inhibition enzymatic browning in food products€¦ · m.sc. fabienne crumière deux inhibiteurs...
TRANSCRIPT
INHIBITION OF ENZYMATIC BROWNING IN FOOD PRODUCTS
USING BIO-INGREDIENTS
Fabienne Crumière
A thesis submitted to the Faculty of Graduate Studies and Research in partial fullilIrnent of the tequiremcois of the degree of Master of Science
Department of Food Science and Agricoltural Cbernistry
McGill University
Montreal, Québec
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SHORT TITLE
M . S c Fabienne Crumière
Two natural enzymatic browning inhibitors, copper-metallothionein (Cu-MT) and
polyphenol esterase (PPE), were obtained fkom A. niger and investigated. Reflectance
measurements, expressed as L (lightness variable) and a (red to green degree of color) were
used to compare, over extended penods of time, the relative inhibitory effectiveness of Cu-
MT and PPE to those observed with the use of selected chemicats including ascorbic acid
(AA), citric acid (CA), ethy lenediaminetetraacetic acid (EDTA), sodium bisul f i te (NaHS03)
and 4-hexylresorcinol (4HR), in the prevention of browning on the cut surfaces of selected
food products such as apple and potato slices as well as fieshly prepared apple juice. The
results indicated that the development of enzymatic browning was directly related to an
increase in the a values and indirectly associated to a decrease in the L values for al1 the
selected food products, with the exception of apple juice which showed an increase in the L
values. Treatrnent of each food product required an optimum concentration of the selected
inhibitor for the inhibition of browning; the optimum concentrations of Cu-MT, PPE, AA,
CA, EDTA, NaHS03 and 4HR were 0.10, 0.10, 2.00, 0.75, 8.00, 0.10, and 0.10% for the
apple slices respectively, 0.025, 0.025, 0.010, 0.240, 0.240, 0.030 and 0.150% with appie
juice respectively and 0.025,0.075, 1.000, 1.000, 0.025,0.025 and 0.005% with potato slices
respectively. The expenmental findings indicated that PPE was more effective than Cu-MT
in controIling enzymatic browning; the PPE inhibitory effect was 6.5, 21 and 297% higher
than the Cu-MT inhibitory effect in apple slices, apple juice and potato slices, respectively.
In addition, Cu-MT and PPE showed similar inhibitory effects, depending on the food
product, to those obtained with AA, CA and EDTA in the prevention of enzymatic browning.
Among the chemicals used, NaHS03 and 4HR showed a higher inhibitory effect compared to
that obtained with Cu-MT and PPE; the inhibition of browning by NaHS03 was greater by
100 to 270% to that obtained with PPE depending on the food product and the inhibitory
effect of 4HR was 7 to 216% greater. The experimental findings obtained fiom the
reflectance spectra in the visible region indicated that the reflectance values decreased as the
browning progressed and that the development of color in the selected food products resulted
mainly fiom the combination of various proportions of yellow (A. = 500 nm) and red (A. = 680
nm) absorbance regions. In addition, the optimum concentration of the inhibitor obtained
fiom reflectance values for the prevention of browning were less accurate and sometimes
varied fiom those detennïned using the tristimulus coordinates L and a.
M.Sc. Fabienne Crumière
Deux inhibiteurs naturels du brunissement enzymatique obtenus à partir d' A. niger,
métallothionéine à cuivre et polyphénol estérase, ont été étudiés. Les mesures de reflectance,
définies selon L (luminance) et a (degré de la couleur variant de vert à rouge) ont été utilisées
afin de comparer, au cours du temps, l'efficacité relative de Cu-MT et PPE à celle des
produits chimiques sélectionnés incluant l'acide ascorbic (AA), l'acid citrique (CA),
I'éthylènediamuietétraacétique acid (EDTA), le sodium bisulfite (NaHS03) et le 4-
hexylrésorcinol, pour empêcher le brunissement à la surface des tranches de pommes et de
pommes de t m e ainsi que dans le jus de pomme fraîchement préparé. Les résultats ont
indiqué que le développement du brunissement enzymatique était directement relié à une
augmentation de la valeur a pour les trois produits alimentaires testés alors qu'il etait
indirectement relié une diminution de la valeur L, sauf pour le jus de pomme où L
augmentait. Chaque traitement a montré une concentration optimale concernant l'inhibition
du brunissement; celle de Cu-MT, PPE, AA, CA, EDTA, NaHS03 et 4HR était 0,lO; 0,lO;
2,OO; 0,75; 8,OO; 0'10 et 0,10% avec les tranches de pommes respectivement; 0,025; 0,025;
0,010; 0,240; 0,240; 0,030 et 0,150% avec le jus de pomme respectivement, et 0,025; 0,075;
1,000; 1,000; 0,025; 0,025 et 0,005% avec les tranches de pommes de terre respectivement.
Les domées expérimentales ont indiqué que PPE était plus efficace que Cu-MT dans le
contrôle du bruissement enzymatique; l'effet inhibiteur de PPE était 6'5; 21 et 297%
supérieur à celui de Cu-MT pour les tranches de pomme, le jus de pomme et les tranches de
pomme de terre respectivement. De plus, selon le produit alimentaire considéré, Cu-MT et
PPE pouvaient présenter un effet inhibiteur similaire à celui obtenu avec AA, CA et EDTA.
NaHS03 et 4HR étaient des inhibiteurs plus efficaces que Cu-MT et PPE; I'inhibition du
bruissement par NaHS03 était, selon le produit considéré, supérieure de 100 à 270% à celle
obtenue avec PPE et l'effet inhibiteur de 4HR était plus important de 7 à 2 16%. Les résultats
expérimentaux obtenus à partir des mesures de reflectance dans le spectre du visible ont
indiqué que les valeurs de reflectance ont diminué alors que le brunissement se développait et
que la sensation de couleur provenait principalement de la combinaison, en proportions
variables, des zones d'absorption jaunes (A k: 500 nm) et rouge (h = 680 nm). De plus, les
concentrations optimales pour l'inhibition du brunissement, obtenues pour chaque traitement
à partir de ces valeurs de reflectance, étaient moins précises et parfois même differentes de
ceIIes obtenues avec les valeurs L et a.
1 would like to thank my supervisor, Dr. Sélim Kermasha, for his scientific guidance,
support, constant encouragement and patience during my graduate studies.
1 would like to express rny gratitude to Dr. Barbara Bisakowski for her precious time
and help for the achievement of this thesis.
A special thanks also to Lassonde Inc. and McCain Food Ltd. who supplied the
apples and potatoes, respectively, and who always had time for me and my questions.
Then, 1 would like to thank my colleagues in the lab as well as al1 my fnends (here
and in France) for their inestimable fiiendship, inexhaustible patience and judicious advise. A
huge thanks to al1 of them, with a special mention to those who supported me throughout the
day, every day, during these three years and who will recognize each other by themselves!
Your presence had been, and is still, so precious to me.
Last but not least, my deepest gratitude goes to my parents for their moral support,
understanding, constant encouragement and, without forgetting, their financial help. 1 would
also like to extend rny thanks to al1 my family that has always been there for me. 1 will never
forget it.
2 . LI~TERATURE REVIEW ..................................................................................................... -4
2.1 . Polyphenol Oxidase (PPO) ....................... ., ......................................................... .... 4
2.1.1. Definition .................................................................................................. 4
2.1.2. CZms flcation ................................................ ...... ................................... 4
........................................................................................................ 2.1.3. Source 5
2.1.4. Natural Substrates ..................................................................................... 5
.................................................................. 2.1.S. Determinniion of PPO Activity 7
2.2. Enzyrnatic Browning in Food ................................... , ....... 7
2.2.1. Introduction ................................... .... ........................................................ 7
2.2.2. Mechanism ............~~.~................................................................................. 8
2.3. Inhibition of Enzymatic Browning ...................................................-....................... 9
2.3.1. Principles of the Prevention of E n v a t i c Browning ............................... 9
2.3.2. Type of Inhibitors ...................................................................................... 9
2.3 .2.1. Practical Methods ........................................................................... 9
............................................................................. 2 -3 -2 .3 . Physical Methods 9
............................................................... 2.3.2.2.1. Effect of temperature 9
2.3.2.2.2. Protection fiom Oxygen .................~..................................... 10
2.3.2.2.3. Supercritical Carbon Dioxide ............................................. 10
2.3.2.3. Use of Chernicals ............................................................................ 10
............................................. . 2.3.2.3.1 Chernical Enzyme Inhibitors 11
2.3.2.3.2. Reducing Agents ................................................................... Il
................................................................... 2.3.2.3.3. Chelating Agents 1 2
2.3.2.3.4. Acidulant Agents ................................................................. 12
..................................... 2 .3.2 -3 .5 . Complexing Agents .................... ... 13
................................................ 2.3.2.3.6. Miscellaneous .... .................. 13
........................................................................ 2.3.2.4. Enzyme Treatments 13
........................................................................... 2.3.2.5. Genetic Approach 14
2.4. Novel Approaches for the Inhibition of Enzymatic Browning ............................... 14 .......................................................................... 2.4.1. Copper MetaIIothionein 14
2.4.1 .1 . Bioiogy of Metallothioneh (MT) ........................ ..... ........ 14 2.4.1.1.1. Definition ........................................................................... 14
2.4.1.1.2. Sources .................................................................................. 15
2.4.1.1.3. Functions ............................................................................... 15
2.4.1.2. Inhibition of PPO using Copper-Chelating Peptide ....................... 15 2.4.1.2.1. Introduction .......................... .. .......................................... 15
.................................................................. 2.4.1 .2.2. Inhibition Studies 15
.................................................................................. ............. 2.4.2. Esterases ,., 16
2.4.1.1. Biology of Esterases ............. ... .................................................. 16 2.4.2.1.1. Definition .............................................................................. 16
.................................................................................. 2.4.2.1.2. Sources 16
........................................................... 2.4.2.1.3. Mechanism of Action 16
.................................. 2.4.2.2. Inhibition of PPO using an Esterase Extract 16
........................................................................... 2.4.2.2.1. Introduction 16
2.4.2.2.2. Inhibition Studies .................................................................. 17 ..... 2.5. Applications of Enzymatic Browning Inhibition in Food and Processed Food 18
.................. ....................... 2.5.1. Methoh for the Evahation of Browning ... 18
..................................................... .......................... . . . 2 5 2 Apple Producrs ... 19
2.5.2.1. Apple Slices .................................................................................... 19
............................................................... ................... 2.5.2.2. Apple Juice .. 20
................................................................. ............................. 5.2.3. Potatoes .. 21
3 . MATERIALS AND METHODS ............................................................................................ 23
3.1. Materials ................... .. ......................................................................................... 23
3.1.1. Raw Products ..... ................................................................................ 23
3.1.2. Inhibitors of Enzymatic Browning ............... ...... .............................. 23
3.1.2.1. Chernicals ....................................................................................... 23
3.1 .2.2. Natural Bio-Xngredients . ...... ................................... ..... ................... 23
3.1 .2.2. 1 . Copper -Meta1100 (Cu-MT) ................................... ...... 23
3.1 .2.2.2. PoIyphenol Esterase (PPE) ................................................ 24 . .
3.2. Protein Determination ........ .. ............................................................................. 24
3.3. Sarnple Preparation ..... ............ ... ....................................................................... 24
3.3.1. Apple Slices ............................................................................................. 24
3.3.2. Apple Juice .............................................................................................. 25
... 3.3.3. Potato SZices ..................... ,,,, ..... ,.., 25
3.4. Inhibitor Treatments .................................... ,.. . . . . . 25
3.4.1. Apple SIices ..................... ... ................................................................ 25
3.4.2. Apple Juice ......................... ,...... ...................................................... 26
3.4.3. Potato Sf ices ............................................................................................ 26
3.5. Color Measurements ............................................................................................... 27
3.6. Evaluation of the Inhibition of Browning ............................................................... 28
..................................................................................... 3.7. Reflectance Measurements 28
3.8. Statistical Analysis .....................................................,......................................... 28
.................................................................... 4 . RESULTS AND DISCUSSION .............. ... ..30
4.1. Apple Slices ............................................................................................................ 30
4.1.1. Measurement of Browning ut the Cut Sur/oce of Apple Slices ............... 3 0
4.1.2. Evafuation of Browning Inhibitor Treatments ut the Cut
SuMace of Apple Slices ......................................................................... 32
4.1.3. Kinetic Study of Enzymatic Browning in Apple SZices using
Reflectunce Spectmm ........................................................................ 45
4.2. Apple Juice ....... .. .................................... .. ........................................................... 50
4.2.1. Mimurement of Browning in Apple Juice ............................................. . 50
4.2.2. Evaluation of Bro wning In hibitor Treatments in Apple Juice .. ............. -52
4.2-3. Kinetic Study of Enrymatic Browning in Apple Juice using
Reflectance Spectrum ........................................................................... 65
4.3. Potato Slices ....................... ....... ....................................................................... 70
4.3.1. Memurernent of Bro wning at the Cut Suflace of Poiato SIices .............. 70
4.3.2. Evaluation of Browning Inhibitor Treatments at the Cut
........................................................................ Surf ce of Potato Slices 72
4.3.3. Kinetic Study of Enzyrnatic Browning in Potafo Slices uring
........................................................................ Reflectance Spec- 85
vii
Page
Table 1. Inhibitory Effect of a Copper-Metallothionein Extract (Cu-MT), nom
A. niger, on Enzymatic Browning in Apple Slices, determined
using a Tristimulus Colorimeter ........................................................ 33
Table 2. Inhibitory Effect of a Polyphenol Esterase Extract (PPE), fiom A. niger,
on Enzymatic Browning in Apple Slices, determined using a . .
Tnstmulus Co lorimeter.. ............................................................... .35
Table 3. Inhibitory Effect of Ascorbic Acid (AA) on Enzymatic Browning in
...................... Apple Slices, detennined using a Tnstimulus Colorimeter.. -36
Table 4. Lnhibitory Effect of Citric Acid (CA) on Enzymatic Browning in
...................... Apple Slices, determined using a Tristimulus Colorîmeter.. -38
Table 5. Inhibitory Effect of Ethylenediamketetraacetic Acid (EDTA) on
Enzymatic Browning in Apple Slices, determined using a . .
Tnstimulus Colorimeter.. .............................................................. - 3 9
Table 6. Inhibitory Effect of Sodium Bisulfite (&HS03) on Enzymatic
Browning in Apple Slices, detennined using a . .
Tnstimulus Colorùneter. ................................................................. 4 1
Table 7. Inhibi tory E ffect of 4-Hexylresorcinol(4HR) on Enzymatic
Browning in Apple Slices, detennined using a . .
Tnstmulus Colorimeter. ................................................................. 42
Table 8. Lnhibitory Effect of a Copper-Metallothioneh Extract (Cu-MT), fiom
A. niger, on Enzymatic Browning in Apple Juice, determined using . . a Tnstunulus Colorimeter. ............................................................. -53
Table 9. Inhibitory Effect of a Polyphenol Esterase Extract (PPE), fiom A. niger,
on Enzymatic Browning in Apple Juice, detennined using a . .
