APPLICATIONS AND EFFECTS OF OHMIC HEATING:

STERILIZATION, INFLUENCE ON BACTERIAL SPORES,

ENZYMES, BIOACTIVE COMPONENTS AND QUALITY
FACTORS IN FOOD

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the

Degree Doctor of Philosophy in the Graduate School of
The Ohio State University

By
Romel Somavat, B. Tech.
Graduate Program in Food, Agricultural and Biological Engineering

The Ohio State University

2011

Dissertation Committee:
Professor Sudhir K. Sastry, Advisor
Professor Gonul Kaletunc
Associate Professor V. M. Balasubramaniam
 Copyright by
Romel Somavat
2011
 ABSTRACT

Industrial applications of ohmic heating are mainly limited to continuous thermal

processing of food. Main objectives of this research were to explore its applications in a

batch type container, and investigate additional non thermal effects of electric field on

bacterial spores, enzymes, carotenoids, flavonoids and quality parameters in food.

An ohmic packet made up of multilayered laminate material and capable of sterilizing

food was developed. A pouch design optimization exercise was conducted to improve the

heating profile and integrity of the hermetic seal at temperature and pressure conditions

associated with a sterilization process. A simulation study in 3D was done for

sterilization in the ohmic pouch. The mathematical model and sterilization were validated

through an inoculated pack study using Geobacillus stearothermophilus spores.

Non thermal effects of electricity at frequencies of 10 kHz and 60 Hz during ohmic

heating were studied on thermophilic bacterial spores of G. stearothermophilus and

Bacillus coagulans. A precise capillary sized ohmic device was invented to eliminate

potential source of experimental errors, and to obtain identical time temperature histories

for both ohmically and conventionally heated samples. Ohmic heating at both frequencies

ii
were found to accelerate inactivation of bacterial spores. It was hypothesized that release

of polar dipicolinic acid molecules and spore proteins at higher temperature conditions

and their vibrations under the external electric field have resulted in an accelerated

inactivation. A linearly increasing temperature analysis of the B. coagulans data

suggested that Z values obtained at isothermal conditions may only be valid over a

narrow range of temperature.

Effects of ohmic heating on pectin methylesterase (PME) and polygalacturonase (PG)

enzymes were evaluated in fresh tomato juice at pH of 3.9 and 4.4. PME at pH 3.9

showed a high variability in the inactivation data possibly due to the interaction of

multiple isozymes with the electric field. PG data at pH 3.9 showed comparatively less

variation and hinted at a higher inactivation rate under ohmic heating. At pH 4.4, PG was

found to show accelerated inactivation under ohmic heating in comparison to literature

values for its resistant isozyme, PG1. Color values (l, a and b) were found to remain

unaffected over the studied pH and temperature ranges.

Carotenoids and phenolic compounds of fresh tomato juice remained unaffected by

ohmic heating. Inherent amounts of β-carotene, lycopene, phenolic acids and quercetin

indicated no or minimal changes at a temperature range from 90 to 110°C at pH of 3.9

and 4.4. Naringenin content showed an interesting behavior with chalconaringenin

iii
converting to naringenin in greater proportions. Naringenin equivalents were found to

increase at the temperature of 105°C under ohmic heating.

iv
Dedicated to my friends and family

v
 ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to my academic advisor Dr. Sudhir K.

Sastry. The opportunity to research in his laboratory is just a fraction of my indebtedness,

for his able guidance, patient and encouraging attitude for new ideas, and scientific

acumen helped in fostering independent thinking. Learning under his advisorship goes

beyond the doctoral research.

I am thankful to Dr. Ahmed E. Yousef for guiding me in the microbiological aspects of

my research, and service on my committee. I am also appreciative of the support and

comments of Dr. V. M. Balasubramaniam, Dr. Gonul Kaletunc, Dr. Steven Schwartz and

Dr. Diane Barrett. I am grateful to Dr. Yoon-Kyung Chung, Dr. Suzanne Kulshrestha, Dr.

Soojin Jun, Rebeka Davis, Rachel Kopec, Dr. Luis Rodriguez-Romo, Dr. Ken Riedl and

Brian Heskitt for research assistance and technical discussions.

I acknowledge NASA Johnson Space Center NRA Grant No. NAG9-1508 for support of

this effort. I am grateful to the Ohio Agricultural Research and Development Center

(OARDC), The Ohio State University for also providing salaries and research support.

vi
Special thanks go to my friends and colleagues in the Cricket Club, Indian Students

Association, Association for India’s Development, FABE Graduate Student

Organization, and International Students Welcome Committee at the Ohio State

University for making my stay memorable. I would also like to acknowledge my old time

friends from India for staying in touch through the years and distance.

Thanks to my sister, brother and mother for their unconditional love and moral support.

Thanks to my better half Isha for her love and understanding, and making the latter half

of my doctorate a better one.

vii
 VITA

October 7, 1983…………………….. Born – Hisar, India

2004…………………………………B. Tech. Agricultural Engineering, CCS

Haryana Agricultural University, India

2005 – 2010………………………....Graduate Research Associate, The Ohio

State University

2010 – Present……………………….Product and Process Scientist, Abbott

Nutrition, Columbus, Ohio

PUBLICATIONS

Sastry, S.K., Jun, S., Somavat, R., Samaranayake, C., Yousef, A., & Pandit, R.B. (2009).

Heating and sterilization technology for long-duration space missions: Transport

processes in a reusable package. Interdisciplinary Transport Phenomena: Annals of New

York Academy of Sciences, 1161, 562-569.

FIELDS OF STUDY

Major Field: Food, Agricultural and Biological Engineering

viii
 TABLE OF CONTENTS

Page
Abstract……………………………………………………………………………............ii
Dedication………………………………………………………………………………....v
Acknowledgements………………………………………………………………….……vi
Vita…………………………………………………………………..……..…………...viii
List of Tables………………………………………………………………..……….….xiii
List of Figures………………………………………………………………..………......xv
List of Symbols……………………………………………………………….…….......xxii

Chapters:

1. Ohmic sterilization inside a multi-layered laminate pouch for long duration

space missions…………………………………………………………………......1
1.1 Abstract……………………………………………………………………1
1.2 Introduction………………………………………………………………..2
1.3 Materials and Methods
1.3.1 Pouch Design……………………………………………………...4
1.3.1.1 Pouch………………………………………………………4
1.3.1.2 Hermetic electrical connection with electrodes…………...5
1.3.1.3 Uniformity of electric field potential……………………...6
1.3.2 System for sterilization……………………………………………8
1.3.3 Seal Integrity………………………………………………………8
1.3.4 Modeling and temperature distribution study……………………10

ix
 1.3.4.1 Model development and simulation……………………...11
1.3.4.2 Temperature distribution study…………………………..13
1.3.5 Microbial experiments…………………………………………...15
1.3.5.1 Spore preparation………...………………………………15
1.3.5.2 Inoculation procedure and microbial analysis……..…….16
1.4 Results and Discussion
1.4.1 Temperature distribution……..…………………………..………16
1.4.2 Simulation……….……………………………………………….17
1.4.3 Inoculated pack study………………………..…………………..20
1.5 Conclusion……………………………………………………………….22
1.6 References………………………………………………………………..23

2. Effect of ohmic heating on inactivation kinetics of Geobacillus

stearothermophilus spores………....…………………………………...……….26
2.1 Abstract……………………………………………………….………….26
2.2 Introduction…………………………………………………..…………..27
2.3 Materials and Methods
2.3.1 System design…………….…………………………...…………32
2.3.1.1 Ohmic and conventional capillary cells…...……..………35
2.3.1.2 Heating and cooling system…………….………………..37
2.3.1.3 Power supply unit……………………….……………….39
2.3.2 Attainment of pure ohmic heating inside ohmic
capillary cells…………………………………………………….39
2.3.3 Preparation of Geobacillus stearothermophilus
spore suspension……...........…………………………………….43
2.3.4 Experimental design……………………………………………..44

x
 2.4 Results and Discussion…………………………………………………..45
2.5 Conclusion……………………………………………………….………49
2.6 References……………………………………………………..…………49
3. Inactivation kinetics of Bacillus coagulans under the effect of
ohmic heating……………………………………………………….……………54
3.1 Abstract………………………………………………………..…………54
3.2 Introduction………………………………………………………..……..55
3.3 Materials and Methods
3.3.1 System design…………………….………………….…………..57
3.3.1.1 Ohmic and conventional capillary cells………….………57
3.3.1.2 Power supply and control…………………………….…..59
3.3.2 Microbiological experiments………………...…………………..61
3.3.2.1 Preparation of Bacillus coagulans spore suspension…….61
3.3.2.2 Enumeration procedure…………………………….…….62
3.3.3 Tomato juice preparation…………………………………...……63
3.4 Results and Discussion………..…………………………………………63
3.5 Conclusion……………………………………………………………….71
3.6 References………………………………………………………….…….72

4. Effect of ohmic heating on kinetics of change of pectin methylesterase,

polygalacturonase and color in tomato juice…………….…………………...….76
4.1 Abstract………………………………………………………..…………76
4.2 Introduction………………………………………………………..……..77
4.3 Materials and Methods………………………………………………...…79
4.3.1 Tomato juice preparation……….…………………....…………..80
4.3.2 System design…………………………………………………....81
4.3.3 Enzyme and color analysis……………………………………….85

xi
 4.4 Results and Discussion………..…………………………………………87
4.4.1 Pectin methylesterase (PME)……………………………….……87
4.4.2 Polygalacturonase (PG)……………………………………….....90
4.4.3 Color…………………………………………………….……….92
4.5 Conclusion……………………………………………………………….94
4.6 References………………………………………………………….…….95
5. Effect of ohmic heating on carotenoids, phenolic compounds and
ascorbic acid in tomato juice………………………………………….………….99
5.1 Abstract………………………………………………………..…………99
5.2 Introduction………………………………………………………..……100
5.3 Materials and Methods……………………………………………..…...105
5.3.1 Tomato juice preparation……….…………………....………....106
5.3.2 System design…………………………………………...……...106
5.3.3 Sample preparation and analysis……………………………….109
5.4 Results and Discussion………..…………………………………..……111
5.4.1 Carotenoids (Lycopene and β-carotene)…………………..……111
5.4.2 Phenolic compounds (Phenolic acids and Flavonoids)…...……115
5.4.3 Ascorbic acid…………………………………………..……….120
5.5 Conclusion………………………………………………………...……122
5.6 References………………………………………………………………123

Bibliography……………………………………………………………………………131

xii
 LIST OF TABLES

Table Page

1.1 Values of the constants used in the simulation study…………………………....13

1.2 Survivor count for the inoculated pack study……………………………………21

2.1 D-values of Geobacillus stearothermophilus (ATCC 7953) at 10 kHz ohmic, 60

Hz ohmic and conventional treatments at the temperatures of 121°C, 125°C and
130°C in minutes per log cfu/ml………………………...…...………………..…47

3.1 D- and Z-values at 95, 100, 105 and 110°C for 10 kHz, 60 Hz and conventional
inactivation of B. coagulans spores……………….…………………………..…66

3.2 Values of Dref and log10 (N0/N) comparing measured and calculated using linearly
increasing temperature method……………….……………………………….…71

4.1 D-value, rate constant (k), and activation energy (Ea) for PME at pH 3.9 at 72, 83

and 88°C………………………………………………………………..………..89

4.2 Comparison of D, Z, k and Ea values for PG inactivation at pH 4.4 and 90°C

under ohmic heating and conventional heating (Anthon et al., 2002)………...…91

xiii
4.3 D-value, rate constant (k) and activation energy (Ea) for PG at pH 3.9……...….92

5.1 Mobile phase gradients used in HPLC analysis………………………………...110

xiv
 LIST OF FIGURES

Figure Page

1.1 Foil electrodes pasted on the laminate cut using food grade adhesive……………5

1.2 Electrical connections were made through the heat sealed patches overlying the
holes to eliminate the chances of short circuiting with the internal aluminum layer
of the pouch…………………………………………………………………….…6

1.3 a. Presence of a straight triangle pipe extending outside of the cross-sectional area
of the electrodes, b. Shoulder flap was sealed to remove the cold zone, thereby
obtaining a flat non-electrode side……………………………………………...…7

1.4 Final design of the rectangular pouch with an external heater……………………7

1.5 Schematic of the ohmic sterilization system………………………………………9

1.6 Maximum error versus the number of tetrahedral elements…………………..…12

1.7 Pouch domain with the refined tetrahedral mesh……………………………..…12

1.8 Positioning of nine thermocouples inside the pouch………………………….…14

1.9 Temperature profile measured at 9 points inside the pouch. External heaters were
fired at the time instant of 243 s and voltage was reduced at 283 s to maintain the
temperature………………………………………………………………………18

xv
1.10 a. Simulated temperature profile right before the firing of the external heaters
showed a maximum temperature difference of 34.4°C, b. 40 s after triggering of
the external heaters the temperature difference reduced to 14.3°C…………...…19

1.11 Comparison of predicted and measured temperatures at center of the pouch; point
G, and bottom edge of the non-electrode side; point B (Figure 1.8)………….…20

1.12 A survivor plot for the inoculated pack study done at 121°C and 130°C……..…21

2.1 Arrangement of capillary cells and cell holder inside the treatment chamber...…34

2.2 a. An ohmic cell, b. a conventional cell………………………………………….37

2.3 Schematic and a picture of the ohmic heating system…………………………...38

2.4 Electrical conductivity of tomato alginate, tomato soup and test chamber fluid
(0.78 % salt solution)………………………………………………………….…41

2.5 Small positive temperature difference (≤3°C) between ohmic cell and test
chamber fluid (average of three) confirmed a pure ohmic heating effect……..…41

2.6 Thermal histories of ohmic and conventional cells and the test chamber fluid,
showing closely matched thermal histories and rapid cooling of the samples..…42

2.7 a. Input voltage during the come-up time, b. Input voltage during the holding
time…………………………………………………………………………...….42

xvi
2.8 Inactivation of G. stearothermophilus spores under ohmic 10 kHz, ohmic 60 Hz
and conventional treatments at 121°C with holding times of 0, 15, 60 and
120s………………………………………………………………………………46

2.9 Inactivation of G. stearothermophilus spores under ohmic 10 kHz, ohmic 60 Hz

and conventional treatments at 125°C with holding times of 0, 15, 30 and

90s…………………………………………………………………………….….46

2.10 Inactivation of G. stearothermophilus spores under ohmic 10 kHz, ohmic 60 Hz

and conventional treatments at 130°C with holding times of 0, 5 and 10 s

(survivor count for 30 s holding time was below the detection limit)………...…47

3.1 Typical arrangement of the capillary cells inside the treatment chamber…….…59

3.2 A representation of the square waveform; 10 kHz frequency and 0.5 duty

ratio………………………………………………………………………………60

3.3 Typical voltage and matching temperature histories of the ohmic and conventional

treatments during the come-up and holding times for the 110°C treatment…..…61

xvii
3.4 Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and

conventional treatments at 95°C with holding times of 0, 10, 20 and 30 min..….64

3.5 Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and

conventional treatments at 100°C with holding times of 0, 2, 4 and 6 min…..….65

3.6 Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and

conventional treatments at 105°C with holding times of 0, 1, 2 and 3 min…..….65

3.7 Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and

conventional treatments at 110°C with holding times of 0, 10, 20 and 30 s….…66

3.8 A Z-value plot showing the D-values at 95, 100, 105 and 110°C for 10 kHz, 60

Hz and conventional treatment………………………………………………......67

3.9 Spore reductions during come up times of 158, 170, 180 and 192s to the

temperatures of 95, 100, 105 and 110°C, respectively, for conventional, 60 Hz

and 10 kHz treatments………………………………………………………..….67

4.1 A glass T-type ohmic heater………………………………………….………….83

xviii
4.2 Typical square waveform with a pulse duty of 0.5 and frequency of 60 Hz…….83

4.3 Time-temperature plot showing the come up time and voltage for a 90°C process

(a) plot for high temperature treatments from 90 to 110°C (b) plot for low

temperature treatments from 72 to 90°C…………………………………………84

4.4 Residual PME activities in tomato juice at pH 3.9 at 72, 78, 83, and 88°C…..…89

4.5 Residual PG activities in tomato juice at pH 4.4 at 88, 90, 95 and 100°C…...….91

4.6 Residual PG activities in tomato juice at pH 3.9 at 72, 78, 83, and 90°C……….92

4.7 Color changes in the tomato juice by ohmic heating at 95, 100 and 105°C for pH

4.4, and 90, 95, 100 and 105°C for pH 3.9………………………...…………….93

4.8 a/b values for the color change for ohmic heating at pH 3.9…………………….94

5.1 A T-type glass ohmic heater……………………………………………………107

xix
5.2 Typical square waveform with a pulse duty of 0.5 and frequency of 60 Hz…..108

5.3 Time-temperature plot showing the come up time and voltage for the 90°C

process……………………………………………………………..………..…..108

5.4 β-carotene content in tomato juice at pH 4.4 after ohmic treatment at 95, 100, 105

and 110°C………………………………………………………………………113

5.5 Amounts of total and all-trans lycopene in tomato juice at pH 4.4 after ohmic

treatment at 95, 100, 105 and 110°C………………………...…………………114

5.6 Total lycopene content in tomato juice at pH 3.9 after ohmic treatment at 90, 95,

100 and 105°C……………………………………………………...…………..115

5.7 Chlorogenic acid equivalents in tomato juice at pH 4.4 after ohmic treatment at

95, 100, 105 and 110°C……………………………………..………………….117

5.8 Naringenin and chalconaringenin in tomato juice at pH 4.4 after ohmic treatment

at 95, 100, 105 and 110°C……………………………………………………....118

xx
5.9 Total flavonoids and quercetin equivalents in tomato juice at pH 4.4 after ohmic

treatment at 95, 100, 105 and 110°C…………………………………………...119

5.10 Ascorbic acid and dehydroascorbic acid content in tomato juice at pH 4.4 after

ohmic treatment at 90, 95, 100, 105 and 110°C……………………………..…121

5.11 Ascorbic acid and dehydroascorbic acid content in tomato juice at pH 3.9 after

ohmic treatment at 90 and 100°C………………………………..……………..122

xxi
 LIST OF SYMBOLS

h Convective heat transfer coefficient W/m°C

ñ Density kg/m3

µ Viscosity Pa.s

Cp Specific heat J/Kg°C

k Thermal conductivity W/m°C

ó Electrical conductivity at 27°C S/m

ó Electrical conductivity at 65°C S/m

V RMS Voltage V

N0 Initial spore concentration log spore cfu/ml

N Final spore concentration log spore cfu/ml

t Come up time (CUT) s

Tref Reference temperature (100°C) °C

T Final temperature °C

D D value min

Dref D value at reference temperature min

Z Z value °C

m and c constants (T= mt +c)

xxii
 D D-value min

Z Z-value °C

k Rate constant s-1

Ea Activation energy KJ/ mole

Co Ascorbic acid concentration (raw) mg/ 100g

C Ascorbic acid concentration (final) mg/ 100g

∆A265 Change in absorbance at 265 nm

V Volume of the sample m3

xxiii
 CHAPTER 1

Ohmic sterilization inside a multi-layered laminate pouch for long

duration space missions

1.1 Abstract

A pouch that can be used to ohmically reheat and sterilize food and later reused to

stabilize waste will significantly reduce the Equivalent System Mass during a long term

space mission. Reheating of food and stabilization of waste have been successfully done

in the past using a V-shaped electrode pouch using pulsed ohmic heating. However,

temperature distribution and simulation studies have shown the inadequacy of a V-shaped

pouch design for the sterilization of food. The main objectives of this study were to

optimize the pouch design to improve the heating profile and to achieve food

sterilization.

The pouch was redesigned to improve the electric potential distribution and hence heating

uniformity. A temperature distribution study on a pouch with 227 g of tomato soup

showed the presence of <0.005 m thick cold regions at the non-electrode sides due to

channeling of current through a hotter and more conductive centre. Polyimide strip

heaters installed externally along these sides yielded a much more uniform temperature

range throughout the pouch. An inoculated pack study using Geobacillus

1
stearothermophilus spores (ATCC5973) was done to validate sterilization. The ohmic

heating process inside the pouch was simulated in 3D to predict possible cold and hot

spots. The simulated heating profile was in good agreement with the measured values

confirming the efficacy of the model. Results of the inoculated pack study confirmed the

mathematical model. This technology provides a new method of sterilization inside

sealed pouches/ packets using ohmic heating.

1.2 Introduction

Manned space flight beyond earth‟s orbit presents new problems and challenges, and

calls for the development of new and improved technology. NASA‟s future long duration

space missions will require high quality food to not only take care of nutritional needs,

but also to help in reducing psychological disorders that can possibly affect well-being of

the crew during extended stay in an unfamiliar and hostile environment (Perchonok and

Bourland, 2002). A mission to Mars will require two intrinsically different food systems

for the inter-planetary transit phase and the stay on a surface habitat, due to variations in

gravity conditions, source of food, disposal of waste, and type and amount of packaging.

Processing and sterilization of the food grown on-board or on a planetary surface will

also be required, unlike the prepackaged food used on current space missions. The key

requirement is the capability to serve safe, palatable and nutritious food to the crew with

minimum energy, volume, mass and waste.

Ohmic heating has potential as an alternative heating technology because of its capability

of delivering high quality food (Kim et al., 1995), rapid heating, compact design, and

minimal heating of the product surroundings (Jun and Sastry, 2005). Since heat is

2
generated and distributed internally in the food, ohmic heating achieves close to 100%

energy transfer efficiency (Salengke and Sastry, 1998) and eliminates the need for an

external heat transfer medium and surfaces that are associated with conventional heating

technologies.

A pouch that can be used to ohmically reheat and sterilize food and later reused to

stabilize waste will significantly reduce the Equivalent System Mass (ESM1) during a

long term space mission. Jun and Sastry (2005) and Sastry et al. (2009) developed such a

pouch by integrating an ohmic heater with foil electrodes inside a basic retort package.

They studied the electric potential and temperature profile inside the pouch with three

different configurations of the foil electrode assembly using a 2D mathematical model.

Jun and Sastry (2007) examined the heating pattern inside a V-shaped electrode pouch

using an improved 3D model. Whereas these experiments successfully proved the

accuracy of the mathematical models and attested to the pouch‟s ability to ohmically

warm the food, the presence of a non-uniform temperature pattern inside of the pouch

suggested the presence of cold and hot spots. Sastry et al. (2009) concluded that to

achieve food sterilization, redesign of the pouch was necessary to improve electric field

distribution and hence the heating profile.

To achieve sterilization with best product quality, it is important that all parts of the food

should be uniformly heated to sterilization temperatures and held for a specific time. The

presence of cold spots can compromise safety and shelf life, whereas hot spots result in

thermal abuse, lowering product quality. Theoretically, parallel and flat electrodes with a

uniform heating chamber present a uniform heating profile, given that the medium is

evenly conductive (Jun and Sastry, 2007). Differences in electrical conductivities of

3
various ingredients of a multi component food system can be reduced by selective

pretreatment during the formulation phase (Tulsiyan et al., 2008; Sarang et al., 2007).

The main goal of this study was to develop an improved pouch design to facilitate

uniform ohmic heating of food, to predict ohmic sterilization with a 3-D mathematical

model, and validate the process through an inoculated pack study using Geobacillus

stearothermophilus (ATCC 7953) spores.

1
ESM is the kg equivalent of the sum of the life support system mass and appropriate fractions of
supporting systems, including pressurized volume, power generation, cooling and crew-time (Levri &
Drysdale, 2003).

