PROPERTIES BY ELECTROPORATION OR PULSED

ELECTRIC FIELDS

Technical Field

[1] The present disclosure generally relates to food processing, food preparations prior to cooking, and more particularly to electroporation of meat for the enhancement of texture, flavor and moisture content.

Background Art

[2] Various methods have long been used to enhance foods to provide a more

enjoyable, and sometimes safer, eating experience for humans or animals. Food enhancement may include a variety of modifications of a food's properties such as, for example, the enhancement or maintenance of flavor, texture, moisture, color, and/or appearance. Examples of such methods include brining, the use of spice and flavor rubs, marinating, tenderizing, and the injection of flavorings and chemicals.

[3] Traditional brining of meat products is known to improve meat flavor and provides moisture control, but has certain drawbacks. For example, brining of large pieces of meat is time consuming. Also, brining an entire turkey can require twelve hours. Large amounts of salt and spices are also required to provide even a modicum of flavoring to the meat, often resulting in waste of the salt and spices.

[4] Spice and flavor rubs are used to provide flavor enhancement to foods. However, rubs only provide flavor to the surface of the food rather than a more desired uniform distribution of flavor through the entire food. Rubs also are not effective in controlling the moisture content of the food. Similarly, marinating primarily provides flavor to the surface of the food, and is further a slow and typically messy process.

[5] For meat products, tenderizing tough meat can be accomplished with aging, the use of chemical meat tenderizers such as papan, or by mechanical means. These approaches have drawbacks. For example, aging meat is time consuming, and in the case of beef as an example it can take several weeks to fully age. Other drawbacks include, moisture loss, the increased chance of developing foul flavors from oxidation of unsaturated fats, and the increased chance of contamination from microorganisms, mold growth, and spoiling. A mechanical method of tenderizing meat breaks up the connective tissue to form a more tender piece of meat, but this method cannot provide moisture control or flavor enhancement.

[6] Chemical meat tenderizers, such as papan, function similarly to the natural

enzymes in meat, which help soften connective tissue as the meat is aged. However, chemical tenderizers tend to be inactive at refrigerator temperatures, only somewhat effective at room temperature, and inactive above 140 °F. Furthermore, chemical tenderizers tend to work on the surface of the meat only, leaving the interior of the meat tough. Overexposure of the meat to papan leads to the breakdown of the connective tissue matrix of the meat and of the protein imbedded in the cell membrane of the cells forming the meat. The proteins are broken down, which undesirably allows additional water to escape through the cell membrane of the meat's cells during cooking, resulting in a drier meat.

[7] Lastly, injection of flavorings and chemicals into a food using a syringe also has drawbacks. For example, for a food such as meat, injection is ineffective in providing a uniform distribution of flavors, and undesirably depends on non-natural chemicals to manipulate properties of the meat.

[8] Accordingly, it is desirable to have an improved method and apparatus for

enhancing the properties of food.

Disclosure

[9]

Summary of Invention

[10]

Technical Problem

[11] Marinating and light brining foods happen at the cellular level, salt and other ions cannot pass thru cell membranes quickly without assistance. Meats exposed to a light brine (3% to 6% of salt to water by weight) require hours to days for the brine to take effect. The ions cannot pass into the cells even under higher concentration gradients until the trans-membrane proteins within the membrane get denatured from the ions first (it is analogous to trying to push a door open when the door is locked and you don't have the key. You can try to cut the hinges to get in or break the lock but this takes time.) The ions must work on denaturing the trans-membrane proteins but this is a difficult task because these proteins have evolved to resist this form of attack with clever structural designs one example is small abrupt hydrophobic to hydrophilic interfaces. The protein structure matches the membrane structure perfectly and resists attack by 'molecular crowbars' making gaining entry time consuming.

[12] Once the trans-membrane proteins finally get denatured the ions flow into the cell and disrupts the structure of the muscle filaments. It dissolves part of the structure that supports the contracting filaments by breaking hydrogen bonds and changing short range interaction forces within the protein resulting in protein unfolding. This unfolding process makes the muscle fibers have a greater water-holding capacity analogous to a sponge. The muscle cells then absorb water salts and flavor from the weak brine the meats weight typically increases by 10% or more. When the meat is cooked it does loose up to 20% of its weight in moisture but this loss is partially balanced by the brine absorbed. The result is a juicer more flavorful meat but at a cost of several hours or even days.

[13] Marinades also take hours to days to affect meat. The diffusion of acid into the meat is even slower than that of a brine. The longer meat is exposed to marinade the deeper the flavor and tenderization gets but unfortunately extending the time results in sour flavor and mushy texture outside and depending on the meat rubbery inside. Marinades typically have an acidic component such as vinegar (acetic acid), wine (tannic acid), fruit juices (citric acid) and buttermilk (lactic acid). These acids help to break down the extra cellular proteins. In the era before refrigeration marinades were used to prevent spoilage but are now primarily used for flavoring.

[14] Chemical meat tenderizers are often added to marinades to try to enhance softening of connective tissue. Tenderizers are protein-digesting enzymes commonly extracted from fruits and plants. The popular tenderizer Papan is an enzyme extracted from the papaya fruit. Enzymes act slowly at refrigerator or room temperatures but up to five times faster at cooking temperatures. Nearly all tenderizing takes place during cooking and unfortunately these enzymes penetrate very slowly (only a few millimeters per day) so the result is a tough rubbery piece of meat with a mushy almost slimy exterior.

