CHAPTER I

INTRODUCTION

1.1 Principles rationale and hypotheses

Browning of vegetables, fruits and flowers alter their appearances, flavors,

textures, and lower their marketing values. Appearance, which is significantly

impacted by color, is one of the first attributes used by consumers in evaluating goods

quality [1]. The browning can be caused by both enzymatic and non-enzymatic

biochemical reactions [2]. Peroxidase (POD) and polyphenol oxidase (PPO) are two

well known enzymes involved in the browning process [2, 3]. The catalytic reactions

of the oxidative enzymes, POD and PPO have been studied in fruits and vegetables

for many years. Both enzymes have some common substrates, but each also has its

specific substrates [4-7]. Their common diphenolic substrates lead to products with

brown colors. Moreover, both enzymes have some common inhibitors and some

specific inhibitors. There are several ways to prevent enzymatic browning such as

using heat treatment or adding reducing agents. However, this method does not work

well for fruits as heat alters their appearances, flavors, textures, and nutritional values.

Reducing agents are sometimes applied in food industry to processed fruits and

vegetables to reduce browning [2].

Inhibition of POD and PPO activities can stop the enzymatic browning

process. A binding of ligand and protein may result in activation or inhibition of the

enzyme [7, 8]. Molecular modeling is a computational technique to model or mimic

the behavior of molecule. In the field of Molecular docking is a frequently used

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method that can predict the binding orientation of small ligand candidates to protein

target in three dimensional view. Molecular docking programs are used to find novel

ligand through virtual screening of compound libraries. Therefore, the aim of

molecular modeling study is to first achieve an optimized conformation and relative

orientation for both the protein and ligand such that the free energy and/or potential

energy of the system are minimized to imitate the real interaction. Such information

may be used for activity prediction of new ligands. In this work, POD and PPO

sequences of Vitis vinifera, commonly known as grape, are used for construction of

models via computational method.

The hypothesis of this research is that POD and PPO share some common

features in the binding site. By examining how common substrates interact with both

enzymes, a design of new common inhibitors may be plausible leading to future

synthesis and applications in inhibition of browning reaction in fruit and vegetables.

1.2 Browning reaction

Browning of foods, fruits and vegetables is one of the main causes of the

quality loss. Browning usually impairs the sensory properties of products due to

associated changes in the color, flavor, and softening besides nutritional properties [9].

In some caseswww the brown flavor is highly desirable and is intimately associated in

our minds with delicious, high-grade product. In coffee, maple syrup, the brown crust

of bread and all baked goods, potato chips, roasted nuts, and in many other processed

foods controlled browning is necessary [10]. The browning reactions appear to be

complicated not only as to the final product but also as to the course of the numerous

reactions. Browning are results from both enzymatic and nonenzymatic oxidation of

phenolic compound [11]. It is difficult to ascertain whether the mechanism has been

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enzymatic or nonenzymatic unless the enzyme in the food that are responsible for the

enzymatic browning is inactivated, then only nonenzymatic reaction is said to occur.

In the past, Hodges (1953) [10] proposed three types of browning reactions

recognized to occur in foods during processing. (1) The reaction of aldehydes and

ketones, among them the reducing sugars, with amino compounds such as amino

acids, peptides, amd proteins. This is the dependent of the presence of oxygen. (2)

Caramelization, the change which occurs in polyhydroxycarbonyl compounds such as

reducing sugars and sugar acids when they are heated to high temperature which is

also independent of oxygen. (3) The oxidative change of polyphenils to di- or

polycarbonyl compounds and possibly the oxidation of ascorbic acid. This may be

partially or wholly enzymatic. Today most evidence suggests that oxidation of phenol

or polyphenols by enzymes is the principal reaction in enzymatic browning.

Browning is a problem of general interest in food technology. The large

number of studies now being conducted both on model systems and on food products

making it possible that these reactions will soon be better understood.

