the climacteric, considerable varia-tions were observed a m o n g the individualglycosides.85

6 Influence of External and Internal Factors during Cultivation and Storage

As in other fruits, the regulation of phenolicmetabolism in apple depends greatly on both ex-ternal and internal factors (light, temperature,ethylene, growth regulators, nutrients, pesticides,

120-a

b

•300 c

FIGURE 3 Changes in the levels of (a) p-coumaroylquinic acid, (b) p-coumaroylglucose, (c) chlorogenic acid, and (d) fresh weight (in grams) during apple growth (A) milligram per 100 g FW; (B) milligram per fruit (From Macheix, J J.,

Physiol Veg., 12, 25, 1974 With permission.)

etc.), as demonstrated by numerous studies

Prob-ably because of its impact on the visual quality of

the fruit, most studies dealt with anthocyanin

bio-synthesis in red-colored apple skins

Although the accumulation level of cyanin is genetically controlled, the requirement

antho-of light for red pigment biosynthesis in apple

skin has been emphasized by many

work-e r s 9.44.154.197.326.393 7 ^ e f f e c t o f temperature on

anthocyanin biosynthesis, which has been ognized for a long time,70 depends largely on thefruit maturity stage.10-103 Thus, for the Jonathancultivar, the optimum temperature increased from12°C in unripe fruit to 16 to 24°C in ripe fruit.103

For these factors, anthocyanin accumulation

ap-peared to be correlated with phenylalanine

am-monia lyase (PAL) activity.415416 Application of

ethylene104 or ethylene-releasing compounds414 to

unripe apples resulted in anthocyanin

accumula-tion to levels similar to those in ripe apples The

same held for the PAL activity when unripe apples

were ethylene treated.104 However, with ripe fruits,

the ethylene treatment had no effect on both

fac-tors, suggesting that during apple ripening,

antho-cyanin accumulation is under the control of the

PAL activity.105 In addition, treatment with

syn-thetic auxins such as a-napthalene acetic acid and

2-[2,4,5-trichlorophenoxy] propionic acid in

combination with Ethephon® (an

ethylene-releas-ing compound) and Alar® (a growth retardant)

enhanced the red color of the skin of some U.S

cultivars.30-97-136 Besides anthocyanin, cultural

prac-tices can also affect other phenolics Thus, a

sig-nificantly lower amount of phenols was found in

apples treated with Melprex 65® (a pesticide

prepa-ration containing dodine).141 Similarly, ciders

obtained from "fed" (NPK fertilizer) Dabinett

apple trees were less bitter and astringent than

those from "unfed" trees, which was related to an

overall decrease of 17% in fruit phenolic

concen-tration.210

During cold storage, carbon dioxide levels

greater than 73% in an in-package, modified

at-mosphere severely destabilized anthocyanins of

the skin of Starkrimson apples after 23 weeks at

2°C and 76% humidity.222

B Polyphenol Oxidase in Apple and

Apple Products

1 Assay of Activity

The correct assay of polyphenol oxidase

ac-tivity is obviously a need, and, in this respect, two

problems have to be overcome The first lies in

the choice of assay method and the second is to

properly differentiate between catecholase and

laccase on the one hand and peroxidase on the

other hand

As already stated, the primary products of the

oxidation of phenols are highly unstable and

un-dergo many secondary reactions with both phenols

and proteins.5230*"310 Therefore, it is difficult inroutine assays to measure precisely either productformation or phenol consumption due to the en-zymatic catalysis Undoubtedly, the use of spec-trophotometry to follow quinone formation is theeasiest method.254-271 However, it is in some ways

an inaccurate method owing to side reactions sulting in nonlinear color formation with time)and enzyme inactivation through a suicide mecha-nism.463 Moreover, the extinction coefficients ofthe enzymically produced quinones are not al-ways accurately known.463 To circumvent theseproblems, some authors have proposed coupledassays in which quinone accumulation is avoided.This is carried out either with ascorbic acid, as inthe "chronometric method",78'313 or with com-pounds forming stable adducts with differentspectral properties such as proline,354 2-nitro-5-thiobenzoic acid,1 0 2 and B e s t h o r n ' s hydra-zone.259-312 Nevertheless, according to Mayer

(re-et al.,257 the polarographic measurement of gen uptake148-249 is the most convenient and accu-rate method for determining polyphenol oxidaseactivity However, although this method hasreplaced the manometric method,467 it sufferssome drawbacks The ratio of oxygen consumed

oxy-to phenol oxidized changes with the reactiontime and depends on the phenolic structureand concentration, p H , and buffer solutionused.53-58-120-243-257-308-333-335-341 The reaction velocity

is dependent on oxygen concentration.504 Becausethe Km O2 of polyphenol oxidase is in the 0.1- to0.5-mM range,180-254 the enzymes are not saturated

by oxygen during the assay and the true values ofVmax are seldom determined Thus, when usingthe polarographic method, it is essential to care-fully control the assay conditions (temperature,

pH, type of buffer, and air saturation) and tomeasure only the initial rate of oxygen uptake.The unique ability to oxidize p-diphenols(andp-diphenylene diamine) is often used as atest for the presence of laccase activity How-ever, it is not sufficient, at least in crude ex-tracts In the latter, the occurrence of endogenouso-diphenols could provoke coupled oxidation ofadded p-diphenols by o-diphenolase, leading tothe false conclusion that laccase is present.Therefore, additional tests with specific inhibi-tors of each activity are required Thus, phenyl-

hydrazine,219 salicylhydroxamic acid,3 lenediol,258 and cinnamic acids475 are specificinhibitors of o-diphenolase, whereas cetyl-trimethylammonium and other quaternaryammonium compounds469-475 inhibit p-dipheno-lase.

