Alternative Food Processing Techniques PDF

CHAPTER 11 Alternative Food Processing Techniques 11.1 Microwave Processing Microwave heating refers to the use of electromagnetic waves of certain frequen cies to generate heat in a material. Typically, microwave food processing uses two frequencies of 2450 and 915 MHz. The 2450 MHz frequency is used for home ovens and both are used in industrial heating. Frequencies of 433.92, 896, and 2375 MHz are also used. There is not much commercial use of these frequencies for food pasteurization or sterilization, although they are used in baking and other processes in the food industry. 11.1.1 Applications of Microwaves 11.1.1.1 Heat Generation with Microwaves Microwave processing takes place in nonconductive materials due to the polar ization effect of electromagnetic radiation. The polarization for heat generation in a material includes rotational responses of polarized molecules to an alternating electric ﬁeld (electronic polarization) and migration of charged ions (ionic polar ization). Microwave heating is widely used for drying, storage of frozen foods for further processing, precooking of meat, pasteurization, and sterilization. Microwaves are part of electromagnetic waves between infrared and radio frequencies. The wavelength of electromagnetic waves in free space decreases with increasing frequency. When microwave energy is incident on a food material, major part of energy is absorbed by the food, resulting in its temperature increase. Microwave heating takes place at molecular and atomic levels, as a result of polar and ionic polarization in food materials when exposed to electromagnetic ﬁelds. Dielectric properties play an important role in heating interactions between electric ﬁelds and food materials. Heating with microwaves involves two mecha nisms: dielectric and ionic. Water in the food is the primary component responsi ble for dielectric heating. Due to their dipolar nature, water molecules try to follow the electric ﬁeld association with electromagnetic radiation as they oscillate at the very high frequencies. Such oscillations of the water molecules produce heat. The Food Microbiology: Principles into Practice, First Edition. Osman Erkmen and T. Faruk Bozoglu. © 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd. 187 188 Chapter 11 second major mechanism of heating with microwaves and radio frequency is through the oscillatory migration of ions in the food that generates heat under the inﬂuence of the oscillating electric ﬁeld. The dielectric constant affects the strength of the electric ﬁeld inside the food. The dielectric properties depend on the composition (or formulation) of the food, moisture, and salt. The subsequent temperature rise in the food depends on the duration of heating, the location in the food, convective heat transfer at the surface, and the extent of evaporation of water inside the food and at its surface. 11.1.1.2 Advantages of Microwave Processing Microwave and radio frequency heating for pasteurization and sterilization are preferred over conventional heating for the primary reason that they are rapid and therefore require less time to come up to the desired process temperature. This is particularly true for solid and semisolid foods that depend on the slow thermal diffusion process in conventional heating. Microwave heating may be relatively more uniform than conventional heating, depending on the particular heating situation. Heating systems can be turned on or off instantly, and the product can be pasteurized after packaging. This processing system can also be more energy efﬁcient and improve food quality. 11.1.1.3 Uses of Microwave Processes Industrial microwave pasteurization and sterilization systems are present. The microwave sterilization process can vary signiﬁcantly among manufacturers. The design of the equipment can inﬂuence the critical process parameters: the location and temperature of the coldest point. Operational systems include batch process ing of yogurt in cups and continuous processing of milk. For in-package pasteur ization or sterilization, packaging materials need to be microwave transparent. Metals reﬂect microwaves, and packages with some metal components can considerably change the food temperatures. In some cases, metals can be added to the package to redistribute microwave energy to achieve heating uniformity. The most common packages for microwaves are transparent rigid ﬁlms, such as polypropylene with an ethylene vinyl alcohol (EVOH) barrier or a crystalline polyethylene terephthalate (CPET) ﬁlm. 11.1.1.4 Antimicrobial Effects on Microorganisms No pathogen is identiﬁed as uniquely resistant to microwave processing. Micro wave heating inactivates pathogenic microorganisms in foods. Bacterial inactivation by microwave heating includes Bacillus cereus, Campylobacter jejuni, Clostridium perfringens, pathogenic Escherichia coli, Listeria monocytogenes, Staphylo coccus aureus, Salmonella, Enterococcus, and Proteus. Microwave heating at 2450 MHz causes greater destruction of Aspergillus, Penicillium, Rhizopus, and aerobic micro organisms in foods than heating alone. Geobacillus stearothermophilus spores have lower D100 °C values when 2450 MHz microwaves are used compared with Alternative Food Processing Techniques 189 conventional heating. Parasite Trichinella spiralis, causing trichinosis, may also be inactivated by microwave heating. Foodborne pathogens can be inactivated by microwave heating in poultry, beef, ﬁsh, milk, and eggs. As with conventional heating, bacteria are more resistant to thermal inactivation by microwave heating than yeasts and molds, and bacterial spores are more resistant than vegetative cells. 