Preservation of Foods with Pulsed Electric Fields

PRESERVATION OF FOODS WITH PULSED ELECTRIC FIELDS

FOOD SCIENCE A N D TECHNOLOGY International Series SERIES EDITOR Steve L. Taylor

University of Nebraska ADVISORY BOARD Daryl B. Lund

Louise Wicker

CorneU University

University of Georgia

Douglas Archer

Mina R. McDaniel

FDA, Washington, DC

Oregon State University

Connie Weaver

Bruce Chassy

Purdue University

University of Illinois

Robert Hutkins

Barbara O. Schneeman

University of Nebraska

University of California, Davis

Howard Zhang

Ohio State University

A complete list of the books in this series appears at the end of this volume.

PRESERVATION OF FOODS WITH PULSED ELECTRIC FIELDS Gustavo V. Barbosa-C~novas M. Marcela G6ngora-Nieto Usha R. Pothakamury Barry G. Swanson Washington State University Pullman, Washington

ACADEMIC PRESS

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London

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Toronto

This book is printed on acid-flee paper. ( ~ Copyright 9 1999 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press a division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com

Academic Press 24-28 Oval Road, London NW1 7DX, UK http://www.hbuk.co.uk/ap/ Library of Congress Card Catalog Number: 99-60092 International Standard Book Number: 0-12-078149-2 PRINTED IN THE UNITED STATES OF AMERICA 99 00 01 02 03 04 MM 9 8 7 6

5

4

Dedication

To our families

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Contents

Preface

xi

Acknowledgments

eoo

Xnll

CHAPTER I

Fundamentals of High-Intensity Pulsed Electric Fields (PEF)

I

Introduction Methods of Applying Electricity High-Intensity PEF Processing System Basics of High-Intensity PEF Energy Requirements Applications of PEF Technology in Food Preservation Some Drawbacks of PEF Final Remarks References

I

Jo

II. III. IV. V. VI. VII. VIII.

3 4

7 II 13 14

17 17

CHAPTER 2

Design of PEF Processing Equipment I. II. III. IV.

Introduction High-Voltage Pulsers Switches Treatment Chambers A. B. C.

Electrode Shape Optimization Static Chambers Continuous Chambers

20 20 20 25 28 31 33 35 vii

Contents

viii

V~

VI. VII. VIII.

Cooling System Typical Measurements in a PEF System Packaging and Storing Final Remarks References

41 43 44 45 45

CHAPTER 3

Biological Principles for Microbial Inactivation in Electric Fields Io Introduction II. Transmembrane Potential III. Electromechanical Compression and Instability IV. Osmotic Imbalance V. Viscoelastic Model VI. Hydrophobic and Hydrophilic Pores VII. Theories Based on Conformational Changes VIII. Electric Field-Induced Structural Changes IX. Final Remarks References

47 47 48 53 55 55 58 60 61 73 73

CHAPTER 4

PEF-Induced Biological Changes I. II. III. IV. V. VI.

VII.

76

Introduction Electropermeabilization Electrofusion Disruption and Biological Alteration Electrical and Thermal Gradients Induced by PEF on Microbial Cell Membranes Main Factors in Microbial Inactivation

79

A.

Factors D e p e n d e n t on T r e a t m e n t Conditions

84

B.

Factors D e p e n d e n t on Microbial Entity Characteristics

98

C.

Factors D e p e n d e n t on T r e a t m e n t Media

Final Remarks References

76 77 77

79 83

I01 103 105

Contents

ix

CHAPTER 5

PEF Inactivation of Vegatative Cells, Spores, and Enzymes in Foods I. II.

Introduction Microbial Inactivation A. Inactivation of Yeasts B. Inactivation of Escherichiacoli C. Inactivation of Staphylococcusaureus D. Inactivation of Lactobacillus E. Inactivation of Bacillus F. Inactivation of Salmonella G. Inactivation of Pseudomonas H. Inactivation of Other Microorganisms I!1. Spore Inactivation IV. Standardization of Inactivation Assessment V. Enzyme Inactivation Vi. Final Remarks References

108 108 108 109 114 125 128 130 132 135 137 138 141 142 151 152

CHAPTER 6

Food Processing by PEF I. ii. !!1. IV. V.

VI.

Introduction Microbial Analysis Chemical and Physical Analyses Sensory Evaluation and Shelf-Life Studies Quality and Shelf-Life Evaluation of PEF Products A. Processing of Apple Juice B. Processing of Orange Juice c. Processing of Milk D. Processing of Eggs E. Processing of Green Pea Soup F. Processing of Brine Solutions and Water in Cooling Systems Final Remarks References

156 156 157 158 158 159 160 162 164 165 167 168 169 170

x

Contents

CHAPTER 7

Hazard Analysis and Critical Control Point (HA CCP) in PEF Processing I. II. III. IV.

V.

172

Introduction Term Definitions in HACCP Systems The HACCPSystem The HACCP System in PEF Processing

172

A. Hazard Assessment B. Critical Control Points C. Record Keeping Final Remarks References

175

173 174 175 177 179 182 183

CHAPTER 8

PEF in the Food Industry for the New Millennium

184

I. II.

Introduction Commercialization

184 185

III.

A. Industrialization and Production Costs B. PEF Implementation in the Food Industry of Today Regulatory Aspects for the Implementation of PEF A. FDA Regulations B. Letters of No Objection from the FDA

IV.

Index

The Future of PEF References

185 186 186 188 189 190 191

193

Preface

Increasing consumer demand for new products with high sensory organoleptic and nutritional qualities has spurred a search for new alternatives to processed foods. For many years, thermal processing was the main technology for producing safe products with long shelf lives, although in most cases losses of fresh flavors, vitamins, and some physicochemical characteristics were the price of safety and long-term stability. Pulsed electric field (PEF) processing as a nonthermal technique has been proven to inactivate microorganisms with minimal losses of flavor and food quality, potentially making it the answer to current consumer demands. In addition, the low processing temperatures used in this nonthermal technology allow the process to be energy efficient, which translates into lower costs and fewer environmental impacts. Encouraging results from the use of electric treatment and application of PEF over the past several years by academia and public and private corporations have spurred increased interest from food processors. Although a number of technical papers, review articles, international patents, and conference proceedings discuss different aspects of PEF techniques, the specificity of such works called for a comprehensive overview comparing different PEF system configurations and their experimental results. This book is the first exclusively dedicated to the preservation of foods by PEF. Readers will discover how the fundamentals of food processing, microbiology, physicochemistry, and some electrical engineering aspects have been combined in the development of this technology. Furthermore, the cost, operation, equipment configurations, and processing conditions that apply to industrial implementation are discussed throughout the eight chapters. The first two chapters focus on the electrical bases, various equipment configurations, and principal components of PEF systems, including fundamental equations and various types of electric circuits and processing chambers. The theories and biological principles believed to explain microbial inactivation are reviewed in Chapter 3. Chapter 4 discusses the biological changes induced by PEF, such as the effect of various factors that lead to such changes. Chapter 5 comprehensively reviews the PEF inactivation of microorganisms and enzymes, and Chapter 6 considers the application of xi

xii

Preface

this technology for processing foods of major interest to food processors and consumers. In response to the increasing demand for safety assurance, Chapter 7 is dedicated to the evaluation of hazard analyses and detection of critical control points in PEF. To encourage future applications of this technology, the concerns of industry and regulatory agencies for food processing in the United States (FDA) are addressed in Chapter 8. Throughout the book the reader will find illustrative pictures, figures, diagrams, and tables with relevant information obtained over the years by different researchers around the world. We sincerely hope this book will be a valuable addition to the food literature and will promote additional research into the PEF preservation of food.

Gustavo V. Barbosa-Cdnovas M. Marcela Gdngora-Nieto Usha R. Pothakamury Barry G. Swanson

Acknowledgments

We express our sincere gratitude to the Honorable Thomas S. Foley, former Speaker of the House; the late Patrick Ormsby, congressional aide to former Speaker Foley; and his staff for their vision, continuous support, and belief in the future of food technology. A special thanks goes to the Bonneville Power Administration (BPA) of Walla Walla, Washington, for believing and investing in our dream to develop nonthermal processing techniques. BPA has contributed significantly to the development of PEF for the preservation of foods and establishment of the Center for Nonthermal Processing of Food (CNPF). It is a pleasure to work with an institution that is committed to the well-being of the community, seeks cooperation with other institutions with common goals, and fosters the best possible use of our valuable energy resources. Our special gratitude is offered to Tom Osborn and Jennifer Eskil, who are excellent examples of how to work in partnership with other organizations for a better tomorrow. We are appreciative of the Washington State University (WSU) International Marketing Program for Agricultural Commodities and Trade (IMPACT) Center for its continuous support in the development of PEF technology. The efforts of its director, Dr. A. Desmond O'Rourke, in sponsoring this technology have been extremely valuable in the promotion of nonthermal food processing, especially PEF. We value the support, guidance, and encouragement provided by the Electrical Power Research Institute-Food Technology Alliance (EPRI-FTA) to develop PEF, especially Director Dr. Donald Quass. We thank Universidad Aut6noma de M~xico (UNAM) and CONACyT (Mexico) for supporting M. Marcela G6ngora-Nieto's doctoral studies at WSU. Our gratitude is also extended to Ms. Dora Rollins, CNPF, for her invaluable editorial assistance.

Gustavo V. Barbosa-Cdnovas M. Marcela Gdngora-Nieto Usha R. Pothakamury Barry G. Swanson oo,

XIII

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CHAPTER

!

Fun dam en ta ls of High-In tens i tY Pulsed Electric Fields (PEF)

I.

Introduction

Consumers are increasingly aware of the taste, color, flavor, and nutritional value of the foods they eat. Most of the time fresh food contains all the nutrients needed for good health, but because it may not always be possible to obtain fresh food, preservation becomes necessary. Methods of preservation have changed from those used in the past. Until recently, thermal processing had been the most commonly used in the food industry to increase shelf-life and maintain food safety by inactivating spoilage and pathogenic microorganisms. However, because studies have shown that color, flavor, and nutrients may be degraded by heat, there is now a demand for alternative methods of food preservation. Nonthermal methods provide such an option because they offer fresh-like minimally processed foods with little loss of color, flavor, and nutrients. The use of high-intensity pulsed electric fields (PEF) is one of the emerging nonthermal processes deserving of attention because it can accomplish food preservation and metabolite release from plant cells with short treatment times and very little heating of the medium. Several research groups from around the world are studying the use of PEF as an alternative to conventional food processing methods (Table 1.1). One of the earliest applications of electricity in food processing was the sterilization of milk. In the early 1900s milk was sterilized with an alternating current known as the Electro-pure process, and microbial inactivation occurred as a result of ohmic heating. The Electro-pure process inactivates Tubercle bacilli, Escherichia coli, and some of the bacteria resistant to traditional food heat processing methods (Palaniappan et al., 1990). Electric pasteurization of milk was composed of pumping the milk through a regenerative (heat exchange) coil, an electrical heating chamber,

2

I. Fundamentals of High-Intensity PEF

T A B L E I. I International Groups Working on the Development of PEF Food Processing

Institution Catholic University of Leuvena University of Guelph AGIR Pernod Ricard Thomson University of Bordeaux University of Montpellier a CPC Europe a Technical University of Hamburg Technical University of Berlin a ICE Tec ~ ATO-DLO TNO Unilever Research Vlaardingen~ University of Aberdeen University of Lleida University of Zaragozaa SIK Goteburg a Tetra Pak a University of Lund Nestle Campden and Chorleywood Food Research Assoc. Natick Laboratories National Center for Food Safety and Technology Ohio State University PurePulse Technologies University of Wyoming Washington State University

Country Belgium Canada France France France France France Germany Germany Germany Iceland The Netherlands The Netherlands The Netherlands Scotland Spain Spain Sweden Sweden Sweden Switzerland Unites Kingdom United States United States United States United States United States United States

aMembers of the European consortium.

a n d a surface h e a t e x c h a n g e r for cooling. T h e electrical c h a m b e r c o n s i s t e d o f a vertical r e c t a n g u l a r t u b e with o p p o s a b l e walls o f c a r b o n e l e c t r o d e s a n d heavy glass for insulation. A 220-V a l t e r n a t i n g c u r r e n t supply with a c o n s t a n t p o w e r o f 15 kW was a p p l i e d to t h e c a r b o n e l e c t r o d e s , a n d raw milk p r e h e a t e d to 52~ was passed t h r o u g h t h e t r e a t m e n t c h a m b e r . T h e electric c u r r e n t passing t h r o u g h t h e milk in t h e t r e a t m e n t c h a m b e r raised its t e m p e r a t u r e to 71~ so it was t h e n c o o l e d to a b o u t 29~ a n d b o t t l e d or c o l l e c t e d for c r e a m s e p a r a t i o n . T h e p r o c e s s has n o t b e e n u s e d in t h e dairy i n d u s t r y since t h e 1960s (Getchell, 1935). F o r m o r e t h a n two d e c a d e s , electrical field pulses have b e e n u s e d to i n d u c e p o r e s or pore-like s t r u c t u r e s within cell p l a s m a m e m b r a n e s to facilitate a t r a n s m e m b r a n e e x c h a n g e o f materials. In g e n e r a l , studies o f t h e t e c h n o l o g y w e r e c o n c e r n e d with g e n e t r a n s f e c t i o n or cell fusion i n d u c t i o n . H o w e v e r , e l e c t r o p o r a t i o n has b e e n p o s t u l a t e d as an i m p o r t a n t m e c h a n i s m in biological cells e x p o s e d to electric fields ( C h e n a n d Lee, 1994).

II. Methods of Applying Electricity

II.

3

Methods of Applying Electricity

The various forms of the electrical pasteurization m e t h o d include ohmic heating, microwave heating, low electric field stimulation, high-voltage arc discharge, low voltage alternating current, and high intensity PEF. Ohmic heating is one of the earliest applications of electricity in food pasteurization. This m e t h o d relies on the generation of heat in a food when an electric current passes through it. Ohmic heating is suitable for viscous products and foods containing particles and is considered a promising technique for aseptic food processing. Microwave heating is now applied extensively in everyday households and the food industry. However, because many food materials possess very low values of static conductivity, subjecting them to microwave fields produces extremely high values of alternating field conductivity and thus considerable energy is consumed. Low electric field stimulation has been explored as a m e t h o d of bacterial control in meat. An electric field of 5-10 V / c m is applied as AC pulses to the sample through electrodes fixed at opposite ends of the long axis of the muscle. Electrical stimulation reduces the bacterial count as well as the thermal resistance of the bacteria. High voltage arc discharges across an electrode gap in liquid foods can destroy the microorganisms suspended in a food. When high voltages are discharged through liquids, a multitude of physical effects (intense shock waves) and chemical compounds (electrolysis) are generated, which cause bacterial inactivation. Enzymes are also inactivated by high-voltage arc discharges, which are attributed to oxidation reactions mediated by free radicals and atomic oxygen. There is no significant temperature rise during the treatment of arc discharges. However, the major drawbacks of this electrical m e t h o d are contamination of the treated food from chemical products of electrolysis and disintegration of food particles by shock waves. This m e t h o d is thus, in principle, not suitable for use in the food industry. The microbicidal action of low-voltage alternating currents (50 Hz) is based on a defined quantity of electricity applied at or above a certain m i n i m u m current density. Pareilleux and Sicard (1970) observed that the m i n i m u m current required to kill E. coli was 25 mA. There was no decrease in the n u m b e r of viable cells immediately after treatment, but the n u m b e r did decrease with holding time. The bactericidal effect d e p e n d e d on the current passing through the suspension, the presence of chloride containing compounds, and the holding time in the medium after treatment. Unlike ohmic heating, which involves the passage of a continuous electrical current through a food material in order to heat it evenly throughout, high-intensity PEF technology is not intended to heat food. Rather, it involves the application of a short burst of high voltage to a food placed between two electrodes. Due to treatment at an ambient or subambient temperature for only microseconds, energy loss due to heating the food is

4

I. Fundamentalsof High-IntensityPEF

minimized. In traditional methods, when electrical energy is converted into thermal energy within a food, it causes microbial inactivation by rapid heating in the product's interior locations. However, when electrical energy is applied in the form of short pulses, it destroys the bacterial cell membrane by mechanical effects with no significant heating of the food. PEF technology has the potential to economically and efficiently improve energy usage, as well as provide consumers with microbiologically safe, minimally processed, nutritious, and fresh-like foods. Potential applications of PEF include cold sterilization of liquid foods such as juices, cream soups, milk, and egg products (Fig. 1.1).

III.

H i g h - I n t e n s i t y PEF Processing S y s t e m

The test apparatus in the continuous system consists of five major components: a high-voltage power supply, an energy storage capacitor, a treatment chamber(s), a pump to conduct food through the treatment chamber(s), a cooling device, voltage, current, temperature measurement devices, and a computer to control operations (Figs. 1.2 and 1.3). The types of highvoltage power supplies that may be used to charge the capacitor are generated by an ordinary source of direct current (DC), which transforms AC power from the utility line (60 Hz) into high-voltage AC power and then rectifies it to high-voltage DC. Another way to generate high voltages is with a capacitor charging power supply, which uses high-frequency AC inputs and provides a command charge with higher repetitive rates than a DC power supply (Zhang et al., 1996). Energy from the high voltage power supply is stored in the capacitor and is discharged through the food material to generate the necessary electrical field in the food. The capacitance [Co (F)] of the energy storage capacitor is given by ~" ~'~rA C0 . . . . , R d

(1.1)

where ~" (sec) is the pulse duration, R (1~) is the resistance, ~r ( S / m ) is the conductivity of the food, d (m) is the gap between electrodes, and A (m 2) is the area of electrode surface. The energy stored in a capacitor [Q (J/m3)] is given by, Q = 0.5c0 V2 ,

(1.2)

where CO is the capacitance and V is the charging voltage. The energy stored in the capacitor can be discharged almost instantaneously (in a millionth of a second) at very high levels of power. The discharge is accomplished using high-voltage switches that must be able to operate reliably at a high power and repetition rate. The type of switches can

III. High-Intensity PEF Processing System

Fig. I. I

5

Raw (a) and treated (b) products at the PEF Washington State University Pilot Plant.

be selected from gas spark gaps, vacuum spark gaps, solid state switches, thyratrons, and high vacuum tubes. The treatment chamber is used to transfer high-intensity PEF to foods. Recirculating cooling water through the electrodes will control the temperature of a sample in the gap formed by the two electrodes in the treatment

6

I. Fundamentals of High-Intensity PEF

Fig. 1.2 Flowchart for processing foods by PEF in a continuous mode.

chamber. A variable speed p u m p regulates the flow rate of the food d u r i n g treatment. Chambers with u n i f o r m electric fields subject all bacterial cells to the same potential. This is advantageous for electroporation, where no a l i g n m e n t or pearl-chaining is required, and can result in high yields, provided the field strength is set at an o p t i m u m value. However, because the yield m i g h t d r o p substantially above or below the o p t i m u m field strength, the o p t i m u m value is not always known. A practical realization of u n i f o r m field chambers is a c h a m b e r with flat parallel electrodes or a coaxial c h a m b e r with a diameter m a n y times larger than the electrode gap. Chambers with n o n u n i f o r m fields allow alignment at low cell densities with an AC field for fusion and reduce the n e e d to have exactly the right field strength to obtain high transfection yields.

Fig. 1.3 Flow chart for processing foods by PEF in continuous mode with more than one treatment chamber (adapted from Yin et al., 1997).

IV. Basicsof High-Intensity PEF

7

Electrical parameters such as voltage and current waveforms applied to fluid foods can be recorded via a digital data acquisition system. A digital storage oscilloscope records outputs from voltage and current monitors. The computer and oscilloscope should be placed in a shielded area to minimize electromagnetic interference, and electrical and flow parameters should be selected so that each unit volume of the food is subjected to the necessary n u m b e r of pulses to create the desired inactivation of microbial cells. After processing, the food is packaged into sterile containers. It is extremely critical to maintain a contaminant-free environment during processing and packaging to avoid cross-contamination of the product. All of the equipment and work area must be thoroughly cleaned and sanitized before processing, and personnel involved in the operation should be properly equipped to maintain a clean working area.

IV.

Basics o f H i g h - I n t e n s i t y

PEF

The principle of applying electric fields for the inactivation of microorganisms is not new. It is reported that electrically treated milk was being supplied to the city of Liverpool as early as 1915. The use of high-voltage pulses for the destruction of microorganisms was first suggested in 1960. Since then, the application for cell disruption in food materials described and promoted by Dovenspeck (1960) has been further developed and expanded to the inactivation of microorganisms. In recent years, PEF technology has also been utilized in electroporation, electrofusion, and food preservation (Knorr et al., 1994). Liquid food materials are usually considered electrical conductors because they contain large concentrations of ions as electrical charge carriers. To generate a high-intensity PEF within a food, a large flux of current must flow in a very short period of time, and because the time between pulses is much longer than the pulse width, the generation of pulses involves slow charging and fast discharging of the capacitor. When an electric field is generated between two parallel-plate electrodes, the electric field strength, is given by

(1.3)

G = V/d,

where V (kV) is the voltage and d (m) is the gap between the electrodes. The electric field strength between coaxial electrodes is given by V Eco =

R1

rln R2

,

(1.4)

8

I. Fundamentals of High-Intensity PEF

where r is the radius at which the electric field is measured and R 1 and R 2 are the radii of the inner and outer electrodes, respectively. The electric field pulses most commonly applied are in the form of exponentially decaying or square waves. An exponential decay voltage wave is a unidirectional voltage that rises rapidly to a maximum value and decays slowly to zero. The circuit in Fig. 1.4 may be used to generate an exponential decay waveform. A DC power supply charges a capacitor bank connected in series with a charging resistor (Re). W h e n a trigger signal is applied, the charge stored in the capacitor flows through the food in the treatment chamber. The R 1 resistor limits the current in case the food sparks over, and R 2 controls the decay time when the food resistivity is larger than expected. The pulse duration of an exponentially decaying pulse is given by T =

RC

o ,

(1.5)

Fig. 1.4 A simplified circuit for producing exponential decay pulses (a) and a voltage trace across a treatment chamber (b) (reprinted from Food Technol., Vol. 49(12), Qin et al., "Food pasteurization using high-intensity pulsed electric fields," pp. 55-60, 1995, with permission from Elsevier Science).

IV. Basics of High-Intensity PEF

9

where R is the load resistance and C o is the capacitance. T h e energy density for exponential decay pulses is a p p r o x i m a t e d as VZCo n Qe =

2v

V2t = 2Rv

(1.6)

where V0 is the initial charge voltage, C O is the capacitance, n is the n u m b e r of pulses, t (sec) is the t r e a t m e n t time, R is the food resistance, and v is the volume of the t r e a t m e n t chamber. A high-voltage transmission line c o n n e c t e d to a m a t c h e d load generates a square pulse. However, it is often inconvenient to m a t c h the load resistance of a food [R or ZF(I~)] with the characteristic i m p e d a n c e of the transmission line [Z0(l~)]. In addition, the real transmission line (a coaxial cable) is not suitable for pulses of microsecond durations. These p r o b l e m s can be overcome by using a pulse forming network (PFN) consisting of an array of capacitors a n d inductors (Fig. 1.5). T h e square waveform is obtained when the t r e a t m e n t c h a m b e r and PFN have m a t c h i n g i m p e d a n c e (Z 0 = ZL). T h e m a x i m u m voltage delivered to the c h a m b e r (VL) is half that of the

Fig. 1.5 Layoutof a square generator using a pulse forming network of three capacitor-inductor units (a) and a voltage trace across a treatment chamber (b) (adapted from Zhang et al., 1994).

I0

I. Fundamentals of High-Intensity PEF

charged voltage (V0) on the capacitors and is a function of the impedance of the load (Z L) given by

( zL )

VL = V 0 Z 0 + Z L

.

(1.7)

The voltage delivered to the chamber (VL) can be equal to the charge voltage by superimposing two PFN. The energy density for square-wave pulses is approximated as VI~ n

V %n

V 2t

(1.8)

where V, I, and z are the voltage, current, and pulse width of the square-wave pulse, respectively (Zhang et al., 1995). W h e n subjected to a PEF, polarization of the dipoles and the bulk m o v e m e n t of ions induce capacitive and resistive currents. Assuming the food has homogeneous dielectric and electric properties, the effective capacitance (C) and resistance (R) of the load are given by

~0e'r A

C = d

R=

(1.9)

d pd

tra

a '

(1.10)

where e0 is the permittivity of free space [8.84 • 10 -8 (/~F/cm)], er is the relative permittivity or dielectric constant of the food, A (m 2) is the electrode area, d (mm) is the gap between electrodes, tr ( S / m ) is the conductivity of the food, and p ( l ~ / c m ) is the resistivity of the food. The dielectric constant of a food increases with increasing water content and decreases with increasing temperature. However, food conductivity increases with an increase in temperature. Table 4.13 presents some food treatment media used in PEF and the corresponding resistivity or conductivity. W h e n an external field is applied to a bacterial cell, a transmembrane potential is induced. This is believed to be the primary event that leads to pore formation. For a spherical cell of radius a in a uniform electric field (E0), the potential difference ( A ~ ) between extracellular and intracellular surfaces of the cell m e m b r a n e is given (in the absence of pores) by Eqs. (1.11-1.13): A ~ = 1.5f*aE0cos0 1 - exp "r -- f * a C m ( ri + r

(t) ~'r

(1.11)

(1.12)

/2)

1

f* =

[1 +

a G m ( r i + re)]

.

(1.13)

V. Energy Requirements

II

m

E0 Fig. 1.6 Induction of the transmembrane potential in a cell exposed to an external electric field (adapted from Chang et al., 1992).

Here t is the time after the constant field is turned o n , C m is the m e m b r a n e capacitance per unit area (assumed to be uniform over the entire cell surface), r i and r e are the specific resistances of intra- and extracellular media, Gm is the m e m b r a n e conductance per unit area (assumed to be uniform over the entire cell surface), ~'r is the relaxation time, f * is a factor that depends on the m e m b r a n e properties (for normal cells with large Gm, f = 1), and 0 is the angle made with the field direction by the radius vector (Fig. 1.6) [i.e., when 0 = 0 ~ or 180 ~ (at the poles of the cell) and cos0 = 1, which gives the maximum value for A~].

V. Energy Requirements The energy required to achieve a certain level of microbial or enzymatic inactivation depends on the treatment volume of the chamber, flow rate of the product, n u m b e r of pulses or treatment time, and system configuration. Qin et al. (1995a) investigated the energy requirements of processing apple juice and found that if short pulses (2.5/zsec duration) are used, energy loss due to heating the product is minimized. The energy input for heating apple juice to the thermal pasteurization temperature (for instance, a 90~ increase) with the high temperature short time (HTST) process (assuming 100% efficiency) is 87 cal/ml. In earlier studies that used a static parallel plate PEF system, 70 to 370 c a l / m l was required to obtain a 6 log reduction, whereas only 6.6 c a l / m l was needed to obtain a 6 log reduction in a continuous chamber. Pulsed electric field processing of some products (i.e., apple juice) does not require energy to keep treatment temperatures low, which makes this technology more energy efficient and less costly than the

12

I. Fundamentals of High-Intensity PEF

HTST method. Continual development of PEF processing has produced even greater energy utilization improvements. One of the latest derivations has been the use of instant-charge-reversal pulses (Fig 1.7) that can drastically reduce energy requirements to as low as 1.3 J / m l (EPRI, 1998). When utilizing PEF, the energy delivered to a product is determined by the properties of the product (i.e., resistivity/conductivity, temperature) and the characteristics of the pulse (i.e., wave shape, width, peak voltage, and current). To guarantee that the required energy is supplied during processing, it is necessary to have appropriate instrumentation that measures the delivered energy by pulse, which can be conceptually easy but practically challenging due to the difficulty of accurately measuring high voltage transients. The energy delivered in each pulse is defined by (1.14)

W = foP(t)dt; P(t) = v(t)*i(t),

where W, the energy in Joules, is the integer over time (t) of the power P in watts, which is the product of the pulse voltage (v) as a function of time, and current (i) as a function of time. The actual measurement of confidential voltage and current transients is rather complex due to the high voltage and small gap time between pulses, which requires transducers and sensing devices with high response times and low noise sensitivity. Furthermore, the measuring devices must be capable of maintaining important frequency components of the pulse signal in order to be able to reproduce the real signal after the sensor has received it. Once the signal has been measured properly, it can be captured easily in a digital oscilloscope. The mathematical calculations are also obtained readily with the use of commercial software or even oscilloscope functions.

d

a

pulse -,--, 0 b

c S

Time Fig. 1.7 A voltage trace of an instant charge reversal pulse where a is a pulse period (2-11 sec), b is a pulse width (2 /zsec), e is a pulse rise time (sec) to reach e (kV), d is a spike width (sec), e is a pulse peak voltage (kV), and f is a spike peak voltage (kV) (Ho et al., 1995).

Vl. Applications of PEF Technology in Food Preservation

VI.

13

Applications of PEF Technology in Food Preservation

When the food engineers at Krupp Maschinentechnik GmbH in Hamburg, Germany, investigated the PEF process (Sitzmann, 1995), they developed the ELCRACK process for the electric cracking of vegetable and animal cells, as well as the ELSTERIL process for electrical sterilization/pasteurization of pumpable electrically conductive media (Sitzmann, 1990). Krupp, in association with the University of Hamburg, claimed successful preliminary results with the application of PEF to fluid foods such as orange juice and milk (Grahl et al., 1992). Test results were obtained in a system consisting of a high voltage generator, capacitors (5 ~F), a high-voltage switch, and two carbon electrodes. The capacitor discharged electric pulses of up to 15 k V / c m through the treatment chamber, which was composed of two plain parallel plate electrodes of 50 cm 2 with a 5- or 12-mm distance between the electrodes. For pulse treatment, sample material was filled into the gap between the electrodes (batch vessel) or pumped through the gap (continuous vessel). PurePulse Technologies, a subsidiary of Maxwell Laboratories located in San Diego, California, owns several U.S. patents to preserve fluid foods such as dairy products, fruit juices, and fluid eggs by treatment with high-voltage pulses (Dunn and Pearlman, 1987; Bushnell et al., 1993, 1995, 1996). The patents comprehensively describe both batch and continuous systems, including chamber characteristics, PFN components, and specific switching arrangements to avoid electrode fouling. Furthermore, it is recommended that liquid foods be treated with PEF at an elevated temperature to enhance microbial inactivation and shelf-life stability. Washington State University (WSU) has a comprehensive program to pasteurize foods by high intensity PEF. The first reported results were obtained using a modified version of an electroporator (International Biotech, Inc., New Haven, CT) to treat 0.1 ml of inoculated simulated milk ultra-filtrate (SMUF) with 20-kV/cm electric pulses (Pothakamury et al., 1995a,b). Later, the WSU group designed and constructed 12- and 25-ml volume temperature-controlled static chambers (Martin et al., 1994; Zhang et al., 1995; Qin et al., 1995b; Vega-Mercado et al., 1997). A 9 log cycle reduction in microbial population (E. coli) was achieved by confining SMUF inside the 12-ml chamber and applying 65 pulses with a peak electric field intensity of 70 kV/cm. Liquid whole eggs (LWE) inoculated with E. coli were exposed to pulses of 38 or 48 kV/cm, and at the latter intensity, more than a 7 log cycle ( > 7 D) microbial inactivation was achieved (Ma et al., 1998). The PEF system designed and constructed by the WSU group includes parallel plate and coaxial continuous treatment chambers and a power supply capable of delivering a peak voltage of 40 kV. The system may be reconfigured to obtain pulses with different characteristics and can transfer energy at frequencies up to 10 Hz. Numerical simulations and experimental

14

I. Fundamentalsof High-Intensity PEF

studies support the chamber's design and system effectiveness. The treatment chamber is temperature stabilized to improve processing and to minimize surface buildup on the electrodes, and the use of electrically conductive polymers such as polyacetylene will prevent buildup without disruption of the generated electric field. A contoured coaxial electrode configuration where relative high and low electric field values are encountered by the flowing media is recommended for low-resistivity products. The advantages of this chamber are (1) the increase in effective electrical resistance across the treatment chamber without reducing the processed fluid path and (2) additional agitation of the fluid being processed (Qin et al., 1997). Another leading group in this technology is working at Ohio State University where they have implemented an integrated pilot plant system with aseptic packaging (Zhang et al., 1997). This system has been applied successfully to process fruit juice, verifying appreciable extended shelf-lives. The PEF system designed by Zhang et al. (1997) has a different treatment chamber configuration, with cofield treatment zones and a PFN capable of delivering energy at rates on the order of kHz that can inactivate more than 99% of a medium's bacterial spores. The refinement of PEF technology has also led to the implementation of different PFN that allow the production of new pulse wave shapes. Ho et al. (1995) developed a pulse power treatment system capable of supplying PEF in a batch process by the application of instant-reverse-charge pulses. Results obtained by this Canadian group show the inactivation of enzymes, spores, and high levels of vegetative cells such as Pseudomonas spp. in foods or model media (Ho et al., 1995, 1997; Marquez et al., 1997). Other possible applications of PEF are the processing of spices and waste brine (Mittal and Choudhry, 1997). Several European groups have also shown interest in the application of PEF technology to process foods (Table 1.1). Determination of the inactivation mechanisms and kinetics of several microorganims in model and real foods are examples of some of the important contributions of these groups (Hamilton and Sale, 1967; Sale and Hamilton, 1967; Hillsheger et al., 1983; Grahl and M~irkl, 1996).

VII.

Some

Drawbacks

of PEF

As an emerging technology, PEF has some shortcomings that must be addressed in future research before proper full-scale implementation can be realized. Some of the more important technical drawbacks are (a) scale up of the system (including treatment chambers and power supply equipment) in such a way that profitable production is possible, (b) the presence of bubbles, which may lead to nonuniform treatment as well as operational and safety

VII. Some Drawbacks of PEF

15

problems, (c) treatment of suspensions with solid particles, with a m i n i m u m risk of breakdown, and (d) availability of commercial units. In a competitive market, one of the most important aspects of implementing a new product, process, or technology are initial and operational costs. In PEF technology implementation, the industry is concerned about which costs are more significant. So far the more expensive c o m p o n e n t is the switcher, due to its specificity and precession to deliver the energy. Although much work has focused on the development of treatment chambers, more work is n e e d e d to allow production flexibility, minimization of buildup in the high-voltage electrode when certain products are processed, and adaptations of configurations that are easy to clean. Major efforts have been made in the determination of treatment conditions that guarantee the inactivation of pathogenic and spoilage flora that may be present in certain products, but for full-scale applications, careful validation of these treatment conditions must be obtained. The PEF process at a commercial scale must also assure the safety of its operators and the consumers, which implies proper plant designs for the power system and the inactivation of pathogens, enzymes, and spores in the product of interest. Because the main concern of those working in a PEF facility is the high voltage, the power supply, capacitors, and treatment chamber must be confined in a restricted access area with interlocked gates. In addition, it is advisable that safety gates be designed to allow the pulser to be turned off when opened, restricted areas marked clearly, emergency switches accessible in case of a process failure, and discharging bars provided to discharge elements in the circuit before maintenance or inspections. A technical issue in PEF processing of particular relevance is the dielectric breakdown of foods, which is characterized by a spark. Preventing the dielectric breakdown of foods is key to the success of the PEF technology. The dielectric breakdown of fluids is attributed to the presence of impurities that substantially enhance the local electric field due to differences in dielectric properties. Dielectric breakdown at the gas-liquid or liquid-solid interface is more frequent than in h o m o g e n e o u s liquids. Breakdown of food is characterized by 9 9 9 9 9

a large electrical current in a narrow channel a bright luminous spark evolution of bubbles formation of pits on the electrodes impulsive pressure through the liquid with an accompanying explosive sound

It has been observed that dielectric breakdown occurs when gas bubbles are present in the treatment chamber. The local electric field inside a gas bubble can be more than 5 times that of the applied electric field. Spherical bubbles elongate in the direction of the electric field, which makes the local electric field at the ends even greater (depending on the ratio of long to short diameter). For a prolated spheroid bubble with a long diameter two

16

I. Fundamentals of High-Intensity PEF

times the short, the local electric field can be enhanced to as high as five times that of the applied field. When the applied electric field exceeds the dielectric strength of the gas bubbles, partial discharges take place inside the bubbles that can volatilize the liquid and produce more vapor so that the bubbles grow even larger. When elongated under the applied electric field, the bubbles become big enough to bridge the gap between the two electrodes and a spark is produced. The presence of gas can be minimized by vacuum degassing or pressurizing the treatment media during processing, which can be done by the use of positive pressure. The r e c o m m e n d e d range is from 25-30 to 1000 psi, but higher pressures may be used to permit safe operation at temperatures above the atmospheric boiling point. However, it is important to note that at these pressures the gas remains in solution (Bushnell et al., 1993; Zhang et al., 1994, 1997). Dielectric breakdown can also be minimized by 9 9 9 9

lowering the temperature of the liquid food using smooth electrode surfaces employing r o u n d electrode edges to prevent field e n h a n c e m e n t designing the chamber to provide a uniform electric field

Although PEF technology exhibits promising results in the pasteurization of homogeneous, low-viscosity fluids without suspended material, it is not yet applicable to the pasteurization of liquid foods containing particles. W h e n applying PEF to a particulate liquid food, the following concerns must be addressed: 9 possible dielectric breakdown produced by electric tracking along the surface of particulates 9 distribution of applied electric fields which is related to uniformity of treatment 9 selection of a treatment chamber and feed p u m p to handle particulates 9 m e a s u r e m e n t and control of electrical heating of foods to ensure nonexcessive temperatures 9 size of the particles because the m a x i m u m particle in the fluid must be smaller than the gap of the treatment region to maintain a p r o p e r processing operation Yet another matter to address is that when a food material exhibits a large electrical conductivity, the peak electric field applied to the food will be reduced because the equivalent resistance of the treatment chamber (R) is smaller. This problem needs to be considered at the treatment chamber design level. In conventional PEF treatment chambers with electric fields generated in a gap between two electrodes, particles in liquid foods may bridge this space and thus increase the chance of dielectric breakdown p r o d u c e d by electrical tracking along the surface of the particulates. Treatm e n t chambers should thus be constructed in such a way that only the food inside the treatment region is subjected to a high-intensity electric field. In

References

17

addition, the electric field at the interface of the electrodes should be maintained at a low level to minimize the dielectric breakdown between the two electrodes and to reduce possible electrolysis in the food. Application of the PEF technology is restricted to food products that can withstand high electric fields. H o m o g e n e o u s fluids provide ideal conditions for continuous treatment with PEF, but solid foods can also be processed by PEF in a batch mode operation as long as dielectric breakdown is prevented. Until now there have been no studies on highly viscous products, which highlights the importance of proper design prior to the application of the technology to meet special requirements for each suitable product. The availability of commercial units is very limited; just one American (PurePulse Technologies, Inc.) and one French (Thomson-CSF) supplier are able to distribute industrial systems. There are many pulse power suppliers capable of designing and constructing reliable pulsers, but except for these two mentioned, the complete PEF systems on the market today must be assembled independently.

VIII.

Final R e m a r k s

This chapter reviewed key aspects of PEF technology as a suitable means to pasteurize food products and found it to be a significant innovation that may be implemented in the near future. Analysis of the various forms of applying electric energy for electrical pasteurization (i.e., ohmic heating, microwave heating, low electric field stimulation, high-voltage arc discharge, and low voltage alternating current) prove that PEF is one of the most promising food processing methods available. The survey of the important components of the PEF system and how the energy from a high-voltage power supply is stored in a capacitor and discharged through a food material contained or flowing through a treatm e n t chamber provide a starting point for understanding the action mechanisms of the technology. The drawbacks of PEF simply present actual challenges. The enormous effort put forth all over the world is a clear indication of the food processing potential and promising future of this technology. The following seven chapters review in more detail the PEF system, mechanisms of inactivation, studied products, inactivated microorganisms and enzymes, safety, and regulatory aspects.

References Bushnell, A. H., Dunn, J. E., and Clark, R. W. (1993). High pulsed voltage systemsfor extending the shelf life of pumpable food products. U. S. Patent 5,235,905.

18

I. Fundamentals of High-Intensity PEF

Bushnell, A. H., Clark, R. W., Dunn, J. E., and Lloyd, S. W. (1995). Prevention of electrochemical and electrophoretic effects on high-strength-electric-field pumpable-food-product treatment systems. U. S. Patent 5,447,733. Bushnell, A. H., Clark, R. W., Dunn, J. E., and Lloyd, S. W. (1996). Process for reducing levels of microorganisms in pumpable food products using a high pulsed voltage system. U. S. Patent 5,514,391. Chang, D. C., Chassy, B. M., Saunders, J. A., and Sowers, A. E., eds. (1992). "Guide to Electroporation and Electrofusion." Academic Press, San Diego. Chen, W., and Lee, R. (1994). Altered ion channel conductance and ionic selectivity induced by large imposed membrane potential pulse. BiophysJ. 67, 603-612. EPRI (1998). Pulsed electric field processing in the food industry: A status report on PEF. Report CR-109742. Industrial and Agricultural Technologies and Services, Palo Alto, California. Dovenspeck, H. (1960). Verfahren und vorrichtung zur gewinnung der einzelnen phasen nus dispersen systemen. German Patent 1,237,541. Dunn, J. E., and Pearlman, J. S. (1987). Methods and apparatus for extending the shelf life of fluid food products. U. S. Patent 4,695,472. Getchell, B. E. (1935). Electric pasteurization of milk. Agric. Eng. 16(10), 408-410. Grahl, T., and M~irkl, H. (1996). Killing of microorganisms by pulsed electric fields. Appl. Microbiol. Biotechnol. 45, 148-157. Grahl, T., Sitzmann, W., and M~irkl, H. (1992). Killing of microorganisms in fluid media by high voltage pulses. Presented at the 10th DECHEMA Biotechnology Conference Series, Frankfurt, Germany, 5B, pp. 675-678. Hamilton, W. A., and Sale, A. J. H. (1967). Effects of high electric fields on microorganisms II. Mechanism of action of the lethal effect. Biochim. Biophys. Acta 148, 789-800. Ho, S. Y., Mittal, G. S., Cross, J. D., and Griffiths, M. W. (1995). Inactivation of Pseudomonas fluorescens by high voltage electric pulses. J. Food Sci. 60(6), 1337-1343. Ho, S. Y., Mittal, G. S., and Cross, J. D. (1997). Effects of high field electric pulses on the activity of selected enzymes. J. Food Eng. 31, 69-85. Hiilsheger, H., Potel, J., and Niemann, E. G. (1983). Electric field effects on bacteria and yeast cells. Radiat. Environ. Biophys. 22, 149-162. Knorr, D., Geulen, M., Grahl, T., and Sitzmann, W. (1994). Food application of high electric field pulses. Trends Food Sci. Technol. 5, 71-75. Ma, L., G6ngora-Nieto, M., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1998). Food pasteurization using high-intensity pulsed electric fields: Promising new technology for non-thermal pasteurization for eggs. In "Proceedings of the Second International Symposium on Egg Nutrition and Newly Emerging Ovo-Technologies" (J. S. Sim, ed.), (in press) CAB International, New York. Marquez, V. O., Mital, G. S., and Griffiths, M. W. (1997). Destruction and inhibition of bacterial spores by high voltage pulsed electric fields. J. Food Sci. 62(2), 399-409. Martin, O., Zhang, Q., Castro, A. J., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1994). Pulse electric fields of high voltage to preserve foods. Microbiological and engineering aspects of the process. Spanish J. Food Sci. Technol. 34, 1-34. Mittal, G. S., and Choudhry, M. (1997). Pulsed electric field sterilization of waste brine solution. In "Proceedings of the Seventh International Congress on Engineering and Food," pp. C13-C16. The Brighton Center, U. K., 13-17 April. Palaniappan, S., Sastry, S. K., and Richter, E. R. (1990). Effects of electricity on microorganisms: A review. J. Food Proc. Pres. 14, 393-414. Pareilleux, A., and Sicard, N. (1970). Lethal effects of electric current on Escherichia coli. Appl. Microbiol. 19(3), 421-424. Pothakamury, U. R., Monsalve-Gonzfilez, A., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1995a). High voltage pulsed electric field inactivation of Bacillus subtilis and Lactobacillus delbrueckii. Rev. Esp. Cienc. Tecnol. Aliment. 35(1), 101-107. Pothakamury, U. R., Monsalve-Gonzfilez, A., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1995b). Inactivation of Escherichia coli and Staphylococcus aureus in model food systems by pulsed electric field technology. Food Res. Int. 28(2), 167-171.

References

19

Qin, B. L., Chang, F., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1995a). Nonthermal inactivation of Saccharomyces cerevisiae in apple juice using pulsed electric fields. Lebensm. -Wiss. Technol. 28, 564-568. Qin, B. L., Pothakamury, U. R., Vega-Mercado, H., Martin, O., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1995b). Food pasteurization using high-intensity pulsed electric fields. Food Technol. 49(12), 55-60. Qin, B. L., Vega-Mercado, H., Pothakamury, U. R., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1995c). Application of pulsed electric fields for inactivation of bacteria and enzymes. J. Franklin Inst. 332a, 209-220. Qin, B. L., Barbosa-Cfinovas, G. V., Pedrow, P. D., Olsen, R. G., Swanson, B. G., and Zhang, Q. (1997). Continuous flow electrical treatment of flowable food products. U. S. Patent 5,662,03. Sale, A. J. H., and Hamilton, W. A. (1967). Effects of high electric fields on microorganisms. I. Killing of bacteria and yeast. Biochim. Biophys. Acta 148, 781-788. Sitzmann, W. (1990). Keimabtotung mit hilfe elecktrischer hochspannungsimpulse in pumpfahigen nahrungsmitteln. Vortrag Anlablich des Seminars "Mittelstansfourderung in der Biotechnologie." Ergebnisse des Indirekt-Spezifischen Programma des BMFT 1986-1989. KFA Julich, Germany, 6-7 February. Sitzmann, W. (1995). High voltage pulse techniques for food preservation. In "New Methods of Food Preservation" (G. W. Gould, ed.), p. 236. Blackie Academic & Professional, London. Vega-Mercado, H., Powers, J. R., Martin-Belloso, O., Luedecke, O. L., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1997). Effect of pulsed electric fields on the susceptibility of proteins to proteolysis and inactivation of an extracellular protease from P. fluorescens M 3/6. In "Proceedings of the Seventh International Congress on Engineering and Food" pp. C73-C76. The Brighton Center, U. I~, 13-17 April. Ym, Y., Zhang, Q. H., and Sastry, S. H. (1997). High voltage pulsed electric field treatment chambers for the preservation of liquid food products. U. S. Patent 5690,978. Zhang, Q., Monsalve-Gonzfilez, A., Qin, B. L., Barbosa-C{movas, G. V., and Swanson, B. G. (1994). Inactivation of Saccharomyces cerevisiae in apple juice by square-wave and exponential-decay pulsed electric fields. J. Food Proc. Eng. 17, 469-478. Zhang, Q., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1995). Engineering aspects of pulsed electric field pasteurization. J. Food Eng. 25, 261-281. Zhang, Q., Qin, B. L., Barbosa-Cfinovas, G. V., Swanson, B. G., and Pedrow, P. D. (1996). Batch mode food treatment using pulsed electric fields. U. S. Patent 5,549,041. Zhang, Q. H., Qiu, x., and Sharma, S. K. (1997). Recent developments in pulsed electric processing. In "New Technologies Yearbook" (D. I. Chandrana, ed.), pp. 31-42. National Food Processors Association, Washington, D.C.

CHAPTER 2

Design of PEF ProcessingEquipment

I.

Introduction

Pulsed electric field processing of food involves the application of short pulses (duration of micro- to milliseconds) of high electric field intensity. Food may be processed at ambient or refrigerated temperatures. In a continuous process the food is subjected to electric field pulses while being pumped through the system. The residence time of the food in the treatment chamber is adjusted so that it is subjected to the required number of pulses. The number of pulses depends on the type of microbes to be inactivated so that the food is safe for consumption. The PEF processing system is composed of a high voltage repetitive pulser, a treatment chamber(s), a cooling system(s), voltage- and currentmeasuring devices, a control unit, and a data acquisition system. A pulsed power supply is used to obtain high voltage from low utility level voltage, and the former is used to charge a capacitor bank and switch to discharge energy from the capacitor across the food in the treatment chamber. Treatment chambers are designed to hold the food during PEF processing and house the discharging electrodes. After processing the product is cooled, if necessary, packed aseptically, and then stored at refrigerated or ambient temperatures depending on the type of food (Qin et al., 1995a; Zhang et al., 1997).

II.

High-Voltage Pulsers

A typical repetitive high voltage pulser (RP1292) manufactured by Physics International in San Leandro, California features a repetitive capacitor discharge modulator (Fig. 2.1) that is designed to drive load resistances 20

21

II. High-Voltage Pulsers Stray

Series Resistor

Switch

Inductance

'W

Output Cables

I

TT

~

I

()

i i ouut Load

High Voltage .. Trigger Generator IR! I~ V Output

Fig. 2. I tances.

T h e circuit of a repetitive pulser with various resistances, capacitances, and induc-

between 2.5 and 15 1~. Some of the other components of the pulser include (a) a 16-kJ/sec switching power supply, (b) an adjustable shunt resistor assembly, (c) a series-limiting resistor that can be bypassed if desired, (d) temperature sensors on the high-power resistor, (e) a fully enclosed interlocked shield cabinet, and (f) an interactive computer control for operating the pulser. The pulsed power supply converts voltage at a normal utility level to a high voltage. The pulser is designed with a set of five capacitors and two shunt resistors to allow adjustment of energy per pulse and pulse shape. Energy stored in the capacitors is discharged almost instantaneously (in a millionth of a second) at power levels higher than 40 kV. The pulser is designed to allow a widely adjustable operating voltage, repetition rate, pulse duration, and pulse energy. The pulse repetition rate f(Hz) depends on the number of pulses desired (n), the treatment chamber volume (v), and the volumetric flow rate of the food (F) (Zhang et al., 1995): nF f = --.

(2.1)

The pulse repetition has an overall limit of 10 Hz and is limited at the higher energy capacitances by the average power rating of the power supply. To expose the food to the electric field pulses, the storage capacitor is charged to a preselected voltage. The full-rated charge voltage is 40 kV. One of the two output monitors is for current and the other for voltage, both of which can be measured with an oscilloscope. The high voltage involved in PEF processing guarantees definite safety measures. The RP1292 pulser is provided with a series interlock loop, which disables the high-voltage output of the power supply and activates the safety crowbar system. The modulator cabinet doors are included in the interlock loop. Opening the power supply cabinet doors disables the high-voltage output and abort switches that are located on the modulator cabinet and

22

Fig. 2.2 system.

2. Design of PEF Processing Equipment

A control panel consisting of an oscilloscope, modulator cabinet, and computer

front control panel (Fig. 2.2). Pushing either of these will activate the safety crowbar system and disable the power supply. The lab scale pulser at Washington State University (WSU) is a repetitive capacitor discharge modulator that supplies energy by switching into an output cable by a pair of triggered series-operated ignitions (Fig. 2.3). Two power supplies arranged in a master-slave configuration provide the highvoltage charging current. In this system the voltage driven into the treatment chamber or load is dependent on the direct current (DC) charge voltage, whereas the pulse width is determined by the R C (resistance-capacitance) decay constant where R is a parallel combination of a shunt resistor and the load resistance. The R C decay constant can be adjusted within the range of 2-30 ~sec with a capacitor combination. This system has been used successfully to study microbial and enzyme inactivation (Qin et al., 1995b, 1996; Mart~n-Belloso et al., 1997; Vega-Mercado et al., 1995, 1996; Pagfm et al., 1998). The GeneZapper (commercial electroporator) manufactured by IBI/ Kodak (or equivalent electoporators) can be used as a bench-top pulsed power supply for basic or preliminary studies of electric field food processing (Fig. 2.4). These units provide pulses with a maximum voltage of 2.5 kV. The instruments consist of a capacitor (7/~F), charge and discharge switches, and a wave controller that can be used to process small volumes of model liquid food. Appropriate voltage and current monitors may be attached to the electoporator to evaluate the energy delivered to the chamber.