2,3-naptha-In crude extracts, it is also preferable to checkthe possible interference of peroxidase activity

This is easily performed by adding catalase andethanol in order to remove residual peroxidesfrom the reaction medium258 or by usingtropolone.188 The latter compound is a very effec-tive inhibitor of polyphenol oxidase189 and canserve as a substrate for peroxidase in the presence

of hydrogen peroxide.190

2 Localization

There is general agreement that polyphenoloxidase is predominantly a plastid enzyme inhigher plants.155-253-254-430-456 In nonsenescent tis-sues, it is mainly located on the thylakoid mem-brane of chloroplasts and in vesicles or otherbodies in nongreen plastid types.457 However, someauthors reported that polyphenol oxidase can alsoexist in mitochondrial fractions or is readilysoluble (nonmembrane associated) in plantcells.38-40-148-256-401 In apple fruit, polyphenol oxi-dase has been located both in organelles (chloro-plast and mitochondria), where it may be tightlybound to the membrane, and in the soluble fraction

of the cell.83-148 As in many other fruits,25-146-192-373the proportion of readily soluble polyphenol oxi-dase activity increases during the ripening of applefruit.17-148-178 It has been proposed that polyphenoloxidase was solubilized from the plastid and re-leased to the cytoplasm, where the enzyme re-mained either soluble or associated with the cell

wall.17 A similar evolution was found with appletissue culture.460-461

3 Extraction

T w o p r o b l e m s are encountered for theoptimization of the extraction conditions of

polyphenol oxidase, full solubilization of the

membrane-bound activity and protection against

phenolic oxidation during and after extraction

As already stated, the strength of polyphenol dase binding to membranes is variable There-fore, in most cases, full extraction of the activityrequires the use of a detergent such as Triton®X100,122-439-501 Triton® XI14,360-361 or SDS.8-25 Forapple fruit, solubilization was achieved by use of

oxi-a detergent, either Triton® X10083-148-149-181-409-474

or digitonin,145 or after preparation of an acetonepowder.132-133-289-349-376-387-405-434-509 Although the lat-ter method avoids the use of a detergent, whichmay cause some problems during further purifi-cation, it also undoubtedly results in modification

of the enzyme properties, the extent of which hasnot always been taken into account

The second problem arises from the neous presence of quinones in crude extracts ofthe enzyme and its endogenous phenolic sub-strates It is therefore essential to avoid, or at leastminimize, the formation of quinones that thenreact with proteins Such reactions may result inactivity losses and the formation of "new" artifac-tual enzymatic forms399 through covalent, hydro-gen, hydrophobic, and ionic bindings betweenproteins and phenolics or quinones.224-225-264 Meth-ods to prevent the reaction of polyphenol oxidasewith phenolics include the use of phenol-bindingagents such as polyethylene-glycol,24 soluble andinsoluble polyvinyl(poly)pyrrolidone (PVP andPVPP),2 2 6 and hydrophobic2 2 7 and anion ex-change399 resins However, as all plant materialshave different types and various amounts of phe-nolics, there is no universal method to effectivelyremove phenols Therefore, it is often advisable

simulta-to add simulta-to the extracting medium compounds thatprevent the formation of quinones or trap thequinones as soon as they are formed In this re-spect, the most frequently used compounds aresodium diethyldithiocarbamate308 (a powerfulchelator of copper) and reducing agents such asascorbic acid,337 mercaptoethanol,435 and cys-teine.19-241-242 Finally, protease inhibitors wereadded to prevent the formation of artifactualmultiple forms of polyphenol oxidase by endog-enous proteolytic enzymes.117-501 In crude enzy-matic extracts of apple, PEG,60-474 PVP,59-132-133-474cysteine45-467-473 (either alone or with mercapto-benzothiazole409), and ascorbic acid have beenused.67-83-150-181-258

4 Purification

Although numerous works have been devoted

to the purification of this enzyme, only a few

polyphenol oxidases have been purified to

appar-ent homogeneity, probably owing to the

difficul-ties encountered in the preparation of active and

stable crude extracts After an initial step, which

most frequently was ammonium sulfate fractional

precipitation, further purification involved the use

of one or several chromatographic steps Apart

from routine chromatographic methods of

separa-tion (adsorpsepara-tion, gel filtrasepara-tion, and ionic

exchange),32>121-292'337 hydrophobic

chroma-tography,I18-182-500 isoelectric focusing,174

con-canavalin-agarose chromatography,485 and

immobilized metal chelate chromatography511-513

have been applied successfully to the purification

of polyphenol oxidase from several origins

Surprisingly, although mushroom polyphenol

oxi-dase was probably the first enzyme to which the

affinity concept for chromatography was

ap-plied,218 there were only a few applications of this

method for other polyphenol oxidases.168-506

Apple polyphenol oxidase was only partially

purified from peel using DEAE cellulose

chroma-tography83149'474 and calcium phosphate

adsorp-tion chromatography,409 from cortex using

hydrophobic chromatography,6-178-181-331-405-434 or

from the whole fruit using gel filtration.45132 After

electrophoresis, the number of isoforms varied

from one132 to two376-474 or three,67-149-181 which

Harel and Mayer145 indicated could be because of

various degrees of subunit aggregation of the same

enzyme Based on the Triton extracts, the

pub-lished purification factors were 260,149 230,409 and

150,181 with a yield ranging from 55 to 35% After

hydrophobic chromatography, Richard-Forget331

further purified polyphenol oxidase from Red

Delicious cortex by immobilized metal chelate

chromatography followed by affinity

chromatog-raphy The former method gave one major peak

containing 75% of the activity with a threefold

purification Affinity chromatography using

p-coumaric acid as ligand on agarose (the gel was

synthesized by coupling p-coumaric acid to

hexamethylenediamine agarose178 via an azo

link-age66'72-73) resulted in one purified fraction

repre-senting 25% of the crude extract activity and a

240-fold purification Nevertheless, silver nitratestaining after SDS electrophoresis revealed onemajor band (90% of the coloration) still accompa-nied by three faint, minor bands (10% of thecoloration) Finally, two major (60 and 35%) andone minor (5%) fraction can be separated by ionexchange chromatography at pH 6.5 using DEAEsepharose CL6B.331 This confirmed a previousexperiment of isoelectric focusing that gave twomajor peaks with a pHi at pH 4.5 and pH 4.8 and

a minor one with a pHi close to pH 6.7.181 Mostpolyphenol oxidases from plants are 40- to