11.1.2 Mechanisms of Microbial Inactivation Microwave heating causes microbial inactivation through thermal effects. Differ ent mechanisms are proposed for inactivation of microorganisms by microwave heating. The selective heating of microwaves involves inactivation of micro organisms by heat, such as denaturation of enzymes, proteins, nucleic acids, or other vital components, as well as disruption of membranes. The selective heating theory states that solid microorganisms are heated more effectively by micro waves than the surrounding medium and are thus killed more readily. The energy absorption from microwaves and radio frequency can raise the temperature of the food high enough to inactivate microorganisms for effective pasteurization or sterilization. The thermal effect is the essential contributor to the destruction of microorganisms as well as the degradation of vitamin B1, thiamin, and others. Microbial inactivation kinetics for microwaves are essentially the same as the inactivation kinetics of conventional thermal processing. There is little or no nonthermal effect of microwaves on microorganisms. Microwave heating at 2450 MHz inactivates E. coli and B. subtilis but is ineffective on B. subtilis spores. Microwave heating causes a greater amount of cellular injury, increases loss of ultraviolet-absorbing cellular material, and extends time for enterotoxin production and catalase oxidative reactions. Both conventional and microwave heating destroy 16S subunit of RNA in sublethally heated cells; only microwave heating also affects the integral structure of 23S subunit. Moreover, when cells are allowed to recover following injury, it will need longer time to restore their 23S RNA. Extensive research on heating uniformity, microbial reduction, product quality, and scaling is still needed for commercialization. 11.1.3 Factors Affecting Microbial Inactivation As with other thermal processes, the primary factors determining the safety with the microwave heating are temperature, time, moisture, ionic content, micro wave frequency, and speciﬁc heat. Other process factors affecting microbial inactivation with microwave heating are given in Table 11.1. 11.1.3.1 Factors Related to Product and Package Food shape, volume, surface area, and composition are critical factors in micro wave heating. These factors can affect the amount of absorbed energy, leading to overheating of corners and edges. Microwaves produce a higher internal rate of heating than near the surface. Such heating patterns can also change with time. 190 Chapter 11 Table 11.1 Critical factors affecting microbial inactivation in microwave heating. Food Shape, size, composition (moisture, salt, etc.), and liquid versus solid. Package Presence of metallic elements, such as aluminum foil. Process Power level, cycling, presence of hot water, air around the food, and equilibration time. Equipment Dimensions, shape, and other electromagnetic characteristics of the oven, frequency, and agitation of the food. Composition of food (such as moisture and salt percentage) has a much greater inﬂuence on microwave processing due to its inﬂuence on dielectric properties. Composition can also change the thermal properties of foods, such as speciﬁc heat, density, and thermal conductivity. These change the magnitude and uniformity of the temperature rise in foods. For example, the temperature of an oil increases at a much faster rate than that of water. Packaging material is also a critical process factor in microwave heating. Metallic components present in a package, such as aluminum foil, can dramatically inﬂuence the heating rates of the packaged food. 11.1.3.2 Factors Related to Process and Equipment Several process and equipment factors are critical in microwave heating. Design (size, geometry, etc.) of the microwave oven can signiﬁcantly affect the heat absorption in the product. The presence or absence of devices to improve uniformity, such as stirrers and turntables, is a major factor affecting temperature distribution. The placement of the food inside the oven can also have a signiﬁcant inﬂuence on the magnitude and uniformity of power absorption. Other factors related to the equipment are the temperature of the medium surrounding the product and the level of food surface evaporation. Cooling effects due to surface evaporation can allow survival of microorganisms or larvae of parasites on meat. Heating of liquids with microwaves without agitation causes ﬂow and thermal stratiﬁcation inside the container. Warmer liquid moves to the top, much like in conventional heating. Due to variations in product characteristics (such as viscosity), package components (such as metal in aluminum foil), and equipment, the patterns of temperature distribution in heating liquids (static or ﬂowing) can be quite complex, and the slowest heating location needs to be determined. 11.2 Ohmic Heating Ohmic heating is an alternative thermal processing method where heat is generated directly inside the food. Ohmic heating can ensure the beneﬁts of conventional thermal processing with respect to food safety, preservation, and retaining vitamins and nutrients. Ohmic heating is deﬁned as a process where electric currents are passed through foods or other materials with the purpose of heating them. The heating occurs in the form of internal energy generation within Alternative Food Processing Techniques 191 the material in terms of joule (1 kWh = 103 Wh = 3.6 × 106 J). Ohmic heating is distinguished from other electrical heating methods with the presence of electro des contacting the food, frequency, and waveform. Ohmic heating may be distinguished from microwave heating by the fre quency and the nature of the electrical current source. Ohmic heating can be used in blanching, evaporation, dehydration, fermen tation, and extraction. The advantage of ohmic heating is its ability to heat materials rapidly and uniformly, including products containing particles. In ohmic heating, particles can be heated