II. High-Voltage Pulsers

23

Fig. 2.3 (a) The pilot plant scale pulser used at WSU for PEF inactivation of microorganisms and enzymes. (b) The main cabinet of the WSU PEF pulser. (c) The current setup of the PEF facility at WSU in which the pulser has a 16-kJ/sec charging power supply, 40-kV peak charging voltage, and 10-Hz pulse repetition rate: C, storage capacitor; D, power supply protection diode; Rc, charging resistor; Rs, series resistor; Rsh, shunt resistor; Rp, voltage-measuring resistor; Imon, current monitor; and Vmon, voltage monitor (adapted from Qin et al., 1995d).

24

2. Design of PEF Processing Equipment

Trigger Rc

lm n

Crowbar

y't)

/~46,

_TD

Power Supplies

--~C

•-Jm--EarthGround

() Treatment Chamber

V,m

f

Fig. 2.3 Continued.

Ho et al. (1995) have demonstrated the effectiveness of a low-cost pulse generator with a maximum power of 30 kV DC. The voltage of the supplier line is transformed and rectified before directed to a 12-/xF capacitor through a series of 6-M l) resistors, and the energy is delivered to the treatment chamber or load by discharging the capacitor through a thyratron switch. In this pulse generator a trigger circuit converts 5-V pulses to 500-V pulses using a silicon control rectifier. A pulse with a 2-/.~sec width and a 2-sec period (Fig. 1.7) characterizes the pulse waveform of this system. A versatile pulse-forming network, capable of delivering square, bipolar and exponential wave-shape pulses has been used successfully by Zhang et al. (1997). The pulse generator includes a 40-kV/8-kV c o m m a n d power supply that charges the system with two 100-1) resistors. A 50-kV/5-kA hollow anode thyratron tube switch delivers the energy at a maximum repetition rate of 1000 Hz. In an effort to uniform the PEF process, leading groups (EPRI, 1998) have established five main characteristics that a laboratory pulse generator should have in order to contain the cost of the system, reduce its complexity, and limit temperature rises above 70~ 9 a laboratory pulser power range between 1.5 and 2 kW and a flow rate of 20 l i t e r / h r 9 output voltages from 20 to 30 kV 9 pulse widths and repetition rates defined by power pulse rates and treatment chamber resistances 9 small pulse widths from 1 to 3 txsec * repetition rates up to 2000 Hz, determined by the energy into the load and pulse shape flexibility

III. Switches

25

Fig. 2.4 (a) The GeneZapper (IBI, Kodak) used for preliminary studies. (b) Major components of the commercial electroporator GeneZapper (reprinted from Food Technol., Vol. 49(12), Qin et al., "Food pasteurization using high-intensity pulsed electric fields," pp. 55-60, 1995, with permission from Elsevier Science). III.

Switches

Generally, two basic circuit configurations are available for the generation of high-power pulses: those that require " o n " and "off" switches and those that need only " o n " switches. This distinction is important because it is rather difficult to turn a machine off at high-power levels. An example of a circuit which requires both " o n " and "off" switches is the partial discharge of a capacitor, which results in a square-shaped wave. Only " o n " switches are n e e d e d for the full discharge of capacitors and pulse-forming networks to generate exponentially decaying and square-like wave-shape pulses, respectively. The switching elements available for this purpose are vacuum tubes, high-power transistors, ignitrons, triggered vacuum gaps, triggered spark gaps, thyratrons, tetrodes, semiconductors, and force-commutated thyristors

26

2. Design of PEF Processing Equipment

TABLE 2. I Switching Devices and Typical Expectations (EPRI, 1997)

Mode Anode U0 Peak current Repetitionrate Ignitron on Gas spark gap on Thyratron on Tetrode on/off Semiconductor on/off

20 kV 40 kV 50 kV 20 kV 1.2 kV

10-100 kA 10-20 kA 5-10 kA 5-10 kA 1 kA

Life(number of pulses)

singleshot to 10 Hz to 10 Hz to 10 Hz to 10 Hz

104 10 6

108 1010

1012

(in which a c o u n t e r c u r r e n t is injected to create zero current). T h e m i n i m a l desirable operation conditions of these switches such as operational modes, m a x i m u m peak voltages, c u r r e n t a n d pulse repetition ranges, as well as a p p r o x i m a t e lifetimes are p r e s e n t e d in Table 2.1. Ignitrons have some restraints, allow just single shots, have the shorter lifetime a m o n g the other switches, a n d p r e s e n t thermal conditioning problems due to the m e r c u r y content. Physics International C o m p a n y developed a spark gap switch (Fig. 2.5) a n d a m a t c h e d series-injection trigger g e n e r a t o r that is suitable for high-energy capacitor b a n k applications (Bhasavanish et al., 1991). Some of the advantages of this switch are its long life, low r e q u i r e d m a i n t e n a n c e , a n d superior p e r f o r m a n c e in terms of p e a k currents. T h e c o m p a n y p u t special care into its design in o r d e r to maximize the life of the switch: a two-electrode gap that operates without a trigger electrode to r e d u c e energy losses, coaxial c u r r e n t returns whose magnetic forces are b a l a n c e d to minimize vibration a n d mechanical loads, graphite electrodes so

Fig. 2.5 A schematic drawing of a spark gap switch (Bhasavanich et al., 1991).

27

III. Switches TABLE 2.2 ST-300 Demonstrated Operating Parameters (Bhasavanich et al., 199 I) operating voltage range peak current charge transfer inductance electrode tip life action integral switching gas size and weight

0-55 kV 280 kA 700 Cb < 200 nH 150,000 Cb (replaceable tips) 120 MJ/I~ air 9 in. dia.• 11 in. high, 20 lbs.

that buildup and pitting are diminished, and insulated housing placed far from the spark channel to lessen heat exposure as much as possible. In addition, the switch has a low prefire pressure and a probability of prefire as low as 10 -5. Because the switch has no separated trigger electrodes and must be directly overvolted, Physics International selected a spiral generator that is conveniently placed at the location of the switch that simplifies cabling and offers ruggedness and low impedance. Table 2.2 lists the operating parameters of this switch. The trigatron gap switch (Fig. 2.6) consists of high-voltage spherical electrodes, a grounded main electrode of spherical shape, and a trigger electrode through the main electrode. The trigger electrode is a metal rod with an annular clearance and is fitted into the main electrode through a bushing. The trigatron is polarity sensitive and requires a proper polarity pulse for correct operation (Naidu and Kamaraju, 1996).

H.V. Electrode

Earthed electrode Bushing

k

Anular gap,

[ii#iiiiiiiii!iiii!ili!il

iJ

li!iiii

Trigger Electrode

Main Gap Fig. 2.6 A schematic drawing of a trigatron gap switch. (Reproduced from Naidu and Kamaraju. "High Voltage Engineering," 2nd Ed, 1996, with permission from The McGraw-Hill Companies.)

28

IV.

2. Design of PEF ProcessingEquipment

Treatment Chambers

A traditional treatment c h a m b e r consists of two electrodes held in position by insulating material to form an enclosure containing the food to be treated. Parallel plate (Figs. 2.7 and 2.8), parallel wire, concentric cylinder

Fig. 2.7

An unassembled (a) and assembled (b) parallel plate static treatment chamber.

IV. Treatment Chambers

Fig. 2.8

29

A parallel plate continuous treatment chamber.

(Fig. 2.9), concentric cone, cofield tube, r o d - r o d , n e e d l e - p l a t e , and r o d - p l a t e are some of the possible electrode configurations. Parallel plates produce uniform electric field strength distribution in a large usable area and are the most practical choice. Concentric cylinders, however, provide smooth and uniform product flow and are attractive in industrial applications. One of the newest electrode configurations includes an e n h a n c e d electric field zone in the t r e a t m e n t c h a m b e r (Fig. 2.20), the implementation of which has led to more configurations (Figs 2.21-2.23) that allow a series of treatment zones with e n h a n c e d electric fields without significantly increasing the complexity of the PEF system. This c h a m b e r design is important to the development of PEF pasteurization technology. Food can be processed using PEF in a batchwise or continuous mode. Laboratory-scale studies of electric fields on foods have been conducted using both, but for industrial-scale operations a continuous m o d e is more economical and efficient. Static chambers are typically used for batchwise operations, whereas continuous and coaxial chambers were designed to facilitate a continuous flow of food during processing. Four major aspects have to be considered in the design of a t r e a t m e n t chamber, including 9 9 9 9

simulation for shape optimization electric field uniform distribution geometry and dimensions mechanical construction and materials

The geometry and dimensions of a c h a m b e r influence the design, construction, and price of the pulsing system in which it will be used. The chamber must permit ease of sterilization, the electrodes have to withstand

30

2. Design of PEF Processing Equipment

Fig. 2.9 An unassembled (a) and assembled (b) coaxial continuous treatment chamber.

the clean in place (CIP) or autoclaving process, a n d the m e c h a n i c a l characteristics n e e d to allow work at pressures of at least 7 bars. Features for cooling the electrodes should be included to prevent t e m p e r a t u r e rises above a target level typically assigned at 70~

IV. T r e a t m e n t Chambers

31

A. ElectrodeShape Optimization The aim in designing a high-voltage PEF treatment chamber is to attain high-intensity, spatially uniform electric fields in the treatment region for maximum microbial inactivation. To obtain high-field intensifies, the electrodes should be designed to minimize local field enhancements as these increase the probability of dielectric breakdown. Nonuniformity less than 10% is highly desirable. Adequate shaping of the electrode configuration is an essential task in high-voltage engineering design. Numerical electric field optimization enables a uniform field distribution within the treatment volume. Qin et al. (1995c) proposed a m e t h o d to optimize electrode configurations by correcting electrode contours to provide a uniform electric field. As presented in Fig. 2.10, point R a o n the electrode surface A moves to R'a in the normal direction while the potential of the electrode Va is kept constant and the field intensity (E a) i n c r e a s e s to E'a or Eta > E a,

where E'a =

Va [Rb -- R'a[

and E a

(2.2)

Va [Rb -- Ra["

Vb and R b are the potential voltage and field points of the electrode with surface B, respectively. Clearly, by moving the electrode contour points in a normal or opposite direction, the field intensity at the electrode surface can be increased or decreased. Electrode shape change is performed by moving the contour points in proportion to the difference between calculated and desired values of field intensity: if A E m _< k oEd

Ed - Ei Agi--- ~ g d - E i AEm

~

( k 0Ed )

~

a

"'",

otherwise

(2.3)

Vb

ER

AIII.I.R,R i >E'a

Ra

Ra

B Fig. 2. l0 Electrode shape optimization to obtain uniform electric fields at electrode surfaces A and B (Qin et al., 1995c).

32

2. Design of PEF Processing Equipment

where the maximum difference between the two electric fields is defined as AE m

=

max{IE a

-

Eil}, i = a, 2 . . . . Arc

(2.4)

and E d, Ei, ko, and Arc are the desired value of electric field intensity, field intensity at the i th point of the electrode contour (a factor that limits the moving distance of the node point), and the total n u m b e r of node points at which the correction is made, respectively. In this case E d was held constant in order to achieve a uniform electric field, and the factor k 0 chosen such that it provided an acceptable finite element mesh. If a node point moves more than the longest side of the triangular element, the meshing information of the element will be incorrect and the finite element m e t h o d (FEM) analysis will cease. Correction of the node points on the electrode contours is given by the following correction vector (Misaki et al., 1982): AEi) rex = r i 1 +

Ei

.

(2.5)

In the optimization based on finite elements, rci and r i are, respectively, the vectors defining the i th node point on the corrected and initial contours. E i was obtained by finite element computation. To simplify electrode optimization, the net space charge due to the movement of charged species (ions, protein, and living cells) in liquid foods may be ignored. The algorithm for electrode shape optimization consists of the following steps (Qin et al., 1995c): 1. Use a FEM to solve for the electric field inside a chamber with an initial contour design and a high-resolution mesh in the region of highintensity electric fields to reduce the numerical error. 2. Move the coordinates of the selected boundary nodes at the surface of the electrode within the treatment region in proportion to the difference in electric fields as described in Eq. (2.2). (This step results in a piecewise linear description of the electrode shape, which may contain n u m e r o u s spikes and edges.) 3. Obtain a smooth approximation of the piecewise linear shape using a spline routine. (Note that the electrode contour change should be conducted in the treatment region only, where only the FEM mesh will be affected.) 4. Regenerate the triangular mesh in the treatment region based on the approximated electrode contour and solve for the electric field intensity on the corrected electrode contour using FEM. The optimization criterion is to find an electrode contour that provides a uniform electric field at the electrode surfaces in the treatment region. If the calculated field distribution is not close enough to the uniform one, return to Step 2. This iteration procedure may end when the optimization criterion is satisfied.

IV. Treatment Chambers

B.

33

Static Chambers

Materials selected to construct a t r e a t m e n t c h a m b e r n e e d to be washable and autoclavable. Polysulfone and stainless steel are r e c o m m e n d e d for the insulation and electrodes, respectively. However, Bushnell et al. (1993) suggested using electrochemically inert materials such as gold, platinum, carbon, or metal oxides to construct the electrodes or electrode surfaces. Parallel plate electrodes with gaps sufficiently smaller than their electrode dimensions can achieve uniform electric field strength. Disk-shaped, roundedged electrodes can minimize electric field e n h a n c e m e n t and reduce the possibility of dielectric breakdown in fluid foods (Zhang et al., 1995). Designing t r e a t m e n t chambers to facilitate sample filling and removal adds to the complexity of their construction. Since a gas bubble is a potential trigger of dielectric breakdown, the filling ports need to facilitate a complete expulsion of air during filling (Zhang et al., 1995). Sale and Hamilton (1967) were a m o n g the earliest researchers to study the inactivation of microorganisms with PEF. At this time carbon electrodes supported on brass blocks were used and placed in a U,shaped polythene spacer as illustrated in Fig. 2.11. Using different spacers regulated the electrode area and a m o u n t of food that could be treated. The m a x i m u m electric field that the c h a m b e r could withstand was limited to 30 k V / c m due to the electrical breakdown of air above the food. The temperature of the food was controlled by the circulation of water through the brass blocks. The chamber designed by D u n n and Pearlman (1987) consists of two stainless-steel electrodes and a cylindrical nylon spacer. The c h a m b e r is 2 cm high with an inner diameter of 10 cm and an electrode area of 78 cm 2 (Fig. 2.12). The c h a m b e r is intended for treating liquid foods, which are introduced through a small aperture in one of the electrodes. The aperture can also be used for temperature m e a s u r e m e n t during the t r e a t m e n t of foods with high-intensity electric fields. The static t r e a t m e n t c h a m b e r designed at WSU is presented in Fig. 2.13. Two round-edged, disk-shaped stainless-steel electrodes were polished to mirror surfaces. Polysulfone or Plexiglas was used as the insulation material. The effective electrode area is 27 cm 2 and the gap between electrodes can

Fig. 2.11 The static chamber designed by Sale and Hamilton (1967).

34

2. Design of PEF Processing Equipment

Fig. 2.12 A cross section of the static chamber designed by Dunn and Pearlman (1987).

be selected at either 0.95 or 0.5 cm. Electric field strengths up to 70 k V / c m have been tested. Cooling of the c h a m b e r is provided by circulating water at preselected temperatures through jackets built into electrodes. An acoustic pressure pulse is observable while PEF is applied. Since a completely sealed t r e a t m e n t chamber is dangerous because of possible sparking and high pressure that could subsequently develop and cause the c h a m b e r to break apart, a pressure release device must still be included to ensure safe operation (Zhang et al., 1995). T r e a t m e n t chambers with parallel plate electrodes offer a uniform electric field distribution along the gap axes and electrode surfaces, but create a field e n h a n c e m e n t problem at the edges of the electrodes. The FEM was used to determine the o p t i m u m position of the insulating spacer in the WSU design so that the food to be treated could be held in the region of the uniform electric field. Section A in Fig. 2.14 represents the region of the uniform electric field, and section B the spacer. The o p t i m u m position for the boundary between sections A and B was d e t e r m i n e d using the FEM. Equipotential lines from the analysis using 4992 elements and 2651 nodes are presented in Fig. 2.15. In an effort to avoid p r o d u c t contact with the electrode wall, Lubicki and Jayaram (1997) proposed the use of a glass coil surrounding the anode

Fig. 2.13 The static chamber designed at WSU.

IV. Treatment Chambers

35

Fig. 2.14 Boundaries of electric field regions in a treatment chamber with parallel plate electrodes (the electric field is symmetrical about the center line, and only the top portion of the configuration is shown) (Qin et al., 1995c).

(Fig. 2.16). T h e s a m p l e v o l u m e o f t h e i r static c h a m b e r is 20 c m ~, w h i c h n e c e s s i t a t e s a filling l i q u i d with h i g h c o n d u c t i v i t y a n d similar p e r m i t t i v i t y to t h e s a m p l e ( m e d i a NaC1 s o l u t i o n tr = 0 . 8 - 1 . 3 S / m , filling l i q u i d w a t e r ~ 10 -a S / m ) u s e d b e c a u s e t h e r e is n o i n a c t i v a t i o n with a n o n c o n d u c t i v e m e d i a (i.e., t r a n s f o r m e r silicon oil). T h i s c o n f i g u r a t i o n m u s t also a d d r e s s t h e q u e s t i o n o f h o w efficiently t h e p u l s e e n e r g y c a n b e t r a n s f e r r e d i n t o t h e sample.

C.

Continuous Chambers

Static c h a m b e r s a r e m a i n l y suitable f o r l a b o r a t o r y use. F o r l a r g e r scale o p e r a t i o n s , c o n t i n u o u s c h a m b e r s a r e m o r e efficient. T o suit this p u r p o s e , D u n n a n d P e a r l m a n (1987) d e s i g n e d a c h a m b e r c o n s i s t i n g o f two p a r a l l e l

20-

z

15-

J

5

. . . . . . . . . .

!

0

!

5

!

!

15

- ~ . /

!

!

J

!

22

!

28

!

j

J

!

!

32

!

!

40

r(mm) ---'-0

& 100 . . . . .

80 - - - 6 0 . . . .

40 - - 2 0 I

Fig. 2.15 Calculated equipotential lines for the half-field region in a parallel plate treatment chamber (the potential distribution is given as a percentage of the electrode voltage) (Qin et al., 1995c).

36

2. Design of PEF Processing Equipment

Fig. 2.16 A treatment chamber with no food exposed directly to electrodes (adapted from Lubicki and Jayaram, 1997).

plate electrodes and a dielectric space insulator (Fig. 2.17). The electrodes are separated from the food by ion conductive m e m b r a n e s m a d e of sulfonated polystyrene and acrylic acid copolymers, but fluorinated hydrocarb o n polymers with p e n d a n t groups would also be suitable. An electrolyte is used to facilitate electrical c o n d u c t i o n between electrodes and ion p e r m e able m e m b r a n e s . Suitable electrolyte solutions include sodium carbonate, sodium hydroxide, potassium carbonate, and potassium hydroxide. These

Fig. 2.17 A continuous chamber with ion-conductive membranes separating the electrodes and food (adapted from Dunn and Pearlman, 1987).

IV. Treatment Chambers

37

Fig. 2.18 A continuous chamber with electrode reservoir zones (adapted from Dunn and Pearlman, 1987). are circulated continuously to remove the products of electrolysis a n d replaced in the event of excess c o n c e n t r a t i o n or depletion of ionic c o m p o nents. A n o t h e r c o n t i n u o u s c h a m b e r described by D u n n a n d P e a r l m a n (1987) is c o m p o s e d of electrode reservoir zones instead of electrode plates (Fig. 2.18). Dielectric spacer insulators that have slot-like openings (orifices) in between where the electric field is c o n c e n t r a t e d a n d liquid food is i n t r o d u c e d u n d e r high pressure. T h e average residence time in each of these reservoir zones is less than 1 min. T h e WSU static parallel plate electrode c h a m b e r was modified by a d d i n g baffled flow channels inside to m a k e it operate as a c o n t i n u o u s c h a m b e r (Fig. 2.19). Two stainless-steel disk-shaped electrodes separated by a polysulfone spacer f o r m the c h a m b e r . T h e designed operating conditions are:

Fig. 2.19 The continuous chamber with baffles designed by WSU: (a) cross section view and (b) top view (reprinted from Food Technol., Vol. 49(12), Qin et al., "Food pasteurization using high-intensity pulsed electric fields," pp. 55-60, 1995, with permission from Elsevier Science).

38

2. Design of PEF Processing Equipment

Fig. 2.20 A cofield treatment chamber (Sensoy et al., 1997).

c h a m b e r volume, 20 or 8 ml; electrode gap, 0.95 or 0.51 cm; a n d food flow rate, 1200 or 6 m l / m i n (Qin et al., 1996; Zhang et al., 1995). T h e c o n c e p t of e n h a n c e d electric fields in the t r e a t m e n t zone was applied by Yin et al. (1997) for the d e v e l o p m e n t of a continuous cofield flow PEF c h a m b e r (Fig. 2.20) with conical insulator shapes to eliminate gas deposits within the t r e a t m e n t volume. T h e conical regions were designed so that the voltage across the t r e a t m e n t zone could be almost equal to the supplied voltage. O t h e r configurations with e n h a n c e d electric fields are p r e s e n t e d in Figs. 2.21 a n d 2.22. In these devices the flow c h a m b e r s can have several crosssection geometries that may be u n i f o r m or n o n u n i f o r m . In this type of c h a m b e r configuration the first electrode flow chamber, insulator flow c h a m b e r , second electrode flow c h a m b e r , c o n d u c t i n g insert m e m b e r s , a n d

Fig. 2.21 A treatment chamber with different electrode geometries and enhanced electric fields in the insulator channel (adapted from Yln et al., 1997).

IV. Treatment Chambers

39

Fig. 2.22 A treatment chamber with enhanced electric fields in the insulator channel and tapered electrodes (adapted from Y'ln et al., 1997).

insulating insert m e m b e r are f o r m e d and configured such that the electrode flow c h a m b e r and insulator flow c h a m b e r form a single tubular flow chamber t h r o u g h the PEF t r e a t m e n t device (Yin et al., 1997). Figure 2.23 is a variant of the flow c h a m b e r configuration where the uniformity of the liquid p r o d u c t flow velocity is improved. In reality this configuration can be a plurality of c o n d u c t i n g m e m b e r s and at least one insulator m e m b e r . W h e n the voltage pulse signal is applied across high and g r o u n d voltage electrodes, an electric field is f o r m e d in the electrode flow channels as well as the insulator channel where the electric field strength is strongest. Therefore, the bactericidal effect of the PEF t r e a t m e n t process of this device occurs primarily in the liquid p r o d u c t flowing t h r o u g h the insulator flow channel. The electrodes of these chambers are of food grade stainless steel and the insulators of policarbonate, but can also be ceramic, glass, or plastic.

Fig. 2.23 A treatment chamber with improved flow characteristics and enhanced electric fields (adapted from Yin et al., 1997).

40

2. Design of PEF Processing Equipment

Coaxial configurations, which provide the advantage of a uniform fluid flow and simple chamber structure, can be easily manufactured and provide well-defined electric field distributions. The field intensity (E) between coaxial electrodes is given by (Zhang et al., 1995):

E = 1//[ r l n ( R 2 / R 1)],

(2.6)

where r is the radius at which the electric field is measured and R 2 and R 1 are the radii of the outer and inner electrodes, respectively. The uniformity of the electric fields in the t r e a t m e n t chambers with coaxial electrodes can be improved when R 2 - R 1 < R 1. Coaxial chambers are basically composed of an inner cylindrical electrode surrounded by an outer annular cylindrical electrode that allows food to flow between them (Bushnell et al., 1993). It is r e c o m m e n d e d that the length of the fluid flow path not be too small or too long c o m p a r e d to the c h a m b e r diameter. The electrical energy (W) consumed in each pulse is given by

W= EZv~'/p,

(2.7)

where E is the electric field (volts/cm), ~- is the pulse duration, v is the volume (ml), and p is the electrical resistivity of the food sample (ohm-cm). The coaxial chamber designed at WSU is based on a modified coaxial cylinder a r r a n g e m e n t (Fig. 2.24). A p r o t r u d e d outer electrode surface enhances the electric field within the treatment zone and reduces the field intensity in the remaining portion of the chamber. The electrode configuration was obtained by optimizing the electrode design with a numerical electric field computation. Using the optimized electrode shape, a pre-

Fig. 2.24 A cross-sectional view of the modified coaxial treatment chamber designed, constructed, and tested for microbial inactivation at WSU (Mart~n-Belloso et al., 1997).

V. Cooling System

41

Fig. 2.25 Electric field distribution in the food region between two electrodes of a coaxial treatment chamber (Qin et al., 1995c).

scribed field distribution along the fluid path without electric field enhancement points was determined. The outer electrode has a p r o t r u d e d contour surface that was obtained by numerical electric field optimization. Figure 2.25 illustrates the electric field distribution of the chamber in its treatment region; to allow for a clear view, the field inside the dielectric spacer is not presented. For numerical simplicity, a 100-V applied voltage was used in the field calculation in order to verify electric field uniformity. In the treatment region between the two electrodes, the potential drop is nearly uniform so a strong electric field is generated. Since outside the treatment region most of the potential drop occurs inside the spacer, the electric field is quite weak. The gap between the chamber's electrodes is adjustable within 2 to 6 m m or more by changing the inner electrode to different diameters. Cooling jackets are built into both electrodes to maintain low temperatures. The whole treatment chamber has an outer diameter of around 13 cm and an approximate height of 20.3 cm. The PEF system in which this chamber is used can handle flow rates from 30 to 120 l i t e r / h r (Qin et al., 1995c).

V.

Cooling System

When no cooling is provided, the increase in temperature (AT) of the fluid subjected to electric field pulses is given by

Q AT =

pfCp

E2n~'o =

pfCp

,

(2.8)

where Q is the energy input and pf a n d Cp are the density and specific heat of the fluid being treated, respectively. The energy input can be defined by

42

2. Design of PEF Processing Equipment

the product of the square of the electric field (E), the n u m b e r of pulses (n), and electrical conductivity (~r) of the fluid being treated. The food temperature is maintained by circulating constant temperature water through the cooling jackets built into the electrodes. A rise in temperature over 70~ must be avoided to preserve natural attributes of food products and to claim a n o n t h e r m a l treatment. Zhang et al. (1994) proposed a one-dimensional finite difference heat transfer model to predict the fluid temperatures in a static chamber. The fluid food is divided into circular disks as illustrated in Fig. 2.26. A disk layer of fluid with thickness Ad, radius r, and volume AV represents each node in the one-dimensional model. The t r e a t m e n t chamber is divided into 2-m nodes, where m is selected to be 20. Using an energy balance, the temperature of each element is related to its adjacent elements as Tr

pfCpAV

- T( =

At

Ad

T?_I - Ti" + ha

Ad

'

(2.9)

where pf is the density, Cp is the specific heat, k is the thermal conductivity of the fluid, T is the temperature, A is the area of heat transfer, At is the time interval of each time step, d is the electrode gap, V is the c h a m b e r volume, and Ad - d / 2 m and AV = V / 2 m are the thickness and volume of fluid for each node, respectively. (Superscripts denote the time step, and subscripts the space coordinate.) The food in contact with the electrodes in the model by Zhang et al. (1994) is assumed to have their same temperature and is therefore considered the boundary condition of the design. Energy input into the food should be uniform within the bulk and is only present at the time of pulse application. Energy input was treated as the initial condition of the model and proved to generate no heat after pulsing was generated. The fluid

/•

_

. ~ .

ermocoupleLocation

TmcontactBottomElectrode /

I

CenterAxis

Fig. 2.26 A one-dimensional finite difference grid of a fluid food inside a parallel plate static treatment chamber (Zhang et al., 1994).

VI. Typical Measurements in a PEF System

43

temperature is measured with a thermocouple inserted 2 m m into the food and halfway between the electrodes, and a thermocouple attached to the cathode gauges the electrode temperature. Figure 2.27 illustrates the predicted fluid center temperatures of apple juice subjected to 20 pulses at 30 sec intervals for energy input levels of 100, 300, and 600 J / p u l s e in the heat transfer model of Zhang et al. (1994). These energy levels correspond to electric fields of 12, 20, and 28 kV/cm, respectively. When repetitive pulses are applied, the fluid center temperature is significantly higher than the electrode temperature. By controlling the interval of time between pulses to longer than 30 sec, the maximum fluid temperature is expected to remain below 25~

VI.

Typical Measurements in a PEF System

Electrical parameters such as voltage and current pulse waveforms applied during PEF treatments should be recorded via a digital acquisition system. Outputs from voltage and current monitors can be recorded by a digital oscilloscope into a computer for future analysis, but it is important that each be placed in a shielded area to minimize electromagnetic interference. To achieve accurate measurements of voltages and currents, all important frequency components must be recorded, which includes specification of the bandwidth of each transducer. With these measurements, further evaluation of the applied energy per volume and spatial average of the electric fields in the treatment region can be computed in order to determine if the process is under its control limits. Direct measurement of the electric fields by

25.00 o

20J30 84 15s IO.O0-

5.oo

0.00

I 100

I 200

I 300

I 400

I 500

600

Time(s) Fig. 2.27 Model-predicted fluid temperatures of apple juice subjected to 20 pulses with energy inputs of 100, 300, and 600 J / p u l s e (the electrode temperature was maintained at 4~ (Zhang et al., 1994).

44

2. Designof PEF ProcessingEquipment

optical fibers in the treatment gap of the chamber would be highly desirable, but this anticipates complexity and increased cost. It is definitely advantageous to take oscilloscope readings with a wellvalidated measuring system. This is especially evident when the frequency content of pulses is desired, as change may indicate a partial discharge that could lead to sparks. The frequency content of pulses is obtained by applying the fast Fourier transform algorithm to the voltage and current as a function of time [v(t), i(t)]. The analog/digital converter is also useful to warn of missed or weak pulses. Because it is important to maintain a treated food temperature at low values to minimize thermal effects, measurements should be made with a high-precision thermocouple, (placed outside the processing chamber) or even the more sophisticated fiber optic transducer. The latter has a precision of up to 0.1~ and a response time of 0.2 sec. Ideally, about four temperature monitoring optical fibers in the interelectrode region, inlet, and outlet of the treatment chamber should be installed. The flow rate and pressure in PEF chambers are critical quantities in the control process and should be measured throughout the system as a function of time and space, respectively. The applied dosage will d e p e n d directly on the flow rate when the pulse frequency and treatment chamber volume are fixed. Although the pressure will not affect the inactivation rate or applied dosage, it will prevent arcing, and m e a s u r e m e n t will detect any plug or leakage in the system.

VII.

Packaging and S t o r i n g

After PEF treatment, foods are cooled if necessary and aseptically packaged. Aseptic technology has been used for liquid milk and fruit juices for more than 30 years and is also known as an effective m e t h o d of producing shelf-stable preserved products that have quality advantages over their conventionally canned or jarred counterparts. Some of these include packaging in a range of materials without limitations on container size and the use of thin plastic membranes or plastic/paper-laminated materials that do not have to withstand high temperatures as in conventional thermal processing, as no retorting of the product is applied after packaging. Additional benefits of such an operation include less damage to the product, shorter processing periods, uniform and improved quality, reduced energy consumption, and utilization of new packaging materials (Singh and Nelson, 1992). After packaging, the bagged foods may be stored at refrigerated or ambient temperatures depending on the type of product. For example, milk may have to be stored at refrigerated temperatures, whereas apple juice has a reasonable long shelf-life even when stored at room temperature. The effectiveness of aseptic packaging has been demonstrated by the extended

References

45

shelf-life of several PEF products (Qin et al., 1995a,d; Zhang et al., 1997). Because c o n s u m e r s d e m a n d freshness, aseptic packaging is the perfect c o m p l e m e n t for PEF to provide a cost-effective way for processors to deliver what markets expect.

VIII.

Final Remarks

This c h a p t e r reviewed key elements for the design of a PEF system a n d the description of the main c o m p o n e n t s that are involved in the p r o d u c t i o n of high-voltage pulses. T h e i m p o r t a n c e of the high-voltage repetitive pulser, switches, t r e a t m e n t chamber(s), cooling system(s), voltage a n d c u r r e n t measuring devices, control units, data acquisition system, a n d packaging system were addressed based on specific system r e q u i r e m e n t s , t r e a t m e n t convenience, a n d future needs. T h e efforts of leading groups a n d institutions to obtain the best processing conditions are p r e s e n t in the d e v e l o p m e n t of new t r e a t m e n t c h a m b e r s a n d PFN configurations. Mathematical simulation used in the design of t r e a t m e n t c h a m b e r s represents a safe a n d m o r e cost-effective tool in the r e f i n e m e n t of new configurations a n d minimization of trial-error steps, so the future i m p l e m e n t a t i o n of m a t h e m a t i c a l software is encouraged. A l t h o u g h there is increasing interest in PEF technology, we detect the n e e d for commercially available high-voltage pulsers a n d switching devices. T h e r e are m a n y suppliers of high voltage i n s t r u m e n t a t i o n but few are involved in the m a n u f a c t u r i n g of complete PEF systems. T h e next c h a p t e r reviews how a n d by which m e c h a n i s m s the pulses p r o d u c e d by the PEF systems discussed in this chapter affect biological materials such as cell m e m b r a n e s , microorganisms, a n d proteins.

References Bhasavanich, D., Hitchcock, S. S., Creely, P. M., Shaw, R. S., Hammon, H. G., and Naff, J. T. (1991). International Pulsed Power Conference, San Diego, California. Bushnell, A. H., Dunn, J. E., and Clark, R. W. (1993). High pulsed voltage systemsfor extending the shelf life of pumpable food products. U. S. Patent 5,235,905. Dunn, J. E., and Pearlman, J. S. (1987). Methods and apparatus for extending the shelf life of fluid food products. U. S. Patent 4,695,472. EPRI (1997). EPRI/Army PEF Workshop II, Chicago, Illinois, 10-11 October. EPRI (1998). Pulsed electric field processing in the food industry: A status report on PEF. Report CR-109742. Industrial and Agricultural Technologies and Services, Palo Alto, California. Ho, S. Y., Mittal, G. S., Cross, J. D., and Griffiths, M. N. (1995). Inactivation of Pseudomonas fluorescens by high voltage electric field pules. J. Food Sci. 60(6), 1337-1343. Lubicki, P., and Jayaram, S. (1997). High voltage pulse application for the destruction of the Gram negative bacterium Yersinia enterocolitica. Bioelectrochem. Bioenergetics 43, 135-141. Mart~n-Belloso, O., Vega-Mercado, H., Qin, B. L., Chang, F. J., Barbosa-C~novas, G. V., and Swanson, B. G. (1997). Inactivation of Escherichia coli suspended in liquid egg using pulsed electric fields. J. Food Proc. Pres. 21, 193-208.

46

2. Design of PEF Processing Equipment

Misaki, T., Tsuboi, Itaka, K., and Hara, T. (1982). Computations of three dimensional electric field problems by a surface charge method and its application to optimum insulator design. 1EEE Trans. Power Appar. Sys. 101(3), 627-634. Naidu, M. S., and Kamaraju, V. (1996). "High Voltage Engineering," 2nd Ed. McGraw-Hill, New York. Pagan, R., Esplugas, S., G6ngora-Nieto, M. M., Barbosa-Canovas, G. V., and Swanson, B. G. (1998). Inactivation of Bacillus subtilis spores using high intensity pulsed electric fields in combination with other food conservation technologies. Food Sci. Technol. Int. 4(1), 33-44. Qin, B. L., Pothakamury, U. R., Vega-Mercado, H., Martin, O., Barbosa-C{movas, G. V., and Swanson, B. G. (1995a). Food pasteurization using high-intensity pulsed electric fields. Food Technol. (Chicago) 49(12), 55-60. Qin, B. L., Vega-Mercado, H., Pothakamury, U. R., Barbosa-C{movas, G. V., and Swanson, B. G. (1995b). Application of pulsed electric fields for inactivation of bacteria and enzymes. J. Franklin Inst. 332a, 209-220. Qin, B. L., Zhang, Q., Barbosa-C{movas, G. V., and Swanson, B. G. (1995c). Pulsed electric field treatment chamber design for liquid food pasteurization using a finite element method. Trans. ASAE 38(2), 557-565. Qin, B. L., Chang, F. J., Barbosa-C~novas, G. V., and Swanson, B. G. (1995d). Nonthermal inactivation of Saccharomyces cerevisiae in apple juice using pulsed electric fields. Lebensm. -Wiss. Technol. 28, 564-568. Qin, B. L., Pothakamury, U. R., Barbosa-C{movas, G. V., and Swanson, B. G. (1996). Nonthermal pasteurization of liquid foods using high intensity pulsed electric fields. Crit. Rev. Food Sci. Nutr. 36(6), 603-627. Sale, A.J.H., and Hamilton, W. A. (1967). Effects of high electric fields on microorganisms. I. Killing of bacteria and yeasts. Biochim. Biophys. Acta 148, 781-788. Sensoy, I., Zhang, Q. H., and Sastry, S. K. (1997). Inactivation kinetic of Salmonella dublin by pulsed electric fields. J. Food Proc. Eng. 20, 367-381. Singh, R. K., and Nelson, P. E. (1992). "Advances in Aseptic Processing Technologies." Elsevier Science, London. Vega-Mercado, H., Powers, J. R., Barbosa-C{movas, G. V., and Swanson, B. G. (1995). Inactivation of plasmin using high voltage pulsed electric fields. J. Food Sci. 60, 1150-1154. Vega-Mercado, H., Pothakamury, U. R., Chang, F.J., Zhang, Q., Barbosa-C~novas, G. V., and Swanson, B. G. (1996). Inactivation of E. coli by combining pH, ionic strength and pulsed electric field hurdles. Food Res. Int. 29(2), 117-121. Ym, Y., Zhang, Q. H., and Sastry, S. H. (1997). High voltage pulsed electric field treatment chambers for the preservation of liquid food products. U. S. Patent 5,690,978. Zhang, Q., Monsalve-Gonz{tlez, A., Barbosa-C~novas, G. V., and Swanson, B. G. (1994). Inactivation of E. coli and S. cerevisiae by pulsed electric fields under controlled temperature conditions. Trans. ASAE 37(2), 581-587. Zhang, Q., Barbosa-C~movas, G. V., and Swanson, B. G. (1995). Engineering aspects of pulsed electric field pasteurization. J. Food Eng. 25, 261-281. Zhang, Q. H., Qiu, X., and Sharma, S. IL (1997). Recent developments in pulsed electric processing. In "New Technologies Yearbook" (D. I. Chandrana, ed.), pp. 31-42. National Food Processors Association, Washington, D.C.

CHAPTER 3

Biological Principles for Microbial Inactivation in Electric Fields

I.

Introduction

Because each preservation technology has its own inactivation mechanism, it is not surprising that PEF researchers are trying to explain this technology's particular mechanisms. This chapter reviews the proposed mechanisms found in the literature, as well as experimental evidence that support some of these theories. Cell electroporation and disruption that lead to cell swelling, shrinking, and lysis have been found to be some of the major degrading changes produced by PEF in bacteria and yeast (Castro et al., 1993). It has therefore been generally accepted that the main effects of PEF are produced in the microbial cell membrane, although the disruption of internal organelles and other structural changes has been also proven, suggesting that electroporation is not the only inactivation mechanism (Harrison et al., 1997). One of the important components of a biological cell is its membrane because it (a) acts as a semipermeable barrier, (b) extrudes extracellular enzymes and cell wall materials, (c) is the site of many complex activities, including RNA, protein and cell wall synthesis, electron transport, and oxidative phosphorylation, and (d) plays an important role in the control of DNA synthesis (Rogers et al., 1980). In a cell subjected to no stress, the membrane acts as a semipermeable barrier. Any damage to the membrane affects its functions and may lead to the inhibition of cell reproduction. PEF can cause electroporation (the permeabilization of the membranes of cells and organelles) or electrofusion (the connection of two separate membranes into one). It can also induce pearl chain formation and the rotation of cells in rotating electric fields. When exposed to electric fields, the cell membrane loses the property of semipermeability, which may be 47

48

3. Microbial Inactivation Principles

reversible. If it is, the cell regains permeability when the electric field is turned off. If not, the cell m e m b r a n e is structurally damaged and microbial inactivation results. Z i m m e r m a n n et al. (1976) described two types of effects of an external electric field on biological cells: the punch-through effect and dielectric breakdown. The first is seen only in larger cells as one of the electrodes should be located inside the cell. It occurs when current pulses m u c h longer than 100 msec are injected into a cell using intra- and extracellular electrodes. A nickel wire grid may be used as the external electrode while the internal electrode consisting of platinum-iridium may be inserted through a longitudinal micropipette into the cell. When an electric field is applied, the m e m b r a n e resistance decreases rapidly while conductance increases beyond the threshold potential. The electrical property changes are due to m e m brane polarization, where the accumulation of negative and positive charges takes place within the cell at the m e m b r a n e areas closest to the cathode and anode; if the stress is high e n o u g h the cell m e m b r a n e may even be mechanically ruptured. However, dielectric breakdown occurs when cells are subjected to pulses only microseconds in duration, the electric field is applied in an indirect manner, and the electrodes are external to the cell. It is believed that the increase in m e m b r a n e conductance due to PEF is caused by the formation of pores in a cell m e m b r a n e when exposed to electric fields. Schoenbach et al. (1997) explains how up to a threshold voltage on the order of several tens of millivolts above the resting m e m b r a n e voltage, the conductance is constant. Increasing the voltage above this threshold, however, causes a nonlinear increase in the c o n d u c t a n c e - - f i r s t in one particular channel and later in others. Further increase of the voltage causes more and more channels of the same type to open. The increased molecular exchange between cells and their environment and the cell stress due to chemical imbalances cause cell death, and the large increase in current due to the open channels is analogous to a dielectric breakdown of the m e m b r a n e . Electropores can be identified as m e m b r a n e defects, cracks, crater-like structures, or partially randomized m e m b r a n e structures that appear as transient blebs (Chang and Reese, 1990). Researchers have explained poration based on an increase of t r a n s m e m b r a n e potential, electromechanical compression of the m e m b r a n e , the viscoelastic properties of the m e m b r a n e , e n h a n c e m e n t of structural defects, and conformational changes in the lipid or protein molecules.

II.

Transmembrane

Potential

In a biological cell, the m e m b r a n e acts as an insulator shell to the cytoplasm, whose electrical conductivity is six to eight orders greater than that of the m e m b r a n e (Chen and Lee, 1994). The cell m e m b r a n e may also be regarded

II. Transmembrane Potential

49

as a capacitor filled with a low dielectric constant material of e - - 2 (Fig. 3.1a). W h e n a cell suspension is exposed to an electric field, the ions inside the cell move along the field until they are held back by the m e m b r a n e . As a result, free charges accumulate at b o t h m e m b r a n e surfaces. T h e accumulation of m o r e surface charges increases the electromechanical stress or t r a n s m e m b r a n e potential (TMP) (Kinosita and Tsong, 1977a; Z i m m e r m a n n , 1986), which is many orders greater than the applied electric field. For example, the i n d u c e d potential is greater in a larger cell with the same magnitude of external electric field, which means larger cells are m o r e susceptible to d a m a g e than smaller cells (Chen and Lee, 1994). The t r a n s m e m b r a n e potential u n d e r the influence of an external electric field could be approximately 500 times as large as the applied field (Kinosita and Tsong, 1977b). The m a x i m u m t r a n s m e m b r a n e potential (Uc) g e n e r a t e d by an external field is given by S c h o e n b a c h et al. (1997) as Uc = f * a o E c ,

(3.1)

Fig. 3. I Electroporation of the cell membrane by compression when exposed to high-intensity electric fields (the membrane is considered a capacitor and is represented by the hatched area; Ec represents the critical electric field intensity). (Reproduced from Rev. Phys. Biochem. Pharmacol., "Electrical breakdown, electropermeabilization and electrofusion," U. Zimmermann, Vol. 105, pp. 176-256, fig. 1, 1986, with permission of Springer-Verlag.)

50

3. Microbial Inactivation Principles

where a 0 is the outer radius of the cell, E c is the critical electric field strength, f * is a form factor. For spheres f * is 1.5, and for cylinders with a length 1 and diameter d it is defined by 1 / ( 1 - d/3). With these simple equations Schoenbach et al. (1997) showed how a critical electric field of 10 k V / c m is necessary to lysis a bacteria 1 /xm in d i a m e t e r with a critical voltage across a m e m b r a n e of 1 V. Teissie and Tsong (1980) f o u n d that an electric field of 2 k V / c m generates a potential of 0.9 V across an erythrocyte m e m b r a n e . Equation (3.2) associates the i n d u c e d TMP to the applied electric field (E), position of the pore in the m e m b r a n e ( M ) , and time of processing after the field is t u r n e d on (t) (Ho and Mittal, 1996):

Uc(E,M,t ) = -f*gErcosO(M)

(

t)

1 - exp - -

,

(3.2)

T

where f * is the cell shape factor; for spheres = 1.5; 0 is the angle between the direction of E and M; T is the characteristic time constant (relaxation time); r is the cell radius; and g is the relative electric permeability of the membrane:

g =

(

(r)

2 oreori

(2ore + orm)(2orm + ori) +

,

(3.3)

~m (orm)(2ore + ori)

where ore, ori, and orm are the electrical conductivities of the external suspending m e d i u m , cytoplasm, and cell m e m b r a n e , respectively; and d m is the m e m b r a n e thickness.

= rC c

(1 1) or1

+ ~ , 2ore

(3.4)

where C c is the cell m e m b r a n e capacitance per unit area. Kinosita and Tsong (1977a) define the m a x i m u m t r a n s m e m b r a n e potential (Umax) as Umax

-- f * ( v , Oo)REo,

(3.5)

where R is the largest semiaxis (i.e., R = a for an oblate and R = b for a prolate) and f * is a geometric factor that d e p e n d s on the axial ratio (v = b/a) and the angle (00) between the symmetry axis (b) and direction of the electric field (E 0) (Fig. 3.2) Hiilsheger et al. (1983) calculated the t r a n s m e m b r a n e potential i n d u c e d by an external electric field u n d e r the assumption of a parallel long particle axis and field vector (Table 3.1). Studies on the TMP and of the e n v i r o n m e n t s u r r o u n d i n g the cell m e m b r a n e have shown that the charges g e n e r a t e d on the m e m b r a n e sur-

Ih Transmembrane Potential

5 1

9

" ,

b

x

Fig. 3.2 A spherical cell exposed to a uniform electric field of E 0 [the cell's two principal axes are a and b (adapted from Kinosita and Tsong, 1997a)].

faces attract each other and are of opposite signs. This attraction gives rise to a compression pressure that causes the m e m b r a n e thickness to decrease (Fig. 3.1b). The electric forces and compression pressure increase with a decrease in the m e m b r a n e thickness. A further increase in the electric field intensity leads to a critical m e m b r a n e potential and subsequently to reversible membrane breakdown or pore formation (Fig. 3.1c). With much greater field strengths, larger and larger areas of the m e m b r a n e are subjected to breakdown (Fig. 3.1d). If the size and n u m b e r of pores become large in relation to

T A B L E 3. I Cell Size and Induced Membrane Potential of Studied Microorganisms a'b

Microorganism E. coli. (4 h) c E. coli. (30 h) ~ K. pseudomona P. aeruginosa S. aureus L. monocytogenes I C. albicans

2R (/zm)

1 (/zm)

V (/zm 3)

f*

Urn(V)

1.15 0.88 0.83 0.73 1.03 0.76 4.15

6.9 2.2 3.2 3.9 m 1.7 m

7.2 1.4 1.7 1.6 0.6 0.8 38.0

1.06 1.15 1.09 1.07 1.50 1.70 1.50

0.26 1.06 1.26 1.25 1.00 0.99 2.63

aReproduced from Radiat. Environ. cells," H. Hiilsheger, J. Portel, and with permission of Springer-Verlag. bum, m e m br a ne potential induced parallel long particle axis and field volume; f * , shape factor. CIncubation time.

Biophys., "Electric field effects on bacteria and yeast E. G. Niemann, Vol. 22, pp. 149-162, table 2, 1983,

by an external field E c u n d er the assumption of a vector; 2R, mean diameter; l, mean length; V, mean

52

3. Microbial Inactivation Principles

the membrane surface, irreversible breakdown associated with mechanical destruction of the cell occurs (Zimmermann, 1986). The primary effect of an electric pulse is to implant pores of limited sizes in cell membranes. In erythrocytes, pores allow the passage of ions and molecules with a molar mass less than 2000 g (such as potassium and sodium ions), but not larger molecules such as hemoglobin or enzymes. The stability of pores formed by electric fields will determine a permanent or reversible poration (i.e., a stable pore has an average size of 1-3 nm, with an area of 0.01 to 0.1% and a lifetime higher than 1 sec) (Kinosita and Tsong, 1977b; Tsong, 1990). Pore formation involves a two-step mechanism of initial perforation followed by pore expansion; the entire process depends on electric field intensity and pulse duration. Treatment suspensions with low-ionic strengths subjected to high-field intensities (Kinosita and Tsong, 1979) favor pore expansion. Pore formation was suggested to happen in the lipid or protein domains of the cell membrane. The primary effects of electric field pulses on cells may be represented by the following pathways (Tsong, 1990): A ~ B ---~C --~

--o B' ---*A

[a]

irrev Pclose ~

[b]

Popen ---0 Pdenatr

[o] Pathway [a] represents a possible mechanism of electric field-induced poration in the lipid domain. A ~ B is the pore initiation step and B --+ C is the pore expansion step, including any conformational changes and dipole reorientation of the lipid molecule. C --+ C' involves the formation of hydrophilic or aqueous pores, and C' --+ B' ~ A are the resealing steps that occur when the electric field is turned off. Pathway [b] represents a possible mechanism for the effects of electric fields on protein channels. Pclose, Popen, Pdenatr, Pirrev, and [O] represent a membrane protein channel in its closed, open, denatured, irreversibly denatured, and excited states, respectively. Pclose to Popen occurs when the transmembrane potential reaches the gating potential of the channel. Because of the high current density passing through the channel, Pdenatr takes place when local heat is generated as this may cause denaturation of the proteins, which could renature slowly or be unable to restore the initial conformation, resulting in an irreversibly denatured membrane protein channel (Tsong, 1990). It is also known that high-voltage pulses introduce electroconformational damage in channel proteins, as well as pores in the membrane (Chen and Lee, 1994).