1.3 Materials and Methods

1.3.1 Pouch design

1.3.1.1 Pouch

A multilayered laminate material with an internal food contact surface of polypropylene

was used as a base material for making the pouches. A polypropylene based laminate was

preferred to polyethylene on the basis of its stability at sterilization temperatures (melting

point of around 170°C). The V-shaped electrode pouch used by Jun and Sastry (2005,

2007) and Sastry et al. (2009) was redesigned into a rectangular pouch. A flat and parallel

electrode configuration helped in improving the uniformity of electric field and hence the

heating profile. Two stainless steel foil pieces (Lyon Industries Chicago, Inc., Chicago,

IL) of dimensions 0.108 m x 0.02 m (thickness 1.27 x 10-5 m) were pasted using Tycel

food grade adhesive (Liofol Company, Cary, NC) on a 0.278 m x 0.178 m cut-piece of

laminate material as shown in Figure 1.1. The ohmic pouch was formed by heat sealing

4
the sides and then the base using an impact sealer (TEW Electric Heating Equipment Co.

Ltd, Taipei, Taiwan) set at 170°C.

Figure 1.1 Foil electrodes pasted on the laminate cut using food grade adhesive.

1.3.1.2 Hermetic electrical connection with electrodes (Patent Applied)

The original V-shaped pouch (Jun and Sastry, 2005) was redesigned into a rectangular

pouch with flat sides. These flat sides served as a base for pasting of the foil electrodes

inside the pouch. To prevent short-circuiting with the aluminum layer of the laminate

material while connecting the electrodes with an external circuit, a hole of 0.004 m

diameter was made at the side of pouch. A patch of 0.012 m x 0.012 m piece of the same

laminate material was heat sealed on the hole inside of the pouch (Figure 1.2). A small

metallic snippet, inserted through the patch overlying the hole, was used to make a

hermetic connect with the outside circuit.

5
Figure 1.2 Electrical connections were made through the heat sealed patches overlying

the holes to eliminate the chances of short circuiting with the internal aluminum layer of

the pouch.

1.3.1.3 Uniformity of electric field potential

While a rectangular pouch geometry was intended to eliminate field nonuniformity,

sealing of the top and base ends of the rectangular pouch resulted in formation of a

straight triangle pipe extending outside the cross section of the electrodes, hence causing

cold zone (Figure 1.3a). To flatten these zones with flat sides, the corner flaps were

pulled outside and sealed as shown in Figure 1.3b. This ensured a perfect rectangular

geometry of the pouch with no food parts extending outside the electrode cross sectional

area. The final design is shown in Figure 1.4.

When stacked together, the rectangular pouches minimized interstitial spaces that are

inherent to currently used MRE pouches with non-identical and irregular thicknesses.

Storage space in the galley area of a space shuttle is limited, thus improved stackability

translates to improved space usage.

6
 Straight Flap seal
triangle pipe

Figure 1.3 a. Presence of a straight triangle pipe extending outside of the cross-sectional

area of the electrodes, b. Shoulder flap was sealed to remove the cold zone, thereby

obtaining a flat non-electrode side.

Electrode tabs

External heater

Figure 1.4 Final design of the rectangular pouch with an external heater.

7
1.3.2 System for sterilization

The pouch was placed inside an enclosure made of Ultem 1000 polyetherimide (AIN

Plastics of Ohio, Inc., Columbus, OH) to make electrical connections. The enclosure had

ports for application of pneumatic pressure (35 psi) during the treatment and for cold

water to cool a sterilized pouch. Heating studies were also conducted using additional

strip heaters (Kapton Polyimide Heater, Watlow, St. Louis, MO) placed externally on the

two non-electrode sides. A temperature controller was used to maintain the temperature

of the external heaters at 120°C (±1). A detailed schematic of the sterilization system is

shown in Figure 1.5.

The pouch was placed inside an enclosure (Jun and Sastry, 2005) to facilitate electrical

connections and application of external pneumatic pressure at 35 psi and water for post

treatment cooling. Pouches were powered using pulsed alternating current at 85 V, with

10 kHz frequency and 0.5 duty ratio to minimize electrochemical reactions at the

electrode food interface (Jun et al., 2007). Cooling water was not used during the

inoculated pack study, to replicate the expected minimal use of the resources during a

real long duration space mission.

1.3.3 Seal integrity

Seal integrity at temperature and pressure conditions associated with a sterilization

process is critical for safety and shelf life of the packaged food. The enclosure facilitated

application of an external pneumatic pressure to enable heating above 100°C. Tests were

conducted at elevated temperatures to check for the worst case scenario. The pouch

withstood the processing temperature of 145°C with minimal or no shape deformation

8
when an external pneumatic pressure of 35psi was applied. Pouch stability was also

tested for a food warming application without the use of external pressure. The desired

temperatures for warming of food for serving purposes range from 65°C to 80°C. The

pouch heated at atmospheric pressure conditions stayed in shape without any leakage

above 90°C.

Cooling Cooling Function

Water In Water Out Generator

Electrode
Enclosure Sides

IGBT Power
Pouch V Circuit Supply
Pressurized
A
Air

External
Heaters
Data
Logger
Temperature
Control
Thermocouples

Power Supply for the Computer

External Heaters

Figure 1.5 Schematic of the ohmic sterilization system

9
1.3.4 Modeling and temperature distribution study

1.3.4.1 Model development and simulation

A 3D mathematical model was developed to simulate the electric field and the heating

pattern. The energy equation for conventional thermal conduction and internal energy

generation was used owing to the chances of a worst case no-convection scenario in zero

gravity:

(1)

where T is temperature, t is time, k is thermal conductivity, ρ is density, CP is specific

heat and S is the source term. The source term for conversion of electrical to thermal

energy depends upon voltage, V, and electrical conductivity, σ, which is a function of

temperature, T. The source term may be represented as:

(2)

The voltage or electrical field potential within an ohmic heater can be calculated by

solving Laplace‟s equation:

(3)

The initial boundary conditions used for the ohmic model were:

where

Electrical boundary conditions were

wall

where

10
 wall

where

And, thermal boundary conditions were:

wall (7)

where

COMSOL Multiphysics (v. 3.5a, COMSOL Inc., Burlington, MA) was used for

simulation in 3D. General Heat Transfer and Conductive Media D.C. modules of the

program were applied to model the ohmic heating process in the pouch. Only half a

pouch was considered due to symmetry. A tetrahedral mesh was created in the domain

after refinement to an adequate detail level. It was observed that the non-electrode sides,

where external heaters were aligned, were a critical part and hence density of mesh was

increased there to an optimum refinement level. A plot of maximum error (°C) versus the

number of tetrahedral elements is shown in Figure 1.6. The maximum error at a

refinement level of n may be represented as:

(8)

where

A mesh with 2105 tetrahedral elements and 124 edge elements was found to optimally

reduce the maximum error and hence was selected (Figure 1.7).

11
 Mesh Refinement
6

Maximum Error, C
4

0
0 1000 2000 3000 4000 5000
Number of tetrahedral elements

Figure 1.6 Maximum error versus the number of tetrahedral elements.

Electrode

Symmetry

Non-electrode
Side

Electrode

Figure 1.7. Pouch domain with the refined tetrahedral mesh

12
For simulation of the process, the thermal boundary condition addressed the potential

convective heat loss from the pouch surface. The value of h could not be estimated by

empirical calculations due to the presence of pressurized air surrounding the multilayered

laminate pouch and formation of condensate on the walls of the enclosure which may

drop on the pouch surface. Jun et al. (2005) used an empirically calculated value of 7.2

W/m°C, but the presence of air (h values range from 10 to 100 W/m°C) and possibility of

water droplets (h values range from 500 to 10000 W/m°C) could make it vary

considerably. Hence, in the current study a value of 35 W/m°C based on trial and error

method was used. Inactivation of Geobacillus stearothermophilus in the pouch was

predicted using the heat transfer model together with the kinetic data from Somavat

(2010).

h Convective heat transfer coefficient 35 W/m°C

ρ Density 1020 kg/m3
µ Viscosity 3000 x 10-6 Pa.s
Cp Specific heat 4020 J/Kg°C
k Thermal conductivity 0.6 W/m°C
σ Electrical conductivity at 27°C 1.02 S/m
σ Electrical conductivity at 65°C 1.87 S/m
V RMS Voltage 85 V

Table 1.1 Values of the constants used in the simulation study

1.3.4.2 Temperature distribution study

The ohmic pouch was filled with 227g tomato soup (Campbell Soup, Camden, NJ)

prepared using an equal volume of condensed soup and sterile tap water as suggested on

the label. A temperature distribution study was conducted using 8-10 T-type

13
thermocouples (Omega Engineering Inc, Stamford, CT) placed inside the pouch to

measure temperature at the locations shown in Figure 1.8. A stuffing box (Ecklund-

Harrison Technologies, Inc, Fort Myers, FL) was used to provide pressure proof passage

to the thermocouples out of the pouch. Thermocouple signals were sent to the data logger

through signal conditioning to eliminate the interference of signals with the electric field.

Inoculated pouch experiments were carried using G. stearothermophilus spores (ATCC

7953) without thermocouples inside the pouch. Experimental data on net power per

pouch (based on time required to heat at a constant voltage) and surface temperature were

used for estimating the temperature inside the pouch without thermocouple inserts.

Electrode

F A
G
H
B
I D

E
Electrode

Figure 1.8 Positioning of nine thermocouples inside the pouch

14
1.3.5 Microbiological experiments

An inoculated pack study using 108 cfu/ pouch of G. stearothermophilus (ATCC 7953)

spores was done to validate ohmic sterilization of food in the pouch. Tests were

conducted at 121°C for holding times of 1, 2, 6 and 12 min, and at 130°C for 1 and 2

minutes (two replicates each).

1.3.5.1 Spore preparation

Geobacillus stearothermophilus ATCC 7953 was obtained from the American Type

Culture Collection (Manassas, VA). The strain was grown in tryptose broth (TB; Difco,

Becton Dickinson and Co., Sparks, MD). Stock cultures were stored at -80°C in TB

containing 40% (v/v) glycerol. Cultures were transferred in TB two times prior to use.

For spore preparation, the method described by Sala et al. (1995) was used. Aliquots of

100 µl overnight cultures of G. stearothermophilus were spread onto nutrient agar (NA;

Difco; Becton, Dickinson and Co., Sparks, MD) supplemented with 10 ppm manganese

chloride. The inoculated plates were incubated at 55°C until >90% of the cells were

sporulated. The plates were packaged during incubation to prevent drying. Sporulation

was verified by observing the refractile spores using a phase-contrast microscope. Spores

were harvested by adding 10 ml cold sterile deionized water on each plate, and releasing

the colonies containing spores from the surface of the agar with the use of a sterile

disposable inoculation loop. Collected spores were washed five times by centrifugation

(8000 x g for 20 min) at 4°C, and the resulting pellet was cleaned to separate free spores

from vegetative cells and debris. The cleaning process consisted of aseptically treating

15
each pellet with lysozyme solution (100 µg/ml for 30 min) and trypsin (200 µg/ml for 2

h). The spore crops were then washed three times in sterile deionized water by

centrifugation at 8000 x g for 10 min at 4°C. After the last centrifugation, the spore

pellets were resuspended in sterile deionized water. The spore suspensions were heated at

80°C for 15 min and checked microscopically to ensure the absence of vegetative cells.

The spore suspension was stored at 4°C until use.

1.3.5.3 Inoculation procedure and microbial analysis

The pouches were filled with 226 g of tomato soup inoculated using 1 ml of 108 cfu/ml of

prepared spore suspension; a concentration of 106 cfu/ml spores in the sample or 108 cfu

spores per pouch. Treated pouches were cleaned from the outside using 1400 ppm

hypochlorite solution and cut open under sterile condition to transfer the sample into

sterile polypropylene tubes containing 0.1% peptone water. A heat shock at 85°C for 10

min was given to the samples to inactivate all vegetative cells. This ensured plating of

only spores from treated samples and untreated controls. Further dilutions were plated on

TSA agar plates. Colonies were counted after 48 hours of incubation at 55°C.

1.4 Results and Discussion

1.4.1 Temperature distribution

The temperature distribution study revealed the formation of thin flat cold regions along

the non-electrode sides of the pouch (Figure 1.9). This temperature nonuniformity

increased with time. At the time of 243 s, the temperature of the main body was at 118°C,

16
while the coldest point on the side was at 78°C. Test runs were also done using an

insulating material around the pouch to reduce the temperature difference due to the heat

loss. This proved ineffective in adequately improving the temperature uniformity and

suggested two negatively synergistic mechanisms- 1) A small amount of heat was lost

through the sides to surroundings, which resulted in formation of a temperature gradient

between the main body of the pouch and the colder edges; and 2) Channeling of the

electric current through a hotter and more conductive center led to a higher heat

generation rate, which further drove the nonuniformity.

The problem was remedied by installing external polyimide strip heaters, physically

aligned along the non electrode sides, to boost ohmic heating of the cold region. The

external heaters, controlled by an on-off circuit at 121°C (±1°C), were fired at the time of

243 s. After around forty seconds of combined heating, at time of 283 s, the temperature

range inside the pouch became more uniform, ranging from 121°C to 136°C (Figure 1.9).

During the holding time, the input voltage was reduced to maintain the desired mean hold

temperature. A voltage reduction from 85 V to 25 V helped in maintaining this

temperature range consistently.

1.4.2 Simulation

The simulated temperature profile of ohmic heating before and after triggering of the

additional external heating at 243 s is shown in Figure 1.10 a and b. It showed a

temperature difference of 34.4°C between the coldest and hottest spots before the start of

external heaters, which subsequently reduced to a more uniform range of 122°C to 136°C

after 40 seconds of combined heating. The simulated temperature profile at the center of

17
the pouch (point G in Figure 1.8) matched very closely with the actual recording data.

However, a maximum error of around 5°C during the come-up time was associated with

the simulated and actual data for the lower corner of the pouch (point E). As the error was

an under prediction, it gave a more conservative estimate of the temperature and hence

did not pose a serious safety issue. Overall the simulated temperature was in agreement

with the results of the temperature distribution study done using nine thermocouples

(Figure 1.11).

Temperature Profile
160

140

120
Edge A
100
Temperature, C

Edge B
Edge C
80
Edge D
60 Edge E
Body I
40 Edge temperatures at Body F
the start of external
heaters Body G
20 Body H

0
0 100 200 300 400 500 600
Time, s

Figure 1.9 Temperature profile measured at 9 points inside the pouch. External heaters

were fired at the time instant of 243 s and voltage was reduced at 283 s to maintain the

temperature.

18
 A.
Electrode

Symmetry

Electrode

B
Electrode

Symmetry

Non-Electrode
Side
Electrode

Figure 1.10 a. Simulated temperature profile right before the firing of the external heaters

showed a maximum temperature difference of 34.4°C, b. 40 s after triggering of the

external heaters the temperature difference reduced to 14.3°C.

19
 Simulated vs Actual Temperatures

140

120
Temperature, C
100
G Actual
80
G Simulated
60
B Actual
40 B Simulated
20

0
0 50 100 150 200 250 300 350 400
Time, s

Figure 1.11 Comparison of predicted and measured temperatures at center of the pouch;

point G, and bottom edge of the non-electrode side; point B (Figure 1.8).

1.4.3 Inoculated pack study

Holding times of 6 min at 121°C and 1 min at 130°C were adequate for killing more than

5 logs of G. stearothermophilus spores. Surviving spore counts were below the detection

limit of 1 log cfu/ml for 12 min treatment at 121°C, and 1 and 2 min treatments at 130°C.

Inactivation of the spores followed the expected linear pattern based on D 121 of 0.88 min

at 121°C and D130 of 0.05 min for 10 kHz ohmic treatment as reported by Somavat

(2010). A survivor plot for the inoculated pack study is presented in Figure 1.12.

20
 Survivor Plot
8
7 121C
130C

Log Spores, cfu/ml

6
5 121C Expected
4 130C Expected
3
2
1
0
0 2 4 6 8 10 12 14
Holding time, min

Note: Minimum detection limit was 1 log spores cfu/ml.

Figure 1.12 A survivor plot for the inoculated pack study done at 121°C and 130°C.

Time, Survivors, St. Dev.

min Log spores cfu/ml
Control 0 7.27 0.12
1 5.57 0.01
121°C 2 4.82 0.35
6 1.35 0.49
12 1.00 0.00
130°C 1 1.00 0.00
2 1.00 0.00
Note: Minimum detection limit was 1 log spores cfu/ml.

Table 1.2 Survivor count for the inoculated pack study.

21
1.5 Conclusion

A rectangular ohmic pouch capable of sterilizing food has been developed. The pouch,

containing flat and parallel electrodes, has shown to have a uniform electric field across

its geometry. External polyimide heaters were used along the non electrode sides to

improve the heating profile and eliminate cold spots. A 3D model was also developed

which successfully predicted the electric potential and temperature inside of the pouch.

Sterilization of the food was validated through an inoculated pack study using

Geobacillus stearothermophilus spores. The pouch concept has presented the food

industry with a novel way of sterilization and heating of the packaged food.

Acknowledgement

The authors gratefully acknowledge support from NASA/JSA NRA Grant No. NAG 9-

1508 USDA-CSREES titled Reheating and Sterilization Technology for Food, Waste and

Water. Salaries and research support also provided by the Ohio Agricultural Research

and Development Center (OARDC), The Ohio State University. References to

commercial products and trade names are made with the understanding that no

endorsement or discrimination from the Ohio State University is implied.

22
1.6 References

Allen, C.S., Burnett, R., Harles, J., Cucinotta, F., Fullerton, R., Goodman, J.R., Griffith,

A.D., Kosmo, J.J., Perchonok, M., Railsback, J., Rajulu, S., Stilwell, D., Thomas, G., Tri,

T., Joshi, J., Wheeler, R., Rudisill, M., Wilson, J., & Simmons, A. (2003). Guidelines and

Capabilities for Designing Human Missions. NASA/TM-2003-210785.

Crew and Thermal Systems Division at JSC (2004). ISS Food Warmer, Part No

SED39114053-309. International Space Station Catalogue of Intervehicular (IVA)

Government Furnished Equipment (GFE) Flight Crew Equipment (FCE). JSC #28533

Rev D.

Hanford, A.J. (2006). Advanced Life Support Research and Technology Development

Metric- Fiscal year 2005. NASA/CR-2006-213694.

Jun, S., & Sastry, S.K. (2005). Modeling and optimizing of pulsed ohmic heating of

foods inside the flexible package. Journal of Food Process Engineering, 28(4), 417–436.

Jun, S., & Sastry, S.K. (2007). Reusable pouch development for long term space

missions: A 3D ohmic model for verification of sterilization efficacy. Journal of Food

Engineering, 80, 1199–1205.

23
Jun, S., Sastry, S.K., & Samaranayake, C. (2007). Migration of electrode components

during ohmic heating of foods in retort pouches. Innovative Food Science and Emerging

Technologies, 8(2), 237-243.

Kim, H.J., Choi, Y.M., Yang, A.P.P., Yang, T.C.S., Tuab, I.A., Giles, J., Ditusa, C.,

Chall, S., & Zoltai, P. (1995). Microbiological and chemical investigation of ohmic

heating of particulate foods using a 5 kW ohmic system. Journal of Food Processing and

Preservation, 20(1), 41-58.

Levri, J.A., Vaccari, D.A., & Drysdale, A.E. (2000). Theory and application of the

Equivalent System Mass Metric. SAE Technical Paper 2000-01-2395. 30th International

Conference on Environmental Systems.

Palaniappan, S., & Sastry, S.K. (1991). Electrical conductivity of selected juices:

influence of temperature, solids content, applied voltage and particle size. Journal of

Food Process Engineering. 14, 247-260.

Samarnayake, C., & Sastry, S.K. (2005). Electrode and pH effects on electrochemical

reactions during ohmic heating. Journal of Electroanalytical Chemistry, 577, 125-135.

Sarang S., Sastry S.K., Gaines J., Yang, T.C.S., & Dunne P. (2007). Product Formulation

for Ohmic Heating: Blanching as a Pretreatment Method to Improve Uniformity in

Heating of Solid–Liquid Food Mixtures. Journal of Food Science, 72(5), E 227-234.

24
Sastry, S.K., & Salengke, S. (1998). Ohmic heating of solid-liuid mixtures: A comparison

of mathematical models under worst-case heating conditions. Journal of Food Process

Engineering. 21, 441-458.

Sastry, S.K., Jun, S., Somavat, R., Samaranayake, C., Yousef, A., & Pandit, R.B. (2009).

Heating and sterilization technology for long-duration space missions: Transport

processes in a reusable package. Interdisciplinary Transport Phenomena: Annals of New

York Academy of Sciences, 1161, 562-569.

Somavat, R. (2010). Applications and effects of ohmic heating: sterilization, influence on

bacterial spores, enzymes, bioactive components and quality factors in food. PhD

Dissertation. The Ohio State University. Columbus, OH.

Tulsiyan, P., Sarang, S., & Sastry, S.K. (2008). Electrical conductivity of

multicomponent systems during ohmic heating. International Journal of Food

Properties, 11, 1–9.

25
 CHAPTER 2

Effect of ohmic heating on inactivation kinetics of

Geobacillus stearothermophilus spores

2.1 Abstract

Ohmic heating is commonly thought to kill microorganisms only through a thermal

effect. Although much literature exists to document non-thermal lethal effects of

electricity under pulsed electric fields, limited literature is available regarding ohmic

heating. Hence the method and conditions for ohmic sterilization are assumed as being

represented by existing thermal kinetics data. The aim this study was to determine the

kinetics of inactivation of Geobacillus stearothermophilus spores (ATCC 7953) under

ohmic and conventional heating. Tests were conducted in a specially constructed test

chamber using capillary sized cells to eliminate potential sources of error from previous

studies and ensure that identical thermal histories were experienced both by

conventionally and ohmically heated samples.

Ohmic treatments at frequencies of 60 Hz and 10 kHz were compared with conventional

heating at 121°C, 125°C and 130°C for 4 different holding times. Both ohmic treatments

showed a general trend of accelerated spore inactivation. It is hypothesized that the

release of polar dipicolinic acid molecules (DPA) and spore proteins at high temperature

26
conditions and their vibration under the external electric field have resulted in accelerated

inactivation.

2.2 Introduction

Little literature exists regarding the non-thermal effect of electricity on bacterial spores

during ohmic heating. In the absence of adequate understanding of spore inactivation

kinetics, the method and conditions for ohmic sterilization are selected on the basis of

existing purely thermal kinetic data (Iciek et al., 2005). New methods are needed for

efficient bio-validation of ohmic heating in order to realize the full potential of the

process. The presence of additional non-thermal effects of electricity, if any, may

facilitate reduction in process severity, thereby improving quality. The aim of this study

was to determine if non-thermal lethal effects of electricity exist during ohmic heating of

Geobacillus stearothermophilus spores (ATCC 7953).

Past studies on non-thermal effects of electricity have investigated microorganisms such

as yeast, vegetative bacterial cells and bacterial spores. Results reflect gradually evolving,

yet inadequate understanding of non-thermal lethality. Palaniappan et al. (1990, 1991)

studying yeasts cells, suggested that the destruction of microorganisms during ohmic

heating was principally because of thermal effects. However, a reduction in thermal

requirement for inactivation of microorganisms was observed at sublethal electrical

treatments by Palaniappan et al. (1992). Sublethal electrical treatments have also shown a

decrease in lag period and modification of metabolic properties during fermentation of

lactic acid bacteria (Cho et al., 1996).

27
Cho et al. (2000) compared the effects of ohmic heating with conventional heating on

inactivation kinetics of Bacillus subtilis at different temperatures and concluded that the

spores heated at 97.2°C showed a significant reduction in D-value under ohmic

treatment. However the Z-value for the two different methods was not statistically

different. A synergistic effect of heat and electrolysis under the influence of low

amperage direct electric current on Saccharomyces cerevisiae was suggested by Guillou

and Murr (2002). The study explored effects of amperage, temperature, pH and ionic

strength. D-values were found to be an inverse function of input current density. The

above two studies used ohmic devices which were considerably larger than the capillary

tube sized sample containers used by conventional microbiologists, and hence lacked

precision in the control of experimental errors.

The basic design of ohmic devices for microbial studies have remained practically the

same since the time when Palaniappan et al. (1990) reviewed the literature on the effect

of electricity on microorganisms. They concluded that the data available on non-thermal

effects of electricity was limited and inconclusive due to inadequate matching of time

temperature histories between electric field treatments and thermal controls. Some of the

basic factors contributing towards the variation in results can be categorized under two

broad categories: engineering design implications and microbiological constraints.

