extending shelf life of juice products by pulsed electric fields

EXTENDING SHELF LIFE OF JUICE PRODUCTS BY PULSED ELECTRIC FIELDS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Seacheol Min, M.S.

* * * * *

The Ohio State University 2003

Dissertation Committee: Approved By

Dr. Q. Howard Zhang, Adviser

Dr. David B. Min Adviser

Dr. Sudhir K. Sastry Food Science and Nutrition

ii

ABSTRACT

Effects of commercial scale pulsed electric field (PEF) processing on the qualities

of tomato juice and orange juice were studied and compared with those of thermal

processing. The inactivation of tomato juice lipoxygenase (LOX) by PEF was studied

using kinetic models.

Tomato juice was prepared by hot break at 88 °C for 2 min or cold break at 68 °C

for 2 min and then thermally processed at 92 °C for 90 s or PEF processed at 40 kV/cm

for 57 µs. Freshly squeezed orange juice was thermally processed at 90 °C for 90 s or

processed by PEF at 40 kV/cm for 97 µs.

Tomato juice was treated by a laboratory scale PEF system with the combinations

of electric field strength (0, 10, 15, 20, 30, 35 kV/cm), PEF treatment time (20, 30, 50,

60, 70 µs), and PEF treatment temperature (10, 20, 30, 40, 50 °C) to study inactivation

kinetics of tomato juice LOX by PEF.

Both thermally and PEF processed tomato juices maintained microbial shelf life

at 4 °C for 112 d. PEF processed tomato juice retained more flavor compounds than

thermally processed or unprocessed control juice (p < 0.05). The lipoxygenase activities

of thermally and PEF processed tomato juices were 0 and 47%, respectively. PEF

processed tomato juice retained more ascorbic acid than thermally processed juice at 4 °C

for 42 d (p < 0.05). PEF processed tomato juice had significantly lower nonenzymatic

iii

browning than thermally processed or control juice (p < 0.05). Sensory evaluations

indicated that PEF processed tomato juice had more preferred flavor and higher overall

acceptability than thermally processed juice (p < 0.01).

Thermally and PEF processed orange juices maintained microbial shelf life at 4

°C for 196 d. PEF processed orange juice retained more ascorbic acid, flavor, and color

than thermally processed juice (p < 0.05). Sensory evaluation of texture, flavor, and

overall acceptability were ranked highest for control orange juice, followed by PEF

processed juice and then by thermally processed juice (p < 0.01).

Laboratory scale PEF treatment at 30 kV/cm for 60 µs at 50 °C inactivated 88.1%

of LOX. The first-order kinetic models, the Hulsheger’s model, and the Fermi’s model

adequately described the LOX inactivation by PEF. Applied electric field strength was

the primary variable for the inactivation of LOX by PEF.

iv

Dedicated to my parents and wife, Kijung

v

ACKNOWLEDGMENTS

I would like to express my sincere appreciation to Dr. Howard Q. Zhang for his

constant support and encouragement during my gradate studies. His keen insight and

systematic guidance have made my Ph. D. studies successful. His advices are always in

my mind and make me wise and strong. I would also like to express gratitude to Dr.

David B. Min and Dr. Sudhir K. Sastry for their valuable input and assistance in my

studies. I have been honored being their one of disciples.

I am very grateful to my colleagues in Ohio State PEF Research Team, specially,

Dr. Tony Jin, Si-Quan Li, Stephen Min, and Rod Caldwell, for their help in my research

and warm friendship. I also wish to thank my friends in the department of Food Science

and Technology at the Ohio State University for their friendship. Special my

appreciation is extended to my family and friends for their love.

Project funding from the U.S. DoD Dual Use Science & Technology program, the

PEF consortium, and the Ohio Agricultural Research and Development Center (OARDC)

are gratefully acknowledged.

vi

VITA

June 3, 1972 ……………………………………. Born – Cheongju, Korea 1992 - 1997………………………………………B.S. Food Science and Technology Chungnam National University Taejon, Korea 1998 - 1998…………………………….………...Graduate Research Associate Chungnam National University Taejon, Korea 1998 - 2000………………………………………M.S. Food Science and Technology

The Ohio State University Columbus, Ohio, U.S.A

1998 - present…………………………………….Graduate Research Associate

The Ohio State University Columbus, Ohio, U.S.A

PUBLICATIONS

Research Publications 1. Min S, Laura R, Zhang QH. 2002. Effects of Water Activity on the Inactivation of Enterobacter cloacae Inoculated in Chocolate Liquor and a Model System. J Food Process Preserv 26:323-337.

vii

FIELDS OF STUDY

Major Field: Food Science and Nutrition

viii

TABLE OF CONTENTS

Page

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

Dedication ……………………………………………………………………………….. v

Acknowledgments ………………………………………………………………...……. vi

Vita ………………………………………………………..…………………………… vii

List of Tables ………………………………..………………………………………… xiii

List of Figures …………………………………………………………………...……... xv

Chapters:

1. Literature Review …………………...…………………………………………… 1

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

Inactivation of microorganisms by PEF……………………....…………………. 2

Mechanism …..………………………………..…………………...…... 2

Critical factors …………………………………..……….………...…... 3

Microbial stability of PEF treated foods…..……………..….………..... 8

Kinetic models …………………………………………..…...….….... 17

ix

Inactivation of enzymes by PEF ……..………………………..................….…..20

Mechanism …..………………………………………..………….…... 20

Critical factors ….……………………………………………..….…... 21

Inactivation of enzymes by PEF ……..………………………..……... 23

Sensory and nutritional properties of PEF treated juices ………………...…..… 27

Packaging issues ……………....………..………...………….…………..…….. 31

Conclusions ……………..…………………………………………….…….…. 34

References …………………………………………………………………..….. 34

2. Effects of Commercial Scale Pulsed Electric Field Processing on the Quality of

Tomato juice………………………………...………………………………………….. 48

Abstract ………………………………..…….………..………….…...………... 48

Introduction ………………………..……………….……..………………..…... 50

Materials and Methods …………..……………….………………...………..…. 51

Results and Discussion …………..…..………………...…………….…...……. 62

References ……………………………………………………..………….……. 79

Acknowledgments ……………………………………………..……………..… 85

3. Effects of Commercial Scale Pulsed Electric Field Processing on Flavor and

Color of Tomato Juice ……………………...……………………………………..…… 86

Abstract ………………………………..……………..……..................……...... 86

Introduction ………………………..……………………………...………..…... 87

Materials and Methods …………..……………….………………...………..…. 88

Results and Discussion …………..…..………………………...……….....…… 97

Conclusions ………………………………………………...……..….……..… 111

References …………………………………………….…………….………….. 111

Acknowledgments ……………………………………………………………… 115

x

4. Effects of Commercial Scale Pulsed Electric Field Processing on the Quality of

Orange Juice ……………………...…………………………………………………… 116

Abstract ………………………………..…………………...…..…....………... 116

Introduction ……………………………...…..………………………………... 117

Materials and Methods …………..…………………...…….…………………. 118

Results and Discussion …………..…..……………………...………….....….. 127

Conclusions …………………………………………………...………...…..… 141

References ………………………………………………..………………….... 141

Acknowledgments ………………………………………..…………………… 144

5. Inactivation Kinetics of Tomato Juice Lipoxygenase by Pulsed Electric Fields.145

Abstract ………………………………..………….………..…..…....………... 145

Introduction ………………………..……………….………..………………... 146

Materials and Methods …………..……………….…………...………………. 148

Results and Discussion ……………...…………………………...……...……. 156

Conclusions ………………………………………...…………………….....… 173

References ………………………………………………..………………….... 173

Acknowledgments ………………………………………..…………………… 176

6. Recommendations for Further Studies …………………………………………177

List of References …………………………………………………………………….. 179

xi

LIST OF TABLES

Table Page

1.1 Overview of the inactivation of microorganisms in foods by PEF treatment ... 13 1.2 Overview of the inactivation of enzymes by PEF treatment …...……………. 25 2.1 Processing conditions for thermally processed, PEF processed, and control tomato juices …...……………………………………………………….…..… 55 2.2 Effects of thermal processing and PEF processing on the concentration of lycopene of tomato juice during the storage at 4 °C for 112 days ……...……. 69 2.3 Effects of thermal processing and PEF processing on the particle size distribution of tomato juice………………………………………………….... 74 2.4 Effects of thermal processing and PEF processing on the viscosity of tomato juice during the storage at 4 °C for 112 days …………………….………..…. 76 2.5 Effects of thermal processing and PEF processing on the sensory properties of tomato juice …………………………………………………………………... 78 3.1 Parameters and temperature of processing …………………………………... 92 3.2 Effects of thermal processing and PEF processing on the retention of flavor compounds (%) of tomato juice during storage at 4 °C for 112 d …………… 99 4.1 Processing conditions for thermally processed, PEF processed, and control orange juices ………………………………………………..………………. 122 4.2 Effects of thermal processing and PEF processing on the total aerobic plate

counts and the yeast & mold counts of orange juice during storage at 4 °C for 196 days ………………………………………………………………..….…129

4.3 Effects of thermal processing and PEF processing on the retention of flavor

compounds (%) of orange juice during storage at 4 °C for 196 days ………. 134

xii

4.4 Effects of thermal processing and PEF processing on the hue angle (θ) of orange juice during storage at 4 °C for 196 days …………………………….138

4.5 Effects of thermal processing and PEF processing on the sensory evaluation of

color, appearance, texture, flavor, and overall acceptability of orange juice ..140 5.1 PEF treatment conditions for (a) the inactivation kinetics of tomato juice LOX

by PEF and for (b) the study determining the primary variable in tomato juice LOX inactivation by PEF ………………………………………………….…152

5.2 Kinetic constants (KE) of the first-order kinetic model, correlation coefficients (R2), p-values of lack-of-fit, and decimal reduction times (D) of the PEF treatment at 15, 20, 30, or 35 kV/cm at 30 °C on tomato juice LOX ………..158 5.3 Kinetic constants (KN) of the first-order kinetic model, correlation coefficients (R2), and p-values of lack-of-fit of the PEF treatment at 10, 20, 30, 40, or 50 °C at 60 µs on tomato juice LOX ………………………..……..165 5.4 Fermi’s equation parameter values and correlation coefficients (R2) for the inactivation of tomato juice LOX by PEF treatment at 30 °C for 20, 30, 50, 60, or 70 µs …………………………………..……………………………… 172

xiii

LIST OF FIGURES

Figure Page

2.1 Flowchart of OSU-6 commercial scale PEF processing system ..…………….. 53 2.2 Effects of thermal processing and PEF processing on the total aerobic plate

counts of tomato juice during the storage at 4 °C for 112 days …………….... 64 2.3 Effects of thermal processing and PEF processing on the yeast & mold counts of tomato juice during the storage at 4 °C for 112 days …………………….…... 65 2.4 Effects of thermal processing and PEF processing on the lipoxygenase activity of tomato juice during the storage at 4 °C for 112 days ……….………………67 2.5 Effects of thermal processing and PEF processing on the retention of ascorbic

acid of tomato juice during storage at 4 °C for 70 days ……………………… 71 3.1 Flowchart of OSU-6 commercial scale PEF processing system ..…………….. 90 3.2 Effects of thermal processing and PEF processing on the retention of trans-2- hexenal of tomato juice during storage at 4 °C for 112 days .……………….... 98 3.3 Effects of thermal processing and PEF processing on the brown color of tomato juice during storage at 4 °C for 112 days …………...……….……….. 102 3.4 Effects of thermal processing and PEF processing on the concentration of 5- hydroxymethyl-2-furfural of tomato juice storage at 4 °C for 112 days ……... 103 3.5 Linear regression plots of the brown color versus the concentration of 5-

hydroxymethyl-2-furfural (a) and the concentration of 5-hydroxymethyl-2- furfural versus the loss of ascorbic acid (b) in PEF processed tomato juice sampled at 0, 7, 14, 28, 35, 42, 49, 56, and 70 days………………………..…106

3.6 Effects of thermal processing and PEF processing on the red-yellow ratio

(Hunter a / b) of tomato juice during storage at 4 °C for 112 days ……….… 108

xiv

4.1 Flowchart of OSU-6 commercial scale PEF processing system ..………...…. 120 4.2 Effects of thermal processing and PEF processing on the retention of flavor compounds (%) of orange juice during storage at 4 °C for 196 days …..…… 131 4.3 Effects of thermal processing and PEF processing on the retention of myrcene of orange juice during storage at 4 °C for 196 days ……………………………. 133 4.4 Effects of thermal processing and PEF processing on the brown color of orange juice during storage at 4 °C for 196 days …………………..……..…………. 135 4.5 Effects of thermal processing and PEF processing on the lightness (L) of orange juice during storage at 4 °C for 196 days ………………………………...….. 137 5.1 A bipolar square wave pulse pair. A peak voltage and a peak current are

indicated. …………………………………………………………………….. 149 5.2 Flow arrangement of four PEF treatment chambers connected to stainless coils

which tomato juice passed through. T1, T2, T3, and T4 are inlet or outlet temperatures. ……………………………………………………………….... 150

5.3 Effects of PEF treatment time on the residual activity of LOX after PEF treatment at 15, 20, 30, or 35 kV/cm at 30 °C. Points are the average of a duplicate with four measurements. Plotted lines correspond to the adjustment of all points at each electric field strength to a first-order kinetic model. …...…. 157 5.4 Figure 4-The effect of electric field strength (E) on the first-order kinetic constant (kE) (a) and the regression line of experimental residual activity of LOX versus predicted residual LOX activity from Equation 10 (b) ....……..………161 5.5 Effects of electric field strength on the residual activity of LOX after PEF treatment at 10, 20, 30, 40, or 50 °C for 60 µs ………..…………………...…163 5.6 The effect of PEF treatment temperature on the first-order kinetic constant (kN)

(a) and the regression line of experimental residual activity of LOX versus predicted residual LOX activity from Equation 11 (b).………………...……. 167

5.7 The regression lines of experimental residual LOX activity versus predicted residual LOX activity from the Hulsheger’s kinetic model (a) and the Fermi’s kinetic model (b) …………………………………………………………….. 170

1

CHAPTER 1

Literature Review

INTRODUCTION

Pulsed electric field (PEF) has been studied as a nonthermal food preservation

method to inactivate microorganisms in foods without significant loss of flavor, color,

taste, and nutrient of the foods (Mertens and Knorr 1992; Dunn 2001). PEF treatment

uses high intensity electric field generated between two electrodes. A large flux of

electrical current flows through foods when a high intensity electric field is generated.

Nonthermal treatment is attained by use of a very short pulse width of treatment time (i.e.

microseconds). The potential of commercialization of PEF technology has drawn

attention from people in food industry, who wishes to satisfy the consumer demands for

fresh food products. A commercial scale PEF system with flow rate of 500-2000 L/h was

constructed and processed orange juice and tomato juice successfully (Min and others

2002a, 2002b; Min and Zhang 2002b).

PEF technology rapidly develops and thus a lot of reports regarding PEF

treatment of foods are being published. There is need to review current publications

reporting effects of PEF treatment on the inactivation of microorganisms and enzymes by

2

PEF and the sensory and nutritional qualities of foods. The benefit of PEF treatment of

foods will be realized when PEF treated foods maintain their initial high quality attributes

including flavor profiles for extended storage time. A packaging issue needs to be raised

to find appropriate packaging materials and methods for PEF treated foods.

INACTIVATION OF MICROORGANISMS BY PEF

Mechanism

Structural damages of cell membrane, which lead to ion leakage, metabolite

losses, protein releases, and increased uptakes of drugs, molecular probes, and DNA,

have been used to explain the microbial inactivation by PEF (Kinosita and Tsong 1977;

Benz and Zimmermann 1980). Chang and Reese (1990) introduced effects of PEF on

microbial cells. Primary effects include structural fatigue due to induced membrane

potential and mechanical stress. Secondary effects include material flow after the loss of

integrity of cellular membrane by the electric field, local heating, and membrane stress.

Tertiary effects include cell swelling or shrinking and disruption due to the unbalanced

osmotic pressure between the cytosol and the external medium.

The electric potential causes an electostatic charge separation in the membrane of

microbial cells due to the dipole nature of the molecules of the membrane (Bryant and

Wolfe 1987). The cell membrane is regarded as an insulator shell to the cytoplasm due to

its electrical conductivity, which is six to eight times weaker than the cytoplasm

(Barbosa-Canovas and others 1999). Electrical charges are accumulated in cell

membranes when microbial cells are exposed to electric fields. The accumulation of

3

negative and positive charges in cell membranes forms transmembrane potential. The

charges attract each other and generate compression pressure, which causes the

membrane thickness to decrease. A further increase in the electric field strength beyond

a critical membrane potential leads to pore formation (electroporation). Sale and

Hamilton (1968) reported that cell lysis with the loss of membrane integrity occurred

when transmembrane potential was approximately 1 volt. This critical electrical potential

varies depending on the pulse duration time, number of pulses, and PEF treatment

temperature (Barbosa-Canovas and others 1999).

Harrison and others (1997) reported that transmission electron microscopy (TEM)

micrographs of PEF treated Saccharomyces cerevisiae in apple juice exhibited disruption

of organelles and lack of ribosomes They proposed that the damaged organelles and

lack of ribosomes after PEF treatment as an alternative inactivation mechanism to the

electroporation theory.

Critical factors

The factors determining the efficiency of the microbial inactivation by PEF can be

classified with treatment parameters, product parameters, and microbial characteristics.

Treatment parameters. The main treatment parameters that affect microbial

inactivation by PEF are electric field strength, PEF treatment time, pulse width, pulse

shape, and treatment temperature. (Knorr and others 1994; Hulsheger and Niemann

1980). Generally, as the intensity of each of theses parameters increase, the microbial

inactivation by PEF also increases (Wouters and others 2001a). A linear relationship

between electric field strength and the inactivation of Escherichia coli was reported by

4

Hulsheger and Niemann (1980). Peleg (1995) showed that the rate of microbial

inactivation by PEF at a constant electric field strength increases as PEF treatment time

increases.

High efficiency of PEF treatment on the inactivation of microorganisms can be

obtained with a large pulse width. PEF treatment time is calculated by multiplying the

number of pulses by the pulse width. As the pulse width increases, the PEF treatment

time also increases, which results in increased inactivation. However, if the pulse width

is too long, a food temperature rise undesirable for PEF treatment can occur (Barbosa-

Canovas and others 1999).

Electric field pulses can be applied in the form of exponentially decay, square

wave, and oscillatory pulses. The pulse shapes commonly used in PEF treatment are

exponential or square wave pulses (Barbosa-Canovas and others 1999). The square wave

pulse minimizes energy absorption in foods (Knorr and others 1994) and is more

effective for inactivating microorganisms than exponential decay pulses (Zhang and

others 1994c). The pulse width for a square pulse is the time that the voltage is kept at

the maximum value. The pulse width of exponential decay pulses is the time needed to

decrease the voltage to 37% of its peak value (Zhang and others 1995a). Bipolar waves

have been used with square wave or exponential pulses. A shielding layer can be formed

on electrodes in PEF treatment chamber when charged molecules such as proteins

migrate to the surface of electrodes. The shielding layer reduces the efficiency of PEF

treatment by altering uniform electric fields. Bipolar pulses can prevent the formation of

the shielding layer (Zhang and others 1995a). Bipolar pulses are also more lethal than

monopolar pulses because a reversal in the orientation or polarity of the electric field

5

changes the direction of charged molecules in the cell membrane, which causes a stress in

the cell membrane of microorganisms and electric breakdown (Barbosa-Canovas and

others 1999).

Synergistic effects between PEF treatment and moderate temperature (45-60 °C)

on the inactivation of microorganisms has been reported. Zhang and others (1995b)

demonstrated that the increase of PEF treatment temperature from 7 to 20 °C

significantly increased the inactivation of E. coli in simulated milk ultrafiltrate. Sensoy

and others (1997) found that increasing the PEF treatment temperature from 10 to 50 °C

increased the sensitivity of Salmonella dublin to PEF treatment. The increased lethal

effects of PEF at the higher PEF treatment temperature might be due to the temperature-

related phase transition of cell membrane from a gel to a liquid-crystalline and the

associated reduction in the bilayer thickness of cell membrane (Stanley 1991). The phase

transition reduces the transmembrane potential needed for the breakage of cell membrane

(Pothakamury and others 1996). Mertens and Knorr (1992) recommended PEF treatment

combined with a moderate thermal treatment for the efficient inactivation of

microorganisms in foods.

Product parameters. Information on the physical properties of foods over a wide range

of temperatures is needed to find optimum PEF treatment condition and to design PEF

processing units (Ruhlman and others 2001). The most critical product parameters

include electric conductivity, density, viscosity, pH, and water activity.

PEF treatment is more effective for the microbial inactivation with the food of

low electrical conductivity. An increase in the electrical conductivity of treatment

medium causes a decrease in inactivation of microorganisms at constant energy input

6

(Vega-Mercado and others 1996). The low electrical conductivity increases the

difference in the electrical conductivity between a medium and a microbial cytoplasm.

This increased difference in the electrical conductivity weakens the membrane structure

of microorganisms due to an increased flow of ionic substances across the membrane

during PEF treatment (Barbosa-Canovas and others 1999). Raso and others (1998a)

showed the dependency of electric field strength on the electrical conductivity of fruit

juices. They reported that the lowest electrical conductivity caused the highest electric

field strength and thus the highest microbial inactivation.

The temperature change during PEF treatment can be expressed as:

pCtET

ρσ2

=∆ (1)

where ∆T is the temperature change in food during PEF treatment (°C), E is the electric

field strength (V/m), t is the PEF treatment time (s), σ is the electrical conductivity of the

food to be processed (S/m), ρ is the density of the food (kg/m3), and Cp is the specific

heat of the food (J/(kg°C)) (Lindgren and others 2002).

Temperature inside PEF treatment chambers increases during PEF treatment. A

low electrical conductivity of foods results in a small temperature change during PEF

treatment (Equation 1). The temperature change during PEF treatment can also be

reduced with high density foods (Equation 1). A high temperature increase during PEF

7

treatment can decrease the solubility of air in foods (Zhang and others 1995a). Air

bubbles can cause dielectric breakdown under electric field (arching). The temperature

increase should be minimized for efficient PEF treatments.

The viscosity of a food determines flow characteristics. A uniform flow profile of

food products in the PEF treatment chamber can provide a uniform PEF treatment

(Ruhlman and others 2001).

The effect of the pH on the inactivation of microorganisms by PEF is not clear

(Wouters and others 2001a). Vega-Mercado and others (1996) and Raso and others

(1998a) found an enhanced efficiency in the inactivation of microorganisms by PEF at

low pH. The inactivation of E. coli by PEF was more effective at pH 5.7 than at pH 6.8

(Vega-Mercado and others 1996). An enhanced lethal effect of PEF on mold spores in

fruit juices at low pH (Raso and others 1998a). However, Hulsherger and others (1981)

and Sale and Hamilton (1967) reported no significant effect of pH on the inactivation of

microorganisms by PEF. The effect of pH on the inactivation of microorganisms by PEF

might depend on the characteristics of microorganisms to be investigated.

Microbial resistance to inactivation treatments including thermal treatment is high

at low water activity (aw) environment (Ababouch and others 1995). Min and others

(2002c) found that the PEF inactivation of Enterobacter cloacae inoculated in chocolate

liquor or a model system increased as aw increased. They reported that E. cloacae

survived from low aw environment had high resistance to PEF. The resistance of

microorganisms to a low aw environment may need to be considered when the

inactivation of microorganisms by PEF is evaluated.

8

Microbial characteristics. Bacteria are generally more resistant to PEF than yeasts

(Barbosa-Canovas and others 1999). Among bacteria, gram positive bacteria are more

resistant to PEF than gram negative bacteria (Hulsheger and others 1983). The higher

resistance to PEF of gram positive bacteria may be related to the rigidity of the teichoic

acids in the peptidoglycan layer of the gram-positive cell wall (Lado and Yousef 2002).

Bacterial spores and mold ascospores are more resistant to PEF treatment than vegetative

cells (Grahl and Markl 1996; Raso and others 1998a).

Microbial cell size or shape may influence the efficiency of PEF inactivation.

Wouters and others (2001b) showed that Lactobacillus species in different sizes or shapes

had different membrane permeabilization by PEF. Larger cells were more easily

permeabilized than smaller cells.

Growth stage of microorganisms is also related to the effectiveness of microbial

inactivation by PEF. Bacteria and yeasts at their logarithmic stage are more sensitive to

PEF than those at the stationary or lag growth stage (Pothakamury and others 1996). The

growth stage of microorganisms needs to be considered when developing mathematical

kinetic models that describe the inactivation kinetics of microorganisms by PEF.