45-kDa proteins, as was first suggested by in vitro

translation experiments.114"116-201 However, 60- to68-kDa forms, containing a transit peptide, thattarget the protein to the chloroplast have alsobeen purified from broad bean,123-344 and recently

a gene coding for a 66.3-kDa polyphenol oxidasewas isolated from tomato.386 Gel filtration resolvedapple enzyme activity into a single peak132-181 with

a molecular weight of 26132 or 46 kDa,181 or intothree peaks83-145 corresponding to molecularweights of 24 to 40,60 to 70, and 120 to 134 kDa,respectively Finally, in apple buds, three isoen-zymes were observed by electrophoresis withmolecular weights of 32, 39, and 77 kDa.478

5 Polyphenol Oxidase Activity

Variations in Apple

The level of polyphenol oxidase activity atharvest and its variation during fruit storage areobviously of great importance from a technologi-cal point of view There is general agreement thatenzyme activity is much higher in young fruitsthan in ripe fruits (at commercial harvest) on a pergram of fresh weight as well as on a fruit ba-sis 148,230.255,488 However, there were some discrep-ancies concerning its evolution in the latter stages

of development and during fresh storage Thus, ifthe amount of freely soluble activity always in-creased in this period17-83-148-178 the total polyphe-nol oxidase activity either decreased17-83-148-181-230

or fluctuated,89'181-445-448 depending on the cultivar.Only in one case516 was total enzyme activityfound to increase during this period During rip-ening of the Royal Delicious variety, activity in-creased in the peel but decreased in the cortex.324

The application of methyl jasmonate, an inhibitor

of ethylene production in postclimacteric apples,strongly stimulated the polyphenol oxidase activ-ity.75 However, this treatment did not induce theformation of new isoenzymes

The level of polyphenol oxidase activity isalso tissue dependent Some authors reportedthat enzyme activity was higher in the peel than

in the cortex,148-409 whereas the reverse was found

by others.193-322-323 With the Grand Alexandervariety, the activity decreased from the core tothe outer part of the cortex and then increased inthe peel.148 Finally, of 12 varieties analyzed atcommercial harvest,181 the levels in the cortexcompared with that in the peel were higher forseven varieties, equivalent for four varieties, andlower for one variety Table 5 shows that polyphe-nol oxidase activity is also cultivar dependent,because the relative levels ranged from 1 to 10 inboth the cortex and the peel.181-193 Similar resultswere found for different cultivars by other work-

e r s 7.12,28.69,150,323,448^16 Compared with other tivars, Red Delicious always had the highestpolyphenol oxidase activity.69-181-193

cul-6 Kinetic Properties

Enzyme specificity for phenolic compounds

has been the subject of several reports, whereasonly a few studies were devoted to the effect ofthe second substrate (i.e., oxygen).253-254-443-489-510Polyphenol oxidases isolated from higher plantsand fungi are able to oxidize a wide range ofmonophenols and o-diphenols However, whereasthe Km values for oxygen only vary between 0.1and 0.5 mM, the kinetic parameters, Vm and Km,

of the different phenolics are highly variable.Moreover, most of the latter constants are onlyapparent, as they were not determined at a saturat-ing concentration in oxygen but most often in air-saturated solutions of the phenolic substrates.Several enzymatic extracts of plantorigin are devoid of monophenolase activ-

1^24,131,151,174,200,300,337,347,486,502,515 w h e n p r e s e n t ,

cresolase activity was often partially or totallylost on purification.19-202-290-388 Moreover, in thesame species, multiple forms may also vary inthe monophenolase-to-o-diphenolase ratio ofactivities.247-301-511

a Phenolic Substrate

Numerous studies have been carried out onthe specificity of apple polyphenoloxidase forphenolic substrates Large differences have beenfound in the apparent Km values for phenolics inair-saturated solutions (Table 6)

TABLE 5 Relative Polyphenoloxidase Activity for Different Apple Cultivars at Commercial Maturity Variety 8

Delicious Jonathan Golden Delicious San Jacinto Peasgood Orleans Reinette Rome Beauty Ben Davis Alex Kyriati

G Alexander Gallia-Beauty

Cortex

100 100 92 85 82 56 52 50 42 37 30 24

Variety"

Red Delicious Mclntosh Fuji Gala Fiorina Golden Delicious Canada

G Smith Mutsu Jonagold Charden Elstar

Peel

100

46 57 30 42 33 48 43 71 43 31 10

Cortex

100

80 71 48 40 30 88 73 54 43 39 20

Variety 6

Richared Delicious Braeburn

Sturmer Pippin Splendor Dougherty Golden Delicious

G Smith Kempton

Peel

100

62 47 71 14 26 65 34

Cortex

100

38 31 30 30 28 10 9

Adapted from Harel et al.; 148 manometric measurement at pH 5.1.

Adapted from Janovitz-Klapp et al.; 181 polarographic measurement at pH 4.5.

Adapted from Klein; 193 spectrophotometric measurement at pH 6.2.

TABLE 6

Km Values of Apple Polyphenoloxidase Toward Different Phenolic Compounds in Air-Saturated Solutions (All Values in Millimoles)

Catechol 4-Methylcatechol Protocatechuic acid o-Dihydroxyphenylacetic acid Dihydrocaffeic acid

Caffeic acid Chlorogenic acid Rosmarinic acid o-Dihydroxyphenylalanine (-)-Epicatechin

(+)-Catechin Procyanidin B2 Procyanidin C1 Quercetin Butein 3-Hydroxyphloridzin

p-Cresol Phloretin Phloridzin

A s s a y method Quinone formation

(spectrophotometry)

o D i p h e n o l s

140, 59 22, 5 0 9 18, 231 S.3 4443 2.1 , 444a 4.7 331

1.6 331

19 331 2331 0.15 331 3.9, 331 1.6" 4 * 26,331 g444b 5.9 3 3 1

5 8 3 3 1 1 2 444a

0.3 331 0.3 331

Monophenols

0.5 33 ' 0.5 33 '

Note: The pH conditions of the assay were the following:

Ref 59 133 509 132

pH 5.5 6 5 4.5

148 467 180 331 5.1 5 4.5 4.5

Oxygen uptake (polarography-manometry)

