Effect of Cyclodextrins on Polyphenol Oxidation Catalyzed by Apple

Chapter 23

Downloaded by KTH ROYAL INST OF TECHNOLOGY on August 24, 2015 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0600.ch023

Effect of Cyclodextrins on Polyphenol Oxidation Catalyzed by Apple Polyphenol Oxidase 1

1

1

2

C. Billaud , E. Regaudie , N . Fayad , Florence Richard-Forget , and Jacques Nicolas 1

l

Chaire de Biochimie Industrielle et Agro-alimentaire, Conservatoire National des Arts et Métiers, 292 rue Saint-Martin, 75141 Paris Cedex 03, France Station de Technologie des Produits Végétaux, Institut National de la Recherche Agronomique, Domaine Saint-Paul, B.P. 91, 84143 Montfavet Cedex, France 2

In order to find nonsulfite antibrowning agents for the food industry, cyclodextrins (CD) have been tested. CD are able to form inclusion compounds with polyphenols and inhibition properties have been studied in model solutions containing one or two phenols and purified apple polyphenol oxidase (PPO). The dissociation constants (K ) of a series of phenolic compounds with α-CD, β-CD and hydroxypropyl-βCD have been determined both by enzymatic kinetic analysis and NMR experiments in solution with one phenol. Large differences have been found among the phenols tested resulting in wide variations in the inhibitory properties of CD. In the mixtures of two phenolic compounds, addition of β-CD can result either in a large inhibition, a slight inhibition, or even a slight activation of oxygen uptake, depending on the kinetic constants and of the phenols present in the solution. Moreover, since β-CD induced a modification of the balance of free substrates in a multiphenolic solution, the colors obtained after enzymatic oxidation catalyzed by PPO can also be modified in different ways by addition of β-CD as demonstrated on the 3 types of mixtures of two phenolic compounds that we have tested, namely chlorogenic acid / (-)-epicatechin, chlorogenic acid / (+)-catechin and 4-methylcatechol / caffeic acid. D

Enzymatic browning which occurs during handling, storage and processing of fruits and vegetables is mainly initiated by the enzyme polyphenol oxidase (odiphenol : oxygen oxidoreductase EC 1.10.3.1) or PPO (7, 2). The loss of cell integrity results in the decompartmentation of phenolic substrates and enzymes and then, in the presence of molecular oxygen, the oxidative production of colored quinones (3). Brown pigmentation following this enzymatic reaction and subsequent non enzymatic 0097^156/95/0600-0295$12.00/0 © 1995 American Chemical Society

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


296

ENZYMATIC BROWNING AND ITS PREVENTION

reactions, is generally considered to be detrimental to food quality from both the organoleptic and nutritional points of view (4). Therefore, the control of enzymatic browning has always been a challenge to food scientists owing to the losses that it causes in many food products (5). Sulfur dioxide (and its derivatives) is by far the most effective inhibitor of browning. It acts at very low concentrations and is inexpensive. However, because of the potential health hazards, the tendency of national and international regulations is to reduce or even ban its use in the fruit and vegetables industry (6). Thus, an active field of research is currently under development to find non-sulfite antibrowning agents for the food industry (7). Enzymatic browning can be controlled by different ways (8). Besides heat and acidification treatments (9,10), several chemical inhibitors have been examined including ascorbic acid and its derivatives (11-14), cysteine (15,16), halide ions (17,18), aromatic carboxylic acids (18), sulfated polysaccharides (79), kojic acid (20), resorcinol derivatives (27), polyphosphates (22) and extracts from honey (23) and ficin (24). These compounds mainly affect the enzyme, polyphenols substrate, or reaction products, although, in some cases, two or three targets can be affected at the same time. Recently, cyclodextrins (CD) have also been proposed for control of the enzymatic browning of apple products (22, 26) and use of CD have been patented (25). CD are macrocyclic torus-shaped non-reducing oligosaccharides of six or more D-glucopyranose residues. The most extensively studied CD are the α-, β-, and γ-CD forms which have respectively six, seven and eight glucose units per macrocycle linked by α (1-4) glycosidic bonds. The central cavity is hydrophobic whilst the outside parts are hydrophilic, due to the location of the primary and secondary hydroxyls at the narrow and wide bases, respectively (27, 28). In aqueous solution, the hydrophobic cavity can accomodate a wide range of guest molecules forming a stable complex. Although the host : guest ratio is usually of 1 : 1, one, two or three CD molecules can contain one or more guest molecules. For phenolic compounds, βCD offers the most suitable cavity size since its dimensions are comparable to those of substituted phenyl groups (29). This, coupled with the fact that β-CD is the cheapest to obtain commercially, explains why it is by far the most extensively studied CD (27, 28). However, β-CD has a very low water solubility (1.85 g / 100 mL at 25 °C) compared with a- and γ-CD (14.5 and 23.3 g / 100 mL respectively). Since this low solubility is mainly due to intra-molecular hydrogen bonding, various derivatives have been proposed with enhanced water solubility (30, 31). Among them, hydroxypropylβ-CD (ΗΡ-β-CD) derivatives are highly water soluble (> 50 g / 100 mL at 25 °C) and more stable in acidic conditions and in the presence of α-amylase than β-CD (32). Only three studies have been carried out on the effects of CD on enzymatic browning of apple (22, 25, 26). Thefirstone showed that β-CD, but not a- or 7-CD, was an effective inhibitor in Granny Smith juice browning (22). The others indicated that insoluble β-CD and derivatized^-CD gave similar results to that of β-CD but the percentage of inhibition which increased with the CD concentration was dependent on the apple variety (25, 26). The latter authors proposed an effect of the phenolic composition. Few studies have been devoted to the interactions of the main phenolic substrates of apple enzymatic browning, i.e. chlorogenic acid and flavan 3-ol derivatives (8), with CD. These works were concerned with the complexation of the



