effect of frequency on polyphenoloxidase activity · ohmic heating is an emerging technology which...

EFFECT OF FREQUENCY ON POLYPHENOLOXIDASE ACTIVITY

DURING MODERATE ELECTRIC FIELD TREATMENT

A Thesis

Presented in Partial Fulfillment of the Requirements for

the Degree Master of Science in the

Graduate School of The Ohio State University

By

Jerry James M. de la Torre

*****

The Ohio State University

2009

Master's Examination Committee: Approved By:

Sudhir K. Sastry, Ph.D., Adviser __________________________________

V.M. Balasubramaniam, Ph.D.

Adviser,

Graduate Program in Food, Agricultural

and Biological Engineering

ii

ABSTRACT

Polyphenoloxidase (PPO, EC 1.14.18.1) is one of the major enzymes in fruits and

vegetables that causes undesirable browning when it reacts with phenolic substrates in the

presence of oxygen to yield dopaquinone and eventually form melanin pigments. In this

study, a purified PPO from mushroom was subjected to a constant electric field strength

(10 V/cm) at different frequencies (60, 600 and 6000 Hz) at three isothermal conditions

(40, 50, 60°C) for 5, 10 and 15 min. To isolate the effect of frequency, samples were also

heated conventionally at the same temperature-time history. Enzyme activity was

measured using spectrophotometric method and compared with that of untreated samples.

Results showed that moderate electric field treatments (MEF) stimulated higher enzyme

activity (p<0.05) compared to conventional heating at 60 and 6000 Hz 40°C 10 min,

6000 Hz 40°C 15 min and at all frequencies at 60°C 15 min. Reduced activity (p<0.05)

was observed at all frequencies but at different conditions in the first 10 min of 60°C

treatments: 60 Hz 5 min as well as 600 and 6000 Hz both at 10 min. The data suggests

that MEF activation is likely to occur at higher frequency (6000 Hz) and at longer

holding periods (15 min). Both the activation and inactivation results can be useful in

medical and food processing applications. Further studies on the isolated effect of

frequency treatments on specific enzyme isoforms and oxidized states may clarify the

response mechanism of PPO to electric field stimulation.

iii

ACKNOWLEDGMENT

The author extends his profound gratitude to those who have helped him in this

research:

Dr. Sudhir K. Sastry, academic and research adviser, for his patient guidance and

brilliant insights not only in this study but throughout the researcher’s academic life. It

was such a priceless experience to learn under such a well-acclaimed, topnotch scientist;

Dr. V.M. Balasubramaniam, for sharing his expertise in food engineering as

member of the Thesis Evaluation Committee;

Dr. Suzanne Kulshrestha, for her enlightening inputs on some biochemical aspects

of the study. She has been very helpful also in reshaping this thesis in this printed form;

Brian Heskitt, for valuable suggestions and technical support in setting up the

experiments;

Fulbright, International Institute of Education (IIE), Ohio State University

Department of Food, Agricultural and Biological Engineering (OSU-FABE) and Bureau

of Postharvest Research and Extension (BPRE) for fund and other forms of assistance;

Josephine M. de la Torre, for being a very supportive and understanding wife;

Rolando S. Asisten, Jr. and his family for their extraordinary forms of support;

and,

Family, relatives and friends for their encouragement.

iv

VITA

July 16, 1975 ……………………………………. Born- Vinzons, Camarines Norte,

Philippines

1992 to 1997 ……………………………………. B.S.Agricultural Engineering,

Camarines Sur State Agricultural

College, Camarines Sur,

Philippines

1999 to 2002 ……………………………………. Science Research Specialist I,

Bureau of Postharvest Research

and Extension, Nueva Ecija,

Philippines

2002 to

present

……………………………………. Science Research Specialist II,

Bureau of Postharvest Research

and Extension, Nueva Ecija,

Philippines

FIELD OF STUDY

Major Field: Food, Agricultural and Biological Engineering

Specialization: Food Engineering

v

TABLE OF CONTENTS

Page

Abstract…………………………………………………………………………………. ii

Acknowledgment………………………………………………………………………. iii

Vita……………………………………………………………………………………... iv

List of Tables…………………………………………………………………………... vii

List of Figures………………………………………………………………………….. ix

Chapters

1. Introduction………………………………………………………………... 1

2. Review of Literature……………………………………………………….. 4

2.1 Nomenclature and Structure of PPO………………………………. 4

2.2 Reaction Mechanism………………………………………………. 8

2.3 Enzyme Activity Assay……………………………………………. 9

2.4 Role of PPO and Melanin…………………………………............. 9

2.5 Substrate Specificity………………………………………………. 10

2.6 Inhibitor Sensitivity……………………………………………….. 11

2.7 pH Dependence……………………………………………………. 13

2.8 Thermal Resistance………………………………………………... 13

2.9 Moderate Electric Field Treatment..………………………………. 15

2.10 Ohmic Heating Effects on PPO and Electromagnetic Field

Treatments of Other Enzymes……………….....…………………..

16

2.11 Effect of Frequency on Other Biological Materials.......................... 17

3. Materials and Methods…………………………………………………….. 19

3.1 Experimental Design………………………………………………. 19

3.2 Experimental Set Up………………………………………………. 21

3.3 Enzyme and Reagents……………………………………………... 22

3.4 Treatments…………………………………………………………. 23

3.5 Enzyme Assay……………………………………………………... 25

3.6 Data Analysis……………………………………………………… 28

4. Results and Discussion…………………………………………………….. 29

4.1 Effect of Frequency on PPO Activity...…………………………… 29

4.2 Enzyme Activity and Variability Factors………………………….. 38

vi

5. Conclusions………………………………………………………………... 43

Appendices……………………………………………………………………………. 44

A List of PPO Names…………………………………………………... 44

B ANOVA for Frequency Effect………………………………………. 46

C Error Analysis for Enzyme Activity………………………………… 53

Bibliography…………………………………………………………………………... 56

vii

LIST OF TABLES

Table Page

1 Oxidized states of PPO from Streptomyces glaucescens………... 6

2 Optimum pH for PPO activity…………………………………………. 13

3 Optimum activity temperature of selected PPO……………………….. 14

4 Reagents used in the experiment………………………………………. 23

5 Volume of reagents in PPO activity assay…………………………….. 27

6 Enzyme activity ratio at 40°C. Error values are ±2 standard deviation.. 32



9 Analysis of variance for frequency effect on enzyme activity

(p =0.05)………………………………………………………………..

47

10 Multiple comparison of treatments for the frequency effect on enzyme

activity at 40°C, 5min using Tukey HSD test………………………….

48


activity at 40°C, 10min using Tukey HSD test…………………………

48


activity at 40°C, 15min using Tamhane Test…………………………..

49



49



50



50

viii



51



51



52

ix

LIST OF FIGURES

Figure Page

1 Experimental design…………………………………………………… 20

2 Block diagram of the experimental set up…………………………….. 21

3 Thermal history of PPO enzyme solution during conventional and

ohmic heating (60, 600 and 6000 Hz)……………………….…….

24

4 Enzyme activity of untreated PPO. Error bars are ±2 standard

deviation ……………………………………………………………….

25

5 Enzyme activity ratio across frequency settings at different holding

times and constant temperature (40°C). Error bars are ±2 standard

deviation…………………………………..……………………………

31



deviation.……………………………………………………….……...

33



deviation…………………….………………………………………….

35

8 Time plots of enzyme activity ratio at different holding temperatures... 37

1

CHAPTER 1

INTRODUCTION

Enzyme activity is one of the major factors affecting organoleptic properties of

fruits and vegetables. Polyphenoloxidase (PPO) for instance, causes browning of plant

tissues when they are cut or bruised and exposed to oxygen (Espin et al., 1995). Such

discoloration, as in the case of banana, apple and other fruits, is usually a sign of loss of

quality and economic value (Billaud et al., 2003; Castro et al., 2004; Dincer et al., 2002;

Lee, 2007; Riener et al., 2008). Sometimes, browning is favorable, especially for

products like coffee, cacao, raisins and tea (Castro et al., 2004; Tepper, 2005). The

International Union of Biochemistry and Molecular Biology (IUBMB) has placed the

PPO nomenclature under enzyme classification (EC) 1.14.18.1.