Some discrepancies among the results

ob-tained by different authors are apparent These

variations could have different origins First, they

could originate from differences in the assay

methods, although Richard-Forget331 obtained

similar apparent Km values for 14 phenols when

she compared the results given by the

spectropho-tometric and polarographic methods Second,

various apple cultivars have been used as the

enzyme source, and small differences have been

found in the apparent values for the

poly-phenoloxidases extracted from Jonathan and

Starking cultivars.444 Third, in the same cultivar,

differences were observed between the crude

ex-tract from chloroplasts and that from

mitochon-dria.148 Moreover, the same authors149 showed thatthe apparent Km values were affected by purifica-tion, since for 4-methylcatechol, the Km of thecrude extract from chloroplasts was 6 mM, whereas

it was 15.4 and 4.9 mM for two fractions isolated

by ionic exchange chromatography on DEAEcellulose However, other authors178-474 using simi-lar methods did not find any variation in the speci-ficity of apple polyphenoloxidase during itspurification Finally, pH undoubtedly affected theapparent Km values.148-444 It was shown181 that theapparent Km values for 4-methylcatechol, chlo-rogenic acid, and (+)-catechin remained almostconstant between pH 3.5 and 5, but increased asthe pH increased above pH 5 When oxygen uptake

was compared for different phenolic substrates,

4-methylcatechol was always found to be the most

rapidly oxidized at saturating

concentra-tions 132.148.149,180,181,331,409,474

However, in terms of efficiency, Vm/Km (thisratio is proportional to the enzyme activity at

substrate concentrations far below Km; the

con-cept is reexamined in Section II.B.7), many

phe-nolic compounds appeared to be better substrates

than 4-methylcatechol (Table 7) This is the case

for compounds with the structure of, or close to

that of, caffeoyl derivatives such as butein and

caffeic, hydrocaffeic, rosmarinic, and chlorogenic

acids The apparent Km of the main phenolics of

the apple fruit cortex, that is, chlorogenic acid, the

two catechin isomers, and the procyanidins B2

and C l , were in the 2 to 6 mM range (Table 6)

These values indicated that the apple enzyme

TABLE 7

Kinetic Parameters of Purified Apple

Polyphenoloxidase Toward Different Phenolic

Compounds in Air-Saturated Solutions at 30°C

Vm

100 80 99 5.8 125 20 67 94 18 52 60 0.8 4.1 6.1 0.7

Vm/Km

20.4 3.9 24.8 38.7 30.5 62.5 35.3 4.7 0.71 9.1 10.2 0.53 15.2 13 1.4 Note: Km values are in millimoles and Vm in percent

of 4-methylcatechol.

Adapted from Richard-Forget, F M., Recherches sur le

brunissement enzymatique Etudes sur I'oxydation de

phenols et sur I'inhibition de la polyphenoloxydase isolee

de la Pomme (Malus sylvestris, var Red Delicious),

Ph.D thesis, University of Paris, 1992, 7 With

permis-sion.

affinity for these natural substrates was not veryhigh, as was usually found for other poly-phenoloxidases from other origins.254-510 More-over, if chlorogenic acid and the two catechinswere rapidly oxidized by apple polyphenoloxidase,the procyanidins B2 and C l were very slowlydegraded Thus, their maximum rates of oxygenuptake represented only 20 and 7%, respectively,

of that obtained with the monomeric flavan-3-ol(i.e., (-)-epicatechin).1 3 3 T h e monophenolaseactivity was either absent59-467 or, when present,much lower compared with the o-diphenolaseactivity obtained with the best phenolic substrate.The monophenolic substrates were p-cresol,148p-chlorophenol,148p-coumaric acid,132 phloretin,431331and phloridzin.43'331 When tested, tyrosine wasnever found to be a substrate of the apple en-zyme.43-59-132-148-289-467 Concerning the apple fla-vonols, the glycosylated derivatives of quercetinwere not substrates,331-387'467 although the enzymewas able to slowly oxidize the aglycone.331-409However, this oxygen uptake did not result in theformation of colored compounds.331-387 Finally,neither cyanidin nor its galactosyl derivative(ideain) was a substrate of apple polyphenol-oxidase.331 Similar results were obtained for theoxidizability of the procyanidins B2 and C l , thephloretin and quercetin derivatives, and antho-cyanins by other polyphenoloxidases of differentorigins.18-295-298-304-314-327

b Oxygen

Only two reports dealt with the effect of gen on apple polyphenol oxidase activity Using4-methylcatechol (7.5 mM) as substrate, Harel et

oxy-al.148 indicated an apparent Km of 25.8% O2 for acrude enzymatic extract from apple chloroplasts

In a more complete study using three phenols(chlorogenic acid, 4-methylcatechol, and (+)-cat-echin) at different concentrations, Janovitz-Klapp

et al.180 found an equilibrium constant of 0.29 mM(independent of the phenolic substrate) betweenoxygen and a purified preparation of the applepolyphenol oxidase, at pH 4.5 and 30°C Thisvalue, close to the oxygen concentration of air-saturated aqueous solutions at this temperature,499confirms that during the routine activity assay,polyphenol oxidase is only approximately half-

saturated by oxygen (as already stated in Section

I.B.I) In the same study,180 the Lineweaver-Burk

representations of the concentration effect of

oxy-gen and phenolic compounds gave a series of

intersecting lines This was characteristic of an

ordered mechanism where one of the substrates is

required to bind to the enzyme before the second

substrate can bind.384 The inhibition mode of

benzoic acid, which was competitive with

4-methylcatechol and uncompetitive with

oxy-gen, permitted the conclusion that oxygen is the

first substrate to be bound by the apple enzyme.180

This is in agreement with results obtained with

polyphenol oxidases extracted from other

sources173-248337 and with the reaction mechanism

proposed for fungal tyrosinases,216-498 if the deoxy

form of the enzyme is arbitrarily chosen as the

first step of the catalysis cycle.510 In contrast,

some reports indicated a ping-pong mechanism, a

random mechanism, or an ordered one where

oxygen does not bind first.90-137'140-220 There are

several explanations for such discrepancies First,

in numerous enzymatic extracts such as those

from fungi,90-140 grape,220 and potato,248 the

monophenolase activity was not negligible In

this case, the mechanism is further complicated,

because o-diphenol has to be considered both a

product of and a substrate for the enzyme

Sec-ond, some authors90 have used a

spectrophoto-metric method to obtain kinetic data, and the part

played by secondary products in the absorption,

which is very difficult to determine, was not taken

into account Third, progress curves of changes in

oxygen concentration obtained in polarographic

assays have also been used to determine the

in-stantaneous velocity at different oxygen

concen-trations.173-220-248 This can lead to erroneous results,

because the initial velocity was not measured for

each oxygen concentration It is well known that

secondary products can play an important role in

oxygen consumption by a nonenzymatic process333

and that polyphenol oxidase undergoes

inactiva-tion during its catalysis.129130

c Kinetics with Two Phenolics—Coupled

Oxidations

Most of the kinetic studies on polyphenol

oxidase were carried out with a reaction medium

containing one phenol However, in natural ucts such as fruits and vegetables, several pheno-lics are present and can be used as a substrate bythe enzyme Few studies have been devoted to theenzymatic oxidation of phenolic mixtures in modelsolutions, and they were mainly concerned withgrape polyphenol oxidase.51-53"55-58-296-334