23.

Effect ofCyclodextrins on Polyphenol Oxidation

BILLAUD ET AL.

297

flavan 3-ol derivatives by a- and β-CD (33, 34) on the one hand and that of chlorogenic acid by α-, β-and γ-CD and polymerized β-CD (35) on the other hand. These studies, mainly using NMR spectroscopy and UV spectrophotometry, showed that the dissociation constants of the phenol-CD complex (assuming a 1:1 stoichiometry) widely varied according to the nature of the phenol and of the CD. In the latter study, the authors concluded that the enzymatic browning control was mainly due to the sequestration of PPO substrates by CD (35). The purpose of this work was mainly focused on the effect of CD on the enzymatic phenolic oxidation catalyzed by purified apple PPO in model solutions. Dissociation constants of the phenol-CD complex have been obtained and compared to NMR data. Since in natural products, PPO acts on a phenol mixture, the enzymatic study has been extended to solutions containing two phenolic compounds. In the latter case, both oxygen uptake and color formation have been studied. Materials and Methods Materials. Apples from the variety Red Delicious picked at commercial maturity were used as an enzyme source. The PPO was 120-fold purified from the cortex in three steps : extraction, fractional precipitation by ammonium sulfate and hydrophobic chromatography with Phenyl Sepharose (Pharmacia) according to the method of Janovitz-Klapp et al. (36). The different phenolic substrates were reagent grade from Sigma (St.Louis) and used without further purification. Native α-CD, β-CD and a ΗΡ-β-CD derivative were kindly provided by Roquette (Lestrem, France). Their specifications were as follows : α-CD > 99.4 %, β-CD 99 % min and ΗΡ-β-CD (non substituted β-CD < 1 %) with a molar substitution between 0.55 and 0.65. All CD were used as received. Assay of PPO Activity. PPO activity was routinely assayed by polarography at 30 °C according to the method of Janovitz-Klapp et al. (37) using 4-methylcatechol (20 mM) as substrate in a McDvaine buffer solution at pH 4.5. Activity was expressed in nmol of oxygen consumed per second (nkat) in the assay conditions. Inhibition of PPO Activity by CD. The phenolic substrate concentrations ranged from 0.5 to 40 mM (the limits were dependent on the Km values) in the control and with two concentrations of the different CD. The CD concentrations ranged from 2 to 20 mM depending on the phenol studied. A constant amount of 30 nkat of purified apple PPO was added to 2 mL of the air saturated phenolic solution. Some inhibition experiments were carried out on binary mixtures of phenols, namely 4-methylcatechol (1 to 20 mM) and caffeic acid (0.1 to 1 mM) or chlorogenic acid (0.5 to 5 mM) and catechin (0.5 to 5 mM), using β-CD (5 or 10 mM). In the latter case, the two isomers of catechin, (+)-catechin and (-)-epicatechin, were tested. Calculation of the K (dissociation constant) of the CD-Phenol Complex from Kinetic Experiments. Assuming a 1: 1 stoichiometry D

K ^ U C D ] [S]}/{[CD.S]}


298


The mass balance equations for S and CD are [S] = [CD.S] + [S] and [CD] = [CD.S] + [CD] where [S]t and [CD] are the total amounts of phenol and CD in the solution, respectively. Hence, Ko = {([CD] - [S] + [S]) [S]} / {[S] - [S]} After rearranging [S]2 + ([CD] - [S] + K ) [S] - K [S] = 0 t

t

t

t

t


t

t

t

D

D

t

The negative solution can be ignored because [S] must be positive [S] = { - ([CD] - [S] + K ) + {([CD] - [S] + K )2 + 4 K [S] }* } / 2 t

t

D

t

t

D

D

t

(A)