Microorganisms at various growth stages would not have identical sensitivity to PEF so

that the distribution of sensitivity to PEF is an issue. A wide range of distribution needs

to be avoided to develop reliable inactivation kinetic models.

Microbial stability of PEF treated foods

An overview of the inactivation of microorganisms in foods by PEF treatment is

shown in Table 1.1. PEF treatment is advantageous for the pasteurization of juice

9

products due to their high acidity and low protein concentration. The high acidity of

juice products retards the growth of bacteria and the germination of bacterial spores

(Raso and others 1998a). Protein and electrolytes may migrate to the surface of

electrodes during PEF treatment. This can form a shielding layer, which reduces

efficiency of PEF treatment. The low protein content of juice products may not cause the

formation of the shielding layer.

Apple juice. Qin and others (1994) reported that a PEF treatment at 12 kV/cm with 20

exponential decay pulses inactivated 4 log cycles of S. cerevisiae in apple juice.

Harrison and others (1997) observed that a PEF treatment at 40 kV/cm reduced

the number of S. cerevisiae inoculated in apple juice from 8 × 107 CFU/mL to 4 × 104

CFU/mL. They showed transmission electron microscopy (TEM) micrographs of PEF

treated S. cerevisiae in apple juice. The PEF treatment disrupted yeast cells and resulted

in the almost total absence of ribosome bodies.

Evrendilek and others (2000) reported that a laboratory scale PEF treatment at 34

kV/cm for 166 µs reduced the number of E. coli O157:H7 in apple juice by 4.5 log cycles.

They also reported that a pilot plant scale PEF processing at 35 kV/cm for 94 µs

increased the microbial shelf life of apple juice and apple cider. Shelf life of an apple

cider treated by a combination of PEF at 35 kV/cm for 94 µs and a thermal treatment at

60 °C for 30 s was more than 67 d at 4 °C and 22 °C.

Cranberry juice. The effects of a pilot plant scale PEF treatment at 35 kV/cm for 195

µs on the inactivation of microorganisms in reconstituted cranberry juice were

investigated by Jin and others (1998). The PEF treatment decreased the numbers of total

Source Food Microorganism PEF system PEF treatment condition Log reduction

Qin and others (1994) Apple juice S. cerevisiae, E. coli Batch parallel plate 12 kV/cm, 20 pulses, exponential decay, < 30 °C S. cerevisiae: 3-4,

E. coli: 3

Zhang and others (1994b) Apple juice S. cerevisiae Batch parallel plate 25 kV/cm, 558 J, exponential decay, < 25 °C 3-4

Qin and others (1995a) Apple juice S. cerevisiae Continuous flow

coaxial 50 kV/cm, square wave, 29.6 °C 6.3

Harrison and others (1997) Apple juice S. cerevisiae

Continuous recirculating parallel

plate 40 kV/cm, 64 pulses, exponential decay, 15 °C 3.3

Evrendilek and others (1999) Apple juice E. coli O157:H7 Co-field flow

tubular 29 kV/cm, square wave 5

Evrendilek and others (2000) Apple juice

Lab scale: E. coli O157:H7, Pilot plant

scale: aerobic microorganisms, yeasts & molds

Co-field flow tubular

Lab scale: 34 kV/cm, 166 µs of treatment time, 1.5 mL/s, 800 ppsa

Pilot plant scale: 35 kV/cm, 94 µs of treatment time,

85 L/h, 952 Hz

Lab scale: 4.5 Pilot plant scale:

aerobic microorganisms –

2.1, yeasts & molds – 1.5

Cserhalmi and others (2002) Apple juice S. cerevisiae Co-field flow

tubular 20 kV/cm, 10.4 pulses, square wave 4

Raso and others (1998a)

Cranberry juice

Byssochlamys fulva canidiospores Coaxial 36.5 kV/cm, 22 °C 5.9

a pps: Pulses Per Second continued Table 1.1: Overview of the inactivation of microorganisms in foods by PEF treatment

10

11

Table 1.1 continued Raso and others

(1998a) Cranberry

juice Neosartorya fischeri Coaxial 51.0 kV/cm, 34 °C Not inactivated

Jin and Zhang (1999)

Cranberry juice

Aerobic microorganisms, yeasts & molds

Co-field flow tubular 40 kV/cm, 150 µs of treatment time, square wave

Aerobic microorganisms:

4.8, Yeasts & molds: 4.9

Qiu and others (1998) Orange juice Aerobic

microorganisms Co-field flow

tubular 29.5 kV/cm, 60 µs of treatment time, square wave 4.2

Sharma and others (1998)

Whey protein fortified

orange juice


Pilot plant scale system, co-field

flow tubular

32 kV/cm, 92 µs of treatment time, 3.3 µs of pulse width, 800 Hz, 79 L/h


0.5 Yeasts & molds:

3.5 Jia and others

(1999) Orange juice



flow tubular

30 kV/cm, 240 µs of treatment time, 2 µs of pulse width, 1000 Hz, 2 mL/s


2.5 Yeasts & molds:

2.5

McDonald and others (2000) Orange juice

L. mesenteroides, E. coli, L. innocua, S.

cerevisiae ascospore

CPS1 system, cathode (PurePulse

Tech)

30 kV/cm or 50 kV/cm (S. cerevisiae ascospores),

100 L/h

L. mesenteroides, E. coli, L. innocua:

5, S. cerevisiae ascospore: 2

Yeom and others (2000) Orange juice



flow tubular

35 kV/cm, 59 µs of treatment time, 1.4 µs of pulse width, 600 pps, 98 L/h

Aerobic microorganisms: 7 Yeasts & molds: 7

Min and others (2002a) Orange juice


Commercial scale system, co-field

flow tubular

40 kV/cm, 97 µs of treatment time, 2.6 µs of pulse width, 1000 pps, 500 L/h


Min and others (2002b) Tomato juice


Commercial scale system, co-field

flow tubular

40 kV/cm, 57 µs of treatment time, 2 µs of pulse width, 1000 pps, 500 L/h


continued

11

12

Table 1.1 continued

Raso and others (1998b)

Apple juice (AJ),

orange juice (OJ),

grape juie (GJ),

pineapple juice (PJ), cranberry juice (CJ)

Zygosaccharomyces balii ascospores

Vegetative cells (V), ascospores (A)

Coaxial

• AJ: 32.3 kV/cm, 19 °C • OJ: 34.3 kV/cm, 20 °C • GJ: 35.0 kV/cm, 20 °C • PJ: 33.0 kV/cm, 20 °C • CJ: 36.5 kV/cm, 22 °C

• AJ (V): 4.8 AJ (A): 3.6 • OJ (V): 4.7 OJ (A): 3.8 • GJ (V): 5.0 GJ (A): 3.5 • PJ (V): 4.3 PJ (A): 3.4 • CJ (V): 4.6 CJ (A): 4.2

Dunn and others (1987) Milk E. coli

Circular parallel stainless steel

electrode 33 kV/cm, 35 pulses, 43 °C 3

Dunn and others (1987) Milk S. dublin


electrode

36.7 kV/cm, 36 µs of treatment time, 40 pulses, 63 °C 4

Zhang and others (1994b)

Simulated milk

ultrafiltrate (SMUF)

E. coli Batch parallel plate 25 kV/cm, 20 pulses, exponential decay, < 25 °C 3

Qin and others (1994) SMUF E. coli Batch parallel plate 40 kV/cm, oscillatory decay, < 30 °C 3

Qin and others (1994) SMUF B. subtilis Batch parallel plate 16 kV/cm, 180 µs of treatment time, bipolar 5.5

Pothakamury and others

(1995a) SMUF L. delbrueckii

ATCC 11842 Batch parallel plate 16 kV/cm, 300 µs of treatment time, 40 pulses, exponential decay, < 30 °C 4-5

continued

12

13

Table 1.1 continued Pothakamury

and others (1995a)

SMUF L. subtilits ATCC 9372 Batch parallel plate 16 kV/cm, 300 µs of treatment time, 50 pulses,

exponential decay, < 30 °C 4-5

Pothakamury and others

(1995a) SMUF

L. delbrueckii ATCC 11842, L.

subtilits ATCC 9372 Batch parallel plate

16 kV/cm, 300 µs of treatment time, 40 pulses or 50 pulses (L. subtilits ATCC 9372), exponential

decay, < 30 °C

4-5

Pothakamury and others (1995b)

SMUF E. coli Batch parallel plate 16 kV/cm, 300 µs of treatment time, < 30 °C 4

Qin and others (1995c) Skim milk E. coli Batch parallel plate 50 kV/cm, 62 pulses, square wave, < 30 °C 2.5

Qin and others (1995c) SMUF E. coli Continuous parallel

plate 50 kV/cm, 48 pulses, square wave, < 30 °C 3.6

Grahl and Markl (1996) UHT milk E. coli Plain parallel carbon

electrode 22.4 kV/cm, 300 µs of treatment time 4.8

Martin and others (1997) Skim milk E. coli Batch parallel plate 40 kV/cm, exponential decay, 15 °C 6

Reina and others (1998) Milk L. monocytogenes Co-field flow

tubular 30 kV/cm, 600 µs of treatment time, square wave,

50 °C 4

Calderun-Miranda and others (1999)

Skim milk L. innocua Continuous concentric cylindrical

50 kV/cm, 64 µs of treatment time, 36 °C 2.5

Dunn and others (1987) Yogurt S. cerevisiae


electrode 18 kV/cm, 55 °C 3

Martin-Belloso and others

(1997) Liquid egg E. coli Coaxial 26 kV/cm, 37 °C 6

Keith and others (1998) Spices (dry) Yeasts Batch cylindrical 65 kV/cm, 750 µs of treatment time 4.2

13

14

aerobic plate count and yeast & mold count of cranberry juice by more than 4 log cycles.

The PEF treated cranberry juice had the shelf life of 8 months, 37 d, and 30 d at 4, 22,

and 37 °C, respectively.

Raso and others (1998a) reported that 6 log cycles of Byssochlamys fulva

conidiospores in cranberry juice were inactivated by a PEF treatment at 36.5 kV/cm.

Jin and Zhang (1999) reported that a PEF treatment at 40 kV/cm for 150 µs

reduced about 5 log cycles in the total aerobic plate count and the yeast & mold count in

cranberry juice. The PEF treatment prevented the growth of yeasts and molds in the

cranberry juice during 14 d of storage at 4 °C.

Orange juice. Qiu and others (1998) reported that a pilot plant scale PEF treatment at

29.5 kV/cm for 60 µs inactivated aerobic microorganisms in reconstituted orange juice by

4.2 log cycles. The PEF treated orange juice had a seven-month shelf life at 4 °C in

aseptic packages.

Sharma and others (1998) reported that a pilot plant scale PEF treatment at 32

kV/cm for 92 µs reduced yeast & mold counts of whey protein fortified orange juice from

1.4 × 105 CFU/mL to less than 40 CFU/mL. The PEF treated protein fortified orange

juice was microbiologically stable for 5 months at 4 °C.

Jia and others (1999) reported that the numbers of the total aerobic plate count

and the yeast & mold count in freshly squeezed orange juice were reduced by a PEF

treatment at 30 kV/cm for 240 µs. They found that the PEF treatment was as effective as

15

the thermal treatment at 90 °C for 1 min in reducing the total aerobic plate count and the

yeast & mold count in single strength orange juice. The total aerobic plate count and the

yeast & mold count of the PEF treated orange juice were < 1 est. CFU/mL at 4 °C for 6

wk.

McDonald and others (2000) inoculated Leuconostoc mesenteroides, E. coli, and

Listeria innocua into orange juice and treated the inoculated orange juice with PEF at 30

kV/cm. They achieved more than 5 log reductions of L. mesenteroides, E. coli, and L.

innocua inoculated in orange juice. They also obtained a maximum 2.5 log reductions of

S. cerevisiae ascospores in orange juice by a PEF treatment at 50 kV/cm.

Yeom and others (2000a) observed 7 log reductions in the total aerobic plate

count and the yeast & mold count in orange juice after a PEF treatment at 35 kV/cm for

59 µ. Yeom and others (2000b) compared effects of PEF treatment by a pilot plant scale

PEF system at 35 kV/cm for 59 µs on the microbial stability of orange juice to those of

thermal treatment at 94.6 °C for 30 s. The PEF treatment maintained the number of

endogenous microorganisms in orange juice at about 1 log cycle at 4, 22, and 37 °C for

112 d, which was as effective as the thermal treatment.

Min and others (2002a) studied effects of a commercial scale PEF treatment at 40

kV/cm for 97 µs on the inactivation of endogenous microorganisms in orange juice.

Commercial scale PEF treatment reduced 6 log cycles in the total aerobic plate count and

the yeast & mold count of orange juice. They reported that fresh orange juice treated by

a commercial scale PEF system showed microbial shelf life (< 4 log cycles of both total

aerobic plate count and yeast & mold count) at 4 °C for 196 d.

16

Tomato juice. Min and others (2002b) reported that a PEF treatment at 40 kV/cm for 57

µs by a commercial scale PEF system inactivated 6 log cycles of endogenous

microorganisms in tomato juice. The PEF treated juices showed microbial shelf life at 4

°C for 112 d (< 4 log cycles of both total aerobic plate count and yeast & mold count).

They observed a higher rate of microbial growth in the PEF treated tomato juice than the

tomato juice thermally treated at 92 °C fro 90 s during storage at 4 °C for 112 d. This

might be due to a relatively lower inactivation of the spores by PEF than the thermal

treatment and the germination of the survived spores during the storage.

Milk. Qin and others (1995b) treated skim milk with PEF at 40 kV/cm for 40 µs and

found that the PEF treated milk had the microbial shelf life of 2 wk at 6 °C.

Grahl and Markl (1996) inoculated UHT milk with E. coli, L. brevis,

Pseudomonas fluorescens or S. cerevisiae and treated the inoculated UHT milk with PEF

at 10-30 kV/cm with 1-22 Hz. They found that 10 kV/cm was the threshold electric field

strength, below which no inactivation of microorganisms occurs, for all the tested

pathogenic microorganisms and high fat content of milk reduced the lethal effect of PEF.

They also treated UHT milk containing different types of spores with PEF at 22 kV/cm.

They reported the PEF treatment did not significantly inactivate the endospores of

Clostridium tyrobutyricum and Bacillus cereus and ascospores of B. nivea.

Reina and others (1998) studied effects of PEF on the inactivation of L.

monocytogenes in milk. Pasteurized whole, 2%, and skim milk were inoculated with L.

monocytogenes Scott A and treated with PEF at 30 kV/cm for 600 µs at 50 °C. A 4 log

reduction of the L. monocytogenes was obtained. The results indicate that the potential

use of PEF for the inactivation of pathogens in milk.

17

Yogurt-based product. Yeom and others (2001b) studied the shelf life of a PEF treated

yogurt-based product. A yogurt-based product similar to a dairy pudding dessert was

formulated with commercial plain low fat yogurt, fruit jelly, and syrup. The yogurt-based

product was processed by a combination of a PEF treatment by a pilot plant scale PEF

system at 30 kV/cm for 32 µs and a thermal treatment at 60 °C for 30 s. The PEF

treatment combined with mild thermal treatment significantly increased the microbial

stability of the yogurt-based products during storage at 4 °C. The yeast & mold count of

the PEF treated product was less 1 log cycle while that of the untreated product was

around 4 log cycles after 90 d storage at 4 °C. The results imply feasibility to process

high viscosity foods by PEF.

Kinetic models

The study of mathematical kinetic models that describe the inactivation of

spoilage and pathogenic microorganisms by PEF is required to find PEF treatment

conditions for desired levels of microbial inactivation. To develop inactivation kinetic

models, microbial inactivation data should be obtained from reliable experiments where

artifacts of the experimental procedures are avoided (Wouters and others 2001a).

18

Microbial inactivation by PEF has been described by first-order kinetic models as

Equation 2 and 3. The natural logarithm of a survivor fraction (S), microbial count after

PEF treatment / microbial count before PEF treatment, is expressed as the function of

PEF treatment time (Equation 2) or electric field strength (Equation 3).

ln(S) = -kEt (2)

where t is the PEF treatment time (µs) and kE is the first-order kinetic constant with t.

ln(S) = -kNE (3)

where E is the electric field strength (kV/cm) and kN is the first-order kinetic constant

with E.

Zhang and others (1994a) showed that the inactivation of E. coli, Staphylococcus

aureus, and S. cerevisae by 5 to 6 log cycles yielded log-linear kinetic data and the data

were fit to the first-order kinetic models of inactivation with respect to PEF treatment

time and electric field strength.

A study on the inactivation kinetics of S. dublin in skim milk by Sensoy and

others (1997) revealed that S. dublin followed first-order kinetics with respect to electric

field strength (R2 = 0.97–0.98) over 4 log cycles of survivor fractions.

Hulsheger and others (1981) proposed a kinetic model for the microbial

inactivation by PEF. The Hulsheger’s kinetic model shown in Equation 4 describes the

kinetics of survival curves that is obtained assuming a linear relationship between the

19

natural logarithm of the survivor fraction and the electric field strength as well as a linear

relationship between the natural logarithm of the survivor fraction and the natural

logarithm of the PEF treatment time.

( ) kEE

c

c

ttS

/−−

= (4)

where t is the PEF treatment time (µs), tc is the critical treatment time below which no

inactivation of microorganism occurs (µs), E is the electric field strength (kV/cm), Ec is

the critical electric field strength below which no inactivation of microorganisms occurs

(kV/cm), and k is the specific rate constant.

The Hulsheger kinetic model accurately predicted the PEF inactivation of E. coli,

L. brevis, and P. fluorescens in sodium-alginate and UHT milk of up to 4 log cycles (R2 =

0.97–1.00) (Grahl and Markl 1996).

Martin and others (1997) showed that the inactivation of E. coli in skim milk by

PEF was successfully described by the Hulsheger’s kinetic model.

In a study by Aronsson and others (2001), the Hulsheger model was applied to the

inactivation data of E. coli, L. innocua, L. mesenteroides, and S. cerevisae. Their results

showed that the kinetic model parameters (Ec, K, tc) were dependent on microbial species.

The Hulsheger’s kinetic model is well established and fits lots of experimental data.

20

Peleg (1995) proposed a kinetic model for the microbial inactivation by PEF

based on Fermi’s equation. The Fermi’s kinetic model shown in Equation 5 represents

the survivor fraction as a function of electric field strength and number of pulses. The

Fermi’s kinetic model provides a sigmoid shaped curve.

aEE heS /)(1

1−+

= (5)

where E is the electric field strength (kV/cm), Eh is the electric field strength (kV/cm)

where RA is 0.5, and a is the parameter indicating the slope of the curve around Eh.

The Fermi’s kinetic model can explain low inactivation rates of microorganisms

after very short PEF treatment times and the tailing effect at long PEF treatment times

due to its sigmoid shaped curve (Peleg 1995). This model is also useful with microbial

inactivation data that spans several log cycles of inactivation (Peleg 1995). Peleg (1995)

tested the Fermi’s kinetic model using published data of the inactivation of L. brevis, S.

cerevisiae, S. aureus, Candida albicans, L monocytogenes I, and P. aeruginosa by PEF.

The testing the model with published data showed very good fits (R2 = 0.973 – 0.999).

Sensory and others (1997) shows that the inactivation of S. dublin in skim milk by

PEF followed the Fermi’s kinetic model (R2 = 0.97-0.98).

The log-logistic kinetic model was proposed by Cole and others (1993). This

model has been applied to lethal agents other than PEF, such as heat and chemicals. The

log-logistic kinetic model is currently studied with PEF inactivation kinetics. Survival

21

curves of S. senftenberg that covered 6-7 log cycles were modeled by the log-logistic

kinetic model (Raso and others 2000). Raso and others (2000) reported that the

experimentally measured inactivation and the estimated inactivation from the kinetic

model showed a very good agreement (R2 = 0.99).

INACTIVATION OF ENZYMES BY PEF

Mechanism

The effects of electric fields on proteins include the association or dissociation of

functional groups, movements of charged chains, and changes in alignment of helices

(Tsong and Astunian 1986).

The conformational changes of enzymes have been suggested as the mechanism

of enzyme inactivation by PEF. Castro and others (2001b) observed that alkaline

phosphatase molecules treated with PEF at 22.3 kV/cm with a pulse width of 0.78 ms

tended to associate and aggregate. The aggregates might be formed by the polarization

created by electrical charges of dipoles on the enzyme. They proposed the polarization

leading to aggregation of the enzyme as the mechanism of enzyme inactivation by PEF.

Castro and others (2001b) also suggested another possible mechanism of the

inactivation of alkaline phosphatase by PEF. They found that PEF preferentially

increased accessibility of aliphatic hydrophobic regions of alkaline phosphatase. The

aliphatic amino acids of alkaline phosphatase are originally buried inside the alkaline

phosphatase, which makes an important contribution to the maintenance of the native

globular structure. When alkaline phosphatase was exposed to PEF, some aliphatic

22

amino acids of alkaline phosphatase moved from the hydrophobic interior to the

hydrophilic surface of the alkaline phosphatase, which resulted in alkaline phosphatase

unfolding.

Yeom and others (1999) worked on the inactivation of papain by PEF. They

reported that oxidation of papain active site is not the major cause of papain inactivation

by PEF. They demonstrated the differences in secondary structures of PEF treated papain

and untreated papain using circular dichroism (CD) analysis. The inactivation of PEF

treated papain was related to the loss of α-helical structure.

Critical factors

The factors that mainly influence PEF enzymatic inactivation are (1) electric

parameters, (2) enzymatic structure, (3) PEF treatment temperature, and (4) suspension

medium (Yeom 2001).

Vega-Mercado and others (1995a) reported that the reduction of enzyme activity

was related to both electric field strength and number of pulses. Ho and others (1997)

suggested that pulse width and pulse waveform might be more important than the electric

field strength in the reduction of enzyme activity.

Min and Zhang (2002a) studied the effect of electric field strength on the

inactivation of tomato juice lipoxygenase (LOX) by PEF treatment with 22 J of energy

input. Three treatment conditions were selected to provide an identical level of energy

input (22 J) to tomato juice, but with different levels of electric field strength. The

temperature change was controlled to 25 °C for the all three PEF treatments. They found

that the LOX inactivation (%) values were 4.7, 46.3, and 60.0% when the values of

23

electric field strength of PEF treatments were 9.0, 17.8, and 30.1 kV/cm, respectively.

The inactivation of tomato juice LOX by PEF increased as the electric field strength

increased with the same level of energy input. Electric field strength was a primary

variable for the inactivation of tomato juice lipoxygenase by PEF (Min and Zhang 2002a).

The enzymatic structure was suggested as a critical factor determining the

efficiency of PEF treatment on the inactivation of enzymes. Ho and others (1997)

proposed that the differences in the secondary or tertiary structure among enzymes result

in the diverse sensitivity of enzymes to PEF. Yeom and others (1999) described that α-

helical structure is susceptible to conformational change by high elelctric fields due to its

dipole moment.

The effects of PEF treatment temperature on the inactivation of enzyme were

reported. Yeom and others (2002) reported a synergistic effect between PEF treatment

and moderate treatment temperature (50 °C) on the inactivation of orange PME. Min and

Zhang (2002a) measured residual activity values of tomato juice LOX after PEF

treatments, which had the same treatment condition other than PEF treatment temperature.

The PEF treatment at 50 °C achieved the 47% higher inactivation of tomato juice LOX

than the PEF treatment at 10 °C.

The effects of suspension medium on the inactivation of enzyme were reported by

Vega-Mercado and others (2001). They reported that casein protected protease against

PEF. They proposed that protease is protected from PEF-induced unfolding by the

presence of casein.

24

Inactivation of enzymes by PEF

An overview of the inactivation of enzymes by PEF treatment is shown in

Table 1.2. Vega-Mercado and others (1995a) reported a 90% reduction of plasmin (milk

alkaline protease) activity in a simulated milk ultrafiltrate after a PEF treatment at 45

kV/cm with 50 pulses. The authors suggested synergistic effects among the electric field

strength, the number of pulses, and the PEF treatment temperature on the inactivation of

plasmin by PEF. The activity of PEF treated plasmin was not restored after 24 h of

storage at 4 °C.

Grahl and Markl (1996) studied the effect of PEF on the inactivation of lipase,

lactoperoxidase, and alkaline phosphatase in raw milk. The inactivation of lipase,

peroxidase, and alkaline phosphatase were 65%, 25%, and < 5%, respectively, after a

PEF treatment at 21.5 kV/cm with 400 kJ/L. They found that a higher fat content of the

milk provide a higher protection effect against PEF to alkaline phosphatase.

Castro and others (2001a) reported that the activity of alkaline phosphate was

reduced by 65% in modified simulated milk ultrafiltrate (MSMUF) and by 59% in raw

milk and pasteurized and homogenized 2% milk after a PEF treatment at 18.8-22.3

kV/cm.