Tnstimulus Colonmeter.. ............................................................... -55
Table 10. Inhibitory Effect of Ascorbic Acid (AA) on Enzymatic Browning in
Apple Juice, detennined ushg a Tristimulus Colorimeter.. ...................... .56
Table 1 1. Inhibitory Effect of Citric Acid (CA) on Enzyrnatic Browning in
Apple Juice, detemined using a Tristimulus Colorimeter.. .................... - 3 8
Table 1 2. Inhibitory E ffect of Ethylenediamineteûaacetic Acid (EDTA) on
Enymatic Browning in Apple Juice, determined using a . . ............................................................... Tnstimulus Colorimeter .59
Table 13. Inhibitory Effect of Sodium Bisulfite (NaHS03) on Enzymatic
Browning in Apple Juice, determined using a Tnstimulus
............................................................................ Colorimeter.. -6 1
Table 14. Inhibitory Effect of 4-Hexylresorcinol(4HR) on Enzymatic
Browning in Apple Juice, determined using a Tnstimulus
............................................................................. Colorïmeter. -63
Table 15. Inhibitory Effect of a Copper-Metallothionein Extract (Cu-MT),
fkom A. niger, on Enzymatic Browning in Potato Slices, detertnined
using a Tristimulus Colorimeter ........................................................ 73
Table 16. Inhibitory Effect of a Polyphenol Esterase Extract (PPE), fiom
A. niger, on Enzymatic Browning in Potato Slices, determined
using a Tristimulus Colorimeter.. .................................................... -75
Table 1 7. Xnhibitory Effect of Ascorbic Acid (AA) on Enzymatic Browning
in Potato Slices, determined using a Tristimulus Colorimeter.. ........... .. ...... 77
Table 18. Inhibitory Effect of Citric Acid (CA) on Enzymatic Browning
in Potato Slices, determhed using a Tristirnulus Colorimeter.. ................... 78
Table 19. Inhibitory Effect of Ethylenediaminetetraacetic Acid (EDTA) on
Enzymatic Browning in Potato Siices, determined using . . .............................................................. a Tnstirriulus Colorimeter 80
Table 20. Inhibitory Effect of Sodium Bisulfite (NaHS03) on Enzymatic
Browning in Potato Siices, detemiined using a Tristimulus
.............................................................................. Colorimeter. -8 1
Table 21. Inhibitory Effect of 4-Hexylresorcinol(4HR) on Enzymatic Browning
in Potato Slices, determined using a Tristimulus Colorhneter.. .................. .83
Page
Figure 1. Reac tions Cataiyzed by Polyphenol Oxidases ......................................... -4
Figure 2. Common Substrates for Polyphenol Oxidases (PPO) ................................ -6
Figure 3. Changes in the L and a Values in Apple Slices, without Inhibitor and
with Different Concentrations in NaHSO3. ......................................... 3 1
Figure 4. Effect of the Concentration of the Seven Selected Inhibitors on
.......................................... Enzymatic Browning in Apple SLices.. ..A4
Figure 5. Scanning of Enzymatic Browning in Apple Slices, over T h e , With
and Without Inhibitor. ................................................................ .46
......... Figure 6. Reflectance Spectra for the Seven Selected Inhibitors in Apple Slices.. -48
Figure 7. Effect of the Concentration of the Seven Selected Inhibitors on
Enzymatic Browning in Apple Slices at 400,460,530,600
and 670 nm. ............................................................................. 49
Figure 8. Changes in the L and a Values in Apple Juice, without Inhibitor and
with Different Concentrations in NaHS03. ........................................ .5 1
Figure 9. Effect of the Concentration of the Seven Selected Inhibitors on
Enymatic Browning in Apple luice.. ............................................. .64
Figure 10. Scanning of Enzymatic Browning in Apple Juice, over Time, With
and Without Inhibitor.. ................................................................ 66
Figure 1 1. Reflectance Spectra for the Seven Selected Inhibitors in Apple Juice.. .......... 68
Figure 12. Effect of the Concentration of the Seven Selected Inhibitors on
Enzymatic Browning in Apple Juice at 400,460,530,600
............................................................................ and 670 nrn.. 69
Figure 13. Changes in the L and a Values in Potato Slices, without Inhibitor and
....................................... with Different Concentrations in NaHS03.. -7 1
Figure 14. Effect of the Concentration of the Seven Selected Inhibitors on
Enzymatic Browning in Potato Slices.. ........................... ,. ............. .A4
Figure 15. Scanning of Enzymatic Browning in Potato Slices, over Time, With
............................................................... and Without Inhibitor.. -86
Figure 16. Reflectance Spectra for the Seven Selected Inhibitors in Potato Slices. ......... -88
Figure 17. Effect of the Concentration of the Seven Selected Inhibitors on
Enzymatic Browning in Potato Slices at 400,460,530,600
.......................................................................... and 670 nm.. ..89
EDTA
a PPE
PPO
Ascorbic Acid
Citric Acid
Copper-met allothioneh
Ethylenediarninetetracetic Acid
4-Hexy lresorcino l
Percent Inhibition Value
Polyphenol Esterase
Pol yp henoi Oxidase
Sodium Bisulfite
The occurrence of browning in raw miits, vegetables and their processed products is a
major problem in the food iridustry and is believed to be one of the main causes of quality
loss during post-harvest handling and processing. The mechanism of browning has been well
characterized and cm be of enzymatic or non-enzymatic origin. Generally, browning causes
many deletenous changes in the appearance and organoleptic properties of food products,
resulting in shorter shelf-life, decreased market value and, in sorne cases, complete exclusion
of the food product fiom certain markets; however, browning of food may also have
beneficial effects since it contributes to desirable color and flavor of such products as raisins,
coffee, tea and cocoa (Walker, 1995).
Enzymatic browning occurs as a result of the oxidation of endogenous phenolic
compounds catalyzed by polyphenol oxidase (PPO). PPO is a term which describes a large
number of related copper-containing enzymes, including tyrosinase, catechol oxidase and
laccase. PPO catalyzes, in the presence of oxygen, the oxidation of mono- and di-phenols to
O-quinones; these products are highly reactive and can either polymerize spontaneously to
form high-molecular-weight compounds or brown pigments, or react with amino acids and
proteins to enhance the brownish color produced (Viamos-Vigyazo, 1981; McEvily et al.,
1992).
The control of enzymatic browning has dways been a challenge to the food industry.
The most widespread process used for the inhibition of browning is the addition of sulfiting
agents; however, the adverse health effects associated with sulfites as well as the increased
regulatory scrutiny (Anon, 1986) have created the need for alternative compounds. In
addition, the use of antibrowning agents in the food industry is constrained by considerations
such as toxicity, effects on taste, flavor, color, texture and cost (McEvily et al., 1992). Many
approaches are available to inhibit enzymatic browning, in particular, the use of ascorbic,
erythorbic and citric acids as well as their sodium salts. A number of other antibrowning
treatments, including reducing agents, aciduiants, chelating agents, PPO inhibitors,
cornplexhg agents, inorganic salts and enzymes, have been investigated (Viamos-Vigyazo,
198 1 ; McEvily et al., 1992; Nicolas et al., 1994; Friedman, 1997). In addition, two novei
approaches were proposed for the control of enzymatic browning, ïncluding the use of a
copper-metallothionein extract (Goetghebeur and Kermasha, 1996) as weU as a partially
puri fied rnicrobial esterase extrac t (Madani et al., 1 997).
Metallotbioneins are ubiquitous c ytosolic proteins, usually characterized b y a high
content of cysteine and selectively bind large amounts of heavy metal ions (Lerch, 1980;
Byrd et al., 1988). induced metallothioneins could be used as highly effixtive demetallizing
agents and exhibit strong reduction activities (Bremner, 1991). Since PPO contains copper in
its active site, metallothioneins could be used as inhibitors of enzyme activity. Using a mode1
system, Goetghebeur and Kermasha (1 996) demonstrated that a copper-metallothionein (Cu-
MT) extract fiom Aspergilf1u.s niger was an efficient inhibitor of the enzymatic activity of a
commercially purified mushroom tyrosinase preparation. A low molecular weight copper-
chelating peptide fiom Dactylium dendroides was also reported to inhibit mushroom PPO
activity (Haret et al., 1967).
Monohydroxylated cinnamic acids are considered to be appropriate inhibitors of PPO
activity (Walker and McCallion, 1980). Walker (1969) suggested that the hydrolysis of the
cinnamoyl derivatives of quinic acid, catal yzed b y the pectinol ytic activity of the Peniciflium
expansum, could result in the formation of cinnarnic acids which could act as cornpetitive and
non-cornpetitive inhibitors of PPO activity, due to their structural sirnilarities with phenolic
substrates (Walker and McCallion, 1980). Recently, Madani et al. (1997) reported that a
polyphenol esterase (PPE), obtained fiom the biomass of Apergilh niger, was capable of
generating cinnamic acids, in particular caffèic acid fiom chiorogenic acid (Kermasha et al.,
1993b). The characterization of tyrosinase and PPE-catalyzed end-products using selected
phenotic substrates indicated that PPE inhibited tyrosinase activity by interacting with the
phenolic substrates (Madani et al., 1999b).
The increasing preference for minimal processing of food products stems fiom the
consumer's desire to have products that retain, as fa . as possible, the nutritional properties
and the organoleptic characteristics of the fksh product. This work is a part of ongoing
research, in out laboratory, aimed at the application of two biotechnological approaches for
the inhibition of enzyrnatic browning in selected food products, particularly in apples and
potatoes.
The specific objectives were:
1. To optimize the inhibition of enzymatic browning in three selected food products (apple
slices, apple juice and potato slices) using two natural bio-ingredients, Cu-MT and PPE.
2. To optimize the inhibition of enzymatic browning in the selected food products using
certain chemicals, including ascorbic acid, citric acid, ethylenediarninetetraacetic acid,
sodium bisul fite and 4-hex y lresorcinol.
3. To investigate the color changes of the treated products by tristimulus colorimetry.
4. To monitor the kinetics of the inhibition of enzymatic browning using reflectance spectra.
2. LITERATURE REVIEW
2.1. Polyphenol Oxidase (PPO)
2.1.1. Defrnition
PPOs, kst reportexi in mushrooms by Schoenbein in 1856, are copper-containing
proteins and belong to the group of oxidoreductases. PPOs are able, in the presence of
oxygen, to act on phenols to produce brown color in damaged fniits and vegetables; this
phenornenon is cailed enzymatic browning. PPOs are also associated with the development
of dark pigmentation in organisms, by beïng involved in the biosynthais of rnelanin
pigments and other polyphenolic compounds which ofien provide a protective fünction
(Whitaker and Lee, 1995).
Figure 1 : Reactions catalyzed by polyphenol oxidases.
(a) Hydroxylation of monophenol to o-diphenol.
@) Dehyârogenation of O-diphenol to oquinone.
Two kinds of enzymes are classifiecl under the trivial name of PPO. Based on substraîe
a specificity, Enzyme Nomenclature (1992) has designatecl as (i) diphenol oxidases, catechol
oxidases or diphenol oxygen oxidoreductases (EC 1.10.3- l), the enzymes which catalyze two
distinct reactions (Fip. 1 ), including the hydroxy lation of monophenols fo O-diphenols
(reaction 1) followed by the oxidation of O-diphenols to O-quinones (reaction 2); and (ii)
laccases, or p-diphenol oxygen oxidoreductases (EC 1.10.3.2), the enzymes that oxidize O-
diphenols as well as p-diphenols to t h e ~ corresponding quinones.
The nomenclature of PPO is somewhat confusing because besides the two types of
PPOs designated as EC 1.10.3.1 and EC 1.10.3.2, a third one exists, designated as EC
1.14.18.1 (Enzyme Nomenclature, 1992); it is referred to as monophenol rnonooxygenase,
cresolase or tyrosinase and corresponds to the same enzyme as EC 1.10.3.1 and catalyzes the
hydroxylation of monophenols.
2.1.3. Source
PPOs are found in most plant tissues including apples (Harel et al., 1964), grapes
(Ivanov, 1966), pears (Rivas and Wtaker, 1973), potatoes (Craft, 1966) and tea (Zawistoski
et al., 1991), as well as certain seeds such as cocoa and coffee, in microorganisms including
bacteria and fùngi (Vamas-Vigyazo, 1981) and rome higher animals such as insects
(Sugumaran, 198 8; 1 WO), arthropods, mammals and humans (Witkop, 1 985). The
. localization of PPO in plant cells depends on the species, age and on maturity of fiuits and
vegetables. In addition, in uncut or not damaged bits and vegetables, the natural phenolic
substrates are separated from PPO by compartmentalization so that browning does not occur
(Marques et al., 1995).
2.1.4. Natu rai Substrates
Fruits and vegetables contain a wide varïety of phenolic compounds with the most
commonly found being benzoic acids, cinnamic acids, flavonols, tannin "precursors" and
anthocyanidins; however only a relatively smaU part of these compounds serves as substrates
to PPO because the enzyme does not act on glycosides. The most important natural substrates
are catechins, cinnamic acid esters including chlorogenic acid and its isomers, 3,4-
dihydroxyphenyl alanine &-DOPA) and tyrosine (Fig. 2) (Macheix et al., 1990).
Catechin
4-Methyl Catechol
L-3,4-Dihydroxyphenylalanine (L-DOPA)
Caffeic Acid Tyrosine
Chlorogenic Acid
Catechol
Figure 2: Common substntes for polyphenol oxidases (PPOs).
The main PPO substrates, in apple, are catechùi, epicatechin and chlorogenic acid
whereas in potato, tyrosine, chlorogenic acid and caffeic acid are found to form colored
products (Viamos-Vigyazo, 198 1).
2.1.5. Determinarion of PPO Activity
PPO activity can be deterrnined by measuring the rate of substrate consumption, or
the rate of product formation. When detennining the rate of substrate consumption, generally
oxygen absorption is measured, either manometrically in a Warburg respirometer, or
polarographicaily with an oxygen electrode (Viarnos-Vigyazo, 1981). The rate of product
formation c m be determined spectrophotornetrically by measurbg the optical density of the
colored compounds, produced fiom the polymerization of O-quinones at specific wavelenghts
(around 400 nm), according to the substrate used (Espin et al., 1997).
2.2. Enzymatic Browning in Food
2.2.1. liitroducîiun
Up to 50% of fiuits and vegetables are lost due to the occurrence of browning. ïhree
different causes (Marqués et al., 1995) can be cited as follows:(i) various technological
processes which constitute the main cause and include wounding (such as cuts, peels),
crushing, extraction, fkeezing, fieeze drymg; (ii) some disorders which may occur during cold
storage; and (iii) phqrsiological evolution related to the maturation.
Enzymatic browning is a significant probIem, leading to major economic losses in
many commodities, especiaily fiesh h i t s such as apples, pears, bananas and papes, and
vegetables such as potatoes, lettuce and mushrooms, and seafood such as shrimp (Whitaker
and Lee, 1995). The deterioration has a great visual impact as enzymatic browning decreases
the commercial quality, the organoleptic acceptance (regarding the appearance of off-flavors
due to the associateci changes in color, flavor and softening (Bajaj et al., 1997)) and the
nutritional value (Osuga et al., 1994). The consumer will therefore not select b i t s and
vegetables that have undergone browning.
However, in certain instances, such as in certain dried f i t s including prunes, black
raisins, black figs, dates as well as in the manufacture of tea, coffee and cocoa, PPO activity
is essential to the manufacturing processes; it contributes to the desirable color and flavor of
the products and thus improves their sensory properties (Walker, 1995). Enzymatic browning
plays an important role in the "fermentation" stage of black tea manufacture because it is
responsible for the biochemical changes that occur (Zawistoski et al., 1991). The beverage
quality of coffee has been shown to be related to the level of PPO activity in green coffee
beans and PPO also plays a part in the development of the h a 1 color of processed cacao
beans which contain large amounts of phenolic constituents such as epicatechin (Walker,
1995).
2.2.2. Mechanikm
PPO is a mixed hc t ion oxidase (Fig. 1) that catalyzes both the hydroxylation of
monophenols at the onho position to O-diphenols (reaction 1) and then the dehydrogenation
of O-diphenols to O-quinones (reaction 2); these two enzymatic reactions consume oxygen
and are referred to as monophenolase (cresolase; EC 1.14.1 8.1) activity and O-diphenolase
(catecholase; EC 1.10.3.1) activity, respectively (Vamos-Vigyazo, 198 1).
The pnmary products, O-quinones, are yellowish, reactive and unstable compounds.
They can (a) react with each other to fom high molecular weight polymers, 0) fom
macromolecular complexes with amino acids or proteins, and (cl oxidize compounds of
lower oxidation-reduction potentials (Varnos-Vigyazo, 198 L ). Further non-enzyrnatic
reactions with oxygen lead to additional reactions to give relatively insoluble and complex
products such as melanins (Whitaker and Lee, 1995). The color of pigments differs widely in
hue and intensity, depending on the phenols fiom which they originate and the environmental
factors of the oxidation reactions (Nicolas et al., 1994).
The most important factors that determine the rate of enzymatic browning in h i t s
and vegetables are the concentrations of active PPO and phenolic compounds, the pH,
temperature and oxygen availability (MarGnez and Whitaker, 1995). Understanding the
details of the enzymatic browning process is therefore necessary in order to control it and to
obtain a final product that is acceptable to consumers.
2.3. Inhibition of Enzymatic Browning
2.3.1 Principles of the Prevention of Enwmatic Browning
The different ways of controlling enzymatic browning can be divided into three
classes, depending on whether they affect the enzymes, substrates, or reaction products
although in some cases, two or three targets can be a f k t e d at the same time. in addition,
enzyme inhibition can be reversible or irreversible; the latter case is often achieved by
physical treatment (heat), while chernicals may act in one or the other way (Nicolas et al.,
1 994).
2.3.2. Type of Inhibitors
There are numerous ways which c m be used reduce enzymatic browning in food. The
choice of one approach over another results fkom an evaluation of the inhibitor's overall
performance, treatment cost, organoleptic impact, and toxicity/regulatory concerns.
2.3.2.1. Practicai Methods
Prevention does not necessarily consist of post-harvest treatment only. Much c m be
done to reduce browning which occurs during storage or processing by selecting cultivars of
slight browning tendency (Amiot et al., 1992) and by using appropriate agicultural
techniques (Mondy and Munshi, 1993). Indeed, the phenol content of h i t s and vegetables
varies naturally according to the degree of rïpeness, the soi1 and climate, but also depends
substantially on intraspeci fic genetic charactenstics.
2.3.2.2. Physical Methods
2.3.2.2.1. Eflecr of Temperaîtire
A cold environment induces a marked decrease in enzymatic browning. Nonetheless,
color changes are still rapid at O°C so that most sensitive products are h z e n by methods
allowing the crystallization temperature of water to be reached as rapidly as possible. Once
fkozen, color changes are practically blocked at the temperature of commercial storage (-
1 SOC).
Heat treatment, such as blanching, constitutes without doubt the simplest and most
direct method of enzyme inactivation. Water blanching was used to prevent enzyrnatic
darkening in fiozen sweet potatoes. Treatment at lûû°C for 3 min or at 94OC for 5 min gave
satis factory protection against darkening without reducing phenol levels (Ma et al., 1992)-
2.3.2.2.2. Protection fi-orn Oxygen
Complete removal of oxygen is the most satisfactory way to control phenolic
oxidation catalyzed by PPO; this method can be applied to dead tissues either by creating a
physical barrier to oxygen diffusion such as dipping h i t s in sugar synip, the use of oxygen-
impermeable packaging or edible fiims (Baldwin et al., 1995), or by using an inert
atmosphere such as partial vacuum or oxygen-poor atmospheres (Nicolas et al., 1994), but it
is inapplicable to living tissues due to the nsk of metabolic deviations caused by anaerobic
conditions.