Enzymatic reactions can proceed in tissues of plants and animals even after

harvest and slaughter, respectively. It has also been observed that enzymes can still

function at refrigerated conditions (Richardson & Hyslop, 1985). To preserve product

quality, it is generally beneficial to inactivate enzymes as soon as possible to prevent

deterioration from enzyme-catalyzed biochemical reactions.

There are several targets to aim at in controlling enzymatic browning in fruits and

vegetables. The common approach is to inactivate the PPO as a whole through heat

treatment and application of acidulants (Richardson & Hyslop, 1985). Sometimes,

2

enzymes have a useful function. In such cases, it may be desirable to activate them.

Conventional heating is limited by heat penetration considerations especially for thick or

large products. Thermal treatments are also known for degradative effects on sensory

qualities (Lee, 2007).

Chemical inhibitors may also mean added cost. PPO has varying sensitivity to a

host of chemical inhibitors, depending on enzyme origin (Billaud et al., 2003;

Chaisakdanugull et al., 2007; Galeazzi & Sgarbieri, 1981). The treatment therefore

requires some degree of specificity and dosage application. Allergy has also been raised

in some instances as an undesirable side effect of inhibitors like sodium metabisulfite

(Chaisakdanugull et al., 2007; Lee, 2007).

Ohmic heating is an emerging technology which may offer potential

improvements in the traditional PPO inactivation. By passing current through a food

material, which usually contains conductive salts, heating is rapidly produced uniformly

from internal tissues (Castro et al., 2004). During ohmic heating, some biological effects

have been observed as in the case of electroporation and diffusion of biochemical

substances at the cellular level (Loghavi et al., 2007; Wang & Sastry, 1993). The effects

of the electric field have been separately described as moderate electric field (MEF)

treatments.

A few studies have been conducted on the effect of moderate electric field

treatments on food and other enzymes but the role of frequency on the inactivation

kinetics has not been fully clarified. Ohmic heating has been found to reduce inactivation

time of some enzymes (Castro et al., 2004) and so it is interesting to see if frequency

contributes some synergistic activation or inactivation effect. PPO may respond to some

3

frequency stimulation due to the metallic property of binuclear copper in its active site.

Thus, the objective of this study was to determine the effect of MEF frequency on

polyphenoloxidase activity.

By exploring some frequency settings, the results could lead to the determination

of resonance levels at which PPO activity may be altered. This information may be useful

in other MEF treatments of structurally similar biological materials.

4

CHAPTER 2

REVIEW OF LITERATURE

2.1 Nomenclature and Structure of PPO

Polyphenoloxidase (PPO) is a binuclear copper-containing enzyme which is

endogenous in bacteria, fungi, plants, animals and humans (Richardson & Hyslop, 1985;

Seo et al., 2003; Tepper, 2005). PPO (EC 1.14.18.1) belongs to the large family of

oxidoreductases and distinguishes itself by acting on a donor compound and

incorporation of one oxygen atom in its two active sites. It also carries out the reaction of

catecholoxidase (EC 1.10.3.1) if 1,2-benzenediols are available as substrate (IUBMB,

2008).

Officially, its accepted and systematic names are monophenol monooxygenase

and monophenol, L-dopa:oxygen oxidoreductase, respectively. However, PPO is also

known by 25 other names (Appendix A) including cresolase, tyrosinase, diphenoloxidase

and catecholase (IUBMB, 2008). The nomenclature appears to be a confusing list but

several characterization studies may offer clarifying identification, as would be seen in

the next section.

Structurally, PPO may also be identified with the metalloproteins group. Copper

proteins perform four basic functions (Tepper, 2005) such as metal ion processing

(storage, transport and uptake), electron transfer, dioxygen processing and catalysis.

5

The presence of copper provides distinct spectroscopic properties which pave the

way for classifying these metalloproteins in seven types (Tepper, 2005). Aside from the

obvious difference in physical configurations among them, there are also variations in

reaction mechanism and functions.

PPO is a Type 3 copper protein. Its binuclear copper ions are generally regarded

as both active sites for binding with substrates and oxygen. It is therefore involved in

both oxygen transport and activation catalysis. Each copper site is designated as Cu(A)

and Cu(B), respectively. In oxidized form, PPO has generally no electron paramagnetic

resonance (EPR) signal due to antiferromagnetic coupling between Cu(II) ions (Tepper,

2005; Tepper et al., 2002). This Type 3 metalloprotein has been found to be structurally

homologous with two other copper proteins: hemocyanin and catecholase (Klabunde et

al., 1998; Tepper et al., 2002). As such, any subsequent finding from one of them is

usually used for modeling and comparative studies with the other two. Currently, it

appears that PPO has the least known properties.

Inspite of structural similarity among these copper proteins, they differ by their

functions. Hemocyanin is involved in the oxygen transport and storage (as in arthropods

and molluscs) but is not capable of catalysis. Catecholase lacks hydroxylase activity but

can oxidize o-diphenols to o-quinones. PPO shows both hydroxylation and oxidation

capabilities of phenolic compounds that yield products for pigmentation (Klabunde et al.,

1998; Tepper et al., 2002).

PPO also varies according to source (species and cultivars) and maturity. Its

distribution also differs with parts of fruits and vegetables (Duangmal & Owusu Apenten,

1999). From the same sample, PPO can still exist in multiple forms called isozymes.

6

These isozymes can exist in several forms of aggregation, ranging from a monomer up to

octamer or even higher. The tetramer however, is the predominant form. (Jolley et al.,

1969).

In other words, a careful analysis is required before generalizations can be made

about the properties of PPO based from previous findings on some samples.

PPO

Derivative

Symbol EPRa Detectable Cu-Cu Distance, Ǻ

Tymet [Cu(II)-OH--Cu(II)] No 2.9

Tyoxy [Cu(II)O22-

-Cu(II)] No 3.6

Tyred [Cu(I) Cu(I)] No 4.4

Tyh-met [Cu(II) Cu(I)] Yes - a electron paramagnetic resonance

Table 1. Oxidized states of PPO from Streptomyces glaucescens.

PPO can also exist in four oxidized states (Table 1) which affect its reaction

mechanism and spectrophotometric properties (Alijanianzadeh & Saboury, 2007; Tepper,

2005; Tepper et al., 2002). The met derivative (Tymet) is the resting form (85-90%) of

PPO at atmospheric pressure, room temperature, neutral pH and absence of substrate.

Each of Cu(II) ions is bound to 3 His residues and Cu2 atom, which is often described as

trigonal pyramidal with one His ligand at the apex.

The oxidized states can be produced by reacting one form with some chemicals.

The half-met (Tyh-met) can be generated by incubating the deoxy form with nitric oxide.

Tyoxy results from the addition of peroxide or two-electron reduction of Tymet to [Cu(I)

Cu(I)] deoxy form. Oxygen binds reversibly as peroxide to give a +2 charge to each

7

copper (Tepper, 2005).

At a resting state, the distance between the copper centers is shorter (2.9 Ǻ for

sweet potato) than the other oxidized states (Tyred = 4.5 Ǻ; Tyoxy = 3.6 Ǻ). The optimal

distance was estimated at 2.9 to 3.2 Ǻ for enzyme activity. The farther apart the copper

centers are, the easier the inactivation becomes.

Spectrophotometric signals may be detectable or not depending on the oxidized

state. Both Tymet and Tyred have no ultraviolet/visible light (UV/VIS) and electron

paramagnetic response (EPR) signal. Tyh-met is EPR active. Tyoxy has no EPR signal but it

is UV/VIS active.

EPR, also known as electron spin resonance (ESR) or electron magnetic

resonance (EMR), is a spectroscopic technique for observing molecular species with

unpaired electrons. An unpaired electron is known for a sensitive spin magnetic moment.

When a magnetic field is applied, such electron can be oriented relative to the field

direction creating a distinct state where microwave energy can be absorbed. Resonance

exists when the magnetic field and microwave frequency match with each other (IERC,

2008).

From Raman Spectroscopy, it has been observed that Tyoxy has a very low O-O

stretching frequency at 750 cm-1

. Using paramagnetic 1H nuclear magnetic resonance

(NMR), Tymet responds at 600 MHz (Tepper et al., 2002).

PPO has a molecular weight ranging from 30,000 kDa (Streptomyces antibioticus)

to 128,000 kDa (mushroom) because it can exist in multiple forms (Jolley et al., 1969;

Seo et al., 2003; Tepper, 2005).