prod-With apple polyphenol oxidase, Klapp et al.180 conducted kinetic studies on modelsystems containing two phenolic compounds Theydeveloped an equation

up-S2) and kinetic parameters (Km^ Vrnj and Km2»

Vm2) of each individual phenolic substrate present.Equation 1 can be extended to a more complexsolution containing more than two phenolics with-out any special difficulty, provided the concentra-tions and kinetic parameters of the additionalcompounds are known However, when the enzy-matic reaction was followed by spectrophotom-etry, the kinetics obtained did not agree with thisequation There are different explanations for thisfact (1) The absorption coefficients of the differ-ent quinones are only partially known351-463 and,

a fortiori, their respective contribution to the final

absorption (2) The quinones are highly unstableand very rapidly undergo nonenzymatic reac-tions.251-333 (3) Some enzymically produced quino-nes are likely to react nonenzymically withphenols.53-269-395 These coupled oxidations can bevery rapid and are dependent on the respectiveredox potential of the different quinone/phenolcouples present53-281 The existence of such coupledoxidations was demonstrated in caftaric (caffeoyltartaric) acid/flavan-3-ol mixtures with grapepolyphenol oxidase,51-54 and chlorogenic acid/cat-echin mixtures with mushroom296 and apple331polyphenol oxidases In all cases, compared withthe oxidation rate of the phenol alone, the flavan-3-ol degradation was faster, while that of thecaffeoyl derivative was slower The mechanismproposed53 involved enzymatic oxidation of thetwo phenols, followed by chemical oxidation of

the flavan-3-ol by the caffeoyl quinone with generation of the caffeoyl derivative Moreover,the o-quinones formed by enzymatic or coupledoxidation can also react with another phenolicmolecule to yield condensation products53-395 in

re-the form of oligomers or copolymers A number

of these secondary products (which can also beobtained by autooxidation) recently have beencharacterized mainly by HPLC using a diode arraydetector.52-61-62-296-331 Besides flavan-3-ols, o-quin-ones of caffeoyl derivatives were able to cooxidizeseveral other phenols such as thiol adducts,56-332anthocyanins,314-327'331 4-methylcatechol,333 fla-

vonols,331 and dihydrochalcones.298-331 According

to Cheynier et al.,53 the redox potential order of

the grape phenolics appeared to be the following:

caftaric acid > epicatechin, catechin > glutathionyl

adduct of caftaric acid > procyanidins >

epicatechin gallate After oxidation studies on

several pairs of phenolics, Richard-Forget331

proposed a similar order: chlorogenic acid >

4-methylcatechol > epicatechin, catechin > cysteinyl

adducts of the four preceding phenols,

anthocya-nins, flavonols, and dihydrochalcones derivatives

Thus, in the two cases, o-quinones of the caffeoyl

derivatives (caftaric and chlorogenic acids) were

the most efficient carrier for coupled oxidation

and seemed to play a determinant role in the

degradation of the other phenolics, including those

that are not a substrate of polyphenol oxidase

d pH and Temperature Effects

Although a pH optimum around 7 was foundfor the polyphenol oxidase activity extracted from

an apple mitochondrial fraction,148'149 most

stud-ies indicated that the bulk of apple polyphenol

oxidase had a pH optimum of activity between

4.5 and 5.5.59,132,181.289.434,467.509 Moreover, the

en-zyme seemed relatively tolerant to acid pH,

be-cause the activity at pH 3 still represented 40% of

the maximum activity.178 Thus, control of

enzy-matic browning by acidification only is difficult

unless a very low pH is obtained

The effect of temperature on both enzymeactivity and stability has been investigated by

numerous workers.13'246-251'272-317'443 Most assays of

the activity of apple polyphenol oxidase are

car-ried out between 25 and 35°C, and it is only for

temperatures higher than 40°C that a significantthermal inactivation occurs during the assay.Compared with other enzymes responsible forfood-quality degradation, polyphenol oxidase isnot a very heat-stable enzyme.1'13-443 Therefore,this activity is rarely used as an index of blanch-ing treatment.495 A partially purified extract ofapple polyphenol oxidase had a half-life of 12min at 65°C and was destroyed at 80°C.467 Inapple juices treated at the same temperature, 30%

of the activity remained after 20 s with theGravenstein variety88 and 1 min with the Reinedes Reinettes variety.82 Both reports stressed theimportance of the pH of the juice for thermosta-bility, because for the same treatment, the re-sidual activity at pH 3 was less than 10% of thatobtained at pH 6 (maximum stability) Likewise,acidification of apple juice to pH 2 by HC1 for 45min followed by a return to the original pH withNaOH resulted in an 88% decrease of polyphenoloxidase activity.514

7 Inhibitors of Polyphenol Oxidase

This section addresses only inhibitors acting

on the enzyme The other approaches used tocontrol enzymatic browning, that is, physicalmethods and chemical agents acting on eithersubstrates or products, are examined in SectionIV.A and B Different modes of action have beenproposed for the inhibitors acting directly onpolyphenol oxidase The classification of Mayerand Harel254 contained two groups Compoundsinteracting with copper are in the first group andthose affecting the active site for the phenolicsubstrate are in the second