[15] Vacuum based marinade systems are an improvement to the traditional marinade process, which is performed at atmospheric pressure. But these so-called 'instant' marinating appliances take up to 30 minutes or more to work depending on the meat size surface to mass ratio. The vacuum based systems are limited by the container size so only smaller cuts of meat work. The vacuum based systems rely on the fact that at atmospheric pressure (101.3kPa) carbon dioxide is readily soluble in aqueous environments approximately .614 mole fractions of water at room temp (25 deg C) and remains as carbonic acid within cellular fluids. At reduced pressures (75 kPa) a portion of the carbon dioxide goes back to gas form until the mole fraction reaches equilibrium at approximately .455 mole fractions of water at room temp. This partial release of carbon dioxide gas slowly creates microscopic bubbles within the cells. These bubbles eventually exit the cell through the membrane thus displacing the gas volume with extra-cellular fluid volume. If the marinade which is usually acidic and diffuses slowly has made it to the cells then marinade gets displaced into the cell. Typically this happens three eights of an inch into the meat and once the gas reaches the equilibrium solubility for the partial pressure in the container the gas escape from the cells stops and the marinating will then resemble the diffusion limited traditional marinade process.

[16]

Solution to the Problem

[17] The solution to the problems described above is to treat the meat with pulsed

electric fields (electroporation). The invention makes the brine and marinade process more efficient in time and in moisture and flavor absorption. By using the exemplary apparatus described in this disclosure pierce; clean uncooked meat with the fork probe electrodes, press the trigger switch and it administers a suitable electroporation pulse (approximately 15-microsecond pulse with electric field strength of approximately 800 volts per centimeter). This pulse will instantly open pores in the cells to allow marinade a direct path to the muscle fibers. Remove the probe and move to adjacent un-treated locations. Continue to administer pulses to treat the entire piece of meat. Immediately thereafter expose the treated meat to your favorite marinade by placing the meat in a bowl or bag of marinade, brine or flavoring.

The marinade or brine will travel down the channels cut by the fork probe electrodes. The process of diffusion will allow the transfer of the agents into the food cells from the channels into the freshly made pores. A marinade with weak brine, less acid and little to no tenderizers (optional on the toughest meats) is all that is required. It is optional to massage the meat for a few seconds this helps the marinade get exposure to all surfaces and to the holes caused by the fork probe electrodes. In approximately six minutes the cell membranes will have resealed and food is ready to cook. Pores in the membranes will stay open longer at cooler temperatures and as an added benefit cooler temperatures will also prevent bacterial growth in the meat and marinade. Cooking can begin sooner than six minutes if desired because the membranes will reseal from kinetic interaction as the temperature rises before cooking really begins.

One object of this invention is to provide a faster, safer, more economical and efficient method and apparatus for transiently forming holes in dielectrics, such as the membranes of the skeletal muscle cells of meat to enable the efficient diffusion of aqueous ions, salts, flavorings, oils, marinades and other agents into the food cells by the application of a controlled electric field (electroporation or pulsed electric fields (PEF)). This is accomplished through improved designs of the electrode assembly and the high voltage pulse generating power supply optimized for the enhancement of food properties. This invention greatly expedites the process of marinating, tenderizing and the brining of food especially meat and other solid foods.

Advantageous Effects of Invention

This invention quickly enhances meats and other foods. Treatment can be done in seconds, the pores formed in the cell membranes allow marinades brines and natural food liquids to quickly diffuse and work in minutes not days or hours. Foods that have been treated by this invention are noticeably more flavorful, tenderer and much juicier after cooking. This tool can assist the cook with nearly instant brining, tenderizing and marinating. This time saving tool allows for more spontaneous and more expansive menu selection. The cook is no longer required to pre-plan for marinated recipes. With pre-preparation time no longer a determining factor, using this invention the cook's menu is only limited by imagination and the availability of ingredients. The embodiments of the invention described here make it safe, convenient and easy to use anywhere; it's compact and fits easily in a standard cutlery drawer.

Description Of Drawing For a more complete understanding of the present disclosure, reference is now made to the following figures, wherein like reference numbers refer to similar items throughout the figures:

FIG. 1 illustrates a hand held electroporation based food enhancement tool according to an exemplary embodiment of the present disclosure;

FIG. 2 is an illustrated part breakdown of the hand held electroporation based food enhancement tool according to an exemplary embodiment of the present disclosure;

FIG. 3 is an illustrated part breakdown of the hand held electroporation based food enhancement tool with additional features according to an exemplary embodiment of the present disclosure;

FIG. 4 is a high-level block diagram of an electronic pulse generation system, according to an exemplary embodiment of the present disclosure, that may be used in the electroporation method described herein;

FIG. 5 is a circuit schematic of a pulse generation system having a battery powered, high- voltage power supply and a single secondary winding on the impulse transformer for a two electrode probe device according to an exemplary embodiment of the present disclosure;

FIG. 6 is a circuit schematic of a pulse generation system having a battery powered, high- voltage power supply with selectable current and primary windings enabling various voltage outputs for multi-electrode, multi fork probe devices according to an exemplary embodiment of the present disclosure;

FIG. 7 is a diagram that illustrates how the electroporation process works on muscle cells and how it enhances the properties of the treated food;

The exemplification set out herein illustrates particular embodiments, and such exemplification is not intended to be construed as limiting in any manner.

Detailed Description

The following description and the drawings illustrate specific embodiments sufficiently to enable those skilled in the art to practice the systems and methods described herein. Other embodiments may incorporate structural, method, and other changes. Examples merely typify possible variations.