8

2.2 Reaction Mechanism

Melanogenesis or enzymatic browning results from three processes

(Alijanianzadeh & Saboury, 2007; Chang, 2007; Concellon, 2004; Espin et al., 1995;

Richardson & Hyslop, 1985; Seo, 2003; Severini, 2003; Tepper, 2005; Tepper et al.,

2002; Weaver, 2004; Xue et al., 2008). These are 1) hydroxylation of monophenols, such

as tyrosine, 2) oxidation of o-quinones, like 3-4-dihydroxyphenylalanine or L-dopa, and

3) polymerization of melanins from the previous oxidation products.

Both hydroxylation and oxidation are catalyzed by PPO or tyrosinase. The third

stage proceeds spontaneously without the aid of enzymes.

Hydroxylation is also called cresolase or monophenolase activity. The next stage,

oxidation, is similarly referred to as catecholase or diphenolase activity. Some authors

prefer to use PPO activity only at the second stage because hydroxylation is technically

confined to monophenols. For a more specific distinction on the process, enzyme and

substrate, this paper will also adopt the same convention. From this point on, PPO will be

viewed from this context; that is to say, PPO activity involves both cresolase (or

monophenolase) and catecholase (or diphenolase) activities.

Melanins, though widely regarded as brown pigments, can actually range from

yellow to black. These are heterogeneous polyphenolic polymers that can be found from

microorganisms, plants, animals and humans (Tepper, 2005).

Enzymatic discoloration can be stopped (Severini et al., 2003) by any of the

following: 1) heat inactivation, 2) exclusion of reacting components, 3) removal or

transformation of substrates (oxygen and phenols), 4) reduction of pH, 5) chelation of

copper with citric acid and similar agents, 6) addition of antioxidants (ascorbic acid,

9

sodium or potassium bisulphate which inhibit PPO or prevent melanin formation), 7)

enzymatic treatments with proteases to hydrolyze PPO.

2.3 Enzyme Activity Assay

The enzyme activity can be measured from the consumption of substrates or the

generation of products.

The product formation can be measured directly (spectrophotometric method) or

indirectly (colorimetric method). Spectrophotometric assay measures the discoloration in

progress due to enzyme-catalyzed pigmentation, which turns a clear mix of reagents into

brownish solution in about 5 minutes of reaction. The change in absorbance is directly

related to the browning reaction (Castro et al., 2004; Weemaes et al., 1997). Indirectly,

PPO activity as manifested in the browning of plant tissues can be measured from the

reflectance of solid samples using a colorimeter (Billaud et al., 2003).

The substrate consumption, like oxygen uptake, can also indicate PPO activity. To

measure this oxygen reaction, the polarographic technique can be used (Billaud et al.,

2003; Weemaes et al., 1997).

In almost all related studies, the spectrophotometric assay is commonly used.

2.4 Role of PPO and Melanin

PPO is the key enzyme in melanin formation which serves several functions in

microorganisms, plants, animals and humans. Hence, it has wide ranging significance in

agriculture, food, health and industrial sectors.

In fungi, melanin production plays a role in the differentiation of reproductive

10

organs, spore formation, virulence and tissue protection (Seo et al 2003).

Among insects, scleoritization involves PPO activity to form a hardened cuticle

which can prevent dehydration and death from injury. For instance, failure of

melanogenesis in Drosophila is lethal (Tepper, 2005).

In plants, browning affects the organoleptic properties which are usually

undesirable (banana, apple, eggplant, etc.) and sometimes favorable, as in the case of

raisin and tea (Seo et al., 2003). This enzymatic darkening of tissues was also observed to

be important in plant defense. During lesion, pigmentation seals off wounds to confine

infection by limiting the spread of pathogens. Quinones, which are products of PPO

activity, also inactivate enzymes produced by pathogens. In banana, PPO and dopamine

was correlated to the resistance against the parasitic nematode Radopholus similis (Wuyts

et al., 2006).

PPO and melanin offer protection to humans against photocarcinogenesis (Seo et

al 2003).

Likewise, PPO is useful in many other processes (Seo et al 2003), such as

biosensing of phenols and catechols, wastewater bioconversion of phenols, antioxidant

synthesis, vitiligo marking, and vector in prodrug therapy.

2.5 Substrate Specificity

Although PPO generally reacts with phenols and oxygen, it exhibits some degree

of relative substrate specificity depending on enzyme source and other factors. This

means for an array of substrates, the same enzyme may exhibit varying degree of activity.

Likewise, for the same substrate, enzymes from different fruits and vegetables may also

11

show different activity levels.

Taro PPO for instance demonstrates substrate specificity in this order: 4-

methylcatechol > chlorogenic acid > L-dopa > catechol > pyrogallol > dopamine >

caffeic acid (Duangmal et al., 1999).

Unlike the taro PPO above, it is interesting to note that another PPO from Longan

fruit does not react at all with chlorogenic acid. Moreover, there was no observed activity

with p-cresol, resorcinol and tyrosine for Longan fruit PPO (Jiang, 1999).

Longan fruit PPO does catalyze pyrogallol, 4-methylcatechol and catechol (Jiang,

1999).

Potato PPO on the other hand, favors the following substrates in this sequence: 4-

methylcatechol > caffeic acid > pyrogallol > catechol > chlorogenic acid > DL-Dopa >

dopamine (Duangmal et al., 1999).

2.6 Inhibitor Sensitivity

The inhibition mechanism at the active site can be competitive, non-competitive,

or mixed. PPO activity can be inhibited by chemicals acting either on the enzyme itself or

on the intermediate tyrosinase reaction products.

Halide salts, carboxylic, chelating and other organic acids interfere with the

browning mechanism by directly acting on the enzyme. On the other hand, reducing

agents such as ascorbic acid (and its derivatives), SH-compounds and sulfites, inhibit

enzymatic discoloration in two ways: reduction of o-quinones back to their precursor o-

diphenols thereby preventing pigment formation or reaction with o-quinones to yield

colorless compounds (Billaud et al., 2003).

12

Some of the known inhibitors are Maillard reaction products, metallothionein

from Aspergillus niger, kojic acid from Aspergillus and Penicillium, thiol, tiron, sodium

metabisulfite, reduced glutathione, L-cysteine, thiourea, FeSO4, SnCl2, and isoflavones

from soybeans (Alijanianzadeh & Saboury, 2007; Billaud et al., 2003; Chang, 2007;

Jiang, 1999).

It was reported that Maillard reaction compounds from glucose and lysine have

inhibited PPO activity in apple by restraining hydrolase activity and modifying reactions

at some tissue xenobiotic enzyme systems. Maillard reaction products can act as reducing

agents, scavengers of reactive oxygen species, hydrogen and electron donors and divalent

cation chelators (Billaud et al., 2003).

Metallothionein chelates copper at the active site while kojic acid is a slow-

binding competitive inhibitor. The latter is also known as a cosmetic whitening agent

(Alijanianzadeh & Saboury, 2007).

It was also observed that Selenium derivatives provide competitive inhibition

against tyrosinase activity (Koketsu et al., 2002).

Some chemicals can either activate or inhibit enzyme activity at certain

conditions. An example is ethyl xanthate (C2H5OCS2Na) which can initiate one or the

other reaction depending on its concentration. PPO has two binding sites for ethyl

xanthate: high affinity activation and low affinity inhibition points. Activation affinity is

decreased by increasing temperature while the reverse is true for inhibition

(Alijanianzadeh & Saboury, 2007).

In Longan fruit, MnSO4 and CaCl2 were also found to enhance activity (Jiang,

1999).

13

2.7 pH Dependence

PPO activity is lost irreversibly below pH 4 or above pH 10 (Richardson &

Hyslop, 1985).

The optimum pH for PPO activity ranges from 4.6 to 8 (Table 2). This is affected

by the PPO origin and substrate, among others.

PPO Source Optimum Activity pH References

Longan fruit 6.5 Jiang, 1999

Mushroom (Agaricus

bisporus)

6.0

7.0

Naidja et al., 1997

Ikehata & Nicell, 2000

Potato 6.8 Duangmal et al., 1999

Streptomyces glaucescens 8.0 Tepper, 2005

Taro 4.6 to 6.5 Duangmal et al., 1999;

Yemenicioglu et al., 1999

Table 2. Optimum pH for PPO activity.

2.8 Thermal Resistance

Generally, PPO activity decreases with increasing exposure to higher

temperatures. This is governed by first order kinetics (Castro et al., 2004). Thermal

inactivation and optimum activity temperature may vary depending on the source of PPO.