Ho and others (1997) treated lipase, glucose oxidase, α-amylase, peroxidase,

polyphenol oxidase, and phosphatase with PEF. Lipase, glucose oxidase, and heat stable

α-amylase exhibited a large reduction in activity from 75% to 85%. Peroxidase and

polyphenol oxidase were inactivated by 30-40%. Alkaline phosphatase was reduced by

5%. PEF treatment at 21.5 kV/cm with 400 kJ/L. They found that a higher fat content of

the milk provide a higher protection effect against PEF to alkaline phosphatase.

25

Source Enzyme Medium PEF system PEF treatment condition Reduction (%)

Hamilton and Sale (1967)

NADH dehydrogenase, succinic dehydrogenase,

hexokinase, acetylcholinesterase,

lipase, α-amylase

• NADH dehydrogenase, succinic dehydrogenase, hexokinase: extract of

pulse treated E. coli 8196 • Acetylcholinesterase:

bovine erythrocytes

Carbon electrode, polyethylene spacer with air contact

Pulse length: 20 µs, 1 Hz • NADH dehydrogenase, succinic

dehydrogenase, hexokinase: 20 – 25 kV/cm

• lipase, α-amylase: up to 30 kV/cm

No inactivation

Castro (1994) Alkaline phosphatase Simulated milk

ultrafiltrate (SMUF), raw milk, 2% milk

Gene electoporator (Kodak), disposable cuvette with air

contact, electrode gap: 0.1 cm

Exponential decay wave, 1/15 Hz, • Alkaline phosphatase in SMUF: 22.3

kV/cm, 0.74 ms of pulse width, 70 pulses • Alkaline phosphatase in raw milk or 2% milk: 18.8 kV/cm, 0.4 ms of pulse width,

70 pulses

• Alkaline phosphatase in modified SMUF: 65% • Alkaline phosphatase in raw milk or 2% milk: 59%

Vega-Mercado and

others (1995a) Plasmin SMUF A continuous flow chamber, two parallel stainless steel

electrodes

30 or 45 kV/cm, 2 µs of pulse width, 50 pulses, 0.1 Hz 90%

Vega-Mercado and others (1995b)

Proteases from Pseudomonas

fluorescens

Tryptic Soy Broth/Yeast Extract (0.6%) media

Pilot plant scale pulse generator 20 or 35 kV/cm, 10 or 20 pulses

20 kV/cm, 10 pulses: 25% 20 kV/cm, 20 pulses: 50% 35 kV/cm, 10 pulses: 60% 35 kV/cm, 20 pulses: 70%

Grahl and Markl (1996)

Lipase, peroxidase, alkaline phosphatase,

lactoperoxidase Raw milk

High voltage pulse generator with 5-15 kV d.c., Two plain

parallel carbon electrodes, electrode gap: 0.5 cm

21.5 kV/cm, 1 – 22 Hz, Energy input (Q) = 400 (kJ/L)

Lipase: 65% Peroxidase: 25%

Alkaline phosphatase: < 5% Lactoperoxidase: 0%

continued

Table 1.2: Overview of the inactivation of enzymes by PEF treatment

25

26

Table 1.2 continued

Ho and others (1997)

Peroxidase, Alkaline phosphatase, α-amylase,

lipase, lysozyme, glucose oxidase,

polyphenol oxidase, pepsin

Buffer solution or deionized water

High voltage pulse generator with ≤ 30 kV d.c., batch circular shape treatment

chamber, Two circular and parallel stainless steel

electrodes, electrode gap: 0.3 cm

2 µs of pulse width, 30 of number of pulses, 0.5 Hz

• Peroxidase: 73.3 kV/cm • Alkaline phosphatase: 83.3 kV/cm • α-amylase: 80 kV/cm • Lipase: 88 kV/cm • Lysozyme: 13.3, 50 kV/cm • Glucose oxidase: 50 kV/cm • Polyphenol oxidase: 50 kV/cm • Pepsin: 40 kV/cm

• Peroxidase: 30% • Alkaline phosphatase: 5% • α-amylase: 85% • Lipase: 85% • Lysozyme: 13.3kV/cm: 60%, 50 kV/cm: 10% • Glucose oxidase: 75% • Polyphenol oxidase: 40% • Pepsin: 150% increase

Yeom and others (1999) Papain 1 mM EDTA solution

Co-field flow tubular PEF treatment chamber, stainless

steel electrode, electrode gap: 0.2 cm

50 kV/cm, 4 µs of pulse width, 1500 Hz, 10 °C

• With activators (L-Cys and DTT): 50%

• Without activators: 90%

Barsotti and others (2002) Lactate dehydrogenase 100 mM potassium

phosphate buffer, pH 7

Batch, stainless steel electrodes, electrode gap: 0.5 cm, volume 5.7 mL

31.6 kV/cm, 0.96 µs of pulse width, 1.1 Hz, 200 of number of pulse, 30 °C,

exponential decay No inactivation

Giner and others (2000)

Pectin methyl esterase (PME) Tomato Gene electroporator

(Bio-Rad Laboratories) 24 kV/cm, 800 µs of treatment time,

exponential decay 93.8%

a pps: Pulses Per Second continued

26

27

Table 1.2 continued

Yeom and others (2000) PME Orange juice

Pilot plant scale system, co-field flow tubular PEF

treatment chamber, stainless steel tubular electrode, electrode gap: 1.0 cm

35 kV/cm, 59 µs of treatment time, 1.4 µs of pulse width, 600 ppsa, 98 L/h 88%

Yeom and others (2002b) PME Orange juice

Co-field flow tubular PEF treatment chamber, stainless

steel electrode, electrode gap: 0.2 cm

20-35 kV/cm, 2.0 or 2.2 of pulse width, 700 pps, 0.42, 0.31 mL/s 90%

Min and others (2002b) Lipoxygenase Tomato juice

Commercial scale system, co-field flow tubular PEF

treatment chamber, boron carbite tubular electrodes,

electrode gap: 1.27 cm

40 kV/cm, 57 µs of treatment time, 2 µs of pulse width, 1000 pps, 500 L/h 54%

Min and Zhang (2002a)

Lipoxygenase Tomato juice Co-field flow tubular PEF

treatment chamber, electrode gap: 0.292 cm

30 kV/cm, 60 µs of treatment time, 3 µs of pulse width, 1 mL/s, 50 °C 88.1%

27

28

Castro and others (2001a) reported that the activity of alkaline phosphate was reduced by

65% in modified simulated milk ultrafiltrate (MSMUF) and by 59% in raw milk and

pasteurized and homogenized 2% milk after a PEF treatment at 18.8-22.3 kV/cm.

Ho and others (1997) treated lipase, glucose oxidase, α-amylase, peroxidase,

polyphenol oxidase, and phosphatase with PEF. Lipase, glucose oxidase, and heat stable

α-amylase exhibited a large reduction in activity from 75% to 85%. Peroxidase and

polyphenol oxidase were inactivated by 30-40%. Alkaline phosphatase was reduced by

5%.

Yeom and others (1999) inactivated papain in a 1mM EDTA solution by a PEF

treatment at 50 kV/cm at 10 °C. A linear relationship between residual activity and

electric field strength was observed.

Vega-Mercado and others (2001) reported a 70% inactivation of the extracellular

protease from P. fluorescens by PEF at 35 kV/cm with 20 pulses. Giner and others

(2000) achieved 94% reduction of PME activity by a PEF treatment at 24 kV/cm for 8 ms.

Yeom and others (2000) observed that a pilot plant scale PEF treatment at 35 kV/cm for

59 µs inactivated 88% PME activity in orange juice. Yeom and others (2002) reported

that a PEF treatment at 25 kV/cm at 50 °C reduced 90% activity of pectin methyl esterase

(PME) in orange juice.

Min and Zhang (2002a) observed that a PEF treatment at 30 kV/cm for 60 µs at

50 °C inactivated 88.1% of tomato juice lipoxygenase. They found that the first-order

kinetic models, the Hulsheger’s model, and the Fermi’s model adequately described the

LOX inactivation by PEF. Calculated D-values for tomato LOX were 161.0, 112.9,

29

101.0, and 74.8 µs at 15, 20, 30, and 35 kV/cm at 30 °C, respectively. Min and others

(2002b) reported that a commercial scale PEF treatment at 40 kV/cm for 57 µs reduced

53 % activity of lipoxygenase in a cold break tomato juice.

The issue of the inactivation of enzymes by PEF is controversial (Yeom and

Zhang 2001a). The diversity of employed PEF systems, such as PEF treatment chamber

design, limits the comparability among inactivation data. The comparison of data from

enzyme inactivation research needs careful consideration on different PEF systems that

may influence enzyme activity differently. While conventional thermal processing

reduces the activity of many enzymes to a large extent, PEF processing is unlikely to

have a similar effect to the thermal treatment (Lelieveld and others 2001).

SENSORY AND NUTRITIONAL PROPERTIES OF PEF TREATED JUICES

Thermal treatment has been widely used to inactivate spoilage & pathogenic

microorganisms and enzymes to extend shelf life of juice products. However, thermal

treatment can lower the sensory and nutritional qualities of juices (Chen and others 1993).

PEF has been intensively studied as a nonthermal agent to inactivate microorganisms in

foods while reducing the loss of flavor, color, and nutrients of juices from heat (Mertens

and Knorr 1992; Dunn 2001).

Orange juice. Conventional orange juice pasteurization either by high temperature short

time (HTST) or ultra high temperatures (UHT) causes significant loss of fresh flavor,

vitamin C, and color (Braddock 1999). Qiu and others (1998) reported that a PEF

treatment at 29.5 kV/cm for 60 µs retained more volatile flavors and vitamin C of orange

30

juice than a thermal treatment at 90 °C for 15 s while providing microbial stability for > 1

year at 4 °C. The PEF treatment reduced 5-9% volatile flavor compounds while the

thermal treatment reduced 25% volatile compounds compared to 100% of freshly

squeezed orange juice. After 90 d of storage at 4 °C, vitamin C content of the PEF

treated orange juice was 68% while that of the thermally treated orange juice was 46%

compared to 100% of freshly squeezed orange juice.

Sharma and others (1998) reported a PEF treatment at 32 kV/cm for 92 µs

reduced the yeast & mold count of a whey protein fortified orange juice by 3.5 log cycles.

The whey protein fortified orange juice treated by the PEF treatment contained more its

natural color than the orange juice thermally treated at 71 °C for 25 s. They also reported

that the PEF treatment caused less protein denaturation and higher retention of vitamin C

in the orange juice than the thermal treatment. The PEF treatment denatured 6-7% whey

protein in the protein fortified orange juice while the thermal treatment denatured 55% of

the whey protein.

Jia and others (1999) reported that a PEF treatment at 30 kV/cm for 240 µs was as

effective as a thermal treatment at 90 °C for 1 min in reducing the total aerobic plate

count and the yeast & mold count. The % reductions of total flavor compounds after the

PEF treatment and after the thermal treatment were 3% and 22%, respectively. The

concentration of ethyl butyrate in orange juice was decreased by 9.7% and 22.4% after

the PEF treatment and the thermal treatment, respectively. Decanal was not reduced by

the PEF treatment while 41% of decanal was reduced by the thermal treatment.

31

Yeom and others (2000b) compared the microbial stability, sensory properties,

and vitamin C contents of orange juice treated by a pilot plant scale PEF system at 35

kV/cm for 59 µs with those of thermally treated orange juice at 94.6 °C for 30 s. Both

PEF and thermal treatments provided microbial stability at 4, 22, and 37 °C for 112 d.

The PEF treated orange juice contained significantly higher concentrations of vitamin C

and flavor compounds than the thermally treated orange juice during storage at 4 °C. The

PEF treated orange juice had lower browning index, higher whiteness (L), and higher hue

angle values than the thermally treated orange juice during storage at 4 °C.

Min and others (2002a) compared the concentrations of vitamin C and volatile

flavor compounds of tomato juice processed PEF at 40 kV/cm for 97 µs with those of

tomato juice thermally processed at 90 °C for 90 s. Both PEF treatment and thermal

treatment inactivated 6 log cycles of aerobic microorganisms and yeasts & molds. The

PEF treatment did not change the concentration of vitamin C significantly while the

thermal treatment reduced 19% of vitamin C. Orange juice processed by the commercial

scale PEF system retained more vitamin C than thermally treated orange juices at 4 °C

for 84 d. Squires and Hanna (1979) reported that orange juice should contain at least 25

mg of vitamin C per 100 mL at the time of expiration date to provide 100% of the U.S.

Recommended Daily Allowances (USRDA) requirement. The concentration of vitamin

C in the PEF treated orange juice decreased to 25 mg/100 mL at 4 °C after 56 d, which is

longer than the 42 d of thermally treated orange juice. The PEF treated orange juice

retained more fresh flavor compounds of α-pinene, octanal, d-limonene, and decanal than

32

the thermally treated orange juice. The PEF treatment decreased 12% myrcene while the

thermal treatment reduced 37% myrcene compared to 100% of freshly squeezed orange

juice.

Apple juice. Qin and others (1995b) reported that a PEF treated apple juice had sensory

characteristics similar to those of freshly squeezed apple juice. Panels could not find

significant differences in sensory properties between the PEF treated apple juice and

freshly squeezed apple juice.

Vega-Mercado and others (1997) reported that PEF treated apple juice showed no

significant changes in physicochemical and sensory properties during the storage at room

temperature for 8 wk.

Evrendilek and others (2000) studied effects of a PEF treatment at 35 kV/cm for

94 µs on the shelf life extensions of apple juice and apple cider. The PEF treatment

reduced 4.5 log cycles of E. coli O157:H7 in the apple juice. They reported that the PEF

treatment did not change the concentration of vitamin C. They conducted a paired

preference test to determine if there is any difference in the sensory properties between

the PEF treated and untreated apple cider samples. The results of the test indicated that

the acceptability of fresh apple juice is not affected by the PEF treatment.

Cranberry juice. Jin and Zhang (1999) studied effects of a PEF treatment on the shelf

life of cranberry juice. They used a PEF treatment at 40 kV/cm for 150 µs, which

reduced the total aerobic plate count and the yeast & mold count of cranberry juice by

about 5 log cycles. They reported that cranberry juice collected immediately after the

PEF treatment showed similar flavor profiles as the untreated cranberry juice. No

significant differences were observed in the content of anthocyanin and L values between

33

PEF treated cranberry juice and untreated cranberry juice. A thermal treatment at 90 °C

for 90 s significantly altered overall flavor profiles and reduced the anthocyanin pigment

content of cranberry juice.

Tomato juice. Min and others (2002b) studied effects of a PEF treatment by a

commercial scale PEF system on the quality of tomato juice. Tomato juice was prepared

by hot break at 88 °C for 2 min and then treated by PEF at 40 kV/cm for 57 µs or treated

thermally at 92 °C for 90 s. The PEF treated tomato juice retained more vitamin C than

thermally treated juice at 4 °C for 42 d. The flavor compounds of trans-2-hexenal, 2-

isobutylthiazole, cis-3-hexanol were retained more in the PEF treated tomato juice than in

the thermally treated or untreated tomato juice during storage at 4 °C for 112 d. The

browning index and the concentration of 5-hydroxymethyl-2-furfural of the PEF treated

juice were significantly lower than those of the thermally treated or untreated tomato

juice during storage at 4 °C for 112 d. No significant differences were observed in the

concentration of lycopene, Brix, pH, and viscosity between the PEF treated and the

thermally treated tomato juices during storage at 4 °C for 112 d. Sensory evaluations

indicated that the PEF treated tomato juice had higher flavor intensity and overall

acceptability than the thermally treated tomato juice.

34

PACKAGING ISSUES

Success in extending shelf life of an initially high quality food would depend on

food packaging. Aseptic food packaging is effective for producing shelf stable food

products that have quality advantages over their conventionally processed products

(Barbosa-Canovas and others). Qin and others (1995b) mentioned the effectiveness of

aseptic food packaging to extend shelf life of PEF treated foods.

Plastics and paper-laminated materials are widely used as packaging materials for

aseptic food packaging. A proper packaging material needs to be selected to extend the

initial high quality of flavor, color, and nutrients of PEF treated foods. Ayhan and others

(2001) investigated the effects of packaging materials on the quality of orange juice

treated by a pilot plant scale PEF system at 35 kV/cm for 59 µs. The PEF treated orange

juice was filled into four different packaging materials, sanitized glass, polyethylene

terephthalate (PET), high-density polyethylene (HDPE), and low density polyethylene

(LDPE) bottles inside a sanitized glove box. They reported that glass bottles and PET

bottles were effective to keep flavor compounds, vitamin C, and color of PEF treated

orange juice during storage at 4 °C for 112 d. The concentrations of vitamin C in glass

bottles and PET bottles were higher than the concentrations of vitamin C in HDPE bottles

or LDPE bottles during storage at 4 °C for 112 d. Polyethylene (PE) has a low barrier

property to oxygen (Baner 1999). Vitamins and the flavor compounds were labile in

polyethylene (HDPE, LDPE) bottles, which might be due to the low barrier property of

polyethylene to oxygen. Packaging materials with low transmission rate of oxygen may

need to be used for the packaging of PEF treated foods to extend shelf life.

35

The presence of oxygen in the package headspace reduces the shelf life of juice

products (Shaw 1992). Marshall and others (1986) reported greater reduction in vitamin

C levels with higher concentration of oxygen in the headspace. To control oxygen in

headspace, modified atmosphere packaging (MAP) can be applied. The shelf life

extension of foods using modified atmosphere packaging has been reported by many

researchers (Jeon and Lee 1999; Bagorogoza and others 2001; Salvador and others 2002).

The modification of atmosphere can be achieved by removing air or replacing air with a

controlled mixture of gases (Robertson 1993). Nitrogen is frequently used as a filler gas

in modified atmosphere packaging to reduce the concentration of other gases in the

packaging material or headspace (Robertson 1993). Modified atmosphere packaging can

be used to extend shelf life of PEF treated foods by limiting oxygen in headspace.

The degradation of flavor compounds can occur not only by oxidation but also by

the permeation of flavor compounds through packaging materials and the absorption of

flavor compounds into packaging materials. A greater absorption rate is found if the

flavor compound has the similar chemical structure or similar polarity to the functional

group of the packaging material (Landois-Garza and Hotchkiss 1987). As an example,

more d-limonene and α-pinene (volatile flavor compounds of orange juice) are absorbed

more easily in low density polyethylene (LDPE) than polyethylene terephthalate (PET),

polyvinylidene chloride (PVDC), or ethyl vinyl alcohol copolymer (EVOH). LDPE

absorbs more d-limonene and α-pinene than the other materials because the non-polar

hydrocarbon of LDPE has a strong affinity to non-polar terpene hydrocarbons of the

flavor (Sheung 1995). Many researchers have investigated absorption and diffusion

phenomena of flavor compounds of foods with various food packaging materials (Baner

36

and others 1991; Ikegami and others 1991; Nielsen and others 1992; Van Willige and

others 2000). To reduce the absorption of the fresh flavor compounds of a PEF treated

food, a packaging material with low diffusivity and solubility for the fresh flavor

compounds would be used.

CONCLUSION

PEF treatment has been studied as a nonthermal food preservation method. Many

publications report that PEF treatment significantly increased shelf life of foods while

reducing the loss of flavor, color, and nutrients of foods. PEF treatment system was

scaled up to commercial processing scale. Commercial scale PEF processing extended

the shelf life of juice products successfully. The initial high fresh qualities of PEF treated

juice products may be extended over time by selecting proper juice packaging materials

and methods.

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the quality of orange juice and comparison with heat pasteurization. J Agric Food

Chem 48:4597-4605.

49

Yeom HW, Zhang QH. 2001a. Enzymatic inactivation by pulsed electric fields: A review.

In: Barbosa-Canovas GV, Zhang QH, editors. Pulsed electric fields in food processing:

Fundamental aspects and applications. Lancaster, PA: Technomic Publishing Company,

Inc. p 57-63.

Yeom HW, Evrendilek GA, Jin ZT, Zhang QH. 2001b. Processing of yogurt-based

product with pulsed electric fields. IFT 2001 Annual Meeting Book of Abstracts, paper

no. 28-7. 47 p.

Yeom HW, Zhang QH, Chism GW. 2002. Inactivation of pectin methyl esterase in

orange juice by pulsed electric fields. J Food Sci 67(6):2154-2159.

Yin Y, Zhang QH, Sudhir KS, inventors; The Ohio State University, assignee. 1997 Nov.

25. High voltage pulsed electric field treatment chambers for the preservation of liquid

food products. U.S Patent 5,690,978.

Zhang Q, Monsalve-Gonzalez A, Barbosa-Canovas GV, Swanson BG. 1994a.

Inactivation of E. coli and S. cerevisiae by pulsed electric fields under controlled

temperature conditions. Trans ASAE 37(2):581-587.

Zhang Q, Chang FJ, Barbosa-Canovas GV, Swanson BG. 1994b. Inactivation of

microorganisms in semisolid foods using high voltage pulsed electric fields. Lebensm

Wiss u Technol 27(6):538-543.

Zhang Q, Monsalve-Gonzalez A, Qin BL, Barbosa-Canovas GV, Swanson BG. 1994c.

Inactivation of Saccharomyces cerevisiae in apple juice by square-wave and

exponential decay pulsed electric fields. J Food Process Eng 17:469-478.

Zhang Q, Barbosa-Canovas GV, Swanson BG. 1995a. Engineering aspects of pulsed

electric field pasteurization. J Food Eng 25:261-281.

50

Zhang Q, Qin BL, Barbosa-Canovas GV, Swanson BG. 1995b. Inactivation of E. coli for

food pasteurization by high-strength pulsed electric fields. J Food Process Preserv

19:103-118.

51

CHAPTER 2

Effects of Commercial Scale Pulsed Electric Field Processing on the Quality of

Tomato Juice

ABSTRACT

Effects of commercial scale pulsed electric field (PEF) processing on the quality of

tomato juice were studied and compared with those of thermal processing. Tomato juice

was prepared by hot break at 88 °C for 2 min or cold break at 68 °C for 2 min and then

thermally processed at 92 °C for 90 s or PEF processed at 40 kV/cm for 57 µs. Thermally

processed, PEF processed, and unprocessed control tomato juices were packed into 50

mL sterilized polypropylene tubes in a sanitary glove box and stored at 4 °C for 112 d.

Both thermally and PEF processed juices showed microbial shelf life at 4 °C for 112 d.

The lipoxygenase activities of thermally and PEF processed juices were 0 and 47%,

respectively. PEF processed juice retained more ascorbic acid than thermally processed

juice at 4 °C for 42 d (p<0.05). No significant differences were observed in the

concentration of lycopene, Brix, pH, and viscosity between thermally and PEF processed

52

juices during the storage (p>0.05). Sensory evaluations indicated that PEF processed

juice had higher flavor intensity and overall acceptability than thermally processed juice

(p<0.01).

Key words: Pulsed electric field, thermal processing, tomato juice, shelf life, ascorbic

acid.

53

INTRODUCTION

Tomato is the second-most consumed vegetable around the world (Hayes and

others 1998). Most tomatoes are consumed as processed products such as tomato juice,

paste, puree, ketchup, sauce, and canned tomatoes (Gould 1992). Processed tomato

products are important sources of minerals and vitamins in diets (Thakur and others

1996). Thermal processing is the most common method to extend the shelf life of tomato

juice by inactivating microorganisms and enzymes. Flavor, color, taste, and nutritive

value are considered as the major quality attributes of foods and influence the consumer’s

choice. Thermal processing can lower the sensory and nutritional qualities of foods

(Braddock 1999; Rouseff and Leahy 1995). Therefore, alternative juice processing

methods have been sought for tomato industries to produce higher quality tomato juice.

PEF processing has been extensively studied as a nonthermal food preservation

method (Mertens and Knorr 1992; Dunn 2001). PEF processing is very effective for the

pasteurization of juices due to their high acidity and low protein concentration. The high

acidity of juices retards the growth of bacteria and the germination of bacterial spores

(Raso and others 1998a). A shielding layer of PEF may be formed on the surface of

electrodes when charged molecules including proteins migrate to the surface of

electrodes and monopolar pulses are successively applied (Zhang and others 1995). The

low protein content and use of bipolar pulses may not cause the formation of shielding

layer.