III. ElectromechanicalCompression and Instability

53

Electroporation is a process that can be defined by a time sequence of three stages: (1) pore formation 3 msec after a pulse application, (2) pore expansion 20 msec after the electric pulse application when the expansion is from 20 to 120 nm in diameter, and (3) pore shrinkage and resealing several seconds after the electric pulse treatment. If the field persists, the size and number of pores (pore density) seem to increase to keep the membrane potential at or below the critical value (Chang et al., 1992). Less than 0.15% of the membrane area would be electroplated at a TMP of 1 V, and the membrane capacitance would remain relatively constant ( ~ 9.61 nF). Other studies (Chen and Lee, 1994) conclude that the area of pore formation would increase with an increase in electric field, whereas the pore radius would increase with an increase in pulse duration. Because pore density is a function of cell orientation where the induced potential is highest, it is possible that sites closest to the electrodes contain the maximum n u m b e r of pores. The membrane potential threshold for damage of voltage-gated channel proteins is greater than that for the damage of phospholipid membranes (Chen and Lee, 1994). This may be due to the structure of the phospholipid membrane, which consists of several lipid molecules held by hydration forces as opposed to chemical bonding, whereas amino acids and subgroups of channel proteins are assembled with chemical bonding. Differences in the charge densities and distributions might also account for the difference in threshold potentials. Some of the secondary effects of electric fields include disintegration of the cytoskeletal network of cells, disruption of membrane properties and cellular processes, and alterations in the chemistry of lipids, proteins, and carbohydrates. If the electric field is much stronger than the critical transmembrane potential, membrane cracks may develop and a large piece of the membrane may even be ripped away from the cell.

III.

Electromechanical and I n s t a b i l i t y

Compression

Ho and Mittal (1996) presented a critical review of the proposed theories to explain membrane permeabilization, which included analyses of the electromechanical instability theory and molecular reorientation theory, which are both based on structural changes of the membrane. The first considers membrane breakdown as a consequence of a decrease in the membrane thickness due to a compressing stress, where the membrane undergoes an increase in area per lipid, which destabilizes the bilayer. The electric compressive force (P~) per unit area of the membrane is given by Coster and

54

3. Microbial Inactivation Principles

Zimmermann (1975): -d al Pe = d 6 fa0 2

e:e~

dx,

(3.6)

where 6 is the membrane thickness, E is the electric field strength, e is the dielectric constant or relative electric permittivity, and e 0 is the electric permittivity of free space. If the electric field within the membrane is uniform and independent of position x, Eq. (3.6) becomes ••0 V2 Pe =

26-------~ ,

(3.7)

where V is the potential difference across the membrane. Compression of the membrane creates elastic strain forces. Assuming the membrane is an ideal elastic material, the mechanical restoring force (Pm) is given by Pm

--

dx

6 = Y in - - ,

o X

~0

Y[ J~

(3.8)

where Y is the elastic compressive modulus of the membrane and 6 o is the original unstrained thickness. When the compressive force and the restoring force are in equilibrium, P m + Pe = 0 or

262

=-Yln

~o "

(3.9)

For sufficiently large compression (i.e., small values of 6), Pe will increase more rapidly with decreasing 6 than Pm, and point membrane breakdown occurs. Assuming the elastic modulus Y remains constant, the critical potential difference (Uc) for electromechanical breakdown is given by 0.36791162 Uc

=

.

(3.a0)

~?~0

One of the drawbacks of the electromechanical instability theory just described is the assumption that the membrane acts as a capacitor containing a perfectly elastic dielectric (Zimmermann et al., 1976). Although supported by experimental evidence, this fails to take into account the subsequent behavior of transmembrane voltage, membrane conductance, electropores, and molecule transportation. It also fails to distinguish between nonreversible membrane rupture and reversible membrane discharge (Ho and Mittal, 1996). Some researchers argue that a better description of the cell membrane may be the fluid mosaic model composed of a lipid bilayer in a largely fluid state with embedded integral proteins (Jacob et al., 1981).

V. Viscoelastic Model

IV.

55

Osmotic Imbalance

The cause of hemolysis or rupture of the membrane is believed to be due to the osmotic imbalance generated by the leakage of ions and small molecules (Kinosita and Tsong, 1977b). Due to the osmotic pressure of the cytoplasmic contents, the cells begin to swell and the pores gradually shrink. When the cell volume approaches 155% of the normal volume, rupture of the cell membrane and lysis of the cell occurs (Tsong, 1990) (Fig. 3.3). The addition of a sufficient amount of impermeant substance to the suspension of pulse-treated erythrocytes retards hemolysis (Fig. 3.4) because cells are prevented from lysis and the membrane spontaneously reseals. The resealing process is temperature dependent, for at 37~ the treated membrane rapidly regains impermeability to cations, whereas at 3~ cells remain highly permeable, even after 20 hr. Two common applications of electroporation and resealing techniques are (1) the alteration of intracellular composition and simplification of experimental design in transport studies and (2) the use of erythrocytes as intravenous drug reservoirs. Gradual release from loaded erythrocytes could help maintain desired drug levels in patients (Kinosita and Tsong, 1977c).

V.

Viscoelastic Model

Electrical breakdown of membranes can be explained by their viscoelastic properties. Although the electromechanical compression theory does not take into account the surface tension and viscosity of the membrane, the Membrane Rupture

Pore Initiation o

o

o

o

o

oo

o

<

~-

< o

o

o

o.

-~.

< o

SWELLING

- o~O,

.

o

o ol

.~,o~o

9

<

-~ o

o

I

HEMOLYSIS

o Ions or Probe Molecules

9 Hemoglobin

Fig. 3.3 Electroporationof a cell membrane based on colloid osmotic swelling(adapted from Tsong, 1990).

56

3. Microbial Inactivation Principles 0 ..0

0

.

. . . .

0

0"0

.0

0

. . . .

9

0"0

o

~.

, ' '" o"-.LL.~o 9~ 9

0

.

.

oo

.

.

>.~o

;o ,

O.

Osmotically Balanced

Erythrocyte Exposed to Electric Field

9

0

.

.

oo

b

No Swelling

Drug Loaded/Resealed

9 Cytoplasmic Macromolecules

o Ions or Probe Molecules

Fig. 3.4 Addition of an i m p e r m e a n t substance to the pulse-treated suspension of erythrocytes retarding hemolysis (the open circles represent molecules of large molecular weight such as insulin or oligosaccharide and the small dots represent a drug being loaded into the erythrocytes) (adapted from Tsong, 1990).

viscoelastic model incorporates these parameters to determine the critical breakdown potential. It considers the m e m b r a n e as a thin viscoelastic film with fluctuating surfaces b o u n d by two semi-infinite bulk phases. Some of the assumptions made in this model include (a) the amplitudes of surface shape perturbations are much smaller than the average m e m b r a n e thickness (h); (b) the surface shape perturbations can be represented as a superposition of surface waves with wavelengths m u c h larger than the m e m b r a n e thickness (Fig. 3.5); (c) the m e m b r a n e is an incompressible body; and (d) the membrane behaves as a viscoelastic, isotropic material represented as a standard solid model composed of a Kelvin body with an elastic modulus (G') and viscosity (/x') in series with a linear spring with its own elastic modulus (Go'). This model is equivalent to the three-element Maxwell fluid model with a

z

A HA

h

Fig. 3.5 Sketch of a m e m b r a n e as a thin viscoelastic film: the m e m b r a n e is considered to have two surfaces (A and B), which fluctuate with amplitudes ~'A,B a r o u n d planes with an average separation of h (the average m e m b r a n e thickness), and H A and H B are the distances from an arbitrary plane of z - 0 to both surfaces. (Reproduced from J. Membr. Biol., "Electric fieldinduced breakdown of lipid bilayers and cell membranes: A thin viscoelastic model," D. S. Dimitrov, Vol. 78, pp. 53-60, fig. 1, 1984, with permission of Springer-Verlag.)

57

V. Viscoelastic Model

--AtTv--

•V•

G'o

G

B O

O ~

~ O

0'"

G

G'

(a)

(b)

Fig. 3.6 Two viscoelastic models to represent membrane dynamics: (a) a Kelvin body (G'0,/z') in series with a spring (G') and (b) a Maxwell body (Go,/z) in parallel with a spring (G). (Reproduced from J. Membr. Biol., "Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic model," D. S. Dimitrov, Vol. 78, pp. 53-60, fig. 2, 1984, with permission of Springer-Verlag.)

viscosity (/~) and elasticity (G 0) in parallel with a restoring spring (Fig. 3.6). It is an extension of the electromechanical model of membrane breakdown because it takes into account surface tension and membrane viscosity. The time (z) at which membrane breakdown occurs is given by Dimitrov (1984) as

~la

"/"---~O~( 2~2V424o' 3~mGh 1)' _

(3.11)

where a is the proportionality constant,/z is the viscosity, G is the elasticity, 8m is the relative dielectric constant of the membrane material, G0 is the permittivity of free space, V is the voltage, o" is the surface tension, and h is the membrane thickness. The critical breakdown potential (Uc) is given by 24o" Gh~ ) 0.25 (3.12) ~'m

Substituting G = E/3 for incompressible bodies where E is the Young's modulus, Eq. (3.12) can be written as 8o')~ Vc2 " -

~-

Eh 2 oo---OOm ~ 9

(3.13)

Equation (3.13) shows that low breakdown potential (Uc) can occur due to low o- even for large E. It can therefore explain decreasing membrane stability with decreasing surface tension. The average membrane thickness may not be changed significantly, but the amplitude of the local shape perturbations can increase and thus lead to local breaking of membrane and pore formation. The Maxwell fluid model predicts experimental data of

58

3. Microbial Inactivation Principles

critical breakdown potential at short times. The process of electric fieldi n d u c e d m e m b r a n e breakdown may be divided into three stages: (1) growing of m e m b r a n e shape fluctuations, (2) molecular r e a r r a n g e m e n t s leading to pore formation, and (3) expansion of the pore resulting in m e c h a n i c a l breakdown of the m e m b r a n e (Fig. 3.7). T h e total time of m e m b r a n e breakdown can be r e p r e s e n t e d as the sum of the times c o r r e s p o n d i n g to each stage. This theory, p r e s e n t e d by Dimitrov (1984), describes the first stage, and if rigorously followed, is valid up to a local thickness change on the order of 0.1 times the average m e m b r a n e thickness (0.1"h) (Fig. 3.5).

VI.

Hydrophobic and Hydrophilic Pores

Due to the polar c o m p o n e n t s of a lipid bilayer, it is highly likely that u n d e r external electrical stresses i n d u c e d by electric fields, electropores will be formed. These pores may have a wall of hydrocarbon lipid tails that are n o m i n a t e d as hydrophobic pores or a wall of reoriented lipid molecules with polar heads known as hydrophilic pores. T h e formation of h y d r o p h o b i c or hydrophilic pores can be explained based on pore energies at different pore radii. Pore energy is the change of free energy resulting from the formation of a cylindrical pore with a radius (r) in the lipid bilayer. A h y d r o p h o b i c pore of zero radius with zero energy represents the m e m b r a n e in an u n d i s t u r b e d state. W h e n r is small, the formation of hydrophobic pores is energetically m o r e favorable because at small radii they possess lower energy than hydrophilic pores, which is generally assumed to be the initial stage of electroporation. W h e n the radius of hydrophobic pores exceeds a critical value (r*) varying between 0.3 and 0.5 nm, the pore energies of both h y d r o p h o b i c and hydrophilic pores b e c o m e equal. W h e n r is greater than r*, the energy of hydrophilic pores is lower than that of hydrophobic pores, which causes a reorientation of the m e m b r a n e pores toward a lower energy configuration; in other words, to form hydrophilic pores, which is called pore inversion (Fig. 3.8). Figure 3.9 explains the formation of hydrophobic and hydrophilic pores d e p e n d i n g on pore radii and energies. T h e formation

(a)

(b)

(c)

Fig. 3.7 Electroporation of cell membranes based on a viscoelastic model: (a) a growing number of membrane fluctuations, (b) molecular rearrangements leading to discontinuity, and (c) expansion of the pore resulting in mechanical breakdown of the membrane. (Reproduced from J. Membr. Biol., "Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic model," D. S. Dimitrov, Vol. 78, pp. 53-60, fig. 4, 1984, with permission of Springer-Verlag.)

VI. Hydrophobic and Hydrophilic Pores

59

Hydrophobic Pore

Hydrophilic Pore

Fig. 3.8 Hydrophobic and hydrophilic pores. (Reprinted from Biochim. Biophys. Acta, 940, Glaser et al., "Reversible electrical breakdown of lipid bilayers: Formation and evolution of pores," pp. 275-287, (1988), with permission from Elsevier Science.)

of hydrophilic pores with an effective radius of 0.6 to 1.0 n m causes reversible electrical breakdown or an increase in m e m b r a n e c o n d u c t a n c e a n d permeability to ions. W h e n the radius of the pores exceeds r d, they will grow indefinitely a n d result in m e c h a n i c a l breakdown of the m e m b r a n e . To f o r m hydrophilic pores, an energy barrier c o r r e s p o n d i n g to the critical size h y d r o p h o b i c pore must be overcome. T h e h e i g h t of the barrier can decrease by an a m o u n t p r o p o r t i o n a l to the square of the m e m b r a n e voltage, which m e a n s the rate of p o r e formation is exponentially d e p e n d e n t on the voltage across the m e m b r a n e . T h e evolution of m e m b r a n e pores was divided into three stages by C h a n g a n d Reese (1990) based on the observed a p p e a r a n c e of discrete pore-like or volcano-shaped structures at the site of electroporation in red blood cells. These intervals are (1) p o r e creation a n d expansion, which occurs in microseconds; (2) a standby p e r i o d where the pore structures basically r e m a i n stable for a few milliseconds; and (3) pore shrinkage due to a resealing process. (It is i m p o r t a n t to note that partially resealed pores have a m u c h longer lifetime than the transient m e m b r a n e openings observed in the second stage.) As m e n t i o n e d earlier, m e m b r a n e p o r a t i o n induces the chances of m e m b r a n e conductivity. Glaser et al. (1988) described three processes with different time constants that d e t e r m i n e the

Ed

Energy E,

r,

rm

rd

Pore Radius Fig. 3.9 Energyof hydrophobic and hydrophilic pores at different pore radii: (a) hydrophobic pore and (b) hydrophilic pore. (Reprinted from Biochim. Biophys. Acta, 940, Glaser et al., "Reversible electrical breakdown of lipid bilayers: Formation and evolution of pores," pp. 275-287, (1988), with permission from Elsevier Science.)

60

3. Microbial Inactivation Principles

changes in membrane conductivity after breakdown: (1) when a steep conductivity decrease (or increase) occurs almost instantaneously ( < 2 /zsec) with a change of applied voltage but a constant number and size of pores; (2) if within approximately 1-10 msec, the mean radius of the pores gradually decreases to 0.5 nm; and (3) the resultant small pores are long-living due to an energy barrier preventing their closure.

VII.

Theories Based on Conformational Changes

The membrane reorientation theory proposed by Neumann and Rosenheck (1972) is based on the electric field-induced displacements of membrane components. Because electric fields induce strong polarization leading to lateral displacements of membrane components such as lipids, transient permeability changes result. Furthermore, due to the transient characteristics of the reoriented polar heads of the pores and the associated increase in membrane conductance, both disappear as soon as the external electric field or triggering potential disappears. Schwarz (1978) described these changes as conformational transitions of integral macromolecular structures such as lipid or protein components of a membrane. Schwarz also agreed that these alterations may lead to electrical a n d / o r mechanical breakdown of the membrane. Electric fields may not only induce conformational changes in membrane components, but phase transitions from gel to liquid-crystalline phases and reorganization of the membrane structure, resulting in pore formation (Teissie and Tsong, 1981; Jayaram et al., 1992) as well. Another approach to explaining pore formation is the concept of the electric fieldinduced enhancement of structural defects present in a membrane (Jacob et al., 1981) Chang and Reese (1990) summarized the chain of effects caused by PEF on the structure of the cell membrane in terms of (a) primary effects, including dielectric breakdown due to induced membrane potential and the structural fatigue caused by mechanical stress; (b) secondary effects, including outcomes created by the movement of ions and molecules after the cell is permeabilized by the electric field, local heating, and membrane stress caused by material flow; and (c) tertiary effects due to changes in the cell as a result of permeabilization, including cell swelling or shrinking and disruption of cytoskeletal structures. After using rapid freezing electron microscopy, Chang and Reese (1990) found that electropore formation is not likely to be determined solely by primary effects because their applied pulse was not enough to override the transmembrane potential, but rather by secondary effects due to the flow of hemoglobin. Furthermore, they determined that the electropores were stabilized by attachments to cytoskeletal proteins, which suggested the formation of electropores by irreversible breakdown of the membrane in a local region. It is important to note,

VIII. Electric Field-Induced Structural Changes

61

however, that this is contradictory to earlier reports assuming that pores are formed by reversible breakdown. The irreversible breakdown of electropores may be as large as 20-120 nm in the first few seconds ( ~ 10 sec), which is big enough to allow long molecules such as DNA ( > 6 nm) to diffuse into cells. This was difficult to explain by the reversible breakdown theory, where pores on the order of 1 nm were suggested. Even though irreversible membrane poration and structural changes are the more appealing explanations for microbial inactivation by PEF, chemical effects may also cause the entry of toxic substances through a temporarily disrupted cell membrane that leaves no evidence of mechanical disruption. However, it is possible that under much stronger field strengths, permanent membrane poration may result in cell death due to mechanical disruptions and chemical effects. Other explanations for the bactericidal effect of PEF are induced mutations, the lysis of protoplasts, the reduction of pH, the release of proteolytic enzymes, the presence of chlorine-containing compounds, or the formation of H202, free oxygen, free hydrogen, hydroxyl and hydroperoxyl radicals, or metal ions from electrodes (Palaniappan et al., 1990).

VIII.

Electric Field-Induced Structural Changes

This section outlines some of the structural changes induced by electric fields on vegetative cells. Earlier researchers pointed out that electric fields induce conductivity changes and loss of the ability to plasmolyze without any global damage to cells. However, this may be due to the application of low electric field intensities and short pulse widths. With the use of higher electric field intensifies, structural damage has been reported in Lactobacillus, Saccharomyces, Escherichia coli, and Staphylococcus spp. Leakage of ninhydrin-positive material and 260-/zm absorbing material occurred when Hamilton and Sale (1967) subjected cells of E. coli 8196 to 10 pulses with a duration of 20 /zsec and electric fields ranging between 5 and 19.5 kV/cm. The electric field treated E. coli cells also lost the ability to synthesize /3-galactosidase when incubated with lactose. Cells subjected to higher intensity electric fields (22 kV/cm) showed a loss of ability to plasmolyze in a hypertonic medium (20 m M phosphate buffer, pH 7.2, with 10% sucrose). A further increase of the electric fields to 25 k V / c m led to destruction of the cell membrane, where the cells appeared flat with an irregular outline due to the loss of intracellular contents. However, there was no evidence that the bimolecular structure of the cell membrane was destroyed. Pothakamury (1995) found that untreated control cells of E. coli (ATCC 11229) exhibited a closely attached cytoplasm and outer membrane (Fig. 3.10). However, exposure to electric fields resulted in the cytoplasm drawing

62

3. Microbial Inactivation Principles

Fig. 3. I0 Untreated (a) and electric field treated (b) cells of Escherichia coli in SMUF using 64 pulses at 60 k V / c m and a processing temperature of 13~ as seen with TEM (adapted from Pothakamury, 1995).

VIII. Electric Field-Induced Structural Changes

63

Fig. 3. I I Untreated (a) and electric field treated (b) cells of E. coli in SMUF as seen with SEM (adapted from Pothakamury, 1995).

64

3. Microbial Inactivation Principles

Fig. 3.12 Cells of E. coli in SMUF as seen with TEM (a) treated with 32 pulses at 60 kV/cm and 13~ (b) 20~ and (c) 30~ (adapted from Pothakamury, 1995).

VIII. Electric Field-Induced Structural Changes

65

Fig. 3.12 Continued.

away from the outer membrane. Electric field treatment also caused the outer membrane to be crenated similar to the edge of a saw. Crenations observed in the PEF-treated cells may be an indication of cell shrinkage because untreated cells exhibited a more uniform distribution of cytoplasm, which was observed to be more granular and clumped than untreated cells. These conclusions may not be obtained with the scanning electron microscopy (SEM) technique, where no significant difference was observed between untreated control and electric field-treated cells (Fig. 3.11). The enhancing effect of the processing temperature after 32 pulses at 60 kV is presented in Fig. 3.12; when electric field-treated cells are compared with untreated and heat treated cells, most of the cytoplasm material appears to leak out of the cells or clump together after the heating process, and the internal organization is completely lost. However, the outer m e m b r a n e is still smooth and intact, without any crenations as occur in electric field-treated cells (Fig. 3.13). Working with S. cerevisiae cells suspended in deionized water, Mizuno and Hori (1988), found them p u n c t u r e d by electric field pulses with an intensity of 20 k V / c m and a pulse width of 160/xsec after 28 msec. Since few cells were punctured, it was assumed that the electric fields inactivated the cells without any structural damage. Zheng-Ying and Yan (1993) also reported an electric field-induced puncture of yeast cells.

66

3. Microbial Inactivation Principles

Fig. 3.13 Cells of E. coli in SMUF as seen with TEM (a) untreated, (b) treated with 64 pulses at 60 k V / c m at 13~ and (c) heat treated 5 min at 75~ (adapted from Pothakamury, 1995).

VIII. Electric Field-Induced Structural Changes

Fig. 3.13

67

Continued.

Additional research conducted by Harrison (1996) and Harrison et al. (1997) with the use of transmission electron microscopy (TEM) revealed that electric field treatment caused important structural changes to S. cerevisiae cells (ATCC 16664)suspended in apple juice. An a b u n d a n t increase in bud scar formation, cell elongation, and surface roughening was also observed (Fig. 3.14). Shrinkage and leakage of cytoplasmic material were detected when the yeast cells were subjected to 60 pulses with an electric field of 40 k V / c m at 10~ Massive damage in the form of a cell wall hole was also noticed (Fig. 3.15). Most of the cellular organelles, such as the nuclear m e m b r a n e and ribosomes, were partially or completely disintegrated. Furthermore, it was concluded that TEM did not support the electroporation mechanisms as the major mode of yeast inactivation, but that PEF treatment resulted in cytological disruption of a large portion of the yeast cellular organelles (especially ribosomal bodies), which was the primary mode of S. cerevisiae inactivation, with electroporation acting as a secondary mechanism of inactivation. Mizuno and Hayamizu (1989) observed cell surface roughening when S. cerevisiae cells were treated with an electric field of 10 or 14 k V / c m . They determined that 1 in every 100 or 200 cells was deformed. The n u m b e r of deformed cells increased with an increase in electric field

68

3. Microbial Inactivation Principles

Fig. 3.14 Untreated (2 and 3) and electric field treated (4 and 5) cells of S. cerevisiae in apple juice using 40 kV/cm as seen with SEM (adapted from Harrison, 1996).

intensity. S o m e of the cells h a d small holes or craters at the c e n t e r with fine debris attached. J a y a r a m et al. (1992) t r e a t e d L . brevis cells with an electric field of 25 k V / c m for 2.4, 6, a n d 16 msec, a n d f o u n d t h a t the e x t e n t of cell d i s r u p t i o n a n d fibril c o n t e n t i n c r e a s e d with an i n c r e a s e in t r e a t m e n t time. Cell inactivation was s u g g e s t e d to be d u e to irreversible cell m e m b r a n e b r e a k d o w n a n d l e a k a g e o f cellular contents. However, L . brevis cells t h a t were autoclave

VIII. Electric Field-Induced Structural Changes

69

Fig. 3.15 Cells of S. cerevisiae in apple juice. (a) Untreated cell: N, nucleus; M, mitochondria; V, vacuole; (arrow) ribosomes; and B, bud scar. (b) Treated cell by PEF (64 pulses, 40 kV/cm): CD, cellular debris; (arrow) decreased cytoplasm; NM, disrupted nuclear membrane; and CW, disrupted cell wall (adapted from Harrison, 1996).

70

3. Microbial Inactivation Principles

Fig. 3.16 Untreated (a) and electric field treated (b) cells of kV/cm seen with SEM (adapted from Pothakamury, 1995).

S. a u r e u s

in SMUF using 60

VIII. Electric Field-Induced Structural Changes

71

sterilized using steam at 121~ for 40 min showed less structural damage than electric field-treated cells. When Pothakamury et al. (1997) treated cells of S. aureus suspended in a SMUF with PEF and observed the effects with SEM, they discovered that those subjected to electric fields exhibited rough surfaces (Fig. 3.16), whereas untreated cells appeared to have smooth surfaces and thick walls (Fig 3.17).

Fig. 3.17 1995).

Untreated cells of

S. a u r e u s

in SMUF as seen with TEM (adapted from Pothakamury,

72

3. Microbial Inactivation Principles

Fig. 3.18 Electric field treated (a) and heat treated (b) cells of 60 k V / c m as seen with TEM (adapted from Pothakamury, 1995).

S. aureus

in SMUF using

References

73

The degree of roughness varied in electric field-treated cells (Fig. 3.16b) and may have been due to rupturing and leakage of their cytoplasmic contents, which was seen as debris in between the cells after t r e a t m e n t with 64 pulses at 60 k V / c m and 13~ (Fig. 3.18a). The effect of heat t r e a t m e n t (10 min at 66~ on S. aureus suspended in SMUF is presented in Fig. 3.18b, where TEM reveals great damage to the cell organelles, between whom no distinction is possible to observe. However, cell wall rupture as induced by electric fields was not observed in heat-treated cells.

IX.

Final R e m a r k s

The primary effect of biological cell exposure to electric fields is an increase of the t r a n s m e m b r a n e potential, which results in electroporation. The formation of pores in the cell m e m b r a n e leads to reversible (electrical) or irreversible (mechanical) breakdown and depends on the magnitude of the t r a n s m e m b r a n e potential. Several theories have been discussed to explain how pores are formed, but it is unclear whether it occurs in the lipid or protein matrices. The molecular mechanism of electroporation is also uncertain, although some researchers postulate that pores are formed due to electroconformational changes in lipid or protein molecules. It has also been suggested that the mechanical breakdown of cell m e m b r a n e s may not be caused by electroporation, but as secondary effects such as osmotic imbalances between cells and their environment. Electric field induced structural changes in microbial cells and m e m branes of different microorganisms give support to some of the reviewed theories, although the microbial inactivation principle seems to vary from microorganism to microorganism. In addition, the same species apparently u n d e r g o different PEF effects, which may be caused by different inactivation mechanisms d e p e n d i n g on t r e a t m e n t conditions and suspending media. Although they have not been considered as factors until now, the technique and protocol followed to evaluate ultrastructural changes may also have an important effect on the observed results, as m e m b r a n e and structural reorganization may be taking place within microseconds after treatment.

References Castro, A., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1993). Microbial inactivation of foods by pulsed electric fields. J. Food Proc. Pres. 17, 47-73. Chang, D. C., and Reese, T. S. (1990). Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. Biophys. J. 58, 1-12. Chang, D. C., Chassy, B. M., Saunders, J. A., and Sowers, A. E., eds. (1992). "Guide to Electroporation and Electrofusion," pp. 9-28. Academic Press, San Diego. Chen, W., and Lee, R. (1994). Altered ion channel conductance and ionic selectivityinduced by large imposed membrane potential pulse. Biophys. J. 67, 603-612.

74

3. Microbial Inactivation Principles

Coster, H. G. L., and Zimmermann, U. (1975). The mechanism of electrical breakdown in the membranes of Valonia utricularis. J. Membr. Biol. 22, 73-90. Dimitrov, D. S. (1984). Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic model. J. Membr. Biol. 78, 53-60. Glaser, R. W., Leikin, S. L., Chernomordik, L. V., Pastushenko, V. F., and Sokirko, A. I. (1988). Reversible electrical breakdown of lipid bilayers: Formation and evolution of pores. Biochim. Biophys. Acta 940, 275-287. Hamilton, W. A, and Sale, A. J. H. (1967). Effects of high electric fields on microorganisms II. Mechanism of action of the lethal effect. Biochim. Biophys. Acta 148, 789-800. Harrison, S. L. (1996). High intensity pulsed electric field and high hydrostatic pressure processing of apple juice. Ph.D. Dissertation, Washington State University, Pullman, Washington. Harrison, S. L., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1997). Saccharomyces cerevisiae structural changes induced by pulsed electric field treatment. Lebensm. Wiss. Technol. 30, 236-240. Ho, S. Y., and Mittal, G. S. (1996). Electroporation of cell membranes: A review. Crit. Rev. Biotechnol. 16(4), 349-362. Hi~lsheger, H., Potel, J., and Niemann, E. G. (1983). Electric field effects on bacteria and yeast cells. Radiat. Environ. Biophys. 22, 149-162. Jacob, H. E., F/Srster, W., and Berg, H. (1981). Microbiological implications of electric field effects. II. Inactivation of yeast cells and repair of their envelope. Z. AUg. Microbiol. 21(3), 225-233. Jayaram, S., Castle, G. S. P., and Margaritis, A. (1992). Kinetics of sterilization of Lactobacillus brevis by the application of high voltage pulses. Biotechnol. Bioeng. 40(11), 1412-1420. Kinosita, K., Jr., and Tsong, T. Y. (1977a). Voltage-induced pore formation and hemolysis of human erythrocytes. Biochim. Biophys. Acta 471,227-242. Kinosita, K., Jr., and Tsong, T. Y. (1977b). Hemolysis of erythrocytes by a transient electric field. Proc. Natl. Acad. Sci. U.S.A. 74, 1923-1927. Kinosita, K., Jr., and Tsong, T. Y. (1977c). Formation and resealing of pores of controlled sizes in human erythrocytes. Nature (London) 268, 438-441. Kinosita, K., Jr., and Tsong, T. Y. (1979). Voltage induced conductance in human erythrocyte membranes. Biochim. Biophys. Acta 554, 479-497. Mizuno, A., and Hayamizu, M. (1989). Destruction of bacteria by pulsed high voltage application. Presented at the Sixth International Symposium on High Voltage Engineering, New Orleans, Louisiana. Mizuno, A., and Hori, Y. (1988). Destruction of living cells by pulsed high-voltage application. IEEE Trans. Ind. Appl. 24(3), 387-394. Neumann, E., and Rosenheck, K. (1972). Permeability changes induced by electric impulses in vesicular membranes. J. Membr. Biol. 10, 279-290. Palaniappan, S., Sastry, S. K., and Richter, E. R. (1990). Effects of electricity on microorganisms: A review. J. Food Proc. Pres. 14, 393-414. Pothakamury, U. R. (1995). Preservation of foods by nonthermal processes. Ph.D. Dissertation, Washington State University, Pullman, Washington. Pothakamury, U. R., Barbosa-C{movas, G. V., Swanson, B. G., and Spence, K. D. (1997). Ultrastructural changes in Staphylococcus aureus treated with pulsed electric fields. Food Sci. Technol. 3, 113-121. Rogers, H.J., Perkins, H. R., and Ward, J. B. (1980). "Microbial Cell Walls and Membranes." Chapman & Hall, London. Schoenbach, K. H., Peterkin, F. E., Alden III, R. W., and Beebe, S.J. (1997). The effect of pulsed electric fields on biological cells: Experiments and applications. FEE Trans. Plasma Sci. 25(2), 284-292. Schwarz, G. (1978). On the physicochemical basis of voltage-dependent molecular gating mechanisms in biological membranes. J. Membr. Biol. 43, 127-148. Teissie, J., and Tsong, T. Y. (1980). Evidence of voltage induced channel opening in Na, K-ATPase of human erythrocytes membranes. J. Membr. Biol. 55, 133-140.

References

75

Teissie, J., and Tsong, T. Y. (1981). Electric field induced transient pores in phospholipid bilayer vesicles. Biochemistry 20, 1548-1554. Tsong, T. Y. (1990). Review: On electroporation of cell membranes and some related phenomena. Bioelectrochem. Bioenergetics 24, 271-295. Zheng-Ying, L., and Yan, W. (1993). Effects of high voltage pulse discharges on microorganisms dispersed in liquid. Presented at the Eighth International Symposium on High Voltage Engineering, Yokohama, Japan. Zimmermann, u. (1986). Electrical breakdown, electropermeabilization and electrofusion. Rev. Phys. Biochem. Pharmacol. 105, 176-256. Zimmermann, U., Pilwat, G., Beckers, F., and Riemann, F. (1976). Effects of external electrical fields on cell membranes. Bioelectrochem. Bioenergetics 3, 58-83.

CHAPTER 4

PEF-InducedBiological Changes

I.

Introduction

This chapter discusses the biological and chemical changes associated with the treatment of various food products by PEF. When an electric field is applied to a food contained between two electrodes in the form of a short burst of high voltage, it is hypothesized that microbial inactivation takes place due to biological changes induced by the high potential developed within the food. Researchers have tried to explain microbial inactivation by means of various theories and inactivation mechanisms based on specific characteristics of microbial cell membranes, media, and processing conditions. Furthermore, experimental studies have identified important factors (i.e., type of microorganism, processing conditions) that control inactivation rates, which in some cases provide supporting evidence for these inactivation theories. Some of the biological changes induced by electric fields include electropermeabilization, electrofusion, motility alteration, and microorganism inactivation. Electropermeabilization is the increase in permeability of a cell when subjected to electric field pulses of high intensity and short duration, whereas electrofusion is the fusion of two or more cells by applying electric field pulses. Chemical changes during high-voltage pulses may also be present and will depend on the type and initial concentration of microorganisms, volume of the medium used, distribution of chemical radicals, and electrode material. Because the interactions of these variables are complex, a high degree of control must be exercised to minimize such changes. When an electric field is applied in the form of high-voltage arc discharges, microbial inactivation occurs mainly due to chemical actions, which include the formation of free oxygen, hydrogen, hydroxyl, hydroperoxyl radicals, and metal ions from electrodes. As discussed later, the interest in changes produced by PEF not only focuses on safety aspects, but on the overall quality of foods submitted to treatment. Research conducted to verify such changes has so far detected 76

III. Electrofusion

77

insignificant detriment of quality attributes, and in some cases, PEF has been shown to preserve those characteristics better than traditional treatments. Examples of this are the results obtained in orange juice, apple juice, milk, liquid eggs, and pea soup, which did not undergo chemical changes during or immediately after electric field processing or storage. Analyzed for their fat, protein, carbohydrate, and sugar contents, these products also showed a minimum 2-week shelf-life (Qin et al., 1995).

II.

Electropermeabilization

Electropermeabilization is the key step in the process of electrofusion or DNA transfection of free suspended cells. If a membrane is considered a capacitor, electric field pulses charge it so that a membrane potential is induced in addition to the intrinsic membrane potential. When the membrane potential exceeds a critical value, electropermeabilization or reversible electrical breakdown occurs. The resealing process is temperature dependent in that at 37~ the cell rapidly regains impermeability to cations, but at 3~ it may remain highly permeable even after 20 hr. Electrical breakdown, which is the primary effect of electric field pulses, causes perturbations in the membrane structure that lead to an increase in membrane permeability. Secondary effects such as changes in membrane properties and the cell interior may also occur (Zimmermann, 1986). These changes are known to depend on cell size; furthermore, the time of exposure to an electric field that causes the electrical breakdown of cells depends on cell size or diameter. Cells such as plant protoplasts that are larger than animal, yeast, or bacterial cells break down with pulses of longer duration (about 40 /zsec). Electropermeabilization allows substances to be incorporated in a cell without destroying its integrity. The first proof of DNA transfer with the aid of electric pulses was obtained using an erythrocyte system, demonstrating that DNA and RNA can be sequestered across a cell membrane and that both can be entrapped in cells at the end of the resealing process (Zimmermann, 1986). Wong and N e u m a n n (1982) described a method to allow increased uptake of DNA by mouse cells using electric pulses with an intensity of 5-10 k V / c m and a duration of 5-10 ~sec. When the conditions leading to electroporation were enhanced, p e r m a n e n t rupturing of the cellular membrane occurred, and irreversible damages in transpiration and compartmentalization were inevitable.

III.

Electrofusion

In vitro cell fusion is a valuable tool in membrane research, genetic mapping, and especially genetic engineering. New strains of bacteria, yeast, fungi, hybrid plants, and hybrid mammalian cell lines have been developed by

78

4. PEF-Induced Biological Changes

chemical- and virus-induced fusion. Although cell fusion was i n t r o d u c e d as early as 1909, the standard conventional techniques have many, and s o m e times severe, limitations. Chemical- and virus-induced fusion is therefore m o r e of an art than a science. To overcome the shortcomings of the conventional techniques, Z i m m e r m a n n and co-workers (1986) developed the electrofusion method. This involves bringing cells into tight m e m b r a n e contact in a h o m o g e n e o u s electric field and fusing the cell pairs or multiples by applying short-duration electric field pulses of high intensity. Electrofusion provides the advantages of selectivity for required fusion products, efficient control of the fusion process, prediction of fusion conditions, and high yields of viable hybrids. As stated earlier, cell m e m b r a n e s must be in close contact for fusion to occur. In the standard electrofusion technique with h o m o g e n e o u s alternating electrical fields of relatively low intensity, cell m o v e m e n t achieves close m e m b r a n e contact, orientation, and alignment. This m o t i o n of neutral (or charged) bodies in a h o m o g e n e o u s field has been termed dielectrophoresis (Fig. 4.1). The presence of electric field leads to the generation of a dipole in cells because of the net force exerted, which pulls the cells in the direction of the highest field intensity (provided the complex dielectric constant of the cell is larger than that of the s u r r o u n d i n g medium). If the cells a p p r o a c h

The external electric field induces a dipole in the particle or cell. In a homogeneous field, particle or cell migration does not occur.

The external electric field induces a dipole in the particle or cell. In an inhomogeneous field, a net force is exerted on the induced dipole.

+

The direction of dielectrophoretic migration is not reversed with the reversal of the external electrical voltage.

The induced dipoles attract each other and the cells adhere to each other and form chains at the electrodes along the electrical field lines. Fig. 4.1 The phenomenon of dielectrophoresis and electrofusion. (Reproduced from Rev. Phys. Biochem. Pharmacol., "Electrical breakdown, electropermeabilization and electrofusion,"

U. Zimmermann, Vol. 105, pp. 176-256, fig. 19, 1986, with permission of Springer-Verlag.)

V. Electrical and Thermal Gradients Induced by PEF on Microbial Cell Membranes

79

each other during migration, it is because the attractive forces arising from the dipoles are much higher than the repulsive forces arising from the net surface charge. The overall result is the formation of pearl chains in which the microbial cells are in very close contact with each other. If the field strength is just high e n o u g h to induce electropermeabilization, electrical breakdown occurs predominantly in the contact zone, which leads to cell fusion (Zimmermann, 1986).

IV.

Disruption and Biological Alteration

Exposure of red blood cells to electric field pulses induces a transmembrane potential across cell membranes, and at a critical point, pores are opened or created in the cells. Pores are smaller in isotonic saline mediums that allow the permeation of potassium and sodium ions and larger in low-ionic strength mediums that may allow the passage of large molecules such as sucrose. The passage of ions a n d / o r leakage causes an osmotic imbalance that leads to the colloidal hemolysis of red blood cells (Kinosita and Tsong, 1977). At least 35% of the electric field-induced pore formation of erythrocytes in a m e d i u m of low ionic strength (30-45 m M NaC1 solution) is related to the opening of Na+/K+-ATPase channels; the remaining 65% of the pores occur at unidentified sites. In contrast, the Na+/K+-ATPase channels are not perforable in a high-ionic strength m e d i u m (Teissie and Tsong, 1980). In addition to the opening of the protein channels, electric fields also cause electroconformational damage (Chen and Lee, 1994). Hamilton and Sale (1967) reported the loss of motility for Pseudomonas and Bacillus megaterium subjected to a DC pulse treatment of 10 pulses at a rate of one pulse per second and a field strength of 22 k V / c m . When flagellation of the Pseudomonas was examined with an electron microscope, the loss of motility was not a result of flagella destruction. However, electric field-treated cells exhibited a flattened appearance with an irregular outline as a result of intracellular content leakage.

VO Electrical and Thermal Gradients

Induced by PEF on Microbial Cell Membranes

As presented in previous sections, microbial inactivation by PEF is partially due to electrical forces generated at the microorganism m e m b r a n e level. Although molecular mechanisms for how electric fields interact with membranes remain unknown, the relevant hypotheses concur that high-intensity

80

4. PEF-Induced Biological Changes

electric fields are the primary source of cell permeation. Therefore, being able to understand and predict changes at the microbial membrane level is a must. Bruhn et al. (1997) undertook such a task when they looked at the environment near and inside model microbes during the application of high-intensity electric field pulses. This research focused on the buildup charges at suspension, liquid/cell membrane, and protoplasm/cell membrane interfaces since it is believed that certain conditions at these interfaces will lead to electroporation and irreversible damage of cell membranes. The main prerequisites for charge accumulation at the suspension liquid/membrane interface and protoplasm/membrane interface are low conductivity for the cell membrane and high conductivities for the suspension liquid and protoplasm. Accordingly, the different conductivities of the regions, along with the inherent disparity in electrical permittivities, resulted in electric fields within the membrane that were much higher than the externally applied field. To simulate the movement of charges surrounding microorganisms suspended in liquid foods exposed to PEF, a one-dimensional model was developed by Bruhn et al. (1997) that accounts for space charge regions formed on both sides of microbial cells (Fig. 4.2). A numerical method that

Applied Electric Field

(a)

Microbe +

_

Suspension Liquid

Electrode

Electrode

(b)

Charge Accumulation

"1 = :11: iqui il:: ill: iqui

Suspension

:

-Protoplasm't" ~.

Suspension

~ Membrane ~

Fig. 4.2 A charge accumulation at the cell membrane and electrolyte boundary: (a) an application of an externally applied electric field and (b) a one-dimensional planar view of a microbe. (Reprinted from Bruhn et al., "Electrical environment surrounding microbes exposed to pulsed electric fields," ~ E E Trans. Dielec. Electric. Insul. 9 1997 IEEE.)

V. Electrical and Thermal Gradients Induced by PEF on Microbial Cell Membranes

81

uses an implicit finite difference scheme was applied to the continuity equation for electrical charges [positive Eq. (4.1) and negative Eq. (4.2)] and Gauss' law [Eq. (4.3)]: 0

Onp

0z (/ZpnpE) -~ 0

0t

- 0

(4.1)

= 0

(4.2)

On n

~ ( / ~ n n n E) 0Z 0 --(E) 0z

0t p = --,

(4.3)

where E is the electric field; /.Lp and /'~n are mobilities; np and n n concentrations of positive and negative ion species, respectively; p is the free volume charge density; and e is the permittivity of the region being considered. The continuity equation describes movement of positive and negative ions, whereas Gauss' law yields the electric field after the movement of ions. One negative ionic species and one positive ionic species are assumed to be in the suspension fluid and protoplasm of the microorganism, whose membrane is modeled as a nonconducting dielectric with zero electrical conductivity. The conduction current and heat source in the m e m b r a n e are therefore zero at all times. This nonconducting m e m b r a n e also causes ions to accumulate at the m e m b r a n e surface during PEF. Figure 4.3 shows the result of Bruhn et al.'s (1997) simulation, which corresponds to the movem e n t and accumulation of positive ions in m e m b r a n e walls. When unidirectional electric fields were applied, free-volume and free-surface charge densities formed along the cell membrane. The formation of free-volume charged density (Fig. 4.4) caused the electric fields to increase in these regions, which in turn caused the charges to be swept out more quickly due to their increased velocity. This sweeping out of charges from the suspension liquid onto the m e m b r a n e surface then created a surface charge density that e n h a n c e d the buildup of voltage across the membrane. Based on previous research, it is assumed that when the buildup of voltage is approximately I V, electroporation results in lysis of the treated microorganism (Sale and Hamilton, 1967), and because the movement of charges controls this buildup of voltage, careful monitoring becomes another challenge for future studies. Because one of the major reasons for using PEF pasteurization is its n o n t h e r m a l nature, special care must be taken so that applications do not result in excessive thermal treatment of foods. Therefore, Bruhn et al. (1998) developed a heat conduction model to simulate heat transfer in microorganisms suspended in liquid foods. A numerical m e t h o d that uses an implicit finite difference scheme was used to investigate heat sources, heat flow, and temperature rise during PEF pasteurization. The boundary conditions at the cell m e m b r a n e interfaces included continuity of temperature [Eq. (4.4)] and

82

4. PEF-Induced Biological Changes Positive ions

x 10 21

v

Positive ions

x 1021

E

v

0ps

r 0

0

5

c 2 10 15 z (pro)

0

20

Positive ions

x 102 1

0

5

10 15 z (pm)

20

Positive ions

x 1021

A

E 4

~

el,.

n

"2

c 2 0

0

5

10 15 z (pm)

0

20

Positive ions

x 1021

0

5

10 15 z (p,m)

20

Positive ions

x 1021 9

P4

-

/

'-2 0~ 0

5

10 15 z (pm)

0

20

0

5

"

10 15 z (pm)

20

Fig. 4.3 A concentration of positive ions for six different times. (Reprinted from Bruhn et al., "Electrical environment surrounding microbes exposed to pulsed electric fields," IEEE Trans. Dielec. Electric. Insul. 9 1997 IEEE.)

heat flow [Eq. (4.5)]: T1 = T2 OT 1

(4.4) OT 2

q~a Oz - q~2 O----z-"

(4.5)

To investigate how the heating would be conducted, simulations were run. The model considered a 0.5-/~m-wide microbe suspended in a liquid with a conductivity of 0.01 S / m that was pulsed with 40 k V / c m for 375 nsec until the drop across the m e m b r a n e reached 4 V. It was thus found that the temperature stayed cooler in and around the membranes for a very short period of time (nsec) when pulsing due to the zero conductivity of the membranes and the time it took for the charge accumulation to occur near the membrane. According to the simulation, the accumulation of charges near the m e m b r a n e will increase the electric fields there (Fig. 4.5), causing the m e m b r a n e to increase in temperature faster than the surrounding environment (Fig. 4.6). Because heat must flow from the hotter regions to the cooler, it will flow initially into the m e m b r a n e and later from the

83

VI. Main Factors in Microbial Inactivation Volume charge density

E ,,,..,

Volume charge density

0.5

03

0

0ps

c~->_0.5

I. 10 15 z (l~m)

5

0.5

o->_0.5 0

20

5

Volume charge density

10 15 z (l~m)

20

Volume charge density

,-, 0.5 E

.-, 0.5

(9

03

E

o ~

o ~

0->_0.5

0->_0.5 0

5

10 15 z (pm)

20

0 5 ~

5

Volume charge density

Q"-

0 0

. 5

5

20

Volume charge density 03

E

0.5

0->_0.5

~

10 15 z (IJm)

10 15 z (pm)

30 ps

20

z (l~m)

Fig. 4.4 A free volume charge density for six different times. (Reprinted from Bruhn et al., "Electrical environment surrounding microbes exposed to pulsed electric fields," 1FEE Trans. Dielec. Electric. Insul. 9 1997 IEEE.)

membrane into p h e n o m e n a are requires further PEF inactivation

VI.

the surrounding environment (Fig. 4.7). Whether these taking place in the same way the model is predicting attention, but each effort provides another piece for the mechanism puzzle.

M a i n F a c t o r s in M i c r o b i a l I n a c t i v a t i o n

Pulsed electric fields have successfully demonstrated the inactivation of microorganisms such as Escherichia coli, E. coli 0157:H7, Salmonella dublin, S. enteritidis, Staphylococcus aureus, Lactobacillus delbrueckii, Bacillus subtilis, Saccharomyces cerevisiae, Klebsiella pneumoniae, Pseudomonas aeruginosa, P. fluorescens, Listeria monocytogenes, Candida albicans, and Zygosaccharomyces bailii. In general, bacteria have proven to be more resistant to electric fields than yeasts at high-electric fields, although spores are the most resistant microbial entities. As mentioned earlier the sensitivity of microbial cells to electric field treatment increases with an increase in the size of the cell. Chapter 3 presented theories and experimental results in support of external electric fields inducing transmembrane potential across cell membranes and causing electroporation a n d / o r damage of cell organelles that lead to cell inactivation.

84

4. PEF-Induced Biological Changes

Fig. 4.5 A free volume charge density from a simulation time of 375 nsec (a); a free volume charge density from a simulation time of 1 /zsec (b); an electric field intensity from a simulation time of 375 nsec (c); an electric field intensity from a simulation time of 1 /zsec (d); a conduction current from a simulation time of 375 nsec (e); and a conduction current from a simulation time of 1 /zsec (f). (Reprinted from Bruhn et al., "Heat conduction in microbes exposed to pulsed electric fields," IEEE Trans. Dielec. Electric. Insul. 9 1998 IEEE.)

T h e s u b s e q u e n t inactivation o f m i c r o o r g a n i s m s by PEF is affected by (a) t r e a t m e n t c o n d i t i o n s , t r e a t m e n t time, electric field s t r e n g t h , t e m p e r a t u r e , pulse waveshape, a n d pulse width; (b) t h e type, c o n c e n t r a t i o n , a n d g r o w t h stage o f t h e m i c r o b i a l entity; a n d (c) t h e physical a n d electrical p r o p e r t i e s o f t h e t r e a t m e n t media. Like any o t h e r p r e s e r v a t i o n t e c h n o l o g y , storage c o n d i tions m a y have a preservative or d e t r i m e n t a l effect o n the m i c r o b i a l c o n t e n t a n d physical characteristics o f t h e P E F - t r e a t e d p r o d u c t . Safety thus b e c o m e s a c o n c e r n w h e n cells t h a t are i n j u r e d b u t n o t inactivated by electric field t r e a t m e n t have the ability to r e c o v e r a n d r e p r o d u c e d u r i n g the s t o r a g e o f f o o d , so it is i m p o r t a n t to store electric field-treated foods at r e f r i g e r a t e d t e m p e r a t u r e s if necessary.

A.

Factors Dependent on Treatment Conditions

Electric field s t r e n g t h , t r e a t m e n t time, t r e a t m e n t t e m p e r a t u r e , a n d pulse w a v e s h a p e are the m a i n factors t h a t d e t e r m i n e PEF t r e a t m e n t c o n d i t i o n s within e x p e r i m e n t s c o n d u c t e d in t h e s a m e type of t r e a t m e n t c h a m b e r (i.e.,

VI.

Main

Factors

in M i c r o b i a l

Inactivation

85 Temperature

Temperature

.~

0 ns

<1:

I

~

I

'

.'.s

'

107 n s:~.... . l iI ..~, . ". - .". . iI ... " ~

,

I

~--lo

E ,,r " 414[ Er

I

I

!

;

z (l~m)

'

~[5

2

~- o

o's

'

,l P"

0

,

0.5

' " I ......... I

,

..

= " I ...... . .":,~,----9. . . . "1 ~ " , /

\.' / \ ' / ,

,

1 z (p,m)

,

i

1.5

2

E 9.95 I .

"

~

9.75[

,

P"

0

9.8t

=

',,J~

"

I

~-

12

"

"

375ns

"

~"~t

o'.s

I

; z (l~m)

=

, 1 z (lJm)

A

"

/',\

9

1'.5

2

~14.1 t0

, 0.5

A

/',\

/;\

/;\

I

, I 1 z (wn)

>= 14.4 [.__.~.~....t...~'....!-

o

2

"

|

1.5

2

Temperature

ls.3[ 151

.~ 12.2 <:

I

0.5

Temperature

E 12.6

~:s

Temperature

Temperature

E ~ " ---- 7.2 [..---,.,.~... .... "~ I 1RR n.q~ < 7.1 t ..... \

; ' z (ptm)

"

, 1.5

2

Fig. 4.6 Temperature profiles of a mathemadcal simulation at six different times. (Reprinted from Bruhn et al., "Heat conduction in microbes exposed to pulsed electric fields," F E E Trans. Dielec. Electric. Insul. 9 1998 IEEE.)

continuous, static, parallel plate electrodes, cofield, and coaxial configurations) with the same process mode (i.e., batch, continuous, recirculation, and stepwise).

