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 ______Adviser, V.M. Balasubramaniam, Ph.D. Graduate Program in Food, Agricultural and Biological Engineering

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. ii 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. iii 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 ……………………………………. Science Research Specialist II, present Bureau of Postharvest Research and Extension, Nueva Ecija, Philippines

FIELD OF STUDY

Major Field: Food, Agricultural and Biological Engineering

Specialization: Food Engineering

iv 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

v 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

vi 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

7 Enzyme activity ratio at 50°C. Error values are ±2 standard deviation.. 34

8 Enzyme activity ratio at 60°C. Error values are ±2 standard deviation.. 36

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

11 Multiple comparison of treatments for the frequency effect on enzyme activity at 40°C, 10min using Tukey HSD test………………………… 48

12 Multiple comparison of treatments for the frequency effect on enzyme activity at 40°C, 15min using Tamhane Test………………………….. 49

13 Multiple comparison of treatments for the frequency effect on enzyme activity at 50°C, 5min using Tukey HSD test…………………………. 49

14 Multiple comparison of treatments for the frequency effect on enzyme activity at 50°C, 10min using Tukey HSD test………………………… 50

15 Multiple comparison of treatments for the frequency effect on enzyme activity at 50°C, 15min using Tukey HSD test………………………… 50 vii

16 Multiple comparison of treatments for the frequency effect on enzyme activity at 60°C, 5min using Tukey HSD test…………………………. 51

17 Multiple comparison of treatments for the frequency effect on enzyme activity at 60°C, 5min using Tukey HSD test…………………………. 51

18 Multiple comparison of treatments for the frequency effect on enzyme activity at 60°C, 5min using Tukey HSD test…………………………. 52

viii 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

6 Enzyme activity ratio across frequency settings at different holding times and constant temperature (50°C). Error bars are ±2 standard deviation.……………………………………………………….……... 33

7 Enzyme activity ratio across frequency settings at different holding times and constant temperature (60°C). Error bars are ±2 standard deviation…………………….…………………………………………. 35

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

ix 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,

1 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

2 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.

3 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.

4 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.

5 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 Symbol EPRa Detectable Cu-Cu Distance, Ǻ Derivative - Tymet [Cu(II)-OH -Cu(II)] No 2.9 2- Tyoxy [Cu(II)O2 -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

6 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).

7 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,

8 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

9 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

10 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).

11 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).

12

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 6.0 Naidja et al., 1997 bisporus) 7.0 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.

13 PPO Source Optimum Activity Reference(s) Temperature, °C 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 14 (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).

15 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

16 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

17 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).

18 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.

19

Figure 1. Experimental design.

20 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). 21 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.

22

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 2.1 mM L-ascorbic acid (A7631, Sigma Aldrich); reagents 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 23 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 24 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.

3000

2500

2000

1500

1000

500

Enzyme units/mg Activity, enzyme 0 0 1 2 3 4 5 Day 1

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 25 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.

26

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:

(T B )( df ) A = 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.

27

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.

28 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

29 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.

30 5 min Holding Time, 40 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

0.2 Enzyme units/mg Activity, 0.0 Conv Heating 60.00 600.00 6000.00 Frequency, Hz

10 min Holding Time, 40 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

0.2 Enzyme units/mg Activity, 0.0 Conv Heating 60 600 6000 Frequency, Hz

15 min Holding Time, 40 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

0.2 Enzyme units/mg Activity, 0.0 Conv Heating 60 600 6000 Frequency, Hz

Figure 5. Enzyme activity ratio across frequency settings at different holding times and constant temperature (40°C). Error bars are ±2 standard deviation.

31

Holding Period, Enzyme Activity Ratio min Conv Heating MEF 60 Hz MEF 600 Hz MEF 6000 Hz 5 0.975 ± 0.135a 1.122 ± 0.183a 1.065 ± 0.034a 1.143 ± 0.365a 10 0.902 ± 0.123a 1.051 ± 0.025b 1.002 ± 0.032a 1.109 ± 0.009b 15 0.980 ± 0.029a 0.970 ± 0.011a,b 1.087 ± 0.344a 1.095 ± 0.015a,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.

32 5 min Holding Time, 50 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

0.2 Enzyme units/mg Activity, 0.0 Conv Heating 60.00 600.00 6000.00 Frequency, Hz

10 min Holding Time, 50 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

0.2 Enzyme units/mg Activity, 0.0 Conv Heating 60 600 6000 Frequency, Hz

15 min Holding Time, 50 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

0.2 Enzyme units/mg Activity, 0.0 Conv Heating 60 600 6000 Frequency, Hz

Figure 6. Enzyme activity ratio across frequency settings at different holding times and constant temperature (50°C). Error bars are ±2 standard deviation.

33

Holding Period, Enzyme Activity Ratio min Conv Heating MEF 60 Hz MEF 600 Hz MEF 6000 Hz 5 1.161 ± 0.592a 1.151 ± 0.534a 0.957 ± 0.838a 1.148 ± 0.816a 10 1.028 ± 0.756a 0.979 ± 0.642a 1.225 ± 0.843a 1.098 ± 0.851a 15 0.967 ± 0.858a 1.223 ± 0.579a 1.147 ± 0.884a 1.126 ± 0.507a a Values within the same row followed by the same superscript are not significantly different (p >0.05)

Table 7. Enzyme activity ratio at 50°C. Error values are ± 2 standard deviation.