1.2.1 Enzymatic browning reaction [2, 12, 13]

Enzymatic browning is a significant problem in a number of important

commodities, specially fruits such as apricots, apples, pears, peaches, banana and

grape; vegetables such as potatoes, mushrooms and lettuce. The discoloration limits

the shelf life of many minimally processed foods and also is a problem in the

production of dehydration and frozen fruits and vegetables. Enzymatic browning is

not always a defect but can contribute to the desirable color and flavor of such

product as raisins, prunes, coffee, tea and cocoa.

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Enzymatic browning is a chemical process which occurs in fruits, vegetables

and also seafood by the polyphenol oxidase and peroxidase, which result in brown

pigment. Enzymatic browning occurs in many tissues whenever they are injured. The

injury can be the result of bruising, cutting, freezing or disease. That part of the

injured fruit or vegetables which is exposed to air undergoes a rapid darkening. In

chemistry, there is a class of phenolic compounds that are present in fruits and

vegetables, known as polyphenols that forms the substrate for this browning enzyme.

Enzymatic browning involving the enzyme PPO and POD is the principal

cause of fruit quality losses during postharvest and processing. The catalytic reactions

of the oxidative enzymes, POD and PPO have been studied in fruits and vegetables

for many years. Both enzymes have some common substrates, but each also has its

specific substrates [4-7]. Their common diphenolic substrates lead to products with

brown colors. Moreover, both enzymes have some common inhibitors and some

specific inhibitors. Inhibition of POD and PPO activities can reduce the browning

process. A binding of ligand and protein may result in the activation or the inhibition

of the enzyme [7, 14].

1.2.2 Nonenzymatic browning reaction

Nonenzymatic browning are maillard reaction and calameilization. The

maillard reaction is a chemical reaction between amino acid and reducing sugar. The

carbon molecules contained in the sugars, or carbohydrates, combine with the amino

acids of the proteins. This combination cannot occur without the additional heat

source. The maillard reaction is named for Louis-Camille Maillard, a French chemist

who studied the science of browning during the early 1900s. The caramelization is

one of the most important types of browning processes in foods. Carmelization leads

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desirable color and flavor in bakery's goods, coffee, beverages, beer and peanuts.

Caramelization occurs during dry heating and roasting of foods with a high

concentration of carbohydrates (sugars). The last series of reactions include both

fragmentation reactions (flavor production) and polymerization reactions (color

production) [15].

Nonenzymatic browning reaction in fruits and vegetables depends on a

number of factors such as (1) product composition, (2) moisture content of the

product, and (3) storage temperature and exposure to oxygen. The compositional

factors include maillard precursors or ascorbic acid [12].

1.2.3 Prevention of enzymatic browning

Browning phenomena are caused after mechanical operations during

processing, enzymes, which are liberated from the tissue, come in contact with

phenolic compounds. The normal approach to inhibit both enzymatic and non-

enzymatic oxidative browning in food has been the application of sulfites. However,

health concerns have limited their application [2]. The proposed mechanisms of anti-

browning additives that inhibit enzymatic browning are (1) direct inhibition of the

enzyme; (2) interaction with intermediates in the browning process to prevent the

reaction leading to the formation of brown pigments; or (3) to act as reducing agents

promoting the reverse reaction of the quinone back to the original phenols [16, 17].

Kojic acid, a �-pyrone derivative, occurs in many fermented Japanese foods,

showed the highest inhibitory activity on apple slide, mushroom, potato, white shrimp

and spiny lobster [18-21]. Kojic acid exhibited a competitive inhibition for the

oxidation of chlorogenic acid and catechol by potato and apple PPO. This compound

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showed a mixed-type inhibition for white shrimp, grass prawn, and lobster PPO when

DL-beta-3,4-dihydroxyphenylalanine and catechol were used as substrates. A mixture

of ascorbic acid and kojic acid has been patented for use as an anti-browning agent in

foods [11].

The mechanism of inhibition differs widely for each of the categories of anti-

browning agents. The combination of different agents may prevent browning better

than a specific chemical alone at the end of the 24 hour storage period [18].