The optimum activity level is a little over room temperature. This is shown in

Table 3 for selected PPO from different sources.

14

PPO Source Optimum Activity

Temperature, °C

Reference(s)

Eggplant fruit 30 Concellon et al., 2004

Mushroom (A. bisporus) 27 Yang & Wu, 2006

Taro 30 Duangmal et al., 1999

Potato 25-40 Duangmal et al., 1999

Yang & Wu, 2006

Longan fruit 35 Jiang, 1999

Table 3. Optimum activity temperature of selected PPO.

The PPO from eggplant fruit lost 18 and 12% activity at 0 and 5°C, respectively.

It retained 48% activity at 60°C (Concellon et al., 2004).

For taro and potato PPO, 75 and 27% respectively, of enzyme activity were

retained at 60°C. Both were inactivated at 70°C, 10 min exposure (Duangmal et al.,

1999).

For temperatures greater than 50°C, mushroom PPO (0.08 mg/mL in 0.1 M

phosphate buffer) showed a steep decline in enzyme activity. At pH 6.5, D53 and D60

were 55 and 5 min, respectively. The isokinetic temperature was subsequently found to

be 49.5°C (Weemaes et al., 1997).

In a related study, there was a pronounced decrease in potato PPO activity above

50°C. Complete inactivation was observed at 80

°C (Severini et al., 2003).

At 50°C and 20 min holding, half of the Longan fruit PPO activity was lost (Jiang,

1999).

In blanching operations, peroxidase (POD) is the preferred target because it is

generally regarded as the most heat resistant enzyme in fruits and vegetables. It appears

that there could be exceptions here. In the case of taro and cabbage, a study

15

(Yemenicioglu et al., 1999) cites that POD was found to be more heat labile than PPO.

2.9 Moderate Electric Field Treatment

Ohmic heating is the process of passing electric current into the food, thereby

producing heat depending on the material resistance, power supplied and holding time. It

uses electrodes which are in direct contact with the food or the surrounding fluid. The

common process variables are the electrical conductivity of the medium and of the food,

sample geometry, pH, electrode material, electric field strength, frequency, waveform,

temperature and residence time. Moderate electric field (MEF) treatment is a process that

relies primarily on electric field effects, rather than heating alone. It is loosely defined as

the application of electric fields between 1 to 1000 V/cm of arbitrary waveform and

frequency for the purpose of inducing desirable effects in biomaterials.

Heat inactivation of enzymes and microorganisms increases with field intensity

and thermal history (Castro et al., 2004; Yildiz & Baysal., 2006; Icier et al., 2008). The

frequency effect on enzyme activity has not been studied yet. Other investigations on the

influence of frequency during MEF treatments were done on cellular diffusion

(Kulshrestha & Sastry, 2003; Kulshrestha & Sastry, 2006; Lakkakula et al., 2004; Lima

& Sastry, 1999; Wang & Sastry, 2002) and stimulation of microbial growth (Loghavi et

al., 2008).

16

2.10 Ohmic Heating Effects on PPO and Electromagnetic Field Treatments of Other

Enzymes

There are currently few studies on ohmic treatment of PPO in literature (Castro et

al., 2004; Icier et al., 2008; Yildiz & Baysal, 2006). From these studies, the effects of

ohmic heating process parameters on PPO activity are not yet fully investigated. The

isolation of electric field factors from the thermal effect is the first big challenge because

of the difficulty in matching the temperature-time history for both ohmic and

conventional heating.

Recently, PPO from grape juice was treated ohmically at different field strengths

(20, 30 and 40 V/cm) from 20°C and heated to varying thermal endpoints (60, 70, 80 and

90°C). Enzyme activity began to drop dramatically at 60°C, 40 V/cm and 70°C, 20-30

V/cm (Icier et al., 2008). This may have been the combined effect of thermal and electric

field because no conventional heat treatment was used to cancel out the temperature

factor.

An earlier study (Castro et al., 2004) showed the isolated effect of electric field in

selected food enzymes. A reduced inactivation time was observed for both PPO and

lipoxygenase, but no significant electric field effect was found for peroxidase, alkaline

phosphatase, β-galactosidase and pectinase. The electric field strength, E, was varied

during preheating (50 < E <90 V/cm) and holding (< 20 V/cm) to match conventional

thermal history. Frequency was held constant at 50 Hz.

In blanching pea puree (Icier et al., 2006), ohmic treatment was applied at 20-50

V/cm and the effect on peroxidase inactivation was studied. Samples were heated both

ohmically and conventionally from 30-100°C. The shortest inactivation was 54 s at 50

17

V/cm.

Ohmic heating was also used in inactivation of pectin methylesterase and

Aspergillus niger at 36-108 V/cm, 50 Hz (Yildiz & Baysal, 2006). Heating was brought

up to 60°C more from 30°C. The enzyme activity decreased with treatment time at 108

V/cm, while microbial inactivation increased with electric field strength. Apparently, no

conventional heating treatment was done to account for the thermal effect.

Other enzyme inactivation studies involved the application of pulsed electric

field, microwave, radio frequency and electromagnetic field (Aguilo-Aguayo et al., 2008;

Blank & Soo, 1997; Byus et al., 1987; Ho et al., 1997; Manzocco et al., 2008 (in press);

Matsui et al., 2007; Riener et al., 2008). A varied number of effects have been observed

and there is not always consensus on thermal contributions to these effects.

2.11 Effect of Frequency on Other Biological Materials

Several studies have been devoted to the effect of frequency in diffusion and

leaching of certain constituents in cellular materials, such as apple juice and rice bran oil

extraction (Imai et al., 2007; Kulshrestha & Sastry, 2003; Kulshrestha & Sastry, 2006;

Lakkakula et al., 2004; Lima & Sastry, 1999; Wang & Sastry, 2002). In the treatment of

orange juice, it was also noted that ohmic heating showed better Vitamin C retention than

microwave at 50, 60, 75 and 90°C (Vikram et al., 2005). The findings generally indicate

that lower frequency enhances heating, tissue permeabilization and extraction.

Waveform may also play a role. The fermentation of Lactobacillus acidophilus

was accelerated at pure sinusoidal waveform (45, 60 and 90 Hz) while the presence of

harmonics at 60 Hz increased bacteriocin production (Loghavi et al., 2008). In the

18

extraction of apple juice, 4 Hz sawtooth wave produced higher yield (Lima & Sastry,

1999).

Moreover, window effects or multiple resonance levels for both frequency and

field intensity have been observed in calcium ion efflux and lymphocytes inhibition. In

the 1 to 75 Hz range, calcium ions responded only at 6 and 16 Hz, with 10 and 56 V/cm

(Bawin & Adey, 1976). In a follow up study at constant frequency (16 Hz), the field

strength window effect was reported. There was enhancement at 5 to 7.5 V/m and 35 to

50 V/m while no significant change was noted at 1 to 2, 10 to 30, and 60 to 70 V/m

(Blackman et al., 1982). On the other hand, lymphocytes were treated in an

electromagnetic field (1, 3, 50, 200 Hz) and inhibition was observed only at 3-50 Hz

(Conti et al., 1983).

19

CHAPTER 3

MATERIALS AND METHODS

3.1 Experimental Design

To determine the effect of frequency on enzyme activity, PPO was treated in

isolation from the substrate. The treatments involved isothermal conditions (40, 50,

60°C), constant electric field strength (10 V/cm (MEF) and 0 V/cm (conventional

heating)) and varying frequency (60, 600 and 6000 Hz). The experiment was designed so

that enzyme samples at conventional and MEF treatment had identical time-temperature

history during heating, holding and cooling. The general experimental design is shown in

Figure 1.

After treatment, the enzyme solution was added to the substrate and other

reagents in a cuvette for subsequent enzyme activity assay using a spectrophotometric

method. Detailed set up and procedural descriptions are presented below.

20

Figure 1. Experimental design.

21

3.2 Experimental Set Up

The experimental set up is shown in the block diagram below (Figure 2.)

Figure 2. Block diagram of the experimental setup.

The ohmic heater was made of a glass tee (2.54 cm dia.) with platinized-titanium

electrodes at 2.96 cm apart. It was directly connected to a transducer which draws power

from an alternating current, variable power supply (Model 1751, Elgar Corp.). It was

mounted on a shaker plate, while immersed in a water bath (Model 3540, 1150 W, Lab

Line Instruments, IL, USA).