2.3.2.2.3. Supercritical Carbon Dioxide
Supercntical carbon dioxide (SC-CO2 treatment was investigated for inactivating
PPO in potato peels, fiesh Flonda spiny lobster and fkesh brown shrimp. Treatment of the
enzymes with hi&-pressure CO2 (58 atm) caused a dramatic loss of activity; however a
treatment of 30 min was required for an activity loss of 9 1 % (Lopez et a l . 1994).
2.3.2.3. Use of Chemicals
Chemicals are the most commonly used for the control of enzymatic browning;
however, their use in processed food products is restricted to compounds that are nontoxic,
wholesome and that do not adversely atrect taste and flavor (McEvily et al., 1992; Sapers,
1993). Chemicals can inhibit or control enymatic browning by sterilely hinderhg the
enzyme or by binding to its active site thereby rendering it incapable of catalyzing the
enzyrnatic reaction; they may also act on the enzymatically-catalyzed products to control
browning. The primaiy reaction products of PPO catalysis, oquhones, are very reactive.
Using chemicai means, the primary products can either be reduced back to the les reactive o-
diphenols or trapped as colorless compounds thereby preventing the occurrence of non-
0 enzymatic condensations to perceptible pigments (Sapers and Miller, 1992).
2.3.2.3- 1. Chernical Enzyme Inhibitors
Chemical enzyme inhibitors act only on the enzyme and are classified in two groups;
those interacting with copper are in the fùst class and those affecting the active site of the
enzyme for the phenolic substrate are in the second one (Mayer and Harel, 1991).
In the first group, inhibition by metal ion chelaton such as &de, cyanide and carbon
monoxide, which are more or less copper specific, is well documented for PPO obtained fiom
different sources (Healey and Strothkamp, 198 1 ; Zawistowski et al., 1988). Similarly,
inhibition by inorganic halide ions, which is strongly pH dependent, has long been
recognized for PPO activity fiom apple (Janovitz-Klapp et al., 1990).
Among inhibitors belonging to the second group, aromatic carboxylic acids of the
benzoic and cinnamic senes have been widely studied (Walker, 1976; Kemasha et ai.,
19936); they act as cornpetitive inhibitors of PPO because of their structural similarities with
phenolic substrates. The substituted resorcinols, such as 4-hexylresorcinol, are also
structurally related to phenolic substrates and are recognized as PPO inhibitors (Monsalve-
Gonzalez et al., 1993).
2.3.2.3.2. Reducing Agents
Probably the most common method of controllhg enzymatic browning, both in
industry and the laboratory, is by the addition of reducing agents such as ascorbic acid, thiol
compounds, sulfites and amino acids (Friedman and Bautista, 1985). Reducing compounds
prevent browning by reducing the enzymatically fonned or endogenous oquinones back to
their parent O-diphenols or by trapping them as colorless addition compounds or by
irreversible inactivation of PPO; however, the effectiveness of these reducing agents is
greatly decreased if their use is delayed until after the enzymatic reaction has starteci.
Ascorbic acid is probably the most used compound for apple products, even if its
effect is only temporary because of its irreversible oxidation. The use of an isomer erythorbic
(d-isoôscorbic) acid or more stable fonns of ascorbic acid such as its phosphorylated
a derivatives, can be an alternative (Sapers et al., 1989b; Sapers et al., 1991; Sapers and Miller,
1992; Sapers, 1993). Sulfites are highly effective in controlling browning in food products
but are h o w n to cause adverse health effects such as asthma (McEvily et al., 1992); they
have therefore been restncted or banned in several countries (Taylor, 1993). Few studies have
been carried out on sulfhydryl-containing amino acids; in this category, cysteine as wel1 as
reduced glutathione have been proven effective in the prevention of browning (Mo lnar-Perl
and Friedman, 1990a;b; Richard et al., 199 1) and their major effect is due to the formation of
colorless thiols-conjugated with O-quinones. -However, a direct inhibition of PPO activity by
cysteine through the formation of stable complexes with copper has also been proposed
(Khan, 1985; Kennasha et al., 1 993a).
2.3.2.3.3. Chelating Agents
Chelating agents including mainly phosphates and ethylenediaminetetraacetic acid
(EDTA), are believed to either bind to the active copper site of PPO or reduce the level of
copper available for incorporation into the holoenzyrne (McEvily et al., 1992); however, they
cm only slow down the enzymatic reaction but do not completely inhibit it.
EDTA is the most known compound and is used to control afler-cooking darkening in
pre-peeled potatoes (Feinberg et al., 1987) whereas Sporix, an acidic polyphosphate product,
is a powerfil chelating agent and very effective in preventing enzymatic browning in apple
juice (Sapers et al., 1989b).
2.3.2.3.4. Acidulant Agents
Since optimum enzyme activity depends on pH, changing the pH of the environment
cm control PPO activity. The pH optimum of PPO activity depending on the source of the
enzyme and the particular substrate, but in most cases, PPO has an optimum pH in the range
of pH 6 to 7 (Aylward and Haisman, 1969). Acidifiers lower the pH of the system to below
3.0 where PPO is efféctively inactive (Richardson and Hyslop, 1985). The main compounds
are citric acicl, organic acids such as malic, tartaric and malonic acids and inorganic acids
such as phosphoric and hyàrochlonc acids. Apart £kom acetic acid used in vinegar
production, ciûic acid, which c m also act as a chelating agent (Santerre et al., 1988), is
vimiaily the only substance authorized for enzymatic browning prevention (McCord and
Kilara, 1983).
2.3.2.3.5. Complexing Agents
The physical elimination of phenolic substrates is realised by specific adsorbents such
as complexing agents; they allow the formation of inclusion complexes which involves the
entrapment of polyphenots, thereby preventing their oxidation by PPO. However, the
formation of the inclusion complex is non-specific resulting at tirnes in the removal of flavor
or color compounds present in low concentrations.
Cyclodextrins, especially P-cyclodextrin, are effective browning inhibitors in apple
j uice (Sapers et al., 1 989b) as well as PVPP (polyviny lpolyp yrollidone), a product permitted
for use as a f ~ n g agent for apple juice (Van Buren, 1989); however, these agents are
resûicted to liquid systems. Sulfated polysaccharides such as carrageenans inhibit browning
in apple juice and apple dices and their inhibitory effect could be synergistically enhanced in
the presence of 0.5% citric acid (Tong and Hicks, 1991); however, the mechanism of
inhibition by these carbohydrate polymers is unknown.
2.3.2.3.6. Miscellaneous
Some studies have been devoted to "natural" inhibitors such as proteins, peptides and
amino acids. The inhibitory properties of honey on different PPO enzymes in mode1 systems
and on browning in grape juice and apple slices were attributed to a small peptide with an
approximate molecular weight of 600 Da (Osmianski and Lee, 1990). Lacfobaciilus
helveticus has been found to secrete a cyclotetrapeptide that inhibits PPO activity (Kawagishi
et aL, 1993). It has also been postulated that the inhibition of browning could result from
both the reducing and the chelating properties of the Maillard reaction products (Nicoli et al.,
1991).
2.3.2.4. Enzyme Treatments
The possibility of an enzyme, itself a protein, being attacked and inactivated by other
enzymes is more than obvious. Contact with kiwi slices or kiwi purée inhibits e n m a t i c
browning due to the presence of a highly active protease, actinidin (Labuza et al., 1990).
Three other plant proteases, ficin fkom fig, papain fkom papaya and bromelain from
pineapple, al1 of which are sulphydiyl enzymes of broad specificity, have proven to be
effective in controlling enzymatic browning (Lozano-de-Gonzales et al., 1993).
Other enzymes prevent the occurrence of browning by removing phenolic substrates
by chernical modification. The use of O-methyltransferase to methylate the O-dihydroxy
phenolics to the corresponding methoxy derivatives has been suggested by Finkie and Nelson
(1963). Kelly and Finkle (1969) ireated apple juice with the bactenal enzyme protocatechuate
3,4-dioxygenase, obtained Erom Pseudomonas aemginosa, which catalyzes the oxidative
~g -open ing reaction and the ortho-fission of catechols; treated juice did not darken when
compared to a control sarnple of untreated juice. However, although enzyme treatments are
elegant processes, they are irnpeded by their high cost and do not appear feasible for
commercial use.
2.3.2.5. Genetic Approach
Molecular biology, through gene manipulation, could be used to inhibit PPO in
transgenic plants by expressing PPO in antisense orientation. For example, the expression of
PPO in potatoes was decreased by using vectors carrying antisense PPO cDNA (Bachem et
al., 1994). It is also possible to develop low-browning f i t s and vegetables using gene
silenchg (Martinez and Whitaker, 1995).
2.4. Novel Approaches for the Inhibition of Enzymatic Browning
2.4.1. Copper Metallothionein
2.4.1.1. Biology of Metallothionein (MT)
2.4.1.1.1. Definition
Fowler et al. (1987) defined metallothioneins (MT) as: "Polypeptides diar resemble
equine renal MT in several oftheir features can be designated as MT." Thus, MT is a genenc
t e m and it is used for a variety of similar metal-thiolate polypeptides. MTs were fint isolated
fiom horse kidney and consist of a group of ubiquitous cytosolic heat stable and low
molecular weight proteins (Turanek et al., 1987). They are characterized by a high cysteine
content, an unique amino acid sequence, a lack of aromatic acids, and exhibit selective
binding wiîh a large amount of heavy metal ions such as A$, Cu2+, Cd2+, ~ g " and Z#
(Hamer, 1 986).
2.4.1.1.2. Sources
MT have been obtained fiom a large variety of eukaryotic species including
vertebrates, invertebrates and plants (Hamer, 1986) as well as microorganisms such as A.
niger (Kermasha et al., 1993~).
2.4-1.1.3. Functions
Several fùnctions have been proposed for MT in eukaryotic cells including cellular
detoxification which is considered as one of the major functions (Ravi et al., 1984, Bremmer,
1991). Lerch (1980) suggested that the MT of N. crassa could fûlfill several important
physiological functions such as copper storage and metal donor to the active site of copper-
containhg enzymes.
2.4.1.2. Inhibition of PPO Using Copper-Chelating Peptide
2.4.1.2.1. Introduction
in contrast to MT fkom mammalian sources, fimgal MT most ofien contains copper
(Lerch, 199 1). A low molecular weight, copper-chelating peptide fiom Dactylium dendroides
was reported to inhibit mushroom tyrosinase activity (Harel et ai.. 1967). Under conditions of
hi& exposure, induced MTs could serve as highly effective demetallizing agents due to their
strong avidity for metal ions (Hamer, 1986). Since PPO contains copper at its active site, MT
could be used as an inhibitor of enzyme activity. In addition, MTs exhibit strong reduction
activities (Bremmer, 1991).
2.4.1.2.2. I n hibition Studies
Lately, copper-metallothionein (Cu-MT) isolated fiom the fiingus A. niger was found
to be an inhibitor, in a mode1 system, of the enzymic activity of a commerciaily purified
mushroom tyrosinase. The type and degree of inhibiton were dependent upon the substrate
used, the degree of purity of Cu-MT and the method used for the determination of PPO
activity. The inhibitory effect of Cu-MT on tyrosinase activity was higher with catechin as
subrtrate compared to that obtained with cMorogenic acid. In addition, the demetallization of
Cu-MT, prior to the inhibition studies, did not increase its inhibitory effect on PPO activity
whereas pre-incubation of the enzyme and Cu-MT increased the inhibitory effect
(Goetghebeur et al , 1996).
2.4.2. Esterases
2.4.2.1. Biology of Esterases
2.4-2.1.1. l?e$nition
Esterases (3.1) catalyze the hydrolysis as well as the synthesis of the ester bonds.
They have a very wide specificity which makes their classification difficult. They are divided
into different groups including carboxylic esterases (3.1. l), thiolesterases (3.1.2),
phosphatases (3.1.3) and sulfatases (3.1.6) (Enzyme Nomenclature, 1992).
2.4.2.1.2. Sources
Esterases are widely distributed in animal and microbial sources. From an economic
and industrial standpoint, microorganisms are abundant in nature and are therefore preferable
to animals as enzyme sources because they can grow in a broad range of environmental
conditions and produce extracellular enzymes which can exhibit optimal activity at high
temperatures or in extreme pH environments (McKay, 1993).
2.4.2.1.3. Mechanism of Action
Depending on the reaction medium, esterases c m catalyze the hydrolysis of ester
bonds. Microbial esterase fiom A. niger was reported to hydrolyze the aliphatic esters of
short chain fatty acids and acetylester of phenols (Okurnura et al., 1983). Pectin
methylesterase catalyzes the hydrolysis of methyl ester groups and has a high specificity for
pectin substrates (Biely et al., 1986).
2.4.2.2. Inhibition of PPO Using an Esterase Extract
2.4.2.2.1. Introduction
Cole and Wood (1961) suggested that PPO activity in apples was inhibiteci in the
presence of Penicilliurn expunsum which exhibited a high level of pectolytic activity. Waker
(1969) reporteci that the hydmlysis of cinnamoyl derivatives h m quinic acid, catalyzed by
the pectinolytic activity of Pexpansum, could result in the formation and accumulation of
phenolic acids such as p-coumark and ferulic acids; these acids were shown to inhibit o-
diphenol oxidase activity and hence the enzymatic browning in apple tissues. CafEeic acid
also inhibited mushroom PPO activity (Kermasha et al., 1993). Multiple foms of ferulic acid
esterase showed different substrate specificities towards a range of methyl ester denvatives of
cinnamoyl and benzoyl acids and have been detected in an A. niger extract (Fauld and
Williamson, 1993; 1994). Carboxylic acids of the cimamic series were demonstrated to be
competitive or non-competitive inhibitors of PPO activity found in different sources such as
potatoes (Macrae and Duggleby, 1968), apples (Walker and Wilson, 1975) and mushrooms
(Kermasha et al., 1993). Recently, Madani et ai. (1997) showed that a polyphenol esterase
(PPE), obtained from the biomass of A. niger, was capable of generating cafZeic acid fiom
chlorogenic acid.
2.4.2.2.2. In hibition Studies
The use of a partially purified PPE extract, obtained fkom A. niger, can reduce the
brown color formation by tyrosinase activity in a mode1 system. The PPE activity in the A.
niger extract exhibited a competitive and a mixed type of inhibition for PPO biocatalysis
using chlorogenic acid and catechin as substrates, respectively (Madani et al., 1997). in
addition, PPE inhibited the rate of oxidation of catechin and chlorogenic acid to brownish
end-products by tyrosinase activity, but not the rate of oxygen uptake. Madani et al. (1997)
suggested that PPE inhibited tyrosinase activity by converting chlorogenic acid into
substances that acted as inhibitors.
Lately , the inhibition of tyrosinase activity b y partial1 y purified and puri fied PPE
&actions was investigated using a wide range of mono- and di-phenols as substrates. The
activity of the purified PPE hction, obtained by ion-exchange chromatography, mmarkly
increased the inactivation of tyrosinase. In addition, the PPE biocatalysis exhibited an
uncornpetitive type of inhibition on tyrosinase activity with catechol as substrate, a
competitive one with 4-rnethylcatechol, and mixed inhibitory effects with 3,4-
dihydroxyphenylacetic acid (DHPAA), cafEeic acid, L-3,4dihydroxyphenylalanine (L-
DOPA), 4-hydroxyp ynivic acid and m- and p-cresol (Madani et al.. 1999a).
Another study showed that the nature and the quantity of end-products formed by
PPE and by the combination of PPE and tyrosinase was substrate-dependent. Both, colored
and colorless end-products were obtained by the oxidation reaction of the substrates by
tyrosinase and/or PPE activity (Madani et al., 1999b).
2.5. Applications of Enzymatic Browning Inhibition in Food and Processed Food
Due to the complexity involvecl in experiments performed with intact or partly
processed fiuits and vegetables, it is difficult to make conclusions concerning the direct effect
of inhibitors on browning so that many studies of enzymatic browning have been restricted to
mode1 systems where an enzyme acts on a single phenolic substrate (Janovitz-Klapp et al.,
1990). Consequently, the mechanisms of apple PPO (Richard-Forget et al., 1992; Goupy et
al., 1995), grape PPO (Cheynier and Ricardo da Silva, 199 1; Guyot et al., 1995) and
commercially prepared tyrosinase (OsPnianski and Lee, 1990; 199 1) in the development of
browning are well known.
2.5.1. Metkods for the Evaluation of Browning
Accurate methods are required for the measurement of browning in tissue slices and
extracts The need for such methods is obvious for the cornparison of different cultivars
designed for their susceptibility to browning or for the evaluation of experimental treatments
studied to control enzymatic browning.
Basically two kinds of methods are available (Macheix et al., l99O). The first method
uses absorbante measurements, generally in the 400 nm region, on solutions obtained af€er
extraction and purification of the brown pigments so that it masures only the soluble
pigments. The second method uses reflectometry or tristimulus cdorimetry and can be
applied directly to surfaces or fkuit purées; it is easy, rapid and nondestructive. Reflectance is
associated with the amount of radiation (light) retumed by a medium so that its value
corresponds to the ratio of reflected to incident radiation. The apparent color of an object
depends on the wavelength of the light that it reflects. In normal or white light, an opaque
object that reflects ail wavelengths appears white whereas one that absorbs al1 wavelengths
appears black (Stenstrom, 1964). Most of the data obtained in tristimulus colorirnetry are
given in L (lightness), a (greeness or redness), and b (blueness or yellowness) values, or a
combination of these three factors. The values obtained are highly dependent on the method
used for measurement and on the state of the surface of the examined object. Durùig
browning, variations in tristimulus data are the result of both, chernical (browning) and
physical changes; the relative importance of the two processes being difficult to assess.