As used herein, the terms 'electroporating', 'electroporation' and 'pulsed electric fields' mean applying, or the application of, an electrical pulse, a high- voltage current, or otherwise electrically treating a cell to create openings or pores or other mass transfer paths in its cell membrane for the movement of material into and/or out of the cell. The openings are usually transient (i.e., reversible electroporation), but in certain applications described herein the electroporation may be irreversible. Electroporation includes, but is not limited to, temporarily permeabilising cell membranes to facilitate the entry of molecules as in, for example, transfection. A specific example of electroporation is the application of an approximately ten-millisecond electrical pulse with electric potential gradients in a food of about 800 V/cm.

[35] As used herein, the term 'agent' or 'food agent' means any material that affects a characteristic or property of a food such as a brine or marinade. Food properties that may be enhanced by a food agent include, but are not limited to, flavor, color, texture, and moisture content. Food agents may include, but are not limited to, charged ions, water-soluble flavorings, chemicals, salts, spices, sugars, water, fats, and the like that can be transported into the cells of the food to provide enhancement or maintenance of flavor, moisture content, texture, color, aroma, stability, and the like. These agents can be derived, for example, from natural and/or man-made sources including extracts of spice, fruit, vegetables and or natural organisms including those produced using genetically-modified organisms (GMOs) and animals. Chemicals may include all food processing chemicals such as, for example, anti-oxidants, bacterial static chemicals, anti-fungal chemicals, enzymes, stabilizers, emulsifiers, and the like. An agent may be formed from a plurality of the foregoing food agents to provide a food agent solution. The agent may be, for example, administered alone or with a food- safe formulant that, for example, enhances transfection efficiency. Suitable formulants may include, but are not limited to, for example, divalent transition metals, polyanionic compounds, and peptides.

[36] The present disclosure describes a method of enhancing one or more food

properties in a food by electroporating the food using electrical pulses. The pulses are typically high-voltage pulses, and the food typically contains cells that become permeable from the electroporation. The food is exposed to food agents, which diffuse into the food's cells to enhance properties of the food.

[37] The present disclosure also describes an electrical pulse generation system that may be used in the electroporation method described herein. For example, this system may form pulses using a high-voltage, pulse forming circuit as described further below. For example, such a circuit may include an electric pulse generator such as a solid-state electronic high voltage pulse generator or a piezo-electric crystal based high voltage igniter and probes for delivering the electrical pulses to a food, all as described below.

[38] Electroporation may be used to facilitate the delivery of food agents such as, for example, charged ions or salts, into the cells of the food more quickly and/or efficiently than prior methods in order to enhance flavor, texture, moisture content, and/ or other food properties. The electroporated meats typically may be processed faster than by traditional curing, brining, basting, and marinating methods. Furthermore, the electroporated meats are typically juicier and/or more palatable. As electroporated foods typically contain higher moisture content, they may be more tolerant of cooking at higher temperatures, and thus more readily allow the reduction or elimination of undesirable bacteria and microorganisms in the food.

[39] Meats that typically may be electroporated as described herein include, for example, beef and beef products, poultry and poultry products, pork and pork products, fish and fish products, and the like. Also, electroporation may be used at various stages of food processing. The disclosure here is primarily describing an appliance to be used on meat to expeditiously enhance the flavor texture and moisture content by the cook just prior to cooking

[40] Foods such as meats are made up of cells, which in turn typically have well- defined cell boundaries. Meats are made up of many anchorage-dependent cells that have extra-cellular proteins that hold the cells in proximity with other adjacent cells. The cell boundaries are typically made up of a lipid bi-layer membrane, which isolates the interior space of the cell from the exterior space. Normally protein channels including trans membrane protiens regulate the passage of molecules in and out of the interior of each cell. Traditional methods of enhancing flavor texture and moisture content such as brining and marinades rely on the slow denaturing of the protected trans membrane proteins with salts and lowered pH. These trans membrane proteins are relatively robust and take time to denature because most of the protein is imbedded within the cell membranes with very little exposed to the aqueous environment. It takes a long time for the salt ions to slowly unwind and eventually denature the protein to create openings into the cells to allow access of more ions into the unprotected proteins within the cell further denaturing and assisting the moisture holding capacity of the cells.

[41] A safe non-chemical method of making the cell membranes of the meat temporarily porous by using electroporation enables the food agents such as marinade to diffuse more quickly into the meat cells expediting the brining and marinating process instantly. The choice of appropriate electrical pulses permits the transport of food agents and/or out of the cell by, for example, temporarily opening the cell membranes to allow the desired food agents to quickly enter the cell (e.g., via natural diffusion). The cell membrane is usually not destroyed in the process, and the cell membrane is in most cases only temporarily opened. Thus, typically, the food's structure remains substantially intact.

[42] Each type of meat has different properties but cell membranes are all approximately the same thickness of about five nanometers. The critical membrane potential for a membrane to become temporarily porous is almost universally one volt but can vary slightly depending on the pulse width and composition of the cell membranes.

[43] Meat that is to be processed using electroporation may be, for example, pierced by a plurality of electrodes used to apply electrical pulses. The user can administer a single pulse or multiple pulses per location. It is most efficient to apply the electric field pulse or pulses perpendicular to the grain of the meat. Smaller close-grained meats such as pork benefit from multiple pulses whereas larger long grained meats such as poultry may require only one pulse. Meats with a lot of connective tissue may need more piercing from the electrode probe array than meats with less connective tissue.