faster than ﬂuids by appropriately formulating the ionic contents of the ﬂuid. The main advantages of ohmic heating are fast and uniform heating of liquid and solid phases by internal heat generation; uniform heating results in less thermal damage; no heat transfer to surfaces; high energy efﬁciency (more than 90% of the electrical energy is converted into heat); reduced fouling and overprocessing of the food that result in less mechanical damage and better retention of nutrients and vitamins; and ohmic heating has simple process control and low cost. Ohmic heating technology also has disadvantages, such as higher initial operational costs and little information available on validation procedures. Ohmic heating may not be easily used due to ionic requirement, such as products with high amounts of fats, oils, and distilled water, since ohmic heating is volumetric and depends on electrical resistance. 11.2.1 Mechanisms of Microbial Inactivation The principal advantage of ohmic heating is the ability to heat materials rapidly and uniformly; thus, overprocessing of foods can largely be prevented. In addition, there is no hot surface in direct contact with the food, thus avoiding fouling problems and thermal damage to the product. Furthermore, there is effective action of the electric ﬁeld per second over microorganisms and enzymes in addition to the effects of temperature in the shorter time. The basic principle of ohmic heating is that heat is generated inside a product when an electric ﬁeld is applied. Every food contains water and ionic salts that are capable of conducting electricity and internal heat is generated when an electric current passes through it. Electrical conductivity increases with temperature; ohmic heating becomes more effective at higher temperatures. The ohmic heating can be used as a thermal process such as pasteurization. A mild electroporation mechanism may occur during ohmic heating. The principal reason for the additional effect of ohmic treatment may be its low frequency (50–60 Hz), which allows cell walls to build up charges and form pores. A mild electroporation mechanism may contribute to cell inactivation during ohmic heating. There is no difference between the effects of ohmic and conventional heat treatments on the death kinetics of cells. The kinetics of inactivation of bacterial spores can be accelerated by an ohmic treatment. 192 Chapter 11 11.2.2 Factors Affecting Ohmic Heating Ohmic heating is fundamentally a thermal process; temperature and time are the principal critical process factors. Ohmic heating is an internal energy generation process, so there is theoretically no upper temperature limit to the process. Thus, if product holdup occurs, it is possible for boiling to occur within the food with a high degree of pressurization. Several other factors signiﬁcantly affect the temperature within an ohmic process: the electrical conductivity of the food, the temperature dependence of electrical conductivity, the design of the heating device, the extent of ﬂuid motion, thermophysical properties of the food, and electric ﬁeld strength. Electric con ductivity is the measure of a substance’s ability to transmit electric charge in siemens per meter (S m 1). It is a ratio of substance density to electric ﬁeld strength and is affected by the chemical composition of a substance. The higher the amount of dissolved salts in a substance, the higher the conductivity. Other product properties that may affect temperature distribution include the density and speciﬁc heat of the food product. High densities and speciﬁc heats are conducive to slower heating. Equipment design is another factor affecting ohmic heating. 11.3 High-Pressure Processing High hydrostatic pressure (HHP) processing is also called high-pressure processing or ultrahigh-pressure (UHP) processing. HHP processing involves exposing of a packed liquid or solid food in water suspension or an unpacked liquid food in a closed chamber to pressure from 100 to 1000 MPa for a desirable period of time at a temperature from 1 to 95 °C. 11.3.1 High Hydrostatic Pressure HHP processing involves subjection of liquid and solid foods, with or without packaging, to pressures between 100 and 1000 MPa for a certain exposure time. Pressures used in the HHP processing of foods have little effect on covalent bonds; thus, foods subjected to HHP treatment at or near room temperature will not undergo signiﬁcant chemical transformations. HHP may be combined with heating to achieve an increasing rate of inactivation of microorganisms and enzymes. Chemical changes in the food will depend on the temperature and exposure time. HHP acts uniformly throughout a mass of food independent of size, shape, and food composition. Thus, package size, shape, and composition are not factors in process determination. The work of compression during high-pressure treatment will increase the temperature of foods through adiabatic heating by approximately 3 °C per 100 MPa depending on the composition of food. For example, if the food contains a signiﬁcant amount of fat, such as butter or cream, the temperature rise can be larger. While the temperature of a homogenous food will increase Alternative Food Processing Techniques 193 uniformly due to compression, the temperature distribution in the mass of food during the holding period at a certain pressure can change due to heat transfer to or from the walls of the pressure vessel. The pressure vessel must be held at a temperature equal to the ﬁnal food temperature increase from compression for truly isothermal conditions. Temperature distribution must be determined in the food for each treatment cycle if temperature is an integral part of the high- pressure microbial inactivation process speciﬁcation. Foods decrease in volume as a function of the imposed pressure. An equal expansion occurs