1. ElectricField Strength As already established, microbial inactivation occurs when the applied electric field exceeds the critical transmembrane potential. Once this happens, microbial inactivation increases with an increase in the applied electric field strength, which is in agreement with the electroporation theory as the induced potential difference across the cell membrane is proportional to the applied electric field. Some empirical or phenomenological models have also been proposed to describe the relation between electric fields and microbial inactivation. For example, Hiilsheger et al. (1981) proposed their model based on the knowledge that the survival ratio (s) is the quantitative measure of microbial inactivation and is defined by the ratio of the living cell count before and after PEF treatment (s = N/No). The model thus relates microbial survival fractions with electric field strength according to the following equation: ln(s) = -bE(E - E c ) ,

(4.6)

86

4. P E F - I n d u c e d Biological C h a n g e s Heat Flow Vector

Heat Flow Vector ~ .

~~

0 ns 0

-1 m

o

I I

I I

!

!

I

os

r

i

0

i . .........

-1000

~s

z (lira)

.....

0

0.5

Heat Flow Vector

•"

"

o4

m

;

~ooo

1 z (pro)

1.5

2

Heat Flow Vector ..-,,.

"

I

"

I

"

I oo0 0

'

.o."

"

,,,

0

....

. . . .

.9. . .

-1 ooo 0

0.5

1 z (pro)

1.5

2

Heat Flow Vector

0

0.5

,oOO ooo "

"~" 2000

~

320 ns

)

E

o

1 z (lira)

1.5

2

Heat Flow Vector -

m

-

u A

-

375 ns

v

~ -2000

,- - 2 0 0 0

-4000

0

0.5

1

z (lira)

1.5

2

i

0

0.5

1 z (lira)

1.5

2

Fig. 4.7 Heat profiles of a mathematical simulation at six different times. (Reprinted from Bruhn et al., "Heat conduction in microbes exposed to pulsed electric fields," TEEE Trans. Dielec. Electric. Insul. 9 1998 IEEE.)

where bE is the regression coefficient, E is the applied electric field, and E c is the critical electric field obtained by the extrapolated value of E for 100% survival or a survival ratio of one. The regression coefficient describes the gradient of the straight survival curves and is a microorganism-media constant. The inactivation kinetics of several microorganisms, kinetic constants, and treatment conditions that exemplify the use of this model are presented in Tables 4.1-4.3. Such kinetic constants give an idea of the microbial susceptibility to PEF: a low value indicates a high inactivation effect whereas the opposite may be due to a protective response of the treatment media (i.e., the smaller kinetic constant of a microbe in solutions of alginate compared with that in 1.5% fat milk suggests the fat particles act as a shield). The critical electric field (Ec) has been found to be a function of cell size and is much lower for bigger cells (Grahl and M~irkl, 1996) (Table 4.1) due to the transmembrane potential experienced by the cell, which is proportional to the cell size. Pulse width also influences the critical electric field. For example, with pulse widths higher than 50 ~sec, E~ is 4.9 k V / c m , while with less than 2 /lsec, E c is 40 k V / c m (Schoenbach et al., 1997). In addition, E~ for gram-negative bacteria is lower than that for gram-positive bacteria (Hiilsheger et al., 1983), which may explain the smaller PEF resistance of the former (Table 4.4). Peleg (1995) proposed a second model [Eq. (4.7)] that describes the sigmoid shape of the survival curves generated by the microbial inactivation

87

VI. Main Factors in Microbial Inactivation TABLE 4. I Calculated H~ilsheger Model Parameters for Different Microorganisms Suspended in Model and Real Foods Treated with Different Electric Field Strengths ~ b

Medium

n

Ec (kV/cm)

bE (cm/kV)

Solution of sodium alginate UHT milk (1.5% fat) UHT milk (1.5 % fat) UHT milk (1.5% fat) UHT milk (1.5% fat) UHT milk (1.5% fat) Solution of sodium alginate UHT milk (1.5% fat) Solution of sodium alginate UHT milk (1.5% fat) Solution of sodium alginate UHT milk (1.5% fat) Orange juice

5 5 10 15 20 20 5 20 5 20 5 5 5

14.0 12.7 14.2 13.9 13.5 11.9 12.1 12.6 11.5 10.7 5.4 4.7 4.7

-0.401 -0.123 - 0.218 -0.347 -0.475 -0.307 -0.348 -0.467 - 0.440 -0.331 - 1.949 - 2.464 - 2.547

Microorganism

E. coli

L. brevis P. fluorescens S. cerevisiae

0.991 0.974 0.994 0.974 0.994 0.996 0.989 0.996 0.990 0.993 0.983 0.983 0.992

Reproduced from Appl. Microbiol. Biotechnol., "Killing of microorganisms by pulsed electric fields," T. Grahl and H. M~irkl, Vol. 45, pp. 148-157, Table 3, 1996, with permission of Springer-Verlag. bE, electric field; n, number of pulses; E~, critical electric field; b E, kinetic constant; and R, regression coefficient.

by PEF. The

model

represents

the percentage

function of the electric fields and number

of surviving organisms

as a

o f p u l s e s a p p l i e d . P e l e g ' s m o d e l is

defined by a critical electric field intensity that corresponds a n d a k i n e t i c c o n s t a n t (a f u n c t i o n o f t h e n u m b e r

to 50% survival

of pulses) that represents

TABLE 4.2 Kinetic Constants of H~ilsheger's Model for E. coli Inactivation in Skim Milk by PEF T r e a t m e n t in a Static Chamber a' b

Model s = e -(E-Ec)/kc Electric field intensity (kV/cm)

Number of pulses

35 40 45 < 45 < 45 < 45

< 64 < 64 < 64 16 32 64

nc 15.2 13.0 11.0 m

Er (kV/cm)

m 18.7 20.4 19.9

k~ (kV/cm)

R2

5.6 6.1 8.0 2.9 3.9 2.7

82.9 95.8 98.5 83.3 86.1 92.4

a From Mart~n-Belloso et al. (1997). b nc, critical number of pulses; E c, critical electric field; k c, inactivation rate constant; and R 2, correlation coefficient for regression analysis (p = 0.05).

88

4. PEF-Induced Biological Changes

TABLE 4.3 Kinetic Constants of Hfilsheger's Model for E. coli Inactivation in Skim Milk by PEF T r e a t m e n t in a Continuous-Flow Chamber ~ b

Model s = e -(E-Ec)/kc Electric field intensity (kV/cm)

Number of pulses

15 20 25 < 30 < 30 < 30

< 30 < 30 < 30 15 20 25

nc

E~ (kV/cm)

k~ (kV/cm)

R2

5,4 1.9 2.7

--

3.9 9.5 5.8 4.3 2.2 2.2

91.8 99.7 95.5 98.5 96.8 93.8

-13.82 14.62 14.44

From Martln-Belloso et al. (1997). b nc, critical n u m b e r of pulses; E c, critical electric field; k~, inactivation rate constant; and R 2, correlation coefficient for regression analysis (p = 0.05).

the steepness of the sigmoid curve. Mathematically,

a b o u t 9 0 % i n a c t i v a t i o n is

attained within the critical electric field plus three times the kinetic constant.

s =

E-ec(,,)

(4.7)

k(n)

l+e

TABLE 4.4 Kinetic Constants of H~ilsheger's Model for Different Microorganisms Suspended in a N a 2 H P O 41KH2PO 4 Buffer with a pH of 7,0 a'b

Model s -

t) -(E-Ec) k

~

Microorganism

E (kV/cm)

t(msec)

E c (kV/cm)

E. coli (4 hr) c E. coli (30 hr) c K. pseudomonia P. aeruginosa S. aureus L. monocytogenes I L. monocytogenes II C. albicans

4-20 10-20 8-20 8-20 14-20 12-20 10-20 10-20

0.07-1.1 0.07-1.1 0.07-1.1 0.07-1.1 0.07-1.1 0.07-1.1 0.07-1.1 0.14-1.1

0.7 8.3 7.2 6.0 13.0 10.0 8.7 8.4

to(/zsec) 11 18 29 35 58 63 36 110

k (kV/cm)

r(%)

8.1 6.3 6.6 6.3 2.6 6.5 6.4 2.2

97.7 97.6 95.7 98.4 97.7 97.2 98.5 96.6

a Adapted from HiJlsheger et al. (1983) bE, electric field; t, treatment time; E c, critical electric field; t c, critical time; k, kinetic constant; and r, regression coefficient. c Incubation time.

89

VI. Main Factors in Microbial Inactivation

w h e r e k ( n ) [ k V / c m ] is the kinetic c o n s t a n t that indicates the steepness of the survival curve a r o u n d the critical electric field E c ( n ) . This is a g e n e r a l i z e d m o d e l since E c ( n ) a n d k ( n ) are expressed as algebraic f u n c t i o n s that n o t only d e p e n d o n the electric field b u t o n the n u m b e r of pulses or t r e a t m e n t time (the n u m b e r of pulses times the pulse width) as well. T h e m o d e l can be simplified by n o t taking into a c c o u n t the relation b e t w e e n the electric field a n d the n u m b e r of pulses; if the a p p l i e d electric field is m u c h h i g h e r than the critical electric field, a s e c o n d simplification can be made:

s l+e

k

In b o t h m o d e l s [Eqs. (4.7) a n d (4.8)] a large value for the kinetic constant [k(n) or k] indicates a wide span in the inactivation rate curve, whereas a small value implies a steep decline or high microbial inactivation rate. T h e r e f o r e , in the c o m p a r i s o n of kinetic constants f r o m different m i c r o o r ganisms, the smaller the kinetic constant, the h i g h e r the susceptibility to PEF. In g e n e r a l then, an analysis of the kinetic constants d e f i n e d by Peleg's (1995) m o d e l (Table 4.5) for different m i c r o o r g a n i s m s leads to the conclusion that the h i g h e r the n u m b e r of pulses, the lower the critical electric field a n d kinetic constant.

T A B L E 4.5 Kinetic Constants of Peleg's Model ~

Number of pulses Ec (kV cm- 1) K (kV cm- 1)

Organism L. brevis S. cerevisiae S. aureus C. albicans

L. monocytogenes

P. aeruginosa

I

m

11.4

1.6

--

13.2 14.1

2.3 2.0

2

21.2

3.1

4 10 3O 2 4 10 30 2 4 10 30

15.3 10.1 7.5 14.9 12.7 10.3 8.5 12.9 10.6 8.3 6.7

3.1 1.3 1.2 2.8 2.0 2.4 2.0 2.6 2.4 2.1 1.8

r 2

Original data source

0.973 Sitzmann (1990) 0.994 Jacob et al. (1981) 0.991 Hamilton and Sale (1967) 0.999 Hiilsheger et al. (1983) 0.993 Hi]lsheger et al. (1983) 0.997 Hi]lsheger et al. (1983) 0.999 Hi]lsheger et al. (1983) 0.981 Hi~lshegeret al. (1983) 0.994 Hi]lsheger et al. (1983) 0.992 Hi~lshegeret al. (1983) 0.999 Hiilsheger et al. (1983) 0.982 HiSlsheger et al. (1983) 0.994 Hi]lsheger et al. (1983) 0.993 Hi~lsheger et al. (1983) 0.999 Hi]lsheger et al. (1983)

a Reproduced from "A model of microbial survival after exposure to pulsed electric fields," M. Peleg (1995). Copyright SCI. Reproduced with permission.

90

4. PEF-Induced Biological Changes

2. Treatment Time P r o p e r PEF t r e a t m e n t time is d e r i v e d f r o m the p r o d u c t o f the n u m b e r o f pulses a n d their d u r a t i o n . In g e n e r a l , an increase in any o f these variables results in an increase in m i c r o b i a l inactivation. However, large pulse d u r a tions may also result in an u n d e s i r a b l e f o o d t e m p e r a t u r e rise (see Chapter 2). O p t i m u m p r o c e s s i n g c o n d i t i o n s s h o u l d t h e r e f o r e be established to o b t a i n the h i g h e s t inactivation rate with the lowest h e a t i n g effect. H i i l s h e g e r et al. (1981) p r o p o s e d an inactivation kinetic m o d e l [Eq. (4.2)] t h a t relates m i c r o b i a l survival fractions (s) with PEF t r e a t m e n t time (t) in the f o r m o f

lns=

-btln

(') ~

,

(4.9)

w h e r e b t is the r e g r e s s i o n coefficient, t is the t r e a t m e n t time, a n d t c is t h e e x t r a p o l a t e d value o f t for 100% survival. Tables 4 . 6 - 4 . 8 p r e s e n t the kinetic c o n s t a n t s t h a t fit this m o d e l for d i f f e r e n t m i c r o o r g a n i s m s . A l t h o u g h it can g e n e r a l l y be stated that inactivation increases with an increase in t r e a t m e n t time, it is i m p o r t a n t to n o t e t h a t in certain cases (i.e., S. cerevisiae), inactivation r e a c h e s a saturation with 10 pulses o f an electric field at 25 k V / c m ( Z h a n g et al., 1994a). Critical PEF t r e a t m e n t time also d e p e n d s o n the electrical field s t r e n g t h a p p l i e d , for w h e n the latter is slightly above the critical electric field, the f o r m e r will be h i g h e r c o m p a r e d with t r e a t m e n t s at h i g h e r electric fields. G r a h l a n d M~irkl (1996) have c o n c l u d e d t h a t for an electrical field s t r e n g t h m o r e t h a n 1.5 times h i g h e r t h a n E c, the critical t r e a t m e n t time will r e m a i n

T A B L E 4.6 Kinetic Constants of Hiilsheger's Model for E. coli Inactivation in L W E by PEF T r e a t m e n t in a Coaxial Continuous C h a m b e r ~ b

Model ln(s)= btln(t~)

Scheme

Pulse rate (Hz)

Pulse duration (/zsec)

bt

Batch Batch Batch Batch Recirculation Recirculation Recirculation Recirculation

1.25 1.25 2.50 2.50 1.25 1.25 2.50 2.50

2 4 2 4 2 4 2 4

5.4441 5.6255 5.2437 6.3429 5.2435 4.8902 5.2705 4.4954

c

R2

12.62 10.09 14.76 13.92 13.54 9.03 10.29 8.63

0.995 0.986 0.956 0.988 0.995 0.979 0.995 0.957

n

a From Martln-Belloso et al. (1997). b bt' constant dependent on field intensity; n c, critical number of pulses; R 2, correlation coefficient for regression analysis (p = 0.05); and tc, critical time = n c X pulse duration.

91

Vh Main Factors in Microbial Inactivation TABLE 4.7 Kinetic Constants of Hfilsheger's Model for E. coli Inactivation in L W E by PEF T r e a t m e n t in a Coaxial T r e a t m e n t Chamber ~ b

Model ln(s) = - b l n b Processing mode Parameter value Standard error CV% r2

48 S c

48 C d

38 S e

38 C f

48 S ~

48 C d

38 S e

38

6.961

4.870

4.225

3.730

4.709

5.607

2.486

2.563

0.4232

0.3998

0.2582

0.3373

0.5235

1.007

0.3713

0.7495

0.06079 0.9036

0.08211 0.8611

0.06112 0.9459

0.09044 0.9251

0.1112 0.9036

0.1795 0.8611

0.1494 0.9459

0.2924 0.9251

Adapted from Ma et al. (1998). kinetic constant; t, treatment c PEF intensity of 48 k V / c m in a d PEF intensity of 48 k V / c m in a e PEF intensity of 48 k V / c m in a f PEF intensity of 48 k V / c m in a b b,

constant.

tc(4Xsec)

However,

microorganism

a

Cf

time; and t c, critical time. stepwise processing mode. continuous circulation processing mode. stepwise processing mode. continuous circulation processing mode.

protective

may increase

state

of

the

treatment

media

over

the

this time.

The consideration of the two critical values of E c and t c led Hfilsheger et al. ( 1 9 8 1 , 1 9 8 3 ) t o p r o p o s e the following empirical equation for the TABLE 4.8 Calculated Hfilsheger Model Parameters for Different Microorganisms Suspended in Model and Real Foods Treated with Different Processing Times ~ t Model log(s) = B t ~-c

Microorganism

E. coli

L. brevis P. fluorescens

Medium

E (kV/cm)

t c (/xsec)

Bt

Solution of sodium alginate U H T milk (1.5% fat) U H T milk (1.5% fat) U H T milk (1.5% fat) U H T milk (1.5% fat) U H T milk (3.5% fat) Solution of sodium alginate U H T milk (1.5% fat) Solution of sodium alginate U H T milk (1.5% fat)

24.8 15.5 17 20.0 22.4 22.4 24.8 22.4 24.8 22.4

4.5 130.2 104.2 46.3 45.7 30.0 10.9 46.1 0.40 19.8

- 3.575 - 3.253 -3.806 -4.149 -5.968 -4.016 - 4.950 - 7.274 - 2.483 - 3.589

0.988 1.000 1.000 0.986 0.975 0.999 0.996 0.988 0.976 0.971

a Reproduced from Appl. Microbiol. Biotechnol., "Killing of microorganisms by pulsed electric fields," T. Grahl and H. M~irkl, Vol. 45, pp. 148-157, Table 4, 1996, with permission of Springer-Verlag. bE, electric field; t, treatment time; t c, critical time; Bt, kinetic constant; and R, regression coefficient.

92

4. PEF-Induced Biological Changes

calculation of the surviving fraction: -(E-E~))

s-

~

(4.10)

where t c is the minimum treatment time that gives s = 1 and k' ( k V / c m ) is a first-order kinetic constant or microorganism constant (Table 4.4). Equation (4.6) is valid for survival fractions lower than 0.5. Sensoy et al. (1997) applied the equation of Hiilsheger et al. to their experimental data (Table 4.9) and in general, found good fits at high-electric field strengths. When Zhang et al. (1994c) evaluated several statistical models to analyze the inactivation of E. coli, S. cerevisiae, and S. aureus suspended in milk and a semisolid medium, they found that E. coli and S. aureus in the semisolid m e d i u m followed first-order inactivation kinetics (Table 4.10) for both the intensity of electric fields (Model 1) and the n u m b e r of pulses (Model 2). When comparing the inactivation of cells in logarithmic and stationary phases, a distinct difference is seen in the threshold values of electric field strength (Ec), whereas the threshold treatment time (t c) remains unchanged. It can therefore be concluded that the alteration of physiological properties a microbial cell undergoes during continuous growth influences its sensitivity to electric fields. 3. Treatment Temperature

Temperature is one of the factors that has a significant effect in all biological processes, and the inactivation of microorganisms by PEF is no exception. Experimental results have shown that the growth temperatures of the cultures used in challenge tests, treatment temperatures, and postprocess temperatures all impact microbial survival and recovery.

TABLE 4.9 Kinetic Constants of Hi~lsheger's Model for S. dublin Inactivation in Skim Milk by PEF T r e a t m e n t in a Cofield Flow High-Voltage Chamber ~ b

Model s =

e -(t-tc)/kt

Electric field intensity (kV/cm)

kt(/xsec)

tc(/xsec)

R 2

40 35 30 25

9.20 11.25 13.86 22.30

0 0 0 0

0.92 0.91 0.97 0.87

a Adapted from Sensoy et al. (1997). bkt, kinetic constant; tc, critical treatment time; and analysis (p = 0.05).

R 2,

correlation coefficient for regression

93

VI. Main Factors in Microbial Inactivation T A B L E 4. I 0 Models for PEF Microbial Inactivation Kinetics a'b

Parameter unit E-E

M o d e l 1" s = e E~ ( k V / c m ) k E (kV/cm) R2

S. cerevisiae

S. aureus

E. coli in PDA

in PDA

in PDA

E. coli in milk

17.5 7.96 0.99

19.3 4.5 0.98 8

18.5 6.8 0.98 16

26.9 31 0.99 16

40

0 2.83 0.95 40

0 9.82 0.95 40

0 16.5 0.85 40

2.0 4.8 0.84 40

1.2 5.3 0.96 40

1.8 4.5 0.83 40

1.4 2.0 0.93 40

35 39 0.38 40

10 10 0.51 40

33 35 0.31 40

29 40 0.39 40

c

kE

n

16 n~n

M o d e l 2: s = e nc kn R2

c

kn 0 10.35 0.96

E (kV/cm) n

M o d e l 3: s

=

(__)-a n c

n c

A R2 E (kV/cm) n - nc Model 4:s=

1 kn

nc kn R2 E (kV/cm)

a F r o m Z h a n g et al. (1994c). bEe, critical electric field; K~., kinetic c o n s t a n t as a f u n c t i o n of electric field; R 2, c o r r e l a t i o n for regression analysis; n, n u m b e r o f pulses; n c, critical n u m b e r of pulses; kn, kinetic c o n s t a n t as a f u n c t i o n of pulse n u m b e r ; E, electric field; a, inactivation index.

During a microbial culture, the bacteria incorporate saturated and long-chain fatty acids into phospholipids, and as the growth temperature is increased, the phase transition temperature (which is 10~ lower than the culture temperature) is affected. The resulting phospholipids are in a rigid gel structure at low temperature, but turn into a less ordered or liquid crystalline structure as the temperature increases. Pulsed electric fields can thus be determined as effective in liquid crystalline structures but not at phase transition temperatures (Ohshima et al., 1997; Ho and Mittal, 1996). G{tskovfi et al. (1996) explained how lower postpulse temperatures allow higher inactivation due to the lifetime of induced pores in lipid membranes a n d / o r the lateral mobility of membrane components, which decreases with decreasing temperatures. They clarify that low postpulse temperatures should therefore aid in maintaining enhanced membrane permeabilities which

94

4. PEF-Induced Biological Changes

p r o m o t e irreversible cell damage, a n d that the high killing efficiency of PEF at low postpulse t e m p e r a t u r e s is lost at h i g h e r strength electric fields. Electric field treatments at m o d e r a t e t e m p e r a t u r e s ( ~ 50-60~ have b e e n proven to exhibit synergistic effects with PEF on the inactivation of microorganisms. With a constant electric field strength, inactivation increases with an increase in t e m p e r a t u r e , although it should be n o t e d that t e m p e r a t u r e s must be held far below those used in pasteurization. However, because the application of electric fields does cause some increase in the t e m p e r a t u r e of foods, p r o p e r cooling should be provided to maintain food t e m p e r a t u r e s far below those g e n e r a t e d by pasteurization. Sufficient time a m o n g pulses, high flow rates, a n d the use of cooling devices will help ensure p r o p e r food temperatures. Sensoy et al. (1997) p r o p o s e d an inactivation kinetic m o d e l [Eq. (4.11)] (Table 4.11) that considered the effect of t r e a t m e n t t e m p e r a t u r e s on the first-order kinetic constant of Eq. (4.10). They also r e c o m m e n d e d the combination of electric field strength a n d t e m p e r a t u r e effect in one equation, b u t f u r t h e r study will be required: $ =gkt

k =kT0(E-Ec)

(4.11)

=kE0 e

EA RT

(4.12)

where k is the survival fraction rate constant (1//zsec), E a is the activation energy ( J / k g mole), kT0 equals cm/kV./zsec; kE0 equals 1//zsec, R is the

T A B L E 4. I I Kinetic Constants of Simplified Model as a Function of T e m p e r a t u r e for S. dublin Inactivation in Skim milk by PEF T r e a t m e n t in a Cofield Flow High-Voltage C h a m b e r ~ b

Model s

=

e -k t

k (1//zsec)

R2

Electric field intensity (kV/cm) c 25 30 35 40

0.045 0.072 0.089 0.109

0.87 0.93 0.91 0.92

Medium temperature(~ 283.15 293.15 303.15 313.15 323.15

0.044 0.064 0.059 0.069 0.083

d

a From Sensoy et al. (1997). bk, kinetic constant; and R 2, correlation coefficient for regression analysis (p = 0.05). cf = 2.06 kHz, r = 1 /zsec T = 24~ dE = 25 kV/cm, f = 1.7 kHz, r = 1 /zsec.

VI. Main Factors in Microbial Inactivation

95

universal gas constant (1.987 J / k g mole ~ and T is the temperature of the media (~ Mfirquez et al. (1997) found a higher lethal effect of PEF treatment after setting the processing temperature at 25~ instead of 5-10~ which might be due to the increase in electrical conductivity of the solution at the higher temperature, making it similar to electrolytic conduction. In the case of a decoated spore, the leakage of mobile ions may increase as the temperature is raised because the change in average kinetic energy of the ions in the core would make them move faster. It may also increase the motion of the solvent molecules in both the surrounding cortex and the core so that they could migrate from one electrode to the other. When the m e d i u m that is surrounding a microorganism contains substances that may attack it (such as organic acids), a higher treatment temperature will increase the cell membrane fluidity. This probably makes it easier for organic acids to transfer into cells, which thus increases the inactivation rate (Liu et al., 1997). 4. Pulse Waveshape

Electric field pulses may be applied in the form of exponentially decaying (Fig. 1.4), square-wave (Fig. 1.5), oscillatory (Fig. 4.8), bipolar (Fig. 4.9), or instant-reverse charges (Fig. 1.7). Oscillatory pulses are the least efficient for microbial inactivation, and square-wave pulses are more energy and lethally efficient than exponentially decaying pulses (Qin et al., 1994). Bipolar pulses are more lethal than monopolar pulses because a PEF causes movement of charged molecules in the cell membranes of microorganisms, and a reversal in the orientation or polarity of the electric field causes a corresponding change in the direction of charged molecules. With bipolar pulses the alternating changes in the movement of charged molecules cause a stress in the cell m e m b r a n e and enhance its electric breakdown. Bipolar pulses also offer the advantages of m i n i m u m energy utilization, reduced

r

D~ D

..~

Time Fig. 4.8 A voltage trace of an oscillatorydecaying pulse wave.

96

4. PEF-Induced Biological Changes

Exponentially decaying 0 0~ 0

Square wave

L_ Time

Fig. 4.9 A voltage trace of bipolar pulse waveshapes.

deposition of solids on the electrode surface, and decreased food electrolysis (Qin et al., 1994). Using a planar lipid bilayer as a model system, Chang (1989) observed the mechanical oscillation of cell membranes exposed to bipolar pulses and noted that a sudden reversal of an applied field orientation will change the direction of the charged groups in a cell membrane. Even when the amplitude of the cell m e m b r a n e motion is not large enough to result directly in a mechanical breakdown, the alternating stress produced by the bipolar pulse results in structural fatigue of the membrane and enhances its susceptibility to electrical breakdown. As explained by Ho and Mittal (1997), the instant-charge-reversal pulse can be described as partially positive at first and partially negative immediately thereafter. Furthermore, they emphasize that the electrical conductivity of treated foods plays an important role in the characteristics of this waveform, where an increase in conductivity decreases the duration of the positive part of the pulse as well as the span of the negative part, which in turn increases the overall peak voltage ratio. Mfirquez et al. (1997) pointed out that with these types of pulses, there is a sharp division between low and high inactivation, which is not that c o m m o n when other pulse shapes are used. The difference between a bipolar and instant-charge-reversal pulse is the relaxation time in between pulses, which is only present in the former. The inactivation effect of an instant reversal charge is believed to be due to a significant alternating stress on the microbial cell that causes structural fatigue. Microbial studies reveal the possibility of a reduction in the critical electric field strength required for electroporation when instant-chargereversal pulses are used. This higher killing effect of instant-chargereversal pulses compared to other pulses can save up to one-fifth or one-sixth off total energy and equipment costs.

VI. Main Factors in Microbial Inactivation

97

So far it is possible to see how a variety of experimental results support the important effects of different waveforms on inactivation rates, but few studies have been conducted to quantify this relationship. O n e of the first attempts was performed by Qin et al. (1994), where the effect of bipolar, oscillatory, exponentially decaying, and square-wave pulses was verified in the final survival fraction. As mentioned earlier, this study illustrated that square-wave pulses generate the smallest survival fraction for identical peak voltage and energy delivered. A later study conducted by Zhang et al. (1997) showed the effect of square-wave, exponentially decaying, and instantcharge-reversal pulses over the extended shelf-life of orange juice, and their results also agree that the square-wave is the most effective pulse shape. Although these two studies gave a good idea of the effect, none presents a quantitative relation of the waveshape factor and its effect over the survival fraction. Love (1998) took on this challenge and found a strong correlation between the frequency components of pulse shapes and survival fractions. The mathematical analysis conducted by Love consisted of an evaluation of important Fourier transform parameters, which was motivated by the observation of relatively small survival fraction changes for fairly high PEF intensities, as these often result in significant differences in amplitudes in the frequency domain. Love obtained his frequency domain from the Fourier transform of a function of time, which in this case was a waveform. The Fourier transform F(co) was thus composed of a real ~'[F(c0)] and imaginary part J'[F(c0)]. These are just two of the six important parameters evaluated by Lovemthe other considered parameters were the magnitude [M(w)] of the F(c0), the physically measurable power of the waveshape pulse [P(c0)], the ratio of the imaginary and real components of the Fourier transform {J[F(o~)]/~'[F(c0)]}, and the ratio of the imaginary term and Fourier transform dC[F(oJ)]/{~[F(co)] + J[F(co)]}. The magnitude and power of the Fourier transform were defined by

M(co)

=

]/(,_~[F(w)]) 2 + (,5[F(w)]) 2

P ( w ) = [M( 0))] 2 = ( , . ~ [ F ( w ) ] ) 2 + ( J ' [ F ( 0 ) ) ] ) 2.

(4.13) (4.14)

Love evaluated the area under the curve of the frequency domain for each of these parameters. In addition to all the parameters for the four waveshapes studied by Qin et al. (1994) (square, exponential, bipolar, and oscillatory), Love also correlated the area ratios, magnitude of the Fourier transform, and area of the pulse with the corresponding survival fraction reported by Qin et al. Table 4.12 summarizes the areas of each of these parameters, as well as the slope and intercept of the log-log correlation equations. Based on his results, Love demonstrated in a quantitative way the stronger inactivation effect of square-wave pulses over other wave shapes

98

4. PEF-Induced Biological Changes

T A B L E 4.12 Fourier Transform Parameters of W a v e f o r m s and Microorganism Survival Fraction Correlations ~

Integrated area Variable Square-wave Exponential Alternating exponential Oscillating exponential Slope Intercept Correlation coefficient

,Y

0.500 0.499 0.499

0.825 0.477 0.471

1.65 0.956 0.944

0.623 0.488 0.482

1.29 0.732 0.933

1.0 X 10 - 4 1.8 X 10 -~ 7.6 X 10 -4

0.368

0.219

0.595

0.373

0.473

2.9 X 10 -2

I

~/J

-5.5 - 2.93 0.98

J/~

+J

111.03 - 6.33 0.98

M(t0)

Survival fraction

~'

-5.56 - 3.38 0.99

m

m

a Adapted from Love (1998).

9 pinpointed the important relation between the imaginary components of the frequency domain and survival fraction 9 determined that since area ratios are intrinsic waveform parameters, they depend on the waveform but not the temporal pulse amplitude and furthermore, that the ratio of imaginary and real components is independent of the maximum peak voltage 9 concluded that the two area ratios [ S / ~ and S / ( J + 2 ) ] give the 9

b e s t c o r r e l a t i o n w i t h survival f r a c t i o n s e s t a b l i s h e d t h a t t h e survival f r a c t i o n is n o t j u s t a f u n c t i o n o f t h e t e m p o r a l p u l s e a r e a by s h o w i n g h o w b o t h b i p o l a r ( a l t e r n a t i n g e x p o nential) a n d e x p o n e n t i a l waves have the s a m e a r e a p e r pulse, yet b i p o l a r waves y i e l d h i g h e r i n a c t i v a f i o n s ( s m a l l e r survival f r a c t i o n s )

This pioneering study not only gave a quantitative measurement of the waveshape effect on survival fractions, but also opened opportunities for new fundamental ideas that may be used beyond the domain of PEF.

0

Factors Dependent on Microbial Entity Characteristics

The type of microorganism (i.e., gram-positive, gram-negative, yeast, or spore former) will define specific characteristics such as cell size and membrane structure that will lead to different inactivation levels of food flora under similar treatment conditions. The growth stage and initial microbial contamination have also proven to be important factors, as several cell changes take place during microbial growth. Therefore, PEF may induce imbalances in the regular paths of reproduction that lead to inactivation.

VI. Main Factors in Microbial Inactivation

99

1. Type of Microorganisms Among bacteria, those that are gram-positive are more resistant than those that are gram-negative. In general, yeasts are more sensitive to electric field pulses than bacteria due to their larger size (Fig. 4.10), although at low-electric fields they seem to be more resistant than gram-negative cells. Inactivation of more resistant microorganisms requires greater electric field strengths, more treatment time, a n d / o r possibly higher temperatures. Because it is important to maintain the quality of foods in addition to inactivating their microorganisms, treatment parameters should be carefully selected so the nutritional components of foods are not drastically affected. The inactivation of B. subtilis and B. cereus spores suspended in NaC1 solutions has been reported to be higher when instant-reverse pulses and a plurality of electric field chambers with high-pulse frequencies are used. Other studies using exponentially decaying pulses have reported no inactivation when spores were suspended in milk or simulated milk ultrafiltrate medium (SMUF) using 22.4 k V / c m (Grahl and M~irkl, 1996), or when using 60 k V / c m (Pag{m et al., 1998). However, experiments using instant-reverse charge and very high pulse frequencies have achieved the inactivation of viable spores up to 5 log cycles (Ho and Mittal, 1996). 2. Concentration of Microorganisms The number of microorganisms in a food may have an effect on its electric field-induced inactivation. For example, the inactivation of E. coli in a model food system consisting of a SMUF was not affected when the concentration of the microorganism was varied from 10 ~ to 108 c f u / m l after being subjected to 16 pulses with a pulse duration of 2 /zsec and an electric field of 70 k V / c m (Fig. 4.11). However, increasing the concentration of S. cerevisiae in apple juice resulted in a slightly lower inactivation with 1 pulse (pulse duration 25 /zsec) at an electric field of 25 k V / c m (Fig. 4.12). It can thus be determined that the effect of microbial concentration on inactiva-

S. aureus

1.Ogm

I~

E. coli

/

I

S. cerevisiae

0.91.trn

""'//"~ .2gm

Fig. 4.10 A cell size comparison (adapted from Qin et al., 1998).

I O0

4. PEF-Induced Biological Changes

g~ 3

0

i 7.14E+08

i

!

1.36E+07

1.22E+05

1.26E+04

1.15E+03

Initial E. coli concentration Fig. 4. I I An initial concentration and inactivation of E. coli in SMUF with 16 pulses of 2 /xsec duration at 70 k V / c m (adapted from Zhang et al., 1995).

tion may be related to the cluster formation of yeast cells a n d / o r possibly concealed microorganisms in low electric field regions (Qin et al., 1996).

3. Growth Stage of Microorganisms Cell and membrane properties are different at different stages of microorganism growth, and logarithmic phase cells are more sensitive than lag and stationary phase cells (Fig. 4.13) (Pothakamury et al., 1996). Microbial growth in logarithmic stage is characterized by a higher number of cells in a state of proliferation, during which the area between the mother and daughter cells and sensitive parts of the cell envelope are susceptible to the

0.05

0.04 r 0.03

9~ 0.02 r~ 0.01

1.00E+06

1.00E+06

1.00E+05

1.00E+05

1.00E+04

1.00E+04

Initial inoculation (CFUImL) Fig. 4.12 An initial concentration and inactivation of S. cerevisiae in apple juice with one pulse of 2 5 / z s e c duration at 25 k V / c m (adapted from Zhang et al., 1994b).

I01

VI. Main Factors in Microbial Inactivation

Survival Fraction 1

0.1 0.01 0.001 0

0.5

1.5

2.5

3.5

4.5

5.5

6

Time of Growth (hours) [ . 2pulse ~ 4 p u l s e I Fig. ,1.13 Cells of E. coli harvested at different growth stages suspended in SMUF and subjected to an electric field of 36 kV/cm at 7~ (Pothakamury et al., 1996). (Reprinted with permission from Journal of Food Protection. Copyright held by the International Association of Milk, Food and Environmental Sanitarians, Inc.) applied electric field. Therefore, the tender cells in the logarithmic phase are more sensitive to electric field action. The effect of the growth stage has been corroborated by Gftskovft et al. (1996), who found the killing effect of PEF on exponentially growing cells to be greater than 30% of those in a stationary stage.

C.

Factors Dependent on Treatment Media

Conductivity, ionic strength, pH, antimicrobials, the presence of particles or gas bubbles, and the dielectric properties of a m e d i u m are all important characteristics that alter the biological changes p r o d u c e d during PEF treatment. In general, conductivity is related to the efficiency of the energy transferred, where a low conductivity leads to a more effective PEF treatment. Conductivity and ionic strength are closely related in that conductivity increases along with ionic strength. Additional factors that e n h a n c e PEF t r e a t m e n t are p H and the presence of antimicrobials, which play the role of hurdles; each of these factors imposes an additional stress to microorganisms, and the result is an increase in the total inactivation, by what is called an e n h a n c e m e n t of the PEF treatment. Particles and gas bubbles suspended in food liquids or semisolids represent an extra challenge to PEF treatment, as the former prevents a n o n u n i f o r m distribution of the applied electric field and the latter causes dielectric breakdown, arcing, or sparks. 1. Effect of Medium pH, Antimicrobials, and Ions

Electric field-induced microbial inactivation increases with a decrease in the ionic strength of a food. Benz et al. (1979) found no effect of a m e d i u m ' s pH on the breakdown of lipid bilayer m e m b r a n e s p r e p a r e d from oxidized

102

4. PEF-Induced Biological Changes

cholesterol. However, Vega-Mercado et al. (1996) reported a slightly greater inactivation of E. coli in a SMUF at a low pH (5.69) than at a high pH (6.82). Likewise, the inactivation of E. coli increased with an increase in the n u m b e r of pulses and an increase in the electric field from 40 to 55 k V / c m and was more significant at a pH of 5.69 than 6.82 (p < 0.05). The pH of the media also plays an important role in inactivation kinetics when PEF treatment is combined with organic acids. The strong synergistic killing effect of the combination of organic acids and PEF treatment at low pH ( ~ 3.4) indicates that the entry of undissociated acids into bacterial cells was enhanced (Liu et al., 1997). The presence of antimicrobials (such as pediocin AcH or nisin) in media has reduced the viability of pathogens such as L. monocytogenes, E. coli O157:H7, and S. typhimurium up to an additional 2.1 log cycles when used in combination with high hydrostatic pressure, another promising n o n t h e r m a l treatment (Kalchayanand et al., 1997). As an example of antimicrobial use in combination with PEF, a population of E. coli treated with one 12.5-kV/cm pulse in a solution containing 1000 ppm of benzoic acid with a pH of 2.4 was reduced by 4 log cycles (Liu et al., 1997). Ions dissolved in cultures and treatment media have been found to produce different effects on inactivation patterns. Ions such as Ca 2+, Na+, K +, and Mg 2+ may influence or interfere with m e m b r a n e and cellular functions. Bacteria cultured in the presence or absence of oxygen does not influence inactivation, nor does Na + and K + in a treatment medium. Ca 2+ and Mg 2+, however, have been shown to induce a protective effect against electric field treatment (Hiilsheger et al., 1981). A clear conjecture about these is not posible yet, since more information is necessary about the specific type of ion used. 2. Conductivity and Medium Ionic Strength

The conductivity of a medium, defined as the ability to conduct electric current, is an important variable in PEF technology. In general, conductivity is symbolically defined by o" and measured in Siems per unit length (S/m). It is the inverse of resistivity, defined by the letter p, and is measured in o h m meters (f~.m). Figure 4.14 is a good example of the effect of media conductivity on the effectiveness of PEF treatment. Table 4.13 presents some treatment media used in PEF and their corresponding resistivities/conductivities. Foods with large electrical conductivities are difficult to work with because they generate smaller peak electric fields across the treatment chamber. Therefore, it is desirable to lower a food's conductivity (if possible) to obtain greater microbial inactivation for the same applied electric field. Because an increase in conductivity increases the ionic strength of a liquid, an increase in the ionic strength of a food results in a decrease in the inactivation rate. Furthermore, an increase in the difference between the conductivities of a medium and microbial cytoplasm weakens the m e m b r a n e structure due to an increased flow of ionic substances across the membrane.

VII. Final Remarks

103

1.0E+00 = o

1.0E-01

0.0092 S/m --4~ 0.1308 S/m ---n---0.4752 S/m • 0.9669 S/m

1.oE-o2

"d .~ 1.0E-03 1.0E-04

",,,,,,.

1.0E-05

1

5

i

15

i

25

I

55

i

95

120

Treatment Time (gs) Fig. 4.14 Effect of conductivity and processing time on a survival fraction of S. dublin after a PEF treatment of 28 kV/cm, 1 /zsec pulse duration, and 3.73-kHz pulse rate (Sensoy et al., 1997).

Therefore, the inactivation rate of microorganisms increases with decreasing conductivity even with an application of equal input pulse energy. In other words, an increase in ionic strength increases the electron mobility through a solution and thus decreases the inactivation rate. Evidence of the relation between the inactivation rate and the ionic strength was verified when Vega-Mercado et al. (1996) obtained a significant difference in the inactivation level of 2.5 log cycles between 0.168 and 0.028 M solutions.

VII.

Final Remarks

This chapter discussed the different biological changes induced by electric fields and how these changes take place in the cell m e m b r a n e of the treated microorganisms. To analyze the m e m b r a n e response to PEF, a capacitor charged by electric field pulses was considered, and an explanation provided for how after the m e m b r a n e had reached a certain potential it may undergo an electrical breakdown or major perturbation in its structure that leads to a permeability increase in the m e m b r a n e . The analysis by mathematical simulation of the electric fields as the primary source of cell p e r m e a t i o n took into account (a) the effect of charge accumulation at the suspension l i q u i d / m e m b r a n e interface and p r o t o p l a s m / m e m b r a n e interface due to differences in conductivities in the cell m e m b r a n e environment and (b) the development of high temperatures at the m e m b r a n e wall evidenced by the simulation. The pulse step provided even more tools for understanding and evaluating the inactivation effect of PEF on microbial cells. Although PEF inactivation depends on several factors acting together, u n d e r some circumstances an i n d e p e n d e n t analysis allowed the identification of specific correlations between certain factors (treatment time, electric fields, n u m b e r of pulses, and waveforms) and the survival fraction. For

104

4. PEF-Induced Biological Changes

TABLE 4.13 Electrical Resistivity and Conductivity of Various Foods and Fluids

Foods/fluids Apple juice c Distilled water (100%, W/W) a Egg white ~ Fluid egg product b Milk (raw) c Milk (raw) c Milk (raw) ~ Milk (skim) ~ Orange juice b Orange juice concentrate ~ Pea soup c Peptone (0.1%, w/w) a Potato dextrose agar c Sodium chloride (0.1%, w/w) a Sodium chloride (0.2%, w/w) a Sodium chloride (0.3%, w/w) ~ Sodium chloride (0.4%, w/w) a Sodium chloride (0.5%, w/w) ~ Sucrose (10%, w/w) a Sucrose (15%, w/w) a Sucrose (20%, w/w) ~ Sucrose (25%, w/w) a Sucrose (30%, w/w) ~ Sucrose (35%, w/w) ~ Tomato ketchup c Yogurt b Xanthan gum (0.2%, w/w) ~ Xanthan gum (0.4%, w/w) ~ Xanthan gum (0.6%, w/w) ~ Xanthan gum (0.8%, w/w) a Xanthan gum (1.0%, w/w) a

Electric resistivity (fl.m)

Conductivity (S/m-1 )

5.7 9,090.90 1.55 1.7 2.2 2.3 2.6 3.1 2.34 3.0 3.8 16.67 7.9 4.17 2.38 1.64 1.23 0.98 555.56 540.54 588.23 588.23 606.06 606.06 0.42 1.69 45.45 25.0 20.0 14.29 11.11

0.175 0.00011 0.645 0.588 0.455 0.435 0.385 0.323 0.427 0.333 0.263 0.065 0.127 0.24 0.42 0.615 0.81 1.02 0.0018 0.00185 0.0017 0.0017 0.00165 0.00165 2.38 0.592 0.022 0.04 0.05 0.07 0.09

Testing temperature

(oc) 15 15 21 25 2O 15 15 42 15 15 15

15 23

m

a From Ho et al. (1995). b From Dunn and Pearlman (1987). c From Zhang et al. (1994c).

waveforms, the evaluation of frequency components opens a new and original way to evaluate the efficiency of the treatment, but although interesting, it is also challenging due to instrumentation and sensor limitations. Furthermore, the study of other factors such as suspending media characteristics and the type, size, and growth stage of a microorganism provide qualitative relations that are in good agreement among research groups. In general, it can thus be concluded that discoveries about individual aspects of PEF lead to better understanding of the technology as a whole, but it is also important to realize that additional studies of higher specificity are necessary to generalize the biological changes produced by PEF.

References

105

References Benz, R., Beckers, F., and Zimmermann, U. (1979). Reversible electrical breakdown of lipid bilayer membranes: A charge-pulse relaxation study. J. Membr. Biol. 48, 181-204. Bruhn, R. E., Pedrow, P. D., Olsen, R .G., Barbosa-C{movas, G .V., and Swanson, B. G. (1997). Electrical environment surrounding microbes exposed to pulsed electric fields. IEEE Trans. Dielec. Electric. Insul. 4(6), 806-812. Bruhn, R. E., Pedrow, P. D., Olsen, R. G., Barbosa-C{movas, G. V., and Swanson, B. G. (1998). Heat conduction in microbes exposed to pulsed electric fields. F E E Trans. Dielec. Electric. Insul. 5(12), 878-885. Chang, D. C. (1989). Cell poration and cell fusion using an oscillating electric field. Biophys. J. 56, 641-652. Chen, W., and Lee, R. C. (1994). Altered ion channel conductance and ion selectivity induced by large imposed membrane potential pulse. Biophys. J. 67, 603-612. Dunn, J. E., and Pearlman, J. S. (1987). Methods and apparatus for extending the shelf life of fluid food products. U. S. Patent 4,695,472. G~skov~, D., Sigler, K., Janderova, B., and Plasek, J. (1996). Effect of high-voltage electric pulses on yeast cells: Factors influencing the killing efficiency. Bioelectrochem. Bioenergetics 39, 195-202. Grahl, T., and M~irkl, H. (1996). Killing of microorganisms by pulsed electric fields. Appl. Microbiol. Biotechnol. 45, 148-157. Hamilton, W. A., and Sale, A . J . H . (1967). Effects of high electric fields on microorganisms II. Mechanism of action of the lethal effect. Biochim. Biophys. Acta 148, 789-800. Ho, S. Y., and Mittal, G. S. (1996). Electroporation of cell membranes: A review. Crit. Rev. Biotechnol. 16(4), 349-362. Ho, S. Y., and Mittal, G. S. (1997). Analysis of two high voltage electric pulse systems for batch and continuous pasteurization of selected food products. Confidential unpublished report, University of Guelph, Canada. Ho, S. Y., Mittal, G. S., Cross, J. D., and Griffith, M. W. (1995). Inactivation of P. fluorescens by high voltage electric pulses. J. Food Sci. 60(6), 1337-1340. Hiilsheger, H., Potel, J., and Niemann, E. G. (1981). Killing of bacteria with electric pulses of high field strength. Radiat. Environ. Biophys. 20, 53-65. Hiilsheger, H., Potel, J., and Niemann, E. G. (1983). Electric field effects on bacteria and yeast cells. Radiat. Environ. Biophys. 22, 149-162. Jacob, H. E., Forster, W., and Berg, H. (1981). Microbiological implications of electric field effects. II. Inactivation of yeast cells and repair of their cell envelope. Z. AUg. Microbiol. 21(3), 225-233. Kalchayanand, S., Dunne, A., and Ray, B. (1997). Effectiveness of hydrostatic pressure in combination with pressurization time and temperature and bacteriocin on viability loss kinetics of foodborne pathogens. Presented at the IFT Annual Meeting, Orlando, Florida. Kinosita, K., Jr., and Tsong, T. Y. (1977). Hemolysis of human erythrocytes by a transient electric field. Proc. Natl. Acad. Sci. 74(5), 1923-1927. Liu, X., Yousef, A. E., and Chism, G. W. (1997). Inactivation of Eschenchia coli 0157:H7 by the combination of organic acids and pulsed electric fields. J. Food Safety 16, 287-299. Love, P. (1998). Correlation of Fourier transform of pulsed electric field waveform and microorganism inactivation. [EEE Trans. Dielec. Electric. Insul. 5(1), 142-147. Ma, L., G6ngora-Nieto, M., Barbosa-C(movas, G. V., and Swanson, B. G. (1998). Food pasteurization using high-intensity pulsed electric fields: Promising new technology for non-thermal pasteurization for eggs. In "Proceedings of the Second International Symposium on Egg Nutrition and Newly Emerging Ovo-Technologies" (J. S. Sim, ed.), in press. CAB International, New York. M~rquez, V. O., Mittal, G. S., and Griffiths, M. W. (1997). Destruction and inhibition of bacterial spores by high voltage pulsed electric fields. J. Food Sci. 62(2), 399-409.

106

4. PEF-Induced Biological Changes

Martln-Belloso, O., Qin, B. L., Chang, F.J., Barbosa-C{movas, G. V., and Swanson B. G. (1997). Inactivation of Escherichia coli in skim milk by high intensity pulsed electric fields. J. Food Proc. Eng. 20(4), 317-336. Ohshima, T., Sato, K., Terauchi, H., and Sato, M. (1997). Physical and chemical modifications of high-voltage pulse sterilization. J. Electrostat. 42, 159-166. Pagan, R., Esplugas, S., G6ngora-Nieto, M. M., Barbosa-C{movas, G. V., and Swanson, B. G. (1998). Inactivation of Bacillus subtilis spores using high intensity pulsed electric fields in combination with other food conservation technologies. Food Sci. Technol. Int. 4(1), 33-44. Peleg, M. (1995). A model of microbial survival after exposure to pulsed electric fields. J. Sci Food Agric. 67, 93-99. Pothakamury, U. R., Vega-Mercado, H., Zhang, Q., Barbosa-C{tnovas, G. V., and Swanson, B. G. (1996). Effect of growth stage and temperature on inactivation of E. coli by pulsed electric fields. J. Food Prot. 59(11), 1167-1171. Qin, B. L., Zhang, Q., Barbosa-C~movas, G. V., Swanson, B. G., and Pedrow, P. D. (1994). Inactivation of microorganisms by pulsed electric fields with different waveforms. IEEE Trans. Dielec. Electric. Insul. 1(6), 1047-1057. Qin, B. L., Pothakamury, U. R., Vega-Mercado, H., Mart~n-Belloso, O., Barbosa-C{movas, G. V., and Swanson, B. G. (1995). Food pasteurization using high-intensity pulsed electric fields. Food Technol. 42(12), 55-60. Qin, B. L., Pothakamury, U. R., Barbosa-C{movas, G. V., and Swanson, B. G. (1996). Nonthermal pasteurization of liquid foods using high intensity pulsed electric fields. Crit. Rev. Food Sci. Nutr. 36(6), 603-627. Qin, B. L., Barbosa-C{tnovas, G. V., Swanson, B. G., Pedrow, P. D., and Olsen, R. G. (1998). Inactivating microorganisms using a pulsed electric field continuous treatment system. 1EEE Trans. Ind. Appl. 34(1), 43-50. Sale, A. J. H., and Hamilton, W. A. (1967). Effects of high electric fields on microorganisms. I. Killing of bacteria and yeast. Biochem. Biophys. Acta 143, 781-788. Schoenbach, K. H., Peterkin, F. E., Alden III, R. W., and Beebe, S.J. (1997). The effect of pulsed electric fields on biological cells: Experiments and applications. IEEE Trans. Plasma Sci. 25(2), 284-292. Sensoy, I., Zhang, Q. H., and Sastry, S. K. (1997). Inactivation kinetic of Salmonella dublin by pulsed electric fields. J. Food Proc. Eng. 20, 367-381. Sitzmann, W. (1990). KeimabtiStung mit hilfe elecktrischer hochspannungsimpulse in pumpf~ihigen nahrungsmitteln. Vortrag anlablich des Seminars "Mittelstansfourderung in der Biotechnologie." Ergebnisse des Indirekt-Spezifischen Programma des BMFT 1986-1989. KFA Julich, Germany, 6-7 February. Teissie, J., and Tsong, T. Y. (1980). Evidence of voltage-induced channel opening in N a / K ATPase of human erythrocytes. J. Membr. Biol. 55, 133-140. Vega-Mercado, H., Pothakamury, U. R., Chang, F. J., Zhang, Q., Barbosa,C{movas, G. V., and Swanson, B. G. (1996). Inactivation of E. coli by combining pH, ionic strength and pulsed electric field hurdles. Food Res. Int. 29(2), 117-121. Wong, T. K., and Neumann, E. (1982). Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107(2), 584-587. Zhang, Q., Monsalve-Gonz{dez, A., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1994a). Inactivation of E. coli and S. cerevisiae by pulsed electric fields under controlled temperature conditions. Trans. ASAE 37(2), 581-587. Zhang, Q., Monsalve-Gonz{dez, A., Qin, B. L., Barbosa-C{movas, G. V., and Swanson, B. G. (1994b). Inactivation of Saccharomyces cerevisiae in apple juice by square-wave and exponential-decay pulsed electric fields. J. Food Proc. Eng. 17, 469-478. Zhang, Q., Chang, F. J., Barbosa-C{movas, G. V., and Swanson, B. G. (1994c). Inactivation of microorganisms in semisolid foods using high voltage pulsed electric fields. Lebensm. Wiss. Technol. 27(6), 538-543.