An electrically insulated thermocouple (type T) sensor was dipped into the glass

tee and connected to a data logger (21X Micrologger, Campbell Scientific, UT, USA).

22

The data logger was also connected to the transducer to monitor voltage across and

current flowing through the ohmic heater. The data was gathered through the data logger

and the computer.

A function generator (GFG 162A, GW Instek, Taiwan) was used to adjust the

power frequency and waveform. To monitor waveform and other power statistics, an

oscilloscope (Tektronix MSO 4034, OR, USA) was attached to the function generator.

During conventional heating, only the water bath, data logger and computer were

running. The set up was the same, with the sample enzyme being heated in the glass tee.

For ohmic heating, all components of the experimental set up were running. The water

bath was set to 15 – 24°C lower than the holding temperature. The extra heat that brought

the sample to the set temperature came from ohmic heating.

After conventional and ohmic heating, the samples were tested for enzyme

activity using a spectrophotometer (Cary 5000, Varian Inc., CA, USA). The change in

absorbance of the enzyme assay solution over time was correlated to enzyme activity

using the spectrophotometric protocol from related studies (Alijanianzadeh & Saboury,

2007; Castro et al., 2004; Chang, 2007; Espin et al., 1995; Koketsu et al., 2002).

3.3 Enzyme and Reagents

A lyophilized PPO powder from mushroom (Tyrosinase, T3824, Sigma Aldrich)

was used as the main subject in this experiment. The PPO in buffer (50 mM KH2PO4, pH

6.5 at 24°C, 0.28 S/cm at 50

°C) was subjected to both conventional and MEF treatments

at different holding temperatures, residence time and frequency. The reagents used for the

assay are given in Table 4.

23

Reagent Description

Enzyme PPO from mushroom, lyophilized powder, 500 to 1000 units activity per mL.

(T3824, Sigma Aldrich)

Buffer 50 mM KH2PO4, adjusted to pH 6.5 with 1 M NaOH at 24°C, 0.28 S/cm at 50

°C

(KH2PO4 monobasic, anhydrous, P5379, Sigma Aldrich; NaOH, S5881, Sigma

Aldrich)

Substrate 5 mM L-3-4-dihydorxyphenylalanine (D9628, Sigma Aldrich)

Other

reagents

2.1 mM L-ascorbic acid (A7631, Sigma Aldrich);

0.065 mM ethylenediaminetetraacetic acid (ED2SS, Sigma Aldrich)

Table 4. Reagents used in the experiment.

3.4 Treatments

The buffer (14 mL, 50 mM KH2PO4, pH 6.5 at 24°C) was preheated first to the

desired isothermal condition and frequency setting. Then, the concentrated enzyme in a

much smaller amount (1 mL tyrosinase, 2.1 mg/mL) was quickly added to the preheated

buffer in the glass tee sample holder using a micropipettor. After 5 minutes of treatment,

0.1 mL of the enzyme solution was taken out and injected into a cold cuvette containing

reagents (2.9 mL) for the spectrophotometric enzyme assay. Two samples from one

replication were obtained. The remaining enzyme buffer was continuously treated at the

same condition for two more 5-minute intervals. Identical replications were done at 10

and 15 minutes holding time. This treatment was repeated in three replicates for all

temperatures and frequencies.

To render preheating time-temperature history insignificant for conventional and

MEF-treated samples, a very small amount of concentrated enzyme solution (1 mL, 2.1

mg solid/mL solution) was added to the preheated buffer (14 mL). Similarly, rapid

cooling was accomplished by withdrawing 0.1 mL of treated enzyme solution and

24

quickly transferring it to a relatively large volume (2.6 mL) of cold (24°C) buffer in a

cuvette. For both preheating and cooling, thermal lags were negligible as verified by

actual temperature measurements in the glass tee ohmic heater and the cuvette. Overall,

the thermal history of treated samples was identical with this technique. The time-

temperature curve for the holding period is shown in Figure 3.

Figure 3. Thermal history of PPO enzyme solution during conventional and ohmic

heating (60, 600 and 6000 Hz).

The electric field strength was maintained at 10 ± 0.84 V/cm all throughout the

experiment. The current flowing through the sample was 0.4 to 0.5 A. The buffer

electrical conductivity was 0.28 S/m at 50°C.

The enzyme activity of untreated PPO was also monitored to see if there was

significant decay over time. At the start of the experiment, the bulk of PPO powder was

dissolved at once for the whole batch of temperature treatments. The experiment was

25

carried out on separate days: day 1, day 2 and day 4. Once dissolved, the solution was

stored at 4°C. During treatment of samples in subsequent runs, the cold enzyme solution

was allowed to warm up freely at room temperature until it reached 24°C. A slight

decline in enzyme activity (Figure 4) was observed over time but this was not statistically

significant.

0

500

1000

1500

2000

2500

3000

0 1 2 3 4 5

Day 1

En

zym

e A

cti

vit

y,

un

its/m

g e

nzym

e

Figure 4. Enzyme activity of untreated PPO. Error bars are ±2 standard deviation.

3.5 Enzyme Assay

The spectrophotometric enzyme assay was carried out similar to the protocol in

related studies (Alijanianzadeh & Saboury, 2007; Castro et al., 2004; Concellon et al.,

2004; Dincer et al., 2002; Duangmal & Apentent, 1999; Espin et al., 1995; Galeazzi &

Sgarbieri, 1981).

PPO reacts with the substrate L-3-4-dihydroxyphenylalanine and ascorbic acid in

26

the presence of oxygen. The products are o-benzoquinone, water and dehydroascorbic

acid. Discoloration immediately follows when melanin pigments are formed. This

reaction takes place at room temperature (24-25°C) and pH 6.5.

The enzyme activity was derived from the change in absorbance between the

blank and test samples in glass cuvette (Z27686-3, Sigma Aldrich), 1 cm light path. The

blank sample, which was used for calibration, consisted of 3 mL solution of L-3-4-

dihydroxyphenylalanine and ethylenediaminetetraacetic acid in a buffer. On the other

hand, the test sample was a 3 mL solution of L-3-4-dihydroxyphenylalanine,

ethylenediaminetetraacetic acid with the addition of L-ascorbic acid and PPO in the same

buffer. The test sample turns brown depending on enzyme activity level while the blank

remains clear.

The buffer was potassium phosphate, 50 mM and adjusted to pH 6.5 with sodium

hydroxide (221465, Sigma Aldrich; CAS 1310-73-2), 1 M. It has a computed electrical

conductivity of 0.28 S/m at 50°C. All reagents, including PPO, were dissolved in this

buffer. The concentration and volume of the reagents are shown in Table 5.

27

Reagents Volume, mL

Test Blank

Buffer: KH2PO4, 50 mM, pH 6.5 2.6 2.8

Substrate: L-3-4-dihydroxyphenylalanine, 5 mM 0.1 0.1

Ethylenediaminetetraacetic acid, 0.065 mM 0.1 0.1

L-ascorbic acid, 2.1 mM 0.1 0

PPO, 750 units activity/mL 0.1 0

Table 5. Volume of reagents in PPO activity assay.

The reagents were mixed by inversion ten times. The change in absorbance was

recorded at wavelength 265 nm for at least 12 min well after the readings leveled off

steadily. The enzyme activity for all samples was later based at 8.25 min readings when

the absorbance reached steady state. The enzyme activity, A, was computed as follows:

A = ( )( )T B df

k

Where:

A = enzyme activity, units per mg enzyme

T = change in absorbance per minute at 265 nm of test cuvette

B = change in absorbance per minute at 265 nm of blank cuvette

k = 0.0001, which is the change in absorbance per minute at 265 nm per unit

of polyphenoloxidase in a 3 mL reaction mixture, pH 6.5, 24°C from 0.1

mL of enzyme

df = 1 for dilution factor

One unit activity is equal to a change in absorbance at 265 nm of 0.001 per min at

pH 6.5 at 24°C in 3 ml reaction cuvette containing L-DOPA and L-ascorbic acid.

28

3.6 Data Analysis

Data were tested for statistical significance using analysis of variance and post

hoc multiple factors comparison. Microsoft Excel 2003 was used for raw calculation.

SPSS version 16 was used in data analysis. Raw enzyme activity data were normalized

by expressing the values as a ratio of treated and untreated enzyme replicates. Outliers

were eliminated using a method similar to the Q-test (UOA, 2008). An outlier was

eliminated if it lay beyond the 2 standard deviation from the mean of the two closer data

points. In the ANOVA post hoc multiple factors comparison, the Tukey HSD Test was

used for data sets with homogeneous variance. Otherwise, the Tamhane Test was used.