on decompression. For this reason, the package for high-pressure treated foods must be considered up to a 15% reduction in volume and return to its original volume, without loss of seal integrity and barrier properties. The pressure level and time of application can cause changes in the appear ance of foods. This is especially true for raw, high-protein foods where pressure induces protein denaturation. HHP can also cause structural changes in foods such as strawberries or lettuce. Cell deformation and cell membrane damage can result in softening and cell serum loss. Usually these changes are undesirable because the food will appear to be processed and no longer fresh or raw. Food products currently employing HHP in their manufacture include fruit jellies, jams, fruit juices, salad dressings, rice cakes, ham, raw oysters, and ﬁsh, among others. Equipment for batch HHP treatment of foods consists of (1) a pressure vessel of cylindrical design, (2) piston restraining the end closures, and (3) a pressure generator. The components of a high-pressure processing system can be arranged to treat unpackaged liquid foods in a semicontinuous manner and packaged foods in a batch conﬁguration. 11.3.1.1 Effects of HHP on Microorganisms Normally, Gram-positive bacteria are more resistant to HHP than Gram-negative bacteria. Bacterial spores are extremely resistant to pressure than vegetative cells, but can be killed at 1214 MPa. Cells sublethally stressed by pressure are more susceptible to inactivation with pressure. Vibrio parahaemolyticus is more sensitive to the effects of high hydrostatic pressure than Listeria monocytogenes. L. mono cytogenes (about 106 colony forming unit (cfu) ml 1) is inactivated within 20 min at 345 MPa in buffer at 23 °C, while a similar number of V. parahaemolyticus are eliminated in 10 min at 173 MPa in fruit juice. Milk, compared with buffer, offers a protective effect for microorganisms. HHP reduces Yersinia enterocolitica by 5 log cycles with 275 MPa for 15 min in phosphate-buffered saline. About 5 log cycles of Salmonella enterica subsp. enterica var. Typhimurium can be reduced within 15 min with 350 MPa, L. monocytogenes with 375 MPa, Salmonella enteritidis with 450 MPa, E. coli O157:H7 with 700 MPa, and S. aureus with 700 MPa. The pressure response of bacteria depends on bacterial strain differences and different suspending media. In milk, 400 MPa pressure at 50 °C for 15 min exposure can reduce E. coli by approximately 5 log cfu g 1, and 500 MPa at 50 °C for 15 min can reduce S. aureus 194 Chapter 11 by approximately 6 log cfu g 1. In minced poultry meat, E. coli can be reduced by approximately 6 log cfu g 1 with 400 MPa at 50 °C in 15 min and S. aureus by 5 log cfu g 1 with 500 MPa at 50 °C in 15 min. The heat-resistant molds, Byssochlamys nivea, Byssochlamys fulva, Aspergillus ﬁscheri, Eupenicillium, and Paecilomyces, can be inactivated by exposure to 300 MPa at 25 °C within a few minutes, while a treatment of 600 MPa at 60 °C eliminates all ascospores within 60 min except ascospores of B. nivea and Eupenicillium. No viable lactobacilli and yeasts can be recovered using a treatment at 294 MPa for 10 min at 25 °C in rice wine. Treatment of freshly minced meat for 20 min at 20 °C from 200 to 450 MPa can reduce total counts up to 5 log cycles. The parasitic worms of Trichinella spiralis are killed by a 10 min exposure to 200 MPa. Pressures of 920 MPa are necessary to inactivate tomato mosaic virus (TMV); human viruses can be reduced by 104–105 viable particles by exposure from 400 to 600 MPa within 10 min, and bacteriophages (DNA virus) at 400 MPa. 11.3.1.2 Mechanism of Microbial Inactivation The various effects of HHP can be grouped into cell envelope-related effects, pressure-induced cellular changes, biochemical changes, effects on genetic mate rials, and morphological changes in cells. Hydrostatic pressures of 100–300 MPa can induce spore germination and resultant vegetative cells are more sensitive to processing conditions. The pressure disrupts the ionic, hydrophobic, and H-bonds (weak bonds) of the cellular molecules without affecting covalent bonds. These chances cause the macromolecules, especially proteins, to unfold and refold in different conﬁgura tions following the pressure release. Large carbohydrate molecules can also undergo similar changes. Pressure can destroy some enzymes as well as increase the activity of some other enzymes. Pressure processing does not have adverse effects on small molecules in a food, such as vitamins, minerals, ﬂavors, and many colors. Pressure enhances reactions that lead to volume decrease. The response of proteins to pressure varies largely due to hydrophobic interactions. Up to pressures of 100 MPa, hydrophobic interactions tend to result in a volume increase, but beyond this pressure, a volume decrease is associated with hydrophobic interactions and the pressure tends to stabilize these bonds. Consequently, the extent of hydrophobicity of a protein will determine the extent of protein denaturation. Additional factors for enzyme inactivation are the alteration of intermolecular structures and conformational changes at the active site. Enzyme inactivation under pressure is also affected by pH and substrate concentration. The primary site of pressure damage is the cell membrane. Pressurization can change membrane permeability. Pressure-induced membrane malfunctions inhibit amino acid uptake probably due to membrane protein denaturation. Alternative Food Processing Techniques 195 Leakages on the cellular membrane and the higher amount of loss from cells correlate with death or injury of microbial cells. Bacteria with a relatively high content of diphosphatidylglycerol (responsible for rigidity of membranes in the presence of calcium) are more susceptible to inactivation by HHP. 11.3.1.3 Factors Affecting