References

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Zhang, Q., Qin, B. L., Barbosa-Cfmovas, G. V., and Swanson, B. G. (1995). Inactivation of E. coli for food pasteurization by high-strength pulsed electric fields. J. Food Proc. Pres. 19, 103-118. Zhang, Q. H., Qiu, x., and Sharma, S. K. (1997). Recent developments in pulsed electric field processing. In "New Technologies Yearbook" (D. I. Chandrana, ed.), pp. 31-42. National Food Processor's Association, Washington, D.C. Zimmermann, U. (1986). Electrical breakdown, electropermeabilization and electrofusion. Rev. Physiol. Biochem. Pharmacol. 105, 175-256.

CHAPTER $

PEF lnactivation of Vegetative Cells, Spores, and Enzymes in Foods

I.

Introduction

This chapter presents how PEF can inactivate microbes, enzymes, and spores in model and real foods. Since the composition of model foods is relatively less complicated than in real foods, lower interference with microbial inactivation is expected. However, real foods are composed of proteins, carbohydrates, and fats, which are likely to influence the effects of electric fields on microbial inactivation, so effective t r e a t m e n t may be more difficult to achieve than in model foods. Research is currently being conducted on the inactivation of microorganisms inoculated in model and real food systems and those naturally present in foods. Microbial challenge tests are conducted to determine the effect of electric fields on the inactivation kinetics of selected microorganisms inoculated in real or model foods. The tests are conducted by applying an electric field, which causes the inactivation of a m a x i m u m n u m b e r of microorganisms without an electrical breakdown of the food.

II.

Microbial Inactivation

Initial studies on the inactivation of microbes were conducted using model foods free of fats, proteins, or sugars easily prepared to obtain media with different electrical and physical properties. This enabled a focus on how t r e a t m e n t conditions and suspending media characteristics affected micro108

109

II. M i c r o b i a l I n a c t i v a t i o n

bial inactivation by PEF with minimum or no interference with other food components. Escherichia coli and Saccharomyces cerevisiae are the most studied microorganisms, not only in the area of PEF processing, but also a m o n g other methods of food preservation. Other relevant microorganisms to be tested and inactivated using PEF include the gram-negative pathogens Salmonella dublin, S. typhimurium, and E. coli O157:H7; the gram-positive Staphylococcus aureus and Listeria monocytogenes; the spore-forming pathogens Bacillus cereus and B. subtilis; and nonpathogenic spoilage flora LactobaciUus bulgaricus, L. brevis, Micrococcus lysodeikticus, and Pseudomonas fluorescens. Sarcina lutea, Bacillus megaterium, Clostridium welchii, and Candida utilis have also been investigated. Further research on the spore-formers Bacillus coagulans, Clostridium

sporogenes, C. botulinum, C. butryricum, Mycobacterium tuberculosium, Aspergillus niger, Candida albicans, Penicillum roqueforti, Bysochlamys fulva, Listeria inocua, and LactobaciUus acidophilus has been recommended. In general, gram-negative bacteria are more sensitive than gram-positive bacteria (Fig. 5.1). Cell shape (i.e., rod, spherical) and size also seem to play an important role in the effect of PEF treatment. Rod-shaped (bacilli) bacteria tend to orient with their longest axis in the direction of the electric field, which causes the oriented rods to migrate at different velocities, and hence inactivation may vary.

A.

Inactivation of Yeasts

One of the more important causes of food spoilage is the presence of yeasts;

Saccharomyces and Candida are two genera of particular relevance. Saccharomyces cerevisiae is used for the leavening of bread and alcoholic fermentation, but in products such as juices, the microorganism causes spoilage due to the production of alcohol and CO 2. In general, S. cerevisiae cells have

100

--

=

9 --

&

=

/I

9

9

A

10

eSc 9

/I

m 9

i

0

5

mCu A

AMp

!

i

i

10

15

20

eMl 25

Electric Field (kV/cm)

Fig. 5. I

Relationship between the survival fraction and electric field strength of 10 pulses of 20 /xsec on S. cerevisiae (Se), C. utilis (Cu), M. pseudomona (Mp), and M. lysodeikticus (MI) (adapted from Sale and Hamilton, 1967).

I I0

5. PEF Inactivation of Cells, Spores, and Enzymes

been found less resistant to PEF treatment than other vegetative cells, which is mostly attributed to their large size (Fig. 4.10). The inactivation of this microorganism has been proven in such food models as water, phosphate buffers, sodium alginate solutions, and semisolid potato dextrose agar (PDA), as well as in such foods as orange and apple juice, UHT milk (1.5% fat), and yogurt. Table 5.1 summarizes most of the research conducted on the inactivation of S. cerevisiae cells with PEF. Zheng-Ying and Yan (1993) studied the inactivation of yeast and Bacilli cells using the r o d - r o d and three-rod electrode systems and found that their survival rates followed the same pattern. However, the inactivation of yeast cells was greater when cell concentrations were lower or the specific resistance of the medium was greater. For the same energy input, the r o d - r o d electrode system produced greater inactivations than the three-rod electrode system, but the inactivation of yeast cells suspended in deionized water increased with an increase in the volume of the test medium. Jacob et al. (1981) found that logarithmic phase yeast cells were more sensitive to electric field treatment than stationary phase cells when 25% of the stationary phase cells of S. cerevisiae (ZIMET H192) were inactivated with an electric field of 30 k V / c m , whereas only 5% of the logarithmic phase cells survived after four pulses with an electric field of 11 k V / c m . These results were confirmed by G{tskovfi et al. (1996), who found that the killing rate in exponentially growing cells increases to nearly 100% when pulse amplitudes exceed about 4 - 1 6 kV/cm. Although a rise in the killing rate in stationary phase cells was observed only at electric fields higher than 1520 k V / c m , it never did surpass 30%. Mizuno and Hayamizu (1989) reported a 3 log cycle reduction of S. cerevisiae in deionized water and a 2.5 log reduction in a 1% NaC1 solution. In each instance the charging voltage was 10 kV, the pulse frequency was 10 Hz, and the electrical energy input was 30 c a l / c m 3. Matsumoto et al. (1991) analyzed the effect of different treatment chamber configurations on the inactivation of vegetative cells and achieved almost 5 log cycles of inactivation in a converged electric field electrode system with an input energy of 20 c a l / c m 3 when cells of S. cerevisiae were suspended in a phosphate buffer. For a fixed treatment time of 30 #sec with an electric field of 25 kV/cm, S. cerevisiae suspended in water, yogurt, and orange juice was inactivated 85.6, 99.7, and 99.97% (0.845, 2.52, and 3.52 log cycles), respectively (Hofmann, 1984). The higher susceptibility of S. cerevisiae cells to PEF is verified by the low critical electric field and critical treatment time values of their first-order inactivation kinetics. Grahl et al. (1992) and Grahl and M~irkl (1996) reported the following values for cells suspended in sodium alginate: E c = 5.4 k V / c m at 5 pulses and t c = 0.40 /zsec at 24.8 kV/cm; for U H T milk, Ec = 4.7 k V / c m at 5 pulses and t c = 19.8 /~sec at 24.8 kV/cm; and for orange juice, E c = 4.7 k V / c m at 5 pulses and t c = 76.1 /zsec at 6 k V / c m . The higher treatment time required for the inactivation in milk confirms the protective role of its fat, while the low pH of the orange juice may favor its

T A B L E 5. I S u m m a r y of S. cerevisiae Inactivation with PEF ~

Source

Log reduction (max)

Suspension media

Jacob et al. (1981) Dunn and Pearlman (1987) Hiilsheger et al. (1983)

0.9% NaC1 Yogurt Phosphate buffer, pH 7.0

Mizuno and Hori (1988)

Deionized water

1.3 3 three stationary cells, four logarithmic cells 6

Matsumoto et al. (1991) Yonemoto et al. (1993)

Phosphate buffer 0.85% NaC1

5 2

Zhang et al. (1994b) Qin et al. (1994)

Potato dextrose agar Apple juice

5.5 4

Qin et al. (1994) Zhang et al. (1994a)

Apple juice Apple juice

4.2 4

Zhang et al. (1994a)

Apple juice

3.5

Zhang et al. (1994a)

Apple juice

3-4

Qin et al. (1995b)

Apple juice

7

Qin et al. (1995b)

Apple juice

6

Grahl et al. (1992); Grahl and M~irkl (1996)

Orange juice

5

B, 3 ml, d = 0.5 cm B B, 4 ml, d = 0.5 cm

3.5 V / ~ m , 20 /.~sec, 4 pulses 55~ 1.8 V / / ~ m 2.0 V / l ~ m , 36/~sec, 30 pulses, t = 1,080/~sec

0.77 c a l / c m ~ / p u l s e , B, parallel plate, 0.5 cm 3, d = 0.8 cm B B, parallel plate, 2 ml, d = 0.55 cm 6 2 J / m l , B, 14 ml 2 7 0 J / p u l s e , B, parallel plate

2.0 V / ~ m , 160/~sec, 175 pulses, exponential decay

2 7 0 J / p u l s e , B, parallel plate 2 6 0 J / p u l s e , B, parallel plate, 25 ml, d = 0.95 cm 260 J / p u l s e , B, Parallel plate, 25 ml, d = 0.95 cm 558 J / p u l s e , B, parallel plate, 25.7 ml, d = 0.95 cm C, coaxial, 29 ml, d = 0.6 cm, 0.2 /~F, 1 Hz 2 8 J / m l , C, coaxial, 30 ml, 2-10 1 / m i n B, 25 ml, d = 0.5 cm, E c = 4.7

Reprinted from Wouters and Smelt (1997), pp. 193-229 by courtesy of Marcel Dekker, Inc. batch; C, continuous. c Temperature, peak electric field, pulse width, number of pulses and shape, and total treatment time (t).

a

b B,

Process conditions c

T r e a t m e n t vessel b

3.0 V / ~ m 0.54 V / l ~ m , 90 ~sec, 10 pulses 15 + 1~ 4.0 V//~m, 3/~sec, 16 pulses < 30~ 1.2 V / i ~ m , 20 pulses, exponential decay < 30~ 1.2 V//~m, 20 pulses, square wave 4-10~ 1.2 V//~m, 90/.~sec, 6 pulses, exponential decay 4-10~ 1.2 V//~m, 60/~sec, 6 pulses, square wave < 25~

2.5 V//~m, 5 pulses

< 30~ 2.5 V / l ~ m , 2-20/~sec, _+ 150 pulses, exponential decay 22-29.6~ 5.0 V//~m, 2.5 ~sec, 2 pulses 0.675 V//~m, 5 pulses

I 12

s. PEF Inactivation of Cells, Spores, and Enzymes

inactivation rate. The studies also revealed that an inactivation of 5 log cycles of S. cerevisiae cells with electric fields as low as 7 k V / c m is possible; this result, in addition to the implied spoilage control benefit, is very important from an economic point of view, where the use of a low electric field and optimal treatment conditions produce energy inputs as low as 10 kJ/liter. Zhang et al. (1994b) reported that the inactivation of S. cerevisiae (ATCC 16664) in a model semisolid system of potato dextrose agar took only 16 pulses with an electric field of 40 k V / c m and 77 J / m l to reduce the bacteria population by 6 logs. Furthermore, when the inactivation kinetic constants were evaluated, the critical electric field was found to be 19.3 k V / c m (at 8 pulses) and the critical treatment time was 5.4 /xsec. The application of 20 pulses at 25 k V / c m and 25~ reduced the population of S. cerevisiae (ATCC 16664) suspended in apple juice by nearly 4 log cycles, but an even greater degree of inactivation was obtained with a reduction in the initial concentration of the bacteria (Zhang et al., 1994a). A comparison by Qin et al. (1994) of the survival fraction of S. cerevisiae subjected to exponential and squarewave pulses in a static parallel plate electrode treatment chamber with an electric field of 12 k V / c m revealed that square-wave pulses produce approximately 60% more inactivation than exponential decay pulses. (The energy efficiency of the square-wave pulse treatment was 91%, whereas that of the exponential pulses was only 64%.) These results led to the conclusion that the intensity of the electric field, treatment time, and n u m b e r of pulses affect the inactivation of S. cerevisiae suspended in different treatment media. Figure 5.2 illustrates the microbial count of S. cerevisiae suspended in apple juice as a function of peak field intensity when in a coaxial treatment chamber u n d e r a continuous system of two pulses with selected field intensifies of 13, 22, 35, and 50 k V / c m (Fig. 5.3). An inactivation of 6 log cycles was achieved after 10 pulses of 35 k V / c m at 22-34~ Ohshima et al. (1997) studied the effects of insulator shape in between two parallel plate electrodes to concentrate relatively low-electric field strengths. The concentration or e n h a n c e m e n t of electric fields was verified

1 0.1 0.01

v

A

0.001

"--..,,

0.0001

0.00001 0.000001 0.0000001

0

|

.

.

|

|

a

10

20

30

40

50

60

Electric Field Intensity (kV/cm) Fig. 5.2 A survival fraction of S. cerevisiae as a function of peak field intensity when two 2.5-/~sec pulses were applied (adapted from Qin et al., 1995b).

II. Microbial Inactivation

I 13

Fig. 5.3 Microbiological count of S. cerevisiae in apple juice as a function of the number of 2.5-/xsec pulses (adapted from Qin et al., 1995b).

by the decrease in the survival ratio of S. cerevisiae. The different insulating plates used in the parallel plate treatment chamber consisted of r o u n d Plexiglas plates with varying amounts of holes of different sizes, which in all cases gave the same area of open space. The sterilization efficiency increased using a spacer with more holes of smaller diameter. The m i n i m u m survival ratio was about ]0 -6 when 300 J / m l of energy was applied at a treatment temperature of 50~ , although the survival ratio at 10~ was only around 10 ~ The authors clarify that sterilization without a pulse treatment below 45~ was not observed. Harrison et al. (1997) assessed the PEF inactivation (64 pulses of 40 k V / c m ) of S. cerevisiae suspended in apple juice by transmission electron microscopy (TEM), and found that the observable ultrastructural changes provided little evidence to support the electroporation inactivation theory as the major mode of yeast inactivation. Transmission electron microscopy micrographs exhibited frequent disruption of S. cerevisiae cellular organelles and almost a total absence of ribosome bodies. Damaged organelles and lack of ribosomes suggested cytological disruption as an alternative inactivation mechanism to the accepted electroporation theory. Additional TEM observations were able to differentiate naturally occurring bud scars formed though the propagation of daughter yeast cells from m o t h e r S. cerevisiae cells as separate from PEF-induced scars (Fig. 3.16). Until now, most of the yeast inactivation research using PEF has been done on S. cerevisiae. However, Hiilsheger et al. (1983) studied the survival rates for C. albicans, which followed a first-order kinetic with a critical electric field of 8.4 k V / c m and a critical time of 110 /xsec. Thirty pulses of 20 k V / c m were used to achieve a survival fraction of almost 10 5, which is higher than in other microorganisms (i.e., L. monocytogenes, which has a final survival fraction of only 10 2) evaluated u n d e r the same conditions. Comparisons between the inactivation of two yeast spp. of different sizes (S. cerevisiae and Kluyveramyces lactis; size ratio 1.5) conducted by G~skov~t et al. (1996) showed that the electric field should be roughly proportional to the cell size, which in this case meant the smaller cells of K. lactis required an intensity 1.5 times higher to achieve the same inactivation level as the S.

I 14

5. PEF Inactivation of Cells, Spores, and Enzymes

cerevisiae cells. Zygosacchromyces bailii is a yeast noted for its exceptional

tolerance to high concentrations of salts, sugars, acidic conditions, and preservatives. This yeast forms ascospores that arise as a result of meiosis so that a typical diploid cell becomes transformed into an ascus containing haploid ascospores. The ascospore protoplast has a structure similar to vegetative cells, but the ascospore wall consists of an outer and inner coat. When Raso et al. (1998) used PEF to inactivate Z. bailii (ATCC 36947) ascospores and vegetative cells suspended in apple, orange, pineapple, cranberry, and grape juices, they found that yeast ascospores were more resistant to physical and chemical agents than vegetative cells, but the treatment was found to be very effective in the inactivation of both vegetative cells and ascospores. In each fruit juice studied, only two pulses with an electric field between 32 and 36.5 k V / c m (depending on the fruit juice) decreased the population of vegetative cells or ascospores between 3.5 and 5 log cycles.

B.

Inactivation of Escherichia coli

Escherichia coli, a gram-negative facultative anaerobic microorganism, is often

an indicator of fecal contamination in food products. This microorganism is found on plants, in soil and water, in the intestinal tract of animals, in animal products, and in prepared foods handled by people. Escherichia coli cells are also recovered from improperly sanitized working surfaces in processing plants (Banwart, 1989). Inactivation studies of different strains (pathogenic and nonpathogenic) of E. coli cells suspended in a variety of media under different treatment conditions in different treatment chambers and electric fields have been reported by various researchers (Hamilton and Sale, 1967; Hi]lsheger and Niemann, 1980; Hiilsheger et al., 1983; Matsumoto et al., 1991; Grahl et al., 1992; Grahl and M~irkl, 1996; Zhang et al., 1994a, 1995; Pothakamury et al., 1995a; Vega-Mercado et al., 1996a,b; Mart~n-Belloso et al., 1997a,b; Schoenbach et al., 1997; Liu et al., 1997; Ohshima et al., 1997). Even though the conditions were diverse, each of the reports was able to corroborate the effectiveness of PEF treatment. Table 5.2 presents a summary of the treatment conditions and inactivation results obtained in several investigation studies on the PEF inactivation of E. coli. One of the earliest studies on E. coli (8196) cells (Hamilton and Sale, 1967) revealed the leakage of essential intercellular material (amino acids, purine and pyrimidine bases) when a suspension of this bacteria was subjected to 10 pulses of 20/~sec at 1 Hz with electric fields ranging between 5 and 19.5 kV/cm. Hamilton and Sale (1967) found ninhydrin-positive material and 260 ~ m of absorbing material in the suspending medium. Alterations in biological activity were also seen when E. coli subjected to 22 k V / c m w a field sufficient to cause more than 99% inactivation--lost the ability to plasmolyse in a hypertonic medium made of 20 m M phosphate

T A B L E 5.2 S u m m a r y of E. coli Inactivation with PEF ~ Log reduction Source

T r e a t m e n t vessel b

Hiilsheger et al. (1983)

0.1% NaC1 17.1 m M saline, N a 2 S 2 0 3, NaH2PO4/Na2HPO, p H 7.0 Phosphate buffer, pH 7.0

D u n n a n d P e a r l m a n (1987) M a t s u m o t o et al. (1991)

Milk Phosphate buffer

three stationary cells, four logarithmic cells 3 5

G r a h l et al. (1992); Grahl a n d M~irkl (1996) G r a h l et al. (1992); G r a h l a n d M~irkl (1996) G r a h l et al. (1992); Grahl a n d M~irkl (1996) G r a h l et al. (1992); G r a h l and M~irkl (1996) G r a h l et al. (1992); Grahl a n d M~irkl (1996) Z h a n g et al. (1994b) Z h a n g et al. (1994b) Z h a n g et al. (1994b) Z h a n g et al. (1994b) Z h a n g et al. (1994c)

Sodium alginate

4-5

B, 25 ml, d = 0.5 cm

U H T milk (1.5% fat) U H T milk (1.5% fat) U H T milk (1.5 % fat) U H T milk (1.5% fat) Potato dextrose agar Potato dextrose agar Skim milk Skim milk SMUF

1

B, 25 ml, d = 0.5 cm

2

B, 25 ml, d = 0.5 cm

3

B, 25 ml, d = 0.5 cm

4

B, 25 ml, d = 0.5 cm

3 6 0.5 3 3

B, 1 4 m l B, 1 4 m l

Sale a n d H a m i l t o n (1967) Hiilsheger a n d N i e m a n n (1980)

m m

(max)

Suspension m e d i a

Process c o n d i t i o n s c 20~ 1.95 V / l ~ m , 20 /xsec, 10 pulses < 30~ 2.0 V / t z m , 3 0 / z s e c , 10 pulses, t = 300 /~sec

2 3-4

B

B, 4 m l , d = 0 . 5 c m

2.0 V / / x m , 36 ~sec, 30 pulses, t = 1080/zsec

B B

43~ 3.3 V / ~ m , 35 pulses 4.0 V / t z m , 4 - 1 0 /xsec, e x p o n e n t i a l decay < 45-50~ 2.5 V / / z m , 5 pulses

B, 4 m l , d = 0 . 5 c m

B B

604J, B, parallel plate, 25.7 ml, d = 0.95 cm

< 45-50~ 2.24 V / / x m , 5 pulses, 5.0 /xF < 45-50~ 2.24 V / t x m , 10 pulses, 5.0 /xF < 45-50~ 2.24 V / / x m , 15 pulses, 5.0 /xF < 45-50~ 2.24 V / / x m , 20 pulses, 5.0 /xF 15 _ 1~ 4.0 V / / x m , 3 /xsec, 16 pulses 15 ___ I~ 4.0 V / / x m , 3 /xsec, 64 pulses 15 _ I~ 4.0 V / / x m , 3 /zsec, 16 pulses 15 _+ 1~ 4.0 V / / x m , 3 /xsec, 64 pulses < 25~ 2.5 V / / x m , 20 pulses

Pothakamury et al. (1995b)

SMUF

4

B, parallel plate, 1 ml, d = 0.1 cm

Qin et al. (1994)

SMUF

1.5

Qin et al. (1994)

SMUF

3

Qin et al. (1994)

SMUF

3

Qin et al. (1994)

SMUF

3

Qin et al. (1995a)

Skim milk

2.5

80 J / p u l s e , B, parallel plate 8 0 J / p u l s e , B, parallel plate 6 0 J / p u l s e , B, parallel plate 6 0 J / p u l s e , B, parallel plate B, parallel plate, 14 ml

Skim milk

3.5

C, parallel plate

Qin et al. (1995a)

SMUF

3.6

Qin et al. (1995a)

SMUF

7

Martln-Belloso et al. (1994)

Nearly 3

Mart~n-Belloso et al. (1994)

Skim milk diluted with water (1:2:3) Skim milk

C, parallel plate 8 cm 3, d = 0.51 cm C, coaxial, 29 ml, d = 0.6 cm, 0.2 /~F, 1 Hz B, Parallel plate, 13.8 ml, 0.51 cm

Martln-Belloso et al. (1994)

Liquid egg

6

Vega-Mercado et al. (1996a) Zhang et al. (1995) Pothakamury et al. (1996) Pothakamury et al. (1996)

Pea soup Modified SMUF SMUF SMUF

6.5 9 3 5

Qin et al. (1995a) m m

2

C, parallel plate with flow-through capability, 45 m l / s e c , v = 8 ml C, coaxial, 11.9 ml, d = 0.6 cm, 0.5 1 / m i n C, coaxial, 0.5 1 / m i n B, parallel plate, 14 ml, d = 0.51 cm B, parallel plate, 12.5 ml, d = 0.5 cm C, parallel plate

a Reprinted from Wouters and Smelt (1997), pp. 193-229 by courtesy of Marcel Dekker, Inc. b B, batch; C, continuous. c Temperature, peak electric field, pulse width, number of pulses and shape, and total treatment time (t).

< 30~ 1.6 (1.2, 1.4, 1.6 t e s t e d ) V / ~ m , 20-300 ~sec, 60 (20, 30, 40, 50, 60) pulses < 30~ 4.0 V//.Lm, 8 pulses, oscillatory decay < 30~ 4.0 V//~m, 8 pulses, oscillatory decay < 30~ 4.0 V//~m, 4 pulses, monopolar < 30~ 4.0 V//.~m, 4 pulses, bipolar < 30~ 5.0 V / / ~ m , 2 /~sec, 62 pulses, square wave < 30~ 5.0 V / i ~ m , 2 /~sec, 48 pulses, square wave < 30~ 5.0 V//~m, 2 /~sec, 48 pulses, square wave < 30~ 2.5 V / ~ m , _+300 pulses, exponential decay

II. Microbial Inactivation

I 17

buffer (pH 7 . 2 ) + 10% sucrose. In addition, these researchers induced /3-galactosidase activity in the/3-galactoside permease-negative mutant E. coli (300 v) when the cell suspension was incubated with a sufficiently high concentration of lactose. Assay of the activity, however, depended on entry into the cell of the substrate 0-nitrophenyl-/3-galactoside (ONPG), which was present in the medium in low concentrations. In the absence of a permease, ONPG hydrolysis was demonstrated only when the permeability barrier had been destroyed. In this study the treatment of the cell suspension with an electric field of 25 k V / c m destroyed the permeability barrier and released fl-galactosidase activity, which demonstrated the effective membrane damage caused by PEF. Escherichia coli cells suspended in NaC1 (17.4 m M ) , Na2S20 3 (8.83 m M ) , and N a H z P O 4 / N a 2 H P O 4 (7.44 m M ) were inactivated when subjected to electric field pulses, and after 10 pulses at 20 kV/cm, a 3.5 log cycle reduction was obtained by Hiilsheger and Niemann (1980). The sulfate and phosphate solutions exhibited no toxic effect, but the chloride solution showed remarkable toxic activity, which was greater when the concentration of E. coli was smaller. With a higher initial concentration of E. coli, the effects of chloride, sulfate, and phosphate were similar. The toxicity of electric field-treated chloride solutions may have been due to the electrolytic production of free active chlorine created by the anodal oxidation of chloride ions. Because hypochloric acid, the bactericidal agent, is produced in a secondary step from a reaction of chlorine with water, it was concluded that electric fields smaller than 3 k V / c m will not have a significant lethal effect on E. coli (Hiilsheger and Niemann, 1980). The presence of organic acids has been proven to have a synergistic effect on microbial inactivation levels when combined with PEF treatment. Liu et al. (1997) investigated the effect of benzoic and sorbic acid (at 1000 ppm), pH, ionic strength of a medium, number of pulses, incubation temperature, and time before treatment (30 min at 0 and 25~ of the inactivation of E. coli 0157:H7 by suspending cells of this microorganism in 10% glycerol, 1% sucrose (nonionic media), 0.1% NaC1, and 5 m M phosphate buffer with a pH of 7 (ionic media). Results showed a higher degree of inactivation (up to 1 log cycle) with the ionic media. Furthermore, a synergistic effect of PEF and organic acids at a pH of 3.5 was found that yielded 5.6 and 4.2 log cycle reductions (benzoic and sorbic) at a pH of 3.5 at 25~ Due to the temporary integrity loss of microbial cell membranes during PEF treatment, the uptake of food preservatives increases when they are added to a food before treatment. The strong synergistic killing effect between organic acids and PEF treatment at a pH of 3.4 indicates that PEF enhanced the entry of the undissociated acids (86% for benzoic and 96.2% for sorbic) into the bacterial cells. Other studies with E. coli suspended in water with 2 ppm, O~, or H202 showed corresponding survival ratios (s = N / N o ) of 10 -15 and 10 -4. Results obtained by Ohshima et al. (1997) confirm that bactericides cause cell membrane wounding that leads to easier pulse sterilization.

I 18

5. PEF Inactivation of Cells, Spores, and Enzymes

The inactivation of E. coli also increases with an increase in the n u m b e r of pulses (0 to 8) and electric fields (from 20 to 55 k V / c m ) or when the treatment medium has a low pH (Vega-Mercado et al., 1996b). Inactivation has been found to be more significant at a pH of 5.69 than 6.82 (p < 0.05) as shown in Figs. 5.4 and 5.5. Vega-Mercado et al. (1996b) found that the temperature effect (10 or 15~ on the inactivation for these experiments was not statistically significant (p > 0.05). The role of pH in combination with PEF in the survival of microorganisms was hypothesized to be in relation to the ability of organisms to maintain cytoplasm pH near neutrality. Membrane permeability should therefore increase due to the formation of pores in cell walls during PEF treatment, and the rate of transport of H + may also increase due to the osmotic imbalance around the cell. Thus, a reduction in the cytoplasm pH may be observed because a higher n u m b e r of H + is available compared to a neutral pH. The change in pH within the cell may induce chemical modifications in fundamental compounds such as DNA or ATP, as well as oxidation and reduction reactions within the cell structure induced by PEF treatment. The effect of temperature was also observed in E. coli suspended in pea soup, where at 55~ a 6.5 log reduction was obtained, and less than 1 at 32~ (Vega-Mercado et al., 1996a). When the effect of bactericides was evaluated at a treatment temperature of 0~ the inactivation was 0.3 log cycles compared with 1.1 log cycles at 25~ (Liu et al., 1997). This can be explained by the higher cell m e m b r a n e fluidity at higher temperatures, where it is presumably easier for the organic acids to transfer into the cells, and higher stress is suffered by the cells in the treated pea soup. However, since PEF treatment is proposed as a nonthermal m e t h o d to pasteurize food, it is important to verify that heat is not the main source of inactivation. Ma et al. (1997) applied different heat treatments of E. coli cells suspended in liquid whole egg (LWE) and found that in a PEF treatment conducted at 32~ the main killing effect was not due to heat (Fig. 5.6).

= 0

I

",.

"~

.,..4

]PET

~"'.::::::::~:= ............ .~ .... "'", ""

0.1

95.7

-O.o. Ooo "Ooo ..o,. ~ -oo~

"'"- ~176176 "!

96.8

*~ "Ooo

0.01 0

i

i

i

2

4

6

~

I

I

8

I0

Number of Pulses

Fig. 5.4 Inactivation of E. coli suspended in SMUF using 40 kV/cm at 10~ with two samples per each experimental condition. (Reprinted from Food Res. Int., Vol. 29(2), Vega-Mercado et al., "Inactivation of E. coli by combining pH, ionic strength and pulsed electric field hurdles," pp. 117-121, (1996b), with permission from Elsevier Science.)

119

ram. Microbial Inactivation I

=

o

0.1

"~

I

"*Ile~.,,, "-',../.:~-.. 9 "":-?~ ::::::::: ....... ! - .

-...... "...... ~&...7.:::- . . . . . . ::::'-2 . . . . . . . . . . . -A

"-.

""-..--..... ",oo

"~.- 0.01

9

............ m

- o.,., ..

"'"'!..'.7".'.-:-.. . . . . . . . . . . . . . . . . .

0.001

.

0

.

2

.

x

T

pH

x I O*C - 5.7

9 15"C - 5.7 9

- 6.8

9 15"C - 6.8

.

4

!

6

10

8

N umber of P ul ses

Fig. 5.5 Inactivation of E. coli suspended in SMUF using 55 kV/cm, with two samples per each experimental condition. (Reprinted from Food Res. Int., Vol. 29(2), Vega-Mercado et al., "Inactivation of E. coli by combining pH, ionic strength and pulsed electric field hurdles," pp. 117-121, (1996b), with permission from Elsevier Science.)

The ionic strength of a solution also plays an important role in the inactivation of E. coli according to Vega-Mercado et al. (1996b). An increase in ionic strength increases the electron mobility through a solution, resulting in a decrease in the inactivation rate. Vega-Mercado et al. (1996b) thus explains the reduced inactivation rate in high ionic strength solutions by the stability of the cell membrane when exposed to a medium with several ions. The effect of ionic strength can be observed in Fig. 5.7 where a difference of 2.5 log cycles was obtained between 0.168 and 0.028 M solutions. 1.00E+08 -

1.OOE+07

1.00E-14~

.-e- 40oc

1.00E-HI5

~45~ --e- 50~ -e-55oc

I.OOE+02

- x - 60~

1.OOE+O1 1.00E+~ 5

10

15

20

25

Time (min) Fig. 5.6 A temperature-time effect on the inactivation of E. coli cells suspended in LWE (adapted from Ma et al., 1997).

120

5. PEF Inactivation of Cells, Spores, and Enzymes 14 I.,-: .....

9 ..... "O.

"

"=

0.1-

"d

0.01 -

"~

........

"O . . . . . . . .

"O...

168 mM "'i. "0"'.

'~O . . . . . . . .

"~176176176176176 ,,o

0.001 -

0.0001 0

I.....

" ' - . , 56mM 28 mM ""'-

',,

"'-

"''!

. 2

.

. 4

. 8

"",,

" *~

. 16

32

Number of Pulses Fig. 5.7 The effect of ionic strength on the inactivation of E. coli suspended in SMUF at 40 kV/cm and 10~ (two samples per each experimental condition). (Reprinted from Food Res. Int., Vol. 29(2), Vega-Mercado et al., "Inactivation of E. coli by combining pH, ionic strength and pulsed electric field hurdles," pp. 117-121, (1996b), with permission from Elsevier Science.)

Resistivity is another important characteristic of treatment media in which microorganisms are suspended. Studies conducted by Schoenbach et al. (1997) in tap water with a resistivity of p = 1.9 Kf~.cm and nutrient broth with p = 0.1 Kf~.cm showed that the higher the resistivity of a m e d i u m , the higher the electric field required to achieve the same population reduction. With a pulse width of ~" = 60 nsec, 100 k V / c m is required for 1 log reduction in water, whereas 70 k V / c m is n e e d e d in nutrient broth, (E c - 4 . 9 k V / c m , E 0 = 6.3 k V / c m , and To = 12 /zsec). However, these results differ from most other findings where a high resistivity of the media will e n h a n c e the inactivation level at the same electric field (Sensoy et al., 1997) (Fig. 4.14). W h e n comparing the inactivation of E. coli cells in logarithmic and stationary phases, a distinct difference is seen in the threshold values of electric field strength (Ec), whereas the threshold treatment time (t c) remains unchanged. It can therefore be concluded that the alterations of physiological properties a microbial cell undergoes during continuous growth influence its sensitivity to electric field action (Fig. 4.13). Microorganism survival rates are also influenced by the chosen concentration of nutrient in cultured media. Low m e d i u m concentration causes a shift in the n u m b e r of viable bacteria up to 10 times as m u c h as obtained with cells from nutrient-enriched cultures. This effect is partly due to altering the time course for the growth behavior because the cell yield is lower in diluted cultures and thus the stationary growth phase is reached earlier. Pothakamury et al. (1995a) reported an inactivation of E. coli (ATCC 11229) suspended in simulated milk ultrafiltrate (SMUF) whereby a 4 - 5 log cycle reduction was obtained after 60 pulses at 16 k V / c m in a c h a m b e r volume of 0.1 ml (Fig. 5.8). Application of 20 pulses at 25 k V / c m and 25~ by Zhang et al. (1994a) resulted in a near 3 log cycle reduction in a c h a m b e r volume of 25 ml, which verified that at higher electric fields, fewer pulses are n e e d e d to achieve almost the same inactivation level. Another example was the inactivation of nearly 9 log cycles of an E. coli population suspended in

II. Microbial Inactivation

121

0.1 o

g~,,,,,,,~

0.01

12 kV/cm (rsq = 0.97)

"~ 0.001 r~

,, .,.

14 kV/cm (rsq = 0.86)

"~

0.0001 """ ,.~

16 kV/em (mq = 0.85) 0.00001 10

100 Number of Pulses

Fig. 5.8 Inactivation of E. coli suspended in SMUF with electric fields of 12, 14, and 16 kV/cm. (Reprinted from Food Res. Int., Vol. 28(2), Pothakamury et al., "Inactivation of Escherichia coli and Staphylococcus aureus in model food systems by pulsed electric field technology," pp. 167-171, (1995a), with permission from Elsevier Science.)

SMUF with five steps o f 16 pulses, 40 k V / c m , a n d 97 k J / l i t e r in a static c h a m b e r (Fig. 5.9) ( Z h a n g et al., 1995). T h e inactivation o f E . coli s u s p e n d e d in LWE was also f o u n d to be h i g h e r w h e n electric fields o f g r e a t e r intensity were used, as Martln-Belloso et al. (1997b) d e t e r m i n e d t h a t 100 pulses at 26 k V / c m were n e e d e d to o b t a i n a 6 log r e d u c t i o n o f E . coli cells (Fig. 5.10), w h e r e a s Ma et al. (1997) was able to achieve m o r e t h a n a 7 log ( > 7D) r e d u c t i o n o f viable E . coli with j u s t 22 pulses at 48 k V / c m (Fig. 5.11). A n o t h e r way to o b t a i n h i g h e r t r e a t m e n t levels in o r d e r to g e t g r e a t e r inactivation is to i n c r e a s e the n u m b e r o f pulses. H o w e v e r , t r e a t m e n t c h a m bers (with set v o l u m e s ) g e n e r a l l y r e q u i r e a selected pulse rate a n d fixed flow rate so the n u m b e r o f pulses a f o o d can receive p e r pass is limited. T o o v e r c o m e this s h o r t c o m i n g , the use o f m u l t i p l e c h a m b e r s c o n n e c t e d in

=

1

"~

0.01

0

Ii

0.0001

iI

1E-06 '~ 1E-08 r~ 1E-10

~t

1

10 Number of Pulses

9 Survival Fraction/8 pulse step

100

A Survival Fraction/16 pulse step

gig. 5.9 Inactivation of E. coli in SMUF using an electric field of 70 kV/cm, 2 psec pulse duration, and treatment temperature of 20 _ I~ (adapted from Zhang et al., 1995).

122

Fig. 5.10 Inactivation of E. Martln-Belloso et al., 1997b).

5. PEF Inactivation of Cells, Spores, and Enzymes

coli

in LWE by PEF at 26 kV/cm and 37~ (adapted from

series (Fig. 1.3), stepwise recirculation (Fig. 1.2), and continuous recirculation (Fig. 5.12) have been suggested. In the stepwise mode, the product is processed completely several consecutive times, including a cleaning step in between treatments. In continuous recirculation the system is never emptied until the theoretical n u m b e r of pulses has been reached (theoretical because in continuous recirculation, perfect mixing and each particle of the product passing through the chamber " n " many times are assumed). The stepwise recirculation mode has been proven to be more effective than continuous recirculation, where to achieve an inactivation level of 7 log reductions 70 pulses are necessary, compared with less than 20 pulses in the stepwise mode with less than five steps (Fig. 5.11).

Fig. 5. I I Inactivationof E. coli in LWE by stepwise and recirculation PEF at 48 and 38 kV/cm and 37~ (adapted from Ma et al., 1997).

II. Microbial Inactivation

123

Fig. 5.12 The WSU PEF recirculation process used to process food products.

In the Washington State University (WSU) parallel plate static chamber, a 2 - 3 log cycle reduction was obtained when E. coli in SMUF was subjected to 50 square-wave pulses with a duration of 2 /xsec and electric field intensity of 45 kV/cm. No inactivation was observed with an electric field smaller than 20 k V / c m , but it was increased to nearly 4 log cycles with an electric field o f 50 k V / c m in a continuous chamber. With a coaxial treatment chamber the inactivation increased to nearly 8 log cycles when the electric field was 25 k V / c m (Qin et al., 1995a). A 4 log cycle reduction was obtained after 5 pulses when E. coli suspended in a sodium alginate solution was subjected to an electric field of 26 k V / c m with a critical electric field strength of 14 k V / c m (Grahl et al., 1992). The inactivation of E. coli (ATCC 11229) in a model semisolid system of potato dextrose agar (PDA) was studied by Zhang et al. (1994b). To conduct this experiment, a suspension of E. coli in SMUF was serially diluted in 0.1% peptone, and a liquid-sterilized potato dextrose agar was inoculated with 1 ml of bacteria suspended in peptone. The inoculated PDA was then placed into a static chamber and allowed to solidify. The solidified PDA containing the bacteria was subjected to electric pulses, and its E. coli was reduced by 6 log cycles after 64 pulses at 40 k V / c m (Fig. 5.13). A pulse rate on the order of 1.25 to 2.50 Hz had no effect on the inactivation rate of E. coli suspended in LWE where a 6 log reduction was obtained with 100 pulses of 26 k V / c m (Mart~n-Belloso et al., 1997b). Further experiments are necessary to verify the effect of this microorganism in

124

5. PEF Inactivation of Cells, Spores, and Enzymes

Fig. 5.13 Inactivationof bacteria in a model semisolid food PDA with an electric field of 40 kV/cm at 15~ (adapted from Zhang et al., 1994b). A, E. coli; 4~, S. aureus; and D, S. cerevisiae.

similar foods. The inactivation of E. coli suspended in whole and skim milk has been studied by Dunn and Pearlman (1987), Grahl et al. (1992), Grahl and M~rkl (1996), and Mart~n-Belloso et al. (1997a). It is hard to make a clear comparison of their results due to differences in treatment conditions, although the inactivation obtained varied between 3 and 4 log reductions. The results of Grahl and MSrkl (1996) and Martln-Belloso et al. (1997a) confirm a first-order kinetic with a critical electric field around 13 kV/cm. When the effects of oscillatory and exponential pulses on the survival of E. coli were compared, oscillatory decay pulses were found to produce the larger survival fraction of E. coli. Oscillatory pulses apparently prevent bacterial cells from being continuously exposed to high-intensity electric fields for extended periods of time, which prevents cell membranes from irreversible rupture over a significantly large area. However, for microbial inactivation and food pasteurization, oscillatory decay pulses were not efficient at 40 kV/cm with 80 J per pulse and a 100-kHz frequency (Qin et al., 1994). The survival fraction of E. coli with monopolar and bipolar exponentially decaying pulses was also compared, where Q,in et al. (1994) showed that bipolar pulses provide a more efficient inactivation (Fig. 5.14). A monopolar pulse has an exponential decay waveform, and a bipolar pulse pair consists of one positive and one negative exponential decay waveform. Because each application of a bipolar pulse pair is equivalent in energy to an application of two monopolar pulses, bipolar pulses produce greater inactivation than monopolar pulses. Furthermore, alternating stress produced by bipolar pulses results in a structural fatigue of the membrane and enhances the susceptibility of the E. coli membrane to breakdown. Pothakamury et al. (1995a) verified the higher inactivation effect of square pulses compared with exponentially decaying pulses in the inactivation of E. coli suspended in SMUF (Fig. 5.15).

II. Microbial Inactivation

125

* Monopolar Bipolar

0.1 0.01 0.001

n

0.0001 0

I

!

!

i

|

2

4

6

8

10

Number of Pulses Fig. 5.14 Inactivationof E. coli using monopolar and bipolar exponential decaying waveshape pulses with a peak electric field of 40 kV/cm and 60 J per pulse (adapted from Qin et al., 1994).

C.

Inactivation of Staphylococcus aureus

S t a p h y l o c o c c u s a u r e u s is a gram-positive p a t h o g e n of high i m p o r t a n c e in food

( processing because it is a toxin that is poisonous to h u m a n s . However, this microorganism represents a health risk only w h e n its c o n c e n t r a t i o n is greater than 10 5 c f u / m l , the level r e q u i r e d to p r o d u c e e n o u g h toxin to cause illness. S t a p h y l o c o c c u s a u r e u s is also formidable in that it is a very resistant m i c r o o r g a n i s m that can grow at high salt concentrations, low water activities (A w as low as 0.83), a n d a relatively low p H (Jay, 1996).

"•

1

~

Exponential Square

0.1

0.01

0.001

u

0.0001 0

|

|

!

!

i

2

4

6

8

10

N u n ~ r of Pulses

Fig. 5.15 Inactivation of E. coli suspended in SMUF using monopolar exponential decaying and square-wave pulses with a peak electric field of 35 kV/cm and 60 J per pulse (adapted from Qin et al., 1994).

126

5. PEF Inactivation of Cells, Spores, and Enzymes

In one of the earliest attempts to verify m e m b r a n e cell damage as the cause of PEF inactivation of S. aureus, Hamilton and Sale (1967) suspended this microorganism in a 20 m M phosphatase buffer (pH 7.2) and subjected the suspension to electric fields between 0 and 27.5 k V / c m . After pulsing, the suspension was treated with the cell wall-dissolving enzyme lysostaphin in a hypertonic medium. They observed the effects of the pulse t r e a t m e n t on the n u m b e r of cells inactivated by a given treatment and the n u m b e r still capable of forming spheroplasts. The direct relationship between the effects of the pulse treatment on cell inactivity and m e m b r a n e damage as measured by poor to no spheroplast formation demonstrated that S. aureus death is a result of m e m b r a n e damage (Table 5.3). Table 5.4 presents a summary of the t r e a t m e n t conditions and inactivation results from a few noteworthy investigations on the PEF inactivation of Staphylococcus spp. and other vegetative microorganisms. Staphylococcus aureus m e m b r a n e damage and ultrastructural changes evid e n c e d by scanning electron microscopy (SEM) and TEM were shown in Chapter 3 (Figs. 3.17-3.19), where cell surfaces were noticeably r o u g h e n e d after treatment with electric fields. Transmission electron microscopy allowed the observation of broken cell walls and cytoplasmic content leakage after exposure to 64 pulses at 60 k V / c m ; in this study the cells of S. aureus were suspended in SMUF. W h e n the same cell suspension was submitted to the effect of heat treatment (10 min at 60~ important changes in the protoplast were observed, but with PEF-treated cells, no cell wall breakdown was evident (Pothakamury et al., 1997). Staphylococcus aureus suspended by Hiilsheger et al. (1983) in a buffer solution with neutral pH and an adjusted resistivity of 600 l~.cm experienced an inactivation of less than 3 cycles after a treatment with 30 pulses of 20 k V / c m . These researchers also obtained the kinetic constants of Eq. (4.10) for other gram-positive and gram-negative bacteria, as well as some yeast cells suspended in the same m e d i u m (Table 4.4). The inactivation kinetic constants for S. aureus and L. monocytogenes cells resulted in good a g r e e m e n t since the critical time was ~ 58 /~sec and the critical electric field was --13 k V / c m . Although there were differences in the microorganism constants, the lower kinetic constant value for TABLE 5.3 S. a u r e u s Activity after PEF Treatment ~

Electric field (kV/cm) 0.00 9.25 14.25 19.50 24.00 27.50

Survivors(%)

Protoplastnot lysed (%)

100

100

100 35 0.9 0.3 0.6

100 43 16 3 1.5

a Reprinted from Biochim. Biophys. Acta, Vol. 148, Hamilton and Sale, "Effects of high electric fields on microorganisms. II. Mechanism of action of the lethal effect," pp. 789-800, (1967), with permission from Elsevier Science.

T A B L E 5.4 S u m m a r y of the Inactivation of Various Microorganisms with PEF ~

Source

Microorganism

Suspension media

Log reduction (max)

T r e a t m e n t vessel b

Process conditions c

Jayaram et al. (1992)

L. brevis

P o t h a k a m u r y et al. (1995a)

L. delbrueckii

P o t h a k a m u r y et al. (1995a)

L. subtilis ATCC 9372

SMUF

4-5

B, parallel plate, 1 ml, d = 0.1 cm

P o t h a k a m u r y et al. (1995a)

S. aureus ATCC 6538

SMUF

3-4

B, parallel plate, 1 ml, d = 0.1 cm

Vega-Mercado et al. (1996a)

B. subtilis ATCC 9372

Pea soup

5.3

C, coaxial, 0.5 1 / m i n

H o et al. (1995)

P. fluorescens

> 6

B, 49.5, 99.1, 148.6 ml, d = 0.3, 0.6, 0.9 cm

Qin et al. (1994) Qin et al. (1994)

B. subtilis B. subtilis

Distilled water, 0.1% peptone, 1 0 - 3 5 % sucrose, 0.1 and 0.5% xanthan, 0.1 and 0.5% sodium chloride SMUF SMUF

2.0 V / l ~ m , 36 /zsec, 30 pulses, exponential decay, t = 1080 /zsec 2.0 V / / z m , 36 /zsec, 30 pulses, exponential decay, t = 1080 /zsec 2.0 V / / z m , 36 /zsec, 30 pulses, exponential decay, t = 1080 /xsec 2.0 V / / x m , 36/zsec, 30 pulses, exponential decay, t = 1080 /xsec 2.0 V / / z m , 36/zsec, 30 pulses, exponential decay, t = 1080 /xsec 63~ 3.67 V / / z m , 36/zsec, 40 pulses 50~ 1.8 V / / x m 1 /zsec, 20 pulses, e x p o n e n t i a l decay 9.0 V / / z m , 1 /xsec, 10 of 6.8 V / / ~ m + 1 o f 7.5 V / / x m + 1 of 8.3 V / / z m + 5 of 9.0 V / / z m 60~ 2.5 V / / ~ m , 46/zsec, 200 pulses, t = 10,000/zsec < 30~ 1.6 V / / z m , 2 0 0 - 3 0 0 / z s e c , 40 pulses, exponential decay, t = 10,000 /zsec < 30~ 1.6 V / ~ m , 2 0 0 - 3 0 0 /xsec, 50 pulses, exponential decay, t = 12,500 /zsec < 30~ 1.6 V / / x m , 2 0 0 - 3 0 0 or sec, 60 pulses exponential decay < 5.5~ 3.3 V / # m , 2 ~sec, 0.5/zF, 4.3 Hz, 30 pulses, e x p o n e n t i a l decay 20~ 1.0 V / / z m , 2 /zsec, 10 pulses, exponential decay, t -- 2 sec

4.5 5.5

B, parallel plate, 100 /xl, d = 0.1 cm B, parallel plate, 100 /zl, d = 0.1 cm

1.6 V / / z m , m o n o p o l a r , 180 /xsec, 13 pulses 1.6 V / / x m , bipolar, 180 /xsec, 13 pulses

Phosphate buffer

3

B, 4 m l , d = 0 . 5 c m

P. auruginosa

Phosphate buffer

3.5

B, 4 m l , d = 0 . 5 c m

S. aureus

Phosphate buffer

3

B, 4 m l , d = 0 . 5 c m

Hiilsheger et al. (1983)

K . pneumoniae

Hiilsheger et al. (1983) Hiilsheger et al. (1983)

ATCC 27736

ATCC 25923 Hiilsheger et al. (1983)

L. monocytogenes

Phosphate buffer

2

B, 4 m l , d = 0 . 5 c m

Hiilsheger et al. (1983)

C. albicans

Phosphate buffer

4.5

B, 4 m l , d -

Dunn Dunn Gupta Gupta

S. L. S. P.

Milk Yogurt NaC1 Milk

4 2 5 4.5

B B

Nail 2PO4/ NazHPO4H20 SMUF

9 4-5

B, parallel plate, 0.5 ml, d = 0.2 cm B, 1 ml, d = 0.1 cm

and and and and

P e a r l m a n (1987) P e a r l m a n (1987) Murray (1989) Murray (1989)

dublin brevis typhimurium fragi

0.5cm

B,d = 6.35mm B,d = 6.35mm

ATCC 11842

a F r o m Wouters and Smelt (1997). b B, batch; C, continuous. c T e m p e r a t u r e , peak electric field, pulse width, n u m b e r of pulses and shape, and total t r e a t m e n t time (t).