This expression representing the amount of tree phenol available for PPO may be substituted in the velocity equation assuming that PPO is inactive on the [CD.S] complex ν = Vm [S] / (Km + [S]) (B) Firstiy, the apparent Km and Vm values were determined from the control experiments by using a non linear regression data analysis program developed by Leatherbarrow (38). Secondly, the KQ values were obtained using an homemade program by fitting experimental and calculated velocities in the presence of CD. Ή NMR Experiments. All studies were carried out at room temperature with a Bruker AC-200 Ε spectrometer operated at 200 MHz. Thefreeinduction decays were accumulated in 16 Κ data points over a 2.4 KHz spectral width, with a 3 ps pulse width (ca 40° flip angle) and operating at LB = - 0.5 and GB = 0.3. The number of scans ranged from 100 to 300. Ή NMR spectra were recorded in 99.8 atom-% D 0 solutions (SDS, France). Chemical shifts were assigned relative to water (4.74 ppm) using 3-(trimethylsilyl)-propionic acid, sodium salt in D 0 as external reference. For the determination of the apparent K value, the β-CD concentration was maintained at 5 mM in D 0 while the phenol concentration varied between 0.5 and 40 mM (10 different concentrations have been tested for each phenol). Spectra were recorded at least one hour after each addition and the values of chemical shift changes Δδ (Δδ = ôf - ô p i i ) which occur for the H-3 and/or H-5 β-CD protons were used for the estimation of K (33, 39). 2

2

D

2

ree

c o m

e x e (

D

L*, a*, b* Measurements. The effect of β-CD on the color variations during the PPO catalyzed oxidation of binary phenolic mixtures was followed using tristimulus colorimetry. Five mL of air saturated phenolic substrates with or without β-CD (4 mM), were placed in small beakers. As soon as 8 nkat of purified apple PPO was added, the L*, a*, b* values of the solution were recorded every minute for 30 minutes using a Minolta CR 300 chromameter. Three mixtures have been tested, namely 4-methylcatechol / caffeic acid, chlorogenic acid / (+)-catechin and chlorogenic acid / (-)-epicatechin.


23.

BILLAUDETAL.

Effect of Cyclodextrins on Polyphenol Oxidation

299


Results and Discussion Effect of CD on PPO Activity in a Single Phenolic Solution. When a constant amount of CD was added to solution containing variable amounts of phenolic substrate, an inhibition of oxygen uptake catalyzed by purified apple PPO was observed. This inhibition decreased as the phenolic concentration increased resulting in a sigmoidal shape for the velocity curve versus substrate concentration. Two typical curves are given in Figure 1 for chlorogenic acid and (+)-catechin in the presence of β-CD. CD are well-known for their ability to form inclusion complexes with phenols (34) resulting in substrate depletion for PPO (26, 35). Assuming a 1 : 1 stoichiometry between CD and phenol (33, 35), an equation containing the dissociation constant ( K ) of the complex CD / Phenol, which gives the amount of free substrate available for the enzyme has been developed (Equation A). The K value can be obtained by fitting the experimental and calculated velocities. For the above experiments with β-CD, the K D values obtained were 2.2 and 0.19 mM for chlorogenic acid and (+)-catechin respectively. With these values, the experimental points were in close agreement with the calculated velocities (dashed and dotted lines in Figure 1). A similar treatment of data was carried out for a series of phenolic compounds in the presence of α-CD, β-CD or ΗΡ-β-CD. The values obtained for these different complexes are given in Table I. With β-CD, the values ranged from 0.2 to more than 30 mM meaning that the binding strength of β-CD widely varied from one phenol to another. Obviously, for low substrate concentrations, high Kjy values resulted in slight inhibition of PPO activity and conversely, low values in strong inhibition. Among the phenols tested, β-CD bound weakly the smallest ones, i.e. pyrocatechol, 4-methylcatechol and o-dihydroxyphenylacetic acid whereas the largest ones, i.e. chlorogenic acid and catechins which are also the most important natural substrates for apple PPO, were bound more strongly. Moreover, the values of (+)-catechin was more than 10 fold lower than that of (-)-epicatechin, meaning that β-CD was stereoselective between these two isomers of catechin. ΗΡ-β-CD exhibited a similar behaviour than β-CD since the KQ values were of the same order of magnitude. Owing to its higher water solubility and since in almost all cases the K D values were slightly lower, ΗΡ-β-CD could be considered as a better inhibitor of apple enzymatic browning than β-CD. α-CD was tested for smaller phenols because of its reduced inner cavity (ca 5 Â ) . However, compared to β-CD or ΗΡ-β-CD, its binding capacity of pyrocatechol and 4-methylcatechol was not improved. Therefore, α-CD cannot be substituted for β-CD (or ΗΡ-β-CD) for inhibition of PPO when the enzyme acted on phenolic substrates of small size. Previous workers have shown that α-CD was not an effective inhibitor of Granny Smith apple juice browning (25, 35). The K values were obtained at pH 4.5, which corresponds to the optimum pH of apple PPO activity (36). Since pH is an important factor of apple enzymatic browning both for the enzyme activity and for the inhibition by aromatic carboxylic acids and halide salts (18), we have examined the pH influence on the complex β-CD / phenol. For the phenols tested, i.e. hydrocaffeic, o-dihydroxyphenylacetic and chlorogenic acids, the K D values did not change between pH 3.5 and 5.5 (data not shown). Therefore, the inhibition properties of β-CD were not affected by pH in the D