28
Engineering design implications

The literature reveals several logistical and technical constraints relative to the design of

ohmic heaters used for studying thermal inactivation kinetics. These factors can either

individually influence results or act synergistically.

a. The size of sample holders used in most ohmic studies has been greater than those

generally used for conventional heating treatments. For example, Guillou and

Murr (2002) used an ohmic heater of capacity of 9.2 ml while Cho et al. (2000)

used a 100 ml vessel. These vessels have higher possibilities for spatial

temperature or cell concentration variations than capillary tubes or tiny thermal

canisters used for conventional thermal death time studies; e.g. Iciek et al. (2006),

who used glass capillary tubes of about 100 µl volume to study inactivation

kinetics of Bacillus stearothermophilus under conventional thermal processing

conditions.

b. The action of convection currents in fluid media can influence uniformity of the

temperature profile inside an ohmic heater or treatment chamber.

c. Thermal stratification of hot and cold parts of the fluid can occur due to gravity.

d. Temperature difference between the treatment chamber and surroundings in both

conventional and ohmic heating can result in different microbial inactivation rates

between the geometric center and the walls of the heater. In ohmically heated

samples, internal temperatures are often higher than (unheated) surroundings; for

conventional heating, the exterior region may heat faster than the interior of

samples. Temperature gradients are minimized during conventional treatments by

29
 use of small samples in capillaries. The elimination of temperature gradients is

critical in attainment of uniform heating treatments.

e. Hot or cold spot formation due to one or more of the above reasons acting

together.

Microbiological constraints and requirements

a. Time-temperature histories of all associated processes should ideally match.

Variation in factors like come-up time, starting temperature, preparation time and

sampling time would result in increased variations in results.

b. Ideally, immediate cooling should be accomplished at the end of the holding time

to ensure that the sample attains a temperature below its optimum range of

activity. This will prevent post-treatment activation in case the study is done on

spores.

c. Study of thermo-resistant microorganisms may require treatment temperatures

above 100°C. In such conditions both the treatment as well as cooling should be

done under pressurized conditions.

d. Microorganisms can survive in significant numbers in micro crevices formed

around seals (o-rings or rubber gaskets) or on the sides of electrodes, influencing

the final enumeration. These parts generally experience a lower temperature than

regions between the cross sectional area of electrodes.

30
The presence of one or more of these factors may result in errors and explain some of the

contrasting results present in the literature. Hence, new methods for efficient bio-

validation of the ohmic heating process are required.

The principal reason for the additional effect of electricity on vegetative microorganisms

is due to charging (and resultant stresses) on the cell membrane during treatment under

alternating electric fields. It has been observed that in particular, low frequencies (50-60

Hz) have been effective in permeabilizing cell membranes. Kulshreshtha and Sastry

(2003) compared extraction of beet dye from beet cubes using moderate electric field

treatment (MEF) in a frequency range from 0 Hz (direct current) to 5 kHz and electric

potential from 0 (conventional heating) to 23.9 V/cm. The study concluded that diffusion

increases with electric field strength and decreases with frequency. Schreier et al. (1993)

found that diffusion of betanin from beetroot was increased under low frequency electric

treatment at 50 Hz. While these studies investigated the effects of electricity on

vegetative micro-organisms and vegetable tissues, limited information is available about

behavior of thermophilic bacterial spores in the presence of a low frequency (50-60 Hz)

electric field.

Electrolysis at electrode-product interface at one time had been a limiting factor in use of

ohmic heating for commercial food processing purposes. New materials and techniques

have been developed in recent years to minimize electrolytic reactions reducing the

phenomenon of electrode fouling. One such technique developed by Samaranayake et al.

(2005) involved use of high frequency pulses with short pulse duration. Square pulsed

waveform at frequencies of the order of 10 kHz minimized electrochemical reactions at

31
the food-electrode interface when used with a duty cycle range of 0.2 to 0.8. Jun and

Sastry (2005) used a frequency of 10 Hz with pulse duty ratio of 0.2 to heat food in

ohmic pouches for NASA‟s long duration space mission. The electric input was found

effective in reducing metal ion migration from the stainless steel foil electrodes below the

upper-level daily dietary exposure limits (Jun et al., 2007). A study of additional non-

thermal effects associated with high frequency (10 kHz) is needed to understand and

evaluate the pros and cons of its usage.

Our study is aimed at comparing microbial inactivation kinetics under ohmic (using two

different frequency values) and conventional heating. Hence the objectives of this study

are:

1. Development of ohmic and conventional capillary cells and a supporting system

to enable a legitimate comparison of conventional and ohmic heating on the

inactivation of Geobacillus stearothermophilus spores (ATCC 7953).

2. To explore the effects of ohmic heating frequency (10 KHz and 60 Hz) on G.

stearothermophilus inactivation kinetics in comparison to conventional heating.

2.3 Materials and Methods

2.3.1 System design

Our test device was designed to eliminate the potential sources of error from previous

studies. To minimize or eliminate spatial temperature gradients, both conventional and

ohmic test cells were made from the same basic capillary cell (construction details

follow). The cells were contained entirely within a larger ohmic test chamber filled with a

32
nearly isoconductive solution (0.78% NaCl) optimized to ensure the appropriate heating

regimes. This ensured that the test chamber fluid and the samples (within cells) heated at

nearly equal rates, essentially eliminating heat losses. All cells, whether conventional or

ohmic, were designed to be mounted with their ends facing the test chamber electrodes

(i.e. parallel to the current path). Figure 2.1 shows a typical arrangement of the capillary

cells and cell holder inside the ohmic treatment chamber.

Ohmic cells were equipped at each end with electrodes constructed of the same material

as the sample, but in the form of alginate gels to prevent sample leakage. To ensure that

the sample would allow the passage of current, the alginate electrodes were made more

conductive than the sample and the test chamber fluid. We subsequently verified that the

sample was indeed being ohmically heated by observing that the temperature of the

ohmic cell was 1-3°C higher than the outside solution (details later). This temperature

gradient in favor of the ohmic cell ensured that any heat flow was entirely from the

sample to the test chamber fluid, and confirmed that the sample was heated by pure

ohmic heating.

Conventional cells were constructed similar to ohmic cells, but with non-conductive end

pieces (clogs). As the clogs were not conductive to electricity, the test samples were

heated by the flow of heat from the surrounding solution. Temperature distribution

studies were conducted to ensure that the samples were heated conventionally. As the

ohmic cells stayed at a slightly higher temperature than the test chamber fluid and

conventional cells, separate experiments had to be conducted for conventional and ohmic

treatments to match the same time-temperature histories in each case. In other words,

33
conventional heating experiments were conducted with test chamber fluid at a marginally

higher temperature than the ohmic experiments, so that sample temperature histories were

the same in each case. Specially designed thermocouple cells, prepared similar to either

ohmic or conventional cells, were used to precisely monitor the temperature inside the

samples.

An elongated sleeve at the lower end of the treatment chamber was used as a cooling

zone. Treated cells were transferred from the heating zone at the end of their respective

holding times to the cooling section to rapidly cool the samples below 20°C.

Figure 2.1 Arrangement of capillary cells and cell holder inside the treatment chamber.

34
2.3.1.1 Ohmic and conventional capillary cells

Glass capillary tubes (Kimble Chase, Vineland, NJ, USA) of internal diameter 0.0011 m

were cut into 0.04 m length pieces for making both ohmic and conventional cells. Each

capillary tube was filled with 37 μl of inoculated tomato soup (Campbell‟s Condensed

Soup; Campbell Soup Company, Camden, NJ, USA). The capillary tubes were then

sealed with tomato alginate by tapping both ends one by one on a tomato alginate gel

prepared using the methods of Brown et al., (1984) and Tulsiyan et al., (2009). Special

precautions were taken to avoid integration of air bubbles, which could have distorted

electric flow through the capillary tube. Ohmic cells (Figure 2.2a.) after preparation were

immediately placed inside the test chamber fluid at 4°C, to avoid warming of the sample,

thereby leading to germination of spores. This also prevented drying and hence shrinking

of the alginate gel ends, which would have created a space filled with air in the dried part

of the alginate clog. Ohmic cells when placed in an external electric field parallel to the

length acted as miniature ohmic heaters.

Conventional cells (Figure 2.2b.) were prepared in the same way as ohmic cells; the only

difference being the use of a non-conductive vinyl plastic compound- critoseal capillary

tube sealant (McCormick Scientific, St. Louis, MO, USA) instead of alginate. The

sample inside such a capillary cell was only heated conventionally by flow of heat from

the test chamber fluid through the walls of the cell. Preparation of conventional capillary

cells of similar dimensions as ohmic cells made it possible to exactly match the

temperature histories and process time of both treatments.

35
To ensure precise temperature monitoring during each experiment, a separate set of

temperature measurement cells (thermocouple cells) were used with each experimental

run. This was done by inserting a T-type thermocouple (Omega Engineering Inc,

Stamford, CT, USA) inside a basic capillary cell tube to measure and monitor

temperature of the sample containing cells. Thereafter these cells were prepared similar

to ohmic and conventional cells. For ohmic thermocouple cells, the junction of the

thermocouple was inserted through a hole on the side of the capillary. Thus, the cross-

sectional area of the capillary cavity (internal diameter of 0.0011 m) had minimal

physical interference in form of the thermocouple junction. The effective electrical

conductivity remained almost the same as the sample without a thermocouple since the

volume of the metallic part (thermocouple junction) was very small (about 0.065 μl) as

compared to that of the cell (37 μl). For conventional thermocouple cells, the

thermocouple was inserted from one end. This provided minimal interference in the path

of heat flow through the capillary walls.

Capillary cell holders were manufactured to hold two cells parallel to each other and to

an external electric field were manufactured by machining molded polypropylene. A

nylon snap button (Dritz, Prym Consumer USA Inc, Spartanburg, SC, USA) was attached

on the base of each cell holder to facilitate holding in place and easy release. Thin

silicone rubber bands were tied to the elongated legs of the cell holder to hold one cell on

each side of the legs.

36
 Tomato alginate clogs Nonconductive crito-seal clogs

a. Inoculated tomato soup b.

Figure 2.2 a. An ohmic cell, b. a conventional cell

2.3.1.2 Heating-cooling system

A schematic of the heater-cooler system is shown in figure 2.3. The ohmic heating

system consisted of a Pyrex glass cross of 0.05 m internal diameter. Two titanium

electrodes (0.05 m diameter) placed 0.055 m from each other formed the ohmic heating

test chamber. Before treatment, capillary cell holders were snapped on a plastic frame in

the center of the ohmic test chamber. The system could accommodate five two-cell

holders at a time in a parallel position to each other. Thus the system contained a total of

8 sample containing and 2 thermocouple cells.

A glass cylindrical sleeve was joined to the lower end of the cross to act as a cooling

section. A port was also provided for the application of external pneumatic pressure in

the system. Rapid cooling after the treatment was achieved by quickly transferring the 4

sample containing cell holders into the cooling section using nylon threads (Coats and

37
Clark, Greenville, SC, USA), which communicated with the exterior via four stuffing

boxes (Ecklund-Harrison Technologies Inc, Fort Myers, FL, USA). Thermocouple cells

were not attached with the nylon threads (except during preliminary temperature

measurement experiments) and stayed at their positions after the end of the process, while

all other sample containing cells were pulled into the cold zone one after another

according to their respective holding times.

Figure 2.3. Schematic and a picture of the ohmic heating system

38
2.3.1.3 Power supply unit

An Integrated Gate Bipolar Transistor (IGBT) chip was used in the circuitry as a high

frequency switching device to generate a high-frequency pulsed square waveform. A

function generator (Instek, Chino, CA, USA) was used to deliver 10 kHz or 60 Hz

frequency input to the IGBT circuit. A square waveform with a duty ratio of 0.5 was used

to reduce the electrochemical reactions on the electrodes (Samaranayake et al., 2005).

Temperature, frequency, voltage, current and time data were recorded by a data logger

(Agilent Technologies Inc, Santa Clara, CA, USA).

2.3.2 Attainment of pure ohmic heating inside ohmic capillary cells

Electrical conductivities of tomato soup, tomato alginate and test chamber fluid were

measured from 24°C to 140°C using a ten-chamber ohmic heater (Tulsiyan et al., 2007).

Electrical conductivity of tomato alginate was adjusted to be higher than that of tomato

soup and test chamber fluid. The test chamber fluid was adjusted to be slightly more

conductive than the tomato soup. Figure 2.4 shows the electrical conductivities of all

three components up to a temperature of 140°C (average of 3).

Optimization tests were conducted to ensure that cells intended for ohmic heating did not

heat conventionally and vice versa. A test chamber fluid with an aqueous solution of

0.78% NaCl succeeded in maintaining the temperature of the ohmic cells slightly higher

(1-3°C) than the surrounding solution.

39
Although the electrical conductivity of the test chamber fluid was higher than tomato

soup, the presence of more conductive alginate clogs in series with tomato soup made the

combination (ohmic cell) achieve a higher temperature. A small amount of convection

heat transfer between the heated test chamber fluid and the cold salt solution present in

the cooling section also helped moderate the temperature and hence electrical

conductivity of the test chamber fluid.

Attainment of a marginally higher temperature by the ohmic cell meant no heat was

flowing from the outside test chamber fluid to the inside of the cell, hence not

contributing towards the heating. A small value of temperature difference (≤3°C) also

ensured minimal heat loss from the sample containing ohmic cell (Figure 2.5). The

arrangement of all cells parallel to each-other in a horizontal plane eliminated chances of

thermal stratification of liquids under gravity from affecting the results. The stability of

the temperature increase showed the lack of eddy currents. These tests ensured that ohmic

cells were heated by a pure ohmic heating effect and essentially all of the electric energy

going in the cell was consumed in inactivation of the spores.

However, whereas a small temperature difference between ohmic cells and test chamber

fluid confirmed the ohmic heating effect, it necessitated treatment of conventional cells in

a separate run. The process temperature, regardless of ohmic or conventional run, was

monitored inside the respective thermocouple cells representing the true temperature of

the samples. Plots for thermal histories of ohmic and conventional cells and the test

chamber fluid are shown in figure 2.6. Input voltages during the come-up and holding

times are shown in figure 2.7a, b.

40
 6

Conductivity, S/m
4

Tomato Alginate
2
Tomato Soup
0.78% Salt Solution

1
20 40 60 80 100 120 140 160

Temperature, C

Figure 2.4. Electrical conductivity of tomato alginate, tomato soup and test chamber fluid

(0.78 % salt solution).

135

Ohmic UCC
130 Test Chamber Fluid
Temperature, C

125

120

115

110
152 155 157 160 162 165 168 170 173 176 178 181 183 186 189 191

Time, s

Figure 2.5. Small positive temperature difference (≤3°C) between ohmic cell and test

chamber fluid (average of three) confirmed a pure ohmic heating effect.

41
Figure 2.6. Thermal histories of ohmic and conventional cells and the test chamber fluid,

showing closely matched thermal histories and rapid cooling of the samples.

Figure 2.7 a. Input voltage during the come-up time, b. Input voltage during the holding

time.

42
2.3.3 Preparation of Geobacillus stearothermophilus spore suspension

Geobacillus stearothermophilus ATCC 7953 was obtained from the American Type

Culture Collection (Manassas, VA. USA). The strain was grown in tryptose broth (TB;

Difco, Becton Dickinson and Co., Sparks, MD, USA). Stock cultures were stored at -

80°C in TB containing 40% (v/v) glycerol. Cultures were transferred in TB two times

prior to use.

For spore preparation, aliquots of 100 µl overnight cultures of G. stearothermophilus

were spread onto nutrient agar (NA; Difco) supplemented with 10 ppm manganese

chloride. Plates were incubated at 55°C until >90% of the cells were sporulated.

Sporulation was verified by observing the refractile spores using phase-contrast

microscopy. Spores were harvested by adding 10 ml of cold sterile deionized water on

each plate, and releasing the colonies containing spores from the surface of the agar with

the use of a sterile disposable inoculation loop. Collected spores were washed four times

by centrifugation (3000 x g for 15 min initially, then 8000 x g for 10 min three times) at

4°C, and the resulting pellet was cleaned to separate free spores from vegetative cells and

debris. The cleaning process consisted of aseptically treating each pellet with 100 ml of

lysozyme solution (200 µg/ml of lysozyme in 0.05 M potassium phosphate buffer

[KH2PO4 and K2HPO4], pH 8.1 and filter sterilized) for 20 min at 55°C. The spore crops

were then washed three times in sterile deionized water by centrifugation at 8000 x g for

10 min at 4°C. After the last centrifugation, the spore pellets were resuspended in sterile

deionized water. The spore suspensions were heated at 85°C for 10 min and checked

43
microscopically to ensure the absence of vegetative cells. The spore suspension was

stored at 4°C until used.

2.3.4 Experimental design

Studies were conducted on spore inactivation kinetics using two ohmic treatments (60 Hz

and 10 kHz) compared to conventional heating. D-values were studied at three different

temperatures of 121°C, 125°C and 130°C. A separate run was performed for each

treatment type. In each run four holding times were studied (2 cells for each holding

time). Experimental time-temperature conditions were 0, 15, 60 and 120 s for the 121°C

treatment, 0, 15, 30 and 90 s for the 125°C treatment and 0, 5, 10 and 30 s for the 130°C

treatment. At least three replicates were done at each condition.

Treated ohmic and conventional cells were crushed inside sterile polypropylene tubes

containing 0.1% peptone water using a sterile glass rod. Before crushing cells their

external surface was washed with 1400 ppm cold hypochlorite solution and rinsed with

cold sterile water. Sterile pieces of stomacher bag material (0.02 x 0.02 m) were used to

hold and close the ends of capillary cells while cleaning the outside surface with

hypochlorite solution, and to prevent it from entering the pores on the alginate surface

and later interfering with the enumeration. Capillary cells were then held by a sterile

stainless steel forceps and rinsed thoroughly with cold sterile water to remove any

remaining hypochlorite. The crushed samples inside peptone water were then given a

heat shock at 85°C for 10 min to inactivate all vegetative cells. This ensured plating of

only surviving spores from treated samples as well as spores from the untreated controls.

44
Further dilutions were plated on TSA agar plates and incubated for 48 hours before

counting.

2.4 Results and Discussion

Figure 2.8, 2.9 and 2.10 exhibit survivor plots for the three treatment types at

temperatures of 121°C, 125°C and 130°C respectively. Ohmic treatment at 10 kHz

frequency resulted in a general trend of higher inactivation than conventional treatment at

all three temperatures. 60 Hz ohmic treatment on the other hand showed a pattern of

improved killing effect as compared to conventional treatment with an increase in the

process temperature.

Regression analysis (p<0.05) confirmed that the D-value of 0.88 min for 10 kHz ohmic

treatment at 121°C was significantly lower than D-values of 1.17 min and 2.53 min

obtained for 60 Hz ohmic and conventional treatments respectively (Table 2.1). At 125°C

both ohmic treatments showed more inactivation with D-values of 0.43 min at 10 kHz

and 0.34 min at 60 Hz, as compared to a D-value of 0.64 min for conventional heating.

Though the trend between ohmic treatments and conventional heating remained the same

with the former producing more inactivation at 130°C also, the difference was not

statistically significant (p>0.05). Z-values for 10 kHz ohmic, 60 Hz ohmic and

conventional treatments were 8.30°C, 7.59°C and 7.44°C, respectively. In a nutshell,

ohmic heating was shown to accelerate the inactivation of the thermophilic bacterial

spores as compared to conventional heating.

45
 121ºC
0.5

0.0

-0.5
Log (N/Nₒ)

-1.0

10 kHz
-1.5
60 Hz

-2.0 Conventional

-2.5
0 15 30 45 60 75 90 105 120

Holding time, s

Figure 2.8. Inactivation of G. stearothermophilus spores under ohmic 10 kHz, ohmic 60

Hz and conventional treatments at 121°C with holding times of 0, 15, 60 and 120 s.

125ºC
1.0 10 kHz
60 Hz
0.0
Conventional
-1.0
Log (N/Nₒ)

-2.0

-3.0

-4.0

-5.0

-6.0
0 15 30 45 60 75 90

Holding time, s

Figure 2.9. Inactivation of G. stearothermophilus spores under ohmic 10 kHz, ohmic 60

Hz and conventional treatments at 125°C with holding times of 0, 15, 30 and 90 s.

46
 130ºC
0.5 10 kHz

0.0
60 Hz
Conventional
-0.5

-1.0
Log (N/Nₒ)

-1.5

-2.0

-2.5

-3.0

-3.5

-4.0
0 5 10

Holding time, s

Figure 2.10. Inactivation of G. stearothermophilus spores under ohmic 10 kHz, ohmic 60

Hz and conventional treatments at 130°C with holding times of 0, 5 and 10 s (survivor

count for 30 s holding time was below the detection limit).

Temperature

Treatment 121°C 125°C 130°C Z-value, °C

Ohmic 10 kHz 0.88a 0.43a 0.05a 8.30

Ohmic 60 Hz 1.17b 0.34a 0.05a 7.59

Conventional 2.53b 0.64b 0.06b 7.42

Note: Different superscripts within the column are significantly different (p<0.05).

Table 2.1. D-values of Geobacillus stearothermophilus (ATCC 7953) at 10 kHz ohmic,

60 Hz ohmic and conventional treatments at the temperatures of 121°C, 125°C and

130°C in minutes per log cfu/ml.

47
Dipicolinic acid (DPA) is a major constituent of a dormant bacterial spore core

accounting for approximately 10-15% of total dry weight (Alimova et al., 2006). High

content of DPA and formation of Ca-DPA complex in bacterial spores are believed to be

important for their exceptional physical & chemical hardiness (Berg & Grecz, 1970) and

core dehydration (Sliemn and Nicholson, 2001) thereby also contributing towards a

decreased electrical conductivity of spores. Carstensen et al. (1971) observed extremely

low electrical conductivities of dormant spores by dielectric measurements done at 50

MHz. The study suggested that initial low concentrations of mobile ions both within the

core and in surrounding integuments were subsequently increased during activation,

germination and outgrowth phases. Heat shock activation of spores probably includes

reversible breakage of sulfide and hydrogen bonds in the tertiary structure of some

enzyme proteins followed by release of spore coat proteins and DPA (Alimova et al.,

2006).

Finley and Fields (1961) examined the activation rates of B. stearothermophilus (1518)

spores over a range of temperature from 80°C to 115°C and found that the activation rate

improved with increasing temperature. Maximal spore activation was observed at 115°C

in 3 min. Interaction of an activated spore with increased electrical conductivity in an

external electric field is not yet fully understood.

It is hypothesized that during heat activation of dormant spores, a synergistic effect of

electric current and temperature might have caused an increase in the release of ionic

compounds like Ca-DPA molecules (Vepachedu et al., 2007) and fragments of denatured

protein enzymes (Alimova et al., 2006) from the core. Interaction of the released ionic

48
molecules with the electric field would have increased the rate of activation and a

simultaneous increase in the electrical conductivity of the spore, making it more prone to

additional non-thermal effects of electricity.

2.5 Conclusion

A precise and versatile capillary cell was developed for studying microbial inactivation

kinetics under ohmic and conventional heating. The device successfully eliminated the

potential source of errors inherent to conventional ohmic heaters and ensured identical

time-temperature histories and uniform heating profiles. Capillary cells may be modified

to accurately study quality related kinetics also.

Ohmic heating at 10 kHz and 60 Hz showed more inactivation of thermophilic bacterial

spores than conventional heating. It is hypothesized that release of ionic materials like

DPA from the spore core and spore coat proteins at higher temperature assisted in

increased lethal effect of electricity.

2.6 References

Alimova, A.A., Katz, A., Gottlieb, P., & Alfano, R.R. (2006). Proteins and dipicolinic

acid released during heat shock activation of Bacillus subtilis spores probed by optical

spectroscopy. Applied Optics, 45(3), 445-450.