Microbial Inactivation Gram-positive bacteria are usually more pressure resistant than Gram-negative bacteria. In general, cells in the exponential growth phase are more pressure sensitive than cells in the stationary phase. The physiological age of bacterial cells would play an important role in the inactivation by HHP. Incomplete inactivation of microorganisms by pressure will result in cell injury and they are capable of recovery under optimal growth conditions. Pressure inactivation rates will be enhanced by exposure to acidic pH. A neutral pH is more protective to the spores than acid pH. pH of liquid foods is reduced by 0.2–0.5 units per 100 MPa increase in pressure. The direction of pH shift and its magnitude must be determined for each food treatment process. As pH is lowered by HHP processing, most microorganisms become more suscep tible to pressure inactivation and sublethally injured cells are killed. Ionic bonds (such as those responsible for folding of proteins) are disrupted by pH under pressure. HHP treatments, in the absence of signiﬁcant temperature increases, do not break covalent chemical bonds. Ionic bonds (responsible for the folding of proteins) can be disrupted under acidic conditions. Treatments from 300 to 600 MPa can inactivate microbial cells at a rate that is increased as a function of acidity increase. Reduction of water activity (aw) from 0.98 to 0.94 can result in a marked reduction in the inactivation rate for microorganisms in a food. Low aw protects microbial cells against inactivation by HHP. Microorganisms may be sublethally injured by pressure but recovery of sublethally injured cells can be inhibited by low aw. Foods are more pressure protective on microorganisms (due to the presence of proteins, fats, and other organic compounds) than buffers or micro biological media. Increasing the pressure, time, or temperature of the pressure process will increase the rate of microbial inactivation. An increase in food temperature can increase the inactivation rate of microorganisms during HHP treatment. The combination of mild heat with hydrostatic pressure produces a synergistic effect. Heat-resistant bacteria are usually more pressure resistant than heat-sensitive types, but there are exceptions. Other important points in the HHP processing are the come-up time (period necessary to reach treatment pressure), pressure release times, and changes in temperature due to compression. Long come-up time increases the total process time and this will also affect inactivation kinetics of microorganisms. The redox potential of the substrate, addition of bacteriocins, and other antimicrobials may inﬂuence the inactivation of microorganisms by pressure. Additionally, the presence of NaCl or glucose provides protection. 196 Chapter 11 11.3.2 High-Pressure Carbon Dioxide The heat treatments reduce the numbers of microorganisms that can cause adverse effects on the ﬂavor and taste, as well as loss of nutrients. As a conse quence, novel nonthermal processes can produce high-quality foods. In accord ance with the current trend, there is a growing interest in the use of high-pressure carbon dioxide (HPCD) for inactivation of microorganisms and its use extends the shelf life of perishable liquid foods. HPCD treatment is an alternative technique to thermal processing of food. CO2 has considerable antimicrobial activity due to the formation of carbonic acid in water. The inhibitory effect of CO2 increases when it is applied under pressure. The principal advantages of pressurized CO2 are (i) the inactivation of both airborne and exposed surface bacteria, as well as easy penetration into the porous materials to affect microbes inside the food, (ii) reduction of the time and temperature required for sterilization, pasteurization, or blanching of foods, and (iii) minimizing thermal degradation of sensitive substances in natural products. Equipment for batch HPCD treatment of foods is shown schematically in Figure 11.1 and consists of a cylindrical pressure vessel, pressure generator, and controller units. 11.3.2.1 Mechanisms of Microbial Inactivation The principal operating parameters inﬂuencing high-pressure CO2 treatment are pressure, temperature, exposure time, initial number of cells, physical and chemical properties of foods, pH of the foods, sample volume, age of cell growth, kind of microorganisms, and others. The value of the equilibrium constant (Khyd) for hydration of CO2 indicates the percentage of carbonic acid present in solution step (i). While the hydration of CO2 to form carbonic acid (H2CO2) proceeds very quickly, the K ´a value for step (ii) shows dissociation of the acid to bicarbonate ion (HCO3 ) and hydrogen ion (H+) slowly. The third dissociation step (iii) produces additional hydrogen ions and carbonate ions (CO3 ) and does not occur to any great extent as evidenced by the K ´´a value of 5.61 × 10 11. The overall dissociation of carbonic acid to the various ionic species depends on the hydrogen ion concentration of the solution. Below Figure 11.1 Schematic diagram of the apparatus for HPCD treatment. (1) CO2 gas cylinder, (2) pressure regulator, (3) needle valve for CO2 inlet, (4) thermostatic controller, (5) water bath, (6) pressure vessel, (7) line ﬁlter, (8) needle valve for CO2 outlet, (9) thermocouple, (10) temperature data logger, (11) pressure transducer, and (12) pressure data logger. Alternative Food Processing Techniques 197 pH 5.0, CO2 in solution exists primarily as CO3 and carbonic acid. Between pH 8 and 9.5, the carbonic acid dissociates (step (iii)) to form CO3 and H. Reactions of CO2 in aqueous solution are as follows: CO2 H2 O ! H2 CO3 (i) H2 CO3 ! HCO3 H (ii) HCO3 ! CO3 2 H (iii) Survival curves under HPCD treatment show two apparent distinct stages in the destruction of microorganisms, suggesting that CO2 inactivation of microbes