[44] Soon after the cell membranes are pulsed using the electroporation probes, a

charge is believed to develop upon the surface of the cell membranes in the food until the cell membranes are compromised. The cell membrane in the cells of a food is typically a lipid bi-layer predominantly formed of phospholipids. These lipids have a polar phosphate head, which is hydrophilic, and a fatty-acid, non-polar hydrophobic tail. These lipids form a lipid bi-layer as the heads face outwards and the tails face in towards one another to form the lowest energy configuration at rest.

[45] The repulsive electrostatic forces built up on the hydrophilic membrane surface from the electroporation are believed to overcome the Van der Waals forces in some of the weaker locations of the cell membrane. The weak forces attracting the phospholipids hydrophobic tails to each other in these local regions of high charge are not sufficiently strong to hold the lipid bi-layer membrane together during electroporation. The result is the formation of porous locations at the weak spots of the cell membrane. The porous locations provide mass transfer paths for food agents move into and out of the cell.

[46] The porous locations formed in the cell membrane typically stay open for a fairly short time (e.g., anywhere from about 30 microseconds to about 8 minutes depending on the electroporation conditions and parameters). The factors that affect the cell membrane's ability to be electroporated and the time that a cell membrane will remain permeable include, for example, the nature of the pulses, the pulse amplitude and duration; the nature of the cells, including, for example, its temperature, pH, and the isoelectric point; and the amount of static-charge buildup on the cell membrane. The food may be exposed to one or more pulses as necessary to provide the desired food properties.

[47] Before or after electroporation, food agents (e.g., salts and water-soluble

flavorings) may be injected into the food via a delivery means so that the food agents are present in the extracellular fluid matrix for entry into the cells of the food once a mass transfer path is provided by electroporation. The opened cells will allow the agents in the extracellular fluid that are typically now at higher concentration gradients than the intracellular fluid to naturally transport into the food cells (e.g., by diffusion). In a fairly short time the charge potentials on both sides of the cell membrane reach charge equilibrium. The charge equilibrium results as the concentration between the intracellular and extracellular fluids reaches concentration equilibrium, typically causing the cell membrane to close. The simplest and most preferred method is to treat the meat to be enhanced by electroporation then transfer the meat into a container or bag containing the marinade or agent of choice.

[48] The mechanism for closing the cell membrane is believed to result from the natural interaction of several small forces on the suspended phospholipids. The random thermal Brownian motion of the phospholipids and other molecules that were previously dislocated attract and bond as the Van der Waals attractive force becomes greater than the now depleted repulsive electrostatic forces. The result is that the phospholipids substantially line back up to the configuration of lowest energy with the hydrophobic fatty acid tails toward the center of the lipid by-layer and the polar hy- drophilic phosphates facing out, thus naturally sealing the cell membrane.

[49] Once the cell membrane has closed, the extracellular fluid may be diluted with a small amount of water. This can be done, for example, through a surface rinse if desired but it is not required. A dilution of the extracellular fluid causes the concentration gradient to be reversed. Higher food agent concentration now is present in the cell, and lower agent concentration is present outside the cell (i.e., in the extracellular fluid). Since the cell membrane is closed and the cell's new contents (i.e., the food agent or agents) cannot be readily transported out of the cell through the cell membrane, substantially the only way that concentration equilibrium can again be reached is if the cells receive more water. Water may enter the cells through osmosis in order for the intracellular fluid to reach concentration equilibrium with the now diluted extracellular fluid. The overall result is that more water, agents, and flavors can be stored within the cells.

[50] When the agents include, for example, mono-valent cations such as sodium (Na ⁺ ) and/or potassium (K ⁺ ) cations, or when treatment by a food agent cleaves the hydrogen bridges, ionic, or hydrophobic bonds, then denaturation of the proteins in the cell is possible. What is typically required to cause water-soluble proteins in a food to denature is heating the food to a bulk temperature in the range of 70 to 90°C (158 to 194°F) and the presence of these ions in the food cells. The result is that the protein's tight spirals unwind to form a tangled mesh during the heating of the cooking process. This collagen protein becomes a gel mesh that helps the food cells retain water and flavors in the food during the cooking process.

[51] Because the electroporated food retains more moisture, the food can usually be cooked at higher temperatures without compromising moisture and tenderness. For instance, it is recommended that a turkey prepared without electroporation should be cooked to an internal temperature of 165°F to ensure destruction of food-borne microorganisms. Unfortunately, the high temperature removes a significant amount of moisture from the turkey. An electroporated turkey often can be cooked to temperatures up to 180°F and still be very moist and flavorful because the gel mesh of proteins acts like a sponge to keep moisture and flavor. The higher temperatures will destroy microorganisms more effectively, with less undesirable impact on food flavor, texture, tenderness and/or moisture content. The higher temperature cooking will generally increase food safety and food quality since higher food temperatures are more effective at killing bacteria.

[52] An exemplary process to enhance the flavor of foods using electroporation includes one or more of the following steps: selecting the food type; piercing the food with a plurality of probes electroporation of the food; short contact exposure food agents marinade; brief diffusion pause time; and optional dilution with water to form an electroporated food. The diffusion pause time will depend upon the food type and temperature. A typical pause time may be less than about eight minutes, specifically less than about five minutes, and yet more specifically less than about one minute. The electroporated food can then be packaged, cooked, stored, and/or transported.

[53]

Probe Structure

[54] In one embodiment, the step of electroporating may comprise contacting and

pulsing the food with an electrode comprising one or more pairs probe electrodes (i.e., one probe to act as an anode and the other probe as a cathode). The electrodes may be made, for example, into a fork. The step of electroporating an entire piece of food may require repositioning of the electrodes.