In the first group, inhibition by metal ionchelators such as azide,153 cyanide,90 carbon mon-oxide,2-252 tropolone,189-438 and sodium diethyl-dithiocarbamate,121-511 which are more or lessspecific for copper, is well documented forpolyphenol oxidases of different sources.Similarly, inhibition by halide ions has longbeen recognized196 for polyphenol oxidasesfrom apple46'84'179'348-349'389-400'419 and other ori-gins.25'240'303-339 In the latter case, the same authorsshowed that inhibition by halides was strongly

pH dependent, increasing when the pH was creased It was proposed that halide and copper

formed a complex after displacement of a

proto-nated histidine from copper.25-303 A mechanism

was also postulated whereby halide binds to the

enzyme in competition with the phenolic

sub-strate only when the enzyme molecule is in the

protonated form.303 Moreover, in a comparative

study of polyphenol oxidases from different

sources, it was suggested that the degree of

en-zyme inhibition by halide ions was the result of

the balance between the stability of the

copper-halide complex responsible for inhibition and the

accessibility of the copper in the active site to the

halide ion.339 For the apple enzyme, the chloride

ion was shown to be noncompetitive,179-389 while

the other halide ions were competitive.179 The

order was F~ » Cl" > Br\I-,175>389 because at pH

4.5 the apparent Ki values were 0.07,20,106, and

HOmM, respectively.179 When the pH was

var-ied, it was postulated that the undissociated form

HF was responsible for inhibition by fluoride (in

agreement with results obtained for broad bean

polyphenol oxidase339) with a Ki close to 4 ^M.179

In addition, the apparent Ki for chloride was shown

to vary according to the equation

with pKa = 3.65 and Ki = 2.4 mil/.179 Thus, for a

pH below pKa, the limiting value of Ki,app is

2.4 mM, whereas for a pH greater than pKa, an

increase of one pH unit corresponds roughly to a

tenfold increase of the apparent Ki for chloride.179

Among inhibitors belonging to the second

group, aromatic carboxylic acids of the benzoic

and cinnamic series have been studied widely

since the first works of Kuttner and Wagreich199

and Krueger.196 Although most authors found that

these compounds were competitive inhibitors of

polyphenol oxidase because of their structural

similarities with phenolic substrates, some

au-thors indicated that the type of inhibition was

dependent on the substrate used for the assay and

was either competitive, noncompetitive, or

mixed.90-139-265-268-293-337-402 For apple, following the

substrate and the enzyme preparation used, Walker

and Wilson477 found different inhibition patterns

(mainly competitive) for substituted cinnamic

acids With 4-methylcatechol as a substrate,

Janovitz-Klapp et al.179 indicated that the 11

aro-matic carboxylic acids tested in the benzoic, namic, phenylacetic, and phenylpropionic serieswere all pure competitive inhibitors (Table 8) Itappeared that cinnamic acids were more potentinhibitors than their benzoic homologs, as the Kivalues were 2 to 30 times lower than in the ben-zoic series Moreover, in both series, the p-hy-droxy substitution slightly enhanced the inhibitoryproperties, whereas adding one or two methoxygroups in the meta position greatly decreasedinhibitor affinity for the enzyme In addition,when the carboxyl group was separated from thebenzene cycle by a methylene group as inphenylacetic acid, the inhibition was greatlyreduced However, it was partially restored by anadditional methylene group, as in phenylpropionicacid, and again enhanced by a p-hydroxy substi-tution (p-hydroxyphenylpropionic acid) There-fore, it is apparent that the substitution pattern forthe aromatic carboxylic acids leads to a similareffect for the affinity of the inhibitors and thecorresponding substrates, because the Ki values

cin-of cinnamic, benzoic, phenylpropionic, andphenylacetic acids (Table 8) are in the same order(although lower) as the Km values of caffeic,protocatechuic (o-dihydroxybenzoic), hydro-caffeic (o-dihydroxyphenylpropionic), and o-dihy-droxyphenylacetic acids (Table 7) Finally, sorbicacid is almost as efficient a competitive inhibitor

as benzoic acid.179 Thus, the presence of the zene nucleus is not an absolute requirement forthe inhibitory effect, because it can be replaced byconjugated double bonds The latter result wasalso found for apricot polyphenol oxidase.402The degree of inhibition by carboxylic acids

ben-is also pH dependent, increasing as the pH creases Following experiments with broad beanpolyphenol oxidase,339 it was proposed that theundissociated form of the acid is responsible forinhibition by forming a complex with copper atthe active center Similar results were obtainedwith a purified apple polyphenol oxidase.179 Onecan speculate that the binding site of the latterenzyme recognized conjugated double bonds thatare contained in the benzene cycle or in an unsat-urated alkyl chain When a carboxyl group waspresent, either directly bound to the benzene cycle(benzoic series) or to the conjugated double bonds(cinnamic series or sorbic acid), it could form acomplex with the copper at the active site When

TABLE 8 Inhibition Effects of Carboxylic Acids on Purified Apple Polyphenol Oxidase at pH 4.5 in Relation to Their Structures

Acid

Benzoic p-Hydroxybenzoic Vanillic

Syringic

Cinnamic p-Coumaric Ferulic Sinapic

Phenylacetic,

Substitution P

Benzoic

OH OH OH Cinnamic

OH OH OH

m m' Series

OCH 3

Series

OCH3 OCH3 OCH3 Phenylpropionic, and Sorbic Phenylacetic

Phenylpropionic p-Hydroxyphenylpropionic OH Sorbic

App Ki (mAf)

0.64 0.57 10 34.5

0.092 0.04 0.29 15 Acids 13 1.4 1.1 0.51 Adapted from Janovitz-Klapp, A H., Richard, F C , Goupy, P M., and

Nicolas, J J., J Agric Food Chem., 38, 926, 1990 With permission.

such a structure is present in the same molecule

together with an o-diphenolic structure, the

inter-action with the o-diphenolic would be greatly

reduced, as shown by the low Vra values obtained

for caffeic and protocatechuic acids (Table 7) In

addition, when the caffeic and chlorogenic acids

are compared, the esterification of the carboxyl

group by quinic acid reduced the affinity of the

caffeoyl moiety for the apple enzyme (increase of

Km) However, it prevented formation of the

com-plex between copper and the vicinal

undissoci-ated carboxyl group, leaving the metal free for the

catalysis of o-diphenol oxidation, as illustrated by

the large increase of Vm (Table 7)

Use of cinnamic acids to control enzymaticbrowning in fruit juices has been suggested.471