involves a complex mechanism. The inactivation rate is slow in the beginning and this indicates that diffusion of CO2 into cells is a rate-limiting step. When the pH reduction level inside cells reaches the critical level due to the formation of carbonic acid, it would exert lethal effects on cells by disturbing or unbalancing biological systems. At any pressure, the earlier stage is characterized by a slow rate of inactivation, which increases sharply afterward, suggesting that the diffusivity of CO2 and carbonic acid into cells is a controlling factor. At the second stage, the inactivation rate increases. Pressure controls both the solubilization rate of CO2 in a suspending medium and cells. Consequently, high pressure enhances CO2 solubilization and facilitates its contact with the cells. CO2 dissolves in aqueous solution under pressure to form carbonic acid. The solubilization of CO2 reduces pH of suspension. Under pressure it is possible that a large number of CO2 molecules pass through the cell membrane and lower the cytoplasmic pH enough to exceed the buffering capacity of the cytoplasm. Lowering pH in cells might denature key enzymes essential for metabolism and inhibit active transport of ions (Figure 11.2). When the pH level inside cells reaches a critical level, it would disturb or unbalance biological systems. The highly pressurized CO2 can extract vital constituents including phospholipids and hydrophobic compounds from the cell walls and membranes. In addition, cells burst in the pressurized stage. Different levels of injury can be produced under pressurized CO2. This would indicate that injury must always be taken in account with this pressure–gas–temperature inactivation system. Figure 11.2 Mechanisms of microbial inactivation with HPCD. 198 Chapter 11 11.4 Pulsed Electric Fields Pulsed electric ﬁeld (PEF) processing involves the application of pulses of high voltage (typically 20–80 kV cm 1) to foods placed between two electrodes. PEF treatment is conducted at ambient, subambient, or slightly above ambient tem perature. For food quality attributes, PEF technology is considered superior to the traditional heat treatment of foods because it avoids or greatly reduces the detrimental changes of the sensory and physical properties of foods. PEF preserves the nutritional components of foods. PEF may be applied in the form of exponentially decaying, square wave, bipolar, or oscillatory pulses. An exponentially decaying voltage wave represents a unidirectional voltage that rises rapidly to a maximum value and decays slowly to zero. Square pulse waveforms are more lethal and more energy efﬁcient than exponentially decaying pulses. A square waveform can be obtained by using a pulse-forming network consisting of an array of capacitors and inductors and solid-state switching devices. Oscillatory decaying pulses are the least efﬁcient, because they prevent the cell from being continuously exposed to a high-intensity electric ﬁeld for an extended period, thus preventing the cell membrane from irreversible breakdown over a large area. 11.4.1 Applications of PEF Technology in Food Preservation A continuous ﬂow diagram for PEF processing of foods is illustrated in Figure 11.3. This processing system consists of the following major components: a high-voltage power supply, an energy storage capacitor, a treatment chamber (such as poly vinyl chloride), a pump to conduct food through the treatment chamber, a cooling device, temperature measurement devices, and a computer to control operations. The RC circuit is composed of a capacitor and a resistor. The capacitor stores the current for each high-voltage pulse. Once it is accumulated, the energy is discharged in a very short time (μs) in the treatment chamber containing food. When the switch is closed, high-voltage pulse generator generates pulses and the food in the treatment chamber is subjected to a high-voltage pulse. The pulse is Figure 11.3 Continuous ﬂow diagram of processing of food. Alternative Food Processing Techniques 199 characterized by the duration at the maximum voltage. The amount of energy is quite high. An undesirable heating is present. A cooling system can be added in the chamber to eliminate the heating effect and ensure an ambient temperature process. PEF is applied to preserve the quality of foods, such as to improve the shelf life of milk, orange juice, liquid eggs, and apple juice, among others. The shelf life of apple juice after treatment with PEF at 50 kV cm 1 with 100 pulses for 2 μs at 45 °C can be about 28 days compared with a shelf life of 21 days of fresh apple juice. PEF treatment does not cause any physical or chemical changes in ascorbic acid or sugars. The ﬂora of orange juice can be reduced by 3–4 log cycles by applying an electrical ﬁeld of 15 kV cm 1 without signiﬁcantly affecting quality. Salmonella enterica subsp. enterica ser. Dublin can be inactivated with PEF at 36.7 kV cm 1 with 40 pulses. Saccharomyces cerevisiae can be inactivated by PEF with 10 pulses of an electric ﬁeld at 25 kV cm 1. The shelf life of raw milk (0.2% milk fat) treated with PEF at 40 kV cm 1 with 30 pulses for 2 μs is about 2 weeks at 4 °C. PEF treatment can decrease viscosity but increase the color (in terms of β-carotene concentra tion) of liquid whole eggs compared with fresh eggs. PEF treatment cannot cause any change in sensory quality of liquid whole eggs. 11.4.2 Factors Affecting Microbial Inactivation Three types of factors affecting the microbial inactivation with PEF are (1) the process (electric ﬁeld intensity, pulse width, treatment time and temperature, and pulse wave shapes), (2) microbial entity (type, concentration, and growth stage of microorganisms), and (3) treatment media (pH, antimicrobials, ionic compounds, conductivity, and medium ionic strength). 