[55] Electroporating of a food may comprise contacting the food with a first electrode probe pair in a first position and contacting the food with a second electrode probe pair in a second position, and then electrically pulsing the food using both electrode pairs. If more than one electrode pair is used, the steps of contacting may be sequential or simultaneous. The number of pulses may vary depending on food type, average cell size (e.g., diameter). Pulse trains may vary in number and duty cycle to minimize joule heating of the food and to maximize electroporation efficiency for each specific food type.

[56] FIG. 1 illustrates a hand held piezo-electric electroporation apparatus for the enhancement of food properties with a pair of three pronged fork style probes that function to pierce meat and act as conducting electrodes 10. The collar 14 made from an insulating material holds the electrodes and the electrode spacer 11 onto the body 20, which provides a rigid non conducting handle for the user. A piezo-electric igniter 30 functioning as a high voltage pulse generator provides the high voltage pulse delivered to the electrodes. The high voltage piezo-electric igniter 30 can be substituted by other suitable pulsed high voltage sources such as a battery powered solid- state module or other high voltage pulsed circuits suitable to perform Electroporation.

[57] FIG. 2 illustrates a simple hand held piezo-electric electroporation apparatus for the enhancement of food properties where the probe assembly is fixed to the body 20 the electrodes 10 are held apart by a non-conductive spacer 11. Note spacer 11 thickness helps determine the electric field strength a larger spacer thickness for a given voltage will produce smaller electric fields compared to thinner spacers for the same voltage. One experienced in the art can recognize by having a adjustable spacer thickness one can change electric field strength without necessarily having to change the voltage applied. Non-conducting screws 8 go through holes 1 and 2 they hold the terminal connectors 7 to the electrodes 10 and bind the assembly together by nuts 9. The fork style electrodes Probes 10 may be electrically coupled to the pulse forming circuit using electrical conductors 5 and 6. The conductors are shown going through the collar 14 and the body 20 where it can connect to the piezo-electric pulse generator 30 using connectors 24 and 25. The body which functions as a handle holds the piezoelectric pulse generator 30 and protects the user from contacting pulse generator circuit terminals and the connectors 24 and 25. The body 20 receives the electrode probe assembly in the slot 4 the probe assembly is fastened onto the body with collar 14 flush with the spacer 11 and the handle stop 3

FIG. 3 illustrates a two-piece hand held piezo-electric electroporation apparatus for the enhancement of food properties where the probe assembly FIG. 3B is removable from the body FIG. 3C. This provides the user the ability to easily remove, change or interchange, probe assemblies for specific food applications or for ease of cleaning.

FIG. 3 A shows a fork style electrode Probe 10 with a reduced conductor area 26 the probe electrode 10 is coated with an inert food grade electrically insulating coating 27 screw holes 1 are not coated 28 to ensure good electrical contact with the wire terminals 7. Electrodes 10 can be made of various grades of food grade stainless steel. Insulating coatings 27 can be any suitable inert food grade material such as non-stick fluoropolymers, ceramic or glass frit coatings.

FIG. 3B The removable probe assembly has a quick disconnect coupling 16 it can have a recessed opening to receive the handle 21 the guide channel 17 receives an alignment pin 19 on the handle 21 allowing the handle to lock onto the probe assembly when rotated. This can insure the electrical contacts 15 of the probe assembly align with the spring loaded electrical contacts 18 of the handle 21.

Electrode spacing is an important factor for control of the electric field. The thickness of the electrode spacer 11 is dependent on the performance of the pulse generator 30 used. Typically electrode spacing will be less than 3cm more than 3mm. Optimum electrode spacing will be dependent on the diameter of the cells and the conductivity of the extracellular fluid. Generally for a given voltage the probe electrode spacing will be directly proportional to the mean cell diameter.

Electroporation Conditions

Factors to consider in determining suitable electroporation conditions may include electrical properties associated with the electroporation such as the following: electric field strength, pulse duration, pulse number, and pulse frequency. Other factors may include physical and/or chemical properties of the food to be electroporated such as one or more of the following: pH, temperature, and conductivity. One of skill in the art is generally able to select appropriate values for the foregoing factors (see, e.g., Chang, Chassy, Saunders and Sowers, Guide to Electroporation and Electrofusion, Academic Press San Diego, Part III pg 429 (1992), which is incorporated herein by reference).

Depending on the food type, the electroporation process is more preferred when the food is at refrigerator temperatures of about 1 to 5°C (34 to 41°F). However, elec- troporation also typically works suitably at temperatures of about 5 to 50°C. There is a balance that may be varied between the amount of time the cell membranes remain open and the mobility of the agents to be delivered.

[65] Generally, as the food is cooler, the cell membranes remain open longer. As the food is warmer, the diffusion rate of the concentrated agents into the cells is greater. It is believed possible, but perhaps fairly slow, to electroporate at 0°C. At temperatures above 50°C, the probability of having permanently damaged cells is fairly high. Such high temperatures typically cause poor texture and moisture control. Because of the physical and chemical properties of the food, and that these factors typically affect how the food is shipped and stored, refrigerator temperatures are expected to offer more control of the electroporation process for the enhancement of food properties.

[66] Electroporation typically may be performed more effectively in food with a

hypotonic extracellular medium (i.e., a solution having a lower osmotic pressure than a comparison solution). This is believed due to the enhancement by osmotic stress of both electrotransfection and electrofusion efficiency in cells. In addition to osmotic effects, low-conductive media (e.g., media whose conductivity is less than that of the cytosol) is believed to increase the electropermeabilization efficiency of suspended cells due to the transient electrodeformation stretching force exerted on the plasma membrane during the field pulse application.