49
Berg, P.E., & Grecz, N. (1970). Relationship of dipicolinic acid content in spores of

Bacillus cereus T to ultraviolet and gamma radiation resistance. Journal of Bacteriology,

103(2), 517-519.

Brown, K.L., Ayers, C.A., Gaze, J.E., & Newman, M. E. (1984). Thermal destruction of

bacterial spores immobilized in food/ alginate particles. Food Microbiology, 1(3), 187-

198.

Carstensen, E.L., Marquis, R.E., & Gerhardt, P. (1971). Dielectric study of the physical

state of electrolytes and water within Bacillus cereus spores. Journal of Bacteriology,

107(1), 106-113.

Cho, H.Y., Sastry, S.K. and Yousef, A.E. (2000). Kinetics of inactivation of Bacillus

subtilis spores by continuous or intermittent ohmic and conventional heating.

Biotechnology and Bioengineering, 62(3), 368-372.

Guillou, S., & Murr, N. El. (2002). Inactivation of Saccharomyces cerevisiae in solution

by low-amperage electric treatment. Journal of Applied Microbiology, 92, 860-865.

Iciek, J., Papiewska, A., & Molska, M. (2005). Inactivation of Bacillus

stearothermophilus sprores during thermal processing. Journal of Food Engineering,

77(3), 379- 471.

50
Imai, T., Uemura, K., Ishida, N., Yoshizaki, S., & Noguchi, A. (1995). Ohmic heating of

Japanese white radish Rhaphanus sativus L. International Journal of Food Science and

Technology, 30(4), 461-472.

Kulshrestha, S.A., & Sastry, S.K. (1999). Low-frequency dielectric changes in vegetable

tissue from ohmic heating. IFT Annual Meeting: Book of Abstracts. p. 211. Chicago, IL.

Kulshrestha, S., & Sastry, S. (2003). Frequency and voltage effects on enhanced diffusion

during moderate electric field treatment. Innovative Food Science and Emerging

Technologies, 4, 189-194.

Lee, C.H., & Yoon, S.W. (1999). Effect of ohmic heating on the structure and

permeability of the cell membrane of Saccharomyces cerevisiae. 1999 IFT Annual

Meeting. Chicago. July 24-28 1999.

Palaniappan, S., Sastry, S.K., & Richter, E.R. (1990). Effects of electricity on

microorganisms: a review. Journal of food processing and preservation, 14(5), 393-414.

Palaniappan, S., & Sastry, S.K. (1991). Electrical conductivity of selected juices:

influence of temperature, solids content, applied voltage and particle size. Journal of

Food Process Engineering, 14, 247-260.

51
Samaranayake, C.P., Sastry, S.K., & Zhang, H (2005). Pulsed ohmic heating- A novel

technique for minimization of electrochemical reactions during processing. Journal of

Food Science, 70(8), E 460-465.

Schreier, P.J.R., Reid, D.G., & Fryer, P.J. (1993). Enhanced diffusion during the

electrical heating of foods. International Journal of Food Science and Technology, 28,

249-260.

Slieman, T.A., & Nicholson, W.L. (2001). Role of dipicolinic acid in survival of Bacillus

subtilis spores exposed to artificial and solar UV radiation. Applied and Environmental

Microbiology, 67(3), 1274-1279.

Somavat, R. (2010). Applications and effects of ohmic heating: sterilization, influence on

bacterial spores, enzymes, bioactive components and quality factors in food. PhD

Dissertation. The Ohio State University. Columbus, OH.

Tulsiyan, P., Sarang, S., & Sastry, S.K. (2007). Electrical conductivity of

multicomponent systems suring ohmic heating. International Journal of Food Properties,

11(1), 233-241.

52
Tulsiyan, P., Sarang, S., & Sastry, S.K. (2009). Measurement of residence time

distribution of a multicomponent system inside an ohmic heater using radio frequency

identification. Journal of Food Engineering, 93(3), 313-317.

Vepachedu, V.R., Hirneisen, K., Hoover, D.G., & Setlow, P. (2007). Studies of the

release of small molecules during pressure germination of spores of Bacillus subtilis.

Letters in Applied Microbiology, 45, 342-348.

53
 CHAPTER 3

Inactivation kinetics of Bacillus coagulans spores under

the effect of ohmic heating

3.1 Abstract

Bacillus coagulans spores are commonly involved in the spoilage of food of pH between

4 and 4.5, and have been shown to increase the pH to values that can allow the

germination of surviving Clostridium botulinum. Recent studies on ohmic heating have

indicated the presence of an additional nonthermal effect of electricity on the bacterial

spores of Geobacillus stearothermophilus and Bacillus subtilis. We investigated the

kinetics of inactivation of Bacillus coagulans spores (ATCC 8038) in fresh tomato juice

under ohmic heating at frequencies of 10 kHz and 60 Hz, and compared it with

conventional heating using a specially designed experimental setup that assured identical

temperature histories for all treatments. Ohmic heating at 60 Hz showed significantly

lower D-values at 95, 100 and 105°C compared to conventional heating. While 10 kHz

also showed a similar trend of higher inactivation compared to conventional heating, the

difference was significant only at 105°C. Both ohmic treatments also showed higher

inactivation than conventional heating during the come-up time. In conclusion, ohmic

54
heating resulted in an accelerated inactivation of the B. coagulans spores compared to

conventional treatment. A linearly increasing temperature analysis suggested that the Z

values obtained at isothermal conditions may only be valid over a narrow range of

temperature.

3.2 Introduction

Bacillus coagulans is a spoilage microorganism commonly associated with food acidified

to between pH 4.0 and 4.5 (Palop et al., 1999). This microorganism is specifically

responsible for flat sour spoilage outbreaks in tomato based products (Sandoval et al.,

1992; York et al., 1975). B. coagulans is a non-pathogenic organism, but it can pose a

safety hazard because of its ability to increase the pH of a high acid food, processed with

a reduced treatment, to a level where surviving Clostridium botulinum spores can

germinate (Anderson 1984; Fields et al., 1977). Hence, it is not only relevant to the

tomato industry because of its higher thermal resistance than the other spore formers

involved with tomato products (Mallidis et al., 1990), but also because in the past it has

been linked to a few cases of botulism through tomato juices (Fields et al., 1977). An

outbreak of C. botulinum in inherently high acid food can only occur with an associated

rise of the pH, which, in the case of a tomato based product, can be linked to the growth

of B. coagulans.

Ohmic heating is an alternate processing method shown to yield higher quality foods than

conventional heating (Kim et al., 1995). It is classified as a purely thermal process,

mainly because of an inadequate understanding of the non thermal effect of electricity on

microorganisms. Several past studies have shown an additional effect of electricity

55
during the ohmic heating of plant tissues (Jemai and Vorobiev, 2002; Kulshrestha and

Sastry, 2003), vegetative microorganisms (Guillou and El Murr, 2002; Guillou et al.,

2003; Loghavi et al., 2007; Loghavi et al., 2009; Palaniappan et al., 1992) and bacterial

spores (Cho et al., 1999). Although studies indicating the additional effects of alternating

current on microorganisms date back to the time of Tracy (1932), they failed to

adequately control sources of errors. Somavat (2010) have presented an assessment of

error in ohmic heating devices used for microbial studies, and concluded that the

relatively large size of ohmic treatment devices and non-identical thermal histories

between conventional and ohmic treatments might have resulted in experimental errors.

The basic design of ohmic devices used for microbial study has remained almost the

same since the time of Tracy (1932), until as recently as Cho et al. (1999) and Guillou et

al. (2003). These devices were considerably larger and more complex than the simple

capillary sized sample holders used by microbiologists.

Somavat (2010) described a capillary sized treatment cell which was able to deliver

identical temperature histories for ohmic and conventional treatments while using similar

sized sample holders. Using these cells they determined the kinetics of inactivation

kinetics of Geobacillus stearothermophilus spores and found additional nonthermal

effects of electricity at the frequencies of 10 kHz and 60 Hz. The frequency of 10 kHz in

pulsed mode with a square waveform is known to reduce electrochemical reactions at the

food-electrode interface (Samaranayake et al., 2005; Jun et al., 2007), whereas the

frequency of 60 Hz is associated with increased inactivation of vegetative

microorganisms (Cho et al., 1999; Guillou and Murr, 2002) and enhanced rupture of plant

tissues (Schreier et al., 1993; Kulshreshtha and Sastry, 2003). A study on inactivation

56
kinetics of the target microorganism of acid food, B. coagulans spores, is required to

further our understanding of the ohmic heating process, as well as to evaluate its potential

for use by the related industry. Hence, the main aim of this study was to investigate the

effect of ohmic heating at the frequencies of 10 kHz and 60 Hz on the inactivation

kinetics of B. coagulans spores (ATCC 8038) in comparison to conventional treatment.

3.3 Materials and Methods

Tomato juice samples inoculated with B. coagulans spores were heated in ohmic and

conventional capillary cells (Somavat, 2010). The samples were treated ohmically at

frequencies of 10 kHz and 60 Hz in pulse mode, or conventionally to the temperatures of

95, 100, 105 and 110°C. The samples were held at four different holding times at each

temperature- 0, 10, 20 and 30 min for 95°C; 0, 2, 4 and 6 min for 100°C; 0, 1, 2 and 3

min for 105°C; and 0, 10, 20 and 30 s for 110°C. Separate runs were conducted for ohmic

and conventional treatments. Two sample-containing capillary cells mounted on each

capillary tube holder were used for each holding time. Three replicates were done at each

condition. Details of experimental protocol are in the following sections.

3.3.1 System design

3.3.1.1 Ohmic and conventional capillary cells

The ohmic and conventional capillary cells and the supporting system described by

Somavat (2010) were used. Capillary tubes holding 37 μl of tomato juice inoculated with

Bacillus coagulans spores were plugged at both ends with tomato alginate (conductive)

57
for ohmic heating, or with nonconductive capillary tube sealant for conventional heating.

The capillary tubes were aligned parallel to the electrical field inside an external ohmic

heating chamber containing an iso-conductive salt solution and designed to provide rapid

cooling and pressurized conditions. To hold capillary cells in place, they were mounted

on cell holders (two cells per holder) snapped on the top part of the treatment chamber

(Figure 3.1). The system accommodated 5 such cell holders, thus containing a total of 8

sample containing cells in addition to 2 thermocouple cells. Thermocouple capillary cells

were prepared by inserting a T-type thermocouple inside a basic cell, which was then

prepared similarly to either ohmic or conventional cells. The achievement of a pure

ohmic heating effect inside the ohmic capillary cells was confirmed through a

temperature distribution study which showed that the ohmic cells always remained at a

slightly higher temperature than the surroundings, thereby confirming the internal ohmic

generation of heat inside the cells. Because of this temperature gradient, separate runs of

ohmic and conventional treatments were conducted to ensure equal temperature histories

in both cases. The system also facilitated rapid post-treatment cooling through pulling of

the treated samples in the cooling section with the help of an attached thread. A detailed

description of the setup and procedures is provided by Somavat (2010).

58
 Ohmic Treatment Chamber
Electrode
Electrode

Cell Holders with

Capillary Cells

Threads

Cooling Section

Figure 3.1 Typical arrangement of the capillary cells inside the treatment chamber.

3.3.1.2 Power supply and control

A square pulsed waveform with a duty ratio of 0.5 was generated using a circuit

containing an integrated gate bipolar transistor (IGBT) serving as a high frequency

switching device. A function generator (Instek, Chino, CA) was used to deliver 10 kHz

or 60 Hz frequency input through the IGBT circuit. The waveform and duty ratio had

been previously shown effective in reducing electrochemical reactions at the electrodes

(Samaranayake et al., 2005). A data logger (Agilent Technologies, Inc., Santa Clara, CA)

was used to record the time, temperature, frequency, voltage and current data. A

waveform plot of the square pulsed waveform at 10 kHz frequency and 0.5 duty ratio is

shown in Figure 3.2.

59
An input voltage of 65 V was used across the electrodes of the external ohmic chamber

(13 V/ cm across the capillary tubes) during the come-up time. Voltage was subsequently

reduced and manually adjusted during the holding period to maintain the specified

temperature. A graph for typical voltage and temperature histories during the come up

and holding time for the 110°C process is presented in Figure 3.3. On average, come-up

times of around 158, 170, 180 and 192s were obtained during heating from room

temperature (21°C) to 95, 100, 105 and 110°C, respectively.

Pulsed Ohmic Waveform (10 kHz)

80
60
40
20
Voltage, V

0
0.00000

0.00003

0.00007

0.00010

0.00013

0.00017

0.00020

0.00023

0.00027

0.00030

-20
-40
-60
-80
Time, s

Figure 3.2 A representation of the square waveform; 10 kHz frequency and 0.5 duty ratio.

60
 Voltage and temperature history
120
Voltage
100
Voltage, V; Temperature, C Ohmic
80 Conventional

60

40

20

0
0 50 100 150 200 250
Time, s

Note: Temperature of the cooling phase was only logged during the process validation and not for

each inactivation run.

Figure 3.3 Typical voltage and matching temperature histories of the ohmic and

conventional treatments during the come-up and holding times for the 110°C treatment.

3.3.2 Microbiological experiments

3.3.2.1 Preparation of the Bacillus coagulans spore suspension

The strain of B. coagulans, ATCC 8038, was obtained from the American Type Culture

Collection (Manassas, VA., USA). The strain was grown in tryptic soy broth (TSB;

Difco; Becton, Dickinson and Co., Sparks, MD, USA) at 37˚C for 48 h under aerobic

conditions and transferred at least three times before spore preparation. Spores of the

microorganism were obtained by plating 500 µl of actively growing culture (24 h at

37˚C) into nutrient agar (NA; Difco; Becton, Dickinson and Co., Sparks, MD, USA)

supplemented with 500 mg/L dextrose (BD, Difco) and 3 mg/L manganese sulfate

61
(Fisher Scientific, Pittsburgh, PA, USA) (Palop et al., 1999). The inoculated plates were

incubated at 50°C for 7 days, where more than 90 % of sporulation was obtained as

verified by observing the refractile spores under phase-contrast microscopy. Spores were

harvested by flooding plates with 5 ml of cold sterile deionized water, and releasing the

colony containing spores from the surface of the agar with the use of a sterile disposable

inoculation loop. Collected spores were washed six times by centrifugation (8000 x g for

20 min) at 4°C. After the last centrifugation, the spore pellets were resuspended in sterile

deionized water. The spore suspensions were heated at 80°C for 15 min and checked

microscopically to ensure the absence of vegetative cells. The spore suspension was

stored at 4°C until used. A volume of 37 μl of tomato juice inoculated with 107 cfu/ml

spores was filled in each capillary cell for treatment.

3.3.2.2 Enumeration procedure

Treated capillary cells were washed with cold 1400 ppm hypochlorite solution and rinsed

with cold sterile water. The capillary washing protocol developed by Somavat et al.

(2010) was followed to eliminate any residual hypochlorite from affecting the final plate

count. The clean capillary cells were then crushed inside sterile polypropylene tubes

containing 0.1% peptone water using sterile glass rods. A heat shock at 80°C for 15 min

was given to inactivate all vegetative cells. Further dilutions in peptone water were

prepared and plated on TSA agar plates. Inoculated plates were incubated for 48 hours at

37˚C and colonies enumerated.

62
3.3.3 Tomato juice preparation

Fresh Roma tomatoes of bright red color (an „a‟ value of 20) bought from a local grocery

store (Kroger Inc.) were used. Tomatoes were cut in four quarters and then were blended

to prepare the tomato juice media. pH of the juice, inherently ranging from 4.1 to 4.3,

was adjusted to a standard value of 4.4 using sodium citrate to eliminate the varying

acidity from affecting the thermal resistance of the organism.

3.4 Results and Discussion

Survivor plots at 95°C, 100°C, 105°C and 110°C are shown in Figure 3.4, 3.5, 3.6 and

3.7, respectively. D-values of 7.96, 1.63 and 0.91 min at the temperatures 95, 100 and

105°C for ohmic heating at 60 Hz were significantly lower than D-values of 10.1, 2.52

and 1.32 min for conventional treatment (Table 3.1). Ohmic heating at 10 kHz frequency

with a square pulsed waveform resulted in significantly lower D-value than conventional

heating only at 105°C with a D-value of 1.03 min; although it showed a general trend of

extra inactivation at other temperatures also. At 100°C, ohmic heating at 60 Hz showed

significantly greater inactivation than the ohmic sample at 10 kHz (D100 of 2.27 min). No

significant difference between ohmic and conventional treatments was observed at the

highest temperature of 110°C. Z-values for 10 kHz, 60 Hz and conventional treatments

were 9.89, 11.18 and 8.68°C, respectively (Figure 3.8).

Ohmic treatment at 60 Hz showed an additional killing effect, significantly over a greater

range of temperature than conventional heating. Whereas, 10 kHz also showed a general

trend of higher inactivation than conventional heating, its effect was only significant at

105°C. The absence of any significant difference between both ohmic treatments and

63
conventional samples at 110°C indicates that at higher temperatures, pronounced thermal

effects overshadow the nonthermal changes of electricity. These results clearly

demonstrate an accelerated inactivation of the B. coagulans spores under the ohmic

treatments as compared to conventional heating.

Both ohmic heating treatments also showed an additional inactivation during the come-up

time from the room conditions to the target temperature (Figure 3.9). Typically, the

come-up times from room temperature to 95, 100, 105 and 110°C were around 158, 170,

180 and 192 s, respectively (also shown in Figure 3.3).

95°C
0.0
-0.5 Conv

-1.0 10 kHz
-1.5 60 Hz
Log(N/No)

-2.0
-2.5
-3.0
-3.5
-4.0
-4.5
0 5 10 15 20 25 30
Holding time, min

Figure 3.4 Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and

conventional treatments at 95°C with holding times of 0, 10, 20 and 30 min.

64
 100°C
0.0 Conv
-0.5
10 kHz
-1.0
60 Hz
-1.5
Log(N/No)

-2.0
-2.5
-3.0
-3.5
-4.0
-4.5
0 1 2 3 4 5 6
Holding time, min

Figure 3.5 Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and

conventional treatments at 100°C with holding times of 0, 2, 4 and 6 min.

105°C
0.0
Conv
-0.5
-1.0 10 kHz
-1.5 60 Hz
Log(N/No)

-2.0
-2.5
-3.0
-3.5
-4.0
-4.5
0 1 1 2 2 3 3
Holding time, min

Figure 3.6 Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and

conventional treatments at 105°C with holding times of 0, 1, 2 and 3 min.

65
 110°C
0.0
-0.5 Conv
-1.0 10 kHz
-1.5 60 Hz
Log(N/No)

-2.0
-2.5
-3.0
-3.5
-4.0
-4.5
0 5 10 15 20 25 30
Holding time, s

Figure 3.7 Inactivation of B. coagulans spores under ohmic 10 kHz, ohmic 60 Hz and

conventional treatments at 110°C with holding times of 0, 10, 20 and 30 s.

D-values, min
Treatment Z-value, °C
95°C 100°C 105°C 110°C

10 kHz 8.81a,b 2.27a 1.03b 0.13a 9.89a

60 Hz 7.96b 1.63b 0.91b 0.15a 11.18a

Conventional 10.13a 2.52a 1.32a 0.16a 8.68a

Note: Different superscripts within the same column are significantly different (p<0.05).

Table 3.1 D- and Z-values at 95, 100, 105 and 110°C for 10 kHz, 60 Hz and conventional

inactivation of B. coagulans spores.

66
 Z value plot
12
10 kHz
10
60 Hz
8 Conv

D value, min 6

0
90 95 100 105 110 115
-2
Temperature, C

Figure 3.8 A Z-value plot showing the D-values at 95, 100, 105 and 110°C for 10 kHz,

60 Hz and conventional treatment.

Reduction During the Come-up Time

8.0 Conv
60 Hz
7.5
10 kHz
log spore, cfu/ml

7.0

6.5

6.0

5.5

5.0
90 95 100 105 110
Temperature, C

Note: The untreated control at 21°C has been represented by the temperature of 90°C in the plot.

Figure 3.9 Spore reductions during come up times of 158, 170, 180 and 192s to the

temperatures of 95, 100, 105 and 110°C, respectively, for conventional, 60 Hz and 10

kHz treatments.

67
The overall trend of higher inactivation of bacterial spores under the effect of ohmic

heating is in agreement with the results observed by Somavat (2010) on Geobacillus

stearothermophilus spores. Thus, our study further confirms the hypothesis presented by

them to explain the non-thermal effects of electricity on bacterial spores. They

hypothesized that during heat activation of dormant spores, a synergistic effect of electric

current and temperature cause an increase in the release of ionic compounds like calcium

dipicolinic acid molecules from the core, and fragments of denatured spore protein

enzymes from the coat. Interaction of these released ionic molecules with the electric

field would further increase the rate of activation through a simultaneous increase in the

electrical conductivity of the spore, making it more prone to additional nonthermal

effects of electricity.

Another interesting point of note is the consistently (with only the 110°C exception)

greater efficiency of 60 Hz treatments over the 10 kHz treatments. This is opposite to the

findings of Somavat (2010) who observed that 10 kHz resulted in comparatively more

inactivation than 60 Hz at lower temperatures for the thermophilic spores of G.

stearothermophilus. However, this ambiguous result is consistent with the hypothesis that

different oscillating electric fields cause different parts of a bacterial spore to react,

thereby resulting in effects which are not yet understood.

We noticed that the heating curve of the samples (Figure 3.3) had a linear come-up phase.

Given that we had available data for inactivation at the end points (come-up time; CUT)

for each temperature of the linearly increasing phase, it was considered worth

investigating whether the kinetic data obtained at constant temperatures could be used to

68
predict inactivation by numerical integration via a linearly increasing temperature

analysis modified from the approach of Rhim et al. (1989). This analysis is presented

here. Mathematically, microbial log inactivation may be represented as:

and

If temperature is constant

When temperature is a function of time

Since, for a linearly increasing temperature plot

T = mt + c

or

Integrating and applying limits:

69
This nonlinear equation in Z was solved analytically for Dref using the known values from

constant temperature data for Z and the measured inactivation during CUT. Dref values

(where Tref = 100°C) were calculated at 95, 100, 105 and 110°C using experimentally

obtained values of Z for conventional and ohmic treatments using the heating curve

shown in Figure 3.3. Another calculation involved calculating log10 (N0/N) using D100

and Z values obtained from constant temperature data.

Calculated D100 values were considerably higher than experimentally obtained values of

2.52, 1.63 and 2.27 min for conventional, 60 Hz and 10 kHz, respectively (Table 3.2).

The predicted inactivation (i.e. log10 N/ N0) using D and Z values from constant

temperature data was much higher than the actual value, suggesting that the D-values at

low temperatures based on experimental data are much lower than expected from

extrapolation of constant temperature data. The dramatic increase in D-value at 95°C

(Figure 3.8) shows that Z varies over the studied temperature range. These data suggest

that tailing effects may occur in kinetic data at low temperature, thus the extrapolation of

log linear inactivation kinetics data obtained at constant process temperatures may

produce inaccurate results.

70
 Dref, min Dref, min log10(N0/N) log10(No/N)
Treatment Temp, C Z Value, C Tref, C (Actual) (Calculated) (Actual) (Calculated)
10 kHz 95 9.89 100 2.27 24.76 0.02 0.22
10 kHz 100 9.89 100 2.27 20.68 0.04 0.36
10 kHz 105 9.89 100 2.27 13.65 0.10 0.60
10 kHz 110 9.89 100 2.27 32.52 0.07 0.95
60 Hz 95 11.18 100 1.63 14.83 0.04 0.36
60 Hz 100 11.18 100 1.63 23.36 0.04 0.57
60 Hz 105 11.18 100 1.63 13.22 0.11 0.89
60 Hz 110 11.18 100 1.63 17.61 0.13 1.33
Conventional 95 8.68 100 2.52 40.53 0.01 0.16
Conventional 100 8.68 100 2.52 36.30 0.02 0.29
Conventional 105 8.68 100 2.52 25.71 0.05 0.51
Conventional 110 8.68 100 2.52 32.86 0.07 0.87

Table 3.2 Values of Dref and log10(N0/N) comparing measured and calculated using

linearly increasing temperature method.