Most authors use the decrease in AL values (Le., the difference in L values before and
aiter browning) to evaluate the extent of browning and recommend the use of the difference
between the initial and final tristimulus values as they are better indices of browning than the
slopes obtained fiom t h e curves (Mastrocola et al., 1990). Sapers and Douglas (1987)
studied enzymatic browning at the cut surface of apple and pear f i t s and showed that the
reflectance L and a values were linear or occasionally bilinear with log t b e and related to the
extent of browning with changes in L values being larger than changes in a values.
2.5.2. Apple Products
Apples, the most popular fhi t in the world, are processed into a variety of products
such as single-strength sweet, fermented or concentrated juices, canned sauces, and fiesh,
canned, dried or fiozen slices (Way and McLellan, 1989). Juice, purée and apple slices that
are badly processed tend to brown intensely and give final products a bad appearance which
is rejected by the consumer.
2.5.2.1. Apple Slices
In order to replace sulfites whose use is restricted in food products (Anon, 1986),
several alternatives have been proposed including, among the more effective, ascorbic acid
and its derivatives (Sapers et al., 1989b), reduced glutathione and N-acetyl-L-cysteine
(Molnar-Perl and Friedman, l99Ob).
The action of ascorbic acid can be enhanced in combination with the use of citric acid
and sodium chloride in concentrations that are ineffective themselves or that even increase
enzyme activity. Pizzocaro et al. (1993) claimed that 90-100% of inhibition occurred when
apple cubes were dipped for 5 min in aqueous solutions of 0.01% ascorbic acid and 0.002%
citric acid, or 0.01% ascorbic acid and 0.0005% sodium chloride. Recently, Gil et al. (1998)
studied the effect of ascorbic acid in combination with low-oxygen atmospheres and showed
that the shelf-life of rninimally processed apple slices could be significantly extended by
storage in oxygen-fiee atmospheres in combination with a 2.0% ascorbic acid dip treatment.
CHexylresorcinol (4HR) belongs to a new generation of antibrowning compounds,
the resorcinol derivatives; it has been used as an active ingredient in various medicines for
more than 40 years (Frankos et al., 1991) and has recently been tested in food applications,
especially in apples (McEvily et al., 1992; Luo and Barbosa-Canovas, 1995). In combination
with ascorbic acid, 4HR is an effective antibrowning agent that compares favorably to
sodium sulfite at storage temperatures of 25°C; a fivefold higher sodium sulfite concentration
was required to produce the equivalent browning inhibition to that obtained with 4HR. 4HR
a could also be used in lower concentrations to preserve color in apple slices processed by a
combination of methods (Monsalve-Gonzales et al., 1993). Walker (1995) proposed another
formulation which consisted of 0.01% 4HR, 0.5% ascorbic acid and 0.2% calcium chloride;
the apple pieces, treated for 5 min with this solution and packaged under partial vacuum,
could be stored at 0S0C for 50 days with acceptable coior and texture.
2.5 -2.2. Apple Juice
Cloudy or unclarified juice has an increasing market potential due to its superior
sensory and nutritional qualities. However, the production of high quality juice is difficult
because of its sensory instability (Lea, 1990). Cloudy apple juice is very sensitive to
enzymatic browning since it contains considerable quantities of polyphenols and polyphenol
oxidase which are bound to suspended particles.
Various methods have been descnbed to control or prevent discoloration in fiuit
juices including maùily ascorbic acid and its derivatives (Sapers et al., 1989b; Janovitz-Khpp
et al., 1990) and sulfiting agents whose use is nowadays very limiteci (Sapers, 1993). Because
none of the currentiy used compounds are as effective and inexpensive as sulfites, a great
deal of research is being done to find alternatives, Lrwin et al. (1994) showed that
cyclodextrins form a complex with PPO substrates such as chloïogenic acid, thereby
protecting the substrate fiom enzymatic oxidation. Hicks et al. (1996) reported that
cyclodextrins, both soluble and insoluble, alone or in combination with organic or inorganic
phosphates, can be used to inhibit enzymatic browning in fiesh nuit juices. Tong et al. (1995)
tested various commercial pectin preparations for their ability to prevent browning in 6esh
Granny Smith apple juice; fiactionation and testing of these preparations showed that the
antibrowning activity was not due to pectin but to the Qresence of a low level of oxalic acid
(<OS%).
Zemel et al. (1990) demonstrated that PPO activity coiilci be keversibly inhibited by
temporarily lowering the pH of apple juice to 2.0 with HCI, however, the formation of salts
greatly affected juice flavor. Electrodialysis, which is based on the dissociation of water
molecules using a bipolar membrane, presents new possibilities for controlhg enzymatic
browning in food Iiquids (Lopez-Leiva, 1988). Tronc et al. (1997) demonstrated that the
acidification of cloudy apple juice to pH 2.7 in an electrodialysis unit partially inhibited the
PPO activity (81%) and slowed enzymatic browning; the same authors (1998) obtained
complete inhibition of PPO activity by reducing the juice pH to 2.0 during electrodialysis.
This treatment enhanced the color of cloudy apple juice during storage without modi@ng the
flavor or sugar content; however, the low level of acidification required the exogenous
addition of K'.
5.2-3- Potatoes
Polyphenolic compounds are secondary plant metabolites found in numerous plant
species including potatoes. Chlorogenic acid is the main component and constitutes up to
90% of the total phenolic content of potato tubers; this content is the same for stewed and
raw potatoes (Friedman, 1997). Sapers et al. (1989a) showed that the tendency for Atlantic
potatoes and related cultivars to brown was related to the total phenolic compounds present,
tyrosine, and to a lesser extent, PPO activity.
The storage life of pre-peeled or cut raw potatoes is limited by the onset of enzymatic
browning. Without inhibitor treatment, potatoes would discolor within minutes by himing
pink, brown, gray or black. Previously, potato processors controlled browning in raw
products by the application of sulfites which were highly effective (Feinberg et al.. 1987);
however, due to the adverse health effects of sulfites on some asthmatics, their use has been
banned by the FDA (Anon, 1990). Various sulfite substitutes are used but they are less
effective and the treated products may require vacuum or modified atmosphere packaging
(Langdon, 1987) or packaging in a preservative cover solution (Santerre et al., 1991) to
achieve the desired storage life of fourteen days.
Molnar-Perl and Friedman (1990b) described the use of rduced glutathione and N-
acetyl-L-cysteine as highly effective alternatives to sulfites for cut potatoes. The
e ffec tiveness of ascorbic acid-based browning inhibitor formulations was greatly improved
by replacing some of the ascorbic acid with ascorbic acid-Zphosphates in diced and pre-
peeled potaoes (Sapers and Miller, 1992). The storage life was extented by 5-7 days over that
obtained with conventional browning uihibitor formulations; however, a problem of leakage
induced by the treatment occured at the cut surface of potatoes. Sapers and Miller (1995)
inhibited potato discoloration for fourteen days at 4OC by using a double treatment consisting
of ascorbic/citric acid solutions plus heat, followed by dipping in a solution containhg
ascorbic and cihic acids plus sodium acid pyrophosphate. The apparent formation of
inclusion complexes of cholorogenic acid with P-cyclodextrins coutd also serve as a basis for
controlling enzymatic browning in potatoes (Zrwin sr al., 1996).
Harrington (1957) developed another type of treatment, the lye digestion, which
removed surface tissues fiom peeled potatoes prior to the use of browning inhibitors. Sapers
and Miller (1993) showed that lye digestion extended the shelf-life of high pressure steam-
and abrasion-peeled potatoes to 13- 15 days at 4*C, wmpared to 3-1 1 days for undigesteci
controls. Thus, lye digestion in conjunction with conventional browning inhibitors represents
a viable alternative to the addition of sulfites to pre-peeled potatoes.
3.1. Materials
3.1.1. Ra w Products
McIntosh apples, harvested in September 1 998, were supplied by Lassonde lnc.
(Rougemont, QC) and kept at 4°C until their use during the Winter 1999. Potatoes of Russer
Burbank's variety, harvested in August 1998, were obtained fiom McCain Food Ltd.
~lorenceville, NB) and stored at 4OC until theu use during the Spring 1999.
3.1.2. Inhibirors of Enqymatic Browning
3.1.2.1. Chernicals
Citric acid, ethylenediamine-tetraacetic acid (EDTA) and sodium bisulfite were
purchased fiom Fisher Scientific Co. (Fair Lawn, NJ) while 4-hexylresorcinol was purchased
fÏom Aldrich Chernical Company hc . (Milwaukee, WI). Ascorbic acid was obtained fiom
Lassonde Inc. (Rougemont, QC),
3.1 -2.2. Naturd Bio-Ingredients
3 - 1 2 2.1. Copper-Metallothionein (Cu-Mn
The fungal strain used in this sîudy as a source of copper-metallothionein (Cu-MT)
was Asper*IZw niger (CBS 131.52), obtained £tom The Central bureau voor
SchimmeIcultures (Baam, The Netherlands). The biomass production of the induced Cu-MT
of A. niger was perfonned according to the procedure descnbed by Kennasha et al. (1993~).
AAer induction, the metalloproteins were extracted fiorn A. niger by homogenization of the
biomass to dismpt the mycelium, using a MSK Braun homogenizer (B. Braun, Melsungen,
Germany); the mycelia were first suspended (8:20, w/v) in Tris-HG1 buffer solution (10 mM,
pH 8.0) and 15 g of g las beads were added, followed by homogeneization for three cycles of
2 min and then by centrifugation at 4OC (20,000 xg, 30 min). The precipitate was discarded
and the supernatant, referred to be as the crude extract, was partially purified by heat
treatment (60°C, 10 min) according to the procedure descnbed by Kermasha et al. (1993~).
The partially purified fraction of Cu-MT was filtrated twice according to the method
0 descnbed by Goetghebeur et al. (1995), then lyophilized and stored at -80°C.
3.1.2.2.2. Polyphenol Esterase ( P m )
The biomass culture of A. niger was also used throughout this study as a source of
polyphenol esterase (PPE). The extraction and purification of PPE were performed according
to the procedure developed in our laboratory (Madani et aL, 1997). The cmde extract was
suspended (1 : 10, w/v) in phosphate citrate buffer solution (0.1 M, pH 5.0). Al1 purification
procedures were performed at 4°C unless otherwise specified. Solid ammonium sulfate was
added at 0-80% of saturation, with continuous stirring, to the enzyme suspension and the
resulting suspension was allowed to settle for 30 min. M e r centrifugation (10,000 xg, 15
min), the precipitate was removed while the supematant was subjected to M e r partial
purification by ammonium sulfate precipitation at 80-100% of saturation. The resulting
supematant, obtained after centrihgation (10,000 xg, 15min), was desalted using a Minitan
ultrafiltration system (MiIlipore, Bedford, MA) equipped with a PLGC UF membrane
possessing a cut-off point of 10,000 daltons. The ultrafiltrateci fiaction, referred to be as the
partially purified PPE extract, was lyophilized and stored at -80°C.
3.2. Protein Determination
The protein content of the Cu-MT and PPE extracts was detemined according to a
modification of the Lowry method (Hartree, 1972). Bovine semm albumin (Sigma Chemical
Co, St- Louis, MO) was used as a standard for calibration.
3.3. Sample Preparation
3.3.1. Apple Siices
Apples were maintained at room temperature for approximately 10 min before
processing. Apples were manually peeled, cored and sliced radially (about 4-5 mm thickness)
with a sharp knife. The apple slices were then immediately immersed for 2 min in a bath
containing one of the selected inhibitors. The slices were subsequently blotted dry with
absorbent paper for around 1.30 min and placed, as compact as possible, onto the quartz
sample cup in order to camy out the color and reflectance measurements. A set of
experiments was carried out for eacb inhibitor, including a control trial (water only) and a
wide range of inhibitor concentrations. To determine, as precisely as possible, the effect of a
browning inhibitor treatrnent, five medium size apples were used in the preparation of each
set of experiments. Three to four slices were taken OB fiom each apple and 15 to 20 slices
were used for each concentration trial.
3.3.2. Apple Juice
Apples were neither peeled nor cored but roughly sliced in quarters pnor to juicing in
a deluxe juice extractor MP 80 (B. Braun). The inhibitor, in powder form, was directly added
to the juice extractor at the time of juicing. To elhinate large particles, the extracted juice
was irnmediately filtered through P8-creped filter paper (Fisher Scientific Co.). A 30 ml
aliquot of thoroughly mixed juice was placed ont0 the quartz opticai cell for tristimulus
reflectance measurements. In order to get an overall view of the efficiency of the browning
inhibitors in apple juice, eight apples were used in each set of experiments. In order to
randornize the samples, one eighth of each apple was used for each concentration trial.
3.3.3. Potato Siices
Potato slices were prepared according to the same expenmental protocol used for the
preparation of apple slices; however, after peeling, potatoes were manually cutting
transversally into 2 to 3 mm thick slices which were then split in halves in order to fit as good
as possible into the quartz sample cup used for the measurements. To precisely evaluate the
effect of the inhibitors on the prevention of browning, each set of experiments was prepared
from five medium size potatoes. Four to six siices were taken off fiom each potato and 9 to
14 half-slices were used for each concentration trial.
3.4. Inhibitor Treatrnents
The fiactions of Cu-MT and PPE, used through this study, contained 30.22 and
3 8.92% of protein, respectively.
3.4.2. Apple Siices
The selected natural and chernical inhibitors were evaluated for their potentiai to
inhibit enzymatic browning in apple slices at a wide range of concentrations: Cu-MT (0.010,
0.025,0.050,0.075,0.100 and 0.250%), PPE (0.025,0.050,0.075, O. LOO, 0.250 and 0.500%),
ascorbic acid (0.250,0.500, 0.750, 1.000, 1.500 and 2.000%), citric acid (0.250,0.500,0.750,
1.000, 1.500 and 2.000%), EDTA (0.250, 0.500, 1.000, 2.000, 4.000 and 8.000%), sodium
bisulfite (0.005,0.010, 0.025, 0.050, 0.075 and 0.100%) and 4-hexylresorcinol(0.005, 0.010,
0.025,0.005,0.075 and O. 100%).
The treatments, c e e d out at room temperature, consisted of 2 min dips, under gentle
stimng, in fieshly prepared solutions where the inhibitor was dissolved in distilled water. The
slices were then imrnediately placed into 250 ml glass beakers containing 200 ml of the
inhibitory solution or into 250 ml glas beakers containing the control solution of 200 ml of
distilled water; however, when using the natural inhibitors Cu-MT and PPE, slices were
placed into 150 ml glas beakers containing 100 ml of the inhibitory solution.
3.4.2. Apple Juice
The selected inhibitors were also used with apple juice, but at different
concentrations: Cu-MT (0.0025, 0.0050, 0.0075,0.0100,0.0250 and 0.0500%), PPE (0.0075,
0.0100, 0.0250, 0.0500, 0.0750 and 0.1000%), ascorbic acid (0.00075, 0.00100, 0.00250,
0.00500, 0.00750 and 0.0 1000%), citric acid (0.0900, 0.1200, 0.1500, 0.1800, 0.2 100 and
0.2400%), EDTA (0.0900, O. 1200, 0.1500, 0.1 800, 0.2 100 and 0.2400%), sodium bisulfite
(0.0010, 0.0025, 0.0050, 0.0075, 0.0100 and 0.0300%) and 4-hexylresorcinol (0.0100,
0.0300,0.0600,0.0900, O. 1200 and O. 1500%).
The treatments, carried out at room temperature, consisted of the addition of a certain
amount of inhibitor in powder fonn, directly into the juice extractor. The experimental tria1
hence ensured the presence of the inhibitor at the beginning of the process as well as its
optimal action. The control solution was carried out in the absence of the inhibitor.
3.4.3. Potato Slices
Potato slices were treated according to the same procedure described previously for
the apple slices; however, the range of concentrations used for the inhibitors was different:
Cu-MT (0.0075,0.0100,0.0250,0.0500,0.0750 and O.lûûû%), PPE (0.0250,0.0500,0.0750,
0.1000, 0.2500 and 0.5000%), ascorbic acid (0.0750, 0.1000, 0.2500, 0.5000, 0.7500 and
1.0000%), citric acid (0.0750, 0.1000,0.2500, 0.5000,0.7500 and 1.0000%), EDTA (0.0050,
0.0075, 0.0 LOO, 0.0250, 0.0500 and 0.0750%), sodium bisulfite (0.00 10, 0.0025, 0.0050,
0.0075, 0.0 100 and 0.0250%) and 4-hexylresorcinol (0.00025, 0.00050, 0.00075, 0.00 100,
0.00250 and 0.00500%).
3.5. Color Measuremen ts
Color measurements as well as reflectance measurements were performed with a
Labscan XE, mode1 L S X E W I spectrocolorimeter (Hunier Associates Laboratory Inc.,
Reston, VA) operated with a D65 illuminant, possessing a diameter of illuminateci area of 44
mm and an angle of observation of 1 OO; this instrument was standardized against a white tile
(X=78.49, Y=83.23, Z=89.3 5) on a daily basis and restandardization was performed before
each measurement.
A quartz sample cup (64 mm i.d., depth of 34 mm) and an opaque sample cup cover
were used for the apple and potato slices. The cover was placed over the filled cup at the
reflectance port and was provided with a light trap to exclude interference fiom external light.