29

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Effect of Frequency on Polyphenoloxidase Activity

Results showed that MEF frequency treatments altered enzyme activity under

certain conditions. The enzyme activity in all the graphs is presented as a ratio of treated

to untreated replicates.

At 40°C (Figure 5 and Table 6), both 60 and 6000 Hz treatments caused some

activating effect (p < 0.05) compared to conventional heating at 10 min holding time.

When held for 15 min, a significant difference (p < 0.05) was observed only between 60

Hz and 6000 Hz with the latter showing a more pronounced higher activity. All other

treatments at the same exposure time showed no significant differences (p > 0.05).

The 50°C treatments (Figure 6 and Table 7) showed some signs of mixed

frequency effects but these were not statistically significant largely due to overwhelming

data variability under all conditions.

At 60°C (Figure 7 and Table 8), some inactivation effects from frequency

treatments were observable in the first 10 min while activation was detected at 15 min.

Specifically, the 60 Hz treatment after 5 min produced significantly lower (p < 0.05)

activity than conventional heating and much lower when compared to 600 Hz. At 10 min,

both 600 and 6000 Hz treatments exhibited significantly lower activity (p < 0.05) than

30

conventional heating. When treated for 15 min, PPO activity was higher (p < 0.05) in all

MEF frequency treatments than conventional heating.

Overall, the 6000 Hz treatment appeared to cause more stimulation of increased

activity compared to other frequency levels as indicated in the results for 40°C 10 and 15

min as well as 60°C 15 min. An MEF inactivation effect was also observed in all

frequencies at 60°C but at different conditions (60 Hz at 5min; 600 and 6000 Hz at 10

min).

In an attempt to get a clearer view of the mechanisms involved in the frequency

response of PPO, the activity ratio of all treatments over time is shown in Figure 8. It is

clear that at 40°C and 50°C, activity changes were relatively small; however, significant

inactivation occurred at 60°C. Enzyme activity at 60°C declined steeply in all treatments

although conventional heating was consistently linear through the 15 min holding time. A

roughly linear fall in activity from MEF treatments was observed only in the first 10 min

while all frequency effects caused some activation at 15 min. Interestingly, all MEF

treatments end up with relatively higher enzyme activity compared to conventional

heating at 15 min of all holding temperatures.

Apparently, the frequency and enzyme activity interaction behaves in a quite

complex manner. The next section attempts to bring together some pieces of information

from the literature which may help clarify the frequency response of PPO.

31

5 min Holding Time, 40 deg C

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Conv Heating 60.00 600.00 6000.00

Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Conv Heating 60 600 6000

Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0


Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e

Figure 5. Enzyme activity ratio across frequency settings at different holding times and

constant temperature (40°C). Error bars are ±2 standard deviation.

32

Holding Period,

min

Enzyme Activity Ratio

Conv Heating MEF 60 Hz MEF 600 Hz MEF 6000 Hz

5 0.975 ± 0.135a 1.122 ± 0.183

a 1.065 ± 0.034

a 1.143 ± 0.365

a

10 0.902 ± 0.123a 1.051 ± 0.025

b 1.002 ± 0.032

a 1.109 ± 0.009

b

15 0.980 ± 0.029a 0.970 ± 0.011

a,b 1.087 ± 0.344

a 1.095 ± 0.015

a,c

a, b, c Values within the same row followed by the same superscript are not significantly different (p >0.05)

Table 6. Enzyme activity ratio at 40°C. Error values are ± 2 standard deviation.

33


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Conv Heating 60.00 600.00 6000.00

Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0


Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0


Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e



34

Holding Period,

min



5 1.161 ± 0.592a 1.151 ± 0.534

a 0.957 ± 0.838

a 1.148 ± 0.816

a

10 1.028 ± 0.756a 0.979 ± 0.642

a 1.225 ± 0.843

a 1.098 ± 0.851

a

15 0.967 ± 0.858a 1.223 ± 0.579

a 1.147 ± 0.884

a 1.126 ± 0.507

a

a Values within the same row followed by the same superscript are not significantly different (p >0.05)


35


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0


Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0


Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0


Frequency, Hz

En

zym

e A

cti

vit

y,

un

its/m

g

en

zym

e



36

Holding Period,

min



5 0.690 ± 0.003a 0.455 ± 0.037

b 0.741 ± 0.154

a 0.578 ± 0.146

a,b

10 0.300 ± 0.013a 0.249 ± 0.043

a,b 0.193 ± 0.040

b 0.220 ± 0.073

b

15 0.077 ± 0.028a 0.172 ± 0.053

b 0.171 ± 0.052

b 0.196 ± 0.072

b

a, b Values within the same row followed by the same superscript are not significantly different (p >0.05)


37

40 deg C Holding Temperature

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10 15

Holding Time, min

En

zym

e A

cti

vit

y R

ati

oConv Heating

60 Hz

600 Hz

6000 Hz


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10 15

Holding Time, min

En

zym

e A

cti

vit

y R

ati

o

Conv Heating

60 Hz

600 Hz

6000 Hz


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10 15

Holding Time, min

En

zym

e A

cti

vit

y R

ati

o

Conv Heating

60 Hz

600 Hz

6000 Hz

Figure 8. Time plots of enzyme activity ratio at different holding temperatures.

38

4.2 Enzyme Activity and Variability Factors

The differences in enzyme activity arising from the treatments applied suggest

that the PPO sample may consist of several constituents with varying physical and

biochemical properties. It was confirmed from the supplier (Sigma Aldrich) that the PPO

sample was extracted from whole mushroom (Agaricus bisporus), and no further

investigation was done to determine isozymes and other variables present in the

commercial product. Even from the same mushroom strain, it was found out that enzyme

activity varies from the skin, flesh, velum and stalk. In one study (van Leeuwen &

Wichers, 1999), 15 latent and active isoforms were detected based from their isoelectric

points.

An error analysis (Appendix C) of the spectrophotometric technique showed that

the error due to the method was only 3.68% of the lowest activity recorded (115.2). Thus,

data variability does not appear to be due to the spectrophotometric technique.

In this experiment, the enzyme activity comparison was confined to the MEF

frequency variables. From related papers, it appears that enzyme activity variations may

be attributed to molecular aggregation, dipole moment, oxidized state, chemical bonding

and isozymes. These factors may also play some roles in the MEF frequency response of

PPO.

Dipole Moment. One of the well-detailed study on the structure and function of

PPO was done on a simple 31-kDa variant from Streptomyces antibioticus (Tepper et al.,

2002). Using spectroscopic and kinetic techniques, it was shown that PPO has closely

spaced (2.9 – 4.4 Ǻ) binuclear copper center as active site. Each copper is attached to

three histidine compounds. As such, this enzyme is roughly symmetric. This translates to

39

a relatively small dipole moment.

Considering that an alternating current is passed through the sample at different

frequency settings, certain PPO species may be induced to some translational motion in

greater magnitude through their electronegativity rather than through some dipole

motion. The PPO sample has an isoelectric point at 4.7 to 5 (Robb & Gutteridge, 1981)

which makes it carry a generally net negative charge because the buffer was at pH 6.5.

Molecular Aggregation. PPO can also exist in various degree of aggregation

although the predominant form is a tetramer (Jolley et al., 1969). It is not clear however

how the four isomers are linked to each other. The structural information could shed light

to symmetry and vibrational mechanics. The bigger the isomer, the lower the resonance

frequency is. Similarly, the smaller isomer is expected to respond to higher frequency

stimulation because of shorter wavelength. What is high or low frequency is still a

subject of investigation. Based from the results in this experiment, it will be noted that all

frequency treatments showed significantly higher enzyme activity at the following

conditions: 60 and 6000 Hz at 40°C 10 min; 6000 Hz at 40°C 15 min and 60°C 15 min. It

would be interesting therefore to compare the molecular aggregation in this activation

conditions over conventionally heated samples. The activating effect of 6000 Hz at

longer holding times may point to the possibility of the predominant tetramer getting

untangled through time so that smaller species are produced later. If true, this smaller

species would in turn respond better to a higher frequency. Or, it may also be probable

that the binuclear copper center was stretched to a favorable distance through some

random vibration and collision of compounds. It was previously found out that latent

PPO has a relatively shorter (≤2.9 Ǻ) copper-copper distance while optimum activity

40

occurs when this gap is stretched between 2.9 to 3.2 Ǻ (Tepper, 2005). X-ray

crystallography or other appropriate techniques may be used to verify the gap between

the copper active sites before and after treatments of activated samples.