D

D



300


Figure 1. Effect of β-CD on phenolic oxidation catalyzed by purified apple PPO. la. With chlorogenic acid as phenolic substrate lb. With (+)-catechin as phenolic substrate (X) Control without β-CD; (+) β-CD = 5 mM (la) or 2 mM (lb); (•) β-CD = 10 mM (la) or 4 mM (lb). Full, dashed and dotted lines corresponded to the calculated values using equations A and Β with Km = 5.6 mM, Vm = 260 nkat.mL" 1 and Kp = 2.2 mM for chlorogenic acid and Km = 7.4 mM, Vm = 140 nkat.mL and K = 0.19 mM for (+)-catechin. -1

D



23.

BILLAUD ET AL.

301 Effect of Cyclodextnns on Polyphenol Oxidation

Figure 1. Continued.


302


range of this study. These statements are in agreement with previous data showing that K D for chlorogenic acid was relatively insensitive to pH in the range 3.5 to 5.5 (35). Table I. Dissociation Constants (K ) of the Complex CD / Phenol for different CD and Phenolic Compounds determined either by Enzymatic Kinetic Analysis or by H NMR Spectroscopy in D,Q Kn[mM] Phenolic Compound ΗΡ-β-CD β-CD α-CD Ή NMR** Kinetic* Kinetic* Kinetic* 16.2 24 Pyrocatechol 27 36 4-Methylcatechol 27 17.5 17 8.9 Caffeic acid 7.7 0-Dihydrophenylacetic acid 11 16 Hydrocaffeic acid 5.8 7 5.6 Chlorogenic acid 2.2 5.2 1.5 (-)-Epicatecbin 2.7 4 1.4 (+)-Catecbin 0.19 0.20 0.22 * Determined kinetically by inhibition of PPO activity in air-saturated McDvaine buffer solution at pH 4.5 and 30 °C. ** Determined by Ή NMR spectroscopy (200 MHz) in D 0 at room temperature using chemical shift changes for H-3 and/or H-5 in β-CD. D


1

2

In order to confirm the Kp values found by enzymatic kinetic analysis, the complex β-CD / phenol was also analyzed by Ή NMR spectroscopy in D 0. For the calculations from the chemical shift changes involving the H-3 and/or H-5 protons of β-CD, two assumptions have been made. FirsUy, the β-CD / phenol complex kinetics are fast on the NMR time scale and secondly, the complex stoichiometry is 1 : 1. Previous workers have shown that these two assumptions were feasible at least for chlorogenic acid and catechins (33, 35). The Kp values obtained with β-CD are given in Table I. Although slight differences were observed between the values given by NMR spectroscopy and kinetic analysis, the two methods gave reasonably similar results. The discrepancies could be due to some differences in the medium used, D 0 in the first case and McDvaine buffer water solution at pH 4.5 in the second case. Moreover, our results are similar to those obtained by other workers, namely 1.9 mM for both chlorogenic acid and (-)-epicatechin (35) and 2.2 and 0.3 mM for (-)-epicatechin and (+)-catechin, respectively (33). 2

2

Effect of CD on PPO Activity in a Binary Mixture of Phenolic Compounds. It has been shown that in the presence of a two phenolic compound mixture, PPO acted independently on both phenolic substrates, S and S (37). Since the amount of oxygen consumed per mole of oxidized phenol is the same for S! and S , the total oxygen uptake is given by x

2

2

ν = {(Vni! [SJ / Km^ + (Vm [S ] / Km )} / {1 + ([SJ / Kn^) + ([S ] / Km )} (C) where Vnij, Km, and Vm , Km are the maximum velocity and the Michaelis constant of S j and S , respectively. 2

2

2

2

2

2

2


2

23.