For the apple juice, a plastic ring was placed inside the quartz sample cup before filling it
with the sample and a ceramic disk was then placed on top of the solution so that its white
portion faced the solution; these accessories were used to exclude extemal light, provide a
consistent white background and maintain a constant volume of apple juice. Ln the presence
of the ring and disk set, the optical ce11 became smaller with a diameter of 59 mm and a depth
of approximately 15 mm.
Approximately 15 to 20 apple slices and 9 to 14 potato half-slices were placed ont0
the sample cup and covered with the opaque cover. Values of the tristimulus coordinates in
the L (lightness variable), a ( r d to green degree of color) and b (yellow to blue degree of
color) system were recorded at 0, 5, 15, 30, 45, 60 and 90 min for the color studies of the
apple slices and at 0, 15, 50 and 100 min for those of the potato slices. Between
measurements, samples were left on the port of the instrument. M e r filtration, the apple
juice was imrnediately placed ont0 the optical cell equipped with the ring and disk set and
data were recorded in the L, a, b system at 0, 15, 30, 45, 60 and 90 min. Between
measwements, samples were iefi on the port of the apparatus.
3.6. Evaluation of the inhibition of browning
To demonstrate the applicability of the tristimulus colorimetry procedure in the
evaluation of browning in food products (slices and juice) treated with inhibitors, the
untreated samples were compared with the treated samples. The degree of inhibition of
enzymatic browning was expressed as the percent difference between the control and the
treatment AL or L\a values after a specified storage time t:
AL control - AL treatment % inhibition Value =
AL control
where AL (or Aa) is the difference between the L (or a ) value at time t and the value at tirne O
min (Sapers and Douglas, 1987); these authors indicated that in order to minimize the
variability in the natural pigmentation of food products, the difference between the fmal and
the initial L values and a values should be calculated.
Positi-;G vaiuzs of % inhibition between O and 100% would indicate that the treatment
was effective as a browning inhibitor. Values greater than 100%, if significant, wodd
indicate the occurrence of sarnple bleaching by the treatment while negative values would
indicate that the treatment promoted browning rather than its inhibition.
3.7. Reflectance Measurements
The reflectance rneasurements were performed with the Labscan XE. The process for
standardization of the instrument and the preparation of different samples were the sarne as
those descnbed previousiy. However, the reflectance data were recorded every min during
the experimental trial period (90 min for apple products and 100 min for potato slices). A
reflectance spectra fÎom 400 to 700 nm was obtained and used for kinetic studies for the
inhibition of enzymatic browning.
3.8. Statistical Analysis
Means and relative standard deviations were caiculated for the percentage of
inhibition, obtained from L and a values, of each treatment and each reaction t h e , with the
selected food products. AU experirnents were carried out in triplicate.
Further statistical analyses were also performed. A completely randomized design
experiment with a factonal arrangement was carried out to determine the reaction time, the
concentration and the inhibitor treatment effect on the prevention of browning. Duncan's
multiple range test was used for cornparison of the means, utilizing SAS software (Statistical
Analysis System for wïndows, 6.12, SAS Institute Inc., Cary, NC). A probability (P) of less
than 0.05 was considered to be significantly different.
4. ~ S U L T S AND DISCUSSION
4.1. Apple Slices
4.1.1. Measurement of Browning at the Cut Surface of Apple Sïices
Figure 3 shows the occurrence of enzymatic browning in apple slices, over a defined
period of time, in the absence and presence of 0.01 and 0.10% sodium bisulfite (NaHSO,) as
inhibitor. The initial tirne, O min, corresponds to the first reflectance measurement made
immediately after the drying of apple slices. The same profiles were obtained with al1 the
inhibitors tested; bowever, the differences between those corresponding to the absence,
intermediate and strong enzymatic browning inhibitions were ciearly demonstrated with the
use of NaHSO,. On the basis of these experirnental results, Figure 3 demonstrates clearly the
degree of enzymatic browning inhibition. Figure 3A shows the change in the L values, the
lightness variable. The results indicate that the L values of the water-dipped control sample
decreased dramatically in 15 min from 69 to 58, and then continued to decline slowly to 54
afler 60 min of reaction before reaching a plateau. The overall change in the L values of the
control sample (17.5) was much higher than those (5 to 10) found in the presence of the
inhibitor for the defined reaction tirne. In addition, an increase in the inhibitor concentration
from 0.01 to 0.10% was directly related to a slower decrease in the L values and a shorter
period of time required to reach the plateau. Using 0.01% NaHS03 as inhibitor, a plateau at
65 was observed afler 40 min of reaction while with O. IO% NaHSO,, a plateau at 80 was
reached after only 5 min. These results suggest that the apple slices lost their brightness with
the developrnent of enzymatic browning as the treated samples presented slight color
differences when compared to that obtained with the control sample. Sapers and Douglas
(1987) reported that as the browning progressed on the cut surface of untreated McIntosh
apple plugs, the t values decreased showing a AL of 6.8 after 180 min of reaction. The
differences in AL values obtained in one experiment to another may be due to the varïability
in natural pigmentation in apples.
The change in the a values, which represent variations in green to red intensities, is
presented in Figure 3B. The a values of the control sample increased markedly fiom 4.0 to
Reaction Time (min)
Figure 3: Changes in the L (A) and a (B) values in apple slica without inhibitor (m), and with 0.01% NaHSO3 (e) and 0.10% N-Os (A ).
9.0 during the fint 15 min, then continued to increase slowly and stabilized at 9.8 after 60
min of reaction. M e r 90 min of reaction tirne, the Au of the control sample (5.8) was greater
than those (4.1 and 3.3) obtained with 0.01 and 0.10% NaHSO,. In addition, as the
concentration of inhibitor increased, the afin,, became lower and stabilized earlier. These
experimental fuidings suggest that in absence of inhibitor the apple slices undenvent severe
browning and quickly tumed red whereas in the presence of 0.01 and 0.100/0 NaHSO,,
browning in the apple slices was moderate or slight, respectively. Sapers and Douglas (1987)
reported that as browning developed at the cut surface of untreated McIntosh apple plugs, the
a values increased exhibiting a Au of 3.8 after 180 min of reaction.
In addition, Mastrocola et al. (1990) indicated that variations in the tristimulus
coordinates L and a were related to color changes in fiuit tissues such as darkening, caused
by enzymatic browning.
4.1.2. Evaluation of Browning Inhibiror Treatments at the Cut Surface of Apple Slices
Tables 1 to 7 report on the use of selected inhibitors for the prevention of enzymatic
browning in apple slices. The percent inhibition values (%IV) for each inhibitor were
calculated using the Aï and Aa values obtained at different times of reaction, 5, 15, 30,45,60
and 90 min. In order to describe and analyze the effects of inhibitors, the different interval
times were selected on the basis of data reported in Figure 3.
Table 1 shows the %IV obtained using the Cu-MT extract at concentrations ranging
fiom 0.01 to 0.25%. The results indicate that similar %IV were obtained at difierent Cu-MT
concentrations based on both AL and Aa values. The %IV were al1 positive with the highest
%IV of 37% obtained after 5 min using 0.25% Cu-MT as inhibitor and ALI as a ba i s of
calculation. These results suggest that the Cu-Mt extract had a relatively moderate efficiency
to act as a browning inhibitor at the surface of apple slices. In addition, the overall %IV
decreased with reaction t h e , thereby indicating that the Cu-MT extract loses its efficiency
over tirne. Goetghbeur and Kermasha (1 996) reported that the degree of inhibition by Cu-MT
was dependent upon the structural nature of the substrate and the degree of purity of the Cu-
MT extract; the same authors reported that the I,, obtained for the purifieci fraction of Cu-MT
Table 1. Inhibitory effect o f a copper metallothionein (Cu-MT) extract, obtained from A. niger, on enzymatic browning in apple slices, determined using a tristimulus colorimeter.
Inhibition (%)O
Tristimulus Reaction Cu-MT concentration (%)
coordinates time (min) 0.010 0.025 0.050 0.075 O. 100 0.250
a Relative percent of inhibition of browning using a wide range of Cu-MT concentrations, detemined with respect to L or a values. b ~ h e percentage of inhibition at a defined tirne (t), with respect to L values, was calculated as (ALcontrol - ALtrepheni) 1 hLconbo~ ~100, w h m AL =
Lt - Linitiab C The percentage of inhibition at a defined time (t), with respect to a values, was calculated as AR,,,^,^ - AatWlmnt) I Auconbol x100, where Au = at - ainitial.
d~elative standard deviation of triplicate samples was determined as the standard deviation divided by the rnean, multiplied by 100.
e '~eans were significantly different with P < 0.05.
on polyphenol oxidase activity in a mode1 system with catechin as substrate was 250 times
lower than that obtained for the partially puified fraction. These results suggest that the
fiiture use of a purified hction of Cu-MT as a browning inhibitor could enhance its
inhibitory effect at the surface of apple slices.
Table 2 presents the %N obtained with the PPE extract at concentrations ranging
fiom 0.025 to 0.500%. The results show that, based on either AL or Aa values, the %IV were
similar with respect to the PPE concentrations used. The %N were al1 positive except those
obtained using 0.50% PPE where two negative values were observed after 90 min. In
addition, the overall %N decreased with reaction t h e , thereby indicating that the PPE
extract loses its efficiency over tirne. The results also showed that only one %IV was superior
to 50% (55.5%) obtauied after 5 min using a 0.25% PPE extract and Au as a basis of
calculation. The majority of %IV ranged between 20-25% suggesting that the PPE extract
had a low inhibitory effect in controlling enzymatic browning at the surface of apple slices.
Madani et al. (1999a) showed that the inhibitory effect of PPE on tyrosinase activity
a increased markedly with the degree of purification and that the I,, value for the purified
fkaction of PPE was 106 times lower than that obtained for the partially purïfied ftaction,
using 3,4-dihydroxyphenylacetic acid as substrate. These fkdings suggest that the use of a
purified PPE fraction could enhance its inhibitory effect at the surface of apple slices.
Table 3 shows the %IV obtained using ascorbic acid (AA) concentrations ranging
fiom 0.25 to 2.0%. The results indicate that the %IV differed considerably depending on
whether AL or & values were used as the basis for calculation. The %IV decreased greatly
with reaction tirne. At a 1.5% AA concentration, the %IV, based on the & values, varied
£iom 101 -4% to -12.9% during 90 min of reaction tirne, thereby suggesting that AA
prevented browning for a iimited period. Negative %IV were also observed, in particular at
AA concentrations lower than 1.0Y0; these tow concentrations seemed to enhance the
discoloration of the apple slice sample which underwent severe browning. However, under
certain conditions such as at 1.5 and 2.0% AA, satisfactory %N were obtained which were
a positive and superior to 50%, d u h g the first 30 min of reaction. These results suggest that
Table 2. Inhibitory effect of a polyphenol esterase (PPE) extract, obtained from A. niger, on enzyniatic browning in apple slices, determined using a tristimulus colorimeter.
Inhibition (%)'
Tristirnulus Reaction PPE concentration (%)
coordinates time (min) 0.025 0,050 0.075 O. 1 O0 0.250 O. 500
24.9 (*4.2fe
20.1 (k5.0fJ
16.0 (ri .cfg
10.6 (11 .9fL
10.4 (11.1)""
8.6 (k1.3)"'
a Relative percent of inhibition of browning using a wide range of PPE concentrations, determined with respect to L or a values. b ~ h e percentage of inhibition at a defined tirne (t), with respect to L values, was calccilated as (Mcontm~ - ALtxatment) 1 ALmnool x100, where AL =
Lt - Linitiab C The percentage of inhibition at a defined time (t), with respect to a values, was calculated as (Aaconirol - Aatrealment) 1 L\aConbol x 100, where Ba = ai - ainitiab
d~elative standard deviation of triplicate samples was detemined as the standard deviation divided by the mean, multiplied by 100. ' ' /~eans were significantly different with P < 0.05.
AA could be an effective browning inhibitor under specific conditions of concentration and
time. Sapen et al. (1989b) reported analogous observations at the cut surface of Red
Delicious plugs dipped in 0.8% AA where %IV of O and -18% were obtained afker 6 hrs of
reaction. In addition, Gil et al. (1998) indicated that AA treatments reduced browning at the
cut swface of apple slices and increased shelf-tife, but only for a short penod of tirne, as the
use of AA softens the tissues and promotes mold growth; however, apple slices treated with
2% AA and held in an oxygen-fiee atrnosphere showed no significant browning or loss of
visual quality for up to 15 days.
Table 4 presents the %IV obtained with citnc acid (CA) as inhibitor at concentrations
ranging from 0.25 to 2.0%. The results show that the %N were al1 positive and sirnilar with
respect to the AL and Au values used as a basis for calculation. The overall %IV were
relatively low (around 20%) with ody one %IV greater than 50% (58.3%) obtained using 2%
CA after 5 min of reaction time and determined with respect to the Aa values. The
expenmental findings also show that the %IV decreased with reaction tirne. These results
suggest that CA loses its efficiency over time and has a low ùihibitory effect on browning.
Vamos-Vigyazo (1995) reported that the immersion of Starking apple slices in 2, 4 and 6%
CA solutions for 1 min, resulted in %N of 0, 60 and 73%, respectively afier 30 min of
reaction. In addition, many studies have shown that when CA was used in combination with
another inhibitor treatment, it could be very emcient in the presence of browning; Pizzocaro
et al. (1 993) showed that 90- 100% inhibition occurred in apple cubes afler being dipped for 5
min in an aqueous solution of 0.01 % AA and 0.002% CA.
Table 5 shows the %IV obtained using ethylenediaminetetraacetic acid (EDTA) at
concentrations of 0.25 to 8.00%. The %N differed according to the AL or Aa values used as
a basis for calculation. For the EDTA concentrations ranging fiom 0.25 to 2.00%, the %IV
increased with reaction time whereas at higher concentrations of 4.00 and 8.00%, the %IV
decreased with reaction tirne; the inhibitory effect of EDTA depended on the concentration
used. The experimental hdings also report that the %IV of EDTA were satisfactory with the
average around 25%; the highest %N of 35.4% was exhibited &er 5 min reaction time using
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C ; o L i i n & Z 4 Y i -a Ya Yix 2 C-
( P m *
4.0 and 8.0% EDTA and with respect to the AL values. These results suggest that EDTA was
poorly effective as a browning inhibitor of apple slices. McEvily et ai. (1992) have rsported
that EDTA was not used alone for the inhibition of enzymatic browning, but generally in
combination with other agents such as AA and CA. In addition, Sapen (1993) indicated that
EDTA does not inhibit enzymatic browning in apple slices.
Table 6 reports the % N O btained with sodium bisulfite (NaHSO,) concentrations
ranging fkom 0.005 to 0.100%. Some variations were obsewed in the %IV obtained using the
AL values and dn values, in particular with 0.005 and 0.010% NaHS03. At low
concentrations, fiom 0.005 to 0.050%, the %IV ciearly decreased with reaction t h e , while
using 0.075 and 0.10% NaHSO,, a slight increase was observed. The experimental findings
also indicate that the %N were higher than 50% at concentrations qua1 to or greater than
0.025% NaHSO, where %IV of 100% were obtained using 0.100% NaHS03. These results
suggest that NaHSO, was highly effective as a browning inhibitor and could provide
complete inhibition for 90 min at the surface of apple slices treated with a concentration of
0.100% NaHSO,; however due to its adverse effect on health, its use was banaed in the food
industry. Similar observations were made by Molnar-Perl and Friedman (1990b) who
reported that apple slices immersed for 2 min in NaHSO, baths ranging in concentration fkom
5 to 50 mM did not undergo browning; an inhibition of 100 and 106% at the cut surface of
Washington Golden and Red Delicious apples, respectively, was obtained for 6 hrs of
reaction using 5 mM NaHSO,.
Table 7 shows the %IV of the inhibitor 4hexylresorcinol(4HR) at concentrations of
0.005 to 0.100%. The results report that the %N varid depending on the basis of calculation
with respect to the AL and Aa values. At low concentrations ranging from 0.005 to 0.025%,
the %N increased with reaction tirne whereas at higher concentrations of 0.050 to O. 100%, it
decreased. In addition, the overall %N were generally high with an average of 80%, except
at the two lowest 4HR concentrations of 0.005 and 0.0 10%. These results suggest that 4HR is
a powerfûl inhibitor of color change at the surface of apple slices. These findings are in
agreement with those reported by Monsalve et al. (1993) who indicated that 4HR
concentrations as low as 0.005% were effective in the inhibition of browning and that the
W P P
n n n
h h h h h h W t + I + * H t + O P P O O P
h h h h h h w l + l + l + w * P P P P ' - . C l -
g g w w w P -3. 3 -q :a -3 0 9 q n -.
h h h h h h ~ * ' + * H H -
P O O 0 0
inhibitory effect of 4HR could be enhanced by the addition of AA at concentrations of 0.2-
0.5%. However, Monsalve et al. (1995) also showed that concentrations of 4HR beyond
0.03% may result in an increase in the residual content in apple tissues and could also
influence apple flavor.