54

Laboratory or pilot plant scale PEF processing has been successfully conducted

and have increased shelf life of juice products, minimizing the loss of flavor, color, and

nutrients of juice products (Evrendilek and others 2000; Jin and Zhang 1999; Qiu and

others 1998; Yeom and others 2000). However, no research has been done with

commercial scale PEF processing. Besides, no information is available about the effects

of PEF on lipoxygenase activity, lycopene, ascorbic acid, physical properties, and

sensory properties of tomato juice. The objectives of this research were (1) to investigate

the effects of commercial scale PEF processing on the inactivation of endogenous

microorganisms in tomato juice and (2) to study and compare the effects of thermal

processing and commercial scale PEF processing on the quality of tomato juice including

the microbial stability, lipoxygenase activity, lycopene content, ascorbic acid content,

particle size distribution, pH, Brix, and viscosity during storage at 4 °C for 112 d and on

the sensory properties of appearance, color, texture, flavor, and overall acceptability.

MATERIALS AND METHODS

Tomatoes

Roma-type Midwest tomatoes were supplied by Hirzel Canning Co. & Farms

(Toledo, OH) throughout the year 2001 tomato season. Tomatoes were processed on the

same day of harvesting.

55

Preparation of tomato juice

The 1,100 kg fresh tomatoes were used to prepare tomato juice. Fresh raw

tomatoes were washed in a soak tank with air agitation and then washed again with 150

psi sprayed water while being conveyed on a roller conveyor. Tomatoes were sorted and

chopped by a mill (Model D, The W. J. Fitzpatrick Co., Chicago, IL) equipped with a

1.91 cm screen. The chopped tomatoes were heated in a tubular heat exchanger (H2187C

type, Specialty Brass Co., Kenosha, WI) for 2 min at 88 °C for hot break and at 68 °C for

cold break. Cold break tomato juice was used only for the study of lipoxygenase activity

during the storage at 4 °C for 112 d. Tomato juice was prepared by a screw type

extractor (CJE-360-D28, Chisholm-Ryder Co., Niagara Falls, NY) with a screen of 1.27

cm diameter.

PEF processing system

The OSU-6 commercial scale PEF processing system is illustrated in Figure 2.1.

The OSU-6 commercial scale PEF processing system consists of an aseptic drink

processor (TetraPak, General-Guisan, Switzerland), a high voltage pulse generator

(Diversified Technology, Inc., Bedford, MA), and co-field tubular PEF chambers (The

Ohio State University, Columbus, OH). The aseptic drink processor monitors and

controls production rates, temperatures, and pressures during PEF processing. The

production rates can be controlled in the range of 400-2000 L/h. The high voltage pulse

generator provides bi-polar squared waveform pulses with a peak voltage of 60 kV and

600 A into multiple PEF chambers. The 60 kV power supplies charge storage

capacitances and these capacitors are partially discharged by a series of solid-state

switches to form the square wave bipolar pulses. The pulse generator operates at a

56

Figure 2.1: Flowchart of OSU-6 commercial scale PEF processing system

Heat exchanger (heating)

Product tank

Pump

Back pressure control

PEF chamber

Sanitary glove box

Waste

Heat exchanger

Holding T

ube

High voltage pulse generator

+/- 60 kV

Heat exchanger

Heat exchanger

Heat exchanger

Heat exchanger

PEF chamber PEF chamber PEF chamber

Aseptic drink processor

57

maximum repetition rate of 2000 pulses per second. Pulses were monitored with a high

voltage probe (attenuation factor 10,000:1, VD-60, Northstar, Albuquerque, NM), current

monitors (attenuation factor 100:1, Model 110, Pearson, Palo Alto, CA), and

oscilloscopes (TDS-210, Tektronix, Beaverton, OR). Each co-field tubular PEF chamber

consists of two boron carbite tubular electrodes and a tubular insulator body made of

ceramic (Yin and others 1997). The inner diameter of the cylindrical processing zone is

0.808 cm and the distance between the electrodes is 1.270 cm. The PEF chambers can be

connected up to 8 in parallel for electrical flow and in series for fluid flow. The peak

current through each PEF chamber was 75 A.

Thermally processed, PEF processed, and control tomato juices. Processing

conditions for thermally processed, PEF processed, and control tomato juices are listed in

Table 2.1. The production rate was 500 L/h for all thermally processed, PEF processed,

and control tomato juices. Tomato juice processing was performed in the following

order: Sterilization-in-place (SIP), thermal processing, PEF processing, control

collection, and clean-in-place (CIP).

The entire fluid handling system was sterilized by SIP at 121 °C for 30 min. The

fluid handling system was cooled to 25 °C after SIP.

For thermal processing, tomato juice was held at 92 °C for 90 s in a holding tube

and then cooled to 25 °C by the heat exchanger following the holding tube (Figure 2.1).

PEF remained off throughout thermal processing. The cooled tomato juice was packaged

inside a sanitary glove box (Figure 2.1). The processing mode was switched from

thermal to PEF after 10 min of filling thermally processed tomato juice.

58

Processing parameter

Thermally

processed

tomato juice

PEF

processed

tomato juice

Control

tomato juice

Production rate (L/h) 500 500 500

Electric field strength (kV/cm) 0 40 0

Pulse duration time (µs) 0 2 0

Total PEF treatment time (µs) 0 57 0

Number of PEF chamber 6 6 6

Holding temperature (°C) 92 45 45

Holding time (s) 90 90 90

Temperature change per a pair of PEF

chamber (°C)

0

8

0

Temperature before filling (°C) 25 25 25

Table 2.1: Processing conditions for thermally processed, PEF processed, and control tomato juices

59

PEF processing conditions were the electric field strength of 40 kV/cm, pulse duration

time of 2 µs, and total PEF treatment time of 57 µs. Tomato juice was pumped to the

PEF chambers without thermal processing. Temperature of tomato juice was controlled

at 45 °C before entering each set of PEF chambers. This temperature control was done

by the heat exchangers at the upstream of each set of two chambers (Figure 2.1). PEF

processed tomato juice was cooled to 25 °C prior to packaging inside the sanitary glove

box. After 10 min of filling PEF processed tomato juice, PEF was turned off in

preparation for control collection.

For control tomato juice, tomato juice was passed through the system without

thermal or PEF processing and packaged inside the sanitary glove box.

CIP was conducted after collecting control tomato juice. The entire fluid

handling system was cleaned by circulating an alkaline detergent (0.5%, Super Alkali

850, Chaska Chemical, Savage, MN, U.S.A) at 70 °C for 20 min. The system was cooled

to 40 °C and rinsed with water. The system was cleaned again by circulating an acidic

detergent (0.5%, AC-30-E, Ecolab, Saint Paul, MN, U.S.A) at 70 °C for 20 min. The

system was cooled to 40 °C and rinsed with water. The flow rates for detergents and

water were 2500 L/h.

Packaging and storage

Thermally processed, PEF processed, and control tomato juices were packaged

into the 50 mL sterilized polypropylene tubes (Corning, Acton, MA) inside a sanitary

glove box filling unit (The Ohio State University, Columbus, OH). The glove box was

prepared by following the method of Ayhan and others (2001a). The glove box consisted

60

of a gas-tight stainless steel box with a glass window, a pair of gloves, a double-door

transfer tunnel, a germicidal UV lamp (Cole Parmer, Vernon Hills, IL), and a HEPA air

filter (Fisher Scientific, Pittsburgh, PA). The glove box was sanitized by spraying and

swabbing 35% hydrogen peroxide and lighting germicidal UV at 254 nm with an

intensity of 76 µW/cm2. The HEPA air filter with 0.3 µm pore size and a 1600 cm2

filtration area was installed to supply positive pressure with bacteria-free air inside the

glove box. Each sample tube was covered with aluminum foil to prevent the exposure of

tomato juice to light. Packaged tomato juices were stored at 4 ºC.

Microbial inactivation study

The hot break tomato juice was incubated at 22 ºC for 3 d to let endogenous

microorganisms proliferate. The microbial counts of total aerobic microorganisms and

yeasts & molds were 6.0 log10 CFU/mL and 5.9 log10 CFU/mL, respectively, after the

incubation.

Plate count agar (PCA) and acidified potato dextrose agar (PDA) were used to

enumerate total aerobic plates and yeasts & molds, respectively, in thermally processed,

PEF processed, and control tomato juices. PCA, PDA, and Peptone water were

purchased from Difco (Detroit, MI). PDA was acidified with 10% tartaric acid (Sigma-

Aldrich, St. Louis, MO). Tomato juice was diluted with 0.1% sterile peptone water up to

10-4 dilution and plated by a spiral autoplater (model 3000, Spiral Biotech Inc., Bethesda,

MD). PCA plates were incubated at 30 °C for 48 h. PDA plates were incubated at 22 °C

for 5 d.

61

Shelf life study

Hot break or cold break tomato juice was cooled to 45 °C and thermally

processed or PEF processed without incubation. Cold break tomato juice was used only

for the study of lipoxygenase activity. Thermally processed, PEF processed, and control

tomato juices were packaged and stored at 4 °C. The shelf life study was conducted for

112 d.

Microbial stability. The microbial stability was determined by the same method

as described for the microbial inactivation study. The initial microbial counts of total

microorganisms and yeasts & molds of control were 1.3 log10 CFU/mL and 1.0 log10

CFU/mL, respectively.

Lipoxygenase activity. Lipoxygenase activity was measured by the methods of

Ben-Aziz and others (1970), Smith and others (1997), and Tangwongchai and others

(2000). The 50 g tomato juice was homogenized with 100 mL 0.1 M phosphate buffer

(pH 6.5), containing 1 mM EDTA and 0.1% (w/v) Triton X-100. The homogenate was

filtered through a double layer of cheese cloth and centrifuged at 20000 × g for 40 min at

5 °C. The enzyme solution was prepared by diluting 0.5 mL supernatant with 1.0 mL 0.1

M phosphate buffer (pH 6.5). A substrate solution containing linoleic acid (2.5 × 10-3 M)

and Tween 20 (0.2%) was prepared according to Ben-Aziz et al. (1970). The substrate

solution was diluted to 2.5 × 10-5 M with 0.2 M phosphate buffer (pH 6.5). The 0.1 mL

enzyme solution was pipetted into the cuvette containing 2.4 mL of the substrate solution

at zero time. The absorbance was measured at 234 nm for 3 min at a 15 s interval using a

Spectronic Genesys 5 spectrometer (Milton Roy, Rochester, NY) at 22 °C. The rate of

62

the reaction was automatically computed from the linear portion of the curve. The 1 unit

of lipoxygenase activity was defined as a change of 0.001 units of absorbance per minute

and milliliter of enzyme extract.

Lycopene analysis. A hexane extract was obtained by using the method of

Chandler and Schwartz (1987) with some modifications. The 5.0 g tomato juice was

homogenized in 50 mL methanol (Fisher Scientific, Pittsburgh, PA) with 1.0 g calcium

bicarbonate (Sigma-Aldrich, St. Louis, MO) and 3.0 g Celite (Sigma-Aldrich, St. Louis,

MO). The homogenate was successively extracted with a 50 mL mixture of 1:1

acetone/hexane (v/v, Fisher Scientific, Pittsburgh, PA) and vacuum-filtered through

Whatman paper No. 1 and No. 42 (Whatman International Ltd., UK). The filtrant was

combined in a separatory funnel. Distilled water was added into the separatory funnel to

induce the separation of the hexane layer.

The lycopene in the hexane extract was analyzed by a high-performance liquid

chromatography (HPLC, Series 1100, Hewlett-Packard, Palo Alto, CA). A reversed

phase C18 column (201TP54, Vydac, Holland, MI), a guard column packed with C18

stationary phase (Vydac, Holland, MI), a diode-array detector (HP 1100 DAD,

Wilmington, DE), and an autosampler were used for all separations. The separation was

performed at 1.0 mL/min using a linear gradient of 32 to 53% methyl-tert butyl ether

(Fisher Scientific, Pittsburgh, PA) in methanol for 60 min. HPLC solvents were certified

HPLC grades. The lycopene in tomato juice was quantified from HPLC profile by using

the lycopene standard from tomato (Sigma-Aldrich, St. Louis, MO).

63

Ascorbic acid analysis. The concentration of ascorbic acid in the tomato juice

was measured by the method of Howard and others (1987) using a high-performance

liquid chromatography (HPLC) system (Hewlett Packard, 1050 series, Wilmington, DE).

The HPLC was equipped with an autosampler and a UV-VIS detector set at 254 nm. The

HPLC chromatograph peak area was calculated using a Hewlett-Packard integrator

(HP3396 series II). A reverse phase C18 column (5 µm particle size, 4.6 mm diameter,

250 mm length, Alltech, Deerfield, IL) along with a Hewlett-Packard C18 guard column

was used to separate ascorbic acid. A mixture of methanol and acidified water (10:90,

v/v) was used as a mobile phase. The acidified water was prepared by adding phosphoric

acid (0.01%, v/v, Sigma-Aldrich, St. Louis, MO) to distilled water. The mobile phase

was filtered through a 0.45 µm membrane filter (Micron Separations Inc., Westboro,

MA) and degassed by helium gas before being passed though the column. The flow rate

of mobile phase was 1.0 mL/min. The L-ascorbic acid from Sigma Chemical Co (St.

Louis, MO) in concentrations ranging from 2 to 100 mg/100 mL was used to obtain a

standard calibration curve. Tomato juice was centrifuged at 12535g for 10 min at 22 °C

in a Beckman Microfuge E (Beckman Instruments Inc., Palo Alto, CA) to remove pulp

and coarse particles. The 10 µL supernatant was injected into the column by the HPLC

autosampler.

Particle size distributions. The particle size of tomato juice was analyzed by a

Mastersizer (Malvern Instruments, Inc., Worcs, U.K.). A 10 mL of tomato juice was

diluted with 500 mL distilled water and circulated in the Mastersizer at 2,000 rpm. A

computer equipped with Mastersizer Micropulus 2.15 (Malvern Instruments, Inc., Worcs,

U.K.) recorded distributions of the particle size of tomato juice.

64

The D[4, 3], D[3, 2], D(v, 0.1), D(v, 0.5), and D(v, 0.9) were reported. The

D[4, 3], D[3, 2], and D(v, 0.5) were used for the comparison of particles sizes of

thermally processed, PEF processed, and control tomato juices. The D[4, 3] is the

volume moment mean of particles and defined as the following equation where d is the

diameter of one unit.

D[4, 3] = ∑∑

3

4

dd

The D[3, 2] is the surface area moment mean of particles and determined as:

D[3, 2] = ∑∑

2

3

dd

The D[4, 3] and D[3, 2] are used to measure particles on the basis of volume and surface

area, respectively. The D(v, 0.1) is the size of particle for which 10% of the sample is

below this size. The D(v, 0.5) is the median of the particle size distribution on the basis

of volume. D(v, 0.9) gives a size of particle for which 90% of the sample is below this

size (Malvern Instruments 1995).

Brix and pH. The Brix of tomato juice was measured using a hand-held

refractometer (Fisher, Pittsburgh, PA). The pH of tomato juice was measured using a pH

meter (Orion, 370, Beverly, MA) at 22 °C.

Viscosity. The viscosity of tomato juice was measured by using a Brookfield

viscometer (LVDVII+, Brookfield Engineering Laboratories, Inc., Stoughton, MA) with

a UL adapter. Viscosity was determined at 22 °C and 4 rpm with 16 mL of tomato juice

placed in the UL adapter.

65

Sensory evaluation

Thermally processed and PEF processed tomato juices, stored at 4 °C for 1 week,

were used for the sensory evaluation. The 1 week was needed for Silliker Laboratories

(Columbus, OH) to confirm the absence of pathogen microorganisms, Salmonella spp.,

Listeria monocytogenes, and Escherichia coli O157:H7 in both thermally processed and

PEF processed tomato juices. A 30-member panel participated in the sensory tests. The

panelists consisted of graduate students in the department of Food Science and

Technology at the Ohio State University and members of the food industry. The

panelists were asked to rate the intensity of appearance, color, texture, flavor, and overall

acceptability by marking on a horizontal line corresponding to the amount of the

perceived stimulus. A hedonic scale of 1 to 9 was used for each attribute. The higher

number represents stronger intensity of attributes. Thermally processed and PEF

processed tomato juices were served in randomly numbered plastic cups on a tray with a

cup of water and a piece of non-salted cracker at the beginning of the evaluation.

Statistical analysis

Analysis of variance and Tukey’s multiple comparisons method at the 1% and 5%

significance levels were performed for the determination of significant differences among

thermally processed, PEF processed, and control tomato juices. The entire analyses were

duplicated with 4 measurements. Minitab 13.31 (Minitab, Inc., State College, PA) was

used for all statistical analyses.

66

RESULTS AND DISCUSSION

Effects of thermal processing and PEF processing on the microbial inactivation

Microbial counts of total aerobic microorganisms and yeasts & molds in tomato

juice incubated at 22°C for 3 d were 6.0 and 5.9 log10 CFU/mL, respectively. Both total

aerobic plate count and the yeast & mold count were reduced to < 10 CFU/mL est. after

either thermal or PEF processing. Thermal processing and commercial scale PEF

processing reduced endogenous microorganisms of tomato juice by 6 logs.

Successful inactivation of yeasts including Saccharomyces cerevisiae and

Zygosaccharomyces Bailii by PEF has been reported (Cserhalmi and others 2002; Qin

and others 1995; Raso and others 1998b; Zhang and others 1994). Microscopic

examination indicated that the major microorganism in control tomato juice was yeast.

Yeasts & molds are the major spoilage microorganisms in juice products due to their

survival and growth at low pH environments and use of sugars and vitamins in juices

(Deak and Beuchat 1996). Yeasts are known to be more tolerant to high temperature than

bacteria (Kimball 1991; Murdock and others 1953). The temperature of tomato juice

increased from 45 °C to 53 °C and maintained at 53 °C for 5 s during the PEF processing.

In order to investigate the effect of thermal treatment at 53 °C for 5 s on the yeast & mold

counts of the tomato juice incubated at 22 °C for 3 d, the incubated tomato juice was only

thermally processed at 53 °C for 5 s and plated on PDA. The numbers of yeasts & molds

on PDA before and after the thermal processing at 53 °C for 5 s were 5.9 log10 CFU/mL

and 5.8 log10 CFU/mL, respectively, and were not significantly different each other

67

(p > 0.05). The temperature increase from 45 °C to 53 °C and the holding at 53 °C for 5

s would not cause the 5.9 log reduction of yeasts & molds. Therefore, the inactivation of

yeasts & molds would be mainly due to PEF. Commercial scale PEF processing was

effective for the inactivation of endogenous microorganisms in tomato juice.

Effects of thermal processing and PEF processing on the microbial stability during

storage

Effects of thermal processing and PEF processing on the total aerobic plate counts

and the yeast & mold counts of tomato juice during the storage at 4 ˚C for 112 d are

shown in Figures 2.2 and 2.3, respectively. The initial total aerobic plate count of the

control hot break tomato juice at 0 d was 1.0 log10 CFU/mL. Both PEF and thermally

processed tomato juices had initially < 10 CFU/mL est. aerobic microorganisms at 0 d.

The number of total aerobes of thermally processed tomato juice was less than 2 log10

CFU/mL during the storage. The number of total aerobic microorganisms of PEF

processed tomato juice was less than 4 log10 CFU/mL during the storage at 4 °C for 112

d while that of control tomato juice reached 5 log10 CFU/mL at 4 °C after 49 d. Control

tomato juice was not sampled after 49 d due to the gas formation by multiplied

microorganisms. The initial yeast & mold count of control hot break tomato juice at 0 d

was 1.3 log10 CFU/mL. The yeast & mold counts of thermally processed tomato juice

was less than 1 log10 CFU/mL and those of PEF processed tomato juice was less than 4

log10 CFU/mL during the storage at 4 °C for 112 d.

68

0

1

2

3

4

5

6

0 20 40 60 80 100 120

Storage time (days)

Log

cfu/

mL

Heat PEF Control

Figure 2.2: Effects of thermal processing and PEF processing on the total aerobic plate counts of tomato juice during the storage at 4 °C for 112 d

69

0

1

2

3

4

5

6

0 20 40 60 80 100 120Storage time (days)

Log

cfu/

mL

Heat

PEF

Control

Figure 2.3: Effects of thermal processing and PEF processing on the yeast & mold counts of tomato juice during the storage at 4 °C for 112 d

70

The higher rate of microbial growth of PEF processed tomato juice than thermally

processed tomato juice during the storage may be due to the relatively lower

inactivationof the spores by PEF and the germination of the survived spores during the

storage. Spores of Bacillus and ascospores of molds and yeasts were detected from the

PEF processed tomato juice by a microscopic examination. Most studies reported that

PEF could not efficiently inactivate bacterial spores (Cserhalmi and others 2002; Grahl

and Markl 1996; Pol and others 2001). Little or no effect of PEF on the inactivation of

mold ascospores, including Byssoclamys nivea ascospores and Neosartorya fischeri

ascospores in tomato juice was also reported (Grahl and Markl 1996; Raso and others

1998a). The higher number of spores of Bacillus and ascospores of molds & yeasts

probably survived in PEF processed tomato juice than thermally processed tomato juice.

PEF processing can be effective for microbial inactivation extending the shelf life of

foods, but not for complete disintegration of microorganisms including spores (Grahl and

Markl 1996).

Effects of thermal processing and PEF processing on the lipoxygenase activity

Effects of thermal processing and PEF processing on the lipoxygenase activity of

tomato juice during the storage at 4 °C for 112 d are illustrated in Figure 2.4.

Lipoxygenase activity was not detected in thermally processed tomato juice throughout

the storage at 4 °C for 112 d. Commercial scale PEF processing inactivated 54% of the

lipoxygenase in the cold break tomato juice. The reduced lipoxygenase activity in PEF

processed tomato juice decreased further for 14 d at 4 °C.

71

Figure 2.4: Effects of thermal processing and PEF processing on the lipoxygenase activity of tomato juice during the storage at 4 °C for 112 days

0102030405060708090

100110

0 20 40 60 80 100 120

Storage time (days)

Rel

ativ

e lip

oxyg

enas

e ac

tivity

(%)

HeatPEFControl

72

Conformational changes of enzymes have been suggested as the mechanism of

enzyme inactivation by PEF (Ho and others 1997; Yeom and others 1999; Castro and

others 2001). Castro and others (2001) found that PEF caused unfolding of alkaline

phosphatase. When alkaline phosphatase was exposed to PEF, some aliphatic amino

acids of alkaline phosphatase moved from the hydrophobic interior to the hydrophilic

surface of the alkaline phosphatase, resulting in alkaline phosphatase unfolding (Castro

and others 2001). The further decrease in the lipoxygenase activity in the PEF processed

tomato juice for 14 d might be related to the gradual denaturation of lipoxygenase which

was unfolded by PEF. Further study is needed for confirmation.

Most desirable fresh flavor compounds in tomato including hexanal, cis-3-hexenal,

trans-2-hexenal, hexanol, trans-2-hexenol, and cis-3-hexenol are generated from the

unsaturated fatty acids such as linoleic and linolenic acid (Galliard and others 1977).

Lipoxygenase plays an important role in the formation of the flavor compounds through

the oxidation of unsaturated fatty acids (Eskin and others 1977). PEF processed tomato

juice may contain fresher flavor than thermally processed tomato juice due to the activity

of the residual lipoxygenase.

Effects of thermal processing and PEF processing on the concentration of lycopene

Effects of thermal processing and PEF processing on the concentration of

lycopene in the tomato juice during the storage at 4 °C for 112 d are shown in Table 2.2.

The concentrations of lycopene in thermally processed and PEF processed tomato juices

73

Concentration of lycopene (mg/100 g) Storage time

(days) Thermally processed

tomato juice

PEF processed

tomato juice

Control tomato

juice

0 11.91 ± 0.29 a 11.92 ± 0.35 a 12.14 ± 0.28 a

7 8.42 ± 0.47 a 9.55 ± 0.48 b 9.75 ± 0.50 b

14 8.41 ± 0.51 a 8.85 ± 0.49 a 9.44 ± 0.52 a

28 6.86 ± 0.28 a 7.60 ± 0.30 b 8.09 ± 0.38 b

35 5.86 ± 0.55 a 6.60 ± 0.47 a 6.95 ± 0.56 a

42 5.78 ± 0.31 a 6.21 ± 0.25 a 6.21 ± 0.28 a

49 5.58 ± 0.50 a 6.03 ± 0.41 a ND

56 5.50 ± 0.24 a 5.80 ± 0.42 a ND

70 5.15 ± 0.37 a 5.61 ± 0.31 a ND

84 4.65 ± 0.52 a 5.66 ± 0.51 a ND

112 4.08 ± 0.25 a 5.73 ± 0.27 b ND

ND: not determined Values are mean ±SD from duplicates of 4 measurements; Different letters in the same row indicate

significant differences (p < 0.05).

