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MEMBRANE PERMEABILITY CHANGES DURING MODERATE ELECTRIC
FIELD PROCESSING OF VEGETABLE TISSUE
DISSERTATION
Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Suzanne A. Kulshrestha, M.S.
The Ohio State University 2002
Dissertation Committee: Professor Sudhir K. Sastry, Adviser Professor David Min Professor Steven J. Schwartz Associate Professor Q. Howard Zhang
Approved by
Food Science and Nutrition GraduateAdviser Program
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precooked potato cylinders were chilled, and then warmed to 25ºC by either allowing them to equilibrate or by MEF treatment. The conductivity from 100 Hz to 20 kHz and apparent dielectric constant from 100 Hz to 5 kHz was initially the same between raw, untreated samples and raw, MEF treated samples, but over 24 hours, that of the raw, MEF treated samples increased while that of the raw, untreated samples remained constant. No such distinct pattern emerged from the thawed or precooked samples. The apparent dielectric constant of raw, MEF treated potato above 5kHz was the same as raw, untreated potato and higher than thawed and precooked potato. None of the samples showed marked changes in dielectric constant at 5-20 kHz over the 24 hour period. Apparently, even mild electrical treatments permeabilize vegetable tissue, permitting enhanced diffusion.
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ACKNOWLEDGMENTS
I would like to thank: ♥ My adviser, Dr. Sudhir Sastry for his time, effort, and patience. He has encouraged intellectual creativity and independence and has asked many challenging questions. He has also provided me with financial support, helped prepare documents for publication, and handled the submission process. ♥ My husband, Dheeraj, who relocated to Columbus in order that I may complete my education, supported me financially, and put forth his time and effort in many ways. ♥ The employees at The Ohio State University Child Care Center and nannies Lori Rutschilling, Tracey Pitts, and Barbara Fox for taking care of my son so well that I could devote my time and mental energy to research. ♥ Dr. Marybeth Lima, who got me started in the lab and gave me the idea for the experiment in Chapter 2. ♥ Brian Heskitt, who was critical in setting up my equipment, which was the most difficult part of the lab work. ♥ My friends in the Department of Food, Agricultural, and Biological Engineering and in the Department of Food Science and Technology, who kept me going through difficult times and helped me get some work done. ♥ My parents, who gave me life, love, and a value for education.
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VITA
August 8, 1967…………………………… Born – Wright-Patterson A.F.B., OH 1989………………………………………. B.S., Biochemistry,The Ohio State University
1993………………………………………. M.S., Food Science and TechnologyTexas A&M University
PUBLICATION Kulshrestha, S.A. and Rhee, K.S. 1996. Precooked reduced-fat beef patties chemical and sensory quality as affected by sodium ascorbate, lactate and phosphate. J. FoodSci. 61(5):1052-1057. FIELDS OF STUDY Major Field: Food Science and Nutrition
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TABLE OF CONTENTS
ABSTRACT........................................................................................................................II
ACKNOWLEDGMENTS ................................................................................................ IV
VITA ................................................................................................................................. VI
LIST OF FIGURES .......................................................................................................... IX
CHAPTER 1 ....................................................................................................................... 1
1.1 Introduction........................................................................................................ 1 1.2 Bibliography……………………………………………………...………….
CHAPTER 2 ..................................................................................................................... 19
FREQUENCY AND VOLTAGE EFFECTS ON ENHANCED DIFFUSION DURING MODERATE ELECTRIC FIELD (MEF) TREATMENT............................................... 19 2.1 Abstract ………………………………………………………………..…. 2.2 Introduction………………………………………………………………….. 2.3 Materials and Methods………………………………………………………. 2.4 Results and Discussion………………………………………………………252.5 Conclusions………………………………………………………………….. 2.6 Bibliography…………………………………………………………………
CHAPTER 3 ..................................................................................................................... 36
LOW-FREQUENCY DIELECTRIC CHANGES IN POTATO FROM OHMICHEATING: EFFECT OF END POINT TEMPERATURE .............................................. 36
3.2 Introduction............................................................................................................. 37
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LIST OF FIGURES
Figure 2.1. Experimental Apparatus ……………………………………………….. Figure 2.2. Nondimensionalized electric field distribution within treatment chamber…………………………………………………………………………….... Figure 2.3. Diffusion of betacyanin from fresh tissue MEF processed for 3 min. at 45Effect of electric field strength and frequency…………………………….………....35°C:
Figure 3.1. Apparatus for dielectric measurements………………………………... 52 Figure 3.2. Conductivity spectrum of samples heated conventionally to various endpointtemperatures………………………………………………………………………….