Figure 4 shows the effect of different concentrations of a wide range of inhibitors,
including Cu-MT, PPE, AA, CA, EDTA, NaHSO, and 4HFt, on enzymatic browning at the
surface of apple slices. In order to compare the efficiency of different inhibitors, the dipping
time was always maintained for 2 min period. The results show that the %N of browning
increased with increasing inhibitor concentrations of Cu-MT (Fig. 4A), AA (Fig. 4C), EDTA
(Fig. 4E), NaHSO, (Fig. 4F) and 4HR (Fig. 4G), while in the presence of the inhibitors PPE
(Fig. 4B) and CA (Fig. 4D), the %N increased to a maximum after which it decreased as the
inhibitor concentration was increased. For each inhibitor treatment, an optimum
concentration for the inhibition of browning was obtained of O. 2 0, 0.10,2.00,0.75, 8.00, 0.10
and 0.10% for Cu-MT, PPE, A& CA, EDTA NaHSO, and 4HR, respectively. The results
also indicate that the %IV slightly differed with respect to the AL and Aa values used for
calculation so that the inhibitory effectiveness of the selected inhibitors was in the order of
NaHSO, > 4HZi > AA > PPE > EDTA > CA > Cu-MT when the AL values were used and
NaHSO, > 4HR > A A > EDTA > Cu-MT > PPE and CA with the Au values.
The different inhibitors were also compared at a specific concentration and reaction
time, with respect to the average of the %IV obtained with the LU and Au values. The results
(Tables 1 , 2 , 4 and 5) showed that d e r 30 min of reaction using an inhibitor concentration of
0.25%, the inhibitory effect of PPE was 5.9, 37.1 and 556.6% higher than that of Cu-MT,
EDTA and CA respectively. The results (Tables 2 and 3) ako show that the %N (25%)
obtained with the PPE extract was also much higher than that obtained with the use of AA (-
1 8 -5%). The inhibitors NaHSO, and 4HR were compared at a concentration of O. 10% and 30
min reaction tirne. The %N of 4HR (Table 6) and NaHSO, (Table 7) was 218.2 and 273.5%
higher, respectively, than that obtained for PPE (Table 2). These hdings suggest that PPE as
a well as Cu-MT could be considered as effective browning inhibitors, with PPE being sliatly
stronger than Cu-MT. In addition, PPE and Cu-MT could compete actively as inhibitors with
0.005 0.010 0.025 0.050 0.075 0.100
Inhibitor Concentration (%)
Figure 4: Effect of inhibitor concentrations on enzymatic browning in apple slices: copper-metallothionein (A), polyphenol esterase (B), ascorbic acid (C), citric acid @), ethylenediaminetetraacetic acid (EDTA) (E), sodium bisuffite (F) and 4-hexylresorcinol (G). The percentage of inhibition was calculated £kom the L values (O) and the a values (O).
chemicals such as &i, CA, and at a lower scale with EDTA while 4HR and NaHSO, were
more eficient than Cu-MT and PPE extracts. Vamos-Vigyazo (1995) reported that the
inhibition of enzymatic browning of apple slices by dipping into 2% A . for 3 min was more
efficient (99%) than that for 2% CA (57%), for the same penod of time. In addition,
Monsalve-Gonzalez et al. (1993) showed that the use of 4HR at storage temperature 2S°C, in
combination with M, was an effective method for preventing enzymatic browning compared
to that obtained with sodium sulfite.
4.1.3. Kinetic Sîudy of Enwmatic Browning in Apple Nices using Rejlectance Spectrum
Figure 5 shows the scanning profile of enzymatic browning at the surface of apple
slices in the absence (Fig. 5A) and presence of 0.1 % NaHSO, (Fig. SB). The results show
also that similar findings were observed with and without the inhibitor; the reflectance values
increased graduaily fkom 400 to 520 nm then increased strongly to form a broad peak in the
yellow-orange absorbance regions of 540 to 640 nm followed by a depression at around 670
nm in the red absorbance region. These results suggest that the color formation present in the
apple slices occurred due to the combination of specific proportions of yellow-red absorbance
regions. Lozano er al. (1994) reported sirnila. results in untreated apples which exhibited a
marked depression in the red absorbance region over a period of time and reduced reflectance
variations. The experimental findings also show that the spectra obtained for apple slices
treated with O. l%NaHSO, were similar at O and 90 min of reaction time while those obtained
for the untreated apple slices indicated an overall decrease in the reflectance spectnim profile
over time. These results suggest that the apple slices treated with 0.1% NaHSO, reminded
bnght in color and pale whereas those in the absence of the inhibitor underwent severe
browning and turned dark and brown. The reflectance values, obtained in presence of 0.1%
NaHSO,, were higher (around 10%) than those obtained in its absence, especially at short
wavelengths. NaHSQ rnight a e c t certain pigments such as flavonoïds and thereby changing
slightly their reflectance values.
Ln addition, the graphs presented in Figure 5 may indicate the presence of a
chiorophyll spectnun associated with other substances. Francis et al. (1955) reported that, in
McIntosh apples, the camtene naîtion, which is the most important yellow pigment, was
480 580
Wavelength (nrn)
Figure 5: Scanning of enzymatic browning in apple slices from O (O) to 90 min (a) without inhibitor (A) and with 0.1% sodium bisulfite (B).
associated to the chlorophyll hction. Moreover, Lozano et al. (1994) explained that negative
o values were given by the green pigmentation of apples.
Figure 6 shows the reflectance spectra obtained for enzymatic browning at the surface
of apple slices in the presence and absence of seven selected inhibitors. Similar profiles to
those described in Figure 5 were observed. The overail results show that the reflectance
spectnun of the control sample gave the highest reflectance values in the visible region fkom
380 to 700 nrn at zero time of reaction whereas at 90 min, the control sample showed the
Iowest reflectance values. The reflectance spectnim obtained for enzymatic browning in
apple slices treated with inhibitor was observed in between the two reflectance spectra
obtained using the control sample at O and 90 min of reaction time. The overall hdings
demonstrated that the closer the reflectance spectra of the samples treated with the inhibitor
were to those obtained in the absence of inhibitor at thne zero, the more effective the
inhibitor was in preventing browning. These results suggest that NaHSO, provided the
highest inhibition followed by 4HR, then by EDTA and AA which seemed to be moderate
browning inhibitors dong with CA and PPE but to a lower degree and then finally by Cu-MT
which appeared to be the ieast effective.
Figure 7 shows the evolution of enzymatic browning, in ternis of reflectance
difference, determined at selected wavelengths including 400,460, 530, 600 and 670 n m as a
hc t ion of inhibitor concentration; this difference of reflectance is the difference between the
reflectance value at the initial time of the reaction and the value at the end of the reaction.
The results indicate that the optimum concentrations for the prevention of browning
detennined with respect to the smallest value of difference of reflectance were 0.10, 0.25,
2.00, 0.75, 2.00, 0.10 and 0.10% for CU-MT, PPE, AA, CA, EDTA, NaHSO, and 4HR
respectively; these values were identical to those obtained in Figure 4 with respect to the AL
and Aa values, except for PPE and EDTA which had exhibited optimum concentrations for
inhibition of 0.10 and 8.00%, respectively. The experimental hndhgs (Fig. 7) also show that
the absorbance profiles obtained at different wavelenghs with respect to inhibitor
concentration were different; the absorbance profiles exhibited at the shorter wavelengths of
1 l I I 1 3 80 480 580 680
Wavelength (nm)
lices: Figure 6: Refl ectance spectra for different inhibitors in apple s copper-met al lothionein (A), pol yphenol esterase (B), ascorbic acid (C), citnc acid @), ethylenediarninetetraacetic acid (EDTA) (E), sodium bisulfite (F) and Chexylresorcùiol (G). Spectra were obtaineâ without inhibitor at O (A) and 90 (+) min and with the highest concentration of inhibitor at 90 min (a).
I l l
O 0.025 0.050 0.075 0.100
Inhibitor Concentration (%)
Figure 7: Effect of inhibitor concentrations on enzyrnatic browning in apple slices detemined at 400 nm (a ), 460 nrn ( O), 530 rm ( l), 600nm (+ ) and 670 nm (O ) for copper-metallothionein (A), polyphenol esterase (B), ascorbic acid (C), citric acid @), ethy lenediaminetetraacetic acid (EDTA) (E), sodium bisul fite (F) and 4-hexylresorcinol (G).
- 400 and 460 nm @lue absorbance area) were sirnilar as well as those found using the longer
wavelengths of 600 and 670 nm (red absorbance area), while the absorbance profile at 530
nm was similar to a îimited extent. These results suggest that the optimum inhibitor
concentrations for the prevention of browning depend on the wavelength selected and are
therefore less accurate than those obtained iising the AL and Aa values. In addition, the
overall results indicate that the absorbance profile observed at 670 nm showed the lowest
values for reflectance difference while that found at 530 nm displayed the highest values for
reflectance di fference.
The overall findings demonstrate that the results obtauied fkom the reflectance values
gave similar conclusions to those obtained fiom the LU and Aa values, however, the
reflectance measurements were nevertheless less accurate.
4.2. Apple Juice
4.2. 1. Meusurement of Browning irr Apple Juice
Figure 8 shows the variations in the L (A) and a (B) values during the development of
enzymatic browning in apple juice in the absence and presence of 0.0025 and 0.0300%
NaHSO,. Al1 the tested inhibitors have showed the sarne profiles; however, the different
degree of inhibition was very clearly demonstrated with NaHSO,; on the basis of the
experimental results, Figure 8 demonstrate representative graphs for the inhibition of
enzymatic browning by NaHSO,. The results (Fig. 8A) show that the plots of the L values
versus time were bilinear during the reaction time of 90 min where an increase in the L values
was followed by a plateau. In addition, an increase in inhibitor concentration was directly
related to a higher steep and shorter penod of time required to reach the plateau. For apple
juice containing 0.0300% NaHSO,, a plateau was observed &er 10 min of reaction while
with apple juice containing 0.0025% NaHSO,, it was reached after 25 min; however, with the
control sample, it was obtained at 35 min. The experimental hdings also show that the AL
values increased as the inhibitor concentration increased; the AL values increased fFom 3 in
the absence of the inhibitor to 8 in the presence of 0.0300% NaHSO, during 90 min of
reaction time thereby suggesting that the apple juice became more brighter in color over tirne
4 6P --.e a.......
Reaction Time (min)
Figure 8: Changes in the L (A) and a (B) values in apple juice without inhibitor (m), and with 0.0025%NaHS03 (0) and 0.0300% NaHSO, (A ).
in the presence of inhibitor than in its absence. In contrast, Sapers and Douglas (1987)
demonstrated that the L values decreased during the occurrence of browning in juice where a
AL of -6.9 was obtained after 30 min of reaction in Cortland apple juice. These variations in
the results could be due to the use of different apple cultivars.
Figure 8B presents the changes that occurred in the a values during the course of
browning in apple juice. The results clearly show that in the absence of the inhibitor, the a
values increased gradually over reaction tirne (& = 6) whereas in the presence of the
inhibitor NaHSO,, the a values decreased slowly before reaching a plateau, In addition, in the
presence of 0.03% NaHSO,, the a,,, reached a negative value of -2 after 20 min reaction
the . These results suggest that the apple juice without the inhibitor tumed brown while with
the addition of inhibitor it becarne lighter. Similar findings were reported in the literature by
Sapers and Douglas (1987) who showed that the a values increased during the browning of
juice in the absence of inhibitor where the A a value was 2.9 after 30 min of reaction in
Cortland apple juice.
In addition, the overall results (Fig. 8) show the occurrence of some nonlinearity
during the first 10 min of reaction which could possibly be due to, in part, the development of
turbidity or dissipation of air bubbles in the apple juice sarnples.
4- 2.2- Evaiuation of Browning I n hibitor Treatments in Apple Juice
Tables 8 to 14 report on the use of selected inhibitors for the prevention of enzymatic
browning in apple juice. The %IV was calculated fiom the respective AL and Aa values
obtained f i e r 15, 30, 45, 60 and 90 min of reaction. In order to characterize the effects of
inhibitors in on the enzymatic browning of apple juice over t h e , these different interval
times were selected on the basis of expenmental data obtained in Figure 8.
Table 8 shows the %N obtained using the Cu-MT extract at concentrations ranging
fiom 0.0025 to 0.0500%. The results indicate that the %N distinctly varied when the AL and
Aa were used as a basis for calculation; based on the AL values, the %IV were higher than
those obtained fiom the Aa values. The overall %IV were almost al1 positive and sornetimes
higher than 50% especially when calculated fiom the AL values; the highest %N (71 -2) was
obtained after 15 min using 0.0500% Cu-MT. These results suggest that the use of the AL
values as opposed to the Aa values is a better indicator to monitor the inhibition of enzymatic
browning in apple juice. The results also demonstrate that the Cu-MT extract had a good
inhibitory effect on browning in apple juice but only for a short period of time (30 min). The
experirnental findings also indicate that the %IV decreased during the 90 min reaction time
thereby suggesting that the Cu-MT extract loses its efficiency with time. However, the use of
a purified Cu-MT fraction as a browning inhibitor has been reporteci by Goetghbeur and
Kermasha (1996) to enhance the inhibitory effect of Cu-MT in apple juice.
Table 9 shows the %IV obtained with the PPE extract at concentrations of 0.0075 to
0.1000%. The results show that the %IV, caiculated on the basis of the &Z values, were
globally higher than those obtained fiom the AL values, especially at concentrations equal to
or higher than 0.0500% PPE. The overall %TV were high, and most of the time greater than
50% and even reached 100% (fkom 45 min of reaction with 0.0250% PPE) which suggests
a complete inhibition. These results suggest the use of the ha values as opposed to the AL
values to monitor the inhibition of enzymatic browning by the PPE extract in apple juice. The
results also demonstrate that the PPE extract was effective as a browning inhibitor, especially
when used at a concentration of 0.0250%. These results also show that the %IV increased
with an increase in reaction time thereby indicating that the PPE extract may undergo a
latence phase before becoming totally active. According to the results presented by Madani et
al. (1999a), the use of a more purified PPE fiaction could fûrther enhance its inhibitory effect
on browning.
Table 10 presents the %IV obtained with AA concentrations ranging fiom 0.00075 to
0.01000%. The results show that the %N differed with respect to the AL and Au values used
as a ba i s for calculation; the %IV obtained with the AL values were in general higher to
those obtained with the Au values. The overall %IV were high at concentrations equal to or
greater than 0.00500% AA, and al1 the %N were greater than 50% reachuig even 100%
using 0.01 000% AA. These results demonstrate that the use of the AL values was preferable
to that of the AQ values to follow the inhibitory effect of AA in apple juice. In addition, the
a 1 3 L i n g g , " h h h h W H - k t l + P w - X X - w w w w q 5 -4 - ! = -
m u , P e <nooz
h h F a a
results indicate that AA exhibited a strong inhibitory effect. The experimental fmdings also
indicate that the %N increased with reaction time; at low concentrations, of 0.00075 to
0.00250%, AA became more efficient after 30 min while at the concentration of 0.00500%
and higher, AA was immediately effective as indicated by the %IV which were higher than
50%. These results suggest that AA required a certain period of t h e in order to exhibit al1 of
its potential. Walrod (1957) proposed the addition of AA to fkeshly pressed apple juice as it
leaves the press, in order to reduce enzymatic browning; AA prevents browning by being
oxidized before the constituents of the apple juice, thus preserving the very light color of the
juice. Sapers et al. (1989b) reported that 100% inhibition was observed after 2 hrs in Granny
Smith juice treated with 1.14 mM AA while Molnar-Perl and Friedman (1990a) showed that
browning in Golden Delicious juice was inhibited by 98% for 2 hrs of reaction after
treatment with 12.40 mM AA. Differences in the relative efficiency of A4 as a browning
inhibitor may have resulted in the literature due to variations in the choice of apple cultivars
and in the range of concentrations compared.
Table 11 presents the %N obtained with CA concentrations ranging fkom 0.09 to
0.24%. The results show that the %N differed according to the basis of calculation used with
respect to the AL and Aa values; the %N obtained from the AL values were clearly supenor .
to those obtained with the Aa values and were higher than 50% with the average being 65%.
Nevertheless, at the lowest concentration of 0.09%, the %N were relatively low, being
inferior to 22.5%. These results demonstrate that the use of the AL values was preferable to
that of the Aa values to follow the iniubitory effect of CA in apple juice and that CA was
effective as browning inhibitor. The experimental hdings also indicate that the %IV
increased with reaction time and that most of the time, 30 min were necessary to reach a %IV
higher than 50% thereby suggesting that CA required a certain time of incubation before
exhibiting al1 of its potential. McEvily et al. (1992) reported that CA is often used in blended
products in combination with other antibrowning agents such as AA, cysteine and Sporïx.
Table 12 shows the %IV obtained using EDTA at concentrations of 0.09 to 0.24%.
The results indicate that the %IV differeû depending on the basis of using the hL and Aa
P w w h )
CIO h h h h K W H # P P P w Y
m
values for calculation and that the %IV obtained from the AL values were lower or higher to
those obtained with the A a values depending on the EDTA concentration used and reaction
time. These results demonstrate that the use of either the AL or the Au values to monitor the
inhibitory effect of EDTA in apple juice is adequate. The experimental findings also show
that the %IV increased with reaction time and that they were al1 relatively hi&, except for
those obtained with 0.09 and 0.12% EDTA. In addition, in the presence of 0.21 and 0.24%,
the %N were equal to or higher than 100%. These results suggest that EDTA needed a
latency phase in order to become totally active and that EDTA had a great potential as a
browning inhibitor especially at concentrations equal to or higher than 0.18%. McEvily et al.
(1992) reported that EDTA was generally used in combination with other agents to eliminate
enzymaûc browning. However, Sapers et al. (1989b) showed that another chelating agent,
Sponx, used at 6% did effectively control the enzymatic browning in apple juice over a
penod of 24 h; in addition, the combination of Sporix with AA represented a highly effective
treatment for prevention enymatic browning of apple juice.