Table 2.2: Effects of thermal processing and PEF processing on the concentration of lycopene of tomato juice during the storage at 4 °C for 112 days

74

decreased from 11.9 to 4.08 mg/100 g and from 11.9 to 5.7 mg/100 g, respectively, after

112 d at 4 °C. The concentration of control decreased from 12.8 to 6.2 mg/100g after 42

d. There was no significant difference in the concentration of lycopene among thermally

processed, PEF processed, and control tomato juices during the storage (p > 0.05). The

concentration of lycopene decreased as storage time increased regardless of processing

methods.

The concentration of lycopene did not change significantly after thermal or PEF

processing (p > 0.05). Lycopene is chemically more stable than other pigments of plant

or animal origin such as chlorophyll, anthocyanin, hemoglobin, and myoglobin (Gould

1992). Lycopene in tomato products is resistant to degradation including thermally

induced trans-cis isomerization reactions (Nguyen and Schwartz 1998). It was proposed

that tocopherols, ascorbic acid, and phenolic antioxidants help stabilize lycopene during

processing (Abushita and others 2000; Takeoka and others 2001).

The main cause of carotenoids degradation in foods is oxidation (Thakur and

others 1996). Oxygen in the headspace of the sampling tube would cause the oxidation

of lycopene in tomato juice. The losses of lycopene of thermally processed, PEF

processed, and control tomato juices for 7 d were most significant throughout the storage

(p < 0.05). This may be due to the high oxygen availability in the headspace of the

sampling tubes during the early storage period. Rodriguez-Amaya (1993) found that the

stability of carotenoids of foods depends on oxygen availability and packaging conditions.

75

Effects of thermal processing and PEF processing on the retention of ascorbic acid

The effects of thermal processing and PEF processing on the concentration of

ascorbic acid during the storage at 4 °C for 112 d are illustrated in Figure 2.5. Ascorbic

acid decreased 10% after thermal processing (p < 0.05) but did not decrease significantly

after PEF processing (p > 0.05). The concentration of ascorbic acid in tomato juice

decreased as the storage time increased regardless of processing methods. However,

higher retention of ascorbic acid in PEF processed tomato juice than thermally processed

tomato juice was observed during the storage (p < 0.05).

Thermal processing in the manufacture of vegetable juices caused noticeable loss

of ascorbic acid (Podgorska and others 1983). Heat generated during thermal processing

initiates and accelerates chemical reactions in foods (Ekasari and others 1988). Ascorbic

acid is a heat sensitive nutrient (Saguy and others 1978). The higher retention of ascorbic

acid of PEF processed tomato juice than thermally processed tomato juice might be due

to the low processing temperature of PEF processing.

The difference between the concentrations of ascorbic acid of thermally processed

tomato juice and PEF processed tomato juice decreased as storage time increased and no

difference was observed after 70 d (p > 0.05). The oxygen in the headspace of package

and the oxygen permeated through package are considered to limit shelf life of food

products (Braddock 1999; Marshall and others 1986). The minimization of oxygen in the

headspace of package and oxygen permeation through package is essential to obtain

minimal oxidative degradations of ascorbic acid, flavor, and color (Sizer and others 1988).

The initially high concentration of ascorbic acid of PEF processed tomato juice can be

76

Figure 2.5: Effects of thermal processing and PEF processing on the retention of ascorbic acid of tomato juice during storage at 4 °C for 70 days

0

20

40

60

80

100

120

0 20 40 60 80

Ret

entio

n of

asc

orbi

c ac

id (%

)

Control

Heat

PEF

Storage time (days)

77

extended over time by selecting proper juice packaging materials such as polyethylene

terephthalate (PET) (Ayhan and others 2001b) and methods such as nitrogen flushing into

packages (Gunes and Lee 1997).

Effects of thermal processing and PEF processing on the particle size distribution,

Brix, pH, and viscosity

Effects of thermal processing and PEF processing on the particle size distribution

of tomato juice are shown in Table 2.3. A particle size distribution is a particle parameter

as a function of the particle size such as volume and surface area (Murphy 1984). The

D[4, 3] and D[3, 2] are means of particles on the basis of volume and surface area,

respectively (Malvern Instruments 1995). The D[4, 3] and D[3, 2] of PEF processed

tomato juice were significantly smaller than those of thermally processed and control

tomato juices (p < 0.05). Yeom and others (2000) found that orange juice processed by a

pilot plant scale PEF system at 35 kV/cm for 59 µs contained significantly smaller

particle size than orange juice thermally processed at 94.6 °C for 30 s. Miki and Akatsu

(1971) reported that the preparation methods of tomato products such as homogenization

and ultrasonication markedly influenced the particle size distributions of the products.

They found that tomato products showed uniform dispersion of lycopene when their

particle sizes are small.

The D(v, 0.5) is the median of the particle size distribution on the basis of volume

(Malvern Instruments 1995). The D(v, 0.5) values of thermally processed, PEF

processed, and control tomato juices were higher than those of D[4, 3] values (Table 2.3).

his indicates that the particle size distributions on the basis of volume were left-skewed.

78

D[4, 3] D[3, 2] D(v, 0.1) D(v, 0.5) D(v, 0.9)

Heat PEF Control Heat PEF Control Heat PEF Control Heat PEF Control Heat PEF Control

Av

(µm) 281.1 273.3 277.1 123.7 115.3 119.4 126.8 120.3 122.8 284.8 276.4 280.7 445.7 437.9 441.7

SD

(µm) 2.2 1.8 1.5 1.8 1.4 2.1 1.4 1.0 1.2 2.4 2.7 1.6 2.7 2.1 1.4

Heat = thermally processed tomato juice; PEF = PEF processed tomato juice; Control = control tomato juice. Av: average of 4 replicates; SD: standard deviation.

Table 2.3: Effects of thermal processing and PEF processing on the particle size distribution of tomato juice

78

79

The D(v, 0.5) values of PEF processed and control juices were not significantly

different (p > 0.05) whereas the D[4, 3] value of PEF processed juice was significantly

smaller than that of control juice. The actual particle size of tomato juice might be not

changed by PEF processing. It is likely there was change in the volume of particles.

Coagulated substances in pulp might be separated by PEF processing, resulting in

significant change in the volume, but insignificant change in the actual particle size.

Thermally processed tomato juice had the largest particle size distribution in all

measurements (p < 0.05). The colloidal materials in juice products are usually

coagulated by heating and settle out readily (Joslyn 1961). The larger particle size of

thermally processed tomato juice than PEF processed and control tomato juices may be

due to the coagulation of colloidal materials in tomato juice. The Brix values for

thermally processed tomato juice and PEF processed tomato juices were 5.20 ± 0.10 and

5.14 ± 0.13, respectively, during the storage at 4 °C for 112 d. There was no significant

difference in the Brix between thermally processed and PEF processed tomato juices (p >

0.05). However, the Brix of control tomato juice decreased from 5.20 to 4.64 during the

storage for 49 d. This significant decrease (p < 0.01) in the Brix of control tomato juice

during the storage may be due to the high growth of microorganisms and their

consumption of soluble solids.

The pH values of thermally processed, PEF processed, and control tomato juices

were 4.27 ± 0.06, 4.30 ± 0.05, and 4.33 ± 0.07, respectively, during the storage at 4 °C

for 112 d. There was no significant change in pH in thermally processed, PEF processed,

and control tomato juices during the storage (p > 0.05). No significant change (p > 0.05)

80

in the Brix and pH of thermally processed and PEF processed tomato juices during the

storage for 112 d may be related to the effective inactivation of spoilage microorganisms

by thermal processing and PEF processing.

Effects of thermal processing and PEF processing on the viscosity of tomato juice

during the storage at 4 °C for 112 d are shown in Table 2.4. There were no significant

differences in the viscosity among thermally processed, PEF processed, and control

tomato juices (p > 0.05). It may be due to the effective inactivation of pectic enzymes

such as pectingalacturonase (PG) and pectin methylesterase (PME) by the hot break. The

hot break might sufficiently inactivate pectic enzymes. Pectic enzymes depolymerize

pectin molecules in tomato pulp or serum and cause decrease in the viscosity of tomato

products (Shomer and others 1984; Thakur and others 1996). There might be no

additional inactivation of residual pectic enzymes by either thermal processing or PEF

processing or there might be no residual activities of pectic enzymes after the hot break

since no significant differences in viscosities of thermally processed, PEF processed, and

control tomato juices were observed during the storage. The interaction between pectins

and proteins, which forms a reversible electrostatic complex, is also an important

contributor to the viscosity of tomato juice (Thakur and others 1996). The maximum

pectin-protein interaction occurs at pH 4.0-4.5 in which all thermally processed, PEF

processed, and control tomato juices ranged during the storage.

81

Viscosity (mPa × s) Storage time

(days) Thermally processed

tomato juice

PEF processed

tomato juice

Control tomato

juice

0 412 ± 45 a 350 ± 52 a 381 ± 42 a

7 400 ± 25 a 373 ± 23 b 416 ± 20 b

14 412 ± 21 a 388 ± 23 a 420 ± 28 a

28 420 ± 18 a 393 ± 22 b 430 ± 25 b

35 444 ± 30 a 391 ± 33 a 449 ± 29 a

42 435 ± 18 a 401 ± 21 a 450 ± 30 a

49 461 ± 25 a 415 ± 30 a ND

56 488 ± 25 a 440 ± 18 b ND

70 480 ± 60 a 460 ± 55 a ND

84 490 ± 28 a 482 ± 39 a ND

98 525 ± 17 a 517 ± 20 a ND

112 536 ± 15 a 518 ± 16 b ND

ND: not determined Values are mean ±SD from duplicates of 4 measurements; Different letters in the same row indicate significant differences (p < 0.05).

Table 2.4: Effects of thermal processing and PEF processing on the viscosity of tomato juice during the storage at 4 °C for 112 days

82

Effects thermal processing and PEF processing on the sensory quality

The results of the sensory evaluation are shown in Table 2.5. The results indicate

that the PEF processed tomato juice had higher flavor intensity and overall acceptability

than thermally processed tomato juice (p < 0.01).

Thermal processing strongly changes the sensory property of tomato products

including fresh tomato flavors (Buttery and others 1990; Rouseff and Leahy 1995). The

higher flavor intensity of PEF processed tomato juice than thermally processed tomato

juice may be related to the higher activity of lipoxygenase of PEF processed tomato juice

(Figure 2.4). The lipoxygenase forms hexanal, cis-3-hexenal, trans-2-hexenal, hexanol,

trans-2-hexenol, and cis-3-hexenol, which are responsible for fresh flavor of tomato juice

(Galliard and others 1977). Freshness is likely to be determined by consumers with their

perceptions (IFT 2001) and flavor is an important element in consumers’ perceptions of

the freshness of juice products. The overall acceptability of foods may be mainly

determined by freshness. Higher ranks of PEF processed tomato juice in the flavor

intensity and overall acceptability than thermally processed tomato juice may be

associated with higher freshness of PEF processed tomato juice than thermally processed

tomato juice.

83

Samples

Attributes Thermally processed

tomato juice

PEF processed

tomato juice

Appearance 5.9 a 6.6 a

Color 6.1 a 6.4 a

Texture 5.7 a 6.4 a

Flavor 4.7 a 6.2 b

Overall acceptability 4.8 a 6.2 b

Different letters in the same row indicate significant differences (p < 0.01).

Table 2.5: Effects of thermal processing and PEF processing on the sensory properties of tomato juice

84

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90

ACKNOWLEDGMENTS

We acknowledge the DoD Dual Use Science and Technology Program for

funding this research. We thank Dr. F. Ozadali (Hirzel Canning Co. & Farms) for

supplying fresh tomatoes. We thank Dr. S. Schwartz, Ms. B. Keller, and Mr. J. Lee for

their technical support. We acknowledge Dr. H. Wang, Mr. S. K. Min, Ms. S. N. Rao,

and Mr. R. Caldwell for the preparation of tomato juice and the operation of the PEF

system.

91

CHAPTER 3

Effects of Commercial Scale Pulsed Electric Field Processing on Flavor and Color of

Tomato Juice

ABSTRACT

Effects of commercial scale pulsed electric field (PEF) processing on the flavor and color

of tomato juice during storage at 4 ºC for 112 d were studied. Tomato juice was prepared

by hot break at 88 °C for 2 min and then thermally processed at 92 °C for 90 s or PEF

processed at 40 kV/cm for 57 µs. PEF processed tomato juice retained more flavor

compounds of trans-2-hexenal, 2-isobutylthiazole, cis-3-hexanol than thermally

processed or unprocessed control tomato juice (p<0.05). PEF processed juice had

significantly lower nonenzymatic browning than thermally processed or control juice

(p<0.05). PEF processed juice had higher redness (p<0.05). Sensory evaluations

indicated that flavor of PEF processed juice was preferred to that of thermally processed

juice (p<0.01).

Key words: Pulsed electric field, tomato juice, tomato juice flavor, tomato juice color,

nonenzymatic browning

92

INTRODUCTION

Flavor and color are considered as the major quality attributes of foods and

influence the consumer’s choice. Many researchers have studied on the flavor and color

of tomato products and emphasized their importance as quality indexes (Kazeniac and

Hall 1970; Miki 1974; Gould 1978; Chung and others 1983; Buttery and others 1990a).

Volatile compounds of fresh tomato flavor are derived from the metabolisms of

fatty & amino acids and breakdown of carotenoids (Buttery and others 1971, 1988).

Approximately 400 volatile compounds were identified from fresh tomatoes (Buttery and

others 1990a). Volatile compounds of fresh tomato flavor need to be carefully studied

for the control of fresh and processed tomato products (Buttery and others 1971). Flavor

and color of tomato products are highly influenced by the nonenzymatic browning

reaction (Servili and others 2000). The nonenzymatic browning results in the

discoloration, off-flavor formation, and nutrient losses of foods and is considered as the

major cause of the quality degradation of foods (Saguy and others 1978; Labuza and

Baisier 1992). The nonenzymatic browning takes place in juice products during

harvesting, transporting, processing, and storage. Juice industries use the nonenzymatic

browning for the quality control (Garza and others 1999). The 5-hydroxymethyl-2-

furfural (HMF) is an intermediate product during the nonenzymatic browning reaction in

an acidic medium including tomato juice (Miki 1974). The HMF has been used as an

indicator of the nonenzymatic browning reaction (Lee and Nagy 1988; Porretta 1991).

The HMF forms during the juice production especially as a result of thermal processing

93

and increases during storage (Elkins and others 1988; Theobald and others 1998). The

level of HMF in processed foods is widely used to evaluate over-processing of juice

products (Elkins and others 1988; Porretta 1991; Hidalgo and others 1998).

Thermal processing is conventionally used to inactivate microorganisms and

enzymes and extend the shelf life of juice products. However, thermal processing can

adversely affect the sensory and nutritive qualities of foods (Rouseff and Leahy 1995;

Braddock 1999). The consumer likes fresh foods (Langlois and others 1996). Pulsed

electric field (PEF), a nonthermal food preservation method, has been extensively studied

to produce high quality juices with freshness (Mertens and Knorr 1992; Dunn 2001).

Laboratory or pilot plant scale PEF processing increased the shelf life of juice products

while reducing the loss of flavor, color, and nutrients (Qiu and others 1998; Evrendilek

and others 2000; Yeom and others 2000). However, there is no publication reporting

commercial scale PEF processing of foods or the effects of PEF on the flavor and color of

tomato juice during storage. The objectives of this research were (1) to study the effects

of commercial scale PEF processing on the flavor and color of tomato juice and (2) to

compare the flavor and color of PEF processed tomato juice with those of thermally

processed tomato juice during storage at 4 °C for 112 d.



Fresh Roma-type Midwest tomatoes were supplied by Hirzel Canning Co. &

Farms (Toledo, OH, U.S.A.) throughout the year of 2001 tomato season. Tomatoes were

94

processed on the same day of harvesting. Tomatoes were washed in a soak tank with air

agitation and then washed again with sprayed water supplied at 1.13 MPa (150 psig)

while being conveyed on a roller conveyor. Tomatoes were sorted manually and chopped

by a mill (Model D, The W. J. Fitzpatrick Co., Chicago, IL, U.S.A.) equipped with a 1.91

cm screen. The chopped tomatoes were heated in a tubular heat exchanger (H2187C

type, Specialty Brass Co., Kenosha, WI, U.S.A.) at 88 °C for 2 min for hot break.

Tomato juice was prepared by a screw type extractor (CJE-360-D28, Chisholm-Ryder

Co., Niagara Falls, NY, U.S.A.) with a screen of 1.27 cm diameter. A total of 1,100 kg

fresh tomatoes were used to prepare 800 L tomato juice.

Thermal processing and commercial scale PEF processing system

The OSU-6 commercial scale PEF processing system is illustrated in Figure 3.1.

The OSU-6 commercial scale PEF processing system was used for both thermal

processing and PEF processing. The system consists of an aseptic drink processor (Tetra

Pak Dairy & Beverage Systems AB, Lund, Sweden), a high voltage pulse generator

(Diversified Technology, Inc., Bedford, MA, U.S.A.), and co-field flow tubular PEF

chambers (The Ohio State University, Columbus, OH, U.S.A.). The aseptic drink

processor monitors and controls production rate, temperatures, and pressure during

thermal processing and PEF processing. The production rates can be controlled from 400

to 2000 L/h. The maximum pulse repetition rate of the high voltage pulse generator is

2000 pulses per second (pps). The high voltage pulse generator provides bi-polar squared

waveform pulses with a maximum peak voltage of ± 60 kV and maximum peak current

95


Recirculation

Heat exchanger 1 (Heating)

Product tank

Pump


PEF chamber

Sanitary glove b

Waste

Heat exchanger 6

Holding T

ube

High voltage pulse

generator +/- 60 kV

Heat exchanger 5

Heat exchanger 4

Heat exchanger 3

Heat exchanger 2


Aseptic drink

Thermal processing

PEF processing

High voltage pulse generator +/- 60 kV

Sanitary glove box

PEF processing section

Thermal processing section

96

of 600 A into multiple PEF chambers. Pulses were monitored with a high voltage probe

(VD-60, Northstar, Albuquerque, NM, U.S.A.), current monitors (Model 110, Pearson,

Palo Alto, CA, U.S.A.), and oscilloscopes (TDS-210, Tektronix, Beaverton, OR, U.S.A.).

Each co-field flow tubular PEF chamber consists of two boron carbite tubular electrodes

and a tubular insulator body made of ceramic (Yin and others 1997). The gap distance

between the electrodes is 1.270 cm and the inner diameter of the cylindrical processing

zone is 0.808 cm. A maximum of eight PEF chambers can be connected electrically

parallel and fluid flow wise in series. A sanitary glove box (The Ohio State University,

Columbus, OH, U.S.A.) was used as a filling unit for this research. The glove box was

sanitized by spraying and swabbing 35% hydrogen peroxide and lighting germicidal UV

at 254 nm for 12 h.

Thermally processed, PEF processed, and control tomato juices

Processing conditions for thermally processed, PEF processed, and control tomato

juices are listed in Table 3.1. The production rate was 500 L/h for thermally processed,

PEF processed, and control tomato juices.

For thermal processing, tomato juice was held at 92 °C for 90 s in a holding tube

and then cooled to 25 °C by Heat Exchanger 2 (Figure 3.1). The high voltage pulse

generator and heat exchangers in PEF Processing Section (Figure 3.1) were not activated

during the thermal processing. The cooled tomato juice was packaged inside the sanitary

glove box (Figure 3.1).

PEF processing conditions were electric field strength of 40 kV/cm, pulse

duration time of 2 µs, pulse repetition rate of 1000 pps, and total PEF treatment time of

97

Processing parameter Heat PEF Control





PEF treatment time (µs) 0 57 0

Pulse repetition rate (pps a) 0 1000 0

Number of PEF chambers 6 6 6

Temperature change per a pair of PEF

chamber (°C)

0

8

0

Heat, PEF, and Control are the thermally processed tomato juice, PEF processed tomato juice, control tomato juice, respectively. a pps: pulse per second

Table 3.1: Parameters and temperature of processing

98

57 µs. Tomato juice was PEF processed while thermal processing was turned off.

Tomato juice was maintained at 45 °C before entering each set of two PEF chambers by

Heat exchangers 3, 4, and 5 (Figure 3.1). PEF processed tomato juice was cooled to 25

°C by Heat exchanger 6 prior to packaging inside the sanitary glove box (Figure 3.1).

For the control tomato juice, tomato juice was passed through the OSU-6

commercial scale PEF processing system without any thermal or PEF processing and

packaged inside the sanitary glove box.


Thermally processed, PEF processed, and control tomato juices were packaged

into the 50-mL sterilized polypropylene tubes (Corning, Acton, MA, U.S.A.) inside the

sanitary glove box. Each tube was covered with aluminum foil to protect tomato juice

from light. Packaged thermally processed, PEF processed, control tomato juices were

stored at 4 ºC in the dark.

Analysis of flavor compound

Volatile flavor compounds of tomato juice were analyzed by the method of

Goodman and others (2002) using a headspace solid-phase microextraction gas

chromatography (SPME-GC) system. Standard flavor compounds of trans-2-hexenal, 2-

isobutylthiazole, and cis-3-hexanol were purchased from Aldrich Chemical Co.

(Milwaukee, WI, U.S.A.). Carboxen/polydimethylsiloxane (Carboxen/PDMS) SPME

fibers, serum bottles, Teflon-coated rubber septa, and aluminum caps were purchased

from Supelco, Inc. (Bellefonte, PA, U.S.A.). The gas chromatography (GC) (5890,

99

Hewlett-Packard, Wilmington, DE, U.S.A.) was baked in an oven at 150 °C for 3 h. A

1.0 mL aliquot of tomato juice was transferred into a sealed 10-mL serum bottles

containing a micromagnetic bar (10 × 3 mm). The SPME fiber was inserted into the

headspace of the 10-mL vial containing tomato juice. The tomato juice in the vial was

incubated at 35 °C for 30 min in a water bath (Isotemp 228, Fisher Scientific, Pittsburgh,

PA, U.S.A.). The tomato juice in the vial was magnetically stirred by an immersed stirrer

(LTE Scientific Ltd., Greenfield, Oldham, U.K.) during the incubation. The SPME fiber

was moved from the vial and immediately injected into the GC with the injector

temperature at 265 °C and kept for 3 min to allow for desorption of flavor compounds.

The desorbed flavor compounds were separated by the GC equipped with a capillary

column (30 m × 0.53 mm i.d.) coated with a 2.65 µm film of 5% phenyl-substituted

methylpolysiloxane and a flame ionization detector. The temperature program held at 35

°C for 4 min, ramped 3 °C/min to 83 °C, then increased 20 °C/min to 150 °C and held for

2 min. The retention percent of selected flavor compound of tomato juice during storage

was calculated as follows:

Retention of flavor compound of tomato juice (%) =

(Concentration of a flavor compound of thermally or PEF processed tomato juice /

Concentration of the flavor compound of control tomato juice at 0 d) × 100

Identification of volatile compounds

The volatile flavor compounds was identified by a combination the GC retention

time of standard flavor compounds and a Hewlett-Packard 5970 Series mass selective

detector (MS) (Wilmington, DE, U.S.A.). The MS was equipped with a Hewlett-Packard

100

59822 B ionization gauge controller. Mass spectra were obtained at 70 eV and 220 °C

ion source temperature. The GC conditions for GC-MS were the identical to the GC

conditions for the analysis of flavor compounds described earlier. Information of the

computer software of NIST Mass Spectral Library (NIST’02. Version 2.0, Gaithersburg,

MD, U.S.A.) was used for the identification.

Determination of the nonenzymatic browning

The nonenzymatic browning was determined by a combination of brown color

and 5-hydroxymethyl-2-furfural (HMF) measurements.

The brown color was measured by the method of Birk and others (1998). The 5 g

ethyl alcohol was added to 5 mL of tomato juice. This mixture was centrifuged at 7800 ×

g for 10 min. Supernatant was clarified using 0.45 µm filter (Gelman Sciences, Ann

Arbor, MI, U.S.A.). The absorbance of the filtrate was measured at 420 nm by a

Spectronic Genesys 5 spectrometer (Milton Roy, Rochester, NY, U.S.A.) at 22 °C.

HMF was determined by the method of Birk and others (1998). The 2 mL of the

supernatant from the browning test, 2 mL (120 g/kg) 3-chloroacetic acid (TCA), and 2

mL (0.025 mol/L) thiobarbituric acid (TBA) were mixed in a 50-mL screw-cap test tube

(Corning, Acton, MA, U.S.A.). The test tubes were placed in a water bath at 40 °C for 50

min and then cooled immediately with tap water to 25 °C. The absorbance measured at

443 nm by a Spectronic Genesys 5 spectrometer (Milton Roy, Rochester, NY, U.S.A.)

was defined as the index to quantify HMF. The concentration of HMF of tomato juice

sample was read from a calibration curve of HMF (Sigma, St. Louis, MO, U.S.A.)

ranging from 0 to 20 mg/kg.