Other acid treatments such as dipping in citric acid or hydrochloric acid

solution (1%) could be a commercial pretreatment for browning control and quality

maintenance of frozen litchi fruit [22]. Although all the fruits contain polyphenolic

compounds, some fruits as peaches, apricots, plums, prunes, cherries, bananas, apples,

and pears show a greater tendency to develop browning very quickly during

processing. Also, some frozen fruits like apples and cherimoya are pretreated by

dipping its slices in sodium chloride solutions (0.1–0.5%) in combination with

ascorbic or citric acid, in order to remove intracellular air and reduce oxidative

reactions [23].

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OH

R

R'

COOH

COOH

OH

R

OH

R

CH=COOH

R=R’=H, p-hydroxybenzoic acid R=OH, R’=H, protocatechuic acid R=OCH3, R’=H, vanillic acid R=R’=OCH3, syringic

R=H, salicylic acid R=OH, gentisic acid

R=H, P-coumaric acid R=OH, caffeic acid R=OCH3, ferulic acid

OOH

OH

OH

R

R'

OH

O

O+HO

OH

OH

R'

OH

R

R=R’=H, kaempferol R=OH, R’=H, quercetin R=R’=OH, myricetin

R=OH, R’=H, cyaniding R=OCH3, R’=H, peonodin R=R’=OH, delphinidin R=OCH3, R’=OH, petunidin R=R’=OCH3, malvidin

OHO

OH

OH

R'

OH

R

OHO

OH

OR

OH

OH

OR=OH, R’=H, catechin, epicatechin R=R’=OH, gallocatechin

R=OH, R’=H, catechin, epicatechin R=R’=OH, gallocatechin

Figure 1.1 Families of phenolic compounds commonly found in fruits and vegetable [9]

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1.3. Enzyme target in browning inhibition process

1.3.1 Polyphenol Oxidase (PPO)

Polyphenol oxidase is a dicopper-containing enzyme, also known as

polyphenolase, catechol oxidase, catecholase in respect to substrate. PPO is present in

some bacteria and fungi, in most plants, some arthropods and all mammals. In all

cases, the enzyme is associated with dark pigmentation in the organism, and seems to

have a protective function [9].They are found in plastids and chloroplasts. They also

occur freely in cytoplasm assisting in senescence and browning of fruits and

vegetable. In some plants these enzymes can either exist in latent (LPPO) or active

soluble form (SPPO) [4].

This enzymatic reaction consists of the oxidation of phenolic substrates as

oxygen is required. Several studies have reported the involvement of polyphenol

oxidase in the oxidation of the polyphenols from plants. PPO activity can be

monitored by oxygen consumption or spectrophotometrically using a variety of

substrates such as pyrogallol, pryocatechol, 4-methylcatechol, 3,4-

dihydroxyphenylacetic acid, 4-tert-butylcatechol and chlorogenic acid [6]. In apples

the phenolic substrates are present in the flesh and peel. The substrate are chlorogenic

acid, caffeic acid, 3,4-dihydroxyphenylalanine (DOPA), p-coumaric acid, flavonol

glycosides, 4-methyl catechol, catechin, 3,4-dihydroxy benzoic acid, p-cresol and

leucocyanidin [9, 11, 18, 24, 25]. The latent polyphenol oxidase from sago log shows

high activity with diphenols such as epicatechin and catechin [4]. The resulting

quinones formed are further polymerized to produce a brown pigment. The general

schematic reaction catalyzed by PPO is as show in Figure 1.1.

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Figure 1.2 The browning reaction of polyphenol oxidase activity. (Figure adapted from Saper [2])

The monophenolic compounds are hydrolyzed to o-diphenols and the latter are

oxidized to o-quinones which can cyclize, undergo further oxidation, and polymerize

to form brown pigments or react with amino acids and proteins that enhance the

brown color produced [26].

The optimal pH for PPO activity is between 5 and 7. The enzyme is relatively

heat labile and can be inhibited by acids, halides, phenolic acids, sulfites, chelating

agents, reducing agents such as ascorbic acid, and quinone couplers such as cysteine

and various substrate-binding compounds. However, blanching cannot be used for

certain products with delicate flavors. Inactivation of PPO can be the mean to control

browning in some fruits and vegetables by blanching. Several inhibitors of PPO have

been used, mainly benzoic acids and their derivatives. Diamine derivatives of

coumarin and 4-hexylresorcinol are effective inhibitors of black spot formation in

shrimp and also inhibit mushroom PPO but not good inhibitors of grape PPO [9].