Table 13 presents the %IV obtained with NaHSO, at concentrations ranging from
0.001 to 0.030%. The results show that the %IV, calculated on the basis of the AL values
were higher than those obtained with the Aa values. In the presence of concentrations ranghg
from 0.005 to 0.030% NaHSO,, the %IV obtained with the AL values increased with reaction
time whereas those obtained with the Au values decreased during 90 min. At the lowest
concentrations of 0.0010 and 0.0025%, the %IV decreased with reaction tirne independently
of the basis used for calculation. These results suggest that the use of the Au values was
preferable to follow the inhibitory action of NaHSO, in apple juice. The decrease in the %IV
also suggests that the inhibitor lost its efficiency over a penod of time. The expenmental
findings show that the overd1 %N were very high, often reaching 100% and sometimes even
200% in the presence of 0.030% NaHSO,, thereby indicating that NaHSO, is a powerful
inhibitor of enzymatic browning. Molnar-Perl and Friedman (1 99Oa) showed similar results
for NaHSO, in apple juice where complete inhibition was still observed after 24 hrs using a
concentration of NaHS03 ranging h m 1.136 to 4.540 mM.
Table 14 shows the %IV obtained using 4HR at concentrations of 0.01 to 0.15%. The
results indicate that the %IV based on calculations using the AL values were generally higher
than those obtained with the Aa values. in addition, the %IV obtained with the AL values
increased with reaction time whereas those obtained wiîh the ALZ values decreased during the
course of 90 min. These results suggest that 4HR reacted differently with the components in
apple juice responsible for the lightness and redness properties. The experimental hdings
also indicate that the overall %IV were very high, almost d l of which were superior to 50%
with the different concentrations used and reached LOO% or more in the presence of 4HR
concentrations of 0.12 and 0.15%. These results demonstrate that 4HR was a powemil
inhibitor of enzymatic browning in apple juice. McEvily et ai. (1992) indicated similar
resuIts for 4HR which largely inhibited enzymatic browning in apple juice.
Figure 9 indicates the effect of different concentrations of a wide range of inhibitors,
including Cu-MT, PPE, AA, CA, EDTA, NaHSO, and 4HR, on erizymatic browning in apple
juice. The results show that the %N generally increased with a concornittant increase in
a inhibitor concentration except in Figure 1OB where the %IV increased to reach a maximum
and then decreased as the inhibitor concentration of the PPE extract continueci to increase.
For each inhibitor treatment, an optimum for the prevention of browning in apple juice
concentration was detennined; the optimal values for maximal inhibition were 0.025, 0.025,
0.010, 0.240,0.240,0.030 and 0.150% for Cu-MT, PPE, AA, CA, EDTA, NaHSO, and 4HR,
respectively. The results also indicate that the %IV differed with respect to the AL and Aa
values used as a basis for calculation; in Figures 10A, 10B, 10D and 10F, broad ciifferences
were observed between the %N obtained with the AL values and those obtained with the Au
values. The effectiveness of the selected inhibitors, based on the optimum concentrations for
inhibition, was in the order of NaHSO, > 4HR > AA > EDTA > PPE > CA > Cu-MT with
respect to the AL values whereas with the Aa values, the classification was in the order of AA
> 4HR > NaHSO, > PPE > EDTA > CA > Cu-MT.
A cornparison between the different inhibitors was performed with respect to a
c. O z h kt- * e Y -9 (b
Inhibitor Concentration (O!)
Figure 9: Effect of inhibitor concentrations on enzymatic browning in apple juice: copper-metallothionein (A), polyphenol esterase (B), ascorbic acid (C), citnc acid @), ethylenediarninetetraacetic acid (EDTA) (E), sodium b isul f i te (F) and 4-hexylresorcinol (G). The percentage of inhibition was calculated fiom the L values ( 0 ) and the a values (O ).
specific concentration and reaction time, using the average of the %IV obtained with both the
LU and Aa values. At an inhibitor concentration of 0.01% and after 30 min reaction time, the
%N obtained with PPE (Table 9) was 21.1% higher than that found with Cu-MT (Table 8)
and 7.1,93 -8 and 1 00.5% lower than those obtained with 4HR (Table 1 4), AA (Table 1 0) and
NaHSO, (Table 13), respectively. For cornparison with EDTA and CA, different
concentrations were used; the %IV obtained using 0.10% PPE (Table 9) was 27.1 and
502.5% higher than those found with EDTA (Table 12) and CA (Table 11) respectively at a
concentration of 0.09%. These results suggest that PPE as well as Cu-MT could be
considered as effective browning inhibitors in apple juice, with PPE being a stronger
inhibitor than Cu-MT. In addition, PPE and Cu-MT could compete with the selected
chemicals, CA and EDTA but not with AA, 4HR and NaHSO, which were distinctly much
more efficient than Cu-MT and PPE. Molnar-Perl and Friedman (1990a) reported that the
inhibition of enzymatic browning in apple juice, obtained with 4.54% (95%), was superior to
that obtained with 4.65% AA (44%) after 2 h of treatment.
4.2.3. Kinetic Study of Entynratic Browning in Apple Juice using Reflectance Spectrum
Figure 10 presents the scanning profile of enzymatic browning in apple juice in the
absence (Figure IOA) and presence of 0.03% NaHSO, (Figure IOB). The results show that
similar profiles were observed in the absence and presence of the inhibitor; however, the
overall reflectance values decreased with respect to reaction time in the absence of the
inhibitor whereas in the presence of NaHSO,, the reflectance values increased with time.
These results suggest that the apple juice, without inhibitor, turned brown due to the
occurrence of enzymatic browning while the apple juice treated with 0.03% NaHSO,
lightened during the 90 min of reaction. The experimental findings also indicate that the
overall reflectance values increased slowly fiom 400 to 490 nrn then increased markedly
fiom 490 to 540 nm and subsequently stabilized in the yellow-orange absorbance regions of
550 and 630 nm; a marked depression was also observed in the red absorbance region at
approximateiy 670 nm. These results suggest that the color intensity of apple juice was
largely due to the yellow-orange absorbance regions where a broad peak was o b s e ~ e d -
Similar results were reported by Lozano et al. (1994) who showed that the occmence of
380 480 580 680
Wavelength (nm)
Figure 10: Scanning of enymatic browning in apple juice fiom O (O) to 90 min (a ) without inhibitor (A) and with 0.03% sodium bisulfite (B).
browning in apple exhibited the same profile with a marked depression at 680 nrn in the red
absorbance region.
Figure 11 shows the reflectance spectra of enymatic browning in apple juice
obtained in the absence and presence of seven selected inhibitors. Similar profiles to those
described in Figure 10 were observed across the visible spectnun. The expenmental fhdings
show that the reflectance spectnun of the control sample obtained at 90 min reaction time
exhibited overall lower reflectance values in cornparison to that obtained at the initial time of
zero; in addition, the former sample also exhibited a narrower peak which shifled towards the
longer wavelengths. The results also show that the reflectance spectrum of the apple juice
containing the inhibitor after 90 min of reaction was either between the two spectra of the
controls of zero and 90 min reaction time such as in Figures 11 A, B, C and E or localized at
higher reflectance values such as in Figures 1 ID, F and G. Since the effectiveness of an
inhibitor depends on the position of its reflectance spectnim in cornparison to those of the
control samples, these results suggest that NaHS03 and 4HR were the most effective
browning inhibitors followed by CA and then by EDTA, AA, PPE and then lastly by Cu-MT;
however, these results obtained using reflectance values differ fiom those (Figure 9) based on
the AL and A a values.
Figure 12 shows the evolution of enzyrnatic browning in apple juice at selected
wavelengths including 400, 460, 530, 600 and 670 nm as a hinction of inhibitor
concentrations. The results indicate that the optimum concentrations for inhibition of
browning were 0.010, 0.025, 0.010, 0.240, 0.240, 0.030 and 0.150% for Cu-MT, PPE, AA,
CA, EDTA, NaHSO, and 4HR respectively; these values were identical to those obtained
fiom Figure 9 with respect to the LU and h values, except for Cu-MT which previously
exhibited a maximum inhibition of browning at a concentration of 0.025%. The expenmental
findings also show that the wavelength curves were not similar; the absorbance profile
obtained at the shorter wavelengths of 400 and 460 nm (blue absorbance area), progressed
together as well as those found at the longer wavelengths of 600 and 670 nm (red absorbance
ma); the absorbance profile obtained using the wavelength of 530 nm was more variable. In
380 480 580 680
Wavelength (nm)
a 11: Reflectance spectra for ditrerent inhibitors in apple juice: copper-metallothionein (A), polyphenol esterase (B), ascorbic acid (C), citric acid @), ethylenediaminetetraacetic acid (EDTA) (E), sodium bisulfite (F) and Chexylresorcinol (G). Spectra were obtained without inhibitor at O 6) and 90 (+) min and with the highest concentration of inhibitor at 90 min (O).
Inhibi tor Concentration (%)
Figure 12:Effect of inhibitor concentrations on enzymatic browning in apple juice determined at 400 nm (a ), 460 nm ), 530 nm (B ), 600 nrn (+ ) and 670 nm ( O ) for copper-metallothionein (A), polyphenol esterase (B), ascorbic acid (C), citric acid @), ethylenediaminetetraacetic acid (EDTA) (E), sodium bisulfite (F) and 4-hexyiresorcinol (G).
addition, the absorbance profile at 670 nm exhibiteci the lowest values for the difference in
refiectance while the cuve at 530 nm exhibited the highest values for the difference in
reflectance. These results suggest that the optimum concentrations of the selected inhibitor
for the prevention of browning Vary depending of the wavelength chosen for analysis and can
therefore be less accwate than those obtained fiom the AL and Au values.
In surnmary, the overall results show that the reflectance measurements generally in
contrast to those obtained using the AL and Au values which give more accurate results.
4.3. Potato Slices
4.3.1. Meusurement of Browning a? the Cut Surface of Potaîo Slices
Figure 13 shows the occurrence of enzymatic browning in potato slices over a penod
of time in the absence and presence of 0.0025 and 0.0250% of NaHSOJhe expenmental
results indicated that regardless the nature of inhibitor tested, the same profiles were
obtained; however, the degree of inhibition was clearly demonstratecl with the use of
NaHSO,. On the basis of these results, Figure 13 is used as clear demonstration for the
inhibition of enzymatic browning in potato slices. Changes in the L values with respect to
time are reported in Figure 13A. The results show that the L values were indirectly related to
the extent of browning at the surface of potato slices. In the absence of inhibitor in the potato
slices the L values decreased imrnediately while in the presence of the inhibitor, a latence
phase was observed after which a slow decrease in the L values occurred. An increase in the
inhibitor concentration resulted in a longer latence phase and a slower steep. In the absence
of the inhibitor, the AL of 29 was relatively high while in the presence of 0.0025% NaHSO,,
it decreased to 15 and reached a value of 8 in the presence of 0.0250% NaHSO,. These
results suggest that the discoloration in potato slices was more intense and faster in the
absence of the inhibitor. A similar trend was reported by Sapers et al. (1989a) who indicated
that the L values were negatively correlated with the extent of browning at the cut surface of
potatoes and who reported a lag tirne of 1 min and a AL value of 12.9 after 6 brs for Russet
Burbank slices. Molnar-Perl and Friedman (1 WOb) reported a AL value of 20.2 f i e r 4 hrs for
control slices of Russet Burbank potatoes as well as a negative correlation between L values
Reaction Time (min)
Figure 13: Changes in the L (A) and a (B) values in potato slices without inhibitor (.), and with 0.0025% NaHS03 (a) and 0.0250% NaHSO, ÇL).
and the extent of browning.
The changes which occurred in the a values are presented in Figure 13B. The results
show that in the presence of 0.0250% NaHSO,, the a values increased with respect to
reaction tirne while in the absence and presence of 0.0025% NaHSO,, a broad peak was
observed during the first 50 and 50 to LOO min of reaction, respectively. These results
indicate that the redness of the color in the potato slices changed during the reaction time,
reaching a maximum in redness after which a purple color became dominant; in addition the
correlation between the a values and the enzyrnatic browning was not obvious. With potato
slices, the enzymatic browning is characterized by a purple-black color while it is
characterized by a orange-red color with apple slices; therefore a value, which describes the
redness, was well related to the extent of enzymatic browning in apple slices but could not
describe precisely the development of enzymatic browning in potato slices. Sapers et al.
(1989a) reported analogous conclusions by indicating that the a values were not related to the
extent of browning at the cut surface of potatoes.
0 4.3.2. Evaluation of Bro wning Inliibiror Treatments at the Cut Surface of Potato Slices
Tables 15 to 21 show the influence of selected inhibitors on the difîerent parameters
(lag t h e , rate and extent of browning) involved in enzymatic browning as well as the %N;
al1 of these data were obtained fiom the L measurements after 15, 50 and 100 min of reaction,
time defined from the Figure 13. In addition, in order to compare our results to those obtained
in the literature and in order to get more information about the inhibition of enzymatic
browning, additional parameters such as lag time, rate and extent of enzymatic browning
were investigated with potato slices; we were able to determine these parameters since the
development of enzymatic browning at the cut surface of potato slices was relatively slow
compared to that in apple products. In addition, Figure 13B showed that the correlation
between a values and the enzymatic browning was not obvious; on the basis of these
experimental findings, only the results obtained fiom L measurements were reported.
Table 15 shows the effect of the Cu-MT extract at concentrations of 0.0075 to
0.1000% on browning formation. The results show the absence of a lag time with al1 the Cu-
Table 15. Inhibitory effect of a copper metallothionein (Cu-MT) extract, obtained from A. niger, on enzymatic browning in potato slices, determined using a tristimulus colorimeter.
-- .- --
Extent of browning Inhibition (%)d
Cu-MT Lag timea slopeb A L' Reaction time (min)
concentration (%) (min) (min") 15 min 100 min 15 50 1 O0
0,0000 O -0.208 -3.1 5 - 17.05 - - -
0.0075 O -0.234 -3.72 - 18.83 -4.4 (I-2.5)eJ 10.6 (12.6)"" 20.7 (1 i .O)"'
0.0 1 O0 O -0.224 -4.12 - 1 7.90 -8.2 ( I - 2~ ) '~ 22.9 (II .9Tg 33.2 (10.8f'~
0.0250 O -0,171 -2.56 -14.18 32.3 (11 .6)eJ 22.9 ( i 1 . 3 ) ' ~ 16.6 (f l . 5 fJ
0.0500 0 -0.222 -3.88 -1 7.52 1 5.7 (13.7)" 20.7 (Il .6)"' 25.5 (10.2)'~
0.0750 O -0.220 -3.77 - 1 7.44 -3.4 (&2.5)Li 25.4(ki.4fJ 41.3(f0.5)~~
O. 1000 O -0.2 1 8 -3.69 -1 7.55 -0.3 (1-2.5)"' 7.4 (II .7)"' 20.7 (u.3Pi
a Tirne characterized by the initial flrt region before onset of browning, based on measurement of the L-values. '~inear region of browning curve, based on measurement of the L-values. C Changes in L- values with respect to the initial values and those obtained afier 15 and 100 min of reaction. d~ercentage of inhibition ût a defined tirne (t), with respect to L values, was calculated as (ALcont,i - ALtreatmenI) I ALconno~ x100, where LU =
Lt - Linitiab e Relative standard deviation of triplicate samples was determined as the standard deviation divided by the mean, multiplied by 100.
f i k ~ e a n ç were significantly different with P < 0.05.
MT concentrations used. The results also show that the slope values were relatively close (-
0.208 to -0.234) except for that obtained with 0.0250% Cu-MT where a value of -1.71 was
obtained. These results suggest that enzymatic browning started immediately and continued
at an equivalent rate in the potato slices treated with the different concentrations. The overall
experirnental findings also indicate that similar LU values at 15 and at 100 min of reaction
were obtained with respect to the Cu-MT concentrations used except for those obtained with
0.0250% Cu-MT where the lowest values of -2 -56 and - 1 4.1 8 were found at 1 5 and 1 00 min
respectively. These results suggest that the browning was less developed in the potato slices
treated with 0.0250% Cu-MT compared to that exhibited with lower or higher Cu-MT
concentrations. In addition, the overall %IV were low with the majonty being below 50%
and increased with reaction tirne; the %IV reached theu maximum at 0.0750% Cu-MT. These
results suggest that the Cu-MT extract had a low inhibitor efficiency as indicated by the trend
observed with the slope and AL values. Goetghbeur and Kemasha (1996) reported that the
use of a more purified Cu-MT fraction as inhibitor could enhance its inhibitory effect at the
surface of potato slices.
The inhibitory effect of PPE at concentrations of 0.025 to 0.500% on browning is
shown in Table 16. The results show that lag times of 2, 6 and 14 min were observed at the
three higher concentrations of 0.100, 0.250 and 0.500% of PPE respectively; however, in the
presence of 0.075% PPE, the lag time was zero and the slope and the LL~ at 15 and at 100 min
had the lowest values of -0.172, -2.79, - 15.1 1 respectively, thereby indicating that enzymatic
browning was reduced. In the presence of 0.500% PPE, the lag tirne were long (14 min) but
the slope and the LU at 100 min were high with values of -0.232 and -21.04 respectively,
thereby suggesting severe browning. These results suggest that there was no correlation
between the lag time and the slope and AL values. The results indicate that the overall %IV
decreased with reaction time and were generally low with o d y one %N greater than 50%
(54.3) which occurred &er 15 min of reaction with 0.500% PPE; however, on average, the
highest %IV were observed in the presence of 0.075% PPE. These results demonstrate that
the PPE extract loses its effectiveness during the 90 min of reaction and exhibits a relatively
moderate inhibitory effect on browning. In addition, the %IV seemed to be correlated with
Table 16. Inhibitory effect o f a polyphenol esterase (PPE) extract, obtained from A. niger, on the enzymatic browning in potato slices, determined using a tristimulus colorimeter.