101

Analysis of ascorbic acid

The concentration of ascorbic acid in the tomato juice was measured by the

method of Yeom and others (2000) using a high-performance liquid chromatography

(HPLC) system (Hewlett Packard, 1050 series, Wilmington, DE, U.S.A.).

Measurement of color

Hunter L, a, and b of tomato juice were measured by a HunterLab colorimeter

(Hunter Associates Laboratory Inc., Reston, VA, U.S.A.). The red-yellow ratio (a / b)

was reported to indicate the redness of tomato juice.

Sensory evaluation

Thermally processed and PEF processed tomato juices, stored at 4 °C for 7 d,

were used for the sensory evaluation. The 7 d was needed for Silliker Laboratories

(Columbus, OH, U.S.A.) to confirm the absence of pathogen microorganisms, Salmonella

spp., Listeria monocytogenes, and Escherichia coli O157:H7 in thermally processed or

PEF processed tomato juice. A 30-member panel participated in the sensory tests. The

panelists consisted of people in the department of Food Science and Technology at the

Ohio State University and food industry. The panelists were asked to rate the preference

of flavor and color. A hedonic scale of 1 to 9 was used for each attribute. Higher

number represents higher preference of attributes. Thermally processed and PEF

processed tomato juices were served in randomly numbered plastic cups on a tray with a

cup of water and a piece of non-salted cracker.

102

A triangle test was conducted with thermally processed, PEF processed, and

control tomato juices to examine which tomato juice had different color from the others.

Data were analyzed with the Triangle Test for Difference Table (Meilgaard and others

1991).



significance levels were conducted to determine significant differences among thermally

processed, PEF processed, and control tomato juices. The entire analyses were

duplicated with 4 measurements. Minitab 13.31 (Minitab, Inc., State College, PA,

U.S.A.) was used for all statistical analyses.


Effects of thermal processing and PEF processing on the flavor compounds

Effects of thermal processing and PEF processing on the retention of trans-2-

hexenal of tomato juice are shown in Figure 3.2 and on the retention of 2-isobutylthiazole

and cis-3-hexanol of tomato juice are shown in Table 3.2.

The retentions of trans-2-hexenal were 98% after thermal processing and 110%

after PEF processing at 0 d (Figure 3.2). The retentions of 2-isobutylthiazole were 99%

after thermal processing and 108% after PEF processing at 0 d (Table 3.2). The

concentrations of trans-2-hexenal and 2-isobutylthiazole of PEF processed tomato juice

were higher than those of control tomato juice at 0 d (p < 0.05). Yeom and others (2000)

103

Figure 3.2: Effects of thermal processing and PEF processing on the retention of trans-2-hexenal of tomato juice during storage at 4 °C for 112 days

0

20

40

60

80

100

120

0 20 40 60 80 100 120

Incubation time (days)

Ret

entio

n of

tran

s-2-

hexe

nal Control

Heat PEF

(days)

104

2-isobutylthiazole cis-3-hexanol Storage

time

(days) Heat PEF Control Heat PEF Control

0 99 a1 108 b1 100 a1 81 a 93 b 100 b

7 96 a 107 b 89 c 61 a 79 b 68 a

14 71 a 96 b 80 c 65 a 70 b 51 c

28 68 a 86 b 75 c 48 a 44 a 9 b

35 68 a 87 b 78 c 45 a 45 a 9 b

42 55 a 87 b 63 c 43 a 38 a 7 b

49 45 a 67 b ND2 42 a 35 a ND

56 41 a 55 b ND 42 a 40 a ND

70 40 a 52 b ND 29 a 29 a ND

84 40 a 49 b ND 22 a 25 a ND

112 30 a 38 b ND 18 a 13 a ND

Heat, PEF, and Control are the thermally processed tomato juice, PEF processed tomato juice, control 1Different letters in the same row of each flavor compound indicate significant differences (p < 0.05). 2ND: not determined because tomato juice was not sampled due to gas formation by multiplied microorganisms. The concentrations of 100 % 2-isobutylthiazole and cis-3-hexanol were 0.051 and 2.078 ppm, respectively. Values are mean of duplicates of 4 measurements.

Table 3.2: Effects of thermal processing and PEF processing on the retention of flavor compounds (%) of tomato juice during storage at 4 °C for 112 d

104

105

also observed that the concentrations of α-pinene, myrcene, limonene, and decanal of

orange juice processed by a pilot plant scale PEF system at 35 kV/cm for 59 µs were

higher than those of unprocessed orange juice at the 0 d of storage at 4 °C. The higher

concentration of the flavor compounds of PEF processed tomato juice at 0 d might be due

to the smaller particle size of PEF processed tomato juice than control tomato juice. A

preliminary study indicated that tomato juice processed by a commercial scale PEF

system at 40 kV/cm for 57 µs contained significantly smaller particles than thermally

processed tomato juice at 92 °C for 90 s and control tomato juice (p < 0.05). The

thermally processed tomato juice had the largest particle size (data not shown). Yeom

and others (2000) also found that orange juice processed by a pilot plant scale PEF

system at 35 kV/cm for 59 µs contained significantly smaller particle size than thermally

processed orange juice at 94.6 °C for 30 s and unprocessed orange juice. The higher

concentration of the flavor compounds of PEF processed tomato juice than control

tomato juice might be due to the decreased particle size of tomato juice during PEF

processing and the consequently increased release of the flavor compounds.

The concentrations of trans-2-hexenal, 2-isobutylthiazole, and cis-3-hexanol in

thermally processed, PEF processed, and control tomato juices decreased as the storage

time increased (Figure 3.2 and Table 3.2). PEF processed tomato juice retained more

trans-2-hexenal and 2-isobutylthiazole than thermally processed tomato juice during

storage at 4 °C for 112 d (p < 0.05). PEF processing did not change the concentration of

cis-3-hexanol significantly (p > 0.05) (Table 3.2). Thermal processing reduced 19% cis-

3-hexanol (p < 0.05). The concentration of cis-3-hexanol of PEF processed tomato juice

106

was significantly higher than that of thermally processed tomato juice at 0, 7, and

14 d (p < 0.05). PEF processed tomato juice retained trans-2-hexenal, 2-isobutylthiazole,

and cis-3-hexanol more than thermally processed and control tomato juices.

Thermal processing easily changes the flavor profile of juice products (Buttery

and others 1990b; Rouseff and Leahy 1995). The heat of thermal processing causes and

accelerates chemical reactions in foods (Ekasari and others 1988). PEF processed tomato

juice was exposed to 53.5 °C for 5 s whereas thermally processed tomato juice was

heated at 90 °C for 90 s. More retention of the flavor compounds in PEF processed

tomato juice than thermally processed tomato juice during the storage might be due to the

lower energy input of PEF processing than thermal processing.

Effects of thermal processing and PEF processing on the nonenzymatic browning

The nonenzymatic browning was determined by a combination of brown color

and 5-hydroxymethyl-2-furfural (HMF) measurements. Effects of thermal processing

and PEF processing on the brown color of tomato juice during storage at 4 °C for 112 d

are shown in Figure 3.3. The values of brown color of thermally processed tomato juice

and PEF processed tomato juice at 0 d were 0.34 and 0.29, respectively. PEF processed

tomato juice showed significantly less browning than thermally processed or control

tomato juice during storage at 4 °C (p < 0.05).

Effects of thermal processing and PEF processing on the concentration of 5-

hydroxymethyl-2-furfural (HMF) in tomato juice during storage at 4 °C for 112 d are

shown in Figure 3.4. The concentrations of HMF of thermally processed tomato juice

107

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 20 40 60 80 100 120


Abso

rban

ce a

t 420

nm

Control HeatPEF

Figure 3.3: Effects of thermal processing and PEF processing on the brown color of tomato juice during storage at 4 °C for 112 days

(days)

Bro

wn

colo

r (A

420)

108

0

2

4

6

8

10

12

0 20 40 60 80 100 120


Con

cent

ratio

n of

HM

F (m

g/kg

)

Control Heat PEF

Figure 3.4: Effects of thermal processing and PEF processing on the concentration of 5-hydroxymethyl-2-furfural of tomato juice storage at 4 °C for 112 days

(days)

HM

F co

ncen

trat

ion

(mg/

kg)

109


tomato juice showed significantly lower concentration of HMF than thermally processed

or control tomato juice during storage at 4 °C (p < 0.05).

A linear regression plot of the brown color versus the HMF concentration in PEF

processed tomato juice sampled at 0, 7, 14, 28, 35, 42, 49, 56, and 70 d is shown in

Figure 3.5 (a). A good relationship between the brown color and the HMF concentration

was observed (R2 = 0.88). The HMF is an intermediate compound during the

nonenzymatic browning reaction. The HMF has been used as an indicator of the

nonenzymatic browning reaction in juice products (Lee and Nagy 1988; Porretta 1991).

The good relationship between the brown color and the HMF concentration indicates that

the HMF can be used to evaluate the browning reaction in tomato juice.

The nonenzymatic browning and the formation of HMF depend on thermal

processing temperature and time and thus are used to evaluate over-processing (Meydav

and others 1977; Elkins and others 1988; Lee and Nagy 1988). The results from the

studies of the brown color and the HMF concentration show that PEF processed tomato

juice had less nonenzymatic browning than thermally processed or control tomato juice

during storage at 4 °C.

A linear regression plot of the concentration of HMF versus the loss of ascorbic

acid in PEF processed tomato juice sampled at 0, 7, 14, 28, 35, 42, 49, 56, and 70 d is

shown in Figure 3.5 (b). The concentration of HMF and the loss of ascorbic acid were

highly correlated (R2 = 0.96). A strong relationship between the formation of furfural

and the loss of ascorbic acid of orange juice was reported by Nagy and Dinsmore (1974).

110

(a)

continued

Figure 3.5: Linear regression plots of the brown color versus the concentration of 5-hydroxymethyl-2-furfural (a) and the concentration of 5-hydroxymethyl-2-furfural versus the loss of ascorbic acid (b) in PEF processed tomato juice sampled at 0, 7, 14, 28, 35, 42, 49, 56, and 70 days

111

Figure 3.5 continued

(b)

112

Ascorbic acid degradation is known as a cause of nonenzymatic browning in foods

(Marcy and others 1984). Destruction of ascorbic acid provides reactive carbonyl groups.

These carbonyl groups undergo the Amadori rearrangement with amino acids such as

lysine and glutamic acid (Kacem and others 1987). Furfurals can be formed from

dehydroreductones from the Amadori rearrangement. Pathways for ascorbic acid

destruction in both aerobic and anaerobic conditions yield furfurals as end products

(Kaanane and others 1988). The nonenzymatic browning in acidic media such as juice

products is primarily due to the ascorbic acid degradation rather than the reaction

between the reducing sugars and amino acids (Kaanane and others 1988). PEF processed

tomato juice retained more ascorbic acid than thermally processed tomato juice for 42 d

at 4 °C (p < 0.05) (data not shown). The slower browning reaction of PEF processed

tomato juice than thermally processed tomato juice (Figure 3.3) would be related to the

more retention of ascorbic acid of PEF processed tomato juice than thermally processed

tomato juice.

Effects of thermal processing and PEF processing on the Hunter L, a, and b

Effects of thermal processing and PEF processing on the red-yellow ratio (Hunter

a / b), indicating the redness of tomato juice during storage at 4 °C for 112 d, are

illustrated in Figure 3.6. The red-yellow ratios of thermally processed, PEF processed,

control tomato juices were above 3 during storage at 4 °C, which is acceptable for

commercial tomato juice (Bontovits 1981). The red-yellow ratios of thermally processed

113

3.0

3.2

3.4

3.6

3.8

4.0

0 20 40 60 80 100 120


a / b

Heat PEF Control

Figure 3.6: Effects of thermal processing and PEF processing on the red-yellow ratio (Hunter a / b) of tomato juice during storage at 4 °C for 112 days

Red

-yel

low

ratio

(a /

b)

(days)

114


tomato juice showed significantly higher red-yellow ratios than thermally processed

tomato juice during storage at 4 °C for 112 d (p < 0.05).

Heat is the most important cause in loss of color during processing (Kattan and

others 1956; Yeom and others 2000). The heat of the thermal processing might

accelerate the color change in tomato juice. Lycopene makes up the 83% of the total

pigment present in tomatoes (Hayes and others 1998). The lycopene is responsible for

red color in tomato and the lycopene degradation results in the decrease in redness and

the loss of color in tomato (Noble 1975). A preliminary study indicated that there was no

significant difference in the concentrations of lycopene between tomato juice processed

by a commercial scale PEF system at 40 kV/cm for 57 µs and tomato juice thermally

processed at 92 °C for 90 s during storage at 4 °C for 112 d (data not shown). Thermally

processed tomato juice had smaller red-yellow ratio than PEF processed tomato juice

during storage at 4 °C for 112 d. This may be due to the higher rate of the nonenzymatic

browning reaction of thermally processed tomato juice than PEF processed tomato juice

during the storage if the concentrations of lycopene of thermally processed and PEF

processed tomato juices were not significantly different during the storage.

Color and clarity are the most important in describing the appearance of tomato

juice (Meilgaard and others 1991). Consumers notice color of tomato juice first and their

observation on its color often provides preconceived ideas about the quality of tomato

juice (Thakur and others 1996). The more color retention of PEF processed tomato juice

than thermally processed tomato juice can be the benefit of PEF processed tomato juice.

115

Effects of thermal processing and PEF processing on the sensory properties of

flavor and color

The panel score for flavor was 4.7 for thermally processed tomato juice and 6.2

for PEF processed tomato juice. The panel score for color was 6.1 for thermally

processed tomato juice and 6.4 for PEF processed tomato juice. The result shows that the

flavor of PEF processed tomato juice was preferred by the panels (p < 0.01). Thermal

processing strongly changes the flavor of tomato (Buttery and others 1990b; Rouseff and

Leahy 1995). The changed flavor of tomato by thermal processing is mainly due to the

formation of reaction products such as volatile carbonyls and sulfur compounds from the

nonenzymatic browning reaction (Buttery and others 1990b). Less retention of the flavor

compounds (Figure 3.2, Table 3.2) and more nonenzymatic browning (Figures 3.3, 3.4)

of thermally processed tomato juice than PEF processed tomato juice may be responsible

for the less preferred flavor of thermally processed tomato juice than PEF processed

tomato juice.

No significant difference was observed in the panel scores for color between

thermally processed and PEF processed tomato juices (p > 0.05). The result from the

triangle test also indicated that the colors of thermally processed, PEF processed, and

control tomato juices are not distinguishable. The sensory evaluation was conducted

after 7 d of storage at 4 °C. The colors of thermally processed and PEF processed tomato

juices might be expected to be different after a longer storage time since the differences

in the nonenzymatic browning (Figures 3.3, 3.4) and in the red-yellow ratio (Figure 3.6)

between thermally processed tomato juice and PEF processed tomato juice increased as

storage time increased.

116

CONCLUSIONS

Commercial scale PEF processing produced tomato juice with more retention of

the flavor compounds, less nonenzymatic browning, and higher redness than thermally

processed tomato juice. Flavor and color of tomato juice are important elements in the

consumer’s perception of the freshness of tomato juice. PEF processed tomato juice

possessed higher fresh quality than the thermally processed tomato juice.

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Chem 48(10):4597-4605.

Yin Y, Zhang QH, Sudhir SK, inventors; The Ohio State University, assignee. 1997 Nov.



ACKNOWLEDGMENTS


funding this research. We sincerely thank Dr. F. Ozadali (Hirzel Canning Co. & Farms)

for supplying fresh tomatoes. We thank Dr. S. Schwartz, Ms. B. Keller, and Mr. J. Lee

for their technical support. We acknowledge Dr. R. Man, Mr. S. K. Min, Ms. S. N. Rao,

and Mr. R. Caldwell for the preparation of tomato juice and the operation of the PEF

system.

121

CHAPTER 4

Effects of Commercial Scale Pulsed Electric Field Processing on the Quality of Orange

Juice

ABSTRACT

Effects of commercial scale pulsed electric field (PEF) processing on the microbial

stability, ascorbic acid, flavor compound, color, Brix, pH, and sensory properties of

orange juice were studied and compared with those of thermal processing. Freshly

squeezed orange juice was thermally processed at 90 °C for 90 s or processed by PEF at

40 kV/cm for 97 µs. Both thermally and PEF processed juices showed microbial shelf life

at 4 °C for 196 d. PEF processed juice retained more ascorbic acid, flavor, and color than

thermally processed juice (p<0.05). No significant differences were observed in Brix and

pH between thermally and PEF processed juices at 4 °C for 196 d (p>0.05). Sensory

evaluation of texture, flavor, and overall acceptability were ranked highest for control

juice, followed by PEF processed juice and then by thermally processed juice (p<0.01).

Key words: Pulsed electric field, nonthermal processing, orange juice, ascorbic acid,

nonenzymatic browning

122

INTRODUCTION

Orange juice is the most popular juice in the United States (Marcy and others

1989). The quality of fresh orange juice is decreased by microorganisms, enzymes, and

chemical reactions (Chen and others 1993). Unpasteurized orange juice can transmit

some pathogens including Salmonella Enteritidis (FDA 2001). Thermal processing has

been widely used to inactivate spoilage & pathogenic microorganisms and enzymes of

orange juice. However, thermal processing can lower the sensory and nutritional

qualities of orange juice (Chen and others 1993; Braddock 1999). Nonthermal

processing including pulsed electric field (PEF) processing and high pressure processing

(HPP) has been intensively studied to minimize the loss of flavor, color, and nutrients of

foods from heat (Mertens and Knorr 1992; Dunn 2001; Nienaber and Shellhammer 2001).

Laboratory scale and pilot plant scale PEF processing increased the shelf life of

juice products by inactivating spoilage and pathogenic microorganisms and by reducing

the loss of flavor, color, and nutrients (Qiu and others 1998; Reina and others 1998;

Evrendilek and others 2000; Yeom and others 2000). Evrendilek and others (2000)

reported that the laboratory scale PEF processing at 34 kV/cm for 166 µs inactivated

Escherichia coli O157:H7 in apple juice by 4.5 logs and the pilot plant scale PEF

processing at 35 kV/cm for 94 µs increased the shelf life of apple juice while maintaining

the natural color and ascorbic acid content. Yeom and others (2000) reported that the

pilot plant scale PEF processing at 35 kV/cm for 59 µs inactivated 88% of pectin methyl

esterase activity and prevented microbial growth in orange juice. They also reported that

PEF processed orange juice had more retention of ascorbic acid for 112 d than thermally

123

processed orange juice. However, there is no publication reporting commercial scale

PEF processing for orange juice pasteurization. Development of a large scale PEF

processing system and the quality evaluation of foods processed by the large scale PEF

processing system are required for the industrial application of PEF processing

(Jeyamkondan and other 1999). The objectives of this research were (1) to study the

effects of commercial scale PEF processing on the inactivation of endogenous

microorganisms in orange juice, (2) to investigate the effects of commercial scale PEF

processing on the quality of orange juice, (3) and to compare the quality of PEF

processed orange juice with that of thermally processed orange juice during storage at 4

°C for 196 d.


Preparation of Orange Juice

Fresh Rohde Valance oranges were purchased from Haines City Citrus Growers

Association (Haines City, FL, U.S.A.). Oranges were washed, waxed, graded, bulked,

and shipped to the Ohio State University (Columbus, OH, U.S.A.). Oranges were stored

at 4 °C until processing. Orange juice was prepared by squeezing fresh oranges using a

FMC Multi-Fruit Juicer (Lakeland, FL, U.S.A.) prior to thermal processing and PEF

processing.

124

Thermal processing and commercial scale PEF processing system

The OSU-6 commercial scale PEF processing system was used for both thermal

processing and PEF processing. The OSU-6 commercial scale PEF processing system is

illustrated in Figure 4.1. The system consists of an aseptic drink processor (Tetra Pak

Dairy & Beverage Systems AB, Lund, Sweden), a high voltage pulse generator

(Diversified Technology, Inc., Bedford, MA, U.S.A.), and co-field flow tubular PEF

chambers (The Ohio State University, Columbus, OH, U.S.A.). The aseptic drink

processor monitors and controls production rate, temperatures, and pressure during

thermal processing and PEF processing. The production rates are from 400 to 2000 L/h.

The high voltage pulse generator provides bi-polar squared waveform pulses with a

maximum peak voltage of ± 60 kV and maximum peak current of 600 A into multiple

PEF chambers during PEF processing. The 60 kV power supplies charge storage

capacitances. The storage capacitances are partially discharged by a series of solid-state

switches to form the square wave bipolar pulses. The high voltage pulse generator

operates at a maximum repetition rate of 2000 pulses per second (pps). Pulses were

monitored with a high voltage probe (VD-60, Northstar, Albuquerque, NM, U.S.A.),

current monitors (Model 110, Pearson, Palo Alto, CA, U.S.A.), and oscilloscopes (TDS-

210, Tektronix, Beaverton, OR, U.S.A.). The co-field flow tubular PEF chamber consists

of two boron carbite tubular electrodes and a ceramic tubular insulator body (Yin and

others 1997). The inner diameter of the cylindrical processing zone is 0.808 cm and the

gap distance between the electrodes is 1.270 cm. A maximum of eight PEF chambers

125


Recirculation

Heat exchanger 1 (Heating)

Product tank

Pump


PEF chamber

Sanitary glove b

Waste

Heat exchanger 6

Holding T

ube

High voltage pulse

generator +/- 60 kV

Heat exchanger 5

Heat exchanger 4

Heat exchanger 3

Heat exchanger 2


Aseptic drink

Thermal processing

PEF processing

High voltage pulse generator +/- 60 kV

Sanitary glove box

PEF processing section

Thermal processing section

126

can be connected electrically parallel and fluid flow wise in series. A sanitary glove box

(The Ohio State University, Columbus, OH, U.S.A.) was used as a filling unit for this

research. The glove box was sanitized by spraying and swabbing 35% hydrogen

peroxide and lighting germicidal UV at 254 nm for 12 h.

Thermally processed, PEF processed, and control orange juices

Processing conditions for thermally processed, PEF processed, and control orange

juices are listed in Table 4.1. The processing conditions were used for microbial

inactivation, shelf life, and sensory studies. The production rate was 500 L/h for

thermally processed, PEF processed, and control orange juices.

For thermal processing, orange juice was held at 90 °C for 90 s in a holding tube

and then cooled to 25 °C by Heat Exchanger 2 (Figure 4.1). The high voltage pulse

generator and the heat exchangers in PEF Processing Section (Figure 4.1) were not

activated during the thermal processing. The cooled orange juice was packaged inside

the sanitary glove box (Figure 4.1).

PEF processing conditions were defined by electric field strength of 40 kV/cm,

pulse duration time of 2.6 µs, and total PEF treatment time of 97 µs. Orange juice was

PEF processed while thermal processing was turned off. Orange juice was maintained at

45 °C before entering each set of two PEF chambers by Heat Exchangers 3, 4, and 5

(Figure 4.1). PEF processed orange juice was cooled to 25 °C by Heat Exchanger 6 prior

to packaging inside the sanitary glove box (Figure 4.1).

127

Processing parameter Thermally processed

orange juice

PEF processed

orange juice

Control orange

juice



Pulse duration time (µs) 0 2.6 0

Pulse treatment time (µs) 0 97 0

Number of PEF chamber 8 8 8



Temperature change per a pair

of PEF chamber (°C)

0

13

0

Temperature before filling

(°C) 25 25 25

Table 4.1: Processing conditions for thermally processed, PEF processed, and control

orange juices

128

For the control orange juice, orange juice was passed through the OSU-6

commercial scale PEF processing system without thermal or PEF processing and

packaged inside the sanitary glove box.


Thermally processed, PEF processed, or control orange juice was packaged into

the 50-mL sterilized polypropylene tubes (Corning, Acton, MA, U.S.A.) inside the

sanitary glove box. Packaged thermally processed, PEF processed, control orange juices

were stored at 4 ºC in a walk-in refrigerator (Thermolinear, Cincinnati, OH, U.S.A.).

Microbial inactivation study

The freshly squeezed orange juice was incubated at 22 ºC for 3 d to grow

endogenous microorganisms. The microbial counts of total aerobic microorganisms and

yeasts & molds were 6.2 log CFU/mL and 5.8 log CFU/mL, respectively, after the

incubation. The incubated orange juice was thermally processed or processed by PEF.