3.5 Conclusion

Ohmic heating at 60 Hz and 10 kHz result in accelerated inactivation of Bacillus

coagulans spores compared to conventional heating. These results further confirm the

presence of the additional non thermal effect of ohmic heating on bacterial spores as

observed by Cho et al. (1999) and Somavat (2010). A linearly increasing temperature

analysis suggested that kinetic data obtained at constant temperature conditions results in

overprediction of inactivation at lower temperatures.

71
Acknowledgement

The authors gratefully acknowledge support from USDA-CSREES Project No: 2009-

55503-05198 titled Quality of Foods Processed Using Selected Alternative Processing

Technologies. Salaries and research support also provided by the Ohio Agricultural

Research and Development Center (OARDC), The Ohio State University. References to

commercial products and trade names are made with the understanding that no

endorsement or discrimination from the Ohio State University is implied.

3.6 References

Anderson, R.E. (1984). Growth and corresponding elevation of tomato juice pH by

Bacillus coagulans. Journal of Food Science, 49, 647-647.

Cho, H., Yousef, A.E., & Sastry, S. K. (1999). Kinetics of inactivation of Bacillus subtilis

spores by continuous or intermittent ohmic and conventional heating. Biotechnology &

Bioengineering. 62(3), 368-372.

Fields, M.L., Zamora, A.F., & Bradsher, M. (1977). Microbiological analysis of home-

canned tomatoes and green beans. Journal of Food Science, 42(4), 931-934.

Guillou, S., & Murr, N. El. (2002). Inactivation of Saccharomyces cerevisiae in solution

by low-amperage electric treatment. Journal of applied microbiology, 92(5), 860-865.

72
Guillou, S., Besnard, V., Murr, N. El., & Federighi, M. (2003). Viability of

Saccharomyces cerevisiae cells exposed to low-amperage electrolysis as assessed by

staining procedure and ATP content. International journal of food microbiology, 88(1),

85-89.

Jean, G.L., Abraham, G., Debray, E., Candau, Y., & Piar, G. (1994). Kinetics of thermal

destruction of Bacillus stearothermophilus spores using a two reaction model. Food

Microbiology, 11, 229-241.

Jemai, A.B., & Vorobiev, E. (2002). Effect of moderate electric field pulses on the

diffusion coefficient of soluble substances from apple slices. International Journal of

Food Science & Technology, 37(1), 73-86.

Jun, S., Sastry, S.K., & Samaranayake, C. (2007). Migration of electrode components

during ohmic heating of foods in retort pouches. Innovative Food Science and Emerging

Technologies, 8(2), 237-243.

Kim, H.J., Choi, Y.M., Yang, A.P.P., Yang, T.C.S., Tuab, I.A., Giles, J., Ditusa, C.,

Chall, S., & Zoltai, P. (1995). Microbiological and chemical investigation of ohmic

heating of particulate foods using a 5 kW ohmic system. Journal of Food Processing and

Preservation, 20(1), 41-58.

Kulshrestha, S., & Sastry S. (2003). Frequency and voltage effects on enhanced diffusion

during moderate electric field (MEF) treatment. Innovative Food Science and Emerging

Technologies, 4(2), 189-194.

73
Loghavi, L., Sastry, S.K., & Yousef, A.E. (2007). Effect of moderate electric field on the

metabolic activity and growth kinetics of Lactobacillus acidophilus. Biotechnology and

bioengineering, 98(4), 872-881.

Loghavi, L., Sastry, S.K., & Yousef, A. E. (2009). Effect of moderate electric field

frequency and growth stage on the cell membrane permeability of Lactobacillus

acidophilus. Biotechnology Progress, 25(1), 85-94.

Mallidis, C.G., Frantzeskakis, P., Balatsouras, G., & Katsaboxakis, C. (1990). Thermal

treatment of aseptically processed tomato paste. International Journal of Food Science

and Technology, 25, 442-448.

Palaniappan, S., Sastry, S.K., & Richter, E.R. (1992). Effects of electroconductive heat

treatment and electrical pretreatment on thermal death kinetics of selected

microorganisms. Biotechnology and Bioengineering, 39(2), 225-232.

Palop, A., Raso, J., Pagan, R., Condon, S., & Sala, F.J. (1999). Influence of pH on heat

resistance of spores of Bacillus coagulans in buffer and homogenized foods.

International Journal of Food Microbiology, 46, 243-249.

Rhim, J.W., Nunes, R.V., Jones, V.A., & Swartzel, K. R. (1989). Determination of

kinetic parameters using linearly increasing temperature. Journal of Food Science, 54(2),

446-450.

74
Samaranayake, C., Sastry, S., & Zhang, H. (2005). Pulsed Ohmic Heating–A Novel

Technique for Minimization of Electrochemical Reactions During Processing. Journal

oWf Food Science, 70(8), E 460-465.

Sandoval, A.J., Barreiro, J.A., & Mendoza, S. (1992). Thermal resistance of Bacillus

coagulans in double concentrated tomato paste. Journal of Food Science, 57(6), 1369-

1370.

Schreier, P.J.R., Reid, D.G., & Fryer, P.J. (1993). Enhanced diffusion during the

electrical heating of foods. International Journal of Food Science and Technology, 28,

249-260.

Somavat, R. (2010). Applications and effects of ohmic heating: sterilization, influence on

bacterial spores, enzymes, bioactive components and quality factors in food. PhD

Dissertation. The Ohio State University. Columbus, OH.

Tracy, R.L.Jr. (1932). Lethal effect of alternating current on yeast cells. Journal of

Bacteriology, 24(6), 423-438.

York, G.K., Heil, J.R., Marsh, G.L., Ansar, A., Merson, R.L., Wolcott, T., & Leonard, S.

(1975). Thermobacteriology of canned whole peeled tomatoes. Journal of Food Science,

40, 764-769.

75
4. Effect of ohmic heating on kinetics of change of pectin methylesterase,

polygalacturonase and color in tomato juice

4.1 Abstract

Ohmic heating shows much promise for processing of tomato pastes, purees and other

particulate containing liquids. This work involved the determination of inactivation

kinetics of pectin methylesterase (PME) and polygalacturonase (PG) in fresh tomato

juice, adjusted to either pH 3.9 or 4.4, under the effect of ohmic heating. All treatments at

pH 4.4 that targeted Bacillus coagulans inactivation showed no residual PME activity.

Thus, studies were conducted at lower temperatures to track the progression of enzyme

levels during inactivation. At pH 3.9, and lower temperatures, inactivation data were

found to be highly variable, despite repeated and specific efforts to minimize variation.

This suggests that different isozymes of PME may respond differently to alternating

electric fields. At pH 4.4 PG inactivation under ohmic heating was found to be

accelerated in comparison to literature values for the most resistant isozyme, PG1. At pH

3.9, PG inactivation data were found to be less variable than for PME, due apparently to

fewer identified isozymes for PG. Some activation effects could be observed at 90°C.

Color values (l, a and b) did were unaffected over the studied temperature and pH ranges.

76
4.2 Introduction

In recent years ohmic heating has generated considerable attention in the food industry

for continuous processing of pumpable viscous liquid and particulate- containing liquid

foods. The capability of ohmic heating to provide uniform heating of liquid- particulate

mixtures at a faster rate, higher energy transfer efficiency than conventional heating

result in products of superior quality (Salengke and Sastry, 1998). Several industrial scale

ohmic treatment plants have recently became functional in Europe for the processing of

fruit and vegetable pieces, purees and juices (Pereira and Vicente, 2009). Many of these

applications include the processing of products such as purees, dices, soups, juices,

ketchup and other sauces. The quality of tomato products can be affected during

processing by the key enzymes: pectin methylesterase (PME) and polygalacturonase (PG)

by influencing the composition of pectin (Sekine et al., 2003). PME catalyses de-

esterification of the methyl group of pectin resulting in the formation of low methoxy

pectin or uronic acid carboxyl, and methanol. These intermediary products are then acted

upon by PG, which by cleaving the glycosidic linkage, decreases viscosity significantly

(Yildiz and Baysal, 2006). PME is the most thermo-resistant pectolytic enzyme known in

tomato, and hence is accepted as the indicator enzyme in heat inactivation studies.

Anthon et al. (2002) and Sekine et al. (2003) studied the thermal resistance of these two

enzymes. They have reported that D70 and Z values for PME range from 5.0 to 10.4 min

and 4.9 to 6.3°C, respectively. This variation is likely due to the presence of labile and

resistant isozymes. The presence of two isozymes of PG, designated as PG1 and PG2,

with considerably different thermal stability, is also well established. The reported D and

77
Z values for PG range from 2.6 to 4.0 min and 10.4 to 10.8°C at 70°C for PG2, and 15.8

to 16.1 min and 7.1 to 7.7 at 90°C for PG1, respectively. Thermal resistance of PME and

PG also vary with pH of the media or homogenate. Smart control of these factors, like in

cold break and hot break processes of tomato homogenate, is done to obtain products of

desired quality and characteristics. The hot break process involves quickly heating tomato

homogenate to 95°C to inactivate endogenous enzymes like PME and PG, thereby

obtaining viscous end products like ketchup and pastes (Anthon and Barrett, 2003). The

cold break process includes heating of the homogenate to 60°C to keep PME and PG

active, which subsequently reduce the viscosity; a desired effect in soups and juices. Cold

break is also known to produce better flavor and color due to lesser thermal abusive and

possible activity of aroma-enhancing reactions of the surviving enzymes (Anthon and

Barrett, 2003).

Ohmic heating has been shown to have additional non-thermal effects of electricity on

biological materials such as vegetable tissues (Kulshrestha and Sastry, 1999, 2003; Imai

et al., 1995; Schreier et al., 1993), vegetative microbial cells (Guillou and Murr, 2002;

Lee and Yoon, 1999) and microbial spores (Cho et al., 2000; Somavat, 2010). This has

been hypothesized as being due to the presence of intracellular metallic and ionic

materials within spores and build-up of charges at cellular locations within vegetative

cells (Somavat, 2010). Ohmic heating has also shown to have interesting effects on

enzymes. Castro et al. (2004) showed that ohmic heating significantly reduced the D-

values of lipoxygenase and polyphenoloxidase as compared to conventional heating,

whereas pectinase, alkaline phosphatase and β-galactosidase remained unaffected. Icier et

78
al. (2006) concluded that the ohmic blanching at 20 V/cm deactivated peroxidase of pee

puree in lower processing time than conventional heating, and also resulted in better color

values. Ohmic blanching at 50 V/cm gave the best color and shortest critical inactivation

time. Icier et al. (2009) further reported that the critical deactivation temperature of

polyphenoloxidase in grape juice was reduced from 70°C at 20 and 30 V/cm treatments

to 60°C at 40 V/cm. Yildiz and Baysal (2006) studied the effect of alternating current on

PME in homogenized tomato juice during the come-up time, using constant input

voltages of 36, 48, 68 and 108 V/cm for pre-defined times. PME activity for 36 V/cm

treatment showed an increase for the first 30 s of processing and until the temperature

reached 55°C. A field strength of 48 V/cm gave the same result after 5 s, when the

treatment temperature was 30°C. These interesting observations suggest the need for

further investigation of the effects of electricity on enzymes to increase the understanding

of ohmic heating process.

The objective of our study was to investigate the effect of ohmic heating on the

inactivation kinetics of pectin methylesterase (PME) and polygalacturonase (PG) and

color in fresh tomato juice at the pH values of 3.9 and 4.4.

4.3 Materials and Methods

Fresh tomato juice samples, pH adjusted to either 3.9 or 4.4, were treated in a glass T-

type ohmic heater using a frequency of 60 Hz in a pulse mode. Treated samples were

quickly transferred into a deep freezer at -20°C, and were stored and transported under -

80°C conditions until analyzed for enzymes and color. Samples were held for five

79
different holding times at 72, 78, 83, 88 and 90°C at pH 3.9 and 90, 95, 100, 105 and

110°C for studying PME inactivation at pH 4.4. PG inactivation was studied at 72, 78, 83

and 90°C for pH 3.9 and 88, 90, 95 and 100°C for pH 4.4. Color changes were

investigated at temperatures of 90, 95, 100 and 105°C and 95, 100 and 105°C for pH 3.9

and 4.4, respectively. Three or more replicates were done at each condition.

Rationale: The above test conditions were developed based in part on experience, and

repeated preliminary experimentation. At pH 4.4, tomatoes need to be processed to

inactivate Bacillus coagulans spores. Our previous experiments on Bacillus coagulans

inactivation kinetics (Somavat, 2010) suggested that temperatures in the range of 100°C

would be necessary. At pH 3.9, processing is primarily for control of yeast and mold;

thus the lower temperature range from 72 to 90°C.

We noticed early on that samples at the higher temperature range showed no PME

activity, hence the lower temperature range (72-90°C) was investigated at pH 4.4 also.

4.3.1 Tomato juice preparation

Fresh Roma tomatoes of bright red color (minimum A-value of 20) were bought from a

local grocery store (Kroger Inc.). Tomatoes were sliced into quarters and blended for 20 s

in an Osterizer blender. The pH of the unfiltered juice was adjusted from the inherent

value of 4.2 to 4.3 to either 3.9 or 4.4 using citric acid or sodium citrate. The juice was

then transferred to the ohmic heater for processing.

Treated samples were quickly transferred to a -20°C freezer after the treatment and were

stored at -80°C until analyzed. The samples were blended, processed and transferred to

80
the deep freezer within ten minutes of slicing, unless otherwise mandated by the

prolonged holding time of a given treatment condition. Fresh juice was prepared for

subsequent experimental runs, using a minimum of four tomatoes to reduce inherent

tomato to tomato variation. Three replicates were done for each time, temperature and pH

combination.

Rationale and Precautions: The above procedures were arrived at after much preliminary

experimentation and refinement. In our early experiments, we completed sample

preparation and processing so that samples were transferred to the freezer within 20

minutes. However, we became concerned about possible gelation of samples, and our

initial results showed significant variability between samples. To minimize sample-to-

sample variation, we blended four tomatoes at a time, and processed them. Any sample

remaining ten minutes after initial preparation was discarded, and a new set of samples,

blending from four tomatoes of the same batch were used.

4.3.2 System design

Samples were processed in two similar ohmic heaters of different capacities made up of a

glass tee and platinized-titanium electrodes. The basic design of the ohmic heaters is

shown in the figure 4.1. The smaller ohmic heater had flat electrodes of 0.012 m diameter

positioned at a distance of 0.012 m from each other (capacity of 6 mL), whereas the

larger ohmic heater had electrodes of 0.025 m diameter placed at a similar distance

(capacity of 25 mL). Process temperature was monitored through a T-type thermocouple

(Omega Engineering Inc., Stamford, CT, USA) inserted in the geometric center of the

81
treatment chamber. A provision to apply pneumatic pressure was provided to enable

heating above 100°C without boiling. Design rationale: The size of the heater was

dictated by the minimum sample size required for analysis of enzymes and color,

although samples could have been processed in a far smaller heater, providing careful

control over product temperature profile, and ensured a short come-up time (CUT).

An input voltage of 65 V (53 V/cm across the sample) was used during the come-up time.

Voltage was subsequently reduced and manually adjusted during the holding phase to

maintain a constant temperature. A square pulsed waveform of duty ratio 0.5 to reduce

electrochemical reactions was used (Samaranayake et al., 2005) (Figure 2). Figure 3

represents typical time, temperature, and voltage plots for a 90°C process both for the

high temperature studies (90-110°C) and the low-temperature treatments (72-90°C).

Differences in come-up rate are due to differences in the field strength used in each case:

we increased field strength for our later studies when the earlier, high temperature studies

showed little or no residual enzyme activity

82
 Air
Pressure

Thermocoupl Treatment
Chamber
e

Electrode Electrode

Figure 4.1 A glass T-type ohmic heater

Pulsed Ohmic Waveform

70

50

30
Voltage, V

10

-10
0.000

0.029
0.004

0.008

0.013

0.017

0.021

0.025

0.033

-30

-50

-70
Time, s

Figure 4.2 Typical square waveform with a pulse duty of 0.5 and frequency of 60 Hz.

83
 (a)
100
90 Voltage

Temperature, C; Voltage, V
80 Temp
70
60
50
40
30
20
10
0
0 50 100 150 200 250
Time, s

(b)
100
90 Temp
Temperature, C; Voltage, V

80 Voltage
70
60
50
40
30
20
10
0
0 10 20 30 40 50 60 70
Time, s

Figure 4.3 Time-temperature plot showing the come up time and voltage for a 90°C

process (a) plot for high temperature treatments from 90 to 110°C (b) plot for low

temperature treatments from 72 to 90°C.

84
4.3.3 Enzyme and color analysis

PME activity was analyzed titrimetrically using procedure mentioned by Anthon et al.

(2002) and Lee and MacMillon (1968). The homogenate (1.0 mL) was added to a 30 mL

aliquot of a solution of 1.0% citrus pectin and 0.2 M NaCl equilibrated to 30°C at pH 7.0.

The pH was maintained at 7.0 for 10 min by adding either 0.05 or 0.005 N NaOH

depending on the activity exhibited by the sample. The rate was calculated in terms of

µmol of NaOH consumed. A homogenate sample boiled for 20 min was used as a blank.

PME activity was listed as percentages of the activities obtained for unheated controls.

To measure PG activity, the homogenate was heated to remove endogenous reducing

sugars and other interfering material and cleared up before assaying using the method of

Pressey (1986). The homogenate was heated and 5 mL of it was transferred to a 50 mL

centrifuge tube. An equal volume of cold water (5 mL) was added to the tube and pH was

adjusted to 3.0 using 0.1 N HCl. The sample was centrifuged for 15 min at 10000g and

resultant pellet was resuspended in 15 mL of water before homogenization with a

polytron. After a second centrifugation, the pellet was resuspended in 7.5 mL of 0.1 M

sodium phosphate buffer at pH 6.5 and was left on ice for 30 min. The buffer contained

either 1.2 M NaCl for extraction of PG1 or 0.5 M NaCl for extraction of PG2. The

sample was again centrifuged and supernatant was collected for assay.

The high salt extracts of the heated homogenate was assayed for the activity of PG1 and

PG2 by measuring the production of reducing sugars from polygalacturnonic acid. A 1%

stock solution of poylgalacturonic acid was prepared in 50 mM citrate buffer and was

adjusted to pH 4.5. Incubations were made with 0.1 M NaCl and 0.25%

85
polygalacturonaic acid and were kept at 37°C for 30 min after addition of 0.2 mL of the

high salt extracts. A boiled and incubated aliquot of the salt-extracted enzyme was used

as a blank. Reactions were terminated using 5.0 mL carbonate buffer and an aliquot of

terminated reaction was colorimetrically analyzed for reducing sugars. Enzyme activity

was calculated based on the difference in absorbance at 560 nm of a boiled enzyme blank

and a standard curve of galacturonic acid.

Color was measured in a Lab Scan XE (Model LSXE, Hunter Associates Laboratory,

Inc., Reston, VA) using a ring and disc set (CMR2926, Hunter Associates Laboratory,

Inc., Reston, VA) to accommodate smaller volumes. The instrument was standardized

using Hunter Lab Color Standard #LX16154 (Hunter Associates Laboratory, Inc.,

Reston, VA) and then hitched with a red tomato paste reference tile (BCR 400, Fluka,

Sigma Alderich, St. Louis, MO) with L, a, and b values of 25.6, 33.4, and 14.7,

respectively. Standardizing in XYZ was done with D65 illuminant, and 10° observer. The

samples were read and the instrument was hitched in Hunter scale (L, a, b) with the C

illuminant and 2° observer. The ring was placed in the 2.5 inch glass sample cup and

juice was added to fill the ring (approximately 10 mL). The disc was firmly placed on top

of the ring in order to read the sample (in triplicates). For comparison, color values were

converted to a/b format and standard deviations were calculated. L values were discarded,

because there was very little variation.

86
4.4 Results and Discussion

4.4.1 Pectin Methylesterase (PME)

All samples at pH 4.4 showed no PME activity from 90 to 110°C, indicating that ohmic

processes that inactivate B. coagulans are more than sufficient for PME inactivation. The

studied temperatures of 90, 95, 100, 105 and 100°C, each at a minimum of five holding

times and three replicates, did not exhibit any residual activity. To verify if this was

consistent with existing PME kinetics, we attempted to predict the expected decrease in

enzyme activity during our come-up time for the high temperature studies. Since the rate

of temperature rise was more or less linear, we used the Linearly Increasing Temperature

Analysis approach of Somavat (2010; Chapter 3); which was a modified version of the

analysis approach used by Rhim et al. (1989). Kinetic data for PME inactivation were

taken from Anthon et al. (2002), which were conducted at the same pH (4.4) and by the

same laboratory that conducted our enzyme analysis. The results predict that this heating

regime would result in negligible residual enzyme activity, as observed. We should note

the limitations of such an analysis: the heating from 20 to 90°C passes through the

temperature zone of enhanced PME activity, thus the simple inactivation model would

only represent the higher, inactivation temperature ranges. Nevertheless, the lack of

enzyme activity in all samples; including those at the CUT would indicate the efficacy of

ohmic treatment in inactivating PME during a B. coagulans targeted process.

At pH 3.9, the inactivation rate was sufficiently slow at the tested temperatures to observe

trends in the plot. However, we noted significant variability in the data, which was not

typical of data in the literature from the same laboratory (Anthon et al., 2002). We

87
revised our procedures to ensure that sample-to-sample variations were minimized (four

tomatoes were blended together) and all samples were processed and frozen within 10

minutes of blending. Despite these precautions, we found the variability to persist. Data

are presented in Figure 4.4, and show little or no inactivation at 72 and 78°C; some

inactivation at 83°C, followed by a leveling-off; and a more rapid initial inactivation at

88oC followed by a leveling to a lower, apparently constant value. While the data

variability suggests prudence in data interpretation, D-values of PME at pH 3.9 were

15.1, 1.05 and 0.75 min for 72, 83 and 88°C. Rate constant (k) and activation energy (Ea)

were also calculated using the method mentioned by Anthon et al. (2002), and were

found to be 0.0025, 0.0366 and 0.0514 s-1, and 493, 440 and 418 KJ/ mole at 72, 83 and

88°C, respectively (Table 4.1).

We hypothesize that the persistent variability in data are due to variability in responses of

different isozymes to the alternating electric field. Since enzymes possess net charge and

a dipole moment, they would be expected to align in response to an electric field.

Different isozymes would be expected to respond differently in their oscillatory behavior,

and may be either activated or inactivated. While there is no specific confirmatory

evidence in this study, we note a number of other studies that have observed the effect of

alternating electric fields on biochemical reactions. Loghavi et al. (2008) have shown that

the course of biochemical reactions may be altered by the presence of high frequency

harmonics superimposed on an alternating waveform. This may be attributed to

oscillation of molecular dipoles in response to external fields. Recent work in our

laboratory (Moon, 2009, unpublished) which shows that lipoxygenase activity may be

88
altered by the frequency of MEF treatment. De La Torre (2009) has hinted that similar

unexplained behavior of enzymes under external electrical field can be because dipole

moment, molecular aggregation, chemical bonding, molecular vibration, state of

oxidization, presence of isozymes and their synergistic actions may contribute to high

variability in activation and inactivation.

Ohmic PME Inactivation at pH 3.9

0.4

0.2
Residual Activity, Log (A/Ao)

0.0

-0.2

-0.4 72C
78C
-0.6
83C
-0.8 88C

-1.0
-10 0 10 20 30 40 50
Time, s

Figure 4.4 Residual PME activities in tomato juice at pH 3.9 at 72, 78, 83, and 88°C.

Ea, KJ/
-1
T, °C D, min k, s mole
72 15.15 0.0025 493
83 1.05 0.0366 440
88 0.75 0.0514 418
Table 4.1 D-value, rate constant (k), and activation energy (Ea) for PME at pH 3.9 at 72,

83 and 88°C.