Effect ofCyclodextnns on Polyphenol Oxidation

BILLAUDETAL.

303

The part of oxygen consumed for the S, oxidation is represented by {Vm, [S,] / Km!} / {1 + ([S,] / Km,) + ([S ] / Km )} and the other part, i.e. {Vm [S ] / Km ) / {1 + ([S,] / Km,) + ([S ] / Km )} 2

2

2

2

2

2

2

corresponds to the oxygen consumed for the S oxidation. Following the nature of the phenol S (i.e. the value of its kinetic constants), its addition to a substrate solution containing the other phenol S, could result in a decrease ("inhibition") or an increase ("activation") of the total oxygen uptake. The first case was apparent when both Vm « Vm, and Km « Km, as shown for the caffeic acid / 4-methylcatechol mixture with purified apple PPO by Janovitz-Klapp et al. (37). When β-CD was added to a binary mixture of phenols, this compound bound both phenols. Obviously, the amounts of free forms S, and S were dependent on the Κβ values of the respective complexes [CD.S,] and [CD.S ]. The concentrations [S,] and [S ], available for PPO and which are useable for equation C, can be calculated by solving the following set of 5 equations representing mass balances and equilibrium constants of the system : 2


2

2

2

2

2

2

[CD] = [CD] + [CD.S,] + [CD.S ] [SJt = [S,] + [CD.S,] [S ] = [S ] + [CD.S ] K o ^ t t S , ] [CD])/[CD.S,] K = ([S ] [CD])/[CD.S ] t

2

2

t

2

D 2

2

2

2

where [CD] , [S,] and [S ] represent the total amounts of the β-CD, thefirstand the second phenolic substrate, respectively. By eliminating [CD], [CD.S,] and [CD.S ], two equations are obtained t

t

2

t

2

[S,]3 {(K / K ,) - 1} + [S,]2 {(K / K ) ([CD] - [S,] ) + K - K -[CD] + 2 [S,] + [S ] } + [S,] [S,] {2 K , - K + [CD] - [S,] - [S ] } - K , [S,] 2 = 0 (D) D2

t

D

2

D2

t

t

D1

D

D 2

t

t

t

t

D 2

2

t

D1

t

D

t

and [S ] = [S ] / {(1 + (K , / K ) ([S,] - [S,]) / [S,]} 2

2

t

D

D2

(E)

t

The equation D can be solved with a computer and gives the [S,] value. This latter concentration can be used in the equation Ε to determine the [S ] value. With these two values of [S,] and [S ] in equation C, we are now able to predict the oxygen uptake in a two phenolic compound mixture in the presence of β-CD. Three types of binary mixtures have been studied, namely 4-methylcatechol / caffeic acid, chlorogenic acid / (-)-epicatechin and chlorogenic acid / (+)-catechin owing to the large differences in their kinetic constants (Km and Vm) with apple PPO and dissociation constants (K ) with β-CD (Table Π). 2

2

D



304


Table II. Kinetic and Dissociation Constants of the Phenolic Compounds used in the Two Phenolic Compound Mixtures Phenolic Vm* Km Ko compound [mM] [mM] (%) 4-Methylcatechol 100 17.5 4.8 Caffeic acid 4.8 0.15 7.7 Chlorogenic acid 93 2.2 5.6 (-)-Epicatechin 50 7.4 2.7 (+)-Catechin 54 7.4 0.19 * Values of Vm are given in % of the value obtained for 4-methylcatechol. All the results are expressed in residual activity, i.e. the ratio of PPO activity found in the presence of β-CD to the PPO activity without β-CD. An excellent correlation has been found between the experimental and the calculated velocities for the 3 binary mixtures studied (Figure 2). For the chlorogenic acid / (-)-epicatechin mixture (Figure 2a), the residual activity steadily increased with both concentrations of the two phenolics. Since both phenols exhibited close KQ values, addition of β-CD resulted in a similar decrease of the free amounts of the two substrates. Thus, the decreases in oxygen uptake for the S, and S oxidations are approximately the same and the effect of β-CD is similar to the one observed with one phenol. For the chlorogenic acid / (+)-catechin mixture (Figure 2b), the behaviour was different. For the low concentrations of chlorogenic acid, the residual activity first decreased and then increased as the (+)-catechin concentration increased. The catechin concentration corresponding to the minimum in residual activity decreased as the chlorogenic acid concentration increased. In this case, owing to the large difference in the Kj, values (Table II), β-CD bound preferentially (+)-catecbin. Therefore, for low chlorogenic acid concentrations and medium (+)-catechin concentrations, addition of β-CD resulted in a large decrease offreesubstrate (mainly (+)-catechin) leading to a decrease in the residual activity. For high chlorogenic acid concentrations, the influence of (+)-catechin concentration on PPO activity was almost nihil (37), thus in these solutions the effect of β-CD is comparable to its effect on pure chlorogenic acid solution. Lastly, another behaviour was observed for the 4-methylcatechol / caffeic acid mixture (Figure 2c). In the presence of caffeic acid, increasing concentrations of 4-methylcatechol between 0 and 5 mM resulted in a rapid increase of the residual activity followed by a plateau for the low caffeic acid concentrations (0.11 and 0.25 mM) or a slight decrease after 10 mM in 4-methylcatechol with 0.5 and 1 mM in caffeic acid. For these caffeic acid concentrations, the maximum residual activity was higher than 1, meaning that the β-CD addition resulted in an activation of the oxygen uptake. This surprising result can be explained by the large difference in the kinetic constants of the two phenols. Since both Vm and Km of caffeic acid are largely lower than that of 4-methylcatechol (Table II), its addition results in a large decrease of oxygen uptake as demonstrated by Janovitz-Klapp et al. (37). The addition of β-CD decreased both phenol concentrations. However, for the oxygen uptake, the decrease 2