[67] Compared to concentrated saline medium, low-ionic solutions generally significantly reduce electrolysis, joule heating, and other DC current related phenomena, thus typically reducing or preventing damage to electromanipulated cells and decreasing post-pulse recovery times. The electroporation pulsing step is typically performed before agent delivery in most cases because of the foregoing.

[68] The electrodes used for electroporation (e.g., the pair of probes 10 illustrated FIG.

1, FIG 2 and FIG. 3) generally emit an electric field strength of less than about 10,000 V/cm. More specific ranges that typically may be used for various foods include, for example, about 1 to 1,000 V/cm, about 25 to 750 V/cm, about 50 to 500 V/cm, about 60 to 300 V/cm, and about 75 to 250 V/cm. Generally, a more desirable electric field strength is greater than about 100 V/cm.

[69] The pulse length may be, for example, from about 50ns (nanoseconds) to 200ms

(milliseconds). More specific ranges include, for example, about 4 to 40us

(microseconds), about 5 to 30us, and about 7 to 25us. As a specific example, suitable electric field strength of about 100 V/cm to 800 V/cm may be used with an electrical pulse length of about 10 to 20us. Pulse lengths are generally kept less than 200ms to minimize permanent cell damage and joule heating.

[70] A suitable number of pulses in a pulse train applied during electroporation is

typically from about 1 to 30 pulses (e.g., about 2 to 20 pulses, about 4 to 15 pulses, about 5 to 12 pulses, or about 5 to 6 pulses). The pulse number may also be increased. As the pulse lengths get smaller, the narrow pulse length typically helps minimize damage from joule heating.

[71] Pulses can be rectangular, triangular or exponential typically quicker rise times of mono phasic pulses work best such as an impulse, but quick fall times from under damped bi-phasic pulses are equally effective.

[72] Mammalian cells like those, for example, of bovine skeletal muscle (beef) are

relatively large and are effectively electroporated with low field strengths (e.g., 600 to 800 V/cm), using pulses of, for example, 2kV amplitude and allow for a wider electrode spacing (e.g., about 2.5 cm). At these modest field strengths, arcing usually does not occur, even in buffers with relatively high ionic strength.

[73]

Food Agent Delivery

[74] The food agent or agents may be administered to the food using any suitable

means. Suitable methods of administration of the agents to the food include, for example injection of the agents into the food using a syringe. Delivery of the agent may be, for example by surface exposure where the electroporated meat is put into a container or bag containing the agent. The agent can contact the food for a short time or a longer time and alowed to soak. The particular delivery system or device is not a critical aspect of the process except that exposure of the food agent to the food should be done promptly (as soon as the electroporation treatment is complete for a particular piece of food or cut of meat) the agent will diffuse into the cells only as long as the cell membranes are porous. A prompt post electroporation exposure will maximize contact time and effectiveness.

[75] According to the approach described herein, electroporation may be used to

enhance the efficiency of agent transfer across the cell membrane. The agent may be administered into the food either before or after the electroporation in preparation for diffusion of the agent into the cells of the food.

[76] The cells of the food may be contacted with more than one electrode probe pair, in which case the contact of the multiple electrode probe pairs may be, for example, simultaneous or sequential. Also, the food may be contacted with the electrodes in multiple positions. For example, the electrodes may be positioned vertically, longitudinally, or horizontally for contacting the food. The electrodes may also be positioned at angles to each other when contacting the food. Suitable angles may include, for example, 45, 60, 75, 90, 120, 160, or 180 degrees.

[77] Usually it is desirable to position the electrodes to ensure that substantially the entire food is exposed to electric pulses. One of skill in the art will appreciate that the position of the electrodes may be adjusted as needed to create an electric field that desirably may extend throughout substantially an entire piece of food during electroporation. As an example, because of the grain of the skeletal muscle in meat, a desirable angle to position the electrode is 90 degrees perpendicular to the food cell grain.

[78] another example, some skeletal muscle cells have a length of several centimeters with diameters ranging from 10 to 100 micrometers. A definite grain structure is typically observed. Therefore, moving the electrode probe pair(s) every few centimeters along the grain of the food to be treated may optimize the electroporation efficiency. Inserting the probes perpendicular to the grain at regular intervals may also aid in improved speed and coverage.

[79] The food may be contacted with a wide variety of structures of electrodes such as, for example, needles, probes, needles with paddles, needles with rotating paddles, and needles with flat plates or calipers. Electrodes also may comprise an array of multiple needles, probes, needles with paddles, and/or flat plates. Certain methods of electroporation for medical uses, which may be adapted for use with the method described herein, are described in U.S. Patent Nos. 6,233,482, 6,135,990, 5,993,434, and

5,704,908, which are incorporated herein by reference.

[80] One of skill in the art will appreciate that the electrode probes can be partially

coated with a dielectric insulator to protect the user from incidental contact and to limit the contact area of the conductive path with the sample to be treated by electroporation.

[81] One of skill in the art will appreciate that the space between two needles on any given electrode (e.g., thickness of spacer 11 shown in FIG. 1, FIG. 2, and FIG 3) may be varied depending on the electric field (e.g., V/cm) requirements. The space between needles or probes may range, for example, between about 0.1 to 5 cm. Other specific examples include spacings of 0.25, 0.4, 1, or 3 cm. Other configurations of the electrodes or electrode arrays, and other angles or shapes of needle arrays, may be used to meet particular size and access needs. One of the factors used in determining suitable electroporation conditions is the electric field strength. In addition, the probe electrode dimensions are also considered in creating the desired electric fields for electroporation.