Studies with mushroom PPO have revealed that ascorbate, bisulfites and thiol

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compounds have a direct inactivating effect on PPO, in addition to their ability to

reduec benzoquinones to o-dihydroxyphenols. The reducing compound sulfite is used

by the industry by placing fruit slices in controlled-atmosphere chambers with

burning sulfur, which reacts with oxygen to produce bisulfite. There is increasing

concern regarding allergic reactions to sulfites in certain individuals, and therefore the

residual concentrations of sulfites have been regulated for different commodities.

Any approach for controlling PPO activity needs to be based on experimental

findings. X-ray crystallography and site-directed mutagenesis may help identify the

complex interactions essential at the active site [27]. Site-directed mutagenesis of

histidine residues 62 and 189 has shown these residues to be important in Cu binding

[28]. Research on the bio- chemical processes that occur on wounding is important to

establish the function of PPO in vivo [29]. Research on genetic engineering methods

such as antisense RNA and gene silencing will help increase our understanding of the

functions of PPO and how to control them to improve crop quality. There are two

conserved amino acid sequence regions in all published PPO sequences. Most of the

histidines are present in these regions (with five conserved histidines in the two

regions of all PPO sequences determined). The two regions seem to correspond to the

active site of the enzyme and show good correlation with the accepted enzymatic

mechanism and previous physicochemical data [9]

1.3.2 Peroxidase (POD)

POD, is a group of enzymes that catalyze oxido-reductions reaction. POD can

be found in plants, animals and microbes. It is one of the most thermostable enzymes

responsible for performing single electron oxidation on a wide variety of compounds,

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in the presence of hydrogen peroxide. They have been classified on the basis of

sequence homology into three types [30]. Class I heme peroxidases contain the

prokaryotic enzymes, including cytochrome c peroxidase (CcP), ascorbate peroxidase

(APX), and the gene-duplicated bacterial catalase-peroxidases. Class II contains the

fungal peroxidase enzymes, including manganese peroxidase and lignin peroxidase.

Class III contains the classical secretory peroxidases, the most notable example being

horseradish peroxidase (HRP).

They reduce H2O2 to water while oxidizing a variety of substrates. The

catalytic process of peroxidase occurs in a multistep reaction. First, the reaction of the

active site with hydrogen peroxide is reduced to form water, followed by the

oxidation of protein by two electrons. The latter state of the protein is called

‘Compound I’ and contains an oxyferryl (Fe (IV)=O) center and organic cation

radicals, these can be located either on the heme or on protein residues, (depending on

the isoenzyme). Next, Compound I oxidizes one substrate(S) molecule to give a

substrate radical (S�) and forms Compound II, when the organic cation radical is

reduced to its resting state. Finally, Compound II is reduced by second substrate

molecule to the resting iron (III) state [5]. This is depicted in following scheme 1.1

[31]:

Heme [Fe(III)] + H2O2 Heme [O=Fe(IV)-R+�] + H2O

(Compound I)

Heme [O=Fe(IV)-R+�] + S Heme [O=Fe (IV)] + S�

(Compound I) (Compound II)

Heme [O=Fe(IV)] + S Heme [Fe(III)] + S�

(Compound II)

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A simplified equation of this chemical reaction is as follows:

POD

H2O2 + 2AH2 2H2O + 2AH�

Which AH2 and AH� represent a reducing substrate and its radical product,

respectively.

The structure of peroxidase is largely �-helices, linked by loop and turns,

while there is also a small region of �-structures [32]. Substrate oxidation on

horseradish peroxidase (HRP) occurs at the exposed heme edge. There are

hydrophobic interactions with amino acid site-chains [33].

1.4 Objectives of this research

1.4.1 To build three dimensional models of peroxidase and polyphenol oxidase

1.4.2 To study common substrate binding with both enzymes by docking method

1.4.3 To propose potential common inhibitors for both enzymes