89
4.4.2 Polygalacturnase (PG) At pH 4.4, some residual PG activity was found (Figure

4.5), which enabled us to determine inactivation kinetics parameters for temperatures

from 88 through 100°C. In general, inactivation over this temperature range was found to

be highly effective, suggesting that a B. coagulans targeting process would also inactivate

PG. We also attempted a comparison between our data and those of Anthon et al (2002),

which were also conducted at pH 4.4, by the same group of researchers that conducted

this analysis. For this comparison, we chose the more heat resistant PG1 isozyme, since

at our test temperatures, we would expect that the heat labile PG2 isozyme would have

been entirely inactivated. Our comparison (Table 4.2) suggest that inactivation under

ohmic heating (D90 value of 5.08 min) is enhanced in comparison to conventional heating

(D90 value of 15.8 min); although it must be noted that the samples compared were not

the same as those in our studies. We further note that such effects have been observed

previously by Castro et al. (2004) who found that the inactivation rates of certain

enzymes, particularly those with metallic prosthetic groups were accelerated by ohmic

heating.

At pH 3.9, the PG inactivation data showed considerably lower variability than PME.

This may be due to the presence of only two isozymes in the case of PG (PG1 and PG2)

as compared to four isozymes for PME (Plaza et al., 2007). While temperatures in the

range from 72 to 83°C resulted in enzyme inactivation, treatment at 90°C was found to

result in some activation after an initial, rapid inactivation. This phenomenon appears

clearly to be related to electric field influences and merits further study. PG inactivation

at pH 3.9 were significantly different at 72, 83 and 90°C with D-values of 9.26, 1.10 and

90
0.21 min, respectively. D-value for the 90°C treatment was calculated only using the

initial inactivation and the later activation phase was excluded from the calculation. Table

4.3 summarizes D, k and Ea values for PG inactivation at pH 3.9.

Ohmic PG Inactivation at pH 4.4

0.0
88C
-0.3
Residual Activity, Log (A/Ao)

90C
-0.6
95C
-0.9 100C
-1.2

-1.5

-1.8

-2.1
-5 0 5 10 15 20 25
Time, min

Figure 4.5 Residual PG activities in tomato juice at pH 4.4 at 88, 90, 95 and 100°C.

D90, min Z, °C k, s-1 Ea, KJ/ mole

Anthon et al., 2002 16.1 7.7 0.0024 324
Current Ohmic Study 5.08 5.14 0.0076 125

Table 4.2 Comparison of D, Z, k and Ea values for PG inactivation at pH 4.4 and 90°C

under ohmic heating and conventional heating (Anthon et al., 2002).

91
 Ohmic PG Inactivation at pH 3.9

0.3

Residual Activity, Log (A/ Ao) 0.0

-0.3

-0.6

-0.9 72C

78C
-1.2
83C
-1.5
90C
-1.8
-10 0 10 20 30 40 50
Time, s
Figure 4.6 Residual PG activities in tomato juice at pH 3.9 at 72, 78, 83, and 90°C.

T, °C D, min k, s-1 Ea, KJ/ mole

72 9.26 0.0041 389
78 4.07 0.0094 366
pH 3.9
83 1.10 0.0348 348
90 0.21 0.1789 322
Table 4.3 D-value, rate constant (k) and activation energy (Ea) for PG at pH 3.9.

4.4.3 Color

No significant color difference was observed between the various time, pH and

temperature combinations in terms of individual L, a and b values. This effect was also

noticed for a/b quantity at all temperatures. The values of a/b for temperatures of 90, 95,

100 and 105°C varied between 2.19 and 1.83 until first 120 s of heating. These results

show the ability of ohmic heating to process tomato juice without affecting color.
92
 Ohmic 95C, pH 4.4 Ohmic 100C, pH 4.4
40 40
L L
20 a 20 a
b b
0 0
1 2 3 4 5 6 Raw 0 12 24 36 48
Time, min Time, s
Ohmic 105C, pH 4.4
40
L
20 a
b

0
Raw 0 45 90 135 180
Time, s
Ohmic 90C, pH 3.9 Ohmic 95C, pH 3.9
40 40
L L
20 a 20 a
b b
0 0
Raw 0 45 90 135 180 Raw 0 20 40 60 80
Time, s Time, s
Ohmic 100C, pH 3.9 Ohmic 105C, pH 3.9
40 40

L L
20 a 20 a
b b
0 0
Raw 0 12 24 36 48 1 2 3 4 5 6
Time, s Time, s

Figure 4.7 Color changes in the tomato juice by ohmic heating at 95, 100 and 105°C for

pH 4.4, and 90, 95, 100 and 105°C for pH 3.9.

93
 Ohmic heating at pH 3.9
3

2.5

2
a/b
1.5
90°C

1 95°C
100°C
0.5
105°C
0
0 50 100 150 200 250
Time, s

Figure 4.8 a/b values for the color change for ohmic heating at pH 3.9.

4.5 Conclusion

Ohmic heating of tomato juice at pH 4.4 resulted in little or no residual activity of PME

at temperatures associated with inactivation of Bacillus coagulans; indicating the

effectiveness of such process conditions. Inactivation of PG at pH 4.4 appears to be more

rapid under ohmic heating than in the literature on conventional heating, although further

studies on the same sample will be necessary. Processing at lower temperatures and pH

3.9 show interesting effects for both enzymes. Despite our best efforts at standardizing

experimental protocol, high variability was observed in the PME data, suggesting that

different isozymes respond differently to alternating electric fields. PG data were less

variable than PME data, possibly due to the presence of only two isozymes; still there

appears to be some activation effect at 9°0C. Color of all samples remained unaffected

by ohmic treatment.
94
4.6 References

Anese M., & Sovrano S. (2006). Kinetics of thermal inactivation of tomato lipoxygenase.

Food Chemistry, 95(1), 131-137.

Anthon G.E., & Barrett D.M. (2003a). Thermal inactivation of lipoxygenase and

hydroperoxytrienoic acid lyase in tomatoes. Food Chemistry, 81(2), 275-279.

Anthon G.E., & Barrett D.M. (2003b). Thermal inactivation of lipoxygenase and

hydroperoxytrienoic acid lyase in tomatoes. Food Chemistry, 81(2), 275-279.

Anthon G.E., Sekine Y., Watanabe N., & Barrett D.M. (2002). Thermal Inactivation of

Pectin Methylesterase, Polygalacturonase, and Peroxidase in Tomato Juice. Journal of

Agricultural and Food Chemistry, 50(21), 6153-6159.

Castro I., Macedo B., Teixeira J.A., & Vicente A.A. (2004). The Effect of Electric Field

on Important Food-processing Enzymes: Comparison of Inactivation Kinetics under

Conventional and Ohmic Heating. Journal of Food Science, 69(9), C696-C701.

De La Torre, J. J. M. (2009) Effect of frequency on polyphenoloxidase activity during

moderate electric field treatment. MS Thesis. The Ohio State University.

95
De Sio F., Dipollina G., Villari G., Loiudice R., Laratta B., & Castaldo D. (1995).

Thermal resistance of pectin methylesterase in tomato juice. Food Chemistry, 52(2), 135-

138.

Icier F., & Ilicali C. (2005). Temperature dependent electrical conductivities of fruit

purees during ohmic heating. Food Research International, 38(10), 1135-1142.

Icier F., Yildiz H., & Baysal T. (2006). Peroxidase inactivation and colour changes

during ohmic blanching of pea puree. Journal of Food Engineering, 74(3), 424-429.

Icier F., Yildiz H., & Baysal T. (2008). Polyphenoloxidase deactivation kinetics during

ohmic heating of grape juice. Journal of Food Engineering, 85(3), 410-417.

Laratta B., Loiudice R., Giovane A., Quagliuolo L., Servillo L., & Castaldo D. (1995).

Thermostability of three pectinesterase isoenzymes in tomato fruit. Food Chemistry,

52(4), 415-418.

Lee M., & MacMillon, J.D. (1968). Mode of action of pectin enzymes. I. Purification and

properties of tomato pectinesterase. Biochemistry, 7, 4005-4010.

96
Loghavi, L., Sastry S.K., & Yousef, A.E. (2008). Effect of Moderate Electric Field

frequency on growth kinetics and metabolic activity of Lactobacillus acidophilus.

Biotechnology Progress, 24(1),148-153.

Mallidis C.G., Frantzeskakis P., Balatsouras G., & Katsaboxakis C. (1990). Thermal

treatment of aseptically processed tomato paste. International Journal of Food Science

and Technology, 25, 442-448.

Moon (2009). Effect of frequency on lipoxygenase during Moderate Electric Field

treatment. (Unpublished internal report). The Ohio State University, Department of

Food, Agricultural and Biological Engineering.

Plaza, L., Duvetter, T., Silvia Monfort, S. Clynen, E., Liliane Schoofs, L., Van Loey,

A.M., & Marc E. Hendrickx, M.E. (2007). Purification and Thermal and High-Pressure

Inactivation of Pectinmethylesterase Isoenzymes from Tomatoes (Lycopersicon

esculentum): A Novel Pressure Labile Isoenzyme. Journal of Agricultural and Food

Chemistry, 55 (22), 9259–9265

Pressey, R. (1986). Extraction and assay of tomato polygalacturonases. HortScience, 21,

490-492.

97
Rhim J.W., Nunes R.V., Jones V.A., & Swartzel K.R. (1989). Determination of kinetic

parameters using linearly increasing temperature. Journal of Food Science, 54 (2), 446-

450.

Sastry S.K., & Barach J.T. (2000). Ohmic and Inductive Heating. Journal of Food Safety,

65, 42-46.

Somavat, R. (2010). Applications and effects of ohmic heating: sterilization, influence on

bacterial spores, enzymes, bioactive components and quality factors in food. PhD

Dissertation. The Ohio State University. Columbus, OH.

Yıldız H., & Baysal T. (2006). Effects of alternative current heating treatment on

Aspergillus niger, pectin methylesterase and pectin content in tomato. Journal of Food

Engineering, 75(3), 327-332.

98
 5. Effect of ohmic heating on carotenoids, phenolic compounds and

ascorbic acid in tomato juice

5.1 Abstract

According to the US Food and Drug Administration (FDA), tomato products in cooked,

raw, dry or canned forms qualify for health claims related to prostate, ovarian, gastric and

pancreatic cancers. These special health benefits are attributed to the presence of

carotenoids and phenolic compounds in the tomato fruit. Ohmic heating is suitable for

processing of tomato based products like purees, ketchup, pastes, sauces and juices, due

to its applicability for continuous processing of viscous and particulate containing foods.

The objective of current study was to investigate the effect of ohmic heating on

carotenoids (β-carotene and lycopene), phenolic compounds (phenolic acids, naringenin,

quercetin and total flavonoids) and ascorbic acid in fresh tomato juice. Results showed

that ohmic heating did not affect the amount of carotenoids and phenolic compounds in

tomato juice. β-carotene, lycopene, phenolic acids and quercetin showed no or minimal

reductions at temperatures of 95, 100, 105 and 110°C at pH 4.4. Lycopene stability was

confirmed at pH 3.9 for 90, 95, 100 and 105°C. Naringenin content showed an interesting

behavior with chalconaringenin converting to naringenin in greater proportions at the

99
temperatures of 95 and 100°C, and remaining unchanged at 105°C. Ohmic treatment at

105°C was found to increase total naringenin equivalents when compared to 0-time

control. Naringenin has shown to reduce oxidative damage to DNA in vitro, to assist in

preventing obesity and to reduce hepatitis C virus. Ohmic heating was also found to

retain the original amounts of ascorbic acid and dehydroxyascorbic acid at 90°C at pH

4.4 and at 90 and 100° at pH 3.9. The rate constants for ascorbic acid degradation under

ohmic heating were found to be lower than those reported in the literature for

conventional heating studies.

5.2 Introduction

In recent years, food has increasingly been viewed as a route to optimal wellness and not

just a vehicle for essential nutrients ensuring proper growth and development. This

paradigm shift has occurred mainly because of increased consumer interest in controlling

their own health, increasing healthcare costs, and confirmation from scientific studies

linking specific diet to chronic disease risk reduction (ADA, 2009). Tomato is one such

food which has been linked with a reduced risk of developing certain types of cancers,

age related macular degeneration and cardiovascular diseases (FDA, 2005; Hwang and

Bowen, 2002; Giovannucci et al., 2002). It is established that these special health benefits

are associated with the presence of important phytonutrients like carotenoids and

phenolic compounds in tomatoes (Beecher, 1998).

100
Development of alternative processing technologies is another field currently attracting

the attention of the food industry. Ohmic heating has generated considerable interest due

to its suitability for continuous processing of pumpable viscous liquid and particulate

containing liquid foods. Other associated advantages include uniform heating, faster

heating rate, and fresher tasting end products than conventional heating (Pereira and

Vicente, 2009; Somavat, 2010). Several industrial scale ohmic treatment plants have

recently been installed in Europe for processing of tomato based products like purees,

dices, soups, juices, ketchup and other sauces, and peeling applications (Pereira and

Vicente, 2009). Ohmic heating has also been shown to have additional non-thermal effect

of electricity on certain bioactive molecules and organisms such as enzymes, plant cells,

bacteria, yeast and molds (Kulshrestha and Sastry, 2003; Icier et al., 2006; Schreier et al.,

1993; Guillou and Murr, 2002; Somavat, 2010). It is therefore of interest to study the

effect of ohmic heating on the components in tomatoes with special health benefits.

Carotenoids are a group of polyunsaturated 40-carbon tetraterpene hydrocarbons present

in the form of red, yellow and orange plant pigments. They possess anticarcinogenic

activity mainly attributed to their intrinsic antioxidant properties and ability as precursors

of vitamin A (Rao, 1999). Antioxidant property of carotenoids reduces the potential stress

caused by reactive oxygen species which are well known causative agents of a number of

degenerative diseases (Sanchez-Moreno et al., 2005). Lycopene and β carotene are the

main carotenoids present in a tomato fruit. Lycopene is one of the most potent

antioxidants among all dietary carotenoids and has been suggested to reduce risk of

chronic diseases such as cancer and cardiovascular disease. Studies have shown its

101
association in decreasing the risk of digestive tract, breast and prostate cancers and

coronary artery disease (Kristenson et al., 1996; Rao, 1999). Lycopene is an acyclic

carotene with 11 conjugated double bonds and is normally present in plants in an all-

trans configuration giving its molecule a long and linear shape (Bramley, 2000; Frohlich

et al., 2006). However these double bonds are subject to isomerisation during processing

steps like heating, blending and chopping resulting in cis isomers. These cis isomers (5,

7, 9, 11, 13, or 15-) have a bend in their structures rather than a straight chain, giving

them a different level of stability depending on the type of isomerisation (Bramley, 2000;

Chasse et al., 2001). Interestingly, human serum and tissue are found to predominantly

contain cis isomers (up to 60%) instead of the naturally dominant all-trans form. It is also

well established that cis isomers are more bio-available than all-trans form. A number of

studies have shown that whereas lycopene is relatively stable to heating and processing, it

is more bioavailable from processed tomato products like ketchup, sauce, puree etc as

compared to a raw fruit or juice (Stahl and Sies, 1992; Furr and Clark, 1997).

Bioavailability further varies with the site and degree of cis isomerization, however the

biological effects of these individual isomers have not yet fully understood (Agarwal and

Rao, 2000).

Dietary β-carotene, a precursor to vitamin A, is essential for normal development, growth

and eyesight (Burri, 1997). In addition to its role in preventing vitamin A deficiency, β-

carotene is also shown to reduce cell mutations and LDL-cholesterol oxidation

(Northrop-Clewes and Thurnham, 2002). Basic structure of β-carotene includes eight

isoprene units cyclised at each end. As a dimer of vitamin A, a β-carotene molecule can

102
be cleaved through the center into two vitamin A units. β-carotene is relatively stable to

heating and processing and shows only minor reductions (George et al., 2011).

Bioavailability of β-carotene is shown to increase through processing of foods (Gartner et

al., 2009; Rock et al., 1998), however, it varies with various other factors as summarized

by Castenmiller and West, 1998. Apart from processing, factors that may affect the

bioavailability of carotenoids include differential absorption of different species of

carotenoids, molecular linkage, concentration of carotenoids in a meal, food matrix,

presence of fats, effectors of absorption and bioconversion, nutrient status of the host,

genetic factors, host-related factors and interactions (Castenmiller and West, 1998;

Yonekura and Nagao, 2007). Owing to their role in human health and the effect of

processing it is interesting to study the effect of electricity during ohmic heating on

lycopene and β-carotene. Yildiz et al. (2008) studied the effect of ohmic heating on β-

carotene content in spinach puree and found that ohmic heating at 60, 70 and 80°C for 10

min resulted in an increased β-carotene content than conventionally heated samples for

the same time-temperature combinations, and the raw sample. According to this author‟s

knowledge, effect of ohmic heating on lycopene content has not been investigated before.

The predominant forms of phenolic compounds present in tomatoes include flavonoids

and phenolic acids. According to the USDA flavonoids database, red tomatoes contain on

a year average about 15 mg of flavonoids mainly in the form of naringenin,its isomer

chalconaringenin (45%) and quercetin (39%) (USDA, 2007; Slimestad and Verheul,

2009). Flavonoids are formed of a C15 (C6 – C3 – C6) flavone nucleus containing two

benzene rings linked though an oxygen-containing pyran or pyrone ring (Caballero et al.,

103
2003). Phenolic acids mainly consist of hydroxybenzoic acids and hydroxycinnamic

acids. Hydroxybenzoic acids have a general structure derived directly from benzoic acid

(C6 – C1) with variations caused by hydroxylations and methylations of the aromatic

rings (Macheix et al., 1990). Hydroxycinnamic acid is the hydroxyl derivative of

cinnamic acid with a base phenylacrylic acid structure (Caballero et al., 2003). Phenolic

compounds reduce lipid peroxidation by scavenging peroxyl radicals. Lipid peroxidation

occurs by oxidation of fatty acids in the presence of certain enzymes and reactive oxygen

species, to aids in free radical chain reaction by the transition of metal ions. This

antioxidant property of the phenolic compounds can be attributed to their capacity to

reduce and chelate ferric iron which catalyses lipid peroxidation (Martinez-Valverde et

al., 2002).

There are contrasting studies showing that phenolic compounds decrease (George et al.,

2011; Sahlin et al., 2004; Re et al., 2002), remain stable (Dewanto et al., 2002) and even

increase (Gahler et al., 2003) during processing. However, most of them suggest that

phenolic compounds are relatively less stable to heating and processing than carotenoids,

and show reduction in significant amounts. Ohmic blanching of artichokes at 40 V/cm

and 85 °C has shown to retain total phenolic content in a greater quantity than a similar

conventional thermal treatment (Icier, 2010).

Tomato is also a good source of Vitamin C. Vitamin C or ascorbic acid is a weak acid

structurally related to glucose with an attached a hydrogen ion, which can also occur in a

mineral ascorbate form. It is an essential nutrient for humans; is required for important

metabolic reactions, provides protection against oxidative stress, supports the immune
104
system and is a natural antihistamine. Ascorbic acid can be lost in significant amounts

during processing of tomatoes, as well as during during storage due to oxidation and

leaching into cooking water (Sahlin et al. 2004). Some studies have compared ascorbic

acid under the effect of ohmic heating with that of conventional heating and concluded

that presence of an electric field does not affect ascorbic acid degradation (Marybeth et

al., 1999; Assiry et al., 2003; Castro et al., 2004; Assiry et al., 2006).

The main objectives of this study were to investigate the effect of ohmic heating of

tomato juice on its components with special health benefits, specificallycarotenoids

(lycopene and β-carotene), phenolic compounds like phenolic acids and flavonoids

(quercetin glycosides, chalconaringenin and naringenin) and ascorbic acid.

5.3 Materials and Methods

Fresh tomato juice was heated inside a glass T-type ohmic heating using a 60 Hz input in

a pulse mode and square waveform. The study was conducted at pH values adjusted to

either 3.9 or 4.4. Treated samples were kept at -80°C until analyzed for carotenoids,

phenolic compounds and ascorbic acid. A total of five holding times (three replicates)

were investigated each at pH 4.4 for 95, 100, 105 and 110°C. Lycopene and ascorbic acid

were also studied at pH 3.9 for the temperatures of 90, 95, 100, 105 and 110°C and 90

and 100°C, respectively.

105
5.3.1 Tomato juice preparation

Fresh Roma tomatoes of bright red color (A-value ≥ 20) were brought from a local

grocery store (Kroger Inc.). Identical cultivar, color and source were selected in an

attempt to minimize the variability in carotenoids, phenolics and ascorbic acid contents

associated with variety, age, color, growing conditions (Salunkhe, 1974). Tomatoes were

sliced into quarters and blended for 20 s in an Ozterizer brand blender. pH of the

unfiltered juice was adjusted to either 3.9 or 4.4 using citric acid or sodium citrate

(inherent values were 4.2 ±1). The juice was then transferred to the ohmic heater for

processing. After treatment samples were quickly transferred to a -20°C freezer and were

moved to -80°C for storage until their analysis. Fresh juice was prepared for each

experimental run using a minimum of four tomatoes to reduce inherent tomato to tomato

variation. Three replicates were conducted for each time, temperature and pH

combination.

5.3.2 System design

Samples were processed in a T-type glass ohmic heater. Platinized-titanium electrodes

with a flat face of 0.025m diameter were positioned at a distance of 0.025m from each

other to form the ohmic treatment chamber (Figure 5.1). Temperature was monitored

through a T-type thermocouple (Omega Engineering Inc., Stamford, CT, USA) inserted

in the geometric center of the treatment chamber. Pneumatic pressure was applied for

treatments at and above 100°C.

106
An input voltage of 75 V (30 V/cm across the sample) was used during the come-up time.

It was reduced and manually adjusted during the holding phase to maintain a constant

temperature. Electrochemical reactions were controlled by the use of a square pulsed

waveform of duty ratio 0.5 shown in Figure 5.2 (Samaranayake et al., 2005). Come up

times from room temperature to 90 and 105°C were 155 and 200 s, respectively. Figure

5.3 represents typical time, temperature, and voltage plots for a 90°C process.

Pressurized
Air

Thermocouple Treatment
Chamber

Electrode Electrode

Figure 5.1 A T-type glass ohmic heater

107
 Pulsed Ohmic Waveform
80

60

40

20
Voltage, V

0.033
0.000

0.004

0.008

0.013

0.017

0.021

0.025

0.029
-20

-40

-60

-80
Time, s

Figure 5.2 Typical square waveform with a pulse duty of 0.5 and frequency of 60 Hz.

Voltage and Temperature

100
90
Temperature, C; Voltage, V

80
Voltage
70
Temp
60
50
40
30
20
10
0
0 50 100 150 200
Time, s

Figure 5.3 Time-temperature plot showing the come up time and voltage for the 90°C

process.
108
5.3.3 Sample preparation and analysis

5.3.3.1 Phenolic acids

Predominant forms of phenolic compounds in tomatoes include phenolic acids, quercetin

glycosides, chalconaringenin and its flavanone form, naringenin. Standards for their

quantitation included chlorogenic acid for the phenolic acids, rutin (quercetin rutinoside)

for the quercetin glycosides and naringenin. Chalconaringenin response was estimated

after adjusting for extinction coefficient differences. The extinction coefficients for

chlorogenic acid, rutin and naringenin were calculated as 19200.0, 20000.0 and 15500.0

cm-1 M-1, respectively.

Tomato juice (-80°C) was thawed at room temperature for approx 60 min. A 3 g sample

of juice was transferred into a 15 mL polypropylene Falcon tube followed by addition of

7 mL methanol and sonication for 20 s with a Brason bath sonicator. The solution was

centrifuged for 10 min at 2400 rpm in a centrifuge (Damon/ IEC model HN-SII) and

supernatant was transferred into a 11 mL glass vial. Final supernatant was collected after

a second centrifugation for 2 min at 10,000 rpm (Microcentrifuge, Corning-costar).