[82] One of skill in the art will recognize more sample can be treated simultaneously with an apparatus that has more electrodes spaced over a wider area this capability can be satisfied with the use of the circuit schematic shown in FIG.6

[83]

Pulse Generation System

[84] As breifly discussed earlier prior art electroporation equipment used

bioscience community is unsuitable for use as an food property enhancer, these systems are large, dangerous complex, expensive and not well suited for adaptation to consumer use. These commercial systems are comprised of large high voltage power supplies with multiple banks of large high voltage capacitors capable of producing substantial current at high voltages at up to 2500 volts, such as that disclosed by Ragsdale on Jun. 7, 1988 in U.S. Pat. No. 4,750,100. [85] Brief pulses of high voltage are required to electroporate cells. A purpose built electroporation system tailored to the specific needs of food enhancement and user safety is needed. These embodiments disclosed here eliminate the problems of storing high voltage energy in long-term high voltage storage capacitors that would otherwise cause safety concerns for the user. By employing piezoelectric elements or by transiently storing the high voltage energy in a magnetic field rather than an electric field makes the electroporation system disclosed here more economical to construct since large expensive high voltage storage capacitors are not required.

[86] The rapid mechanical deformation of piezo-electric crystals provides an ideal high voltage pulse. This invention provides a system more compact and safe, yet much less complicated and expensive than other electroporation systems. Piezo-electric igniters typically produce a voltage pulse of lOkv to 20kv (strong enough to jump a 5mm spark gap) typically output is 17kv across a 3 pico farad lOOmega Ohm load. When the voltage pulse is discharged into a 1000-Ohm load (representing a typical meat resistance measured between electrodes) the voltage pulse peak to peak amplitude is approximately 700V. The duration of this pulse is typically 10 to 15 microseconds providing a quality electroporation pulse protocol for the membranes enclosing muscle fiber bundles in meat provided electrode spacing is approximately on centimeter.

[87] Impulse transformers also provide an ideal high voltage pulse with more power and flexibility than the piezo electric systems. Instead of storing the energy in large dangerous and expensive high voltage capacitor banks. The energy can be stored in smaller lower voltage capacitors the current impulse from the capacitors into the primary winding of an impulse transformer will provide the required current to produce a magnetic field large enough so that when the magnetic field collapses it produces an output pulse in the secondary with current, voltage and duration characteristics suitable for proper electroporation of meat. The impulse transformer powered electroporator high voltage pulse source provides a safe compact system that allows for multiple fork probe electrode systems. Multiple fork probe electrode systems naturally require more power than two fork probe-electrode electroporation systems. In order to increase output power one needs to increase input power by increasing voltage and or current methods to control the current and voltage are discussed in following sections.

[88] FIG. 4 is a high-level block diagram of a pulse generation system that may be used in the electroporation method described herein. System comprises a battery a boost circuit a charge pump storage capacitor a trigger circuit pulse forming transformer. A pulse or a pulse train of electrical pulses is delivered to a food using fork probe electrodes (e.g., probe 10 of FIG. 1, FIG. 2, FIG. 3).

[89] FIG. 5 Is a circuit schematic of a pulse generation system for use in the hand held battery powered apparatus for the enhancement of food properties by electroporation or pulsed electric fields according to an exemplary embodiment of the present disclosure; the circuit contains more detail introduced by the high-level block diagram of a pulse generation system shown in FIG. 4.

[90] FIG. 5 is a circuit schematic of a pulse generation system having a battery 40 as a possible DC power supply. The feedback boost oscillator comprises the primary windings of transformer 43 the primary winding 44. The feedback winding 45 is connected to the capacitor 47 forming a tank oscillator. Switch 42 starts the oscillations in transformer 43 resistor 41 provides the initial time constant. The primary winding 44 and secondary winding 46 are one hundred eighty degrees out of phase from each other when the magnetic field collapses in the transformer induces a current in the secondary winding 46 connected to the base of transistor 48 inducing a pulse in the primary winding 44 to sustain oscillations. Capacitor 49 and secondary winding 46 form a tank circuit the charge pump comprising the secondary of the transformer 43 and diode 50 rectify the current pulses and store the charge on storage capacitor 51. The charge stays until the trigger switch (momentary switch) 56 is closed and opened. The rapid drop in current causes the magnetic field to collapse in the pulse transformer 53 causing a high voltage current spike suitable for electroporation. The target food is represented by the RC network 58 through electrodes 57 FIG. 5 is only provided as specific example for purposes of illustration and not of limitation.

[91] The key feature in FIG. 5 is the pulse generating circuit comprising the

components and circuit topology to the right of the storage capacitor 51. The feedback boost circuit to the left of storage capacitor 51 can be replaced with other suitable DC- to-DC boost converter circuits. Those familiar in the art will note many possible circuit designs using discrete and integrated circuits exist to perform DC to DC boost converter and voltage regulation functions.