34 5 min Holding Time, 60 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

0.2 Enzyme units/mg Activity, 0.0 Conv Heating 60 600 6000 Frequency, Hz

10 min Holding Time, 60 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

Enzyme units/mg Activity, 0.2 0.0 Conv Heating 60 600 6000 Frequency, Hz

15 min Holding Time, 60 deg C

2.0 1.8 1.6 1.4 1.2 1.0

enzyme 0.8 0.6 0.4

0.2 Enzyme units/mg Activity, 0.0 Conv Heating 60 600 6000 Frequency, Hz

Figure 7. Enzyme activity ratio across frequency settings at different holding times and constant temperature (60°C). Error bars are ±2 standard deviation.

35

Holding Period, Enzyme Activity Ratio min Conv Heating MEF 60 Hz MEF 600 Hz MEF 6000 Hz 5 0.690 ± 0.003a 0.455 ± 0.037b 0.741 ± 0.154a 0.578 ± 0.146a,b 10 0.300 ± 0.013a 0.249 ± 0.043a,b 0.193 ± 0.040b 0.220 ± 0.073b 15 0.077 ± 0.028a 0.172 ± 0.053b 0.171 ± 0.052b 0.196 ± 0.072b a, b Values within the same row followed by the same superscript are not significantly different (p >0.05)

Table 8. Enzyme activity ratio at 60°C. Error values are ± 2 standard deviation.

36

40 deg C Holding Temperature

1.4 1.2

1.0 Conv Heating 0.8 60 Hz 0.6 600 Hz 0.4 6000 Hz

Enzyme Ratio Activity 0.2 0.0 0 5 10 15 Holding Time, min

50 deg C Holding Temperature

1.4 1.2

1.0 Conv Heating 0.8 60 Hz 0.6 600 Hz 0.4 6000 Hz

Enzyme Ratio Activity 0.2 0.0 0 5 10 15 Holding Time, min

60 deg C Holding Temperature

1.4 1.2

1.0 Conv Heating 0.8 60 Hz 0.6 600 Hz 0.4 6000 Hz

Enzyme Ratio Activity 0.2 0.0 0 5 10 15 Holding Time, min

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

37 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

38 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

39 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, 40 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.

41 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.

42 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.

43

APPENDIX A

LIST OF EQUIVALENT NAMES OF POLYPHENOL OXIDASE (EC 1.14.18.1)

44 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

45

APPENDIX B.

ANALYSIS OF VARIANCE AND MULTIPLE COMPARISON OF FACTORS FOR THE FREQUENCY EFFECT ON ENZYME ACTIVITY

46

Treatment Sum of Degrees Mean Square F-ratio F p- Squares of critical value Freedom 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, Between 0.046 3 0.015 14.6 6.59 0.013 10mins Within 0.004 4 0.001 Total 0.050 7 40°C, Between 0.030 3 0.010 0.836 5.41 0.529 15min 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, Between 0.081 3 0.027 0.182 4.35 0.905 10min Within 1.03 7 0.147 Total 1.11 10 50°C, Between 0.089 3 0.030 0.208 5.41 0.887 15min Within 0.712 5 0.142 Total 0.801 8 60°C, Between 0.138 3 0.046 15.6 4.76 0.003 5mins Within 0.018 6 0.003 Total 0.156 9 60°C, Between 0.015 3 0.005 7.88 4.35 0.012 10mins 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).

47

Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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.

Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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 0.190 600 Hz 0.106 0.032 0.097 -0.025 0.234 s means significant at p = 0.05

Table 11. Multiple comparison of treatments for the frequency effect on enzyme activity at 40°C, 10 min using the Tukey HSD test.

48 Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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 0.206 600 Hz 0.008 0.099 1.00 -1.05 1.07 s means significant at p = 0.05

Table 12. Multiple comparison of treatments for the frequency effect on enzyme activity at 40°C, 15 min using Tamhane test.

Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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 0.922 600 Hz 0.191 0.289 0.908 -0.734 1.17 s means significant at p = 0.05

Table 13. Multiple comparison of treatments for the frequency effect on enzyme activity at 50°C, 5 min using the Tukey HSD test. 49 Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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

Table 14. Multiple comparison of treatments for the frequency effect on enzyme activity at 50°C, 10 min using the Tukey HSD test.

Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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 1.30 600 Hz -0.021 0.377 1.00 -1.41 1.37 s means significant at p = 0.05

Table 15. Multiple comparison of treatments for the frequency effect on enzyme activity at 50°C, 15 min using the Tukey HSD test. 50 Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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 0.296 600 Hz -0.162 0.050 0.062 -0.334 0.010 s means significant at p = 0.05

Table 16. Multiple comparison of treatments for the frequency effect on enzyme activity at 60°C, 5 min using the Tukey HSD test.

Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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 0.039 600 Hz 0.027 0.021 0.592 -0.041 0.095 s means significant at p = 0.05

Table 17. Multiple comparison of treatments for the frequency effect on enzyme activity at 60°C, 10 min using the Tukey HSD test. 51

Frequency Comparison (I versus J) 95% Confidence Interval Mean Standard p-value Lower Upper Difference Error Bound 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 0.093 600 Hz 0.025 0.022 0.682 -0.045 0.094 s means significant at p = 0.05

Table 18. Multiple comparison of treatments for the frequency effect on enzyme activity at 60°C, 15 min using the Tukey HSD test.

52

APPENDIX C

ERROR ANALYSIS FOR ENZYME ACTIVITY MEASUREMENT

53 The enzyme activity, A, is calculate from the absorbance of the test and blank cuvettes using the equation:

(T B )( df ) A  (C1) k

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:

A df 4 ET    1x 10 (C2) Tk

A df 4 EB   -  -1x 10 (C3) Bk

54 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:

22 AA    E dT   dB  (C4) TB   

where:

dT, dB = p = 3 x 10-4 (Varian Inc., 2002)

Hence,

22 4 4   4 4  E1 x 10 3 x 10     1 x 10 3 x 10   (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.

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61