11.4.2.1 Electric Field Intensity Electric ﬁeld intensity is one of the main factors that inﬂuences microbial inactivation. The microbial inactivation increases with an increase in the electric ﬁeld intensity. Pulse width also inﬂuences the critical electric ﬁeld. 11.4.2.2 Treatment Time Treatment time is deﬁned as the product of the number of pulses and the pulse duration. An increase in treatment time increases microbial inactivation. Pulse width inﬂuences microbial reduction by affecting Ec. Longer widths decrease critical electric ﬁeld (Ec), which results in higher inactivation; however, an increase in pulse duration may also result in an undesirable food temperature increase. Critical treatment time also depends on the electric ﬁeld intensity applied. Critical treatment time decreases with higher electric ﬁelds. 11.4.2.3 Pulse Wave Shape Electric ﬁeld pulses may be applied in the form of exponentially decaying, square wave, oscillatory, bipolar, or instant reverse charges. Oscillatory pulses are the 200 Chapter 11 least efﬁcient for microbial inactivation, and square wave pulses are more lethal on microorganisms than exponentially decaying pulses. Bipolar pulses are more lethal than monopolar pulses because a PEF causes movement of charged molecules in the cell membranes of microorganisms. Square wave pulses are more effective, yielding products with longer shelf lives than those products proceeding with exponentially decaying pulses. 11.4.2.4 Treatment Temperature PEF treatments at moderate temperatures (50–60 °C) can cause synergistic effects on the inactivation of microorganisms. With constant electric ﬁeld strength, inactivation increases with an increase in temperature. The application of electric ﬁeld causes increase in the temperature of the foods; proper cooling is necessary to maintain food temperatures. The lethal effect of PEF treatment increases with increase in the process temperature. This may be due to the increase in the electrical conductivity of the solution at the higher temperature. Additional high treatment temperatures change cell membrane ﬂuidity and permeability of microorganisms. 11.4.2.5 Product Factors The electrical conductivity of a medium (σ, S m 1), which is deﬁned as the ability to conduct electric current, is an important variable in the PEF process. Foods with large electrical conductivities generate smaller peak electric ﬁelds across the treatment chamber and therefore are not feasible for PEF treatment. An increase in conductivity results from increase in the ionic strength of a liquid and an increase in the ionic strength of a food results in a decrease in the inactivation rate of microorganisms. Thus, the inactivation rate of microorganisms increases with decreasing conductivity. At 55 kV cm 1 (with eight pulses), as the pH is reduced from 6.8 to 5.7, the inactivation ratio increases. The PEF treatment and ionic strength are responsible for electroporation and compression of the cell membrane. 11.4.2.6 Microorganisms Gram-positive bacteria are more resistant to PEF than Gram-negative. In general, yeasts are more sensitive to electric ﬁelds than bacteria due to their larger size. The number of microorganisms in food may have an effect on their inactivation with electric ﬁelds. Increasing the number of cells in foods can slightly lower inactivation. The effect of microbial number on inactivation may be related to releasing organic compounds from inactivated cells. In general, logarithmic phase cells are more sensitive to stress than lag and stationary phase cells. For the cells undergoing division, the cell membrane is more susceptible to the electric ﬁeld. The killing effect of PEF in the logarithmic phase is about 30% greater than that in the stationary phase of growth. Alternative Food Processing Techniques 201 11.4.3 Mechanisms of Microbial Inactivation The application of electrical ﬁeld to biological cells causes buildup of electrical charges on the cell membrane. Membrane disruption occurs when the membrane potential exceeds a critical value of 1 V in many cellular systems. This corresponds to an external electric ﬁeld of 10 kV cm 1. Several theories are available to explain microbial inactivation by PEF. Two of them are breakdown and electroporation or disruption of cell membranes. 11.4.3.1 Electrical Breakdown Electrical breakdown of cell membrane is shown in Figure 11.4. The membrane can be considered as a capacitor ﬁlled with a dielectric (Figure 11.4a). Opposite charges on the membrane create 1 V potential difference across the membrane. Potential difference is proportional to the ﬁeld strength Ec and radius of the cell membrane. The increase in the membrane potential leads to reduction in the cell membrane thickness (Figure 11.4b). Breakdown of the membrane occurs if the critical breakdown voltage (Vc; 1 V) is reached by a further increase in the external ﬁeld strength. Breakdown causes the formation of transmembrane pores (ﬁlled with conductive solution), which leads to an immediate discharge on the mem brane. Breakdown is reversible if the product pores are small in relation to the total membrane surface (Figure 11.4c). Above critical ﬁeld strengths with long exposure times, larger areas of the membrane are subjected to breakdown (Figure 11.4d) and pores become large in relation to the total membrane surface. This is associated with mechanical destruction of the cell membrane. 11.4.3.2 Electroporation Electroporation is the exposure of cells to high-voltage electric ﬁeld pulses and this destabilizes the lipid bilayer and proteins of cell membranes (Figure 11.5). The plasma membranes of cells become permeable to small molecules, causing swelling and eventual rupturing of the cell membrane. Therefore, the main effect of an electric ﬁeld on a microbial cell is to increase membrane permeability due to membrane compression and poration. Large pores are obtained by increasing the intensity of the electric ﬁeld and pulse duration or reducing the ionic strength of the medium. Figure 11.4 Schematic diagram of membrane breakdown with electrical breakdown exposed to the PEF process. (a) Cell membrane with an electrical potential, (b) membrane compression, (c) reversible breakdown with small pores, and (d) irreversible breakdown with large pores. 