[92] FIG. 6 is a circuit schematic of a pulse generation system with modifications over the previous schematic (FIG. 5) for flexible output power, multi-electrode, multi fork probe devices. This Circuit schematic also shows a battery 40 but can be powered by another DC source. The high voltage pulse circuit to the right of storage capacitor 51 shows the high- voltage pulse generator impulse transformer 53 with selection switches 59 and 63 enabling various voltage outputs. Switch 59 allows the selection of current into the primary winding of the impulse transformer with resistor 60, resistor 61 or wire 62. The lower the resistance the more current will flow into the primary winding of the pulse transformer. Switch 63 allows the selection of different taps on the primary winding for the selection of the number of turns in the primary winding of the impulse transformer. The top tap 64 has the largest inductance and the most turns. The middle tap 65 has a lower inductance and smaller number of primary turns than tap 64 but more than the lowest tap 66 which has the least turns and lowest inductance. The trigger switch 56 is shown with an optional arc suppression capacitor 69.

[93] In FIG. 6 the impulse transformer 53 has multiple secondary windings 55, 70, 71 and 72 this design shows a device with 5 fork probe electrodes 73, 74, 75, 76 and 77. A 3-electrode fork probe system would be the same except secondary windings 71 and 72 and electrode probes 76 and 77 would be omitted. More secondary windings can be added to increase the number of fork probe electrodes. Fork probes can be larger and wider fork probes can have more tines added. Higher or lower power requirements will alter the impulse transformer design parameters such as dimension, wire size, winding number etcetera. FIG. 6 is only provided as specific example for purposes of illustration and not of limitation it is also obvious many of the features in FIG. 6 can be implemented in FIG. 5 such as the switches 59, 63 and capacitor 69.

[94]

Explanation of electroporation steps

[95] FIG. 7 illustrates the electroporation process steps demonstrated on skeletal mussel cells for the enhancement of food properties FIG. 7A shows a cutaway cross section of a myofibril (cell) highlighting the lipid bi-layer membrane. It is this membrane that the electroporation process causes to be temporarily porous. FIG. 7B shows the crossection of the intact cell before electroporation. FIG. 7C shows the cell after the electroporation event the pores form due to the temporary dielectric breakdown of the lipid bi-layer caused by the electric field pulse. FIG. 7D shows the exposure of the cell to food agents. FIG. 7E shows the high concentration of food agents diffusing into the cell thru the pores formed in step C by electroporation. FIG. 7F shows the cell membranes closing as the concentration of agents reach equilibrium. FIG. 7G shows the pores have all healed closed due to random molecular movements of the lipid membrane molecules. FIG. 7H shows agents denaturing proteins within the now closed cell causing an expansion of the water holding capacity of the cell.

[96] The process illustrated in FIG. 7 happens to all adjacent cells exposed to the electroporation electric field pulse. By moving the probe from one area to the next all sections of a food can be enhanced thus treating the entire cut of meat and enhancing the properties of the meat so it will be ready to cook, the resulting cooked meat is more tender, moist and flavorful due to this enhancement process.

[97]

Definitions

[98]

Detachable Designed to be unfastened or disconnected without damage.

Electrode probes A conductor used to make physical and electrical contact with the food. The term Electrode probes or probe

Electrodes are functionally equivelent. fork probe electrodes are a preferred embodiment but can be a conductor made in any shape or style, examples include; flat blades, round pins or needles.

Electroporator Is a device capable of creating pulsed electric fields suitable to perform electroporation. Electroporation Is a mechanical method used to introduce charged molecules into a host cell through the cell membrane. In this procedure, a pulse electric field temporarily increases the permeability of cell membranes allowing molecules like ions to pass into or out of the cell by diffusion depending on concentration gradients.

Macroscopic Large enough to be perceived or examined by the unaided eye.

Nanoscopic So small as to be invisible or obscure; extremely small; minute on the order of nanometers billionths of a meter.

Piezo-electric The property of certain crystals and polarized ceramic

materials that causes them to produce voltage when a mechanical pressure is applied to them, such as the force impact of a mass spring apparatus.

Piezo-electric Pulse Generator A pulse generator Having the ability to

generate a voltage when a mechanical force is applied.

Plurality The condition of being plural or numerous; a great number;

multitude

Pulse Transformer A transformer capable of operating over a wide range of frequencies, used to transfer nonsinusoidal pulses without materially changing their waveforms.

Self-contained Having all working parts of machinery, complete with motive power, in an enclosed unit: of said machinery.

[99]

Conclusion

[100] As was discussed above, several properties of food that may be enhanced or

maintained by electroporation include, for example, flavoring, coloring, stabilizing, moisture enhancement, texture, and the like. By adding agents to the food using electroporation, the properties of the food cells may be changed both internally and/or externally.

[101] Several food processes may be expedited using electroporation, typically saving both time and money. The slow process of brining can be accelerated with increases in tenderness and moisture content. In addition to making juicy, succulent food, the process herein may contribute to an increase in food safety, as the electroporated food may be able to handle higher temperatures that would normally overcook and texturally degrade untreated foods. This time saving device encourages the use of marinade which intern increases safety. It has also been shown that using marinades reduces the amount of heterocyclic amines (HCAs) by 90%* (HCAs are known cancer causing compounds found in grilled meats) *J. Smith, F. Ameri and P. Gadgil 'Effect of marinades on the formation of heterocyclic amines in grilled beef steaks' Journal of Food Sci. 2008 Aug;73(6):T100-5.

[102] Several methods are introduced to create a suitable electric field across cell

membranes to cause a temporary dielectric breakdown of the cell membranes thus creating pores. Therefore, such adaptations and modifications are within the meaning and range of equivalents of the disclosed embodiments. The phraseology or terminology employed herein is for the purpose of description and not of limitation. The key advantages in the disclosure here are safety for the user (no high voltages stored in capacitors no lethal currents) Ease of use and inexpensive to manufacturer.

[103]

CITATION LIST

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Aug;73(6):T100-5