HPLC analysis was done using a symmetrical 4.6x75 mm, 3.5μm C18 column (Waters

Alliance 2695 with 2996 photodiode array (PDA) detector). Mobile phase were a)

0.1%(v/v) formic acid in water and (B) 0.1%(v/v) formic acid in acetonitrile. Column

temperature was set at 30°C and sample temp at 15°C. Flow rate, injection and volume

were 1.0 mL/min and 20 µL. Mobile phase gradients are summarized in Table 5.1. Data

acquisition was done at detection wavelengths of- 320 nm, 354 nm, 290 nm and 365 nm

for phenolic acids, quercetin glycosides, naringenin, and chalconaringein, respectively.

109
 Time,
min %A %B curve
0 95 5 6
12 63 37 6
13 5 95 6
16 95 5 1

Table 5.1 Mobile phase gradients used in HPLC analysis

5.3.3.2 Carotenoids

To extract carotenoids significant modifications were made to the method published by

Ferruzzi et al. (1998). Carotenoids analysis was done following the HPLC method of

Barona et al. (2010). Details of extraction and analysis methods are not presented here, as

they will be published in separate research articles.

5.3.3.3 Ascorbic acid

Ascorbic acid was determined using a spectrophotometric method based on that of

Tsumura et al. (1993). Approximately 1 mL of the homogenized sample was centrifuged

for 8 min at 16.1 rcf. The supernatant was transferred to another centrifuge tube.

Ascorbic acid concentration was determined by reading the absorbance at 265 nm of a 3

mL cuvetter containing 0.1 M sodium phosphate pH 6.5 buffer, sample, and horseradish

peroxidase (Sigma Type II, St. Louis, MO). It was followed by additional of a 150 mM

110
hydrogen peroxide solution to activate the horseradish peroxidase and oxidize all of the

ascorbic acid. After ten seconds OD 265 was read again, and ascorbic acid concentration

was calculated from the difference in absorbance. To determine dehydroascorbic acid

concentration, phosphate buffer and sample were mixed and the absorbance was read at

265 nm. Next a 30 mg/mL dithiothreitol solution was added, and the sample incubated

for 20 minutes at room temperature to allow all the dehydroascorbic acid to be reduced.

Absorbance was read again at 265nm and dehydroascorbic acid concentration was

calculated from the difference in absorbance. Duplicates were made for each sample.

Ascorbic acid degradation kinetics were calculated from the slopes of the degradation

time courses:

-1/DT = dLog(C/C0) /dt (1)

Where, C0 is ascorbic acid concentration (mg/ 100g) of the raw tomato juice, and C is the

concentration after ohmically processing it for time t.

C0 = (∆A265*3*176.1)/ (14.4*V) (2)

Where, molecular weight of ascorbic acid is 176.1, extinction coefficient at 265 nm for

ascorbic acid is 14.4 mM-1, final volume is 3 mL, V is the volume of the sample, and

∆A265 is the change in absorbance at 265 nm.

Rate constant (k) and activation energy (Ea) calculations were done using the relations:

111
5.4 Results and Discussion

5.4.1 Carotenoids (Lycopene and β-carotene)

β-carotene did not show any major change at temperatures of 95, 100, 105 and 110°C for

holding times of 24, 10, 3 and 0.8 min at pH 4.4 (Figure 5.4). Its values remained

between 0.24 to 0.35 mg/ 100 g of tomato juice for all temperatures. Although the 110°C

treatment seemed to show some reduction, the effect was negligible as the lowest

recorded value of 0.24 mg/ 100 g was comparable to other treatments. The results of our

study complement the findings of Yildiz et al. (2008), who observed an increase in β-

carotene during ohmic heating at the temperatures of 60, 70 and 80°C when compared to

conventional heating. Literature shows that a majority of the studies on β-carotene show a

minor reduction upon conventional heating (Perez et al., 2009; George et al., 2011), with

a few concluding that it remains unaffected (Sanchez-Moreno et al., 2006).

Similar to β-carotene, lycopene values also remained unaffected by ohmic heating at 95,

100, 105 and 110°C for holding times of 24, 10, 3 and 0.8 min at pH 4.4 (Figure 5.5).

The difference between all-trans lycopene and total lycopene contents remained

unchanged during the complete holding time at each temperature condition. Values of

total and all-trans lycopene stayed between 2.7 to 4.2 mg/ 100g and 2.5 to 4.0 mg/ 100g,

respectively, without showing any particular pattern over the holding periods. Data for

tomato juice at pH 3.9 also showed that amounts of lycopene before and after ohmic

treatments at temperatures of 90, 100 and 105°C for holding times of 3, 0.8 and 0.4 min,

respectively, remained unaffected (Figure 5.6). Only 95°C treatment showed a minor

112
reduction of lycopene from 3.6 mg/ 100 g in untreated control to 2.5 mg/ 100 g. These

results are similar to the effect of conventional heating on lycopene content observed by

others (Perez et al., 2009; George et al., 2011). These results are not surprising, since

lycopene is known to be heat stable, and is a nonpolar molecule, which would not be

affected by an oscillating electric field.

β-carotene at pH 4.4
0.40
0.35
β-carotene, mg/ 100g

0.30
0.25
0.20
0.15
0.10 95C
0.05 100C
0.00
-8 0 8 16 24 32
Time, min

β-carotene at pH 4.4
0.40
0.35
β-carotene, mg/ 100g

0.30
0.25
0.20
0.15
0.10 105
C
0.05
0.00
-50 0 50 100 150 200
Time, s

Figure 5.4 β-carotene content in tomato juice at pH 4.4 after ohmic treatment at 95, 100,

105 and 110°C.

113
 Lycopene at pH 4.4
4.0
Lycopene, mg/ 100g 3.5
3.0
2.5
2.0 95C, Total
1.5 95C, All-trans
1.0 100C, Total
0.5 100C, All-trans

0.0
-8 0 8 16 24 32
Time, min

Lycopene at pH 4.4
4.5
4.0
Lycopene, mg/ 100g

3.5
3.0
2.5
105C, Total
2.0
105C, All-trans
1.5
110C, Total
1.0
0.5 110C, All-trans

0.0
-50 0 50 100 150 200
Time, s

Figure 5.5 Amounts of total and all-trans lycopene in tomato juice at pH 4.4 after ohmic

treatment at 95, 100, 105 and 110°C.

114
 Lycopene at pH 3.9
5.0
4.5 90C
4.0

Lycopene, mg/ 100g

95C
3.5
100C
3.0
2.5 105C
2.0
1.5
1.0
0.5
0.0
-50 0 50 100 150 200
Time, s

Figure 5.6 Total lycopene content in tomato juice at pH 3.9 after ohmic treatment at 90,

95, 100 and 105°C.

5.4.2 Phenolic Compounds (Phenolic Acids and Flavonoids)

Chlorogenic acid equivalents in tomato juice at pH 4.4 were found to be unaffected after

ohmic treatment at 95, 100, 105 and 110°C (Figure 5.7). The observed values of

chlorogenic acid equivalents, representing phenolic acids, were between 46 and 56 mg/

kg FW showing minimal or no effect of ohmic heating.

Figure 5.8 shows interesting behaviour of chalconaringenin and its flavone form

naringenin under the effect of ohmic heating. Chalconaringenin converted to naringenin

upon heating, resulting in reduction in its value from 0.019 to 0.001 mmol/ kg after 24

min of ohmic heating at 95°C, and from 0.022 to 0.016 mmol/ kg after 10 min at 100°C.
115
Corresponding values of naringenin increased from 0.001 to 0.013 at zero time and 0.003

to 0.013 mmol/ kg at 95 and 100°C after the same holding times. This conversion was

reduced for higher temperature treatments, with chalconaringenin values remaining

constant at 0.021 mmol/ kg at 105°C after 3 min, and showing minor reduction from

0.017 to 0.009 mmol/ kg at 110°C after 0.8 min. Total naringenin equivalents showed

minor reductions from 5.5 to 3.9, 6.6 to 4.4, and 6.3 to 4.8 mg/ kg FW at 95, 100 and

110°C, respectively. However, it showed an increase (statistically not significant) at the

105°C ohmic treatment from 7.3 to 8.8 mg/ kg FW. Naringenin is shown to reduce

oxidative damage, to assist in controlling cholestrol and to reduce hepatitis C virus (Lee

et al., 1999; Mulvihill et al., 2009; Nahmias et al., 2008).

Figure 5.9 shows the effect of ohmic heating on quercetin and total flavonoid content.

Quercetin too, like other phenolic compounds remained unaffected by ohmic heating at

the four time-temperatures combinations. The plot of total flavonoids shows the stability

of flavanoids at these temperatures and shows minor elevation at 105°C reflecting the

increase in the amount of total naringenin.

These results show that ohmic heating resulted in retaining a higher quantity of inherent

phenolic compounds, whereas according to a majority of studies conventional heating

negatively affects the total phenolic content (Re et al., 2002; Perez et al, 2006; George et

al., 2011).

116
 Chlorogenic acid at pH 4.4
75
70
65
mg/ kg FW 60
55
50
45
40 95C
35 100C
30
-10 0 10 20 30
Time, min

Chlorogenic acid at pH 4.4

75
70
65
60
mg/ kg FW

55
50
45
40 105C
35 110C
30
-50 0 50 100 150 200
Time, s

Figure 5.7 Chlorogenic acid equivalents in tomato juice at pH 4.4 after ohmic treatment

at 95, 100, 105 and 110°C.

117
 Naringenin at pH 4.4
0.028
95C, Naringenin
0.024
95C, Chalconaringenin
0.020 100C, Naringenin
100C, Chalconaringenin
mmol/ kg

0.016

0.012

0.008

0.004

0.000
-8 0 8 16 24 32
Time, min

Naringenin at pH 4.4
0.060
105C, Naringenin
0.050
105C, Chalconaringenin
110C, Naringenin
0.040
110C, Chalconaringenin
mmol/ kg

0.030

0.020

0.010

0.000
-50 0 50 100 150 200
Time, s

Figure 5.8 Naringenin and chalconaringenin in tomato juice at pH 4.4 after ohmic

treatment at 95, 100, 105 and 110°C.

118
 Total Flavonoids and Quercetin at pH 4.4

25 95C, Total Flavonoids

100C, Total Flavonoids
20 95C, Quercetin
100C, Quercetin
mg/ kg FW

15

10

0
-8 0 8 16 24 32
Time, min

Total Flavonoids and Quercetin at pH 4.4

25

20

15
mg/ kg FW

10
105C, Total Flavonoids
110C, Total Flavonoids
5
105C, Quercetin
110C, Quercetin
0
-50 0 50 100 150 200
Time, s

Figure 5.9 Total flavonoids and quercetin equivalents in tomato juice at pH 4.4 after

ohmic treatment at 95, 100, 105 and 110°C.

119
5.4.3 Ascorbic Acid

Ascorbic acid (AA) and dehydroxy ascorbic acid (DHA) remained stable at the

temperature of 90°C and pH 4.4 (Figure 5.10). AA at 95°C was found to increase during

the come-up time from around 34 to 60 μg/ mL, and reduced to 20 μg/ mL at the end of

18 min holding time. Dewanto et al. (2002) reported 15.79 and 28.95% loss of ascorbic

acid after 15 and 30 min of conventional heating of tomato at 88°C. The rate constants

for degradation of AA under ohmic heating were 0.006, 0.022, 0.108, 0.116 and 0.620

min-1 for temperatures of 90, 95, 100, 105 and 110°C, respectively at pH 3.9. In

comparison, Marfil et al. (2008) reported a rate constant of 0.0035 min-1 at only 70°C for

tomato halves, and Bineesh et al. (2005) reported 0.058 min-1 for ascorbic acid in

drumstick leaves at the temperature of 90°C. Activation energy was found to be 33.35

kcal/ mol. Lima et al. (1999) found activation energies for ohmic and conventional

heating of tomatoes to be 12.6 and 12.5 kcal/ mol, respectively. DHA showed reductions

of around 15%, 65%, 70% and 100% at the temperatures of 95, 100, 105 and 110°C. AA

and DHA values at a pH of 3.9 and temperatures of 90 and 100°C showed no significant

reductions after heating for 3 and 1 min, respectively (Figure 5.11).

Ohmic heating‟s ability to retain AA and DHA at 90°C and pH 4.4 and at 90 and 100° at

pH 3.9 is significant, as the literature on the effect of conventional heating on ascorbic

acid shows marked reductions at these treatment conditions (Sanchez et al., 2006).

120
 Ascorbic Acid at pH 4.4
90C, AA
80 90C, DHA
95C, AA
70 95C, DHA
100C, AA
60
100C, DHA
50
μg/ mL

40

30

20

10

0
-5 0 5 10 15 20 25
Time, min

Ascorbic Acid at pH 4.4

80
105C, AA
70 105C, DHA
60 110C, AA
110C, DHA
50
μg/ mL

40

30

20

10

0
-5 0 5 10 15 20 25
Time, min

Figure 5.10 Ascorbic acid and dehydroascorbic acid content in tomato juice at pH 4.4

after ohmic treatment at 90, 95, 100, 105 and 110°C.

121
 Ascorbic acid at pH 3.9
120

100

80
μg/ mL

60

AA 90C
40
DHA 90C
AA 100C
20
DHA 100C

0
-50 0 50 100 150 200
Time, s

Figure 5.11 Ascorbic acid and dehydroascorbic acid content in tomato juice at pH 3.9

after ohmic treatment at 90 and 100°C.

5.5 Conclusion

Ohmic heating at 95, 100, 105 and 110°C at pH 4.4 showed no or minimal reductions of

carotenoids and phenolic compounds. Ohmic treatment at 105°C was found to increase

total naringenin equivalents when compared to 0-time control. Naringenin is shown to

assist in human health by lowering oxidative damage to DNA, cholesterol and hepatitis C

virus. Ohmic heating was also found to have no or minimal effect on ascorbic acid and

dehydroxyascorbic acid at 90°C for pH 4.4 and at 90 and 100° for pH 3.9.
122
The results of this study when compared to conventional heating studies in the literature

suggest that ohmic heating is efficient in retaining inherent amounts of carotenoids and

phenolic compounds. Ohmic heating has shown to increase total naringenin content at

105°C, and requires further investigation.

5.6 References

Agarwal, S., & Rao, A.V. (2000). Tomato lycopene and its role in human health and

chronic diseases. Canadian Medical Association Journal, 163(6), 739- 744.

Barona, J., Jones, J.J., Kopec, R.E., Comperatore, M., Andersen, C., Schwartz, S.J.,

Lerman, R.H., & Fernandez, M.L. (2010). A mediterranean-style low-glycemic-load diet

increases plasma carotenoids and decreases LDL oxidation in women with metabolic

syndrome, Manuscript in Preparation.

Beecher, G.R. (1998). Nutrient content of tomatoes and tomato products. Proceedings of

the Society for Experimental Biology and Medicine, 218, 98–100.

123
Bineesh, N.P., Singhal, R.S., & Pandit, A.B. (2005). A study on degradation kinetics of

ascorbic acid in drumstick (Moringa olifera) leaves during cooking. Journal of the

Science of Food and Agriculture, 85, 1953-1958.

Caballero, B., Trugo, L.C., & Finglas, P.M. (2003). Encyclopedia of food sciences and

nutrition. San Diego, CA.

Castenmiller, J.J.M., & West, C.E. (1998). Bioavailability and bioconversion of

carotenoids. Annual Review of Nutrition, 18, 19-38.

Chasse, G.A., Mak, M.L., Deretey, E., Farkas, I., Torday, L.L., Papp, J.G., Sarma,

D.S.R., Agarwal, A., Chakravarthi, S., Agarwal, S., & Rao., A.V. (2001). An an initio

computational study on selected lycopene isomers. Journal of molecular structure

(Theochem), 571, 27-37.

Dewanto, V., Wu, W., Adom, K.K., & Liu, R.H. (2002). Thermal processing enhances

the nutritional value of tomatoes by increasing total antioxidant activity. Journal of

Agriculatural and Food Chemistry, 50, 3010-3014.

FDA Qualified Health Claims: Letter regarding “Tomatoes and prostate, ovarian, gastric

and pancreatic cancers (American Longevity Petition)”. Docket No. 2004Q-0201.

Accessed on December 2, 2010

124
http://www.fda.gov/Food/LabelingNutrition/LabelClaims/QualifiedHealthClaims/ucm072760.ht

Ferruzzi, M.G., Sander, L.C., Rock, C.L., & Schwartz, S.J. (1998). Carotenoid

determination in biological microsamples using liquid chromatography with a

coulometric electrochemical array detector. Analytical Biochemistry, 256, 74-81.

Fröhlich, K., Kaufmann, K., Bitsch, R., & Böhm, V. (2006). Effects of ingestion of

tomatoes, tomato juice and tomato purée on contents of lycopene isomers, tocopherols

and ascorbic acid in human plasma as well as on lycopene isomer pattern. British Journal

of Nutrition, 95(4), 734-741

Furr, H.C., & Clark, R.M. (1997). Intestinal absorption and tissue distribution of

carotenoids. Nutritional Biochemistry, 8, 364-377.

Gahler, S., Otto, K., & Bohm, V. (2003). Alterations of vitamin C, total phenolics, and

antioxidant capacity as affected by processing tomatoes to different products. Journal of

Agricultural and Food Chemistry, 51, 7962- 7968.

Gartner, C., Stahl, W., & Sies, H. (1997). Lycopene is more bioavailable from tomato

paste than from fresh tomatoes. The American Journal of Clinical Nutrition, 66, 116-

122.

125
George, S., Tourniaire, F., Gautier, H., Goupy, P., Rock, E., & Caris-Veyrat, C. (2011).

Changes in the contents of carotenoids, phenolic compounds and vitamin C during

technical processing and lyophilisation of red and yellow tomatoes. Food Chemistry, 124,

1603- 1611.

Giovannucci, E., Rimm, E.B., Liu, Y., Stampfer, M.J., & Willett, W. C. (2002). A

prospective study of tomato products, lycopene, and prostate cancer risk. Journal of the

National Cancer Institute, 94, 391–398.

Guillou S., & El Murr N. (2002). Inactivation of Saccharomyces cerevisiae in solution by

low-amperage electric treatment. Journal of Applied Microbiology, 92(5), 860-865.

Hwang, Eun-Sun1; Bowen, Phyllis E. Can the Consumption of Tomatoes or Lycopene

Reduce Cancer Risk? Integrative Cancer Therapies, Volume 1, issue 1 (June 01, 2002),

p. 121-132.

Icier F., Yildiz H., & Baysal T. (2006). Peroxidase inactivation and colour changes

during ohmic blanching of pea puree. Journal of Food Engineering, 74(3), 424-429.

Institute of Food Technologists. Functional foods: Opportunities and challenges. Institute

of Food Technologists Web site. [Accessed on October 20, 2010]

126
http://members.ift.org/IFT/Research/IFTExpert Reports/functionalfoods_report.htm.

Kristenson, M., ZiedEn, B., Kucinskien, Z.E., Abaravicius, A., Razinkovien, L.E.,

Elinder, L.S.A., Bergdahl, B.O., Elwing, B., Calkauskas, H., & Olsson, A. G. (1996).

Antioxidant state and mortality from coronary heart disease in lithuanian and swedish

men: concomitant cross sectional study of men aged 50. British Medical Journal, 314,

629-633.

Kulshrestha S., & Sastry S. (2003). Frequency and voltage effects on enhanced diffusion

during moderate electric field (MEF) treatment. Innovative Food Science and Emerging

Technologies, 4(2), 189-194.

Lee, S.H., Park, Y.B., Bae, K.H., Bok, S.H., Kwon, Y.K., Lee, E.S., & Choi, M.S.

(1999). Cholestrol-lowering activity of naringenin via inhibition of 3-Hydroxy-3-

Methylglutaryl Coenzyme A reductase and acyl Coenzyme A: Cholestrol acyltransferase

in rats. Annals of Nutrition and Metabolism, 43, 173- 180.

Marfil, P.H.M., Santos, E.M., & Telis, V.R.N. (2008). Ascorbic acid degradation kinetics

in tomatoes at different drying conditions. Food Science and Technology, 41, 1642-1647.

127
Martinez-Valverde, I., Periago, M.J., Provan, G., & Chesson, A. (2002). Phenolic

compounds, lycopene and antioxidant activity in commercial varieties of tomato

(Lycopersicum esculentum). Journal of the Science of Food and Agriculture. 82, 323-

330.

Mulvihill, E.E., Allister, E.M., Sutherland, B.G., Telford, D.E., Sawyez, C.G., Edwards,

J.Y., Markle, J.M., Hegele, R.A., & Huff, M. W. (2009). Naringenin prevents

dyslipidemia apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-

Null mice with diet-induced insulin resistance. Diabetes. 59(10), 2198- 2110.

Nahmias, Y., Goldwasser, J., Casali, M., van Poll, D., Wakita, T., Chung, R.T., &

Yarmush, M.L. (2008). Apolipoprotein B-dependent hepatitis C virus secretion is

inhibited by the grapefruit flavonoid naringenin. Hepatology. 47(5), 1437- 1445.

Northrop-Clewes, C.A., & Thurnham, D.I. (2002). Vitamins. The Nutrition Handbook for

Food Processors (C.J.K. Henry and C. Chapman, eds) CRC Press, Boca Raton, FL.

Perez, D.C., Garcia, J.A., Garcia, V.V., Iniesta, M.D., Jacob, K., Sanchez, L.M.S., Ros,

G., & Periago, M. J. (2009). Changes in bioactive compounds and antioxidant activity

during homogenization and thermal processing of tomato puree. Innovative Food Science

and Emerging Technologies, 10, 179-188.

128
Rao, A.V., & Agarwal, S. (1999). Role of lycopene as antioxidant carotenoid in the

prevention of chronic diseases: A review. Nutrition Research, 19(2), 305–323.

Re, R., Bramley, P.M., & Rice-Evans, C. (2002). Effects of food processing on

flavonoids and lycopene status in a Mediterranean tomato variety. Free Radical

Research. 36(7), 803-810.

Rock, C.L., Lovalvo, J.L., Emenhiser, C., Ruffin, M.T., Flatt, S.W., & Schwartz, S.J.

(1998). Bioavailability of beta carotene is lower in raw than in processed carrots and

spinach in women. The Journal of Nutrition. 128, 913-916.

Sahlin, E., Savage, G.P., & Lister, C.E. (2004). Investigation of the antioxidant properties

of tomatoes after processing. Journal of Food Composition and Analysis, 17, 635-647.

Salunkhe, D.K., Jadhav, S.J., & Yu, M.U. (1974). Quality and nutritional composition of

tomato fruit as influenced by certain biochemical and physiological changes. Plant Foods

for Human Nutrition (formerly Qualitas Plantarum). 24(1/2), 85-113.

Samaranayake C., Sastry S., & Zhang H. (2005). Pulsed Ohmic Heating–A Novel

Technique for Minimization of Electrochemical Reactions During Processing. Journal of

Food Science, 70(8), e460-e465.

129
Sanchez-Moreno, C., Plaza, L., de Ancos, B., & Cano, M. P. (2006). Impact of high-

pressure and traditional thermal processing of tomato puree on carotenoids, vitamin C

and antioxidant activity. Journal of the Science of Food and Agriculture. 86(2), 171-179.

Sanchez-Moreno, C., Plaza, L., de Ancos, B., & Cano, M. P. (2006). Nutritional

characterization of commercial traditional pasteurized tomato juices: carotenoids, vitamin

C and radical-scavenging capacity. Food Chemistry, 98, 749–756

Schreier P.J.R., Reid D.G., & Fryer P. J. (1993). Enhanced diffusion during the electrical

heating of foods. International Journal of Food Science and Technology, 28, 249-260.

Slimestad, R., & Verheul, M. (2009). Review of flavonoids and other phenolics from

fruits of different tomato (Lycopersicon esculentum Mill.) cultivars. Journal of Science of

Food and Agriculture, 89, 1255- 1270.

Somavat, R. (2010). Applications and effects of ohmic heating: sterilization, influence on

bacterial spores, enzymes, bioactive components and quality factors in food. PhD

Dissertation. The Ohio State University. Columbus, OH.