128

5. PEF Inactivation of Cells, Spores, and Enzymes

S. a u r e u s cells (k = 2.6 k V / c m ,

L . monocytogenes k = 6.5 k V / c m ) w a s attributed to their spherical shape. A comparison between the inactivation of gram-negative E . coli and gram-positive S. a u r e u s can be observed in Fig. 5.16 (Pothakamury et al., 1995a). A more effective inactivation is evident in E. coli cells, which is attributed to their bigger size compared with S. a u r e u s cells (Fig. 4.10). Qin et al. (1998) noticed the same tendency of better inactivation of E . coli over S. a u r e u s in a continuous treatment chamber. Their study revealed less than 6 log cycles of S. a u r e u s cells suspended in SMUF inactivated with 40 pulses of 36 k V / c m , whereas a similar treatment of an E . coli suspension resulted in the inactivation of 8 log cycles. The effect of electric fields on the inactivation of S. a u r e u s cells (ATCC 6538) suspended in SMUF was reported by Pothakamury et al. (1995a), whereby a 4 - 5 log cycle reduction was obtained after 50 pulses with an electric field of 16 k V / c m , but less than 3 log cycles with an electric field of 12 k V / c m (Fig. 5.17). In an attempt to determine the effect of PEF on the inactivation of microorganisms in semisolid and solid foods, Zhang et al. (1994b) used a potato dextrose agar model to ascertain the PEF inactivation kinetics of S. a u r e u s . Results s h o w e d a first-order PEF inactivation kinetics where the critical electric field and kinetic constant in the PDA were 18.5 and 6.8 k V / c m , respectively. The microbial count was reduced by 6 log cycles after 64 pulses at 40 kV/cm, and the energy required for the inactivation was 232 J / m l .

D.

Inactivation of Lactobacillus

These microorganisms are gram-positive, nonsporulating rod-shaped cells that can be very long or in the form of coccobacilli; they may also appear by