Figure 3.3. Conductivity spectrum of samples heated ohmically to various endpoint temperatures………………………………………………………………………… 54 Figure 3.4. Conductivity at 100 Hz of conventionally and ohmically heated potato cores heated to various endpoint temperatures……………………………………............... Figure 3.6. Conductivity at 20 kHz of conventionally and ohmically heated potato cores heated to various endpoint temperatures…………………………………...…………. Figure 3.7. Apparent dielectric constant spectrum of samples heated conventionally to various endpoint temperatures………………………………………………………….
Figure 3.8. Apparent dielectric constant spectrum of samples heated ohmically to various endpoint temperatures …………………………………………………………………. Figure 3.9. Apparent dielectric constant at 100 Hz of conventionally and ohmicallyheated potato cores heated to various endpoint temperatures…………………………..
Figure 3.10. Apparent dielectric constant at 20 kHz of conventionally and ohmically heated potato cylinders heated to various endpoint temperatures…………………......... Figure 4.1. Apparatus for dielectric measurements……………………………….......... Figure 4.2. Beet before ohmic treatment…………………………………………......... 76 Figure 4.3. Beet after ohmic treatment………………………………………………….
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- 3.3 Methods...................................................................................................................
- Preparation of isoconductive KCl solution.
- 3.4 Results and Discussion 433.6 Bibliography
- CHAPTER
- TREATED VEGETABLE TISSUE OVER TIME........................................................... CHANGES IN PERMEABILITY OF MODERATE ELECTRIC FIELD (MEF)
- 4.1 Abstract
- 4.2 Introduction.................................................................................................................
- 4.3 Methods.......................................................................................................................
- Microscopy.
- Dielectric changes over time.........................................................................................
- 4.4 Results And Discussion 67Preparation of isoconductive KCl solution.
- 4.6 Bibliography
- CHAPTER 5……………………………………………………………………………..
- CONCLUSIONS ……………………………………………………..………………....
- Bibliography ……………………………………………………………………..……...
- Figure 4.4. Conductivity of potato over 24 hours at 100 Hz…………………………..
- Figure 4.5. Conductivity of potato over 24 hours at 20 kHz……………………………
- Figure 4.6. Initial conductivity spectrum of potato…………………………………….
- Figure 4.7. Conductivity spectrum of potato after 24 hours…………………………….
- Figure 4.8. Initial apparent dielectric constant low-frequency spectra………………….
- Figure 4.9. Low-frequency apparent dielectric constant spectra after 24 hours………..