Stahl, W., & Sies, H. (1992). Uptake of lycopene and its geometrical isomers is greater

from heat-processed than from unprocessed tomato juice in humans. The Journal of

Nutrition, 2161-2166.

130
Tsumura, F., Ohsako, Y., Haraguchi, Y., Kumagai, H., Sakurai, H., & Ishii, K. (1993).

Rapid enzymatic assay for ascorbic acid in various foods using peroxidase. Journal of

Food Science, 58(3), 619-622.

USDA, Agricultural Research Service Database for the Flavonoid Content of Selected

Foods. Release 2 (2007). [Accessed on 10/25/2010]

http://www.ars.usda.gov/Services/docs.htm?docid=6231

Yildiz, H., Icier, F., & Baysal, T. (2010). Changes in β-carotene, chlorophyll and color of

spinach puree during ohmic heating. Journal of Food Process Engineering, 33, 763-779.

Yonekura, L., & Nagao, A. (2007). Intestinal absorption of dietary carotenoids.

Molecular Nutrition and Food Research, 57, 107-115.

131
 BIBLIOGRAPHY

Agarwal, S., & Rao, A.V. (2000). Tomato lycopene and its role in human health and

chronic diseases. Canadian Medical Association Journal, 163(6), 739- 744.

Allen, C.S., Burnett, R., Harles, J., Cucinotta, F., Fullerton, R., Goodman, J.R., Griffith,

A.D., Kosmo, J.J., Perchonok, M., Railsback, J., Rajulu, S., Stilwell, D., Thomas, G., Tri,

T., Joshi, J., Wheeler, R., Rudisill, M., Wilson, J., & Simmons, A. (2003). Guidelines and

Capabilities for Designing Human Missions. NASA/TM-2003-210785.

Alimova, A.A., Katz, A., Gottlieb, P., & Alfano, R.R. (2006). Proteins and dipicolinic

acid released during heat shock activation of Bacillus subtilis spores probed by optical

spectroscopy. Applied Optics, 45(3), 445-450.

Anderson, R.E. (1984). Growth and corresponding elevation of tomato juice pH by

Bacillus coagulans. Journal of Food Science, 49, 647-647.

Anese M., & Sovrano S. (2006). Kinetics of thermal inactivation of tomato lipoxygenase.

Food Chemistry, 95(1), 131-137.

132
Anthon G.E., & Barrett D.M. (2003a). Thermal inactivation of lipoxygenase and

hydroperoxytrienoic acid lyase in tomatoes. Food Chemistry, 81(2), 275-279.

Anthon G.E., & Barrett D.M. (2003b). Thermal inactivation of lipoxygenase and

hydroperoxytrienoic acid lyase in tomatoes. Food Chemistry, 81(2), 275-279.

Anthon G.E., Sekine Y., Watanabe N., & Barrett D.M. (2002). Thermal Inactivation of

Pectin Methylesterase, Polygalacturonase, and Peroxidase in Tomato Juice. Journal of

Agricultural and Food Chemistry, 50(21), 6153-6159.

Barona, J., Jones, J.J., Kopec, R.E., Comperatore, M., Andersen, C., Schwartz, S.J.,

Lerman, R.H., & Fernandez, M.L. (2010). A mediterranean-style low-glycemic-load diet

increases plasma carotenoids and decreases LDL oxidation in women with metabolic

syndrome, Manuscript in Preparation.

Beecher, G.R. (1998). Nutrient content of tomatoes and tomato products. Proceedings of

the Society for Experimental Biology and Medicine, 218, 98–100.

Berg, P.E., & Grecz, N. (1970). Relationship of dipicolinic acid content in spores of

Bacillus cereus T to ultraviolet and gamma radiation resistance. Journal of Bacteriology,

103(2), 517-519.

133
Brown, K.L., Ayers, C.A., Gaze, J.E., & Newman, M. E. (1984). Thermal destruction of

bacterial spores immobilized in food/ alginate particles. Food Microbiology, 1(3), 187-

198.

Caballero, B., Trugo, L.C., & Finglas, P.M. (2003). Encyclopedia of food sciences and

nutrition. San Diego, CA.

Carstensen, E.L., Marquis, R.E., & Gerhardt, P. (1971). Dielectric study of the physical

state of electrolytes and water within Bacillus cereus spores. Journal of Bacteriology,

107(1), 106-113.

Castenmiller, J.J.M., & West, C.E. (1998). Bioavailability and bioconversion of

carotenoids. Annual Review of Nutrition, 18, 19-38.

Castro I., Macedo B., Teixeira J.A., & Vicente A.A. (2004). The Effect of Electric Field

on Important Food-processing Enzymes: Comparison of Inactivation Kinetics under

Conventional and Ohmic Heating. Journal of Food Science, 69(9), C696-C701.

Chasse, G.A., Mak, M.L., Deretey, E., Farkas, I., Torday, L.L., Papp, J.G., Sarma,

D.S.R., Agarwal, A., Chakravarthi, S., Agarwal, S., & Rao., A.V. (2001). An an initio

computational study on selected lycopene isomers. Journal of molecular structure

(Theochem), 571, 27-37.

134
Cho, H.Y., Sastry, S.K. and Yousef, A.E. (2000). Kinetics of inactivation of Bacillus

subtilis spores by continuous or intermittent ohmic and conventional heating.

Biotechnology and Bioengineering, 62(3), 368-372.

Crew and Thermal Systems Division at JSC (2004). ISS Food Warmer, Part No

SED39114053-309. International Space Station Catalogue of Intervehicular (IVA)

Government Furnished Equipment (GFE) Flight Crew Equipment (FCE). JSC #28533

Rev D.

De La Torre, J. J. M. (2009) Effect of frequency on polyphenoloxidase activity during

moderate electric field treatment. MS Thesis. The Ohio State University.

De Sio F., Dipollina G., Villari G., Loiudice R., Laratta B., & Castaldo D. (1995).

Thermal resistance of pectin methylesterase in tomato juice. Food Chemistry, 52(2), 135-

138.

FDA Qualified Health Claims: Letter regarding “Tomatoes and prostate, ovarian, gastric

and pancreatic cancers (American Longevity Petition)”. Docket No. 2004Q-0201.

Accessed on December 2, 2010

135
http://www.fda.gov/Food/LabelingNutrition/LabelClaims/QualifiedHealthClaims/ucm072760.ht

Ferruzzi, M.G., Sander, L.C., Rock, C.L., & Schwartz, S.J. (1998). Carotenoid

determination in biological microsamples using liquid chromatography with a

coulometric electrochemical array detector. Analytical Biochemistry, 256, 74-81.

Fields, M.L., Zamora, A.F., & Bradsher, M. (1977). Microbiological analysis of home-

canned tomatoes and green beans. Journal of Food Science, 42(4), 931-934.

Fröhlich, K., Kaufmann, K., Bitsch, R., & Böhm, V. (2006). Effects of ingestion of

tomatoes, tomato juice and tomato purée on contents of lycopene isomers, tocopherols

and ascorbic acid in human plasma as well as on lycopene isomer pattern. British Journal

of Nutrition, 95(4), 734-741.

Furr, H.C., & Clark, R.M. (1997). Intestinal absorption and tissue distribution of

carotenoids. Nutritional Biochemistry, 8, 364-377.

Gahler, S., Otto, K., & Bohm, V. (2003). Alterations of vitamin C, total phenolics, and

antioxidant capacity as affected by processing tomatoes to different products. Journal of

Agricultural and Food Chemistry, 51, 7962- 7968.

136
Gartner, C., Stahl, W., & Sies, H. (1997). Lycopene is more bioavailable from tomato

paste than from fresh tomatoes. The American Journal of Clinical Nutrition, 66, 116-

122.

George, S., Tourniaire, F., Gautier, H., Goupy, P., Rock, E., & Caris-Veyrat, C. (2011).

Changes in the contents of carotenoids, phenolic compounds and vitamin C during

technical processing and lyophilisation of red and yellow tomatoes. Food Chemistry, 124,

1603- 1611.

Giovannucci, E., Rimm, E.B., Liu, Y., Stampfer, M.J., & Willett, W. C. (2002). A

prospective study of tomato products, lycopene, and prostate cancer risk. Journal of the

National Cancer Institute, 94, 391–398.

Guillou S., & El Murr N. (2002). Inactivation of Saccharomyces cerevisiae in solution by

low-amperage electric treatment. Journal of Applied Microbiology, 92(5), 860-865.

Guillou, S., Besnard, V., Murr, N. El., & Federighi, M. (2003). Viability of

Saccharomyces cerevisiae cells exposed to low-amperage electrolysis as assessed by

staining procedure and ATP content. International journal of food microbiology, 88(1),

85-89.

Hanford, A.J. (2006). Advanced Life Support Research and Technology Development

Metric- Fiscal year 2005. NASA/CR-2006-213694.

137
Hwang, Eun-Sun1; Bowen, Phyllis E. Can the Consumption of Tomatoes or Lycopene

Reduce Cancer Risk? Integrative Cancer Therapies, Volume 1, issue 1 (June 01, 2002),

p. 121-132.

Iciek, J., Papiewska, A., & Molska, M. (2005). Inactivation of Bacillus

stearothermophilus sprores during thermal processing. Journal of Food Engineering,

77(3), 379- 471.

Icier F., & Ilicali C. (2005). Temperature dependent electrical conductivities of fruit

purees during ohmic heating. Food Research International, 38(10), 1135-1142.

Icier F., Yildiz H., & Baysal T. (2006). Peroxidase inactivation and colour changes

during ohmic blanching of pea puree. Journal of Food Engineering, 74(3), 424-429.

Icier F., Yildiz H., & Baysal T. (2008). Polyphenoloxidase deactivation kinetics during

ohmic heating of grape juice. Journal of Food Engineering, 85(3), 410-417.

Imai, T., Uemura, K., Ishida, N., Yoshizaki, S., & Noguchi, A. (1995). Ohmic heating of

Japanese white radish Rhaphanus sativus L. International Journal of Food Science and

Technology, 30(4), 461-472.

138
Institute of Food Technologists. Functional foods: Opportunities and challenges. Institute

of Food Technologists Web site. [Accessed on October 20, 2010]

http://members.ift.org/IFT/Research/IFTExpert Reports/functionalfoods_report.htm.

Jean, G.L., Abraham, G., Debray, E., Candau, Y., & Piar, G. (1994). Kinetics of thermal

destruction of Bacillus stearothermophilus spores using a two reaction model. Food

Microbiology, 11, 229-241.

Jemai, A.B., & Vorobiev, E. (2002). Effect of moderate electric field pulses on the

diffusion coefficient of soluble substances from apple slices. International Journal of

Food Science & Technology, 37(1), 73-86.

Jun, S., & Sastry, S.K. (2005). Modeling and optimizing of pulsed ohmic heating of

foods inside the flexible package. Journal of Food Process Engineering, 28(4), 417–436.

Jun, S., & Sastry, S.K. (2007). Reusable pouch development for long term space

missions: A 3D ohmic model for verification of sterilization efficacy. Journal of Food

Engineering, 80, 1199–1205.

139
Jun, S., Sastry, S.K., & Samaranayake, C. (2007). Migration of electrode components

during ohmic heating of foods in retort pouches. Innovative Food Science and Emerging

Technologies, 8(2), 237-243.

Kim, H.J., Choi, Y.M., Yang, A.P.P., Yang, T.C.S., Tuab, I.A., Giles, J., Ditusa, C.,

Chall, S., & Zoltai, P. (1995). Microbiological and chemical investigation of ohmic

heating of particulate foods using a 5 kW ohmic system. Journal of Food Processing and

Preservation, 20(1), 41-58.

Kristenson, M., ZiedEn, B., Kucinskien, Z.E., Abaravicius, A., Razinkovien, L.E.,

Elinder, L.S.A., Bergdahl, B.O., Elwing, B., Calkauskas, H., & Olsson, A. G. (1996).

Antioxidant state and mortality from coronary heart disease in lithuanian and swedish

men: concomitant cross sectional study of men aged 50. British Medical Journal, 314,

629-633.

Kulshrestha, S.A., & Sastry, S.K. (1999). Low-frequency dielectric changes in vegetable

tissue from ohmic heating. IFT Annual Meeting: Book of Abstracts. p. 211. Chicago, IL.

Kulshrestha, S., & Sastry, S. (2003). Frequency and voltage effects on enhanced diffusion

during moderate electric field treatment. Innovative Food Science and Emerging

Technologies, 4, 189-194.

140
Laratta B., Loiudice R., Giovane A., Quagliuolo L., Servillo L., & Castaldo D. (1995).

Thermostability of three pectinesterase isoenzymes in tomato fruit. Food Chemistry,

52(4), 415-418.

Lee, C.H., & Yoon, S.W. (1999). Effect of ohmic heating on the structure and

permeability of the cell membrane of Saccharomyces cerevisiae. 1999 IFT Annual

Meeting. Chicago. July 24-28 1999.

Lee M., & MacMillon, J.D. (1968). Mode of action of pectin enzymes. I. Purification and

properties of tomato pectinesterase. Biochemistry, 7, 4005-4010.

Lee, S.H., Park, Y.B., Bae, K.H., Bok, S.H., Kwon, Y.K., Lee, E.S., & Choi, M.S.

(1999). Cholestrol-lowering activity of naringenin via inhibition of 3-Hydroxy-3-

Methylglutaryl Coenzyme A reductase and acyl Coenzyme A: Cholestrol acyltransferase

in rats. Annals of Nutrition and Metabolism, 43, 173- 180.

Levri, J.A., Vaccari, D.A., & Drysdale, A.E. (2000). Theory and application of the

Equivalent System Mass Metric. SAE Technical Paper 2000-01-2395. 30th International

Conference on Environmental Systems.

141
Loghavi, L., Sastry, S.K., & Yousef, A.E. (2007). Effect of moderate electric field on the

metabolic activity and growth kinetics of Lactobacillus acidophilus. Biotechnology and

bioengineering, 98(4), 872-881.

Loghavi, L., Sastry, S.K., & Yousef, A. E. (2009). Effect of moderate electric field

frequency and growth stage on the cell membrane permeability of Lactobacillus

acidophilus. Biotechnology Progress, 25(1), 85-94.

Mallidis, C.G., Frantzeskakis, P., Balatsouras, G., & Katsaboxakis, C. (1990). Thermal

treatment of aseptically processed tomato paste. International Journal of Food Science

and Technology, 25, 442-448.

Martinez-Valverde, I., Periago, M.J., Provan, G., & Chesson, A. (2002). Phenolic

compounds, lycopene and antioxidant activity in commercial varieties of tomato

(Lycopersicum esculentum). Journal of the Science of Food and Agriculture. 82, 323-

330.

142
Moon (2009). Effect of frequency on lipoxygenase during Moderate Electric Field

treatment. (Unpublished internal report). The Ohio State University, Department of

Food, Agricultural and Biological Engineering.

Mulvihill, E.E., Allister, E.M., Sutherland, B.G., Telford, D.E., Sawyez, C.G., Edwards,

J.Y., Markle, J.M., Hegele, R.A., & Huff, M. W. (2009). Naringenin prevents

dyslipidemia apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-

Null mice with diet-induced insulin resistance. Diabetes. 59(10), 2198- 2110.

Nahmias, Y., Goldwasser, J., Casali, M., van Poll, D., Wakita, T., Chung, R.T., &

Yarmush, M.L. (2008). Apolipoprotein B-dependent hepatitis C virus secretion is

inhibited by the grapefruit flavonoid naringenin. Hepatology. 47(5), 1437- 1445.

Northrop-Clewes, C.A., & Thurnham, D.I. (2002). Vitamins. The Nutrition Handbook for

Food Processors (C.J.K. Henry and C. Chapman, eds) CRC Press, Boca Raton, FL.

Palaniappan, S., Sastry, S.K., & Richter, E.R. (1990). Effects of electricity on

microorganisms: a review. Journal of food processing and preservation, 14(5), 393-414.

Palaniappan, S., & Sastry, S.K. (1991). Electrical conductivity of selected juices:

influence of temperature, solids content, applied voltage and particle size. Journal of

Food Process Engineering. 14, 247-260.

143
Palaniappan, S., Sastry, S.K., & Richter, E.R. (1992). Effects of electroconductive heat

treatment and electrical pretreatment on thermal death kinetics of selected

microorganisms. Biotechnology and Bioengineering, 39(2), 225-232.

Palop, A., Raso, J., Pagan, R., Condon, S., & Sala, F.J. (1999). Influence of pH on heat

resistance of spores of Bacillus coagulans in buffer and homogenized foods.

International Journal of Food Microbiology, 46, 243-249.

Perez, D.C., Garcia, J.A., Garcia, V.V., Iniesta, M.D., Jacob, K., Sanchez, L.M.S., Ros,

G., & Periago, M. J. (2009). Changes in bioactive compounds and antioxidant activity

during homogenization and thermal processing of tomato puree. Innovative Food Science

and Emerging Technologies, 10, 179-188.

Plaza, L., Duvetter, T., Silvia Monfort, S. Clynen, E., Liliane Schoofs, L., Van Loey,

A.M., & Marc E. Hendrickx, M.E. (2007). Purification and Thermal and High-Pressure

Inactivation of Pectinmethylesterase Isoenzymes from Tomatoes (Lycopersicon

esculentum): A Novel Pressure Labile Isoenzyme. Journal of Agricultural and Food

Chemistry, 55 (22), 9259–9265

144
Pressey, R. (1986). Extraction and assay of tomato polygalacturonases. HortScience, 21,

490-492.

Rao, A.V., & Agarwal, S. (1999). Role of lycopene as antioxidant carotenoid in the

prevention of chronic diseases: A review. Nutrition Research, 19(2), 305–323.

Re, R., Bramley, P.M., & Rice-Evans, C. (2002). Effects of food processing on

flavonoids and lycopene status in a Mediterranean tomato variety. Free Radical

Research. 36(7), 803-810.

Rhim, J.W., Nunes, R.V., Jones, V.A., & Swartzel, K. R. (1989). Determination of

kinetic parameters using linearly increasing temperature. Journal of Food Science, 54(2),

446-450.

Rock, C.L., Lovalvo, J.L., Emenhiser, C., Ruffin, M.T., Flatt, S.W., & Schwartz, S.J.

(1998). Bioavailability of beta carotene is lower in raw than in processed carrots and

spinach in women. The Journal of Nutrition. 128, 913-916.

Sahlin, E., Savage, G.P., & Lister, C.E. (2004). Investigation of the antioxidant properties

of tomatoes after processing. Journal of Food Composition and Analysis, 17, 635-647.

145
Salunkhe, D.K., Jadhav, S.J., & Yu, M.U. (1974). Quality and nutritional composition of

tomato fruit as influenced by certain biochemical and physiological changes. Plant Foods

for Human Nutrition (formerly Qualitas Plantarum). 24(1/2), 85-113.

Samaranayake, C.P., Sastry, S.K., & Zhang, H (2005). Pulsed ohmic heating- A novel

technique for minimization of electrochemical reactions during processing. Journal of

Food Science, 70(8), E 460-465.

Samarnayake, C., & Sastry, S.K. (2005). Electrode and pH effects on electrochemical

reactions during ohmic heating. Journal of Electroanalytical Chemistry, 577, 125-135.

Sanchez-Moreno, C., Plaza, L., de Ancos, B., & Cano, M. P. (2006). Impact of high-

pressure and traditional thermal processing of tomato puree on carotenoids, vitamin C

and antioxidant activity. Journal of the Science of Food and Agriculture. 86(2), 171-179.

Sanchez-Moreno, C., Plaza, L., de Ancos, B., & Cano, M. P. (2006). Nutritional

characterization of commercial traditional pasteurized tomato juices: carotenoids, vitamin

C and radical-scavenging capacity. Food Chemistry, 98, 749–756

146
Sandoval, A.J., Barreiro, J.A., & Mendoza, S. (1992). Thermal resistance of Bacillus

coagulans in double concentrated tomato paste. Journal of Food Science, 57(6), 1369-

1370.

Sarang S., Sastry S.K., Gaines J., Yang, T.C.S., & Dunne P. (2007). Product Formulation

for Ohmic Heating: Blanching as a Pretreatment Method to Improve Uniformity in

Heating of Solid–Liquid Food Mixtures. Journal of Food Science, 72(5), E 227-234.

Sastry S.K., & Barach J.T. (2000). Ohmic and Inductive Heating. Journal of Food Safety,

65, 42-46.

Sastry, S.K., & Salengke, S. (1998). Ohmic heating of solid-liuid mixtures: A comparison

of mathematical models under worst-case heating conditions. Journal of Food Process

Engineering. 21, 441-458.

Sastry, S.K., Jun, S., Somavat, R., Samaranayake, C., Yousef, A., & Pandit, R.B. (2009).

Heating and sterilization technology for long-duration space missions: Transport

processes in a reusable package. Interdisciplinary Transport Phenomena: Annals of New

York Academy of Sciences, 1161, 562-569.

147
Schreier, P.J.R., Reid, D.G., & Fryer, P.J. (1993). Enhanced diffusion during the

electrical heating of foods. International Journal of Food Science and Technology, 28,

249-260.

Slieman, T.A., & Nicholson, W.L. (2001). Role of dipicolinic acid in survival of Bacillus

subtilis spores exposed to artificial and solar UV radiation. Applied and Environmental

Microbiology, 67(3), 1274-1279.

Slimestad, R., & Verheul, M. (2009). Review of flavonoids and other phenolics from

fruits of different tomato (Lycopersicon esculentum Mill.) cultivars. Journal of Science of

Food and Agriculture, 89, 1255- 1270.

Somavat, R. (2010). Applications and effects of ohmic heating: sterilization, influence on

bacterial spores, enzymes, bioactive components and quality factors in food. PhD

Dissertation. The Ohio State University. Columbus, OH.

Stahl, W., & Sies, H. (1992). Uptake of lycopene and its geometrical isomers is greater

from heat-processed than from unprocessed tomato juice in humans. The Journal of

Nutrition, 2161-2166.

Tracy, R.L.Jr. (1932). Lethal effect of alternating current on yeast cells. Journal of

Bacteriology, 24(6), 423-438.

148
Tsumura, F., Ohsako, Y., Haraguchi, Y., Kumagai, H., Sakurai, H., & Ishii, K. (1993).

Rapid enzymatic assay for ascorbic acid in various foods using peroxidase. Journal of

Food Science, 58(3), 619-622.

Tulsiyan, P., Sarang, S., & Sastry, S.K. (2007). Electrical conductivity of

multicomponent systems suring ohmic heating. International Journal of Food Properties,

11(1), 233-241.

Tulsiyan, P., Sarang, S., & Sastry, S.K. (2009). Measurement of residence time

distribution of a multicomponent system inside an ohmic heater using radio frequency

identification. Journal of Food Engineering, 93(3), 313-317.

USDA, Agricultural Research Service Database for the Flavonoid Content of Selected

Foods. Release 2 (2007). [Accessed on 10/25/2010]

http://www.ars.usda.gov/Services/docs.htm?docid=6231

Vepachedu, V.R., Hirneisen, K., Hoover, D.G., & Setlow, P. (2007). Studies of the

release of small molecules during pressure germination of spores of Bacillus subtilis.

Letters in Applied Microbiology, 45, 342-348.

Yıldız H., & Baysal T. (2006). Effects of alternative current heating treatment on

Aspergillus niger, pectin methylesterase and pectin content in tomato. Journal of Food

Engineering, 75(3), 327-332.

149
Yildiz, H., Icier, F., & Baysal, T. (2010). Changes in β-carotene, chlorophyll and color of

spinach puree during ohmic heating. Journal of Food Process Engineering, 33, 763-779.

Yonekura, L., & Nagao, A. (2007). Intestinal absorption of dietary carotenoids.

Molecular Nutrition and Food Research, 57, 107-115.

York, G.K., Heil, J.R., Marsh, G.L., Ansar, A., Merson, R.L., Wolcott, T., & Leonard, S.

(1975). Thermobacteriology of canned whole peeled tomatoes. Journal of Food Science,

40, 764-769.

150