I : E. coli- 8 k V / c m

~~~[~[~,--I--E.

coli - 12 k V / c m

0.1

aureus- 9 kV/cm

aureus - 16 k V / c m

o c~

0.01

-9

0.001 0.0001 .

0.00001 0

20

.

.

40 Number of Pulses

. 60

80

Fig. 5.16 Inactivation of E. coli and S. aureus in SMUF by PEF (adapted from Pothakamury et al., 1995a).

II. Microbial Inactivation

129

,,,,~ "',.

0.1

-

0.01

-

~ a

14 kV/cm (rsq = 0.94)

12 kV/cm (rsq = 0.92) \

"i,e

"" "*~. ~ '=,,

0.001

-

0.0001

-

9

/4 "',.,-

I

16 kV/cm (rsq - 0.91)'"

I

0.00001

lO

1 Number

100

of Pulses

Fig. 5.17 Inactivation of S. aureus suspended in SMUF with electric fields of 12, 14, and 16 kV/cm. (Reprinted from Food Res. Int., Vol. 28(2), Pothakamury et al., "Inactivation of Escherchia coli and Staphylococcus aureus in model food systems by pulsed electric field technology," pp. 167-171, (1995a), with permission from Elsevier Science.)

themselves or in chains. In addition, the species is facultative anaerobic, usually nonmotile and mesophilic (some psychrotrophs), and homo- or heterolactic. It can be found in milk, meat, and feces. Lactobacillus delbrueckii subsp, bulgaricus, L. helveticus, L. p l a n t a r u m , L. acidophilus, L. reuteri, and L. casei subsp, casei are some of the many Lactobacillus spp. used in food processing (Jay, 1996). One of the first attempts to analyze the effect of PEF on the inactivation of Lactobacillus spp. was the t r e a t m e n t of yogurt with 18 k V / c m where almost 1.5 log cycles of inactivation were obtained (Dunn and Pearlman, 1987) (Table 5.4). Lactobacillus brevis is heterofermentative, and due to its aciduric characteristics can grow rapidly when the acidity of its suspending m e d i u m is 1%. It also produces lactic acid, acetate, and ethanol. Its presence is necessary for sauerkraut manufacturing, but undesirable in products such as mayonnaise and salad dressings where it causes spoilage and in wine because it produces a defect called "calle t o u r n e , " which is an increase in acidity due to the fermentation of glucose and fructose. Jayaram et al. (1991, 1992) exposed cells of L. brevis suspended in a phosphate buffer solution o f N a z H P O 4 / N a H 2 P O 4 9H 2 0 (0.845/0.186 m M ) to different electric fields (up to 30 k V / c m ) , pulse n u m b e r s (30 to 60), t r e a t m e n t times (0-15 ms), and t r e a t m e n t temperatures. W h e n 60 pulses of 30 k V / c m at 24, 30, and 45~ were applied, the inactivation of L. brevis was a r o u n d 6 log cycles, whereas a 9 log cycle reduction was obtained when the t r e a t m e n t t e m p e r a t u r e was increased to 60~ To verify the m e m b r a n e damage, SEMs were taken of cells with different t r e a t m e n t times (0-16 msec) of 25-kV/pulses at 24~ Micrographs revealed an increase in the cellular fibril matrix that coincided with the reduction in the n u m b e r of surviving

130

5. PEF Inactivation of Cells, Spores, and Enzymes

1 I 9 kV/a'n (rsq = 0.85)

r~

0.00011

o

O.O00Ol 10

N u m b e r of Pulses

1O0

Fig.

5.18 Inactivation of L. delbrueckii suspended in SMUF with electric fields of 9, 12, and 16 k V / c m (adapted from Pothakamury et al., 1995b).

L. brevis cells (109-10~). Furthermore, the increase in concentration of C1-

of the suspending test medium (3.8-7.2 ppm) indicated increasing cellular content leakage with the increased electric field strength, pulse number, and medium temperature for a given field strength. This leakage was from the lysis of L. brevis cells, as confirmed by the high concentration of C1- (9 ppm) when cells were suspended in a hypoosmotic medium (deionized water). The anionic concentration of C1 in the buffer containing no microorganisms was the same whether subjected to pulse treatment or not. Further studies report more than 5 log cycles of inactivation for L. brevis suspended in milk after the ESTERIL process with 20 pulses of 20 ~sec (width) at 20 k V / c m (Sitzmann, 1990). Grahl and M~irkl (1996) observed a first-order kinetic for the inactivation of L. brevis suspended in a solution of sodium alginate and 1.5% fat UHT milk with critical treatment times of 10.9 and 46.1 /zsec, respectively, and critical electric fields around 12 kVcm in both media. A 4-5 log cycle reduction of L. delbrueckii (ATCC 11842) in SMUF was obtained after 40 pulses with an electric field of 16 k V / c m (Pothakamury et al., 1995b) (Fig. 5.18).

E.

Inactivation of Bacillus

Bacillus spp. is a gram-positive, endospore-forming microorganism that can

be mesophilic, psychrotrophic, aerobic, or facultative anaerobic with or without motility. Cells can also be in chains, rodshaped, straight, and variable in size (0.5 to 1 X 2 to 10/zm). The endospores produced by Bacillus can be spherical or oval (one per cell), but are always highly heatresistant. Bacillus cereus causes foodborne diseases, whereas B. coagulans is often a source of

13 ]

II. M i c r o b i a l I n a c t i v a t i o n

food spoilage. Bacillus subtilis produces spoilage in mayonnaise and canned foods due to the production of acid and gas (CO2). Similarly, quality attributes lost in bread caused by B. subtilis are characterized by a soft, stringy, brown mass with a fruity odor that is due to the formation of amylases, proteases, and acids. However, the ability of Bacillus spp. to produce certain enzymes and to hydrolyze carbohydrates, proteins, and lipids is useful in food bioprocessing (Jay, 1996). One of the first studied subspecies was B. subtilis, where PEF proved to produce a good level of inactivation under different treatment conditions and media. Gilliland and Speck (1967) reported the inactivation of vegetative cells of B. subtilis in an aqueous media by the electrohydraulic treatment, although this process is not considered strictly a PEF treatment. Sale and Hamilton (1968) later found a lysis of B. subtilis protoplasts under the influence of electric fields up to 20 kV/cm. (The lysis was measured from the change in turbidity of the suspension containing the protoplasts.) A more recent inactivation study by Pothakamury et al. (1995b) resulted in a 4-5 log cycle reduction of B. subtilis vegetative cells (ATCC 9372) suspended in SMUF after 40 pulses at 16 k V / c m (Fig. 5.19). The pulse waveshape effect on the inactivation rate of B. subtilis was studied by Qin et al. (1994), who conducted experiments in a small-scale bench-top electroporator unit with a cuvette characterized by a 0.1-cm gap and 100 /xl of volume. The results showed that 30 bipolar exponentially decaying pulses gave a more efficient inactivation (3 log cycles) of B. subtilis cells compared with 30 monopolar pulses (less than 2 log cycles) of 16 k V / c m at a 280-/xsec pulse width. Additional studies in a pilot plant with a continuous PEF system and coaxial chamber allowed even higher inactivation. After 30 pulses, B. subtilis inoculated in pea soup was reduced by 5 log cycles with an electric field of 33 kV/cm. However, the effectiveness of the microbial inactivation de-

q= 0.81)

0.1 o

v

0.01 0.001

o -"~q~ = 0.952) 16 kV/crn (rsq = 0.997)

0.0001 -

0.00001 10

100 Number of Pulses

Fig. 5.19 Inactivation of B. subtilis suspended in SMUF with electric fields of 9, 12, and 16 kV/cm (adapted from Pothakamury et al., 1995b).

132

5. PEF I n a c t i v a t i o n of Cells, Spores, and E n z y m e s

8

9Ec-25 kV/cm - 32~

7

cO

9Ec-33 kV/cm - 55~

6

9Bs-28 kV/cm - 40~

LL

9Bs-33 kV/cm - 55~

.5 4 P

=3

03

3 2 1 0

5

10

15

20

25

30

35

Number of Pulses Fig. 5 . 2 0 Individual inactivation of microorganisms suspended in pea soup as a function of treatment temperature and electric fields (Ec is E. coli and Bs is B. subtilis) (Vega-Mercado et al., 1996a).

creased almost 2 log cycles when both E. coli and B. subtilis were inoculated in the soup. A 4 log cycle reduction of the combined populations of E. coli and B. subtilis was obtained after 30 pulses with an electric field of 33 k V / c m (Vega-Mercado, 1996a) (Figs. 5.20 and 5.21). Pag{tn et al. (1998) corroborated these higher rates of inactivation with higher electric fields when they achieved more than a 5 log cycle reduction of B. subtilis cells in SMUF after a PEF treatment with 5 pulses of 60 k V / c m .

F.

Inactivation of Salmonella

Salmonella spp. are gram-negative facultative anaerobes with m e d i u m rod

shapes (1 • 4 / x m ) that are usually motile and mesophilic. While there are more than 2000 species of this microorganism, only a small n u m b e r of them

4.5

4

9Ec/Bs-28 kV/cm - 40~

o 3.5 Fs

14.

~ Ec/Bs-30 kV/cm - 550C

3

Ecx-28 kV/cm - 40~

-- 2.5

X Ecx-30 kV/cm - 53~ Bsx-30 kV/cm - 53~

o,

1

0.5 0

5

10

15

20

25

30

35

Number of Pulses Fig. 5.21 Inactivation of a microorganism mixture suspended in pea soup (Ec is E. coli, Bs is B. subtilis, E c / B s is the overall inactivation for the mixture of microorganisms, Ecx is the inactivation of E. coli in the mixture, and Bsx is the inactivation of B. subtilis in the mixture) (Vega-Mercado et al., 1996a).

II. Microbial Inactivation

133

have been associated with foodborne illnesses, although all are regarded as h u m a n pathogens. Salmonella are found in the gastrointestinal tracts of humans, animals, and birds. The major causes of foodborne diseases are S. enteritidis and S. typhimurium, the former of which has been associated with outbreaks caused by contaminated grade A shell eggs and products containing raw eggs that were not fully cooked. The inactivation of Salmonella spp. has been investigated in NaC1 buffers, distilled water, fluid egg products, and milk. The inactivation of 5 log cycles of S. typhimurium from an initial inoculum of 10 ~ c f u / m l was achieved after Gupta and Murray (1988) applied a treatment to the microorganism suspended in a NaC1 buffer (1000 l~.cm resistivity) with 20 exponentially decaying pulses of 83 k V / c m . This inactivation level is 0.5 logs higher than when 60 k V / c m was used. The treatment chamber used was static, with parallel electrodes of stainless steel separated by a lexan insulator with a gap of 6.35 mm. Dunn and Pearlman (1987) used the static parallel electrode treatment chamber (10 cm diameter and gap of 2 cm) to process milk inoculated prior to treatment with S. dublin (No = 3.8 X 103 cfu/ml). The PEF treatment consisted of 40 exponentially decaying pulses with a 36.7 peak voltage ( ~ 18.3 k V / c m ) and a decaying time of about 20 /zsec. No viable cells of Salmonella were found immediately after treatment or during a follow-up of 192 hr (8 days, u n d e r refrigeration at 7-9~ Furthermore, the results suggest a selective treatment with a preferential inactivation of S. dublin over milk bacteria. The synergistic effect of temperature was demonstrated when low levels of kill (10% survival) were obtained with a treatment conducted at less than 40~ whereas a treatment at 50~ yielded greater kill (0.01% survival). Untreated control samples that were held at 50~ during PEF testing showed substantially no loss of microbial viability. It can thus be concluded that a synergism with temperature was also corroborated in this gram-negative species. D u n n and Pearlman (1987) found no S. dublin in several liquid egg products after PEF treatments of 25 pulses at 34.8-36.3 kV and 30 pulses at ~ 17-18 kV/cm. Whether the products were initially inoculated was not reported, but control samples without PEF treatment were found to be contaminated with S. dublin. Ohshima et al. (1997) studied the sterilization of S. typhimurium u n d e r controlled temperatures with and without PEF treatment (32 k V / c m ) in a recirculation system of parallel plates and found that survival ratios at 50~ with and without PEF were 10 -4 and 10-1, respectively. The experiments at different temperatures showed an inflection point around 10~ below the culture temperature, which seems to indicate that the phase transition temperature of the cell m e m b r a n e consisted of a lipid bilayer. The phase transition temperature was about 10~ below the culture temperature because bacteria incorporate increasing proportions of saturated and long-chain fatty acids into phospholipids as growth temperatures are increased. Above the phase transition temperature, bilayer phospholipids are less ordered and membranes have a liquid crystalline structure that causes a larger reduction in viable cells. It was thus concluded that PEF sterilization may not be as

134

5. PEF Inactivation of Cells, Spores, and Enzymes

efficient when conducted at processing temperatures below the phase transition temperature. The effect of medium conductivity on the inactivation of S. dublin was tested by Sensoy et al. (1997), who treated cells in solutions with different concentrations of KC1 at 28 k V / c m with pulses 1-~sec wide at a rate of 3.73 kHz. With 25 /zsec of treatment time and m e d i u m conductivities from 0.00925 to 0.9669 S / m , the achieved inactivation was 5 and less than 2 log cycles, respectively (Fig. 4.14). These results can be explained by the transmembrane potential being d e p e n d e n t on the conductivities of the cytoplasm, m e m b r a n e , and suspending medium. Lowering the conductivity of the liquid m e d i u m thus increases the difference between the conductivities of the m e d i u m and the microbial cytoplasm and weakens the m e m b r a n e structure due to an increased flow of ionic substances across the membrane. Inactivation kinetic models of S. dublin to predict PEF treatment dosage as a function of electric field strength (15-40 kV/cm), treatment time (12-127/~sec), and temperature (10-50~ in skim milk were based on previous models by Hiilsheger et al. (1981) [Eqs. (4.10) and (5.1)] and Peleg (1995) [(Eq. (4.8)]. In the study by Sensoy et al. (1997), a third model [Eqs. (4.11) and (4.12)] was proposed to evaluate the change in the kinetic constant of Hiilsheger's model with temperature. The inactivation kinetic models with the best fits for S. dublin in milk were Eqs. (5.1) and (5.2), with a critical electric field between 12.57 and 13.59 k V / c m (evaluated from 25 to 40 k V / c m ) and a critical treatment time of 0 /zsec. Therefore, Eq. (5.1) is simplified to Eq. (5.2). Equation (5.4) evaluates the kinetic constant of Eq. (5.2) as a function of temperature (283.15 to 323~ using an electric field of 25 k V / c m , a pulse frequency of 1.7 kHz, and a 1-~sec pulse width for the inactivation of S. dublin in milk. Equation (5.5) evaluates the kinetic constant as a function of 25- to 40-kV/cm electric fields and a 2.06-kHz pulse rate at 24~ s

-

s = e -k'

(5.2)

t

s=

7c

(5.3) EA

k = kv ~e

RT

k = k~0(E - E~)

(5 4) (5.5)

where t c is the m i n i m u m treatment time that gives s - 1, k is the survival fraction rate constant (1//zsec), E a is the activation energy ( J / k g mol), kT0 is a temperature d e p e n d e n t rate constant ( c m / k V / ~ s e c ) , kv ~ is an electric field d e p e n d e n t rate constant (1//zsec), R is the universal gas constant (1.987 J / k g mol ~ and T is the temperature of the m e d i u m (~

II. Microbial Inactivation

135

From the previous analysis and experimental results, it can be concluded that a linear increase in treatment time causes an exponential decrease in the survival ratio of S. dublin and microorganisms in general. Almost 4 log cycles of S. dublin inactivation were obtained after 120 /xsec of treatment with 30-kV/cm pulses 8-/xsec wide at a frequency of 3 kHz. The temperature effect followed a similar pattern, increasing the inactivation rate almost 2 log cycles when the treatment temperature was 50~ instead of 10~ with 100 /xsec of treatment at 25 kV/cm, 1.74 kHz, and 1 /xsec pulse width (Sensoy et al., 1997). The effect of media conductivity is another factor of the inactivation of Salmonella spp. that has been studied. Sensoy et al. (1997) proved that media conductivity is an important factor in the inactivation of Salmonella despite the application of equal input pulse energies. With a medium conductivity of 0.475 S / m , less than 2 log cycles of the bacteria were inactivated; with ~ 280 /xsec of treatment, 5 log cycle reductions were obtained in a media with 0.00925 S / m in 25 /xsec (Fig. 4.14). [This is especially important to consider since food conductivities are between 0.1 and 2.4 S / m (Table 4.13).]

G.

Inactivation of Pseudomonas

Pseudomonas spp. belong to the gram-negative aerobic group, can be straight or curved motile rods (0.5 X 5 /xm), and are an important spoilage flora capable of metabolizing a wide variety of carbohydrates, proteins, and lipids in foods. Some important species with relevance in foods are P. fluorescens, P. aeruginosa, P. putrida, and P. fragi. Because each of these is psychrotrophic, they can grow at 5~ and below and multiply quite rapidly at 10 to 25~ Many foods are stored under aerobic and refrigerated conditions, which provide an ideal environment for the growth of this spoilage flora. In general, Pseudomonas spp. can taint milk, liquid eggs, fruit, vegetables, fish, and meats by contamination before or after processing. It will cause detectable flavor defects at populations of 10 6 cfu/ml, and under favorable conditions, may be able to grow from 101 to 106 c f u / g in 12 days at 2~ (Jay, 1996). Hiilsheger et al. (1983) studied the inactivation kinetics of P. aeruginosa cells suspended in phosphate buffer solutions [NaHzPOa/NazHPO4 (7.44 m M , 7 pH)] with an electrical resistivity of 600 l).cm at 20~ within a working electric field of 2 to 20 k V / c m using between 2 and 30 pulses in a static chamber. For the highest pulse number and electric field, the inactivation of this microorganism was more than 3 log cycles, compared with less than 1 for treatments at 8 k V / c m and 30 pulses or less. Within these treatment conditions, the first-order inactivation kinetics had a critical electric field of 6 kV/cm, a critical time of 35 /xsec, and a kinetic constant of 6.3 kV/cm, which are near those of other gram-negative flora such as E. coli and Klebsiella pneumoniae when both are in stationary phase.

136

5. PEF Inactivation of Cells, Spores, and Enzymes

Grahl and M~irkl (1996) evaluated the inactivation kinetics of P. fluorescens in sodium alginate and UHT milk with 1.5% fat and found that in both media the critical electric field was around 11 kV/cm. However, the critical treatment time was much higher in milk (19.8 ~sec) compared to alginate (0.40 ~sec), which demonstrated the protective effect of the complex milk system. It may be helpful to visualize these results by considering the number of pulses necessary to obtain 4 log cycles of P. fluorescens inactivation, where less than 5 pulses are needed for an alginate solution and more than 20 for UHT milk. The inactivation of 4.3 log cycles of P. fragi from an initial inoculum of 106 c f u / m l was achieved by Gupta and Murray (1988) after a treatment (in a static chamber) of the microorganism suspended in a NaC1 buffer (with a resistivity of 1000 l~.cm) with 10 exponentially decaying pulses of 68 kV/cm, plus 1 pulse at 75 kV/cm, another at 85 kV/cm, and 5 more at 90 kV/cm. However, due to the combination of different electric fields and number of pulses, it is difficult to make any conclusions or comparisons with the PEF inactivation kinetics of other gram-negative bacteria. The microbial reduction of P. fluorescens in five different media (disfilled water, peptone, sucrose, xanthan gum, and NaC1) with different conductivities and rheological properties was studied by Ho et al. (1995) using a static chamber with stainless-steel parallel disk electrodes, Derlin as an insulator, and variable gaps of 0.3, 0.6, and 0.9 mm. The killing rate was found to be either extremely high or drastically low. With a gap of 0.3 mm, the microbial reduction was between 6.6 and 7.3 log cycles; at higher gaps the microbial inactivation was less than 1 log cycle. In both cases, no significant effect of electric field strength (10-45 kV/cm), pulse period (2 and 4 sec, pulse width of 2 ~sec), and number of applied pulses (10-30 pulses at 0.3-mm gap and 10-100 pulses at 0.6- and 0.9-mm gaps) was observed. These extreme inactivation results were explained by the differences in pulse waveforms caused by the gap differences given that under the same fluid medium and electrical conditions, a decrease in electrode distance to 0.3 cm produces sudden reversal voltages or spikes (Fig. 1.7). A solution with a high conductance generates high spike electric field strength. Ho et al. (1995) concluded that the reversed voltage might have produced a high alternating stress on the cell membrane causing structural fatigue, which took place only after a few pulses. Furthermore, no higher than a 6 log cycle microbial inactivation was obtained with an increase in field strength from 15 to 35 k V / c m when sucrose and NaC1 solutions were used. In xanthan solutions the inactivations were very low (0-0.5 log cycles) at 15 kV/cm, but very high (6.4-6.7 log cycles) at 25 and 35 kV/cm, which implies that a protective shield for the microorganisms due to a high degree of interaction between polymer chains resulted in a network of molecular aggregates held together by secondary valence forces that require a higher electric field strength compared with that used on NaC1 solutions to rupture cell membranes. In other studies (Hofmann, 1984) where no spikes were present, a lower inactivation rate was obtained for Pseudomona spp, whereby

II. Microbial Inactivation

137

a suspension of the bacteria in water after a PEF treatment of 90/zsec using exponential decaying pulses of 25 and 40 k V / c m had an inactivation level of 99.67% (2.48 log cycles) that was not even a function of the electric field applied.

H.

Inactivation of Other Microorganisms

Although the inactivation of microorganisms has been focused on a relatively small group of species, less extensive studies can be found on such important h u m a n pathogens as Streptococcus spp., Micrococuss spp., gram-positive L. monocytogenes, gram-negative Yersinia enterocolitica, and enteric K. pneumoniae. However, the food industry is now requesting more and more information about the inactivation attributes of PEF on pathogens of all kinds suspended in real foods. In order to compare the inactivation after PEF of similar strains with different cell size, two serovars of L. monocytogenes were employed by Hiilsheger et al. (1983). The first strain had regular rod-shaped cells and the second a rough form with threads up to 100 ~m. Both were harvested in stationary growth phase and suspended in phosphate buffer solutions [ N a H z P O 4 / N a z H P O 4 (7.44 m M , 7 pH)] with an electrical resistivity of 600 l].cm at 20~ The inactivation kinetics were studied within a working electric field of 2 to 20 k V / c m using between 2 and 30 pulses in a static chamber. The lower critical electric field and critical treatment time for the second strain (Table 4.4) were attributed to a physiological defect in the m e m b r a n e assembly and enlarged cell size that led to stabilization of the stressed cells. U n d e r the same treatment conditions, the inactivation kinetics of the gram-negative, rod-shaped K. pneumoniae exhibited an interesting deviation from other bacteria (i.e., P. fluorescens and E. coli) in that the inactivation curves as a function of electric field and pulse n u m b e r bent at a field strength of about 14 k V / c m for all the pulse numbers. Because cells of K. pneumoniae were capsula forming, it was assumed that the capsula may provide an additional protecting mechanism that leads to a lower decline of the survival curves until a certain field strength is exceeded. The inactivation of Micrococcus lysodeikticus observed in Fig. 5.1 shows the important effect of small cell size in reducing the inactivation effect of PEF. Whereas in other species (i.e., S. cerevisiae and E. coli) the percentage of survivors is less than 1% after a treatment of 10 pulses of 20 ~sec at low electric fields (5 kV/cm), more than 50% of the survivors of Micrococcus spp. were found after a treatment with electric fields higher than 20 k V / c m in a study conducted by Sale and Hamilton (1967). T h e higher efficiency of longer treatment times was verified by H o f m a n n (1984) when Streptococcus thermophilus suspended in water was treated with pulses of 15 and 90 ~sec in a continuous coaxial treatment chamber with an electric field of 40 kV/cm. The inactivation of this bacteria was lower than 1.5 log cycles at short treatment times, whereas a 4 log cycle reduction was obtained with 90/zsec.

138

s. PEF Inactivation of Cells, Spores, and Enzymes

The effect of peak voltage (between 10 and 70 kV), the number of exponentially decaying pulses (between 0 and 250 pulses), the rise time (between 500 and 1300 nsec), and the use of a treatment chamber with no food exposed directly to electrodes (Fig. 2.16) were studied by Lubicki and Jayaram (1997) to evaluate the inactivation of Y. enterocolitica. Results showed that at high-peak voltages and pulse numbers, 7 log reductions were achieved with an energy input of 120 J / c m 3. Furthermore, an analysis of TEM micrographs showing alterations in cell shape and disruption of membranes provided additional evidence of the effectiveness of PEF. Although these inactivation results are not quite comparable with other research, the configuration of the chamber used in this study meant that a uniform electric field between electrodes in the treatment zone could not be guaranteed. Another disadvantage of this chamber configuration was that it made it difficult to address how efficiently the pulse energy was transferred to the sample.

III.

Spore Inactivation

Compared to vegetative cells, microbial spores are resistant to extreme ambient conditions such as high temperatures and osmotic pressures, high and low pHs, and mechanical shocks. Their resistance is associated not only with their small size (which makes them more difficult to destroy than larger cells), but also dehydration and mineralization. Those bacterial spores that survive heat treatment may severely restrict the shelf-life of thermally processed foods because of spoilage or poisoning. Marquez et al. (1997) pointed out that spore size, mechanisms of spore activation, initiation processes, ability of spores to recover, and the way electric fields and spores interact are important factors in the destruction of spores by PEF. Waveshape and pulse frequency may be two of the most critical factors in the inactivation of bacterial spores by PEF. Low frequency exponentially decaying waveshape pulses have been shown not to be as effective as instant-reverse-charge waveshapes (Marquez et al., 1997) or pulse frequencies on the order of kHz (Yin et al., 1997). Hamilton and Sale (1967) found that spores of B. cereus and B. polymyxa suspended in 1% NaC1 were completely resistant to DC exponential pulse treatment with field strengths up to 30 kV/cm. During germination and outgrowth, however, the spores became sensitive. Whereas spores of B. cereus began to develop sensitivity as soon as they germinated, spores of B. polymyxa only showed appreciable sensitivity once the vegetative cells began to grow out of the spore integument. This may have been due to the core membrane of the spore that became the plasma membrane of the vegetative cell lying inside the thick cortex and coat layers and therefore was protected from potentially damaging agents such as electric field pulses. The germination and outgrowth of B. cereus spores are also characterized by the immediate disappearance of the cortex and swelling of the cell accompanied by a

III. Spore Inactivation

139

gradual dissolving of spore coat layers. Removal of the protection afforded to the membrane by the cortex and coat layers is therefore a continuous process throughout the germination and outgrowth of the spore, as is an increase in the sensitivity of the cells to electric pulses. In contrast, Hamilton and Sale (1967) detected no alterations in the cortex or coat structure in germinated spores of B. polymyxa and B. subtilis and little evidence of the dissolution of these layers even during outgrowth. However, when they incubated these bacteria for approximately 1 hr in a yeast glucose broth, the outer layers of the spore split open and the developing vegetative cell began to emerge. It was only at this point that the unprotected membrane was exposed to the electric field and cells showed sensitivity toward this treatment. Simpson et al. (1995) investigated the effect of PEF in combination with other hurdles such as heat shock, lysozyme, ethylenediaminetetraacetate (EDTA), and pH for the inactivation of B. subtilis spores suspended in SMUF. No inactivation was achieved when the pH of the medium was varied from 4 to 7, the lysozyme concentration was 0-1000 U I / m l , or when 5 ~g in combination with 10 m M EDTA was used prior to PEF treatment (32 pulses at 15 kV to 45 kV/cm, depending on the treatment). However, a 2 to 4 log reduction in the n u m b e r of spores was observed when the spores were heatshocked at 80~ for 10 min prior to treatment with lysozyme, followed by PEF at 60~ The difficulty of inactivating spores was verified in other studies using 75 exponentially decaying pulses of 2 /zsec at 60 k V / c m in a continuous coaxial treatment chamber (Pag{m et al., 1998). After the combination of PEF and moderate temperatures around 60~ the activation of spore suspensions prior to PEF treatment, a n d / o r the use of up to 5000 U I / m l lysozyme, no inactivation of spores was achieved. It was thus concluded that an intermediate step that allows the outgrowth of spores to vegetative cells was necessary. Pag{m et al. (1998) also r e c o m m e n d e d the use of a hurdle approach to inactivate bacterial spores by the combination of high hydrostatic pressure (HHP) and PEF as an alternative to inactivating the spores with heat. Y m et al. (1997) inactivated up to 2 log cycles of B. subtilis spores with pulse durations of 1, 2, 4, and 6/zsec at frequencies of 3000, 1500, 750, and 500 Hz, respectively, with an applied electric field strength of 30 k V / c m at 36~ With the same energy input and an increased pulse duration time from 1 to 6 ~sec, the inactivation of spores was shown to increase. However, with frequencies between 2000 and 4000 Hz, the inactivation rate of the bacterial spores decreased as the frequency increased. The researchers hence suggested that an optimal PEF treatment frequency to cause resonance would result in a loosening of the rigid bacterial spore structure such that the applied PEF could, in effect, punch through the spore structure and inactivate spores, showing an almost 1 log cycle reduction when a frequency of 2000 Hz was applied. At this optimum frequency, Yln et al. (1997) evaluated the electric field intensity effect and found that up to 98% ( ~ 1.7 log cycles) inactivation was achieved with 40 kV/cm. The inactivation at

140

5. PEF Inactivation of Cells, Spores, and Enzymes

different temperatures revealed that at 36~ more bacterial spores t e n d e d to germinate and be inactivated as the treatment time was extended. The presence of 0.01 L-alanine (a germinating agent) and 0.2% NaC1, with a t r e a t m e n t time of 1800 /zsec at 30 k v / c m , achieved almost 2 log cycles of inactivation, whereas less than 1 log cycle was inactivated without the germinating agent. W h e n treatment times were smaller than 300 /zsec, very little difference in inactivation was found in the different treatment m e d i a (Fig. 5.22). Yin et al. (1997) thus concluded that bacterial spores tend to germinate and be inactivated by PEF as treatment times increase. Marquez et al. (1997) evaluated the effect of electric fields between 20 and 50 k V / c m in the inactivation of viable spores of B. subtilis and B. cereus when 15 to 50 instant-charge-reversal pulses were used. Bacillus cereus spores were suspended in 0.1% NaC1 and B. subtilis in 0.15% NaC1 within a t r e a t m e n t chamber with two parallel 145-mm-diameter, round-edged, stainless-steel disk electrodes 3 m m apart. After the treatment the samples were kept for 30 days at 6~ to demonstrate that the damage by PEF was not reversible. With a treatment at 50 k V / c m at 25~ up to 3.4 log cycles of B. subtilis spores were inactivated with 30 pulses, whereas 50 pulses inactivated at least 5 log cycles of the B. cereus spores. However, the same t r e a t m e n t on the same bacteria at 5 to 10~ resulted in only a 1.2 log cycle reduction. In the case of a decoated spore, the leakage of mobile ions may increase as the temperature is raised because the change in average kinetic energy of the ions in the core would make t h e m move faster. It may also increase the motion of solvent molecules in both the surrounding cortex and the core so that they could migrate from one electrode to the other. Marquez et al. (1997) used lower pulse frequencies with time gaps between pulses from 2 to 3 or 5 to 6 sec, and showed that PEF treatment of B. cereus spores can be more effective at longer times between pulses. Spore

O

_

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0.02% NaCI + 0.01% L.a 0.02% NaC1

0.01

i

i

i

300

600

900

!

i

i

1200 1500 1800 2100

Treatment Time (microseconds) Fig. 5.22 Inactivation of B. subtilis spores using 30 k / c m , 1000 Hz, and 6-msec pulses with L-alanine (L.a) (Ym et al., 1997).

IV. Standardization of Inactivation Assessment

141

inactivation was explained to be due to pulse polarities that favor the immobilized ions in the spore core and form a shielding layer instead of penetrating the core, and thus shorter PEF exposure times result in less effective inactivation. Accordingly, SEMs of B. cereus showed the damage of the spore by the formation of holes in the surface. In addition, some spores were reported to be completely destroyed or enlarged. Although the time in which the spores were damaged by PEF was not clearly identified, Marquez et al. (1997) agree that PEF does cause structural changes to B. subtilis spores.

IV.

Standardization of Inactivation Assessment

The near future implementation of PEF technology demands reliable and comparable inactivation microbial data among the results of different research groups. Some guidelines to conduct future research that yield comparable data recommend following standardized procedures for the preparation of the inocula, sample collection, dilution, plating, and data recording (EPRI, 1998). Preparation of inocula as indicated by American Type Cell Culture (ATCC) procedures includes activating the culture into 10 ml of an appropriate medium followed by an appropriate incubation period under required conditions (i.e., agitation and temperature). The second activation step is cell harvesting and cell concentration by centrifugation (i.e., 10 min at 7000 g) or other proper method to obtain inoculum levels of approximately 106-107 cfu/ml. Additional ATCC requirements include relevant and sufficient samples for microbiological analysis that must be taken before and after inoculation (i.e., six samples), and during the experiment at least twice for each pulse frequency in duplicate or triplicate. If a configuration with multiple chambers is used, sampling of the product should be made after each treatment chamber, at the final outlet of the product, and at the product tank. Counting procedures should include proper 10-fold serial dilutions of each sample and replications using either 0.9% saline solution or 0.1% peptone; also important are the use of standard plating techniques and incubation under appropriate time and temperature conditions. Finally, careful recordkeeping procedures are vital for future analyses. Because of the increasing interest in this technology, international groups with different areas of expertise have gotten together to start a firm platform for PEF. As an example of this effort, determinations have been made as to what microorganisms are of major consequence, and plans for PEF treatments are now in existence (Table 5.5). Although some of the bacteria of focus have already received a significant degree of attention, it is evident that there is still a great need for additional research on each (EPRI, 1997).

142

5. PEF Inactivation of Cells, Spores, and Enzymes

TABLE 5.5 Microorganisms of Possible Interest to Test in PEF Treatments ~

Type of microorganism G - pathogens G + pathogens Spore-forming pathogens

Fungi (vegetative and spores)

Nonpathogens/spoilage

Spore formers

Acid-fast organisms

Microorganism S. enteridius E. coli O157:H7 b L. monocytogenes b S. aureus B. cereus C. botulinum b C. perfringens a. niger C. albicans P. roqueforti S. cerevisiae B. fulva L. plantarum L. innocua L. mesenteroides Micococcus spp. L. acidophilus b B. coagulans C. sporogenes C. butyricum M. tuberculosis

a Adapted from EPRI (1997). b Key species.

V.

Enzyme Inactivation

To prevent denaturation, an enzyme has to maintain its native structure. Hydrophobic interactions, hydrogen bonding, van der Waal interactions, ion pairing, electrostatic forces, and steric constraints stabilize the three-dimensional molecular structure of globular proteins (secondary, tertiary, and quaternary). Since a change in the magnitude of any of these forces could cause denaturation, the application of PEF may affect them. Furthermore, external electric fields influence the conformational state of a protein through charge, dipole, or induced dipole chemical reactions (Tsong and Astunian, 1986). The charged groups and structures are highly susceptible to various types of electric field perturbations. Association and dissociation of ionizable groups, movements of charged side chains, changes in the packing and alignment of helices (helical or sheet content), and the overall shape of a protein may all be induced by external electric fields (Tsong, 1990). Because pulsed electric fields affect the conformafional state of proteins and enzymes, they can be used to prevent detrimental reactions that produce oxidation, off flavors, and color changes in food products. Proteases

V. Enzyme Inactivation

143

and lipases are heat-stable enzymes that cause spoilage of ultrahigh temperature-processed milk during storage at 3 to 6~ Proteases cause the breakdown of milk protein, which leads to the development of a bitter flavor or coagulation. Some of the earliest studies on the application of electricity to inactivate enzymes are those of Gilliland and Speck (1967) using electrohydraulic shock on trypsin, lactic dehydrogenase, and protease from B. subtilis with an electric field of 31.5 kV/cm. Hamilton and Sale (1967) used PEF for the inactivation of benzene-treated E. coli 300-V/3-galactosidase and found that the activity was not affected by a DC pulse treatment of the cells prior to activity assay. The lysing of bovine erythrocytes by DC pulses also did not reduce the activity of acetylcholinestearase. Another relatively unsuccessful treatment was assayed in NADH dehydrogenase (EC 1.6.99.3) activity as a measure of electron transport system activity in extracts from pulse-treated and untreated E. coli 8196, succinic dehydrogenase (EC 1.3.99.1), and hexokinase (EC 2.7.11)activities. Although the pulse treatments were sufficient to kill more than 90% of the cell population, no significant inhibition of individual enzyme activities was achieved. Another important proteolytic enzyme is plasmin (fibrinolysin EC 3.4.21.7) or milk alkaline protease, which is an indigenous enzyme in bovine milk. When present after heat treatment, it will cause spoilage (characterized by bitter flavor) during storage. Because of its significance, Vega-Mercado et al. (1995) studied the effect of PEF inactivation on plasmin and found that its proteolytic activity promotes several changes in milk. Some of these include a decrease in viscosity of casein dispersions, an increase in the amount of soluble protein due to the formation of peptides, and an increase in rennet coagulation time. Although pasteurization decreases the initial plasmin activity in milk, it does not prevent proteolytic activity from developing during storage. Vega-Mercado et al. (1995) also reconstituted a freeze-dried sample of plasmin (fibrinolysin EC 3.4.21.7) from bovine plasma with 10 ml of 1 m M HC1 and then froze it in 1-ml vials at-35~ until used. For inactivation studies, the reconstituted plasmin was mixed in a SMUF to obtain a plasmin concentration of 100 /xg/ml. The pH and ionic strength of SMUF were found to be 6.11 and 0.056 M, respectively. The plasmin/SMUF solution was then passed through a continuous flow parallel plate chamber at a flow rate of 45 m l / m i n and subjected to electric fields of 15, 30, and 45 kV/cm. Plasmin activity decreased by 90% with electric fields of 30 and 45 k V / c m after 50 pulses at a temperature of 15~ and up to 60% at 10~ (Figs. 5.23 and 5.24). Electric field-treated plasmin solutions showed no significant changes in activity after 24 hr of storage at 4~ Pseudomonas spp. is one of the protease-producing psychrotrophic bacteria in milk. Thermal treatment for 60 min at 60~ causes 56-60% inactivation of the proteolytic enzymes produced by this microorganism. VegaMercado et al. (1997)isolated an extracellular protease from P. fluorescens M 3 / 6 by chromatofocusing and gel-exclusion chromatography and studied its inactivation by PEF. Figure 5.25 illustrates the elution, pH, and activity

144

5. PEF Inactivation of Cells, Spores, and Enzymes 70.00 o

60.00

o~ 50.00 40.00 o

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5

10

15

20

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Number of Pulses

Fig. 5.23

PEF inactivation of plasmin at 10~ (Vega-Mercado et al., 1995).

profiles for the chromatofocusing separation of the tryptic soy broth enriched with a yeast extract (TSB/YE) and protease. A pool of the active protein fractions from the chromatofocusing procedure, once mixed and concentrated, was loaded onto a gel-exclusion column for molecular weight classification of the proteins as illustrated in Fig. 5.26. Figure 5.27 depicts the distribution by SDS-PAGE of the proteins for each step of the purification procedure. The isolated protease had a molecular mass of 45-50 kDa and an isoelectric point of 8.0 (Vega-Mercado, 1996). Pulsed electric field treatments were also conducted in triplicate using a constant electric field of 6.2 k V / c m and 0, 10, and 20 pulses of 700/~sec in a bench-top electroporator (GeneZapper, IBI-Kodak, Rochester, NY) with six SMUF formulations. Pulse electric field inactivation of the protease was studied by suspending it in three different media: (1) tryptic soy broth enriched with yeast extract, (2) skim milk, and (3) a casein-Tris buffer solution. The protease activity was found to decrease by 80% with an electric field of 18 k V / c m after 20 pulses in the tryptic soy broth medium, and the skim milk's protease was inactivated up to 65% with an electric field of 15 k V / c m (pulsing rates of 1 to 2 Hz) after 98 pulses (Fig. 5.28). However, both the proteolytic activity and the susceptibility of the casein to proteolysis increased with an electric field of

100.00-

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5

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Number of Pulses Fig. 5.24

PEF inactivation of plasmin at 15~ (Vega-Mercado et al., 1995)'

,,,i

45

i

50

V. Enzyme Inactivation

145

Fig. 5.25 Enzymaticactivity assay procedure (adapted from Vega-Mercado, 1996).

25 k V / c m at a pulsing rate of 0.6 Hz. It is interesting to note that no significant inactivation of protease or any significant change in the susceptibility of casein to proteolysis was obtained in the casein-Tris buffer m e d i u m . This indicates that casein has a protective effect on protease against electric field treatment, and the three-dimensional structure of the protease may be protected from unfolding by the presence of casein (Vega-Mercado et al., 1997).

Fig. 5.26 Elution and activity profile for gel-exclusion chromatography of a partially purified TBS/YE-protease mixture from P. fluorescens M3/6 (adapted from Vega-Mercado, 1996).

146

5. PEF Inactivation of Cells, Spores, and Enzymes

Fig. 5.27 Silverstaining of an electrophoresis gel: (a) TSB/YE-protease mixture; (b) polybuffer 96 pool, (c) gel-exclusion pool; and (d) molecular weight standards (adapted from Vega-Mercado, 1996).

F u r t h e r studies c o n d u c t e d by Vega-Mercado (1996) a p p l i e d the conclusions of Barach et al. (1976) a n d Barach a n d A d a m s (1977) a b o u t the sensitivity of P. fluorescens to EDTA. Using E D T A as an additional h u r d l e to PEF, Vega-Mercado showed a significant inhibitory effect on the proteolytic activity of a protease p r o d u c e d by P. fluorescens M 3 / 6 (Fig. 5.29). T h e proteolysis rate a n d specific activity were d e t e r m i n e d as net change absorbance rate =

(5.6)

min net change absorbance

specific activity =

(5.7)

/zg p r o t e i n - m i n

80 70 r

TSB/YE - 18 kV/cm, 24~ TSB/YE - 11 kV/cm, 20~

"--- Skim Milk - 15 kV/cm, 50~

E

3(1 10 0 0

20

40

60

80

100

N u m b e r of P u l s e s

Fig. 5.28 Inactivation of a protease from P. fluorescens M3/6 in tryptic soy broth enriched with a yeast extract (TSB/YE, pulsing rate of 0.25 Hz) and skim milk (pulsing rate 2 Hz) using 2 /zsec pulses (adapted from Vega-Mercado et al., 1997).

147

V. E n z y m e Inactivation 500 o T" r

400

o

-E ro

n-

200

100

0

2

4

6

8

10

12

14

16

18

20

EDTA (mM)

Fig. 5.29 Inhibitory effect of EDTA on a protease from P. fluorescens M3/6 (adapted from Vega-Mercado, 1996).

The inactivation was evaluated as the percentage reduction in the specific activity after PEF treatment, where the beneficial effect of EDTA in combination with PEF to inactivate the P. fluorescens protease was also verified in SMUF (Fig. 5.30). The inactivation of the protease when exposed to PEF did not d e p e n d on the presence of calcium in media containing the protease, as illustrated in Fig. 5.31. Furthermore, the inactivation was the same for the three solutions containing 0, 10, and 15 m M calcium, where the proteolytic activity of the protease was consistently reduced 30% after exposure to 20 pulses of 700 /xsec at 6.2 k V / c m and 15-20~ Alkaline phosphatase (ALP) is another enzyme present in milk that is used as an indicator of the adequacy of pasteurization as ALP has 100% activity in raw milk and 0% in pasteurized milk. The activity of ALP in pasteurized milk products has public health significance as it indicates inadequate pasteurization or contamination of pasteurized milk with raw

Fig. 5.30 PEF inactivation of a protease from P. fluorescens M3/6 in SMUF with EDTA (adapted from Vega-Mercado, 1996).

148

5. PEF Inactivation of Cells, Spores, and Enzymes 500

- - ~ - - 0 mM Ca ---Bl- 10mM Ca

~" T" 4OOl

15mM Ca

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n-

200

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Number of Pulses

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Fig. 5.31 Effect of calcium in the PEF inactivation of protease from P. fl~orescens M3/6 at 6.2 kV/cm (adapted from Vega-Mercado, 1996).

milk. Alkaline phosphatase is present in raw milk in association with the membrane of fat globules and in the form of lipoprotein particles in skim milk. In milk pasteurized at high temperatures for short periods of time (165~ sec), ALP is inactivated but often regenerates during storage. Aging of milk prior to pasteurization, the absence of air, pasteurization at high temperatures for short periods of time, and the presence of magnesium and calcium all enhance the regeneration of ALP. However, raw and regenerated ALP are not identical. Native ALP from raw cream exhibits an electrophoresis pattern of at least three isoenzymes ( a , t , and y) with ALP activity, but the electrophoresis pattern of regenerated ALP shows only the fl isoenzyme. Grahl et al. (1992) reported no inactivation of ALP in milk after 20 pulses with a pulse duration of 39/~sec and an electric field of 26 kV/cm. Castro-Castillo (1994) studied the PEF inactivation of ALP in raw whole milk, 2% milk, nonfat milk, and a SMUF media and was able to reduce the activity of ALP in nonfat milk by 65% after 70 pulses with a duration of 0.74 msec and an electric field of 18.8 k V / c m using a commercial electroporator (Gene-Zapper, Kodak). It took 70 pulses with a duration of 0.74 msec and an electric field of 22 k V / c m to reduce the ALP activity by 65% in SMUF (Fig. 5.32). After 70 pulses with a duration of 0.4 msec and an electric field of 18.8 kV/cm, the ALP activity was reduced by 59% in the raw and pasteurized 2% milk (Fig. 5.33). The activity of ALP in 1 ml of raw milk mixed in 100 ml of pasteurized 2% milk was reduced by 96% with an electric field of 13.2 k V / c m after 70 pulses. The temperature of the milk at the end of this treatment was 43.9~ A comparison between heat- and electric field-induced inactivation of ALP revealed that when 1 ml of raw milk mixed in 100 ml of pasteurized 2% milk was heated to ~ 44~ the ALP activity was reduced by 30%, whereas with PEF treatment the reduction was 96% (Fig. 5.34). In contrast, ALP suspended in 1000 m M diethanolamine and 0.5 m M magnesium chloride buffer, which was PEF treated using 30 instant-charge-

149

V. Enzyme Inactivation lOO

m

MSMUF [] 22.0kV/cm

9o

.~"

80

Milk 9 18.0kV/cm

_ . Nonfat -

~

~

so 30 20 10 0

!

0

10

,---

I

20

30 Number of

40

50

60

70

Pulses

Fig. 5.32 PEF i n a c t i v a t i o n o f ALP d i l u t e d in S M U F or n o n f a t m i l k ( a d a p t e d f r o m CastroCastillo, 1994).

reversal pulse waveshapes of 2 /~sec width and 25 kV/cm, yielded just 5% inactivation (Ho et al., 1997). The difference in the 44% ALP inactivation after 20 pulses at 22 kV/cm (Fig. 5.32) and the 5% after 30 pulses at 25 kV/cm obtained by Castro-Castillo (1994) (Fig. 5.32) indicates that the number of pulses and electric field strength are not the only important factors in PEF inactivation. Ho et al. (1997) submit pulse width and waveform as additional influences, and the treatment media might be another important factor to consider. Pulsed electric fields not only cause a change in the activity of ALP but also in its structure. Alkaline phosphatase is a globular protein with activity dependent on a specific internal structure. An increase in the susceptibility of a PEF-treated ALP to trypsin proteolysis indicates degradation of the enzyme's secondary structure. A decrease in the UV absorption of ALP treated with electric fields and digested by trypsin suggests that both the intramolecular linkages of the active center and the entire globular configuration of ALP are altered by electric fields. In an experiment conducted by Castro-Castillo (1994), the increase in percentage of digestion by trypsin was shown to parallel the increase in optical density at 298 nm. Native ALP exhibited a maximum absorption at 214 and 278 nm before and after trypsin 1oo 90

!"1 14.8kV/cm

~"

so

9 18.8kV/cm

"~"

70

9

~

.

50 30 20 10 0

0

I0

20

V-1

I

30

40

50

60

70

N u m b e r of Pulses

Fig. 5.33 PEF i n a c t i v a t i o n o f A L P d i l u t e d in U H T Castro-Castillo, 1994).

pasteurized 2% milk (adapted from

150

5. PEF Inactivation of Cells, Spores, and Enzymes

Fig. 5.34 Inactivation of alkaline phosphatase by PEF or heating at 44~ for 17.5 min (adapted from Castro-Castillo, 1994).

digestion, respectively, and the resulting maximum absorption of electric field treated ALP shifted to 276 nm. Castro-Castillo (1994) also found that polyacrylamide gel electrophoresis of electric field-treated and -untreated ALP resulted in essentially identical bands with similar intensifies. This observation ruled out the possibility of chemical changes being responsible for the loss of ALP activity, but suggested electric field induced conformational changes. A mechanism was then proposed to explain the loss of ALP activity. Pulses of high-intensity electric fields caused the polarization of ALP and increased the dipole movement of the protein, and polarized molecules tended to associate to form aggregates. Untreated ALP exhibited a helical conformation with polar residues aligned in one direction, positive charges at the N terminus of the helix, and negative charges at the C terminus parallel to the helical axis. Electric fields caused unfolding of polypeptides with electric dipoles oriented at random. Dipole-dipole interactions also played an important role in stabilizing protein a-helices, so when electric field pulses disrupted the dipole-dipole interactions of enzyme, an unstable enzyme was produced that denatured and formed aggregates. In the search for a better understanding of the effect of PEF on enzymes of major importance in the food industry, Ho et al. (1997) applied instantcharge-reversal pulse waveshapes to inactivate different enzymes suspended in buffer solutions. Thirty pulses of 2-~sec width and 13-87 k V / c m yielded inactivations up to 70-85% for lipase, glucose oxidase, and a-amylase, whereas only 30-40% was obtained for peroxidase and phenol oxidase (Fig. 5.35). In contrast, lysozyme and pepsin presented an increased activity in certain ranges of the applied voltage. Pepsin and lysozyme also exhibited an inhibitory effect once a particular field was reached. Because the degree of denaturation varied from enzyme to enzyme, it can be concluded that more research is necessary to determine if the inactivation level is due to the

VI. Final Remarks

15 1 .o 120 : 100

m.

0

.~

80

~

60

--,--L ~G.O. A.A. P. P,O.

~9 40 20 =~

0

i

0

i

i

i

5 10 15 20 Voltage Supplied (kV)

i

25

Fig. 5.35 Changes in enzyme activity after a PEF treatment with 30 instant-charge-reversal pulses at a 2-/zsec pulse width for a period of 2 sec [lipase (L), glucose oxidase (G.O.), a-amylase (A.A.), peroxidase (P), and phenol oxidase (P.O.)] (adapted from Ho et al., 1997).

structure of the protein, the creation of active sites, the concentration of the treated enzyme, local heating effects, or some combination of each of these factors.

VI.

Final Remarks

Globalization of the results presented in this chapter is quite difficult, although there is evidence of the PEF lethality over a great variety of microorganisms, some enzymes, and even a few bacterial spores. The high efficiency of PEF to inactivate yeast cells (mainly S. cerevisiae in buffer solutions and fruit juices) is especially encouraging. Yeast inactivation was not only found to be effective at low temperature processing conditions, but the low conductivity of one particular suspending media (i.e., 0.175 S / m , apple juice) even allowed a successful PEF treatment without any heat generation. These findings p r o m p t the use of this technology for the processing of fruit juices, so the feasibility of industrialization is presented in Chapter 6. The high inactivation levels obtained over enteric and pathogenic bacteria such as E. coli and Salmonella spp. permit confidence in the pasteurization abilities of PEF. Accordingly, data from PEF studies of other microorganisms present the first steps toward the characterization of this technology. Although results from the work on enzymatic activity reduction by PEF are positive so far, more attention is still necessary as it is not possible to describe certain inactivation patterns due to variations in enzymes. Spore inactivation is still a fairly new area where few inactivation levels have been recorded, but researchers still seem to be convinced of the inactivation efficiency of some waveshape pulses applied at very high frequencies. Based on the experimental results of different research groups, it is possible to confirm that structural changes suffered by various microorganisms are present where high levels of inactivation are detected, which

152

5. PEF Inactivation of Cells, Spores, and Enzymes

supports the inactivation theories p r e s e n t e d in Chapter 3. Although these findings provide additional supporting evidence of PEF inactivation capabilities, in some cases the inactivation levels a n d structural changes that were observed in the same species were not alike. This suggests that m o r e work is r e q u i r e d to verify w h e t h e r the inactivation m o d e of PEF changes as a function of processing conditions. T h e usefulness of kinetic models to c o m p a r e the sensitivity of different microorganisms and species u n d e r the same t r e a t m e n t conditions has also b e e n d e m o n s t r a t e d . Although until now most inactivation studies have b e e n p e r f o r m e d via challenge tests on specific microorganisms, there is no evid e n c e that inactivation kinetics with a c o m b i n a t i o n of microorganisms in the same t r e a t m e n t m e d i a will follow the original inactivation of individual species. This gap must e n c o u r a g e research in the area, where not just buffer solutions a n d m o d e l m e d i a should be used, but food products as well. All the research d o n e in the past 20 years strongly supports the importance of each identified factor in the PEF process, where electric field, t r e a t m e n t time, waveshape, t r e a t m e n t media, a n d t r e a t m e n t system characteristics define the inactivation kinetics of vegetative cells, spores, a n d enzymes. F u r t h e r m o r e , the discoveries p r e s e n t e d by those working in the field allow the quantitative identification of each factor over a variety of biological entities. In addition, there is a g r e e m e n t that obtaining a high killing efficiency with a m i n i m u m of adverse effects on PEF-treated products is possible with only m i n o r adjustments on operating conditions. T h e insights f r o m the inactivation a n d challenge tests described in this chapter lead to the utilization of PEF to process several food products, so C h a p t e r 6 presents a detailed look at these possibilities.

References

Banwart, G.J. (1989). "Basic Food Microbiology." AVI Van Nostrand-Reinhold, New York. Barach, J. T., and Adams, D. M. (1977). Thermostability at ultrahigh temperatures of thermolysin and protease from a psychrotrophic Pseudomonas. Biochim. Biophys. Acta. 485, 417-423. Barach, J. T., Adams, D. M., and Speck, M. L. (1976). Stabilization of a psychrotrophic Pseudomonas protease by calcium against thermal inactivation in milk at ultrahigh temperature. Appl. Env. Microbiol. 31,875-879. Castro-Castillo, A. J. (1994). Pulsed electric field modification of activity and denaturation of alkaline phosphatase. Ph.D. Dissertation, Washington State University, Pullman, Washington. Dunn, J. E., and Pearlman, J. S. (1987). Methods and apparatus for extending the shelf life of fluid food products. U. S. Patent 4,695,472. EPRI (1997). EPRI/Army PEF Workshop II, Chicago, 10-11 October. EPRI (1998). Pulsed electric field processing in the food industry: A status report on PEF. Report CR-109742. Industrial and Agricultural Technologies and Services, Palo Alto, California. G~skov~t, D., Sigler, K., Janderova, B., and Plasek,J. (1996). Effect of high-voltage electric pulses on yeast cells: Factors influencing the killing efficiency. Bioelectrochem. Bioenerg. 39, 195-202.

References

| 53

Gilliland, S. E., and Speck, M. L. (1967). Mechanism of the bactericidal action produced by electrohydraulic shock. Appl. Microbiol. 9, 1033-1044. Grahl, T., and M~irkl, H. (1996). Killing of microorganisms by pulsed electric fields. Appl. Microbiol. Biotechnol. 45, 148-157. Grahl, T., Sitzmann, W., and M~irkl, H. (1992). Killing of microorganisms in fluid media by high voltage pulses. Presented at the 10th DECHEMA Biotechnology Conference Series, Frankfurt, Germany, 5B, pp. 675-678. Gupta, R. P., and Murray, W. (1988). Pulsed high electric field sterilization. ~EE Pulsed Power Conference Record, pp. 58-64. Gupta, R. P., and Murray, W. (1989). Pulsed high electric field sterilization. Seventh ~EE Pulsed Power Conference, Monterey, California 11-14 June. Hamilton, W. A., and Sale, A.J.H. (1967). Effects of high electric fields on microorganisms. II. Mechanism of action of the lethal effect. Biochim. Biophys. Acta 148, 789-800. Harrison, S. L., Barbosa-Cfinovas, G. V., and Swanson, B .G. (1997). Saccharomyces cerevisiae structural changes induced by pulsed electric field treatment. Lebensm. Wiss. Technol. 30, 236-240. Ho, S. Y., Mittal, G. S., Cross, J. D., and Griffith, M. W. (1995). Inactivation of P. fluorescens by high voltage electric pulses. J. Food Sci. 60(6), 1337-1343. Ho, S. Y., Mittal, G. S., and Cross, J. D. (1997). Effects of high field electric pulses on the activity of selected enzymes. J. Food Eng. 31, 69-85. Hofmann, G. A. (1984). Microflora reduction in liquids with pulsed electric fields. Internal Technical Report. Biotechnologies & Experimental Research Inc., San Diego. Hfilsheger, H., and Niemann, E. G. (1980). Lethal effects of high-voltage pulses on E. coli K12. Radiat. Environ. Biophys. 18, 281-288. Hfilsheger, H., Potel, J., and Niemann, E. G. (1981). Killing of bacteria with electric pulses of high field strength. Radiat. Environ. Biophys. 20, 53-65. Hfilsheger, H., Potel, J., and Niemann, E. G. (1983). Electric field effects on bacteria and yeast cells. Radiat. Environ. Biophys. 22, 149-162. Jacob, H. E., Forster, W., and Berg, H. (1981). Microbiological implications of electric field effects. II. Inactivation of yeast cells and repair of their cell envelope. Z. Allg. Microbiol. 21(3), 225-233. Jay, J. M. (1996). "Modern Food Microbiology," 5th Ed. Van Nostrand-Reinhold, New York. Jayaram, S., Castle, G. S. P., and Margaritis, A. (1991). Effects of high electric field pulses on Lactobacillus brevis at elevated temperatures. ~EE 5, 674-681. Jayaram, s., Castle, G. S. P., and Margaritis, A. (1992). Kinetics of sterilization of Lactobacillus brevis by the application of high voltage pulses. Biotechnol. Bioeng. 40(11), 1412-1420. Liu, X., Yousef, A. E., and Chism, G. W. (1997). Inactivation of Escherichia coli 0157:H7 by the combination of organic acids and pulsed electric fields. J. Food Safety 16, 287-299. Lubicki, P., and Jayaram, S. (1997). High voltage pulse application for the destruction of the Gram negative bacterium Yersinia enterocolitica. Bioelectrochem. Bioenerg. 43, 135-141. Ma, L., Chang, F. J., and Barbosa-Cfinovas, G. V. (1997). Inactivation of E. coli in liquid whole eggs using pulsed electric fields technology. In "Proceedings of the Fifth Conference of Food Engineering" (G. V. Barbosa-Cfinovas, S. Lombardo, G. Narishman, and M. Okos, eds.), pp. 216-221. American Institute of Chemical Engineers, New York. Marquez, V. O., Mital, G. S., and Griffiths, M. W. (1997). Destruction and inhibition of bacterial spores by high voltage pulsed electric fields. J. Food Sci. 62(2), 399-409. Martln-Belloso, O., Zhang, Q., Castro, A.J., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1994). Pulsed electric fields of high voltage to preserve foods. Microbiological and engineering aspects of the process. Spanish J. Food Sci. Technol. 34, 1-34. Martln-Belloso, O., Qin, B. L., Chang, F. J., Barbosa-Cfinovas, G. V., Swanson B. G. (1997a). Inactivation of Eschevichia coli in skim milk by high intensity pulsed electric fields. J. Food Proc. Eng. 20(4), 317-336. Mart{n-Belloso, O., Vega-Mercado, H., Q i n B. L., Chang, F. J., Barbosa-C{movas, G. V., and Swanson B. G. (1997b). Inactivation of Escherichia coli in liquid egg using pulsed electric fields. J. Food Proc. Pres. 21, 193-208.

154

5. PEF Inactivation of Cells, Spores, and Enzymes

Matsumoto, Y., Satake, T., Shioji, N., and Sakuma, A. (1991). Inactivation of microorganisms by pulsed high voltage applications. Conference Record of F E E Industrial Applications Society Annual Meeting, pp. 652-659. Mizuno, A., and Hayamizu, M. (1989). Destruction of bacteria by pulsed high voltage application. Presented at the Sixth International Symposium on High Voltage Engineering, August 28-September 1, New Orleans, Louisiana. Mizuno, A., and Hori, Y. (1988). Destruction of living cells by pulsed high-voltage application. IEEE Trans. Ind. Appl. 24(3), 387-394. Ohshima, T., Sato, K., Terauchi, H., and Sato, M. (1997). Physical and chemical modifications of high-voltage pulse sterilization. J. Electrostat. 42, 159-166. Pagfin, R., Esplugas, S., G6ngora-Nieto, M. M., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1998). Inactivation of Bacillus subtilis spores using high intensity pulsed electric fields in combination with other food conservation technologies. Food Sci. Technol. Int. 4(1), 33-44. Peleg, M. (1995). A model of microbial survival after exposure to pulsed electric fields. J. Sci. Food Agric. 67, 93-99. Pothakamury, U. R., Monsalve-Gonz~dez, A., Barbosa-C{movas, G. V., and Swanson, B. G. (1995a). Inactivation of Escherichia coli and Staphylococcus aureus in model food systems by pulsed electric field technology. Food Res. Int. 28(2), 167-171. Pothakamury, U. R., Monsalve-Gonzfdez, A., Barbosa-C~novas, G. V., and Swanson, B. G. (1995b). High voltage pulsed electric field inactivation of Bacillus subtilis and LactobaciUus delbrueckii. Rev. Esp. Cienc. Technol. Aliment. 35(1), 101-107. Pothakamury, U. R., Vega-Mercado, H., Zhang, Q., Barbosa-C{movas, G. V., and Swanson, B. G. (1996). Effect of growth stage and temperature on inactivation of E. coli by pulsed electric fields. J. Food Prot. 59(11), 1167-1171. Pothakamury, U. R., Barbosa-C{movas, G. V., Swanson B. G., and Spence, K. D. (1997). Ultrastructural changes in Staphylococcus aureus treated with pulsed electric fields. Food Sci. Technol. Int. 3, 113-121.

Qin, B. L., Zhang, Q., Barbosa-C{movas, G. V., Swanson, B. G., and Pedrow, P. D. (1994). Inactivation of microorganisms by pulsed electric fields with different voltage wave-forms. F E E Trans. Dielec. Electric. Insul. 1(6), 1047-1057. Qin, B. L., Zhang, Q., Barbosa-C{movas, G. V., Swanson, B. G., and Pedrow, P. D. (1995a). Pulsed electric field treatment chamber design for liquid food pasteurization using the finite element method. Trans. ASAE 38(2), 557-565. Qin, B. L., Chang, F., Barbosa-C{movas, G. V., and Swanson, B. G. (1995b). Nonthermal inactivation of Saccharomyces cerevisiae in apple juice using pulsed electric fields. Lebensm. Wiss. Technol. 28, 564-568. Qin, B. L., Barbosa-C{movas, G. V., Swanson, B. G., Pedrow, P. D., and Olsen, R. G. (1998). Inactivation of microorganisms using pulsed electric field continuous treatment system. FEE Trans. Ind. Appl. 34(1), 43-49. Raso, J., Calder6n, M. L., G6ngora, M., Barbosa-C~novas, G. V., and Swanson, B. G. (1998). Inactivation of mold ascospores and conidiospores suspended in fruit juices by pulsed electric fields. Lembensm. Wiss. Technol. 31 (7/8), 668-672. Sale, A.J.H., and Hamilton, W. A. (1967). Effects of high electric fields on microorganisms. I. Killing of bacteria and yeast. Biochim. Biophys. Acta 148, 781-788. Sale, A. J. H., and Hamilton, W. A. (1968). Effects of high electric fields on microorganisms. II: Lysis of erythrocytes and protoplasts. Biochim. Biophys. Acta 163, 37-43. Schoenbach, K. H., Peterkin, F. E., Alden III, R. W., and Beebe, S.J. (1997). The effect of pulsed electric fields on biological cells: Experiments and applications. FEE Trans. Plasma Sci. 25(2), 284-292. Sensoy, I., Zhang, Q. H., and Sastry, S. K. (1997). Inactivation kinetic of Salmonella dublin by pulsed electric fields. J. Food Proc. Eng. 20, 367-381. Simpson, M. V., Barbosa-C{movas, G. V., and Swanson, B. G. (1995). The combined inhibitory effect of lysozyme and high voltage pulsed electric fields on the growth of Bacillus subtilis spores. Presented at the Institute of Food Technologists Annual Meeting. Anaheim, California.

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Sitzmann, W. (1990). Keimabtotung mit hilfe elecktrischer hochspannungsimpulse in pumpfahigen nahrungsmitteln. Vortrag Anlablich des Seminars "Mittelstansfourderung in der Biotechnologie." Ergebnisse des Indirekt-Spezifischen Programma des BMFT 1986-1989. KFA Julich, Germany, 6-7 February. Tsong, T. Y. (1990). Electrical modulation of membrane proteins: Enforced conformational oscillations and biological energy and signal transductions. Annu. Rev. Biophys. Chem. 19, 83-106. Tsong, T. Y., and Astunian, R. D. (1986). Absorption and conversion of electric field energy by membrane bound ATPases. Bioelectrochem. Bioenerg. 15, 457-476. Vega-Mercado, H. (1996). Inactivation of proteolytic enzymes and selected microorganisms in foods using pulsed electric fields. Ph.D. Dissertation, Washington State University, Pullman, Washington. Vega-Mercado, H., Powers, J. R., Barbosa-C{movas, G. V., and Swanson B. G. (1995). Plasmin inactivation with pulsed electric fields. J. Food Sci. 60, 1143-1146. Vega-Mercado, H., Martln-Belloso, O., Chang, F.J., Barbosa-C~novas, G. V., and Swanson B. G. (1996a). Inactivation of Escherichia coli and Bacillus subtilis suspended in pea soup using pulsed electric fields. J. Food Proc. Pres. 20, 501-510. Vega-Mercado, H., Pothakamury, U. R., Chang, F. J., Barbosa-C~novas, G. V., and Swanson, B. G. (1996b). Inactivation of E. coli by combining pH, ionic strength and pulsed electric field hurdles. Food Res. Int. 29(2), 117-121. Vega-Mercado, H., Powers, J. R., Mart[n-Belloso, O, Luedecke, O. L. Barbosa-C{movas, G. V., and Swanson, B. G. (1997). Effect of pulsed electric fields on the susceptibility of proteins to proteolysis and inactivation of an extracellular protease from P. fluorescens M 3/6. In "Proceedings of the Seventh International Congress on Engineering and Food," pp. C73-C76. The Brighton Center, U. K., 13-17 April. Wouters, P. C., and Smelt, J. P. P. M. (1997). Inactivation of microorganisms with pulsed electric fields: Potential for food preservation. Food Biotechnol. 11(3), 193-229. Yln, Y., Zhang, Q. H., and Sastry, S. H. (1997). High voltage pulsed electric field treatment chambers for the preservation of liquid food products. U. S. Patent 5,690,978. Yonemoto, Y., Yamashita, T., Muraji, M., Tatebe, W., Ooshima, H., Kato, J., Kimura, A., and Murata, K. (1993). Resistance of yeast and bacterial spores to high voltage electric pulses. J. Ferment. Bioeng. 75, 99-102. Zhang, Q., Monsalve-Gonz~dez, A., Barbosa-C~novas, G. V., and Swanson, B. G. (1994a). Inactivation of E. coli and S. cerevisiae by pulsed electric fields under controlled temperature conditions. Trans. ASAE 37(2), 581-587. Zhang, Q., Chang, F. J., Barbosa-C{movas, G. V., and Swanson, B. G. (1994b). Inactivation of microorganisms in semisolid foods using high voltage pulsed electric fields. Lebensm. Wiss. Technol. 27(6), 538-543. Zhang, Q., Qin, B. L., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1995). Inactivation of E. coli for food pasteurization by high-intensity-short duration pulsed electric fields. J. Food Proc. Pres. 19, 103-118. Zheng-Ying, L., and Yan, W. (1993). Effects of high voltage pulse discharges on microorganisms dispersed in liquid. Presented at the Eighth International Symposium on High Voltage Engineering, 23-27 August, Yokohama, Japan.

CHAPTER 6

Food Processing by PEF

I.

Introduction

To determine the feasibility of pasteurizing a product by PEF, different stages are required. The first two were accomplished by the studies and results presented in Chapter 5, which are defined as (1) the conduction of inactivation studies or challenge tests to establish the a m o u n t of t r e a t m e n t n e e d e d and (2) the treatment of raw foods with native microbial populations. The third and fourth stages of the process, which are delineated in this chapter, are (3) examination of food microbial, chemical, and physical characteristics and (4) sensory evaluation and shelf-life studies. In the previous chapter, the outlined results from various inactivations of microbes, spores, and enzymes in model and real foods encouraged research on the quality effects on liquid foods processed by this method. The overall quality can be assessed by the evaluation of the microbiological and physicochemical attributes of a product, which are related to hygiene and safety or nutritional, sensorial, and functional characteristics, respectively. Furthermore, the high inactivation levels obtained for some microorganisms with relatively short treatments suggest that PEF could ensure a safe, high-quality product with an extended shelf-life. Accordingly, this chapter will present how the inactivation of naturally present flora is accomplished to increase the shelf-life of real food products without decreasing their quality attributes. Also discussed will be how the use of this technology may even be extended to other areas such as waste treatment and fouling prevention. Because of the advantages that PEF presents as a n o n t h e r m a l process, products such as fruit juices, milk, and beaten eggs have been u n d e r extensive research in order to i m p l e m e n t the process at an industrial level. Flavor freshness, low energy utilization, and extended shelf-life are some of the virtues attributed to PEF treatment, but it has also been proven that the

156

II. Microbial Analysis

157

textural and functional attributes of some products can be preserved at safe microbiological levels (Dunn, 1995). Pulsed electric field-treated beaten eggs are just one example of the appeal this technology has, as egg products processed by traditional heat treatments exhibit operational problems due to protein coagulation in heat exchangers. However, because PEF enables microbial inactivation at lower temperatures, it represents an important alternative to the egg industry. Significant shelf-life extensions with minimum changes in the physical and chemical properties of certain other foods have also been demonstrated. Experimental results have shown that, in general, the sensory properties of foods are not degraded by PEF, and electric field-treated products such as green pea cream soup and fluid eggs were even preferred by panelists in sensory tests over at least one commercial brand product. As mentioned earlier, electric field treatment is also associated with energy efficiency. In the treatment of apple juice, energy utilized with the PEF technology is 90% less than the amount needed for the high-temperature short-time (HTST) processing method (Qin et al., 1996a). Furthermore, the development and utilization of low-energy, instant-charge-reversal pulses by one of the leading groups at Guelph University allow energy consumption of less than 7 J / m l for the PEF processing of products such as waste brine, orange juice, and apple cider (EPRI, 1998).

II.

Microbial Analysis

One of the important factors in the quality control of foods is microbial analysis because all foods may not have the same type of microbial contamination. Depending on raw materials and processing conditions, each food should be tested for different microbes. Standard procedures are specified for microbial analyses by food inspection and approval authorities. The concentration of these microbes should not exceed the standards set by the Food and Drug Administration (FDA) in order for the food to be approved for human consumption. Microbial analyses of apple juice consist of aerobic plate counts, detection of yeasts and molds, acidic bacteria, and Salmonella. Five prescribed routine tests chosen for milk are aerobic plate count, coliform count, Salmonella detection, the Moseley quality test, and the Listeria test. The microbiological analysis for beaten eggs should follow the methods recommended by the FDA Bacteriological Analytical Manual (AOAC, 1992), which includes an aerobic plate count at 35~ a coliform count at 35~ yeast and mold counts, and detection of Salmonella. For pea soup, aerobic plate, coliform, yeast, and mold counts are to be determined along with the detection of Streptococci, Salmonella, and Staphylococcus (Qin et al., 1996b).

158

III.

6. Food Processing by PEF

Chemical and Physical Analyses

Chemical analyses of processed foods include estimation of their protein, carbohydrate, fat, and ash contents. The protein content may be calculated by determining the nitrogen value of the food. Using an appropriate procedure for the Kjeldahl method, the nitrogen value is converted into a protein content. The fat and ash contents are determined using the standard procedures specified by the Association of Official Analytical Chemists (AOAC), who have determined that the carbohydrate content can be computed by subtracting 100 from the sum of the protein, fat, and ash contents. Physical analyses of foods include determination of pH, acidity, water activity, moisture content, color changes, and viscosity. The pH may be measured using a simple pH meter, and acidity may be measured by titrating the food sample against 0.1 N NaOH to a pH of 8.3 according to AOAC standards (AOAC, 1990). Water activity is determined using a water activity meter. The color of a food product may change after processing, therefore, this calculation is a bit more complicated. For example, eggs are evaluated in terms of a /~g /3-carotene/g sample extracted with acetone whose content is measured using a spectrophotometer. Viscosity can be simply calculated with a viscometer such as the standard Haake concentric cylinder model.

IV.

Sensory Evaluation and Shelf-Life Studies

Sensory evaluation is conducted to determine the relative acceptance of foods processed by electric field treatment as compared to foods available at the supermarket processed by other methods. A typical sensory evaluation study may include a triangle test and an affective test to quantify the acceptability of the food. Some of the major attributes considered in the sensory evaluation include color, clarity, texture, sweetness, sourness, bitterness, saltiness, astringent mouthfeel, and if the product tastes fresh, natural, cooked, rancid, or like sulfur. Like any other processed foods, PEF-treated products may undergo changes during processing and storage that may adversely influence their quality attributes. Furthermore, the modern competitive market and increasing consumer interest in food quality, date marking, and safety demand proper quality and shelf-life evaluations that must be grounded on sound scientific principles and supported by up-to-date techniques. If shelf-life is considered as the extent of time a product is considered suitable for consumption, the first question asked should be "what makes the product suitable for consumption?" The answer is not straightforward as it depends on the deterioration of one or more quality attributes. After a certain storage period these attributes can be lost due to chemical, physical, and biological

V. Quality and Shelf-Life Evaluation of PEF Products

159

changes that are caused by limiting factors. Proper identification and evaluation of such factors is thus the first step to shelf-life evaluation. When considering both safety and spoilage, it is important to remember that the attribute of interest to be maintained during storage varies from product to product. As mentioned earlier, PEF is considered to retain good quality attributes after processing, and in some cases, for longer periods of time than compared to other preservation methods. Examples of such desirable characteristics are retention of fresh flavors, vitamin content, fresh color, and good inactivation of microbial flora. Shelf-life determination of PEF products was initially based on the microbiological quality of foods due to safety reasons, but today there is more and more interest in comprehensive analysis of products in such a way that the stored foods are tested periodically for their microbiological quality and physical and chemical changes. Microbiological quality assessment includes determination of the total counts of aerobic and anaerobic bacteria, yeasts, molds, and detection of coliform, Salmonella, Staphylococcus, and Streptococcus spp. When the concentration of microorganisms exceeds specified standards set by regulatory agencies [in the United States, the FDA and Department of Agriculture (USDA)], the product is no longer considered safe for consumption. The maximum microbial load varies among microorganisms and from product to product: for example, pasteurized grade A milk can have standard plate counts of 20,000 colony forming units per milliliter (cfu/ml) and a coliform count with less than 10 cfu/ml, whereas the presence of pathogens such as Salmonella spp., E. coli O157:H7, or Clostridium botulinum in any marketed foods is illegal. It is for this reason that in some cases the period until when the concentration of microorganisms becomes unacceptable is considered the shelf-life of the product. The evaluation of sensory and physical changes may provide another parameter to estimate the shelf-life of certain products where there is no specific microbial count limit of nonpathogenic bacteria, as in the case of liquid egg products.

VO Quality and Shelf-Life Evaluation of PEF Products The next sections describe electric field processing conditions and systems used to treat foods with PEF. Continuous and batch processing systems were used to conduct these studies under specific conditions of electric field, treatment time, temperature, and flow rate for each product. Different attributes to assess the food quality were utilized depending on the product. The modern food industry has already learned of promising results for apple, grape, and orange juices, as well as those for milk, eggs, salad dressings, and pea soup products. Some of these PEF-treated foods present

160

6. Food Processing by PEF

an extended shelf-life of more than 8 weeks without refrigeration, which is one of the most attractive results of the technology so far. In addition, some groups have proposed the use of PEF in other areas of food processing such as the development of new products and the treatment of subproducts or waste material from the food industry.

A. Processingof Apple Juice Juices are one of the most important areas in beverage production, but as m e n t i o n e d earlier, one of the drawbacks has been the loss of fresh-like flavor. However, the nonthermal attributes of PEF allow treatments at low temperatures which do not require the harmful effects of hot filling, as is the case in traditional processing. Qin et al. (1995) reported some of the earliest studies on the effect of PEF pasteurization on the quality attributes and shelf-life of five different products (Table 6.1). They processed two commercially available juices (a reconstituted from concentrated apple juice and freshly squeezed apple juice) that had both been stored at 4-6~ beforehand. The concentrate was reconstituted with one part concentrated juice and six parts water at room temperature (22-25~ before processing and then subjected to 10 pulses with an electric field intensity of 50 k V / c m and pulse duration of 2 /xsec. The initial process temperature was 8.5 _ 1.5~ and the maximum temperature during the process increased to 45 _+ 5~ After treatment, the apple juice was aseptically filled into packages of 250 ml for shelf-life studies. The bags were opened and filled directly from the treatment chamber outlet while being flushed with purified nitrogen gas due to product sensitivity to oxygen. The commercially available fresh apple juice was allowed to sit for 24 hr at 4-6~ before processing to allow for sedimentation of particulates. Bulky dregs settled on the bottom of its container, and then the upper layer of