Cinnamic acid added to Granny Smith juice at

concentrations greater than 0.5 mM resulted in an

inhibition of browning for over 7 h.471 However,

apple plugs dipped in 10 mAf sodium cinnamate

were protected for several hours but then

exhib-ited a severe browning over extended storagetimes.369 It has been proposed that cinnamic acidmay undergo gradual conversion at the cut sur-face to a polyphenol oxidase substrate bycinnamate hydroxylase and other enzymes in-volved in the biosynthesis of phenols.369 Similarresults obtained with sodium benzoate led theauthors to recommend not using these aromaticcarboxylic acids as components of antibrowningformulations.369

The substituted resorcinols, which are alsostructurally related to phenolic substrates, wererecently recognized as polyphenol oxidase inhibi-tors.262-263 In a structure-activity study on a series

of 4-substituted resorcinols, the highest inhibitionwas obtained for hydrophobic substituents in the4-position such as 4-hexyl, 4-dodecyl, and4-cyclohexyl resorcinols.263 The same authors in-dicated that 4-hexyl resorcinol can be used for thebrowning control of fresh and hot-air dried appleslices as well as of apple juice.263 In addition, they

claimed that 4-hexyl resorcinol has several

ad-vantages over sulfites for the prevention of shrimp

melanosis and for use in foods in general

Kojic acid

[5-hydroxy-2-(hydroxymethyl)-4-pyrone] has been shown to inhibit fungal

tyrosi-nase.375 Recently, it was found that it had an

inhibitory effect on plant and crustacean

poiyphe-noi oxidases.49 It is a competitive inhibitor for the

oxidation of chlorogenic acid and catechol by

apple poiyphenoi oxidase.49 In addition, this

com-pound can reduce pigments or pigment precursors

to colorless compounds.48

Oxalate has been shown to inhibit poiyphenoi

oxidase in two ways: (1) upon incubation, it slowly

inactivated the enzyme following a first-order

kinetic in the enzyme and (2) it acted as a

com-petitive inhibitor of o-diphenol oxidation by

fun-gal507 and apple331 poiyphenoi oxidases The

addition of copper can restore the activity lost in

the first process, as was shown for the fungal507

and spinach377 enzymes

Finally, a mixed type of inhibition by several

natural aliphatic alcohols was reported for grape

poiyphenoi oxidase.440 Inhibition seemed mainly

related to the hydrophobic chain rather than to the

alcohol function.440 To our knowledge, no study

was carried out on these compounds with the

apple enzyme

8 Secondary Reactions Affecting

o-Quinones

The primary products of enzymatic oxidation

are o-quinones These molecules have different

spectral properties, and their color mainly pends on the pH and phenol from which theyoriginate.421 Thus, after oxidation, catechin isbright yellow with a maximum absorbance at 380

de-nm, chlorogenic acid is a dull orange-yellow with

a maximum at 420 nm, and DOPA droxyphenylalanine) is pink with a maximum close

(o-dihy-to 480 nm The molar extinction coefficients atthe maximum wavelengths are given in Table 9and show a wide range of variation

Moreover, the o-quinones are reactive pounds,110-269-350 as illustrated in Figures 4 and 5.o-Quinones can react with another molecule ofphenol, resulting in dimers of the original phe-

com-no126i,395,508 (reaction 1, Figure 4) These dimers,having an o-diphenolic structure, can be subject

to reoxidation52-395 either enzymically or by other o-quinone, resulting in the formation oflarger oligomers with different color intensities.The o-quinones can also react with a differentphenol molecule, either leading to a copolymer orregenerating the original phenol and giving a dif-ferent o-quinone by coupled oxidation (reactions

an-2 and 3, Figure 4).51-54-296-331Reactions can also occur with nonphenoliccompounds (Figure 5) Thus, another coupledoxidation was observed with ascorbic acid (reac-tion 1), giving the regeneration of the phenol withformation of dehydroascorbic acid.420 With sulfites(reaction 2), colorless addition compounds areformed99-378-395-479 together with regenerated phe-nol,317 although the latter possibility remainedquestionable The o-quinones also form additioncompounds with thiol groups by nucleophilic

TABLE 9 Molar Extinction Coefficient of Quinones from Different o-Diphenolic Substrates of Poiyphenoi Oxidase

(Wavelength at Maximum of Absorbance)

o-Diphenolic Substrate

Pyrocatecho!

4-f-Butylcatechol L-DOPA

3,4-Dihydrophenylacetic acid 4-Methylcatechol

Hydrocaffeic Chlorogenic acid

Wavelength

(nm)

390 400 480 390 400 412 420

Extinction coefficient

1417 1150 3388 1311 1400 1124 2000

Ref

463 463 463 463 463 463 351

FIGURE 4 Reactions of o-quinones with phenolic compounds (All reactions are nonenzymatic except those with polyphenol oxidases; reactions 2 and 3 are able to regenerate the original phenol.) Products with different color

intensities are indicated by asterisks (From Rouet-Mayer, M A., Philippon, J., and Nicolas, J., Encyclopaedia in

Food Science, Food Technology and Nutrition, McRae, R., Robinson, R K., and Sadler, M J., Eds., Vol 1, Academic

Press, London, 1993, 499 With permission.)

additions (reactions 3 and 4) Cysteine, either

free 91.243,275,308,341,359 Q r b o u n d i n s m a U p e p

tides57-156-309 or large proteins,310 gives colorless

compounds Although these compounds have an

o-diphenolic structure, they are not a substrate of

polyphenol oxidase58-329-359'397 but can either be

oxidized by laccase355 or react with an excess of

o-quinones by a coupled oxidation mechanism332

and form intensely colored products Nucleophilic

additions also occur with amino groups of amino

acids or peptides.243-309 Both primary (e.g., serine39)

and secondary (e.g., proline437) amines form

addi-tion compounds with o-quinones (reacaddi-tions 5 and

6) Moreover, further substitutions with thiol

(re-action 4) or amine (re(re-action 7) groups contained

in proteins may occur, leading to the formation of

intra- or intermolecular cross-links.250-264-311

Fi-nally, water can be added slowly to the ones79*333 to form triphenols that are readily oxi-dized by polyphenol oxidase or by excesso-quinones, leading to the p-quinones (reaction8) The last pathway was favored by acid

o-quin-p J J 124,125,183,333

The reactivity (or in other terms, the stability)

of the o-quinones in these different pathways ishighly variable The nature of the phenol oxidized

as well as the oxidation conditions (medium, pH,temperature, etc.) are determinant Thus, it wasshown that in the same conditions, o-quinonesfrom 4-methylcatechol were more stable than thosefrom chlorogenic acid, which in turn were morestable than those from catechins.333 Obviously,the presence of reactive molecules, with amino orthiol groups, in the medium can greatly affect the