Chemical Bonding and Molecular Vibration. Compounds and molecules vibrate

at a certain frequency depending on mass, bond, symmetry and other factors. A

compound may behave similar to a spring which stretches and bends from and toward a

center of mass (Skoog et al., 2006). From this spring analogy, the stretching frequency,

V, was derived as

where:

k = equivalent spring constant for single (500 N/m),

double (1000 N/m) and triple (1500 N/m) bonds.

m1, m2 = is the mass of any two adjacent molecules.

In infrared spectroscopy, this equation may be used to estimate the resonance

frequency. It was claimed that this was validated experimentally (Skoog et al., 2006).

It was further suggested that by estimating the stretching frequency of

neighboring molecules from one end to another of a compound, a common frequency

band may be obtained to give some resonant response for that compound (Skoog et al.,

2006).

For symmetric compounds, the vibration is most likely that of symmetric

stretching, in-plane scissoring and out of plane wagging. For asymmetric compounds,

41

alternate stretching and out of plane twisting may be observed. Moreover, the structural

asymmetry of samples leads to periodic dipole moment and hence, multiple resonance

frequency levels (Skoog et al., 2006). This is also called a window effect in various

papers. Based from this, it may be possible that the isolated effect of 6000 Hz at certain

temperature and holding time could be due to the response of symmetric and smaller

compounds. On the other hand, the window effect of 60 and 6000 Hz at 40°C 10 min for

instance may be due to the response of asymmetric compounds.

Oxidized State. The differences in frequency response of the PPO may also be

influenced by constituent derivatives of the enzyme. In its resting form or native state,

PPO mainly (85-90%) consists of the oxidized met or Tymet. This oxidized state reacts

with diphenolic substrates, but not with monophenolic ones. On the other hand, the

oxygenated derivative, Tyoxy, of the enzyme can bind with both monophenols and

diphenols (Tepper, 2005; Tepper et al., 2002). From this information, it would therefore

be interesting to determine if MEF at 6000 Hz may be converting more Tymet into Tyoxy.

This can give insights on what makes Tymet more responsive to a certain frequency than

the others.

Isozymes. PPO has been found to exist in several isozymes (Jolley et al., 1969;

Weemaes et al., 1997) which may behave differently at varying conditions. A detailed

study on PPO from mushroom revealed 15 isoforms with varying isoelectric points (van

Leeuween & Wichers, 1999). This paper reported that the most abundant latent isoform

has pI 5.5 while the predominant active isoform has pI 4.7. Both of these were present in

all tissues of the mushroom. The term isoform rather than isozymes was used in this case

because amino acid sequencing was not done in conjunction with the isoelectric focusing.

42

From the dissected portion of the mushroom, it was found out the gills have the least

active PPO species while the skin has the highest activity at the topmost area of the

crown. The inner flesh has greater activity from the center (van Leeuween & Wichers,

1999). This inherent variability of PPO may cause unclear trends in experimental

replicates especially when the heterogeneity of PPO from different parts of the mushroom

is not well accounted for.

In another paper (Weemaes et al., 1997), it was suggested that mushroom

isozymes may vary in thermal stability because as indicated by different heat inactivation

curves. Aside from distinct thermal sensitivity, the isozymes were distinguished by

isoelectric focusing. Fifteen isozymes were identified with pI between 3.65 and 6.84. The

most abundant isozyme has pI 3.65. Based from thermal treatments, it was deduced that

the isokinetic temperature of mushroom PPO at pH 6.5 is 49.5°C.

Going back to the MEF treatments in this experiment, it will be noted that MEF

was not significantly different from conventional heating at 50°C at all holding periods.

The role of the isokinetic temperature and frequency interaction however is not

immediately clear especially with such highly variable results. Perhaps further

investigation on different MEF treatments at the isokinetic temperature may provide

better clarification.

43

CHAPTER 5.

CONCLUSIONS

The results showed that MEF has both significant activating and inactivating

effect on PPO at certain treatment conditions. MEF activation was observable at 60 and

6000 Hz, 40°C 10 min; 6000 Hz, 40°C 15 min and at all frequencies at 60°C, 15 min. In

stimulating increased activity, MEF was apparently most effective at higher frequency

(6000 Hz) and longer holding period (15 min). Reduced activity occurred at all

frequencies but at different conditions: 60 Hz, 40°C 15 min and 60°C 5 min; 600 and

6000 Hz at 60°C 10 min. The data variability and lack of very strong activity pattern for a

certain frequency suggest that PPO isoforms with time-temperature sensitivity may be

present in the samples. These findings have major implications in either enhancing the

use of enzymes in industrial applications or on inactivation of enzymes particularly in

food processing.

44

APPENDIX A

LIST OF EQUIVALENT NAMES OF POLYPHENOL OXIDASE (EC 1.14.18.1)

45

Accepted name: monophenol monooxygenase

Systematic name: monophenol, L-dopa:oxygen oxidoreductase

Other names [IUBMB, 2008]:

1 catechol oxidase

2 catecholase

3 chlorogenic acid oxidase

4 chlorogenic oxidase

5 cresolase

6 diphenol oxidase

7 dopa oxidase

8 monophenolase

9 monophenol dihydroxy-L-phenylalanine oxygen oxidoreductase

10 monophenol dihydroxyphenylalanine:oxygen oxidoreductase

11 monophenol monooxidase

12 monophenol oxidase

13 N-acetyl-6-hydroxytryptophan oxidase

14 o-diphenolase

15 o-diphenol:O2 oxidoreductase

16 o-diphenol oxidase

17 o-diphenol oxidoreductase

18 o-diphenol:oxygen oxidoreductase

19 phenolase

20 phenol oxidase

21 polyaromatic oxidase

22 polyphenolase

23 polyphenol oxidase

24 pyrocatechol oxidase

25 tyrosinase

26 tyrosine-dopa oxidase

46

APPENDIX B.

ANALYSIS OF VARIANCE AND MULTIPLE COMPARISON OF FACTORS FOR THE

FREQUENCY EFFECT ON ENZYME ACTIVITY

47

Treatment Sum of

Squares

Degrees

of

Freedom

Mean Square F-ratio F

critical

value

p-

value

40°C, 5min Between 0.039 3 0.013 0.882 4.76 0.501

Within 0.089 6 0.015

Total 0.128 9

40°C,

10mins

Between 0.046 3 0.015 14.6 6.59 0.013

Within 0.004 4 0.001

Total 0.050 7

40°C,

15min

Between 0.030 3 0.010 0.836 5.41 0.529

Within 0.059 5 0.012

Total 0.089 8

50°C, 5min Between 0.088 3 0.029 0.233 4.07 0.871

Within 1.00 8 0.125

Total 1.09 11

50°C,

10min

Between 0.081 3 0.027 0.182 4.35 0.905

Within 1.03 7 0.147

Total 1.11 10

50°C,

15min

Between 0.089 3 0.030 0.208 5.41 0.887

Within 0.712 5 0.142

Total 0.801 8

60°C,

5mins

Between 0.138 3 0.046 15.6 4.76 0.003

Within 0.018 6 0.003

Total 0.156 9

60°C,

10mins

Between 0.015 3 0.005 7.88 4.35 0.012

Within 0.004 7 0.001

Total 0.019 10

60°C, Between 0.025 3 0.008 11.6 4.07 0.003

15mins

Within 0.006 8 0.001

Total 0.031 11 s means significant at p = 0.05

Table 9. Analysis of variance for frequency effect on enzyme activity (significance level p = 0.05).

48

Frequency Comparison (I versus J) 95% Confidence Interval

Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz -0.147 0.111 0.580 -0.532 0.237

600 Hz -0.090 0.122 0.878 -0.511 0.331

6000 Hz -0.168 0.111 0.486 -0.552 0.216

60 Hz ConvHeating 0.147 0.111 0.580 -0.237 0.531

600 Hz 0.057 0.111 0.952 -0.327 0.441

6000 Hz -0.020 0.099 0.997 -0.364 0.323

600 Hz ConvHeating 0.090 0.122 0.878 -0.331 0.511

60 Hz -0.057 0.111 0.952 -0.441 0.327

6000 Hz -0.078 0.111 0.894 -0.462 0.306

6000 Hz ConvHeating 0.168 0.111 0.486 -0.216 0.552

60 Hz 0.020 0.099 0.997 -0.323 0.364

600 Hz 0.078 0.111 0.894 -0.306 0.462 s means significant at p = 0.05

Table 10. Multiple comparison of treatments for the frequency effect on enzyme activity at 40°C, 5

min using the Tukey HSD test.


Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz -0.150 s 0.032 0.034 -0.282 -0.171

600 Hz -0.101 0.032 0.113 -0.233 0.031

6000 Hz -0.208 s 0.032 0.011 -0.340 -0.075

60 Hz ConvHeating 0.150 s 0.032 0.034 0.017 0.282

600 Hz 0.048 0.032 0.517 -0.084 0.181

6000 Hz -0.058 0.032 0.396 -0.190 0.074

600 Hz ConvHeating 0.101 0.032 0.113 -0.031 0.233

60 Hz -0.048 0.032 0.517 -0.181 0.084

6000 Hz -0.106 0.032 0.097 -0.234 0.026

6000 Hz ConvHeating 0.208 s 0.032 0.011 0.075 0.340

60 Hz 0.058 0.032 0.396 -0.074 0.190




49


Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz 0.011 0.011 0.979 -0.350 0.371

600 Hz -0.107 0.100 0.951 -1.14 0.930

6000 Hz -0.115 0.011 0.136 -0.348 0.118

60 Hz ConvHeating -0.011 0.011 0.979 -0.371 0.350

600 Hz -0.117 0.099 0.930 -1.18 0.946

6000 Hz -0.126 s 0.006 0.023 -0.206 -0.044

600 Hz ConvHeating 0.107 0.100 0.951 -0.930 1.14

60 Hz 0.117 0.099 0.930 -0.946 1.18

6000 Hz -0.008 0.099 1.00 -1.07 1.05

6000 Hz ConvHeating 0.115 0.011 0.136 -0.118 0.348

60 Hz 0.126 0.006 0.023 0.044 0.206



min using Tamhane test.


Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz 0.001 0.289 1.00 -0.916 0.935

600 Hz 0.205 0.289 0.891 -0.721 1.13

6000 Hz 0.013 0.289 1.00 -0.912 0.939

60 Hz ConvHeating -0.010 0.289 1.00 -0.935 0.916

600 Hz 0.195 0.289 0.904 -0.730 1.12

6000 Hz 0.004 0.289 1.00 -0.922 0.929

600 Hz ConvHeating -0.205 0.289 0.891 -1.13 0.721

60 Hz -0.195 0.289 0.904 -1.12 0.730

6000 Hz -0.191 0.289 0.908 -1.12 0.734

6000 Hz ConvHeating -0.013 0.289 1.00 -0.939 0.912

60 Hz -0.004 0.289 1.00 -0.929 0.922




50


Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz 0.049 0.313 0.999 -0.988 1.08

600 Hz -0.197 0.350 0.940 -1.36 0.962

6000 Hz -0.070 0.313 0.996 -1.11 0.967

60 Hz ConvHeating -0.049 0.313 0.999 -1.09 0.988

600 Hz -0.246 0.350 0.893 -1.41 0.914

6000 Hz -0.119 0.313 0.980 -1.16 0.918

600 Hz ConvHeating 0.197 0.350 0.940 -0.962 1.36

60 Hz 0.246 0.350 0.893 -0.914 1.41

6000 Hz 0.127 0.350 0.982 -1.03 1.29

6000 Hz ConvHeating 0.070 0.313 0.996 -0.967 1.11

60 Hz 0.119 0.313 0.980 -0.918 1.16

600 Hz -0.127 0.350 0.982 -1.29 1.03 s means significant at p = 0.05




Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz -0.256 0.344 0.876 -1.53 1.02

600 Hz -0.180 0.344 0.950 -1.45 1.09

6000 Hz -0.159 0.344 0.964 -1.43 1.11

60 Hz ConvHeating 0.256 0.344 0.876 -1.02 1.53

600 Hz 0.076 0.377 0.997 -1.32 1.47

6000 Hz 0.096 0.377 0.993 -1.30 1.49

600 Hz ConvHeating 0.180 0.344 0.950 -1.09 1.45

60 Hz -0.076 0.377 0.997 -1.47 1.32

6000 Hz 0.021 0.377 1.00 -1.37 1.41

6000 Hz ConvHeating 0.159 0.344 0.964 -1.11 1.43

60 Hz -0.096 0.377 0.993 -1.49 1.30




51


Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz 0.235 s 0.050 0.013 0.063 0.407

600 Hz -0.052 0.050 0.736 -2.22 0.120

6000 Hz 0.111 0.054 0.272 -0.077 0.299

60 Hz ConvHeating -0.235 s 0.050 0.013 -0.407 -0.063

600 Hz -0.286 s 0.044 0.003 -0.440 -0.132

6000 Hz -0.124 0.050 0.159 -0.296 0.048

600 Hz ConvHeating 0.052 0.050 0.736 -0.121 0.223

60 Hz 0.286 s 0.044 0.003 0.132 0.440

6000 Hz 0.162 0.050 0.062 -0.010 0.334

6000 Hz ConvHeating -0.111 0.054 0.272 -0.299 0.077

60 Hz 0.124 0.050 0.159 -0.048 0.296





Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz 0.051 0.023 0.211 -0.025 0.127

600 Hz 0.107 s 0.023 0.010 0.031 0.183

6000 Hz 0.080 s 0.023 0.040 0.004 0.156

60 Hz ConvHeating -0.051 0.023 0.211 -0.127 0.025

600 Hz 0.056 0.021 0.107 -0.012 0.124

6000 Hz 0.029 0.021 0.522 -0.039 0.097

600 Hz ConvHeating -0.107 s 0.023 0.010 -0.183 -0.031

60 Hz -0.056 0.021 0.107 -0.124 0.012

6000 Hz -0.027 0.021 0.592 -0.095 0.041

6000 Hz ConvHeating -0.080 0.023 0.040 -0.156 -0.004

60 Hz -0.029 0.021 0.522 -0.097 0.039




52


Mean

Difference

Standard

Error

p-value Lower

Bound

Upper

Bound

I J I-J

ConvHeating 60 Hz -0.095 s 0.022 0.011 -0.164 -0.025

600 Hz -0.094 s 0.022 0.011 -0.163 -0.024

6000 Hz -0.118 s 0.022 0.003 -0.188 -0.048

60 Hz ConvHeating 0.095 s 0.022 0.011 0.025 0.164

600 Hz 0.001 0.022 1.00 -0.069 0.071

6000 Hz -0.024 0.022 0.707 -0.094 0.046

600 Hz ConvHeating 0.094 s 0.022 0.011 0.024 0.163

60 Hz -0.001 0.022 1.00 -0.071 0.069

6000 Hz -0.025 0.022 0.682 -0.094 0.045

6000 Hz ConvHeating 0.118 s 0.022 0.003 0.048 0.188

60 Hz 0.024 0.022 0.707 -0.046 0.093




53

APPENDIX C

ERROR ANALYSIS FOR ENZYME ACTIVITY MEASUREMENT

54

The enzyme activity, A, is calculate from the absorbance of the test and blank

cuvettes using the equation:

( )( )T B dfA

k

(C1)

where:

A = enzyme activity in units per mg enzyme

T = absorbance of the cuvette containing the test sample

B = absorbance of the cuvette containing the blank sample

df = dilution factor = 1

k = 0,0001, which is the change in absorbance per minute at 265 nm per unit

of PPO in a 3 ml reaction mix, pH 6.5, 24C, containing 0.1 mL of enzyme solution.

The sensitivity coefficients from the test, ET, and blank, EB, readings are:

4TE 1 10

A dfx

T k

(C2)

4BE - -1 10

A dfx

B k

(C3)

55

Considering the photometric error, p, of the instrument (Varian Carry 5000), the

total error, E, in enzyme activity due to the absorbance of both the test and blank samples

is:

2 2A A

E dT dBT B

(C4)

where:

dT, dB = p = 3 x 10-4

(Varian Inc., 2002)

Hence,

2 2

4 4 4 41 10 3 10 1 10 3 10E x x x x

(C5)

E = 4.24 units of activity

The lowest recorded enzyme activity was 115.2. Thus, the total error translates to

3.68% of such recorded enzyme activity.

56

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