TABLE 6. I Pulsed Electric Field Processing Conditions for Selected Liquid Foods a

Food Peak electric field Pulse duration Pulse number (/xsec) Initial temperature (~ Maximum treatment temperature (~ Storage temperature (~ Shelf-life (days) a From Qin et al. (1995).

Apple juice from concentrate

Fresh apple juice

Raw skim milk

Beaten eggs

Green pea soup

50 2 10 8.15 _+ 1.5 45 _+ 5

50 2 16 8.15 _+ 1.5 45 _+ 5

40 2 20 1 0 _ 1.5 50_+4

35 2 10 8.5_+ 1.5 45_+5

35 2 32 22+_2 53_2

22-25 28

4-6 21

4-6 14

4-6 28

4-6 10

V. Quality and Shelf-Life Evaluation of PEF Products

161

clear juice was collected for PEF treatment. The processing required three steps of PEF exposure to prevent an increase in temperature beyond 45~ during treatment. The first step consisted of six pulses, and the two additional steps were five pulses; each utilized an electric field intensity of 50 kV/cm and pulse duration of 2 /zsec. The processed juice was then filled into bags directly from the outlet tubing of the treatment chamber. Simpson et al. (1996) evaluated the physical and chemical attributes of PEF-treated apple juice from concentrate stored at 4~ and found no physical or chemical changes in ascorbic acid or sugars (glucose, fructose, sucrose) (Table 6.2). However, the pH of treated and untreated control juices varied from 4.1 to 4.36, and the conductivity of untreated apple juice was slightly higher than the treated juice (1097/zS/cm vs 1300-/zS/cm). The difference in conductivites may be attributed to the greater mineral concentration (Ca, Mg, Na, and K) in the untreated juice (Table 6.3). The shelf-life of the treated apple juice from concentrate was as long as 4 weeks, whereas that for fresh-squeezed apple juice was extended by 3 weeks. A sensory panel found no significant differences between untreated and electric field-treated juice from concentrate or fresh-squeezed juices. Improvements to PEF techniques and expertise are even more evident when comparing these results with those obtained by Vega-Mercado et al. (1997), where apple juice from concentrate after PEF treatment was stored at room temperature (22-25~ for more than 8 weeks with no apparent change in its physicochemical and sensory properties. Likewise, fresh apple juice also remained unaffected after being processed with 16 pulses and stored for 32 days.

TABLE 6.2 pH, Titratable Acidity, Ascorbic Acid, and Mean Sugar Content in PEF-Treated Apple Juicea

Apple juice

pH

Titratable acidity (malic acid) (mg/100 g)

Control 40 kV/cm, 32 pulses 40 kV/cm, 16 pulses 20 kV/cm, 32 pulses 40 kV/cm, 16 pulses

4.10 _+ 0.02 4.36 • 0.03

2.63 _+ 0.02 2.67 _+ 0.02

1.15 _+ 0.01 1.02 _+ 0.02

2.91 _+ 0.33 2.87 _+ 0.06

4.95 _+ 0.64 2.18 _+ 0.25 4.96 _+ 0.11 2.25 +_ 0.06

4.09 _+ 0.01

2.63 +_ 0.02

1.02 _+ 0

3.01 _ 0.34

5.08 +_ 0.67 2.21 +_ 0.31

4.18 _ 0.01

2.75 _ 0.07

1.12 _+ 0

2.90 _+ 0.09

4.89 _+ 0.13 2.13 _+ 0.06

4.23_+0.01

2.61_+0

1.15 _+ 0.24

2.57 _+ 0.25

4.33 _+ 0.47 2.43 _+ 0.13

a Adapted from Simpson et al. (1996).

Ascorbic acid (mg/100 g)

Glucose (mg/100 g)

Fructose Sucrose (mg/100 g) (mg/100 g)

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6. Food Processing by PEF

TABLE 6.3 Total Solids and Selected Mineral Composition of PEF-Treated Apple Juice (mg / 100 g)l, 2 Apple juice

% solids

Ca

Mg

Na

Control

11.2 _+ 0.5 11.2 _+ 0.1

18.5 _+ 1.0 d 11.6 _+ 1.0 a

13.8 + 0.4 g 9.9 q- 1.0 b

18.2 q- 1.1 j 10.6 +_ 1.7 c

9.0 _+ 1.4 e

6.6 + 0.7 h

8.3 _+ 1.6 k

84.5 _+ 1.3 ~

4.4 -F 0.2 r

11.32 _+ 0.1

14.1 _+ 0.9 f

10.4 ~ 0.5 b

13.6 _+ 1.11

115.1 q- 1.9 p

4.6 -F 0.1 r

11.45 _+ 0.1

11.0 -F 0.9 a

8.2 _+ 01.2 i 10.1 _+ 1.7 c

90.7 -k- 0.9 q

4.5 -F 0.1 r

40 kV/cm, 32 pulses 40 kV/cm, 16 pulses 20 kV/cm,

11.4 _+ 0

K

C1

157.9 q- 1.0 m 4.4 _+ 0.6 r 100.5 _+ 2.3 n 4.7 -F 0.3 r

32 pulses

40 kV/cm, 16 pulses

1 A d a p t e d f r o m S i m p s o n et al. (1996). D a t a p r e s e n t e d are m e a n values o f two e x p e r i m e n t s e a c h c a r r i e d o u t in d u p l i c a t e . V a l u e s with t h e s a m e letters were n o t f o u n d to be significantly d i f f e r e n t (p < 0.05).

B. Processingof Orange Juice The main disadvantages of traditional orange juice heat treatments either by HTST or ultrahigh temperatures ( U H T ) a r e the loss of vitamin C, changes in color, and destruction of fresh flavor. Dunn and Pearlman (1987) conducted a study of PEF effects on commercial freshly squeezed high pulp orange juice with a limited shelf-life by subjecting it to 35 exponentially decaying pulses of electric fields ranging between 33.6 and 35.7 kV/cm. Its native microbiological population consisted of a mixture of yeasts, molds, and bacteria, but after PEF treatment a 5 log inactivation was obtained and its shelf-life was extended by more than a week. The electric field-treated orange juice was also acceptable in terms of taste and odor after 10 days, whereas the untreated juice was unacceptable after only 4 days. Zhang et al. (1997) evaluated the shelf-life of orange juice treated with an integrated PEF pilot plant system. Single strength (ll.8~ pulp free orange juice reconstituted from frozen concentrate was processed in a PEF system with a series of cofield chambers at a flow rate of 75-85 liter/hr; to maintain treatment temperatures near ambient (22 to 25~ cooling devices were used in between the chambers. Three different waveshape pulses were used to compare the effectiveness of the processing conditions: (a) square waves with a peak electric field of 35 kV/cm, an effective pulse width of 37.22 /zsec, and a pulse rise time of 60 nsec; (b) exponentially decaying waves with a peak electric field of 62.5 k V / c m , an effective pulse width of 0.57 ~sec, and a pulse rise time of 40 nsec; and (c) charge-reversal waves with a peak electric field of 37 kV/cm, an effective pulse width of 0.96 ~sec, and a pulse rise time of 400 nsec. After treatment the juice was aseptically packaged (200-ml plastic cups) and stored u n d e r refrigeration at 4, 22, and 37~ for microbial evaluations of total aerobic plate and fungi counts, as well as vitamin C and color retention levels. The PEF processing assessment was

V. Quality and Shelf-Life Evaluation of PEF Products

163

TABLE 6.4 Accelerated Shelf-Life Study Data ~

Waveform

Square-wave

Ep (kV/cm)

~'e (/zsec) Rise time (nsec) Shelf-life at 37~ Shelf-life at 22~

35 0.93 60 8 days 34 days

Exponential decay

Chargereversal

62.5 0.57 40 4 days 7 days

37 0.96 400 5 days 26 days

a From Zhang et al. (1997).

achieved by c o m p a r i n g these attributes to a control sample of orange juice pasteurized in a conventional HTST plate heat e x c h a n g e r with a m i n i m u m shelf-life of 5 m o n t h s at a storage t e m p e r a t u r e of 4~ Not surprisingly, the square-wave pulses were f o u n d to be most effective, yielding products with longer shelf-lives than those products treated with exponentially decaying and charge reversal pulses (Table 6.4). The follow-up analysis of vitamin C loss was higher in juices that were heat treated c o m p a r e d with those that were PEF treated. The authors evaluated the kinetics of degradation and c o n c l u d e d that it followed a pseudo first-order reaction as described in Eq. (6.1). F u r t h e r m o r e , they evaluated the relation between the reaction rate constant k in Eq. (6.1) and activation energy using Arrhenius' equation (6.2). Values for the kinetic constant at three different temperatures are presented in Table 6.5, where the activation energy (E a) that defines Eq. (6.2) was f o u n d to be 28.6 k J / m o l with a Qa0 equal to 1.3: C=

Coexp(-k*t

(6.1)

)

k = A exp(-Ea//RT),

(6.2)

TABLE 6.5 Reaction Rate Constant, D a y - i Values of Vitamin C Degradation in Heat, and PEF-Processed Orange Juice at Different Storage Temperatures ~

Treatment

4~

22~

37~

Heat processed R2 PEF processed

0.0058 0.81 0.0032 0.86

0.008 0.89 0.0042 0.95

0.011 0.81 0.126 0.89

R2

a From Zhang et al. (1997).

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6. Food Processing by PEF

TABLE 6.6 Comparison of Orange Juice Pasteurized by PEF and Heat ~

Relative % content lost after treatment Processing type

d-Limonene

Ethyl butyrate

Control, fresh squeezed PEF (35 kV/cm, 240/~sec) PEF (35 kV/cm, 240/zsec) Heat pasteurization (91~ 30 sec)

0 12 16.7 44.9

0 0 1.2 21.5

a From Zhang (1997).

where C is the vitamin C concentration at time t, C o is the initial vitamin C concentration at t = 0, k is the reaction rate constant (1/day), A is the frequency factor, E a is the activation energy ( k J / m o l ~ R is the regression coefficient and T is the absolute temperature (~ Another part of this comprehensive study by Zhang et al. (1997) was a color evaluation of the orange juice, which revealed a better preservation for the PEF-treated product compared to the heat-treated samples during the initial storage period. The authors declare the color change to be insignificant between the two processes during subsequent periods and explain the increase in the a* values during storage to be due to the degradation of ascorbic acid to furfural, which is a browning product. In another study conducted by Zhang (1997), a much better flavor was found in orange juice processed by PEF compared to heat-treated juice when the flavor evaluation was based on the relative content and loss percentage of key components such as d-limonene and ethyl butyrate (Table 6.6).

C.

Processingof Milk

D u n n and Pearlman (1987) worked with homogenized and pasteurized milk to combine a challenge test and shelf-life study where milk samples were inoculated with Salmonella dublin and treated with 40 pulses of 36.7 kV over a 25-min time period. Pathogenic bacteria were not detected after treatment, nor even after the milk was chilled and stored at 7-9~ for 8 days. The milk bacteria population increased to 107 per ml in the untreated milk, whereas treated milk showed approximately 400 per ml (Table 6.7). Further study by D u n n (1995) indicated less flavor degradation and no chemical or physical changes in enzymatic activity, fat or protein integrity, starter growth r e n n e t clotting yield, cheese production, calcium distribution, or casein structure. The author suggests that these attributes should be considered to promote the manufacture of dairy products such as cheeses, butter, and ice cream with fresher flavors.

V. Quality and Shelf-Life Evaluation of PEF Products TABLE 6.7 Pasteurized Milk Inoculated with

Time after treatment

Salmonella(Peak Voltage

40 kV) ~

Untreated(counts/ml)

Treated(counts/ml)

3.8 S b

20 Bc OS 6B 100 B 100 B 400 B

0 24 72 144 192

165

4.6 S + B 1.2 X 106 S + B 2.7 X 107 B 107 B

a From Dunn and Pearlman (1987). b S. dublin.

c Milk bacteria.

Raw milk was preprocessed by Qin et al. (1995) to remove fat prior to PEF treatment. The milk had a 2% fat content and was stored at 4~ before and after the processing, which required three steps of PEF exposure to prevent an increase in temperature beyond 55~ during treatment. The milk was subjected to two steps of seven pulses each and one step of six pulses with an electric field of 40 k V / c m . PEF-processed milk was then aseptically filled in packaging bags and was found to have a shelf-life of 2 weeks. Its physical and chemical properties were not influenced by the electric fields. Further sensory evaluation studies conducted with PEF on heat-pasteurized milk showed significant differences between heat-pasteurized and PEFtreated, prepasteurized milk, and the shelf-life was extended to more than 3 weeks.

D.

Processingof Eggs

Some of the earliest studies in egg products were conducted by Dunn and Pearlman (1987) in a static parallel electrode treatment c h a m b e r with a 2-cm gap using 25 exponentially decaying pulses with a peak voltages of a r o u n d 36 kV. Tests were carried out on liquid eggs from which a portion of the yolk was removed, heat-pasteurized liquid egg products, and egg products with potassium sorbate and citric acid added as preservatives. Eight products were eventually c o m p a r e d to evaluate the effectiveness of PEF and the hurdle approach (PEF in combination with pasteurization and preservatives) at shelf-life extension. Comparisons were made with regular heat-pasteurized egg products with and without the addition of food preservatives when the eggs were stored at low (4~ and high (10~ refrigeration temperatures; the effect of processing temperature (50 and 60~ was also evaluated. This study revealed the important effect of the hurdle approach in shelf-life extension, whose effectiveness was even more evident during storage at low temperatures where an egg p r o d u c t with a final count a r o u n d 2.7 log c f u / m l stored

166

6. Food Processing by PEF

at 10~ maintained a low count for 4 days, but when stored at 4~ the initial count was kept for up to 10 days. This is especially significant since the control samples were unable to hold their microbial limits for more than a few hours. Fresh eggs purchased by Qin et al. (1995) from a local supermarket were washed with alcohol and sterilized water, cracked in a clean environment, and then mixed using a sterile beater. Citric acid in the a m o u n t of 0.15% (w/v) was added to the beaten egg mixture. To maintain the temperature of the eggs during PEF treatment at a level such that no coagulation occurred (PEF at or below 50~ the process was performed in three steps of exposure. Each step consisted of four pulses at an electric field of 35 k V / c m , with the treatment chamber cleaned within 10 min of each step. The treated liquid eggs were then aseptically filled into packaging bags for shelf-life study. Ma et al. (1997) compared the effect of PEF, high hydrostatic pressure (HHP), and thermal processing on the sensory, physical, chemical, and microbiological attributes of liquid whole eggs (LWE) after PEF treatment in a coaxial continuous chamber with 20 pulses of 48 k V / c m and 2 /zsec in five steps. After treatment the products were bagged and refrigerated to allow a microbial shelf-life study. The results led to the conclusion that PEF will be suitable for industrial implementation due to the better quality attributes of the LWE treated by this m e t h o d compared to the others that were studied. In experiments conducted by Qin et al. (1995) and Ma et al. (1997), beaten eggs pasteurized with electric fields were found to have a shelf-life of 4 weeks. Their microbial counts were evaluated at 1-week intervals throughout the 4 weeks of storage, which included total plate counts in plate count agar (PCA), yeast and mold counts in acidified potato dextrose agar (APDA), and coliform counts in violet red bile agar (VRB). In addition, the products were verified to be free of Salmonella by following the procedures specified in the FDA Bacteriological Analytical Manual (AOAC, 1992). The viscosity and color of the fresh whole eggs were selected as physical attributes that should be maintained after PEF treatment in order to keep a high-quality product. Viscosity was evaluated by means of a Brookfield viscometer and color by the whiteness calculated from the pictures taken by a Minolta color spectrophotometer. The read values from the Minolta were lighmess (L*) from 0 to 100 on a scale from black to white; redness-greenness (a*); and yellowness-blueness (b*). Results showed that PEF treatment decreased the viscosity but increased the color (in terms of the/3-carotene concentration of the liquid eggs when compared to fresh eggs. After a sensory panel evaluation with a triangle test Qin et al. (1995) found no differences between scrambled eggs prepared from fresh eggs and electric field-treated eggs, the latter of which were preferred over a commercial brand (frozen Egg Beaters, Nabisco Foods Inc., East Hanover, NJ). In addition to microbial and color analyses of egg products, Ma et al. (1997) evaluated the density of fresh and PEF-treated LWE, since it is an indicator of egg protein foaming ability (the higher the density the lower the

V. Quality and Shelf-Life Evaluation of PEF Products

167

foaming ability). Because an egg product's functional properties in baking are of major importance, the texture of sponge cakes made with raw and PEF-processed eggs was evaluated by measuring cake strength as defined by the force times the penetration distance through the cake of a probe attached to a TA.XT2 texture analyzer. Furthermore, a sensory test was performed using a triangle difference test to look for h u m a n detectable differences in texture and flavor due to PEF processing. The evaluation of physical attributes indicated that the stepwise process used by Ma et al. (1997) did not cause differences in density or whiteness between the PEFtreated and control LWE in a paired comparison. The strength of the sponge cakes prepared with PEF-treated eggs was found to be greater than the one made with nonprocessed eggs, which was attributed to the lower volume obtained, after baking. The statistical analysis of the sensory evaluations revealed no differences between cakes prepared from processed and control LWE.

E. Processingof Green Pea Soup Processing green pea soup is challenging because of the presence of insoluble particles suspended in a continuous phase constituted of pregelatinized starch, pea powder, and sugar, as these ingredients may interfere with the microbial inactivation. Vega-Mercado et al. (1996) prepared green pea soup with split pea powder, pregelatinized starch, regular corn starch, hickory smoke flavor, 168 g granulated sugar, monosodium glutamate, and distilled water. The ingredients were mixed in boiling water, and after the emulsification of the ingredients the soup was allowed to cool to room temperature and then subjected to electric field processing that required two steps of PEF exposure to prevent an increase in temperature beyond 55~ during treatment. Each step consisted of 16 pulses at an electric field of 35 kV/c, with the treatment chamber cleaned in the 30-min interim between each step. Vega-Mercado et al. (1996) carried out microbial and chemical analyses as well as shelf-life studies on green pea soup that had been stored after PEF treatment at refrigeration temperature, room temperature (22-24~ and high temperature (32-34~ The shelf-life of the treated pea soup stored at refrigeration temperature was more than 4 weeks, but both 22 and 32~ were found inappropriate for storage of the product. There were no apparent changes in the physical and chemical properties of the pea soup directly after PEF processing nor during the 4 weeks of storage at refrigeration temperatures, and a sensory panel found no differences between heatpasteurized and PEF-treated soup. A commercial brand of pea soup (Cup-aSoup, Lipton Inc., Englewood, NJ) received the lowest score in the acceptability test.

168

6. Food Processing by PEF

TABLE 6.8 Microbiology of Waste Brine ~

Microorganism Total plate count with 10% NaC1 Total plate count Staphylococcus/Micrococcusspp. Pseudomonas spp. Lactic acid bacteria

Enterobacteriaceae

Incubation temperature (~

Incubation Colonies time (hr) Mediab Aerobic (cfu/ml)

25 25 37 24 30 35

48 48 36 24 48 24

10 PCA PCA MSA CFC MRS VRBG

+ + + + +

1.04 X 10s 9.2 X 102 7.6 X 102 5.0 X 10 2.8 X 10 NDc

a From Mittal and Choudhury (1997). bCFU, colony-forming units; PCA, plate count agar; MSA, mannitol salt agar; CFC, cefelolrodin fucidin cetrimide agar; MRS, deman rogosa sharp agar; VRBG, violet red bile glucose agar. c Not detected.

Processing of Brine Solutions and Water in Cooling Systems Pulse electric field t r e a t m e n t attributes have f o u n d applicability n o t only in the possible pasteurization of food products for h u m a n c o n s u m p t i o n , b u t also in the t r e a t m e n t of subproducts g e n e r a t e d during food processing. T h e food industry is known for the p r o d u c t i o n of large a m o u n t s of waste with organic c o m p o u n d s susceptible to d e g r a d a t i o n by microbial flora, which may r e p r e s e n t a significant biological c o n t a m i n a t i o n hazardous to h u m a n health. Mittal a n d C h o u d h u r y (1997) evaluated the effect of PEF on the inactivation of native flora f o u n d in waste brine solution from bacon curing by following specific p r o c e d u r e s to d e t e r m i n e the type a n d n u m b e r of microorganisms p r e s e n t in the brine. T h e identified microorganisms were Staphylococcus, LactobaciUus, Pseudomonas, a n d Enterobacter spp. Table 6.8 presents the microbial analysis of the brine waste, where the naturally occurring flora in the waste was treated in a parallel static c h a m b e r using 10 t o 50 instant reversal pulses with 40 and 50 k V / c m . After PEF treatment, a total plate c o u n t a n d a Staphylococcus plate c o u n t were p e r f o r m e d (Table 6.9). Based on the experim e n t a l results, the authors suggested 50 k V / c m a n d the 10 to 20 pulses as o p t i m u m t r e a t m e n t conditions. To assure the total lethality of these conditions a n d irreversible inactivation of the bacteria, a microbial analysis was c o n d u c t e d that verified that after 14 days of storage no survivors were found. O t h e r appealing applications of PEF t r e a t m e n t are in the prevention of pipe a n d cooling system biofouling, tap water debacterialization, a n d medical operations as suggested by the results obtained by S c h o e n b a c h et al. (1997). In a biofouling study the authors used a Blumlein pulse f o r m i n g network (PFN) that provided a 6.45 k V / c m in the 2-cm gap of a r e c t a n g u l a r t r e a t m e n t cell with titanium electrodes a n d a pulse frequency of 12 Hz. T h e load consisted of water f r o m the Elizabeth River in Norfolk flowing at

VI. Final Remarks

169

TABLE 6.9 Mean Fraction ( N / N 0) of Total Plate Count and

Number of pulses 10 15 20 25

Staphylococcusspp.

Total plate count

Staphylococcus spp.

Level of electric field (kV/cm) 40 50 N/N 0 SD N/N 0 SD

Level of electric field (kV/cm) 40 50 N/N 0 SD N/N 0 SD

0.32 __+0.01 0.23 • 0.01 0.24 __ 0.01 0.18 __+0.01

0.06 + 0.08 0.00 __+0.00 0.00 __+0.00 0.00 __+0.00

0.200 __+0.009 0.179 __ 0.006 0.133 __+0.008 0.036 • 0.006

0.000 __+0.000 0.000 + 0.000 0.000 __+0.000 0.000 __+0.000

a From Mittal and Choudhury (1997).

4 g a l / m i n with a resistivity of a p p r o x i m a t e l y 50 1). [A m i s m a t c h e d load (9 1~ vs 7 1)) was used to p r e v e n t a voltage reversal in case of a salt c o n t e n t (conductivity) increase.] A l t h o u g h the a u t h o r s f o u n d the efficiency of the t r e a t m e n t low, blue mussels, barnacles, a n d h y d r a z o n a biofouling in the pipes were a p p a r e n t l y e l i m i n a t e d as a result. T h e y h i g h l i g h t e d the advantages o f PEF over o t h e r t e c h n i q u e s used for b i o f o u l i n g p r e v e n t i o n to be 9 i n d e p e n d e n c e f r o m chemicals 9 possibility to stun r a t h e r than kill, allowing the preservation o f valuable species 9 n o g e n e r a t i o n of shock waves that could affect the structure of the cooling system 9 possible installation of a filter in f r o n t of an existing cooling system In debacterializing the tap water, S c h o e n b a c h et al. (1997) f o u n d that only 1.5 J / c m 3 was n e e d e d to treat this m e d i u m , which has a resistivity of 1.8 kf~. T h e e n e r g y r e q u i r e d was equivalent to 1 k W / h r for the debacterialization of 2400 liters o f tap water, which w o u l d be a highly efficient processing system.

VI.

Final Remarks

As e v i d e n c e d by this a n d previous chapters, the application of PEF has b e e n successful in processing selected fluid foods w h e r e b y f o o d t e m p e r a t u r e s d u r i n g t r e a t m e n t are h e l d below traditional t h e r m a l processing t e m p e r a tures. However, the application of PEF processing is limited to food p r o d u c t s that can withstand high electric fields. H o m o g e n e o u s fluids have p r o v e n to provide ideal conditions for c o n t i n u o u s t r e a t m e n t with PEF. N o n f l u i d foods a n d foods c o n t a i n i n g particulates can also be processed, p r o v i d e d that dielectric b r e a k d o w n is p r e v e n t e d by m e d i a pressurizing a n d degassing a n d the m a i n t e n a n c e of a gap b e t w e e n the electrodes is larger than the particle size.

170

6. Food Processing by PEF

It should now be easier for the reader to see that for each food product, PEF processing design includes four stages: (1) microbial challenge tests to establish the amount of treatment needed; (2) treatment of the native microbial population in the food; (3) microbial, chemical, and physical analyses of the food after processing; and (4) sensory and shelf-life studies of the processed food product. The results presented in this chapter should provide a starting point for more comprehensive research on other products, as well as encourage the further development of technology toward full-scale implementation. To be widely applicable in the food industry, PEF processing still requires the execution of approved manufacturing practices and procedures to produce safe foods. The next chapter discusses these aspects of PEF technology.

References AOAC (1990). "Official Methods of Analysis," 15th Ed. Association of Official Analytical Chemists, Washington D. C. AOAC (1992). "FDA Bacteriological Analytical Manual," 7th Ed. Association of Official Analytical Chemists, Washington D. C. Dunn, J. E. (1995). Pulsed light and pulsed electric field for foods and eggs. Poultry Sci. 75, 1133-1136. Dunn, J. E., and Pearlman, J. S. (1987). Methods and apparatus for extending the shelf life of fluid food products. U. S. Patent 4,695,472. EPRI (1998). Pulsed electric field processing in the food industry: A status report on PEF. Report CR-109742. Industrial and Agricultural Technologies and Services, Palo Alto, California. Ma, L., Chang, F.J., and Barbosa-Cfinovas, G. V. (1997). Inactivation of E. coli in liquid whole eggs using pulsed electric fields technology. New frontiers in food engineering. In "Proceedings of the Fifth Conference of Food Engineering" (G. V. Barbosa-Cfinovas, S. Lombardo, G. Narsimhan, and M. Okos, eds.), pp. 216-221. American Institute of Chemical Engineers, New York. Mittal, G. S., and Choudhury, M. (1997). Pulsed electric field sterilization of waste brine solution. In "Proceedings ICEF 7," (R. Jowitt, ed.) pp. O13-O16. Academic Press, Midsomer Norton, Bath, UK. Qin, B. L., Pothakamury, U. R., Vega-Mercado, H., Martln-Belloso, O. M., Barbosa-Cfmovas, G. V., and Swanson, B. G. (1995). Food pasteurization using high-intensity pulsed electric fields. Food Technol. 12, 55-60. Qin, B. L., Chang, F. J., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1996a). Nonthermal inactivation of Saccharomyces cerevisiae in apple juice using pulsed electric fields. Lebensm. -Wiss. Technol. 28, 564-568. Qin, B. L., Pothakamury, U. R., Barbosa-Cfmovas, G. V., and Swanson, B. G. (1996b). Nonthermal pasteurization of liquid foods using high intensity pulsed electric fields. Crit. Rev. Food Sci. Nutr. 36(6), 603-627. Schoenbach, K. H., Peterkin, F. E., Alden III, R. W., and Beebe, S.J. (1997). The effect of pulsed electric fields on biological cells: Experiments and applications. IEEE Trans. Plasma Sci. 25(2), 284-292. Simpson, M. V., Qin, B. L., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1996). Pulsed electric field processing and the chemical composition of apple juice. Internal Research Report, Washington State University, Pullman, Washington.

References

171

Vega-Mercado, H., Martln-Belloso, O., Chang, F.J., Barbosa-C{movas, G. V., and Swanson, B. G. (1996). Inactivation of Esherichia coli and Bacillus subtilis suspended in pea soup using pulsed electric fields. J. Food Proc. Pres. 20, 501-510. Vega-Mercado, H., Qin, B. L., Belloso, O. M., Chang, F.J., Ma, L., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1997). Nontheral food preservation by pulsed electric fields, In "Proceedings of ICEF 7," (R. Jowitt, ed.), pp. C81-C84. Academic Press, Midsomer Nofton, Bath, UK. Zhang, Q. H. (1997). Integrated pasteurization and aseptic packaging using high-voltage pulsed electric fields. In "Proceedings of ICEF 7," (R. Jowitt, ed.) pp. K3-K15. Academic Press, Midsomer Norton, Bath, UK. Zhang, Q. H., Qiu, x., and Sharma, S. K. (1997). Recent developments in pulsed electric field processing. In "New Technologies Yearbook," pp. 31-42. National Food Processors Association, Washington, D. C.

CHAPTER 7

Hazard Analysis and Critical Control Point (HACCP) in PEF Processing

I.

Introduction

Although the hazard analysis and critical control point (HACCP) system is based on the principle that food safety issues can be eliminated or minimized by prevention during production rather than detection in the finished product, the concept of hazard and operability (HAZOP) is used to identify hazardous working conditions in a specific step of a processing system that jeopardizes the safety of employees. Both concepts may be used in the design construction and troubleshooting of a PEF processing facility. The systems are also adopted to ensure food safety through development, implementation, and effective management of a hazard-controlled process. It is important to note, however, that successful implementation of HACCP and HAZOP begins with executive management commitment, because without this it is just another program with little or no importance in daily operations (Vail, 1994). The design of a PEF facility based on the HACCP and HAZOP concepts is a preliminary step toward approval by regulatory agencies such as the U. S. Food and Drug Administration (FDA) and the U. S. Department of Agriculture (USDA). The HACCP concept was used by Vega-Mercado et al. (1996) to define the following points in a PEF facility: (a) the raw material receiving area, (b) treatment chamber, and (c) packaging line. Based on the information presented in the previous chapters, it is now possible to identify the fundamental parameters and conditions that must be considered in the design of the PEF process. The challenge of the 90s has been to provide better assurance that the foods consumers eat are safe and wholesome, while maintaining a reasonable 172

II.

Term

Definitions in HACCP Systems

173

price. The concept of HACCP emerged as a result of the efforts of the Pillsbury Company to manufacture safe food products for the National Aeronautics and Space Administration (NASA) who wanted a "zero defects" program to guarantee safety in the foods astronauts consume in space. The HACCP system guarantees food safety while reducing or eliminating the need for sample testing the finished product by eliminating microbiological, chemical, and physical hazards through anticipation and prevention rather than inspection. To establish and design an effective HACCP program, the following requirements must be fulfilled (National Food Processors Association, 1992): * 9 9 9

management, leadership, and commitment expert knowledge in the design of an HACCP program employee training and operator control effective verification program

The National Advisory Committee on Microbiological Criteria for Foods (NACMCF) developed seven HACCP principles for food safety. 1. Assess hazards and risks associated with growing and harvesting raw materials and ingredients, processing, manufacturing, distribution, marketing, preparation, and food consumption. 2. Determine critical control points (CCP) required to control identified hazards. 3. Establish critical limits that must be met at each identified CCP. 4. Establish procedures to monitor CCP. 5. Establish corrective action to be taken when there is deviation from the critical limits. 6. Establish effective record-keeping systems that document the HACCP plan. 7. Establish procedures for verification that the HACCP system is working correctly. Following the first principle is critical to applying the others. Before beginning the hazard analysis, an HACCP team consisting of individuals who have specific knowledge and expertise appropriate to the product and process must be formed. It is the responsibility of this team to describe the food and method of its distribution, identify the intended use and consumers of the food, develop a flow diagram describing the process, and verify the flow diagram with the actual processes occurring in the facility.

II.

Term

D e f i n i t i o n s in H A C C P

Systems

The language used in HACCP is very specific, so it is important to differentiate between words that can give a different context or meaning. The following is a list of the most common definitions used in this area [National

174

7. HACCP in PEF Processing

Advisory Committee on Microbiological Criteria for Foods (NACMCF), 1992]:

Hazard: Any microbiological, chemical, or foreign material that if consumed could cause harm to consumers. Risk: An estimate of the likely occurrence of a hazard. Process hazard analysis: The methodology and procedures of detailed examination and evaluation of all processes from the field to the final consumer for the purpose of identifying the location and severity of food safety hazards. Control point (CP): A position in the food processing or handling system where inadequate control would result in food contamination, but managem e n t programs, procedures, and practices function to prevent such adulterated food from reaching the consumer. Critical control point: A position in the food processing or handling system where inadequate control would result in food contamination, but no m a n a g e m e n t programs, procedures, or practices exist to prevent the adulterated food from reaching the consumer. Critical control point specification: Acceptable performance criteria at any CCP. Critical control point deviation: Failure to meet specification acceptance criteria at any CCP. Corrective action: The procedure to be followed when a deviation occurs. Deviation response: An action taken to maximize the control of products that do not meet specification acceptance criteria at any CCP. HACCPplan: The written d o c u m e n t based on the principles of HACCP that delineates the procedures to be followed to assure the control of a specific process or procedure. Verification: The methodology used to assess and evaluate the effectiveness of the hazard control plan. Validation: The methodology used to assess the effectiveness of the entire HACCP process referred to as procedures.

III.

The

HACCP

System

The first step in the development of an HACCP system is to put together a team consisting of a group of individuals representing each operating d e p a r t m e n t charged with the responsibility of developing and implementing the HACCP system. Because HACCP is an analytical process involving all departments at every employee level, it is important to have these areas represented during all phases of development. Each section or area of a processing facility will have particular situations and conditions that are best understood and managed by those working in that area. The next step in the development of the HACCP system is hazard analysis, which begins with a current flow chart of the production process. It

IV. The HACCP System in PEF Processing

175

is advisable to include the source of ingredients, distribution, and marketing of the product in the flow chart. The next step is to evaluate each operation for situations that, if not properly managed, could result in contaminated products. After determining the location of the potential hazards, the next step is to decide where the CCPs exist, establish a safe operating standard, and agree on monitoring procedures, frequencies, and responsibilities for checking the CCPs. Clear deviation responses are also n e e d e d when monitoring indicates that the standard is not being m e t and unsafe processing conditions exist. W h e n CCP deviations occur it is i m p o r t a n t to take immediate corrective actions. Many HACCP programs fail because CCP examination results are not adequately evaluated and appropriate actions are not taken when deviations occur. In addition, it is important to eliminate or drastically minimize the potential for recurrence. Documentation of CCP determination and corrective actions taken is also vital.

IV.

The HACCP

S y s t e m in PEF Processing

A typical PEF process is represented by the flow chart in Fig. 7.1. The primary operations in the process include receiving the raw materials, PEF treatment, aseptic packaging of the product, storage, and distribution of the product. Based on the seven principles set by the NACMCF, the first step in developing an HACCP system for the PEF process is assessment of the hazards involved. A.

Hazard Assessment

Hazard assessment consists of a systematic evaluation of a specific food and its raw materials or ingredients to determine the risk from biological,

I

Storage Area

I

I C~

~

Pu('ser I

I

.J Treatment I

Reservoir

'

.J CoolingArea .,Product

~

Packaging

Pump FinishedProduct Storage Area

Distribution Fig. 7. I A flow chart representing the PEF processing of foods.

[~"

176

7. HACCP in PEF Processing

chemical, or physical hazards. Microbial hazards are the main concern throughout the PEF operation, and these may be identified in (a) raw materials containing pathogens and spoilage microbes, which under poor storage conditions will be able to reproduce and increase the risk of microbial contamination; (b) the processing equipment cleanliness that may prevent cross-contamination problems; and (c) the packaging system, which should be properly sterilized and leakage free to avoid any postcontamination. Chemical and physical hazards must also be taken into account to guarantee no foreign matter in a treated food. The hazard analysis is a two-step procedure involving an analysis of the hazard and assignment of risk categories. The NACMCF defines six hazard characteristics (Corlett, 1990): Hazard A: Nonsterile products designated and intended for consumption by at-risk populations (i.e., infants, the aged, the infirm, or immunocompromised individuals). Hazard B" Products containing sensitive ingredients in terms of microbiological hazards. A "sensitive ingredient" is defined as any ingredient historically associated with a known microbiological hazard. Some of these are meat, poultry, eggs, milk, cheese, dairy products, fish, spices, nuts, and chocolate. Foods that may be occasionally contaminated with hazardous microorganisms but are not considered sensitive include salt, sugar, chemical preservatives, food gums, and synthetic colors (Corlett, 1990). Hazard C: Processes that do not contain a controlled step that effectively prevents, destroys, or removes toxic chemical or physical hazards. Examples include steps for preventing the formation of toxic or carcinogenic substances during processing; destruction of cyanide-containing compounds; and removal of toxic processing chemicals such as lye or dangerous foreign objects such as sharp pieces of metal. Hazard D: Products subject to recontamination after manufacturing before packaging (i.e., when a manufactured product is bulk-packed, shipped, and packaged in another facility). Hazard E: Situations in which there is substantial potential for chemical or physical contamination in distribution or consumer handling that could render the product harmful when consumed. Examples are contamination of a food from container or vehicle compartments that previously contained toxic chemicals or selling food in open containers. Hazard F: Situations in which there is no way for the consumer to detect, remove, or destroy a toxic chemical or dangerous physical agent. Examples are the presence of toxic mushrooms, paralytic shellfish toxins, or sharp metal objects buried in a food.

The second step is to assign risk categories (0 through VI, in ascending order) to the food. In PEF processing, physical hazards include foreign matter in raw materials such as soil, stones, rubber or plastic pieces, and shells in the case of eggs. When an arc is generated during the treatment of a food, there is a possibility of electrode damage, and the resulting particles

IV. The HACCP System in PEF Processing

177

may pose a physical hazard. Any debris from a damaged plastic or rubber seal can also be considered a hazard. One of the chemical hazards involved in PEF processing is from the antibiotic and pesticide residues remaining in raw materials. Electrochemical reactions in a food may result in the formation of toxic compounds, so thorough cleaning of the processing equipment is strongly r e c o m m e n d e d for the production of safe foods. However, excessive residues of the detergent or sanitizer used in cleaning may also pose a chemical hazard. Microbiological hazards are of primary concern throughout PEF processing. Spoilage microbes a n d / o r pathogens present in raw materials are two examples. Furthermore, the presence of soil, water, and airborne microorganisms in the storage area may increase the risk of microbial contamination in raw materials. Inadequate cleaning of the processing equipment may cause contamination of the food being processed, especially when the same equipment is used for different types of foods. Cross-contamination of processed food during packaging and improper handling of the processed food during storage are additional causes of microbial contamination. B.

Critical Control Points

It is essential that hazard analysis and risk assessment always be conducted correctly before attempting to apply the other HACCP principles. Failure to conduct a risk assessment may lead to omission of CCP and result in serious gaps in a food safety assurance program. The second step in the development of an HACCP system involves identifying the CCP, setting limits for the CCPs, describing monitoring procedures, and determining corrective actions when the CCPs deviate from the set limits. Quality assurance procedures must be developed for the approval or rejection of PEF-treated products based on the CCP limits and corrective actions imposed by each processor. Table 7.1 suggests some CCPs and actions to take. The information gathered during hazard analysis enables the HACCP team to identify which steps in the process are CCPs; a decision tree similar to the one presented in Fig. 7.2 may be used for this purpose. However, it should be noted that different facilities preparing the same food can differ in the risk of hazards and CCPs due to variations in layout, equipment, selection of ingredients, or processes employed (NACMCF, 1992). In a PEF processing facility, CCPs are the receiving, storage, treatment, and packaging steps. The main factors to be considered and monitored for each CCP are handling of the raw material and finished product, processing conditions, and cleanliness of the equipment and containers or utensils used during processing. Processing conditions consist of electric field intensity, pulsing rate, treatment time, input voltage and current, and temperature of the treatment chamber, all of which should be monitored and recorded on a continuous basis (Vega-Mercado et al., 1996). Furthermore, a uniform PEF treatment requires the design and construction of a pulser, which as was

178

7. H A C C P in PEF Processing

TABLE 7. I Critical Control Point Deviations and Possible Corrective Actions in a PEF Operationa

Critical control point Raw material Receiving and storage

PEF processing unit

Finished Product Packaging/storage

Control parameter

Deviation

Spoilage/particulate Storage time Storage temperature

Detected Exceeded Above specifications

Electric field intensity

Below specifications

Pulsing rate

Below specifications

Flow rate

Above specifications

Arcs or sparks

Detected

Package seal

Leaks

Storage temperature

Above specifications

Storage time Spoilage/particulate

Exceeded Detected

Action Reject Reject Adjust temperature Reject Adjust electric field Reprocess product Laboratory analysis Adjust pulsing rate Reprocess product Adjust flow Reprocess product Reject Adjust packaging machine Reprocess product Reject Adjust temperature Laboratory analysis Reject Reject Reject

a From Vega-Mercado et al. (1996). (Reprinted with permission from Dairy, Food and Environmental Sanitation. Copyright held by the International Association of Milk, Food and Environmental Sanitarians, Inc.) reviewed in previous chapters will control charging rates, voltage settings, pulse widths, and pulse shapes. Pulser c o m p o n e n t s such as the power source, control interfaces, triggering system, and t r e a t m e n t c h a m b e r must comply with defined specifications, including high reliability, which can be m e a s u r e d in terms of the n u m b e r of pulses with the required energy level per unit of time. The purpose of this strict selection of variables is to eliminate all potentially hazardous microorganisms that may not be inactivated to the desired level if the appropriate t r e a t m e n t was n o t delivered. The flow sheet p r e s e n t e d in Fig. 7.3 may be used to identify potentially hazardous microorganisms in foods, both infectious and pathogenic. In the case of pasteurization, only vegetative cells are r e d u c e d by a certain factor. Thus, the conditions of pasteurization may be critical, and no relevant microorganism should be ignored. Recontamination of processed products is a c o m m o n feature in food production; organisms such as S. aureus and L . monocytogenes

179

IV. The H A C C P System in PEF Processing

Do preventive measure(s) exist for the identified hazard? Yes

No

Modify step or

process

+

$

?

Does this step eliminate

Is control at this step

or reduce the likely occurrence of a hazard to an acceptable level?

necessary for safety? ~ Yes ,1, No ---) Not a CCP

,L ~ No +

Yes --~ Is a CCP

Could contamination with an identified hazard occur in excess of acceptable levels or could these increase to unacceptable levels? --~

+

No --~ Not a CCP

Yes

Will a subsequent step eliminate identified hazard(s) or reduce the likely occurrence to an acceptable level?

+

--> No--> I s a C C P

Yes --~ Not a CCP Fig. 7.2 A decision tree to identify the CCPs in a food processing operation (adapted from NACMCF, 1992).

can easily colonize processing equipment. Food handlers may be carriers of several pathogens such as S. aureus and ShigeUa, so it is essential to avoid such recontamination by applying good manufacturing practices (GMP) during processing (Vega-Mercado et al., 1996). In the application of HACCP, the use of microbiological testing is seldom an effective means of monitoring the CCPs because of the time required to obtain the results. In most instances, monitoring can best be accomplished through the use of physical and chemical tests, visual observations, or rapid methods of microbial detection when applicable. In accordance with the National Academy of Sciences r e c o m m e n d a t i o n , the HACCP system must be developed by each food establishment a n d tailored to its individual products, processing, and distribution conditions (NACMCF, 1992).

C. RecordKeeping It is extremely essential to maintain written d o c u m e n t a t i o n of the daily events related to processing. Record keeping should cover all the processing steps, from receipt of the raw material to distribution of the finished product. Table 7.2 is a simplified PEF operation checklist suggested by

180

7. HACCP in PEF Processing

list of microorganisms causing food diseases

present in raw material

yes

no

~-- eliminateorganisms from the list

production process eliminatesmicroorganismscompletely

no 1~

ces

eliminate organisms from the list

pathogenicorganisms contaminating the product after processing

Did these organismscause problems in the past with identical or related products (literaturereview)?

no

yes

infectious

anisms

eliminate, organisms

from the list

toxinogenic organisms

growth of organisms in product ~es

nc

~ eliminateorganisms from the list

potentially hazardous microorganisms Fig. 7.3 A flow chart used to identify potentially hazardous microorganisms (adapted from Vega-Mercado et al., 1996).

Vega-Mercado et al. (1996), in c o m b i n a t i o n with setup and CCP m o n i t o r i n g procedures. Records utilized in the HACCP system must include the following (NACMCF, 1992): Record of the HACCP plan: A listing of the HACCP team and assigned responsibilities, description of the p r o d u c t and its i n t e n d e d use, flow diag r a m of the entire m a n u f a c t u r i n g process indicating the CCPs, hazards associated with each CCP and preventive measures, critical limits, m o n i t o r ing system, corrective action plans for deviations from the critical limits,

IV. The H A C C P System in PEF Processing

181

TABLE 7.2 Batch Record for a PEF Operation ~

Checkpoints

Yes

No

By

Time

Product Raw Materials Item Description ID number Amount Equipment and utensils Treatment chamber Sterile utensils Totally disassembled With detergent With chlorine (200 ppm) Rinsed with sterile water Autoclaved (121~ min) Processing system Cleaned with detergent With chlorine (200 ppm) Rinsed with sterile water Autoclaved (121~ min) Aseptic package Number of bags UV light exposed With H 2 0 2 (%) Rinsed with sterile water Work bench UV light exposed With chlorine (200 ppm) Rinsed with sterile water Dry and clean Pulser setup Safeguards in place Compressor "on" Setup voltage (kV) Setup frequency for pulsing (Hz) Process parameters Flow rate (liters/min) Pulsing rate (Hz) Set voltage (kV) Peak field (kV/cm) Peak current (kA) Temperature in treatment chamber Temperature out of treatment chamber a Adapted from Vega-Mercado et al. (1996).

record keeping procedures, and procedures for verification of the HACCP system. Record of raw materials: Name of the supplier, date received, condition of the raw material when received, storage temperature, and shelf-life.

182

7. HACCP in PEF Processing

Record of processing: Data of all monitored CCPs to establish the safe shelf-life of the product and prove the adequacy of the process to manufacture safe foods. Record of packaging: Records to indicate compliance with packaging material and sealing specifications. Record of storage and distribution: Temperature data and records indicating when temperature-sensitive products were shipped. Record of deviation(s) and corrective action(s) Record of HACCP system validation: Modification of the HACCP plan (if any) indicating approved revisions and changes in ingredients, formulations, processing, packaging, and distribution as needed.

Record of employee training In a PEF processing facility, standard operating procedures (SOP) should be in place to define aspects such as receiving, storing, and preparing raw materials to ensure proper handling and to reduce the risk of contamination. A batch record must be maintained to reflect the raw materials used, processing conditions employed, and packaging and distribution procedures for each processing cycle. These records are extremely useful in the event of a deviation from the CCP limit. Record keeping also includes specifying procedures for the assembly and disassembly of pulsing and packaging units. Cleaning specifications such as frequency and type of detergents or sanitizers should be established to prevent cross-contamination between products, and corrective actions must be specified in case of deviations from CCP limits. The verification step is an important component of the HACCP system. Verification procedures include ensuring that the critical limits of the CCPs are satisfactory, the HACCP plan is functioning effectively, documented periodic revalidation remains free of audits, and the HACCP system is functioning in compliance with government regulations (NACMCF, 1992). This step may also include consideration of any consumer complaints about the food in question (Notermans et al., 1994).

V.

Final Remarks

The HACCP system is an essential part of the food manufacturing industry. Proper implementation of the system ensures safety of the food without the necessity of testing the finished product. Like any other food processing operation, the HACCP system must be adopted for foods processed using the PEF technology. Once the HACCP system is developed, commitment to its proper implementation is crucial. Education and training are also important elements of the HACCP concept. Employees who will be responsible for the HACCP program must be trained adequately in its principles, application, and implementation. Educational and training programs should be designed

References

183

to address the n e e d s o f industry, g o v e r n m e n t , and academic personnel, as well as consumers.

References Corlett, D. A., Jr. (1990). Importance of hazard analysis and critical control point system in food safety evaluation and planning. ACS Symp. Ser., Am. Chem. Soc. 484, pp. 120-130. National Advisory Committee on Microbiological Criteria for Foods (1992). Hazard analysis and critical control point system. Int. J. Food Microbiol. 1(1), 1-23. National Food Processors Association (1992). HACCP and total quality management--Winning concepts for the 90's: A review. J. Food Prot. 55(6), 459-462. Notermans, S., Zwietering, M. H., and Mead, G. C. (1994). The HACCP concept: Identification of potentially hazardous microorganisms. Food Microbiol. 11,203-214. Vail, R. (1994). Fundamentals of HACCP. CerealFoods World 39(5), 393-395. Vega-Mercado, H., Luedecke, L. O., Hyde, G. M., Barbosa-Cfinovas, G. V., and Swanson, B. G. (1996). HACCP and HAZOP for a pulsed electric field processing operation. Dairy, Food, Environ. Sanit. 16(9), 554-560.

CHAPTER 8

PEF in the Food Industry for the New Millennium

I.

Introduction

Like any emerging food process, PEF technology will face challenges at different stages of its implementation in the food industry in the new millennium. Demands of consumers, producers, and governmental organizations will have to be fulfilled with safer and better quality products. Even now producers are interested in such commercialization aspects as initial investment, process cost, and benefits of a milder treatment that will allow them to obtain safe and fresher products with an extended shelf-life and high marketability. Governmental considerations include specific regulatory aspects that food processors have to consider before the implementation and gaining of approval to commercialize PEF products. To be able to compare different PEF processes, a standard method to evaluate their effectiveness in microbial inactivation, processing conditions, and product attributes is needed. Also essential is the development of protocols to conduct the process during experimental research. Many researchers are already developing these protocols; leading groups in the area have identified different technical issues that will allow the comparison of ongoing project results all over the world. Several research groups are also devoting resources to the commercialization of PEF. The Electric Power Research Institute (EPRI) in Palo Alto, California, through its Food Technology Alliance (FTA) in St. Paul, Minnesota, and Toledo, Ohio, has organized a nonthermal pasteurization initiative and a consortium of scientists who are investigating this electrotechnology. The most influential research groups are found in the United States, Canada, Japan, and several European countries. The goal of these food

184

II. Commercialization

185

engineers and scientists is to get the acceptance of PEF for the preservation of foods, and their strategy starts with the consideration of product and employee safety, as well as consumer and regulatory acceptance. These researchers are making efforts to narrow the limitations and challenges that PEF now faces, and because of this shared objective, the most critical variables of the process are being put in place, including the determination of the future direction for the technology and even the best way to ensure this takes place. As a result of this international interaction, the identification of critical microorganisms of interest for additional inactivation studies ( T a b l e 5.5) has been made so that the results of a variety of different research groups can be compared (EPRI, 1997).

II. A.

Commercialization Industrialization and Production Costs

The major concern of industrial consortiums interested in the application of PEF is the initial investment. Cost estimates of a commercial-scale PEF pulse generator with a production scale of 1000 to 10,000 l i t e r / h r indicate a range between $500,000 and $1,000,000 U.S. dollars. However, these expenses are estimated for a custom design, so the initial cost of future equipment may be less expensive. The pilot plant size pulser commercially available at the PurePulse Company in San Diego, California, can process 100-300 l i t e r / h r and apply square pulses of 2 - 3 /.Lsec at a repetition rate of 1000 Hz. It is designed to process orange juice at a flow rate of 100-300 liter/hr. PurePulse also has estimated costs for a 10 liter/hr, 50-kV/cm, 100-J/ml, and 2-/zsec square-wave laboratory-scale system available u p o n request. Also important to note is that these systems have estimated operating costs of $0.2/liter (EPRI, 1998). Even despite such seemingly astronomical start-up costs, the allocation for energy once the system is in place would be much lower than with conventional thermal systems because of the conservative nature of PEF in this regard. In those processes where no cooling is necessary, the process becomes even more attractive. Operational costs include maintenance and electrical power costs, the latter of which would be more focused in the treatment chamber, with an estimate of $5 per working h o u r at a flow rate of 5000 l i t e r / h r . The expected lifetime of a high-pulse voltage generator is around 4 - 5 years with 20 h r / d a y of operation. Researchers have estimated that 42% of the operating costs are those related to electricity. Energy consumption for the pasteurization step has been reported as low as 1.3 J / m l , which is particularly appealing compared with almost 100 J / m l used in heat pasteurization.

186 B.

8. PEF for the New Millennium PEF

Implementation in the Food Industry of Today

Implementation of the PEF technology in the middle of a rapidly changing food industry where consolidation is key and mergers and acquisitions are delimiting the market in the hands of a smaller number of globally based conglomerates are additional challenges promoters of this emerging technology will have to overcome. Competition with PEF processing will take place in the growth markets of the United States, Asia, Europe, and Central and South America. It appears that commercial PEF processing of fresh juices with low pH is on the horizon. The Food and Drug Administration's (FDA) notice of intent for fresh juice published on August 28, 1997, will be followed by a more encompassing advanced notice of proposed rule making. For this type of product, there are three fundamental regulatory issues that are described outside the code of federal regulations (CFR): Title 114, including compliance with good manufacturing practices (GMP); demonstration of a minim u m 5 log cycle reduction of pathogenic microorganisms of usual concern; and insurance that no harmful substances are added to the product by the process. It is not u n c o m m o n that foreign countries commercialize food products produced with nonthermal technologies before they are introduced or approved in the United States. An example is the Australian dairy industry interested in incorporating the CoolPure technology within their thermal processing line to extend the shelf-life of milk for export. Because milk is one of the most regulated processes in the world, PEF by itself is not yet accepted as a pasteurization process. However, in conjunction with a thermal process it can minimize the heat required for pasteurization and destroy vegetative organisms not killed by heat, thus extending the product's shelf-life (Morris, 1997). Based on a utility/customer partnership, the EPRI-FTA is promoting interaction between food processing customers and the nonthermal pasteurization initiative. Some of the major aspects include long-term customer relations at the corporate level, equity investments, and project advisory input; marketing and strategic decisions; workshops, access to university scientists and relevant government agencies, progressive equipment vendors, and worldwide trade groups; and provision of a technical advisory council.

III.

Regulatory Aspects for the Implementation of PEF

The difficulty of demonstrating the equivalency of nonthermal methods to existing thermal processes is one of the main reasons for the slow commercialization of these new preservation technologies (Cole, 1997). In the

III. Regulatory Aspects for the Implementation of PEF

187

United States the regulatory aspects for an "alternative pasteurization" process cannot be treated lightly. The potential indirect food additive implications associated with presenting a high voltage across two electrodes immersed in a product thus need to be addressed by each manufacturer. Although the petition put forth by PurePulse Technologies to the FDA does not consider PEF to be a food additive when used in the m a n n e r specified by the manufacturer, this does not mean that under different conditions PEF would not be considered as such. Before the commercialization of processed foods by a nonthermal technology, each process must comply with the appropriate safety regulations set forth by the FDA according to type of product. The lengthy n u m b e r of regulations for thermally processed low-acid foods is an example of the implications to be contended with. Regulations for thermally processed low-acid foods packaged in hermetically sealed containers are contained in the CFR under Title 21, Part 113. Other regulations that may be addressed for the industrialization of PEF are GMP (21 CFR 100), Grade A Pasteurized Milk Ordinance (21 CFR 133, 21 CFR 1240.61), Food, Drug and Cosmetic Act [Section 402(a)(1) and (4)], and Electronic Records: Electronic Signatures (21 CFR 11) (Larkin and Spinak, 1997). To determine which regulations apply to a new product, the first step is to verify if the product attributes fulfill the requirements of the regulation. For example, a low-acid canned food product has to have a pH of 4.6, a water activity greater than 0.8, be shelf-stable (not in need of refrigeration after processing), packaged in a hermetically sealed container, and thermally processed to a commercially sterile end point. It is this very last characteristic which brings to light that the lethality of the thermal treatment has to be such that the most resistant organism of public concern will be inactivated. Therefore, to fulfill such regulations PEF must demonstrate that it can inactivate these types of microorganisms. Cole (1997) exposed the concerns of the food industry about showing the impossibility that a new sterilization process is capable of delivering an equivalent level of processing to the traditional one (12-D process). Furthermore, the real reduction factor afforded by a process of 3 min at 121~ would be a little over 7 logs rather than 12, so that only by defying the limits to safety would it be possible to approach them with confidence under the risk assessment concept. If the new product has the characteristics of an acidified food as described in Part 114 of the CFR, then no thermal treatment is required, but the processor will still need to demonstrate that the process is able to render a product free of any pathogenic microorganisms that may reproduce during storage, distribution, retail, or consumer use. Before food processed by PEF gains approval from the FDA, the extension of shelf-life for products such as fruit and vegetable juices and refrigerated and acid foods will need to be addressed under Part 110 of the section entitled GMP in the CFR. On August 28 of 1997, the FDA published their intention to regulate some or all the fruit and vegetable juice industry by way of HACCP (Larkin and Spinak, 1997). One of the major concerns with

188

8. PEF for the New Millennium

extending the shelf-life of products is temperature abuse after processing, so producers have been asked to demonstrate that a product obtained under GMP can be safe and does not present a health hazard under temperature abuse conditions. In addition, inoculation studies have to be conducted to verify that the products do not support the growth of psychrotrophic pathogens at extended refrigeration times. Larkin and Spinak (1997) pointed out that the regulations for grade A pasteurized milk leave an " o p e n door" to new pasteurization processes that have to be recognized as "equally efficient" to other FDA-approved processes before gaining their own approval by the agency. But there is no approved alternative procedure yet. If someone wanted to get an alternative pasteurization process approved for milk products, they would need to submit all the pertinent information regarding safety and process control.

A.

FDA Regulations

The process review objective of the FDA is to conduct a scientific evaluation of a particular process to determine if the product poses a potential public health hazard. They are also concerned with ensuring that all of the critical factors necessary to render the product commercially sterile are monitored and controlled. To meet the requirements of the FDA in "filing a new and a novel process" (Larkin and Spinak, 1996), it is necessary to (a) establish an active and continuous dialogue with the FDA during process development, (b) meet with the FDA to describe the process, (c) invite the FDA to a site visit (pilot and production facility), (d) draft and provide the FDA with an outline of the proposed filing, and (e) identify the most resistant organism of public health concern, the most resistant organism for commercial viability, and the least lethal treatment zone of the system. In regard to the novelty of the process, the FDA is interested in reviewing equipment design, product specification, process design, and process validation: a. Equipment design: a description of the system, control mechanisms used, and fail-safe procedures. b. Product specifications: a full description of the product, including physical/chemical aspects, critical factors, and influence of processing on the critical factors. c. Process design: a complete description of the critical/processing conditions used in the manufacture of the product. d. Validation: a physical demonstration of the accuracy, reliability, and safety of the process.

III. Regulatory Aspects for the Implementation of PEF

189

Apple juice is the most likely to be the first commercialized product, when it will have to face three fundamental regulatory issues as described in 21 CFR 114: 1. Good manufacturing practices as described in 21 CFR 110. 2. A minimum 5 log cycle reduction of pathogenic microorganisms of usual concern. 3. Demonstration that no harmful substances are added to the product by processing. Not only do food safety requirements have to be addressed, but also the safe equipment operation under high voltages. On August 21 of 1997 a new regulation about using electronic signatures went into effect (21 CFR 11). This allows any Title 21-regulated system to use an electronic signature in place of a handwritten one. This is very important in reference to process automation because it means that the processor will now be responsible for demonstrating that the regulation is being satisfied when requested to do so.

B.

Letters of No Objection from the FDA

Under current regulations, it would not be necessary to "file a process" because of the potential indirect food additive issue (due to electrode erosion). In this case a letter of no objection for each PEF process is recommended. An example of this occurred in July of 1995 when the FDA cleared the CoolPure cold-pasteurization process developed by PurePulse Technologies for the antimicrobial treatment of liquids and pumpable foods. The following are suggested steps to obtain a " n o objection" letter: 1. Contact the office of premarket approval at the FDA. 2. Schedule a preliminary meeting in Washington, D.C. 3. Establish a confidential file after describing the intent and testing protocol. 4. Record data, including specific information about the physical and microbiological characteristics of the product to be treated. 5. Record the PEF treatment operation conditions and physical and microbiological characteristics of the treated product, as well as the refrigerated product. 6. Contact the office of premarket approval at the FDA and schedule a follow-up meeting. 7. Create a letter describing how the process will be monitored and controlled, what conditions and material handling techniques will be used, the impact of processing on the product, and conclusions. 8. Present the results and conclusions, answer the questions from the FDA, and submit your letter to the confidential file.

190

IV.

8. PEF for the New Millennium

The Future of PEF

Researchers all over the world still have many possible project development designs that need to be focused on the better understanding of this technology. The projects must be related to aspects of the PEF product and process that have not been addressed yet and are of relevance to implementation at a commercial level. The results obtained up to now are not enough for a complete generalization of different aspects dealing with the quality, microbiological, and nutritional characteristics of products as well as their processing conditions. The establishment of unknown destruction kinetics of many microbial pathogens (especially Clostridium botulinum) and the identification of proper indicator organisms for each specific product that consider the handling and storage conditions of raw products and final products are examples of where microbiological experience will be of major contribution. The uniformity of the delivered treatment and the means to assess the process are still challenging food and electrical engineers. The impact of processing conditions such as temperature, pH, moisture, and lipid content on the safety and quality aspects of new products leaves an area that is still open to food chemists. Furthermore, the implementation of hurdle technology and the use of food additives suggest even more new alternatives. To facilitate the analysis and comparison of future investigations, as well as fully utilize the potential of PEF for food processing, researchers are aware that there are many technical issues that need to be addressed: a. Reliable generation of high-strength PEF. b. A data acquisition system to record critical processing parameters such as flow rate, waveform, electric field, n u m b e r of pulses, and temperature; a computerized control system must also be integrated with this monitoring process. c. Reliable measuring devices to evaluate the energy delivered per cubic meter by each pulse during processing, which includes evaluating the frequency content of the pulse, identifying possible partial discharges and spark impedances, detecting missed pulses, and calculating the spatial average of the electric field in the treatment chamber. d. The critical, maximum, and optimum field strengths appropriate to inactivate given microorganisms essential to food storage. e. The flow rate and dosages. f. The heat induced during treatment and temperature control. g. The interference of air bubbles or suspended particles and backpressure implementation. h. Treatment chamber design for full-scale processing. i. Arcing at the electrode-interface, liquid followed by the creation of chemicals.

References

191

j. Electrode fouling. k. Inactivation kinetics for pathogens (not only pure cultures) in various food media. It is not clear yet if the food industry will fully accept PEF as a processing technology. Nevertheless, its tremendous potential to replace or complement conventional methods is clear, which provides the basis for ongoing studies on PEF that can be regarded as both meaningful and worthy of consideration to all those in the field of food processing.

References Cole, M. B. (1997). The oudook for novel preservation technologies: A food industry perspective. Presented at the Fifth Conference of Food Engineering, Los Angeles. EPRI (1997). EPRI/Army PEF Workshop II, Chicago, October 10-11. EPRI (1998). Pulsed electric field processing in the food industry: A status report on PEF. Report CR-109742. Industrial and Agricultural Technologies and Services, Palo Alto, California. Larkin, J. W., and Spinak, S. H. (1996). Regulatory aspects of new/novel technologies. In "New Processing Technologies Yearbook" (D. I. Chandarana, ed.), pp. 86-92. National Food Processors Association, Washington, D.C. Larkin, J. w., and Spinak, S. H. (1997). Aspects of new technology implementation in the U. S. Presented at the New Technologies Symposium NFPA Convention. 29 October, Chicago. Morris, C. E. (1997). Processing technologies for the 21st century: New thermal and nonthermal technologies are available now for food pasteurization and sterilization. Food Eng. 69(1), 41-46.

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Index

acetylcholinestearase, 143 alkaline phosphatase, 147, 149 ALP, see alkaline phosphatase antimicrobials, 101, 102 benzoic acid, 102, 117 citric acid, 163 EDTA, 139 nisin, 102 sorbic acid, 117 apple juice concentrated, 160 effect of PEF, 78 energy requirements, 11, 157 freshly squeezed, 160 processing temperature, 43 S. cerevisiae inactivation, 64, 101, 110-114 shelf-life, 159-161 storage, 44 applications, 169 arc discharges, 3, see sparks ascospores, 114 Aspergillus niger, 109 Bacillus, 83 cereus, 99, 138, 140, 141 coagulans, 109

inactivation, 109, 130, 131, 140 megaterium, 79, 109 subtilis, 83, 99, 131,132, 139-141,143

bactericidal effect, 39, 61 benzoic acid, 102, 117 breakdown bubbles, 14, 15, 101 cell, see cell breakdown due to particles, 16

electrical, 77, 96 food, 15, 33 mechanical, 96 membrane, 48, 51, 54, 57, 58, 68 brine microbial analysis, 168 microbial flora, 168 processing, 157, 168

Candida, albicans, 83, 109, 113 utilis, 109

capacitance, 4, 9, 10 carbohydrate, 77 casein protective effect, 143-145 proteolysis, 144 cell breakdown, 15 conductivity, 80 culture, 141 damage, 7, 113 electroporation, 47 fusion, 78, 79 growth stage, 84 leakage, 44, 55, 67, 68 lysis, 47, 50, 55, 61, 130, 131 membrane potential, 51, 53, 60 membrane role, 47 osmotic imbalance, 118 rupturing, 73 shrinking, 47, 60 size, 87, 114, 137 structural fatigue, 125, 137 swelling, 47, 60

193

194 transmembrane potential, 10, 48-50, 52, 53, 60, 73 ultrastructural changes, 113, 126 chamber coaxial, 6, 7, 9, 13, 14, 28, 84, 112, 123, 131, 137, 139, 166 co-field, 29, 38, 84, 162 continuous, 35, 84, 123, 166 design, 16, 28, 29 electrode optimization, 31 multiple, 121, 141 parallel plate, 6, 7, 11, 13, 28, 34, 84, 112, 113, 123, 143 resistance, 16 static, 33, 84, 121,123, 135-136, 168 treatment, 2, 4-6, 8, 9, 13-15, 17, 84 volume, 9 changes biological, 76, 77, 95, 97, 101,103, 104 chemical, 76, 77 charge accumulation, 80, 82 build-up, 80 electrical, 81 movement, 80 cheese, 165 HACCP, 176 chemical analyses, 158 citric acid, 166 Clostridiu m botulinum, 109, 190 butyricum, 109 sporogenes, 109 welchii, 109

commercialization aspects, 184 implementation, 15, 186 scale, 15 concentric cylinders, see coaxial chamber conductivity, 10, 101, 102, 103 food, 96 liquid, 82 medium, 134, 135 membrane, 59, 134 static, 3 costs energy, 98 equipment, 96 operational, 15, 185 production, 185 pulse generator, 185 cranberry juice, 114 critical control points, 177

Index

current conduction, 81 measurement, 4, 22, 43

degradation kinetics of vitamin C, 163 dehydrogenase, 143

EDTA, 139, 146, 147 egg, 77, 133, 135, 157-159, 166, 167, 176 color, 167 functional properties, 167 HACCP, 176 hurdle aproach, 166 processing, 166 products, 4 sensory evaluation, 167 viscosity,167 electric current, 2, 3 electric field, 84 coaxial chamber, 7, 39 critical, 86, 89 effect on microbial cells, 48, 77 enhancement, 33, 38, 112 measurement, see oscilloscope optimization, 31 parallel plate chamber, 7, 34 stimulation, 3, 17 electrode configurations, see chamber design electrofusion, 47, 76, 77, 78 electropermeabilization, 76, 77, 79 electroporation, 2, 6, 7, 47, 55, 58, 59, 67, 73, 80, 83 electroporator, 13, 131, 144, 148 Electro-pure process, 1 energy capacitor, 4 consumption, 157, 185 costs, 98 delivered, 12, 24 density, 9 electrical, 4 input, 11, 112, 128, 169 loss, 3, 11 requirements, 11, 12 thermal, 4 enzymes, 3, 14, 15, 17 inactivation, 108, 142, 143, 150, 151 Eschedchia,

O157:H7, 83, 102 coli, 1, 3, 13, 61, 83, 92, 102, 109, 114,

118-124, 128, 132, 135, 137, 143, 159 inactivation, 114

Index fat, 77, 87 protective role, 110, 136, 148, 158 flavor milk, 165 food resistance, 9 Fourier transform, 97

195 ionic strength, 79, 101-104, 119, 120, 143

juices, 44, 77, 97, 99

Klebsiella pneumoniae, 83 Gram

negative, 86, 98, 99 positive, 86, 98, 99, 109, 125, 126, 128, 130, 137 grape juice, 114, 159 growth stage, 84, 92, 100, 101 exponential phase, 110 logarithmic phase, 110 stationary phase, 110, 120, 135, 137

HACCP, see Hazard Analysis and Critical Control Point concept, 173 definitions, 173 principles, 173 hazard assessment, 175 characteristics, 176 Hazard Analysis and Critical Control Point, 172 hexokinase, 143 hurdle approach, 166

ignitrons, 25 impedance, 10 characteristic, 9 matching, 9 inactivation, see model Bacillus, 130 Escherichia coli, 114 glucose oxidase, 150 kinetic constants, 112, 126 kinetics, 108, 110, 128, 135-137, 152 LactobaciUus, 128 lipase, 143, 151 mechanism, 47, 83, 113 microorganism, 76 Pseudomonas, 135 peroxidase, 150 plasmin, 143 rates, 76, 89 Salmonella, 132 Staphylococcus aureus, 125 synergistic, 94, 117, 120, 133 yeast, 109

Lactobacillus, 61, 109, 129, 168 brevis, 68, 109, 129 bulgaricus, 109 liquid whole egg, see egg Listeria monocytogenes, 83, 102, 113, 126, 128, 137,

178 logarithmic phase, see growth stage lysozyme, 139 inactivation, 150

membrane capacitance, 11 conductance, 11 damage, 117, 126, 129 fluidity, 119 permeability, 77, 118 potential, 77 microbial analysis, 157, 168, 169 inactivation factors, 84 microscopy scanning electron, 126 transmission electron, 113 milk, 1, 4, 7, 13, 44, 77, 86, 92, 99, 124, 129, 130, 133-136, 143, 147, 157, 159 commercialization after PEF, 186 HACCP, 176 microbial load, 159 PEF processing, 165 raw, 165 UHT, 110, 130, 136, 143 model heat conduction, 81 Hi~lsheger, 85, 90 Maxwell, 56, 57 Peleg, 87 Sensoy, 94

nisin, 102 nonthermal methods, 1

196 PEF, 82

ohmic heating, 1, 3, 17 orange juice, 13,77, 97, 110, 114, 157, 159, 162-164, 185 color, 164 flavor, 164 processing, 162 vitamin C, 162 organic acids synergistic inactivation, 117, 119 oscilloscope, 7, 12, 21, 43, 44

packaging, 7, 14, 20 aseptic, 44, 162, 167 particles suspended in foods, 3, 15, 16 suspended in the medium, 86, 101 pea soup, 119, 131,155, 159, 168 analysis, 168 processing, 168 pediocin, 102 permittivity of free space, 10 pH, 101, 102, 117, 118, 125, 126, 135, 137, 139, 143, 158 apple juice, 161 effect, 110 phosphate buffer, 110, 117, 129, 135, 137 pineapple juice, 114 plasmin, 143 pore expansion, 52, 53 formation, 51, 53, 57, 58, 59, 60, 74 shrinkage, 53, 59 potassium sorbate, 166 power supply, 20 protease, 142, 143 protein channels, 77, 79 Pseudomonas, 14, 79, 83 fluorescens, 83, 135-137, 143, 146, 147 inactivation, 109, 135, 143, 168 pulse waveform bipolar, 95-98, 124, 131. effect, 136 energy density, 9 exponential decay, 8, 9, 95, 112, 124, 131, 133-139, 162, 166 frequency components, 97 instant-reverse-charge, 12, 14, 95 number, 89 oscillatory, 95, 124 square-wave, 8, 9, 112, 123, 162

Index

waveshape, 95 width, 7, 10, 86 pulser design, 21 high voltage, 20 pulsing rate, 145, 177 record-keeping, 141, 173 regulations, 17 FDA, 188 resistance, 3, 4, 9, 10, 14 chamber, 16 resistivity, 8, 10, 12, 14, 120 water, 169 Saccharomyces, 61, 83 cerevisiae, 65, 83, 90, 92, 99, 109, 110-115, 137 safety, 1, 14, 15, 17, 21 regulations, 187 salad dressing, 159 Salmonella, 83 dublin, 83, 109, 133, 134, 135, 165 enteritidis, 83, 133 inactivation, 132 typhimurium, 102, 109, 133 SEM, see microscopy sensory evaluation, 158 egg, 167 shelf-life, 1, 13, 138, 156, 157, 159-161, 165, 166, 170, 184 determination, 182 egg, 167 extension, 14 importance, 158 milk, 165 orange juice, 162 pea soup, 168 PEF products, 160 simulated milk ultra-filtrate, see SMUF SMUF, 13, 99, 102, 120, 123, 124, 126, 128, 130, 131,139, 143, 144, 147, 148 sodium alginate, 110, 123, 130, 136 sorbic acid, 117 soup cream, 4 see pea soup spark, 5, 15, 16 gap, 25, 26 spore, 95 inactivation, 108, 138-141,152, 154 Staphylococcus, 61, 92, 109, 125, 126, 157, 159, 168

Index

67, 109, 126, 127 inactivation, 125-128 storage, 84 refrigerated, 84 switch, 5, 15 operating parameters, 27 thyratron, 24 types, 25 system PEF, 20 aureus,

TEM, see microscopy temperature control, 5 effect, 119, 133, 140 measurement, 4 thyratrons, 25 transducers, 12 transmembrane potential, 10, 79, 134, see cell treatment, see chamber conditions, 84 media, 84, 91 non-uniform, 14 temperature, 42, 84, 92

197

time, 84, 90, 110, 112, 120, 134-137, 140, 152, 159, 177 trigatron, 27

ultrastructural changes,

see

cell

voltage buildup, 81 measurement, 4, 43

water, 110, 114, 117, 120, 125, 130, 133, 136, 137, 158, 166, 169 microbial control, 169 microbial hazard, 177 waveshape pulse, see pulse waveshape

yeast, 109 yogurt, 110, 129

Zygosaccharomyces bailii,

83, 114

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