- Figure 4.10. Apparent dielectric constant of potato over 24 hours at 100 Hz…………
- Figure 4.11. Apparent dielectric constant of potato over 24 hours at 20 kHz…………
CHAPTER 1
1.1 INTRODUCTION
Ohmic heating is a thermal food process whereby the electrical resistance of the food itself generates heat as current is passed through it. Unlike conventional retort processes, ohmic heating allows high-temperature/short-time processing of particulates, thus avoiding excessive destruction of nutrients and sensory properties (Anonymous, 1990). In addition, a high-volume ohmic heater may be more cost-effective for low-acid foods than canning or freezing (Allen et al., 1996). De Alwis and Fryer (1990) have reviewed technological developments in direct resistance heating up to 1989. Since then, researchers have developed and refined mathematical models for ohmic process design, which have been reviewed by Sastry and Palaniappan (1992). Other studies have focused on phenomena observed during ohmic heating, including changes in electrical conductivity (Halden et al., 1990), enhanced diffusion (Schreier et al., 1993), and microbial death kinetics (Palaniappan and Sastry, 1992). Electrical treatments of vegetable and fruit tissue, such as ohmic heating and high voltage pulsed electric field (PEF) treatments, have been investigated for a variety of food processing applications. Such treatments have been investigated in attempts to
improve recovery of secondary metabolites (Hunter and Kilby, 1988; Dörnenburg and Knorr, 1993), dehydration (Rastogi et al., 1999; Sensoy, 2002; Lima and Sastry, 1999; Wang and Sastry, 2000), blanching (Sensoy, 2002; Mizrahi et al., 1975), and juicing (Lima and Sastry, 1999; Bazhal and Vorobiev, 2000) processes, with mixed results. The blanching studies have shown clearly that ohmic heating has the advantage of a rapid heating rate. However, effects on product quality have not been deeply investigated. Electric pretreatments have had positive effects on dehydration and juice extraction compared to raw tissue (Rastogi et al., 1999; Bazhal and Vorobiev, 2000; Lima and Sastry, 1999; Wang and Sastry, 2000), but results of comparisons of ohmically and conventionally heated tissue heated to temperatures above 60ºC have been inconsistent (Sensoy, 2002; Wang and Sastry, 2000). This is probably due to the increase in permeability that is only seen at temperatures below 60ºC. It is unlikely that yield increases over conventional heating will be substantial in processes that heat the tissue to over 60ºC, because thermal permeation is complete at such high temperatures. A deeper understanding of the permeation mechanisms should enable better design and prediction of these processes. With ohmic heating, greater electropermeabilization is seen at low frequencies (Imai et al., 1995; Lima et al., 1999; Chapter 2), so they should be used where electropermeabilization is desired. In addition, the chemical environment is likely to have a large effect on the electrical properties of cells (Osterhout, 1922), so it may be possible to further optimize processes by choice of chemical additives. Electric treatments for fruit and vegetable have great potential for improving product quality and
The authors suggested four possible mechanisms for the electrical enhancement of conductivity:
- Electroosmosis. This occurs when an immobile charged matrix, surrounded by mobile oppositely-charged ions, is placed in an electric field. The counterions move toward one electrode, causing a convection current which moves the solvent and other solutes with it. (Hunter, 1981).
- Plasmolysis. This probably refers to irreversible dielectric breakdown of the cell membrane, also known as electroporation or punch-through (Zimmerman et al., 1974; Coster, 1965). Holes are formed in the cell membrane when it is exposed to an external electric field of sufficient intensity and duration. The membrane can reseal under mild conditions, but plasmolysis results under more extreme conditions.
- Overcoming diffusion limitation in carrier mediated transport as per Athayde and Ivory (1985). If a chemical species is transported across a membrane by a charged carrier, its flux can be increased by application of an alternating current. The carrier moves from one interface to the other in response to the electric field, which is faster than by diffusion.
- Electrohydrodynamic mixing (Hoburg and Malihi, 1978; Plonski et al., 1979). Mass transfer of fluids of different conductivity separated by a non-selective barrier was enhanced by application of a static electric field (Hoburg and Malihi, 1978). The effect was attributed to an accumulation of free charge in the barrier due to the conductivity gradient. Plonski et al. (1979) also observed enhanced diffusion between two solutions of different conductivities across an octanol membrane when an alternating current was
applied. They describe electrohydrodynamic mixing as a type of convection driven by the electrical forces of accumulated charge at the membrane-solution interface. The effect is independent of the orientation of the electrodes. Enhanced diffusion was further investigated by Schreier et al. (1993), who observed it from both beetroot and Visking semipermeable tubing. Electroporation is not a possible mechanism for enhanced efflux from tubing. Their data were consistent with equations for electroosmosis which predict a linear relationship between dye flux and electric field. However, the authors ruled out membrane effects because they found the diffusion enhancement to be the same regardless of tube orientation. Sims et al. (1991) have proven that electroosmotic effects occur across semipermeable tubing. They discuss the factors which affect molecular mobility across natural and artificial membranes, which include both electroosmotic and electrophoretic effects. Electrophoretic enhancement is a general increase in ion motion in response to the electric field. Diffusion of cations, anions, and neutral molecules across membranes are all affected by an electric field. Schreier et al. (1993) discussed an electrophoretic mechanism, but the authors felt that more research was necessary to make a definite conclusion. It can not be the primary mechanism for enhanced flux from beetroot because living beet cell membranes are normally impermeable to betacyanins (Zhang et al., 1992). The cell membrane must be permeabilized in order for the dye to effuse from the cell.