23.

BILLAUD ET AL.


2

305

3

(-)-Epicatechin (mM)

Figure 2. Effect of β-CD on the oxidation of a two phenolic compound mixture catalyzed by purified apple PPO. 2a. Chlorogenic acid / (-)-Epicatechin with β-CD = 5 mM 2b. Chlorogenic acid / (+)-Catechin with β-CD = 5 mM 2c. 4-Methylcatechol / Caffeic acid with β-CD = 10 mM (•) chlorogenic acid 0 mM (2a and 2b), caffeic acid 0 mM (2c) ( · ) chlorogenic acid 0.5 mM (2a and 2b), caffeic acid 0.1 mM (2c) (X) chlorogenic acid 1 mM (2a and 2b), caffeic acid 0.25 mM (2c) (O) chlorogenic acid 2.5 mM (2a and 2b), caffeic acid 0.5 mM (2c) (•) chlorogenic acid 5 mM (2a and 2b), caffeic acid ImM (2c). The different lines corresponded to the calculated values using equations C, D and Ε with the Km, Vm and K values given in Table Π. Continued on next page D




306

Figure 2. Continued.


23.

BILLAUDETAL.



of free caffeic acid results in a greater activation than the inhibition due to the decrease of free 4-methylcatechol leading to an apparent activation of PPO. Effect of β-CD on Color Formation during PPO Oxidation in Binary Mixture of Phenols. Following the nature of the phenols involved in the oxidation, the resulting pigments varied widely in color intensity and hue (5). Thus, it has been established that the relative proportion among the different classes of phenolic substrate was probably the most important factor that contributed to the extent of enzymatic browning in apple varieties (40). Since β-CD binds the different phenols according to their Kp values, this compound is able to modify the balance among the different free phenolic substrates available for the PPO and therefore the resulting color of the oxidized solution. In this respect, the color variations of two phenolic compound mixtures have been compared during PPO oxidation with or without β-CD. The color variations were followed by tristimulus colorimetry for the 3 mixtures described in the preceeding paragraph. The amounts of free substrates in the presence of β-CD have also been calculated and the measurement of color variation of the resulting solution (equivalent solution) has been included in this study. The obtained results are given in Figure 3 for one example of each binary mixture. An excellent correlation was observed between the L*, a* and b* variations of the solutions with β-CD and the equivalent solutions. Moreover, the two latter solutions gave results which differed from the solution without β-CD. Concerning the chlorogenic acid / catechin mixtures (Figures 3a and 3b), the L* values decreased rapidly in the first 3 minutes, then increased until 10 minutes and slowly decreased between 10 and 30 minutes. During thefirstpart of the reaction, the dissolved oxygen was consumed (data not shown) and the enzymatic reaction probably stopped owing to the lack of oxygen. With β-CD, the inhibition (the residual activity was close to 0.64 in both cases) slowed down the enzymatic reaction which stopped later (after 5 minutes). Thus, darker solutions were obtained after 5 minutes (and later on) although the initial darkening rates were lower. Moreover for the chlorogenic acid / (+)-catechin mixture (Figure 3b), the hue was modified by the presence of β-CD since the a* values were higher and the b* values were lower corresponding to solutions less yellow and more red. The catecbins mainly gave yellow pigments whereas chlorogenic acid gave pink to red pigments (8). Since β-CD preferentially bound (+)-catechin (the ratio chlorogenic acid / (+)-catechin was equal to 7 without β-CD and more than 40 with β-CD), this explains that the color turned from yellow to red. However, a preferential binding of the catechin o-quinones cannot be ruled out since β-CD induced also a similar change,although less marked, in hue for the (-)-epicatechin / chlorogenic acid mixture. Concerning the 4-methylcatechol / caffeic acid mixture, addition of β-CD gave darker solutions after 15 minutes of oxidation with higher a* values and almost no effect on the b* values. The chosen conditions (3 mM in 4-methylcatechol and 1 mM in caffeic acid) corresponded to a slight activation (residual activity was equal to 1.06) of oxygen uptake which could explain the darkening of the solution. In conclusion of this study, CD were always inhibitors of PPO activity in a single phenolic solution. These compounds acted by complexation of the substrate. However, in multiphenolic solutions, the effects were variable. Thus, following the