Plate count agar (PCA) and acidified potato dextrose agar (PDA) were used to

enumerate total aerobic microorganisms and yeasts & molds, respectively. PCA, PDA,

and Peptone water were purchased from Difco (Detroit, MI, U.S.A.). PDA was acidified

with 10% tartaric acid (Sigma-Aldrich, St. Louis, MO, U.S.A.). Orange juice was diluted

with 0.1% sterile peptone water up to 10-4 dilution and plated by a spiral autoplater

(model 3000, Spiral Biotech Inc., Bethesda, MD, U.S.A.). PCA plates were incubated at

30 °C for 48 h. PDA plates were incubated at 22 °C for 5 d.

129

Shelf life study

Freshly squeezed orange juice was thermally processed or PEF processed for

shelf life study. Thermally processed, PEF processed, and control orange juices were

packaged and stored at 4 °C. Thermally processed and PEF processed orange juices were

sampled for 196 d. Control orange juice was sampled for 21 d for the microbial stability

study and for 14 d for the other shelf life studies due to gas formation by multiplied

microorganisms.

Microbial stability. The microbial stability was determined by the same method

as used for the microbial inactivation study. The initial microbial counts of total aerobic

microorganisms and yeasts & molds of control orange juice were 3.5 log CFU/mL and

3.3 log CFU/mL, respectively.

Ascorbic acid. The concentration of ascorbic acid in the orange juice was

measured by the method of Yeom and others (2000) using a high-performance liquid

chromatography (HPLC) system (Hewlett Packard, 1050 series, Wilmington, DE,

U.S.A.).

Flavor compounds. Volatile flavor compounds of orange juice were analyzed by

the method of Jia and others (1998) using a headspace solid-phase microextraction gas

chromatography (SPME-GC) system. Standard flavor compounds of α-pinene, myrcene,

octanal, d-limonene, and decanal were purchased from Aldrich Chemical Co.

(Milwaukee, WI, U.S.A.). Polydimethylsiloxane (PDMS) SPME fibers, 10-mL vials,

130

Teflon-coated rubber septa, and aluminum caps were purchased from Supelco, Inc.

(Bellefonte, PA, U.S.A.). The gas chromatography (GC) (5890, Hewlett-Packard,

Wilmington, DC, U.S.A.) was baked in an oven at 150 °C for 3 h. A 1.0 mL orange juice

was transferred into a sealed 10-mL vial containing a micromagnetic bar (10 × 3 mm).

The SPME fiber was inserted into the headspace of the 10-mL vial containing orange

juice. The orange juice in the vial was incubated at 60 °C for 20 min in a water bath

(Isotemp 228, Fisher Scientific, Pittsburgh, PA, U.S.A.). The orange juice in the vial was

magnetically stirred by an immersed stirrer (LTE Scientific Ltd., Greenfield, Oldham,

U.K.) during the incubation. The SPME fiber was moved from the vial and immediately

injected into the GC injection port at 220 °C and kept for 2 min to allow for desorption of

flavor compounds. The desorbed flavor compounds were separated by the GC equipped

with a capillary column (30 m × 0.53 mm i.d.) coated with a 2.65 µm film of 5% phenyl-

substituted methylpolysiloxane and a flame ionization detector. The temperature of the

GC was programmed from 60 to 120 °C at a rate of 10 °C/min, increased to 140 °C at a

rate of 4 °C/min, and then increased to 200 °C at a rate of 20 °C/min and held for 5 min.

The GC chromatograph peak area was calculated using a Hewlett Packard integrator (HP

3396A, Wilmington, DE, U.S.A.).

Identification of volatile compounds. The volatile flavor compounds was

identified by a combination of the GC retention time of standard flavor compounds and a

Hewlett-Packard 5970 Series mass selective detector (MS, Wilmington, DE, U.S.A.).

The MS was equipped with a Hewlett-Packard 59822 B ionization gauge controller.

Mass spectra were obtained at 70 eV and 220 °C ion source temperature. The GC

131

conditions for GC-MS were the identical to the GC conditions for the analysis of flavor

compounds described earlier. Information of the computer software of NIST Mass

Spectral Library (NIST’02. Version 2.0, Gaithersburg, MD, U.S.A.) was used for the

identification.

Brown color. The brown color was measured by the method of Yeom and others

(2000). Orange juice was centrifuged at 12500 g for 10 min at 22 °C by a Beckman

Microfuge E (Beckman Instruments Inc., Palo Alto, CA). Supernatant was clarified

using 0.45 µm filter (Gelman Sciences, Ann Arbor, MI, U.S.A.). The absorbance of the

filtrate was measured at 420 nm by a Spectronic Genesys 5 spectrometer (Milton Roy,

Rochester, NY, U.S.A.) at 22°C to determine brown color.

Hunter L, a, and b. Hunter L, a, and b were determined by a HunterLab

colorimeter (Hunter Associates Laboratory Inc., Reston, VA, U.S.A.). The lightness (L)

and hue angle (arctangent of b / a × 57.3) were reported.

Brix and pH. The Brix of orange juice was measured using a hand-held

refractometer (Fisher Scientific, Pittsburgh, PA, U.S.A.). The pH of orange juice was

measured using a pH meter (370, Orion, Beverly, MA, U.S.A.) at 22 °C.

Sensory analysis

The sensory evaluation was performed immediately after processing. A 30-

member panel participated in the sensory tests. The panelists were from the department

132

of Food Science and Technology at the Ohio State University and food industry. The

panelists were asked to rate the preference of color, appearance, texture, flavor, and

overall acceptability. A hedonic scale of 1 to 9 was used for each attribute. The higher

number represents higher preference of attributes. Thermally processed, PEF processed,

and control orange juices were served in randomly numbered plastic cups on a tray with a

cup of water and a piece of non-salted cracker.



significance levels were conducted to determine significant differences among thermally

processed, PEF processed, and control orange juices. The entire analyses were

duplicated with 4 measurements. Minitab 13.31 (Minitab, Inc., State College, PA,

U.S.A.) was used for all statistical analyses.


Effects of thermal processing and PEF processing on the microbial inactivation

The total aerobic plate count and the yeast & mold plate count for the incubated

orange juice at 22 °C for 3 d were 6.2 logs and 5.8 logs, respectively. After either

thermal or PEF processing, the total aerobic plate count and the yeast & mold count were

all < 10 CFU/mL est. Both thermal processing and commercial scale PEF processing

inactivated about 6 logs of endogenous microorganisms in the orange juice.

133

Yeasts are known to be more tolerant to high temperature than bacteria (Kimball

1991). Sadler and others (1992) reported that pasteurization at 66 °C for 10 s inactivated

yeasts in orange juice from 3.6 to 3.1 log CFU/mL and could not prevent the growth of

yeasts at 4 °C. The temperature of orange juice increased from 45 °C to 58 °C and

maintained at 58 °C for 5 s during the PEF processing. The heat generated at 58 °C for 5

s is not considered as a major cause of yeast inactivation in our study. Dunn (2001)

mentioned that the microbial reductions of Escherichia coli in a model nutritive medium

by below 80 J/mL energy during PEF treatment would be resulted from the effect of PEF

alone. The energy of the PEF processing, which was calculated using the equation of

Wouters and others (2001), was 6.9 J/mL. The 6 log reduction of microorganisms by the

PEF processing in this study would be mainly due to PEF.

Effects of thermal processing and PEF processing on the microbial stability during

storage

Effects of thermal processing and PEF processing on the total aerobic plate counts

and the yeast & mold counts of orange juice during storage at 4 ˚C for 196 d are shown in

Table 4.2. The initial total aerobic plate counts and yeast & mold counts of control

orange juice were 3.5 log CFU/mL and 3.3 log CFU/mL, respectively. After either

thermal or PEF processing, the total aerobic plate count and the yeast & mold count were

all < 10 CFU/mL est. Both thermally processed and PEF processed orange juice

maintained the microbial stability of microorganisms at less than 1 log at 4 °C for 112 d.

Both total aerobic plate count and yeast & mold count of control reached 5.2 logs after

134

Total aerobic plate counts

(log CFU/mL)

Yeast & mold counts

(log CFU/mL)

Storage

time

(days) Heat PEF Control Heat PEF Control

0 < 10a < 10 3.5 < 10 < 10 3.3

7 < 10 < 10 3.7 < 10 < 10 3.3

14 < 10 < 10 3.9 < 10 < 10 3.7

21 < 10 < 10 5.2 < 10 < 10 5.2

28 < 10 < 10 NDb < 10 < 10 ND

56 < 10 < 10 ND < 10 < 10 ND

112 < 10 < 10 ND < 10 < 10 ND

133 < 10 1.0 ND < 10 1.2 ND

154 < 10 3.2 ND < 10 3.1 ND

196 < 10 3.8 ND < 10 3.7 ND

Heat = thermally processed orange juice; PEF = PEF processed orange juice; Control = control orange juice. a< 10 log CFU/mL est. bND: not determined because tomato juice was not sampled due to gas formation by multiplied microorganisms. Values are mean of duplicates of 4 measurements.

Table 4.2: Effects of thermal processing and PEF processing on the total aerobic plate counts and the yeast & mold counts of orange juice during storage at 4 °C for 196 days

135

21 d. The numbers of total aerobic microorganism and yeast & mold of thermally

processed and PEF processed orange juice were less than 1 log and 4 logs, respectively,

during storage at 4 °C for 196 d.

A microscopic examination showed that orange juice sampled immediately after

PEF processing had ascospores of molds and yeasts. Little effect of PEF on the

inactivation of mold and yeast ascospores, including Byssoclamys nivea ascospores,

Neosartorya fischeri ascospores, and Zygosaccharomyces Bailii ascospores in orange,

apple, pineapple, cranberry, and grape juices was reported (Grahl and Markl 1996; Raso

and others 1998a; Raso and others 1998b). The increase in the microbial counts of PEF

processed orange juice after 112 d might be due to the growth of yeasts and molds from

the ascospores initially present in the PEF processed orange juice.

Effects of thermal processing and PEF processing on the ascorbic acid

The effects of thermal processing and PEF processing on the concentration of

ascorbic acid in orange juice during storage at 4 °C for 196 d are shown in Figure 4.2.

PEF processing did not change the concentration of ascorbic acid significantly (p > 0.05).

Thermal processing reduced 19% of ascorbic acid (p < 0.05). The concentrations of

ascorbic acid in thermally processed, PEF processed, and control orange juices decreased

as storage time increased. However, PEF processed orange juice retained more ascorbic

acid than thermally processed orange juices for 84 d at 4 °C (p < 0.05).

Ascorbic acid retention has been used as an indicator of orange juice shelf life

(Shaw 1992). Squires and Hanna (1979) reported that orange juice should contain at

least 25 mg of vitamin C per 100 mL at the time of expiration date to provide 100% of

136

0

10

20

30

40

50

60

0 20 40 60 80 100 120 140 160 180 200

Storage time (days)

Con

cent

ratio

n of

Asc

orbi

c Ac

id

(mg/

100m

L)HeatPEFControl

Figure 4.2: Effects of thermal processing and PEF processing on the retention of ascorbic acid of orange juice during storage at 4 °C for 196 days

(days)

Asc

orbi

c ac

id c

once

ntra

tion

(mg/

100m

L)

137

the U.S. Recommended Daily Allowances (USRDA) requirement. The concentration of

ascorbic acid in PEF processed orange juice decreased to 25 mg/100 mL at 4 °C after 84

d, which is longer than the 56 d of thermally processed orange juice.

Effects of thermal processing and PEF processing on the flavor compounds

Effects of thermal processing and PEF processing on the retention of myrcene of

orange juice during storage at 4 °C for 196 d are shown in Figure 4.3 and on the retention

of α-pinene, octanal, d-limonene and decanal of orange juice are shown in Table 4.3.

Thermal processing and PEF processing decreased myrcene by 37% and 12%,

respectively. PEF processed orange juice retained more myrcene than thermally

processed orange juice during storage at 4 °C for 154 d (p < 0.05). PEF processed orange

juice also retained more α-pinene, octanal, d-limonene, and decanal than thermally

processed orange juice, especially during the early storage (Table 4.3) (p < 0.05).

Thermal processing degrades the flavor compounds of orange juice (Shaw 1992;

Braddock 1999) including myrcene, α-pinene, octanal, d-limonene, decanal, citral,

acetaldehyde, and ethyl butanoate (Ahmed and others 1978; Chen and others 1993). The

heat of thermal processing initiates and accelerates chemical reactions (Ekasari and

others 1988). PEF processed orange juice was exposed to 58 °C for 5 s whereas

thermally processed orange juice was heated at 90 °C for 90 s (Table 4.1). The more

retention of flavor compounds in PEF processed orange juice than thermally processed

orange juice may be due to the low processing temperature and heat load during PEF

processing.

138

0102030405060708090

100110

0 20 40 60 80 100 120 140 160 180 200

Storage time (days)

Rete

ntio

n of

myr

cene

(%)

HeatPEFControl

Figure 4.3: Effects of thermal processing and PEF processing on the retention of myrcene

of orange juice during storage at 4 °C for 196 days

(days)

139

Heat = thermally processed orange juice; PEF = PEF processed orange juice. 1Different letters in the same row of each flavor compound indicate significant differences (p < 0.05). The concentrations of 100 % α-pinene, octanal, limonene, and decanal were 0.041, 1.602, 680.7, and 0.707 ppm, respectively. Values are mean of duplicates of 4 measurements.

Table 4.3: Effects of thermal processing and PEF processing on the retention of flavor compounds (%) of orange juice during storage at 4 °C for 196 days

α-pinene Octanal d-limonene Decanal Storage

time (days) Heat PEF Heat PEF Heat PEF Heat PEF

0 80 a1 98 b1 69 a 81 b 71 a 87 b 77 a 93 b

7 73 a 83 b 65 a 78 b 65 a 75 b 72 a 80 b

14 69 a 78 b 63 a 71 b 62 a 74 b 64 a 74 b

21 67 a 78 b 59 a 67 b 58 a 73 b 61 a 70 b

28 66 a 72 a 55 a 60 a 58 a 63 a 59 a 63 a

35 61 a 67 a 50 a 58 b 55 a 60 a 56 a 56 a

42 60 a 66 a 42 a 52 b 51 a 55 a 53 a 48 a

56 53 a 62 b 39 a 44 a 43 a 50 a 40 a 43 a

70 50 a 61 b 38 a 42 a 38 a 44 a 37 a 38 a

112 46 a 55 a 35 a 39 a 36 a 44 b 35 a 37 a

133 43 a 53 a 32 a 35 a 35 a 43 a 33 a 33 a

154 42 a 50 a 27 a 33 a 35 a 41 a 32 a 31 a

196 42 a 49 b 15 a 21 a 32 a 35 a 29 a 30 a

139

140

Effects of thermal processing and PEF processing on the brown color

Effects of thermal processing and PEF processing on the brown color of orange

juice during storage at 4 °C for 196 d are shown in Figure 4.4. The brown color of

thermally processed orange juice increased from 0.20 to 0.34 and that of PEF processed

orange juice increased from 0.18 to 0.31 during storage at 4 °C for 196 d. PEF processed

orange juice showed significantly less browning than thermally processed orange juice

during storage at 4 °C for 196 d (P < 0.05).

The browning reaction in orange juice depends on thermal processing temperature

and time and is considered a useful indicator of over-processing (Elkins and others 1988;

Lee and Nagy 1988). The less browning of PEF processed orange juice than thermally

processed orange juice may be due to less heating during PEF processing than thermal

processing.

Effects of thermal processing and PEF processing on the lightness and the hue angle

Effects of thermal processing and PEF processing on the lightness (L) and the hue

angle (θ) of orange juice during storage at 4 °C for 196 d are shown in Figure 4.5 and

Table 4.4, respectively. The L values at 0 d were 48.6, 49.2, and 50.7 and the hue angles

were 35.4, 54.0, and 54.5 for thermally processed, PEF processed, and control orange

juices, respectively. PEF processed orange juice showed significantly higher L values

and hue angles than thermally processed orange juice during the storage time including 0

d (p < 0.05).

141

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0 20 40 60 80 100 120 140 160 180 200

Storage time (days)

Abso

rban

ce a

t 420

nm

HeatPEFControl

Figure 4.4: Effects of thermal processing and PEF processing on the brown color of orange juice during storage at 4 °C for 196 days

(days)

Bro

wn

colo

r (A

420)

142

40

42

44

46

48

50

52

0 20 40 60 80 100 120 140 160 180 200

Storage time (days)

L

Heat

PEF

Control

Figure 4.5: Effects of thermal processing and PEF processing on the lightness (L) of orange juice during storage at 4 °C for 196 days

(days)

Ligh

tnes

s (L

)

143

Hue angle (θ) Storage time

(days) Heat PEF Control

0 53.4a1 54.0b1 54.5c1

7 53.5a 54.1a 54.3a

14 53.6a 54.2a 54.0a

21 53.2a 53.8a ND2

28 54.2a 54.7a ND

56 54.0a 54.8a ND

84 55.0a 55.6a ND

112 52.9a 54.5a ND

133 52.4a 55.1a ND

154 52.0a 54.0a ND

196 51.7a 53.8a ND Heat = thermally processed orange juice; PEF = PEF processed orange juice; Control = control orange juice. 1Different letters in the same row of each flavor compound indicate significant differences (p < 0.05). 2ND: not determined because tomato juice was not sampled due to gas formation by multiplied microorganisms. Values are mean of duplicates of 4 measurements.

Table 4.4: Effects of thermal processing and PEF processing on the hue angle (θ)

of orange juice during storage at 4 °C for 196 days

144

Color study indicates that PEF processed orange juice had brighter color than

thermally processed orange juice during the storage, which also can be drawn from the

result of the brown color study (Figure 4.4). Detrimental changes in the color of orange

juice are primarily caused by the nonenzymatic browning reaction (Klim and Nagy 1988).

Effects of thermal processing and PEF processing on the Brix and pH

The Brix values of thermally processed and PEF processed orange juices were

about 12 during storage at 4 °C for 196 d. The Brix of control orange juice decreased

significantly from 12.1 to 10.9 during storage for 14 d (p < 0.05). The pH values of

thermally processed and PEF processed orange juices were about 4 at 4 °C for 196 d.

The pH values of control orange juice were about 4 at 4 °C for 14 d.

The significant decrease in the Brix of control orange juice may be due to the

multiplication of microorganisms and their consumption of soluble solids such as

carbohydrates. No significant differences in Brix and pH between thermally processed

and PEF processed orange juice during storage at 4 °C for 112 d were observed (p >

0.05). The Brix and pH of thermally processed and PEF processed orange juices did not

change significantly during the storage (p > 0.05). This no significant change in the Brix

and pH of thermally processed and PEF processed orange juices during the storage may

be due to the effective inactivation of spoilage microorganisms by thermal processing and

commercial scale PEF processing.

145

Samples

Attributes

Heat PEF Control

Color 6.1 a1 6.8 a1 7.0 a1

Appearance 5.8 a 6.2 a 6.8 a

Texture 4.8 a 6.0 b 7.3 c

Flavor 3.7 a 5.9 b 7.8 c

Overall acceptability 4.2 a 6.1 b 7.4 c

Heat = thermally processed orange juice; PEF = PEF processed orange juice; Control = control orange juice. 1Different letters in the same row indicate significant differences (p < 0.01).

Table 4.5: Effects of thermal processing and PEF processing on the sensory evaluation of color, appearance, texture, flavor, and overall acceptability of orange juice

146

Effects of thermal processing and PEF processing on sensory quality

The sensory evaluation results are shown in Table 4.5. The control orange juice

had the highest preference on texture, flavor, and overall acceptability (p < 0.01). PEF

processed orange juice had a higher preference on texture, flavor, and overall

acceptability than thermally processed orange juice (p < 0.01). Thermal processing can

strongly change the sensory properties of orange juice including fresh juice flavor (Chen

and others 1993; Braddock 1999). Texture and flavor are important elements in

consumers’ perception of the freshness of orange juice. Also, the overall acceptability of

orange juice may be mainly determined by freshness. The results from the sensory

evaluation may imply that PEF processed orange juice was fresher than thermally

processed orange juice.

CONCLUSIONS

Commercial scale PEF processing was effective in inactivating endogenous

microorganisms in orange juice and extended the shelf life of orange juice while reducing

the degradation of the major qualities of orange juice including ascorbic acid, flavor, and

color. Commercial scale PEF processing also provided better sensory properties of

orange juice than thermal processing. PEF processed orange juice possessed higher fresh

quality than the thermally processed tomato juice. It is commercially feasible to

pasteurize orange juice by PEF technology.

147

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Yin Y, Zhang QH, Sudhir SK, inventors; The Ohio State University, assignee. 1997 Nov.



150

ACKNOWLEDGMENTS


funding this research. We thank Dr. R. Man and Mr. R. Caldwell for the preparation of

orange juice and the operation of the PEF system.

151

CHAPTER 5

Inactivation Kinetics of Tomato Juice Lipoxygenase by Pulsed Electric Fields

ABSTRACT

Pulsed electric field (PEF) inactivation kinetics for tomato juice lipoxygenase (LOX)

were studied and the primary variable for the inactivation of LOX by PEF was found.

Tomato juice was treated by PEF with the combinations of electric field strength (0, 10,

15, 20, 30, 35 kV/cm), PEF treatment time (20, 30, 50, 60, 70 µs), and PEF treatment

temperature (10, 20, 30, 40, 50 °C). The PEF treatment at 30 kV/cm for 60 µs at 50 °C

inactivated 88.1% of LOX. The first-order kinetic models, the Hulsheger’s model, and

the Fermi’s model adequately described the LOX inactivation by PEF. Calculated D-

values were 161.0, 112.9, 101.0, and 74.8 µs at 15, 20, 30, and 35 kV/cm at 30 °C,

respectively. The activation energy for the inactivation of LOX by PEF was 35.7 kJ/mole.

Applied electric field strength was the primary variable for the inactivation of LOX by

PEF.

Key words: Pulsed electric field, lipoxygenase, tomato juice, kinetics, temperature

152

INTRODUCTION

Lipoxygenase (LOX), a nonheme iron-containing dioxygenase, is widely present

in plant tissues (Wong 1995). LOX catalyzes the oxidation of polyunsaturated fatty acids

containing a cis, cis-1,4-pentadiene system, which produces 9- or 13- cis, trans-

hydroperoxides. The hydroperoxides generally decompose into acids, ketones, and

aldehydes (Leoni and others 1985). Tomato juice LOX initiates the formation of the

fresh flavor compounds of tomato. Most desirable fresh flavor compounds of tomato

including hexanal, cis-3-hexenal, trans-2-hexenal, hexanol, trans-2-hexenol, and cis-3-

hexenol are generated by LOX from the unsaturated fatty acids such as linoleic acid and

linolenic acid (Galliard and others 1977). However, tomato juice LOX can destruct

essential fatty acids and develop off-flavor in tomato juice during storage. Also, the

hydroperoxides and free radicals produced from the fatty acid by LOX can degrade

vitamins and proteins in tomato juice during storage. The desired amount of LOX

activity in the final tomato juice may depend on the use of the juice. If a tomato juice

needs to be shelf stable, a minimum LOX activity in the tomato juice may be desirable.

If a tomato juice needs to have fresh flavor and will be stored for a short time at

refrigerated temperature, the LOX activity in the tomato juice may remain higher than

that of the shelf stable tomato juice.

Thermal treatment is the most common method to extend the shelf life of tomato

juice by inactivating microorganisms and enzymes. However, thermal treatment can

degrade the sensory and nutritional qualities of foods (Rouseff and Leahy 1995;

Braddock 1999). PEF treatment has been extensively studied as a nonthermal food

153

preservation method (Mertens and Knorr 1992; Dunn 2001). PEF treatment increases the

shelf life of juice products while reducing the loss of their flavor, color, and nutrients

(Qiu and others 1998; Jin and others 1999; Evrendilek and others 2000; Yeom and others

2000).

Investigation on the effects of PEF on the enzymes responsible for the quality

alteration of foods during storage is needed for the commercialization of PEF processing

(Raso and others 1998). PEF may be effective for the inactivation of some selected

enzymes. Vega-Mercado and others (1995) reported the 90% reduction of plasmin

activity in a simulated milk ultrafiltrate by a PEF treatment at 30 kV/cm for 100 µs. Ho

and others (1997) reported significantly reduced activities of lipase, glucose oxidase,

heat-stable α-amylase, peroxidase, and polyphenol oxidase after PEF treatment. Yeom

and others (1999) observed a 90% inactivation of papain by PEF at 50 kV/cm. Giner and

others (2000) achieved a 94% reduction in the activity of tomato pectin methyl esterase

(PME) with a PEF treatment at 24 kV/cm. They applied first-order kinetic models, the

Hulsheger’s kinetic model, and the Fermi’s kinetic model to the inactivation data of

tomato PME by PEF and showed that these models fit experimental data adequately.