A living cell has a thin, dielectric membrane with high resistance (about 104^ Ω
cm2) and a capacitance (C) of about 1 μf/cm2 (Williams et al., 1964). When a cell of
The maximum electric field within the cell membrane (Em) is simply the ∆ψmax
divided by the membrane thickness (d), which is about 10 nm for plants (Fensom, 1985). Thus, Em = (∆ψnat -1.5 R E)/d Beet cells have a ∆ψnat of -154 mV (Zhang et al., 1992) and an approximate
diameter of 45 μm (Joersbo et al., 1990). The Em of a beet cell will increase from about
1.54 × 105 V/cm to 2.36 × 105 V/cm when placed in an electric field of 24 V/cm, which is typical of our laboratory ohmic heating conditions. This corresponds to a hyperpolarization of 83 mV, for a ∆ψmax of 233 mV.
Coster (1965), while charging the membranes of giant algal cells with a microelectrode, observed that hyperpolarizing currents caused a sudden increase in permeability when the membrane potential reached about -300 mV. He called the phenomenon "punch-through", but it is now known as “electroporation” or “electropermeabilization”. Zimmerman et al. (1974) used an externally applied high- voltage field to cause "dielectric breakdown" of red blood cell membranes. Most research on electroporation uses a pulsed electric field method which is used to insert DNA into cells during transformation. Molecules, such as DNA, that are to be inserted are included in a suspension of cells which are electroporated. Under the right conditions, pores are formed in the membrane through which diffusion occurs. The pores close up, and the cell recovers (Tsong, 1989). The mechanism of electroporation involves aqueous pores (Weaver, 1987), which are channels, about 0.4 nm in width, naturally present in biological membranes (Kotyk
and Janacek, 1975). The surface of a pore is continuous with the inner and outer surfaces of the cell membrane, which is composed of phospholipids (Stein and Danielli, 1956). Therefore, they have a negatively charged surface surrounded by hydrogen ions. They are normally permeable to water, but not to most ions or other molecules larger than water (Kotyk and Janacek, 1975). When a cell is exposed to an electric field, the ions accumulating at the membrane surface are preferentially drawn to the aqueous pores, which have a much higher capacitance than the lipid fraction of the membrane (Weaver, 1987). The ions pushing on the pore cause a pressure which expands it. When the hole has become large enough for ions to pass through, a decrease in resistance is measured. Experiments which used single pulses of 10 nsec to 1000 μsec revealed that the threshold breakdown voltage is inversely proportional to pulse duration (Zimmerman and Benz, 1980; Joersbo et al., 1990). That is, a longer pulse can cause breakdown at a lower field strength. Because it takes several minutes for the cell membrane to recover completely (Zimmermann and Benz, 1980), pulses given in quick succession (every 0. sec) have a cumulative effect (Lindsey and Jones, 1987). Ohmic heating, because it uses alternating current, is analogous to a sequence of pulses. The number of pulses per second is simply the frequency. The duration of an equivalent pulse would be the time spent above the threshold voltage and is therefore a function of the voltage amplitude, the threshold voltage, and the frequency. Pulsed radio- frequency (Chang, 1989) and 50 Hz alternating current (Joersbo and Brunstedt, 1990) pulses have been used for electroporation.