FIGURE 5 Reactions of o-quinones with nonphenolic compounds (All reactions are nonenzymatic except those

with laccase; reactions 1 to 3, 6, and 8 are able to regenerate the original phenol.) Products with different color intensities are indicated by asterisks Ox, further oxidation reactions by oxygen or o-quinone; Pr-SH and Pr-NH 2 , proteins; Pro-NH, Proline; AA-NH 2 , amino acids; Asc A, ascorbic acid; DHA, dehydroascorbic acid; FTSH, small thiol

compounds (e.g., cysteine or glutathione) (From Rouet-Mayer, M A., Philippon, J., and Nicolas, J., Encyclopaedia

of Food Science, Food Technology and Nutrition, McRae, R., Robinson, R K., and Sadler, M J., Eds., Vol 1,

Academic Press, London, 1993, 499 With permission.)

reactivity of o-quinones The secondary products

formed may or may not be good substrates for

polyphenol oxidase and may exhibit differences

in their reactivities with o-quinones

C Other Factors

1 Ascorbic Acid in Apple and Apple

Products

Ascorbic acid is a powerful reducing agent

and in its presence the enzymically formed

o-quinones are converted back to their precursor

Therefore, this compound could play a role in theextent of tissue browning after bruising.236 Com-pared with many other fruits, apples are not veryrich in vitamin C Recent surveys gave a meancontent of 50 mg/kg (FW basis).214496 However,large differences were found among samples, asvalues varied between 30 and 370 mg/kg Manyfactors can be responsible for these variations, forexample, variety214 and maturity stage before488and after harvest.496 The peel contains more ascor-bic acid than the pulp as well as the side exposed

to the sun compared with the shaded side.100 nally, because of oxidation losses during process-ing, the ascorbic acid content in apple juice isalmost zero, - although some retention was

found in freeze-concentrate enriched juice.417 In

apple puree, a rapid decrease of ascorbic acid

content was shown after blending, with a

con-comitant increase in dehydroascorbic acid,

result-ing in almost no loss in total vitamin C content.274

Nevertheless, it seemed that endogen ascorbic

acid levels did not play a major role in limiting

the extent of browning.230-488 Recently, a HPLC

procedure was developed for the determination of

ascorbic acid, dehydroascorbic acid, and ascorbic

acid-2-phosphate in raw apple samples

supple-mented with ascorbic acid or ascorbic

acid-2-phosphate to prevent browning.365

2 Peroxidase in Apple and Apple

Products

Peroxidases are ubiquitous enzymes in fruitsand vegetables that are able to oxidize a large

number of molecules.342'343-443 However, few

stud-ies have been devoted to the peroxidase system of

apple Peroxidase activity was proposed as a

pos-sible parameter of ripening and senescence of

Golden Delicious apples,134 although the

isoen-zyme patterns did not change during this period.31

Activity was found to vary significantly with

cul-tivar, picking date, and storage period, but

with-out any regularity for the three cultivars tested.447

Peroxidase appeared to be more concentrated in

the peel than in the pulp.285-330 For eight cultivars

picked at commercial maturity, the lowest

peroxi-dase activity (Mclntosh var.) in peel represented

22% of the highest (Red Delicious var.).330

After partial purification, at least three zymes were characterized from either peel330 or

isoen-pulp.286 The major isoenzyme, the less heat

stable,286 was cationic, with a pHi close to 9.286330

Although separated by ion exchange

chroma-tography, all isoenzymes eluted in a single peak

in gel filtration corresponding to a molecular

weight of 40 kDa.330 The optimum for activity

was close to pH 5.8, a value that is in the range

given for peroxidase activity extracted from other

fruits.272-443 Moreover, apple peroxidase was able

to oxidize not only chlorogenic acid and

(+)-catechin, but also several quercetin

glyco-sides by a ping-pong, bireactant mechanism.330

Thus, the glycosylated flavonols, which are not a

substrate for polyphenol oxidase, can be degraded

by apple peroxidase in the presence of hydrogen

peroxide Note that horseradish peroxidase is alsoable to oxidize kaempferol,273 quercetin,383 andother flavonoids.77-382 However, to our knowledge,

no direct correlation has been found betweenperoxidase activity and the browning susceptibil-ity of apple varieties

Other works were not directly concerned withthe fruit, but, rather, the embryo,353 root-stocks,267-458-459-482-483 or cell cultures.26 All thesestudies used electrophoretic methods in order tocharacterize the peroxidase polymorphism for theidentification of apple cultivars

III RELATIONSHIP BETWEEN INTENSITY OF BROWNING AND BROWNING PARAMETERS

A Methods for Evaluation of Browning

Accurate methods are required for the surement of browning in tissue slices and ex-tracts This need is obvious when different cultivarsare compared for susceptibility to browning or forevaluation of experimental treatments designed

mea-to control enzymatic browning.12Basically, two kinds of methods are avail-able.236 The first uses absorbance measurements,usually in the 400-nm region, on solutions afterextraction and purification of the brown pig-ments.488 The second uses reflectometry ortristimulus colorimetry and can be applied di-rectly to cut surfaces or fruit puree.364-442 Althoughboth methods are easy and rapid, they do haveserious disadvantages

Absorbance measurements evaluate only thesoluble pigments It is well known that, as thereaction proceeds, polymerization occurs and thesolubility of a large part of the brown pigmentsdecreases.395 The insoluble entities are eliminatedduring the filtration and centrifugation steps inthe purification process Moreover, depending onthe kind of pigments, which in turn depend on theoriginal phenols and their relative proportions,the wavelength of maximum absorbance rangesbetween 360 and 500 nm Therefore, absorbancemeasurements at a single wavelength correlatepoorly with visual evaluation of browning

In order to obtain information on the relativeimportance of browning parameters, it has been