Extent of browning Inhibition (%)d
PPE Lag tirne* slopeb A L' Reaction time (min)
concentration (%) (min) (min-') 15 min 100 min 15 50 100
0,000 O -0,254 -4.1 3 -2 1.78 - - -
' ~ i m e characterized by the initial flat region before onset of browning, based on rneasurement o f the L-values. b Linear region of browning curvc, based on measurement of the L-values. C Changes in L- values with respect to the initial values and those obtained afier 15 and 100 min of reaction. d~ercentage of inhibition at a defined tirne (t), with respect to L values, was calculated as (ALcontrol - ALtreatment) f Mconhol ~ 1 0 0 , where AL =
Lt - Linitiab e Relative standard deviation of triplicate samples was detennined as the standard deviation divided by the mean, multiplied by 100. '&~eans were significantly different with P < 0.05.
the slope and LU values. A s indicated by Madani et al. (1999a), the use of a more purified
fraction as inhibitor could enhance the inhibitory effect of PPE at the surface of potato slices.
Table 17 shows the effect of AA at concentrations of 0.075 to 1.000% on the
inhibition of browning. The results show that the onset of enzymatic browning was delayed
from 3 to 30 min in the presence of inhibitor concentrations ranging fiom 0.100 to 2 -000%
AA. In addition, the lowest values for the slope and AL were obsewed with 1.000% M.
These results suggest that there was a correlation between the Iag time and the slope and AL
values as well as with the inhibitor concentration; the results indicate that as the inhibitor
concentrations increased, enzymatic browning was delayed (longer lag time), and occurred
more slowly (significantly smailer slope) and to a lesser extent (smaller LX). In addition, the
%IV increased with time and very low AA concentrations while at concentrations equal to or
greater than 0.250% AA, the %N decreased with time. The ovetall %IV were high and
greater than 50% only during the first 15 min of reaction for tU concentrations higher than
0.250%, while otherwise, the %IV were more or less low. These results suggest that AA
could be an efficient browning inhibitor only under certain experimental conditions; in
addition, a correlation was observed for the %N and the values of LU characterizing the
extent of browning. The literature reported that AA has a great potential as an inhibitor of
browning especially when used in combination with other antibrowning agents; Sapers et al.
(1995) indicated that the treatment of potatoes with hot water containing 1.0% AA and 1.0%
CA uihibited discoloration for 9 days and showed a AL vaiue of 2.4.
Table 18 presents the inhibitory effect on browning obtained with CA at
concentrations ranging from 0.075 to 1.000%. Similar observations were found as those
obtained for AA as inhibitor. The results show that as the inhibitor concentrations increased,
the onset of enzymatic browning was delayed for longer periods of time (longer lag tirne) and
occurred more slowly (significantly smaller slope) and to a lesser extent (smatler AL). In the
presence of 1.000% CA, the longest lag time of 25 min was exhibited as weil as the lowest
slope value of -0.023 and hL. values at 15 and at 100 min of 0.14 and - 1.62 respectively while
the %IV were the highest, ai around 100%. In addition, the overall %N
Table 17. lnhibitory effect of ascorbic acid (AA) on enzymatic browning in potato slices, determined using a tristimulus colonmeter.
Extent of browning Inhibition (%f
Ascorbic acid Lag timea slopeb A L' Reaction tirne (min)
concentration (%) (min) (min-') 15 min 100 min 15 50 100
a Time characterized by the initial flat region before onset of browning, based on measurement of the L-values.
b Linear region of browning curve, based on measurement of the L-values. C Changes in L- values with respect to the initial values and those obtained after 1 5 and 100 min of reaction. d~ercentnge of inhibition at a defined time (t), with respect to L values, was calculated as (MCont,i - ALireûtment) I hl-,ontrol ~ 1 0 0 , where AL =
Lt - Linitial. e Relative standard deviation of triplicate samples was determined as the standard deviation divided by the mean, multiplied by 100.
f k ~ e a n s were significantly different with P < 0.05.
Table 18. Inhibitory effect of citric acid (CA) on enzymatic browning in potato slices, detemined using a tristimulus colorimeter.
Extent of browning Inhibition (%f
Ciric acid Lag timea slope A L~ Reaction time (min)
concentration (%) (min) (min") 15 min 100 min 15 50 100
a Time characterized by the initia1 flat region before onset of browning, based on measurement of the L-values.
b~inçar region of browning curve, based on measurernent of the L-values. C Changes in L- values with respect to the initial values and those obtained af'ter 15 and 100 min of reaction. d Percentaye of inhibition at a defined time (t), with respect to L values, was calculated as (ALeoncroi - ALtreatment) 1 Uconirol x100, wherc LU. =
LI - Liniiiab c Relative standard deviation of h-iplicate samples was detemined as the standard deviation divided by the mean, multiplied by 100. F k ~ e a n s were significantly different with P < 0.05.
decreased with respect to reaction time. These results suggest that CA is an effective
browning inhibitor but loses its efficiency over time; in addition those parameters including
lag tirne, rate and extent of browning and the %IV were correlated to another. The literature
reported that CA has a great potential as an inhibitor especially when used in combination
with other antibrowning agents; Sapen et al. (1995) indicated that the treatment of potatoes
with hot water containing 1% LM and 1% CA inhibited discoloration for 12 days and showed
a AL value of 0.0.
The results obtained for the effect of EDTA at concentrations of 0.005 to 0.075% on
enzymatic browning are reported in Table 19. The results indicate the occurrence of only one
lag time of 2 min observed at 0.075% EDTA which is in agreement with the low value of - 0.003 obtained for the slope but in contrast with the high vaiues found for the AL as well as
the moderate %IV. In addition, the highest %N was associated with the lowest AL value
obtained after 100 min (-16.84) with the use of 0.0250% EDTA; however, the lowest values
for the slope and AL after 5 min were not obtained at this EDTA concentration. These results
suggest that the parameters related to the extent of browning were not related to each other or
to the %IV as they changed independently of each other. In addition, the overall %N
decreased during the 100 min of reaction and were al1 less than 50%. These results indicate
that EDTA loses its effectiveness over the course of the reaction and has a Iow inhibitory
effect. Nevertheless, Feinberg et al. (1987) reported that EDTA could be used with sodium
acid pyrophosphate to control after-cooking darkening in pre-peeled potatoes.
Table 20 shows the inhibitory effect of NaHSO, at concentrations of 0.001 to 0.025%
on browning in potato slices- The results show that the strongest %N (101.7), the longest lag
tirne (50 min) as well as the lowest slope value (-0.058) were observed in the presence of the
highest concentration of NaHSO, (0.025%); however, at this concentration, the AL values
were not the lowest. These resuIts suggest that the %N and slope values as well as the lag
tirne and inhibitor concentration were in good correlation with each other while the AL values
were obtained independently. The experimentd findings also indicate that the overall %IV
decreased with time and that the highest %IV were obtained at 15 min reaction time.
P P O O
O P c. O 0 O O -l
O tri
O tn O z Cn
ô z CD 3 CL
g: ,C. O 1 h
ort w
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3 5- V
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- tri
3 5-
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O O 3 3-
L
ui
These results demonstrate that NaHS03 is an effective browning inhibitor but loses its
effectiveness over t h e . A similar trend was reported by Molnar-Perl and Friedman (1 99Ob)
who indicated that potato slices immersed for 2 min in different sodium bisulfite bath
ranging in concentrations kom 5 to 50 mM, exhibited after 4 hrs, showed excellent %IV
ranging fiom 93 to 105%.
Table 2 1 indicates the influence of 4HR in the range of concentrations of 0.00025 to
0.00500% on browning. The results show that the strongat %IV (80.9%), the longest lag
time (12 min) as well as the lowest values for the slope (-0.069) and AL obtained afier 100
min (-7.63) were noticed at the highest concentration of NaHSO, (0.00500%); only the AL
value obtained after 5 min disagreed with these observations. These results suggest that the
%IV and dope values as well as the lag tirne and the general extent of browning (LU afier
LOO min) were in good correlation with each other and with the increase of inhibitor
concentration. The experimentd hdings also indicate that the %N decreased with time so
that they were especially hi& during the first 15 min of reaction and in the presence of 4HR
concentrations equal to or higher than 0.00100%. These results demonstrate that 4HR has a
great potential as a browning inhibitor but it loses its effectiveness over a penod of time.
Figure 14 demonstrates the effect of different concentrations of a wide range of
inhibitors, including CU-MT, PPE, AA, CA, EDTA, NaHSO, and 4HR, on enzymatic
browning at the surface of potato slices. In order to compare the efficiency of different
inhibitors, the same dipping time of 2 min penod was used. The results show that the %N
either increased with inhibitor concentration (Figures 14C, 14D, 14F and 14G) or the %TV
reached a maximum and then decreased as the inhibitor concentration continued to increase
(Figures 14B and 14E); however, in Figure 14A, two maxima for %IV were obtained at two
inhibitor concentrations. For each inhibitor treatment, an optimum concentration for maximal
inhibition of browning on po tato slices was determined; the inhibition concentrations were
0.025 and 0.075, 0.075, 1 -000, 1 -000, 0.025, 0.025 and 0.005% for Cu-MT, PPE, AA, CA,
EDTA, NaHSO, and 4HR, respectively. The effectiveness of selected inhibitors in controlling
the enzymatic browning was in the order of CA > NaHSO, > AA > 4HR > PPE > EDTA
10 I O 0.002 0.004
hhibitor Concentration (%)
Figure 14: Effect of inhibitor concentrations on enymatic browning in potato slices: copper-metallothioneh (A), polyphenol esterase (B), ascorbic acid (C), citic acid (D), ethylenediaminetetraacetic acid (EDTA) (E), sodium bisulfite (F) and 4-hexylresorcinol (G). The percentage of inhibition was calculated h m the L values (m ).
Based on a specific inhibitor concentration of 0.100% and reaction time 50 min, the
inhibitory effect of 29.4% exhibited by the PPE extract (Table 16) was much higher
compared to that obtained with CA (Table 18) which was negative (-20.3%) and 297 and
230% higher than those found with the Cu-MT extract (Table 15) and AA (Table 17)
respectively. An inhibitor concentration of O . O Z % was used for cornparison of the inhibitory
effect of EDTA and NaHSO, with the PPE extract; the PPE inhibitory effect was 52- and
104- fold lower than those found with EDTA (Table 19) and NaHSO, (Table 20)
respectively. With respect to 4HR (Table 21), the PPE %N, obtained at 0.0075% of
concentration, was 488% lower than that found with 4HR at 0.0050% of concentration. These
results suggest that PPE as well as Cu-MT could be considered as effective browning
inhibitors, with PPE being clearly stronger than Cu-MT. In addition, PPE and Cu-MT could
compete with the selected chemicals, CA and AA while EDTA, 4HR and M O , were
much more efficient than the two bio-ingredients, Cu-MT and PPE.
0 4.3.3. Kinetic Study of Enwmatic Browning in Potaîo Slices using Rejlecrance Spectrum
Figure 1 5 shows the scanning of enzymatic browning at the surface of potato slices in
the absence (Fig. 1 SA) and presence of 0.025% NaHSO, (Fig. 15B). The results show sirnilar
profiles were observed for enzymatic browning in potato slices with and without inhibitor;
the reflectance values increased gradually across the visible spectrum ranging fkom 400 to
700 nrn; this increase was slightly stronger in the green absorbance region of 490 to 530 nm
and then gradually decreased at longer wavelengths. These results suggest that the color
sensation of potato slices was a combination of green-yellow-red absorbance regions in
various proportions. The expenmental findings also report that, in the absence of the
inhibitor, the overall reflectance values of the spectra decreased during the reaction t h e of
90 min after which a AL value of 19 was obtained at 680 nm. In the presence of 0.025%
NaHSO,, the difference between initial and final spectra was reduced during the reaction time
of 90 min as indicated by a lower AL value of 3 at 680 nm. These results suggest that the
a decrease in reflectance values was directly related to the development of enzymatic browning
480 580
Wavelength (nm)
Figure 15: Scanning of enzyrnatic browning in potato slices fiom O (0)
to 90 min (a) without inhibitor (A) and with 0.025% sodium bisulfite (Bk
and that the potato slices underwent severe browning in the absence of the inhibitor whereas
only slight browning was observed in the presence of 0.025% NaHSO,.
Figure 16 shows the reflectance spectra of enzymatic browning at the surface of the
selected inhibitors. The overail results demonstrate that al1 of the reflectance spectra
exhibited a similar profile as that shown in Figure 15. The reflectance spectrum of the control
sample, obtained at the initial tirne of zero, showed higher reflectance values across the
visible spectnim than that obtained after 90 min of the reaction. The reflectance spectnun oc
the sample treated with the inhibitor, obtained after 90 min of reaction, was always in
between the two spectra of the control sarnple found afier O and 90 min of reaction. The
results indicate that the closer the reflectance spectrum of the inhibitor was to that of the
control at the initiai time of zero, the more the inhibitor was efficient. These results suggest
that AA was the most effective browning inhibitor followed by NaHS03 and CA and then by
4HR, EDTA? PPE and fïnally by Cu-MT which exhibited the lowest inhibitory effect; this
classification was different to that obtained with L values in Figure 14.
Figure 1 7 shows the development of enzymatic browning at selected wavelengths
including 400, 460, 530, 600 and 670 nm as a function of the concentration of selected
inhibitors. The results indicate that the optimum concentration for the prevention of browning
were 0.0250, 0.0750, 1.0000, 1.0000, 0.0250, 0.0250 and 0.0025% for Cu-MT, PPE, AA,
CA, EDTA, NaHSO, and 4HR respectively; these values for maximum inhibition of
browning were identical to those obtained with Figure 14, except for 4HR which previously
exhibited optimum inhibition of browning at a concentration of 0.0050%. With respect to Cu-
MT, the optimum concentration for inhibition of 0.0250% differed to that obtained in the
Figure 14 (0.075%). The experimental findings also show that the reflectance profile at
different wavelengths was similar with the exception of CA in Figure 17D where some
irregularities were observed.
The experimental fïndings obtained h m the reflectance measurements show that the
results differed with respect to the representation used for the anaiysis of data including the
reflectance percentage as a function of wavelengths or inhibitor concentrations; in addition
1 1 l
8 O 480 580 680
Wavelength (nm)
Figure 16: Reflectance spectra for different inhibitors in potato slices: copper-metallothionein (A), polyphenol esterase (B), ascorbic acid (C), citric acid (D), ethylenediaminetetraacetic acid (EDTA) (E), sodium bisul fite (F) and 4-hexylresorciwl (G). Spectra were obtained without inhibitor at O (a) and 90 (* ) min and with the highest concentration of inhibitor at 90 min (a) .
1
O 1
0.002 0.004
Inhibitor Concentration (%)
Figure 17: Effect of inhibitor concentrations on m a t i c browning in potato slices determined at 400 nm (a ), 460 nrn (0 ), 530 nm (a), 6 0 h (6 ) and 670 nm (O ) for copper-metallothionein (A), polyphenol esterase (B), ascorbic acid (C), citric acid @), ethylenediaminetehaacetic acid (EDTA) (E), sodium bisulnte (F) and Chexylresorcinol (G).
these results generally disagree with those obtained with L values which showed more
accuracy.
The results gathered in this study indicated that the partially purified fiactions of and
polyphenol esterase (PPE), obtained fiom A. niger, were able to inhibit, in part, the
occurrence of enzymatic browning in food products such as apple skes , apple juice and
potato slices. The experimental findings confimed the inhibitory effect of Cu-MT and PPE
on browning previously demonstrated in our laboratory at the time of the investigation of the
inhibition of tyrosinase activity in mode1 solutions using selected substrates. The strong
affùlity of the induced Cu-MT to bind the copper could explain the ùihibitory effect of this
protein, acting as a chelating agent, on the PPO activity, In addition, the experimental
findings suggest that the mechanistic action of PPE and its role in the inhibition of PPO
activity could be due either to its cornpetition with the PPO for the use of same substrate or to
the consumption of end-product, generated by the PPO activity.
Tristirnulus colorimetry analysis was appropnate to follow, over selected penods of
a tirne and in selected food products, the development of enzymatic browning as well as its
inhibition by different treatrnents (chemical and enzymatic). For each inhibitor tested, an
optimum concentration was determined. The findings also showed that in the selected food
products used, two chemical compounds, sodium bisul fite and 4-hexylresorcinol, were
always the most efficient inhibitors of browning and their inhibitory effect was much higher
than those obtained for PPE or Cu-MT. On the other hand, the two natural bio-ingredients,
Cu-MT and PPE, codd compete with the chernicals tested including ascorbic acid, citric acid
andor EDTA, with respect to the food product tested.
The study of the reflectance spectra in the visible region was also related to enzymatic
browning but at a lower scale than the colorirnetry method which was more accurate. The
reflectance measurements showed that the color sensation found in the food products tested
resulted mainly from the combination of various proportions of yeilow and red regions as
browning progressed.
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