Energy input is considered as the cause of the inactivation of microorganisms and

enzymes by PEF. However, a question can be raised if the inactivation rates are all the

same as long as the amounts of energy input are same. The primary variable in the

inactivation of microorganisms and enzymes by PEF needs to be found to obtain

optimum PEF treatment conditions for desired levels of inactivation. The objectives of

this study were (1) to study the effects of PEF on the inactivation of tomato juice LOX,

154

(2) to evaluate the use of kinetic models for the inactivation of tomato juice LOX by PEF,

and (3) to find the primary variable responsible for the inactivation of tomato juice LOX

by PEF.



Tomatoes (Red Bear, Ruthven, Canada) were purchased from a local grocery

store (Kroger, Columbus, OH, U.S.A.) during August, September, and October in 2002.

Tomato juice was prepared from the tomatoes by a juice maker (JM-I, Juiceman Jr., Mt.

Prospect, IL, U.S.A.).

PEF treatment system

OSU-4A bench scale PEF system (The Ohio State University, Columbus, OH,

U.S.A.) was used for the PEF treatment of tomato juice. OSU-4A can generate voltage

up to 15 kV and provide bipolar or unipolar pulses with a frequency of 0 – 2000 pulses

per second (pps) and a pulse width of 2-15 µs. A signal generator (Model 9310 Quantum

Composers, Bozeman, MT, U.S.A.) controlled pulse width and pulse delay time (time

between the positive and negative pulses). The waveforms of pulses and the dosages of

voltage and current were monitored by a two channel digital oscilloscope (Model TDS-

210, Tektronix Inc., Beaverton, OR, U.S.A.). Bipolar square wave pulse pairs were used

for this research and shown in Figure 5.1. Four (two pairs) co-field tubular PEF

treatment chambers were connected to OSU-4A. Figure 5.2 shows the flow arrangement

155

Figure 5.1: A bipolar square wave pulse pair. A peak voltage and a peak current are indicated.

Current

Voltage Peak voltage

Peak current

156

• • • •

Figure 5.2: Flow arrangement of four PEF treatment chambers connected to stainless coils which tomato juice passed through. T1, T2, T3, and T4 are inlet or outlet temperatures.

High Voltage

Ground

Electrode

Insulator

One set of PEF chamber

Water bath

T1 T2 T3 T4

Gap distance

Inner diameter

157

of four PEF treatment chambers. The inner diameter and gap distance of the PEF

treatment chamber are 0.23 cm and 0.29 cm, respectively. Temperatures, T1, T2, T3, and

T4, were measured at the inlet and outlet of each pair of chambers by placing

thermocouples (Type K, 1.6 mm diameter, Fisher Scientific, Pittsburgh, PA, U.S.A.) to

the surface of stainless steel coils (Figure 5.2). The thermocouples were connected to

temperature readers (Fluke 52, Fluke Corporation, Everett, WA, U.S.A.).

PEF treatment conditions and experimental design

The PEF treatment conditions for the kinetic study of the LOX inactivation by

PEF and for the study determining the primary variable in the LOX inactivation by PEF

are shown in Table 5.1. PEF treatment temperature was T1 (Figure 5.2). A blocked full

factorial design with six electric field strength levels (0, 10, 15, 20, 30, 35 kV/cm), five

PEF treatment time levels (20, 30, 50, 60, 70 µs), and five PEF treatment temperature

levels (10, 20, 30, 40, 50 °C) was used. Experiments at the same PEF treatment

temperature level were executed within 1 block.

Measurement of lipoxygenase activity

Lipoxygenase activity was measured by the method of Morales-Blancas and

others (2002) with some modifications. The 10 mL tomato juice was homogenized with

10 mL cold (4 °C) 0.2 M potassium phosphate buffer (pH 6.5) for 1 min by a

homogenizer (M133/1281-0, Biospec Products Inc., Bartlesville, OK). The 5 mL

homogenate was centrifuged at 18,000 × g for 30 min at 4 °C. The supernatant from the

158

(a)

Treatment parameter Condition

Electric field strength (kV/cm) 10, 15, 20, 30, 35

PEF treatment time (µs) 20, 30, 50, 60 ,70

PEF treatment temperature (°C) 10, 20, 30, 40, 50

Pulse width (µs) 3

Pulse delay time a (µs) 20

Flow rate (mL/s) 1 a Pulse delay time = time between two pulses

(b)

Treatment parameter Condition 1 Condition 2 Condition 3

Electric field strength (kV/cm) 30.1 17.8 9.0

Current (A) 40 24 13

PEF treatment time (µs) 60 165 572

PEF treatment temperature (°C) 20 20 20

Pulse width (µs) 3 8 32

Pulse delay time a (µs) 2.4 2.4 2.4

Flow rate (mL/s) 1 1 1

∆ T c 25 25 25 a Pulse delay time = time between two pulses ∆ T = T4 – T3

Table 5.1: PEF treatment conditions for (a) the inactivation kinetics of tomato juice LOX by PEF and for (b) the study determining the primary variable in tomato juice LOX inactivation by PEF

159

centrifugation was filtered through a 0.45 µm syringe filter (Gelman Sciences, Ann Arbor,

MI, U.S.A.). The filtered supernatant was used as the enzyme extract. To prepare the

LOX substrate solution, 300 µL linoleic acid (Sigma-Aldrich, St. Louis, MO, U.S.A.),

300 µL Tween 20 (Sigma-Aldrich, St. Louis, MO, U.S.A.), and 15 mL distilled water

were mixed. The mixed solution was clarified by adding 1.5 mL sodium hydroxide (1 N)

(Fisher Scientific, Pittsburgh, PA, U.S.A.). Before assay, the 15 mL clarified mixed

solution was diluted to 60 mL with 0.2 M potassium phosphate buffer (pH 6.5). The 60

mL diluted solution was shaken manually for 2 min and then left to settle in the dark at

22 °C for 10 min. The 60 mL solution settled for 10 min was used as the substrate

solution. The 0.5 mL enzyme extract was pipetted into a quartz cuvet (Standard, Fisher

Scientific, Pittsburgh, PA, U.S.A.) containing 3.0 mL substrate solution and mixed with

the substrate solution for 3 s by a vortex (Genie 2, Fisher Scientific, Pittsburgh, PA,

U.S.A.). The absorbance was measured at 234 nm for 3 min at 22 °C by a

spectrophotometer (UV-2401PC, Shimadzu Scientific Instruments, Inc. Columbia, MD,

U.S.A.) immediately after the mixing. A blank was prepared with 0.5 mL distilled water

and 3.0 mL substrate solution. The volume of the 60 mL substrate solution was prepared

for 15 measurements. The spectrophotometer reading was recalibrated with a fresh

substrate solution after each time of the preparation of substrate solution. The rate of the

reaction was automatically computed from the linear portion of the absorbance curve.

The 1 unit of lipoxygenase activity was defined as a change of 0.001 units of absorbance

per minute and milliliter of enzyme extract.

160

Kinetics of LOX inactivation by PEF

The residual activity (RA) of LOX obtained after each PEF treatment was defined

as:

RA = A / A0 (1)

where A is the LOX activity after PEF treatment and A0 is the initial LOX activity before

PEF treatment.

Experimental data were fit to the first-order kinetic models in Equations (2) and

(5), the Hulsheger’s kinetic model (Hulsheger and others 1981) in Equation (6), and the

Fermi’s kinetic model (Peleg 1995) in Equation (7) by Minitab 13.31 (Minitab, Inc., State

College, PA, U.S.A.). The inactivation rate constant of kE or kN was obtained from the

slope of the regression of ln(RA) versus PEF treatment time (t) or versus electric field

strength (E), respectively.

ln(RA) = -kEt (2)

where t is the PEF treatment time (µs) and kE is the first-order kinetic constant with t.

161

Decimal reduction times (D) were calculated using the formula:

D = 2.303 / kE (3)

The activation energy (Ea) (J/mole) was calculated from the slope of the regression of ln

(kE) versus 1/T:

ln (kE) = -Ea / RT ⋅ 1/T (4)

where RT is the universal gas constant (8.3144 J/mole K-1) and T is the PEF treatment

temperature (K).

ln(RA) = -kNE (5)

where E is the electric field strength (kV/cm) and kN is the first-order kinetic constant

with E.

( ) kEE

c

c

ttRA

/−−

= (6)

where t is the PEF treatment time (µs), tc is the critical treatment time below which no

inactivation of LOX occurs (µs), E is the electric field strength (kV/cm), Ec is the critical

electric field strength below which no inactivation of LOX occurs (kV/cm), and k is the

specific rate constant.

162

aEE heRA /)(1

1−+

= (7)

where E is the electric field strength (kV/cm), Eh is the electric field strength (kV/cm)

where RA is 0.5, and a is the parameter indicating the slope of the curve around Eh.

Determination of energy input

The peak power and the energy input during PEF treatment were calculated using

following equations:

Peak power (W) = Peak voltage (V) × Peak current (A) (8)

Energy input (J) = Peak power (W) × PEF treatment time (s) (9)

The peak voltage and the peak current were read from waveforms using the oscilloscope

(Figure 5.1).


Analysis of variance and regression analysis were conducted with Minitab 13.31

(Minitab, Inc., State College, PA, U.S.A.) to estimate the model parameters (kE, kt, tc, Ec,

k, Eh, a).

163


First-order kinetic models

Effects of PEF treatment time on the residual activity (RA) of LOX after PEF

treatment at 15, 20, 30, or 35 kV/cm at 30 °C are shown in Figure 5.3. The LOX activity

decreased with any increase in PEF treatment time or electric field strength. A 80 %

reduction of LOX activity was observed when LOX was exposed to PEF at 35 kV/cm for

50 or 60 µs at 30 °C.

The RA values as a function of PEF treatment time at 30 °C at the different levels of

electric field strength were fit to the first-order kinetic model of Equation 2. The

regression parameters of the first-order kinetic model are listed in Table 5.2 (p = 0.05).

The high R2 values indicate that the first-order kinetic model is valid for describing the

inactivation of tomato juice LOX by PEF at the different levels of electric field strength.

The p-value of lack-of-fit, which is a formal assessment of the adequacy of simple linear

regression use, was 0.882, 0.978, 0.940, or 0.867 at 15, 20, 30, or 35 kV/cm, respectively.

The high p-values of lack-of-fit indicate that ln (RA) and PEF treatment time at 30 °C at

the different levels of electric field strength had a linear relationship.

The effect of electric field strength on kE is shown in Figure 5.4(a). The result

showed a positive and exponential dependence (R2 = 0.91) between electric field strength

and kE. As the electric field strength increased from 15 to 35 kV/cm, the kE values

164

0

20

40

60

80

100

0 20 40 60 80

Treatment time (ms)

RA

%

15 kV/cm

20 kV/cm

30 kV/cm

35 kV/cm

Figure 5.3: Effects of PEF treatment time on the residual activity of LOX after PEF treatment at 15, 20, 30, or 35 kV/cm at 30 °C. Points are the average of a duplicate with four measurements. Plotted lines correspond to the adjustment of all points at each electric field strength to a first-order kinetic model.

Treatment time (µs)

165

Electric field

(kV/cm) kE (1/µs) R2

Lack-of-fit

(p-value) D (µs)

15 0.0163 ± 0.0018 0.940 0.882 161.0

20 0.0204 ± 0.0016 0.976 0.978 112.9

30 0.0228 ± 0.0027 0.946 0.940 101.0

35 0.0308 ± 0.0051 0.901 0.867 74.8

Table 5.2: Kinetic constants (KE) of the first-order kinetic model, correlation coefficients (R2), p-values of lack-of-fit, and decimal reduction times (D) of the PEF treatment at 15, 20, 30, or 35 kV/cm at 30 °C on tomato juice LOX

166

increased from 0.0163 to 0.0308 µs-1. This indicates that the LOX inactivation by PEF

increased as electric field strength increased. The effect of electric field strength on kE

can be described as:

kE = 0.0110 × e(0.0277)E (10)

The experimental RA was plotted against the predicted RA from Equation 10 and the plot

is shown in Figure 5.4(b). The slope and intercept obtained by simple regression (p =

0.05) agreed with their predicted values (R2 = 0.96).

D values decreased from 161.0 to 74.8 µs as electric field strength increased from

15 to 35 kV/cm (Table 5.2). The D value is defined as the PEF treatment time required to

reduce the activity by 90% at a given electric field strength and at a PEF treatment

temperature. A 90% inactivation of tomato juice LOX is expected after a PEF treatment

at 35 kV/cm with the PEF treatment time of 74.8 µs.

The temperature dependence of the inactivation of tomato juice LOX by PEF can

be explained with the concept of activation energy (Ea). The calculated activation energy

167

(a)

continued Figure 5.4: The effect of electric field strength (E) on the first-order kinetic constant (kE) (a) and the regression line of experimental residual activity of LOX versus predicted residual LOX activity from Equation 10 (b)

y = 0.011e0.0277x

R2 = 0.9115

0.010

0.015

0.020

0.025

0.030

0.035

10 15 20 25 30 35 40

E (kV/cm)

k (1

/ms)

Electric field strength (kV/cm)

k E (1

/µs)

168


(b)

Predicted RA

Expe

rimen

tal R

A

169

of the LOX inactivation by PEF was 35.7 kJ/mole. Yeom and others (2002) reported

that the activation energy of orange pectin methyl esterase (PME) by PEF was 36.4

kJ/mole. Activation energy is the energy required for a reaction to occur. The higher the

activation energy, the slower the reaction (Rawn 1989). Inactivation of orange PME may

be slightly more difficult than the inactivation of tomato juice LOX by PEF treatment.

Effects of electric field strength on the RA of tomato juice LOX after PEF

treatment at 10, 20, 30, 40, or 50 °C for 60 µs are shown in Figure 5.5. The LOX activity

was not changed significantly after PEF treatment at 10 kV/cm at 10, 20, or 30 °C (p >

0.05), but decreased significantly at 15, 20, 30, and 35 kV/cm at any level of PEF

treatment temperature (p < 0.05). A lower RA% was observed at a higher PEF treatment

temperature at 10, 20, 30, or 35 kV/cm. A maximum 88.1% inactivation of tomato juice

LOX was observed with the PEF treatment at 30 kV/cm for 60 µs at 50 °C.

There was about 47% difference between RA% values obtained after PEF

treatment at 10 °C (62.2%) and 50 °C (15.4%) at the same electric field strength of 20

kV/cm (Figure 5.5). The effects of PEF treatment temperature on the PEF inactivation of

microorganisms and PME were reported (Jayaram and others 1992; Zhang and others

1995; Reina and others 1998; Yeom and others 2002). They reported the synergistic

effect between PEF treatment and moderate temperature (45-60 °C). The inactivation of

microorganisms and PME by PEF increased markedly with the moderate treatment

temperature. The synergistic effect between PEF treatment and PEF treatment

temperature was observed in the LOX inactivation by PEF.

The RA was not significantly changed when electric field strength increased from

20 to 30 kV/cm at 40 or 50 °C. Biphasic inactivation models were proposed to describe

170

0

20

40

60

80

100

120

0 10 20 30 40

Electric field strength (kV/cm)

RA

%

10 C

20 C

30 C

40 C

50 C

Figure 5.5: Effects of electric field strength on the residual activity of LOX after PEF treatment at 10, 20, 30, 40, or 50 °C for 60 µs

171

the thermal inactivation kinetics of LOX of green beans, broccoli, green asparagus, and

carrots (Indrawati and others 1999; Morales-Blancas and others 2002). The biphasic

inactivation by thermal treatment was caused by a heat labile fraction and a heat resistant

fraction of LOX isoenzymes (Indrawati and others 1999; Morales-Blancas and others

2002). The tailing in the inactivation of tomato juice LOX by PEF might be due to the

presence of PEF resistant isoenzymes.

The RA values as a function of electric field strength at 60 µs at the different

levels of PEF treatment temperature were fit to the first-order kinetic model of Equation 5.

The regression parameters of the first-order kinetic model are shown in Table 5.3

(p = 0.05). The high R2 values indicate that the first-order kinetic model describe

successfully the inactivation of tomato juice LOX by PEF treatment at the different levels

of PEF treatment temperature. The p-value of lack-of-fit was 0.871, 0.906, 0.898, 0.854,

or 0.865 at 10, 20, 30, 40, or 50 kV/cm, respectively. These high p-values of lack-of-fit

indicate a linear relationship between ln (RA) and electric field strength.

The effect of the PEF treatment temperature on kN is shown in Figure 5.6(a). The result

showed a positive and exponential dependence (R2 = 0.93) between the PEF treatment

temperature and kN. The kN values increased from 0.0225 to 0.0730 cm/kV as the PEF

172

Water bath

temperature (°C) kN (cm/kV) R2

Lack-of-fit

(p-value)

10 0.0225 ± 0.0041 0.908 0.871

20 0.0326 ± 0.0045 0.947 0.906

30 0.0521 ± 0.0076 0.923 0.898

40 0.0726 ± 0.0226 0.838 0.854

50 0.0730 ± 0.0199 0.869 0.865

Table 5.3: Kinetic constants (KN) of the first-order kinetic model, correlation coefficients (R2), and p-values of lack-of-fit of the PEF treatment at 10, 20, 30, 40, or 50 °C at 60 µs on tomato juice LOX

173

(a)

continued Figure 5.6: The effect of PEF treatment temperature on the first-order kinetic constant (kN) (a) and the regression line of experimental residual activity of LOX versus predicted residual LOX activity from Equation 11 (b)

y = 0.0178e0.0315x

R2 = 0.9327

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0 10 20 30 40 50 60

Water bath temperature (C)

kN (c

m/k

V)

PEF treatment temperature (°C)

k N (c

m/k

V)

174


(b)

Predicted RA

Expe

rimen

tal R

A

175

treatment temperature increased from 10 to 50 °C. This indicates that the LOX

inactivation by PEF increased as the PEF treatment temperature increased. The effect of

PEF treatment temperature on KN can be described as:

kN = 0.0178 × e(0.0315)T (11)

The experimental RA was plotted against the predicted RA from Equation 11 and this

plot is displayed in Figure 5.6(b). The slope and intercept obtained by simple regression

(p = 0.05) agreed with their predicted values by Equation 11 (R2 = 0.86).

Hulsheger’s kinetic model

The model parameters, Ec, tc, and k, obtained from the statistical analysis, were

1.72 kV/cm, 12.83 µs, and 33.44 kV/cm, respectively. The Hulsheger’s kinetic model fit

the experimental data with a very good agreement (R2 = 0.93, p < 0.001).

The experimental RA were plotted against the predicted RA from the Hulsheger’s

kinetic model (Equation 6) and shown in Figure 5.7(a). The slope and intercept obtained

by simple regression (p = 0.05) agreed with their predicted values from the Hulsheger’s

kinetic model (R2 = 0.91). The p-value for the lack-of-fit was 0.782. This p-value

suggests that a simple linear regression model can explain the variability between

experimental RA and predicted RA values.

Hulsheger and others (1981) proposed a model describing the inactivation kinetics

of microorganisms by PEF. Giner and others (2000) used the Hulsheger’s kinetic model

for the inactivation kinetics of tomato PME by PEF and found that the model fit

176

(a)

continued

Figure 5.7: The regression lines of experimental residual LOX activity versus predicted residual LOX activity from the Hulsheger’s kinetic model (a) and the Fermi’s kinetic model (b)

Predicted RA

Expe

rimen

tal R

A

177

Figure 5.7 continued (b)

Predicted RA

Expe

rimen

tal R

A

178

experimental data with a good agreement. They reported Ec of 0.7 kV/cm for PME

inactivation by PEF. The Ec of LOX (1.72 kV/cm) is higher than that of PME. This

indicates that the LOX inactivation began at higher electric fields than the tomato PME

inactivation.

Fermi’s kinetic model

The parameters of the Fermi’s kinetic model for the inactivation of tomato juice

LOX by PEF at 30 °C for 20, 30, 50, 60, and 70 µs are shown in Table 5.4. The Fermi’s

kinetic model fit the experimental data with a good agreement at different levels of PEF

treatment time (R2 > 0.85). The Eh, the electric field strength where RA is 0.5, decreased

from 54.59 to 10.01 kV/cm as the PEF treatment time increased from 20 to 70 µs.

A regression line of experimental RA versus predicted RA from the Fermi’s

kinetic model (Equation 7) is illustrated in Figure 5.7(b). The predicted RA from the

Fermi’s kinetic model agreed with the experimental RA well (R2 = 0.92).

Peleg (1995) reported that the Fermi’s kinetic model successfully described the

survival curves of microorganisms exposed to PEF. Giner and others (2000) applied the

Fermi’s kinetic model to the inactivation of tomato PME by PEF and showed feasibility

to use the Fermi’s model to predict the inactivation of enzymes. The regression analysis

of this study indicates that the Fermi’s kinetic model adequately described the

inactivation of tomato juice LOX by PEF.

179

PEF treatment time

(µs) Eh (kV/cm) a (kV/cm) R2

20 54.59 ± 6.27 40.44 ± 3.82 0.987

30 21.84 ± 3.30 14.85 ± 3.25 0.946

50 15.04 ± 2.71 13.55 ± 2.14 0.934

60 11.39 ± 2.18 15.88 ± 2.32 0.952

70 10.01 ± 1.54 22.68 ± 2.80 0.851

Table 5.4: Fermi’s equation parameter values and correlation coefficients (R2) for the inactivation of tomato juice LOX by PEF treatment at 30 °C for 20, 30, 50, 60, or 70 µs.

180

Effect of electric field strength on inactivation of LOX by PEF treatment with 22 J

The effect of electric field strength on the inactivation of tomato juice LOX by

PEF treatment with 22 J of energy input was studied. The three treatment conditions

were determined to provide 22 J of energy input to tomato juice, but with different levels

of electric field strength (Table 5.1(b)). The ∆T, the temperature difference between T4

and T3, was 25 °C for the all three PEF treatments. The LOX inactivation (%) values

were 4.7, 46.3, and 60.0 % when the values of electric field strength were 9.0, 17.8, and

30.1 kV/cm, respectively. As the electric field strength increased in the three treatments,

the inactivation of tomato juice LOX by PEF also increased even though the levels of

energy input for the three treatments were same as 22 J. Electric field strength can be

considered as the primary variable for the LOX inactivation by PEF.

CONCLUSIONS

The inactivation kinetics of tomato juice LOX by PEF showed that electric field

strength, PEF treatment time, and PEF treatment temperature significantly affected the

efficiency in the inactivation of tomato juice LOX by PEF. The first-order kinetic

models, the Hulsheger’s kinetic model, and the Fermi’s kinetic model adequately

described the inactivation of tomato juice LOX by PEF. Applied electric field strength

was the primary variable for the inactivation of tomato juice LOX by PEF. Information

from this research would be useful in determining remaining LOX activity in tomato

juice after PEF treatment for the quality control of tomato juice.

181

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orange juice by pulsed electric fields. J Food Sci 67(6)2154-2159.

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184

ACKNOWLEDGMENTS


funding this research.

185

CHAPTER 6

Recommendations for Further Studies

Success in extending shelf life of initially high-quality processed food would

depend on food packaging. It was observed that the initially significant differences in

flavor and ascorbic acid contents between thermally processed and PEF processed tomato

and orange juices decreased as storage time increased. The high contents of flavor and

ascorbic acid of PEF processed products can be maintained if appropriate packaging

materials and methods are used. Polyethylene terephthalate (PET) may be considered as

an alternative packaging material for PEF processed food due to their low gas

permeability and appropriateness for aseptic packaging. Modified atmosphere packaging

(MAP) may be applied to control oxygen in headspace and thus to minimize oxidation of

PEF processed foods. Packaging for PEF processed foods need to be further studied and

improved.

The shelf life of foods is extended by inactivating microorganisms and enzymes.

The effects of PEF on the inactivation of enzymes should be further investigated for

commercial juice production by PEF. The conformational change of enzymes, induced

by PEF, has been suggested as the mechanism of enzyme inactivation by PEF. However,

186

the issue of the inactivation of enzymes by PEF is still controversial. This might be due

to the diversity of the systems being used for PEF research. The PEF system needs to be

considered when PEF research is compared. The mechanism for the inactivation of

enzymes by PEF needs to be further studied to elucidate the effect of PEF on enzymes.

Such mechanism is also needed for PEF processing optimization. Primary PEF

processing parameters, directly related to the inactivation, may be enhanced to increase

the microbial or enzymatic inactivation with minimum energy input.

Kinetic models that describe the inactivation of important food enzymes need to

be determined and developed for the commercial juice production by PEF. A kinetic

model that incorporates all major PEF processing parameters, such as electric field

strength and PEF treatment time and temperature needs to be further developed. The

evaluation of kinetic models for the inactivation of enzymes needs to be conducted with

respect to isoenzymes.

187

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extending shelf life of juice products by pulsed electric fields

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