307


0

10

20

30

10

20

30

Figure 3. Effect of β-CD on L*, a* and b* variations of a two phenolic compound mixture during the oxidation catalyzed by purified apple PPO. 3a. Chlorogenic acid (2 mM) / (-)-Epicatechin (2 mM) with β-CD = 4 mM 3b. Chlorogenic acid (3.5 mM) / (+)-Catechin (0.5 mM) with β-CD = 4 mM 3c. 4-Methylcatechol (3 mM) / Caffeic acid (1 mM) with β-CD = 4 mM Full lines were control experiments without β-CD. Dotted lines were experiments with β-CD = 4 mM. Dashed lines were experiments with equivalent solutions in which the concentrations of the phenolic compounds were: chlorogenic acid 1.02 mM and (-)-epicatechin 1.12 mM (3a); chlorogenic acid 1.87 mM and (+)-catechin 0.045 mM (3b); 4-methylcatechol 2.53 mM and caffeic acid 0.70 mM (3c) (equivalent solutions were without β-CD).

Tim© (min)

20

70

Time (min)

25

75

h 30

6

Li 80

b

35

40

10

14

85

90


20 Time (min)

10

30

Ο 2

00

ο

BILLAUDETAL.



23.


309



310


23.

BILLAUD ET AL.


311


phenolic compounds present in the solution, addition of β-CD can result in a large or a small decrease or a slight activation of the oxygen uptake catalyzed by PPO. Moreover, since the balance among the available phenolic substrates can be highly modified, C D can induce great changes in the color of the oxidized solutions. This could probably explain the effects observed by Crouzet et al. (26) who indicated that the inhibition of enzymatic browning by C D in apple juice was variety dependent. Literature cited 1. Whitaker, J.R. In Chemical Changes in Food during Processing; Richardson, T.; Finley, J.W., Eds.; AVI Publishing: Westport, C T , 1985, pp 121-176. 2. Zawistowski, J.; Biliaderis, C.G.; Eskin, N . A . M . In Oxidative Enzymes in Foods; Robinson, D.S.; Eskin, N . A . M . , Eds.; Elsevier: London, 1991, pp 217-273. 3. Macheix, J.J.; Fleuriet, Α.; Billot, J. Fruit Phenolics ; C R C Press: Boca Raton,FL, 1990, pp 1-378. 4. Rouet-Mayer, M.A.; Philippon, J.; Nicolas, J. In Encyclopaedia of Food Science, Food Technology and Nutrition; McRae, R.; Robinson, R.K.; Sadler, M.J., Eds.; Acad. Press: London, 1993, pp 499-510. 5. Nicolas, J.; Cheynier, V . ; Fleuriet, Α.; Rouet-Mayer, M . A . In Polyphenolic Phenomena; Scalbert, Α., Ed.; INRA Editions: Paris, 1993, pp 165-175. 6. Taylor, S.L.; Highley, N.A.; Bush, R.K. Adv. Food Res. 1986, 30, 1-76. 7. Sapers, G . M . ; Hicks, K.B. In Quality Factors of Fruits and Vegetables: Chemistry and Technology; Jen, J.J., Ed.; ACS Symp. Ser. No 405; American Chemical Society: Washington, D C , 1989, pp 29-43. 8. Nicolas, J.J.; Richard-Forget, F.C.; Goupy, P.M.; Amiot, M.J.; Aubert, S.Y. CRC Crit. Rev. Food Sci. Nutr. 1994, 34, 109-157. 9. Vamos-Vigyazo, L . CRC Crit. Rev. Food Sci. Nutr. 1981, 15, 49-127. 10. Zemel, G.P.; Sims, C.A.; Marshall, M.R.; Balaban, M . J. Food Sci. 1990, 55, 562563. 11. Sapers, G . M . ; Ziolkowski, M.A. J. Food Sci. 1987, 52, 1732-1733, 1747. 12. Sapers, G . M . ; El-Atawy, Y.S.; Garzarella, L . ; Hicks, K.B. J. Food Sci. 1989, 54, 1096-1097. 13. Sapers, G . M . ; Miller, R.L.; Douglas, W.F.; Hicks, K.B. J. Food Sci. 1991, 56, 419-422, 430. 14. Santerre, C.R.; Cash, J.N.; VanNorman, D.J. J. Food Sci. 1988, 53, 1713-1716, 1736. 15. Richard-Forget, F.C.; Goupy, P.M.; Nicolas J.J.; Lacombe, J.M.; Pavia, A . A . J.Agric Food Chem. 1991, 39, 841-847. 16. Richard-Forget, F.C.; Goupy, P.M.; Nicolas J.J. J.Agric Food Chem. 1992, 40, 2108-2113. 17. Rouet-Mayer, M.A.; Phillipon, J. Phytochemistry 1986, 25, 2717-2719. 18. Janovitz-Klapp, A . H . ; Richard, F.C.; Goupy, P.M.; Nicolas J.J. J. Agric. Food Chem. 1990, 38, 926-931. 19. Tong, C.B.S.; Hicks, K.B. J. Agric. Food Chem. 1991, 39, 1719-1722. 20. Chen, J.S.; Wei, C.I.; Marshall, M.R. J. Agric Food Chem. 1991, 39, 1897-1901. 21. McEvily, A.J.; Iyengar, R.; Otwell, W.S. CRC Crit. Rev. Food Sci. Nutr. 1992, 32, 253-273.



312


22. Sapers G . M . ; Hicks, K.B.; Phillips, J.G.; Garzarella, L . ; Pondish,D.L.; Matulaitis,R.M.; McCormack, T.J.; Sondey, S.M.; Seib, P.A.; El-Atawy, Y.S. J. Food Sci. 1989, 54, 997-1002, 1012. 23. Oszmianski, J.; Lee, C.Y. J. Agric Food Chem. 1990, 38, 1892-1895. 24. Taoukis, S.L.; Labuza, T.P.; Lillemo, J.H.; Lin, S.W. Lebensm. Wissen. Technol. 1990, 23, 52-54. 25. Hicks, K.B.; Sapers, G . M . ; Seib, P.A. U.S. Patent 1990, 4,975,293. 26. Crouzet, J.; Bastier, P.; Signoret, Α.; Mentink, L . In Food Ingredients Europe ; Conference Proceedings, Expoconsult Publishers: Maarsen, 1991, pp 293-295. 27. Szejtli, J. Pharm Technol. Int. 1991, 3 (2), 15-22. 28. Szejtli, J. Pharm Technol. Int. 1991, 3 (3), 16-24. 29. Demarco, P.V.; Thakkar, A . L . J. Chem Soc. Chem. Commun. 1970, 2-4. 30. Duchêne, D.; Wouessidjewe, D. J. Pharm Technol. Int. 1990, 2 (6), 21-29. 31. Szejtli, J. J. Incl. Phenom Mol. Recogn. Chem. 1992, 14, 25-36. 32. Yoshida, Α.; Yamamoto, M . ; Irie, T.; Hirayama, F.; Uekama, K. Chem. Pharm. Bull. 1989, 37, 1059-1063. 33. Cai, Y . ; Gaffney, S.H.; Lilley, T.H.; Magnolato, D.; Martin, R.; Spencer, C . M . ; Haslam, E . J. Chem. Soc. Perkin Trans. 1990, 2, 2197-2209. 34. Martin, R.; Cai, Y . ; Spencer, C.M.; Haslam, E . Bull. Liaison Groupe Polyphénols 1990, 15, 304-318. 35. Irwin, P.L.; Pfeffer, P.E.; Doner, L.W.; Sapers, G . M . ; Brewster, J.D.; Nagahashi, G.; Hicks, K.B. Carbohyd. Res. 1994, 256, 13-27. 36. Janovitz-Klapp, Α.; Richard, F.; Nicolas J. Phytochemistry 1989, 28, 2903-2907. 37. Janovitz-Klapp, A . H . ; Richard, F.C.; Goupy, P.M.; Nicolas J.J. J. Agric. Food Chem. 1990, 38, 1437-1441. 38. Leatherbarrow, R.J. In Enzfitter, A Non Linear Regression Data Analysis Program for the IBM PC; Elsevier: Amsterdam, 1987, pp 1-91. 39. Foster, R.; Fyfe, C.A. Trans. Faraday Soc. 1965, 61, 1626-1631. 40. Amiot, M.J.; Tacchini, M . ; Aubert, S.; Nicolas, J. J. Food Sci. 1992, 57, 958-962. RECEIVED May 26,

1995


Effect of Cyclodextrins on Polyphenol Oxidation Catalyzed by Apple

Recommend Documents