202 Chapter 11 Figure 11.5 Electroporation of a cell membrane exposed to PEF. 11.5 High-Voltage Arc Discharge High-voltage arc discharge (HVAD) is a method to pasteurize liquid foods by applying rapid discharge voltages through an electrode gap below the surface of aqueous suspensions of microorganisms. Microorganisms and enzymes can be inactivated by HVAD. This process involves passing an electrical current through carbon electrodes. When rapid high voltages are discharged through liquids, the microbial cells are exposed to a multitude of physical effects and electrolysis. This is referred to as electrohydraulic shock. This shock inactivates the microorganisms. Enzyme inactivation is caused by oxidation reactions mediating free radicals and atomic oxygen. There is no signiﬁcant temperature increase during treatment by HVAD. High-voltage electrical impulses are discharged at a rate of 1 s 1. HVAD with 200 shocks of 182 J per discharge to a gap of 1.6 mm at 4.5 kV can reduce microorganisms in liquids (with pH 7.2) in a static treatment chamber. This treatment can inactivate at least 95% of the vegetative cells of microorganisms, such as E. coli and Enterococcus faecalis. E. faecalis and E. coli are less resistant, whereas Micrococcus radiodurans and Bacillus subtilis are more resistant to high- voltage shock. HVAD cell break cannot occur. More than 90% of the bacterial vegetative population can be killed with 10 discharges. The chemical reactions are the major contributors to bacterial inactivation and thermal effects are insigniﬁcant, since the temperature rise is only a few degrees (10 discharges increase the temperature by only 0.5 °C). Endotoxins and microorganisms (such as L. monocytogenes, Clos tridium sporogenes, Salmonella enterica subsp. enterica ser. Typhimurium, Lactobacillus lactis, E. coli O157:H7, Aspergillus niger, and Penicillium digitatum) can be reduced by HVAD. The process can cause cell reduction in milk and orange juice without effecting taste and color. This process consumes little energy compared with thermal pasteurization of juices and is more energy efﬁcient than other non- thermal processes (such as high pressure and pulse electrical ﬁelds). The process can also be used to disinfect process water. The inactivation of microorganisms by HVAD is due to the hydraulic shock wave generated by an electrical arc. Arc discharges initiate the formation of highly reactive free radicals from chemical compounds in foods (such as oxygen). These Alternative Food Processing Techniques 203 free radicals are toxic compounds that inactivate certain intracellular components required for cellular metabolism, such as inactivation of lactic dehydrogenase, trypsin, and proteinases. The enzyme inactivation occurs due to free radical oxidation reactions. Membrane damage can occur by the lyses of protoplasts, leakage of intracellular contents, the loss of ability of bacteria to undergo plasmolysis in a hypertonic medium, and the release of galactosidase activity. Bacterial inactivation does not occur due to heating, but mainly due to irreversible loss of semipermeable barrier of the cell membrane and the formation of toxic compounds (oxygen radicals and other oxidizing agents). Microbial inactivation depends on the voltage applied, the type of microorganism, initial number of cells, volume of the medium, distribution of chemical radicals, and electrode material. Oxygenation is a critical part of the process since the sub merged arc discharge actually takes place within the gas bubbles. This partial breakdown of gas causes ionization, resulting in reactive ozone and UV radiation. 11.6 Pulsed Light Technology Pulsed light is a method of food preservation that involves the use of intense and short-duration pulses of broad-spectrum “white light.” The spectrum of light for pulsed light treatment includes wavelengths in the ultraviolet (UV) to the near- infrared region. The material to be processed is exposed to at least one pulse of light (typically 1–20 ﬂashes per second) with a duration range from 1 μs to 0.1 s and an energy density in the range of about 0.01–50 J cm 2 at the surface. A wavelength distribution of at least 70% of the electromagnetic energy is within the range from 170 to 2600 nm. Pulsed light is produced by accumulating electrical energy in an energy storage capacitor over relatively long times and releasing this storage energy to do work in a much shorter time magniﬁes the power applied. Pulsed light is applicable mainly in reducing the microbial population on the surface of packaging materials, food products, and other surfaces, extending the shelf life, and improving the quality of products. Pulsed light provides shelf-life extension and preservation of foods. Pulsed light inactivates microorganisms on the surface of foods and packaging surfaces. The process can be effective for inactivating molds in a variety of baked goods and extending their shelf life. More than 7 log cycles of Aspergillus niger spore inactivation can result with a minimal number of pulsed light ﬂashes with 1 J cm 2. A variety of microorganisms including E. coli, S. aureus, B. subtilis, and S. cerevisiae can be inactivated by using 1–35 pulses of light with an intensity ranging from 1 to 2 J cm 2. The temperature of food can be increased by 5 °C with 16 J cm 3 and a pulse duration of 0.5 ms. Light characteristics (wavelength, intensity, duration, and number of the pulses), packaging, and food attributes (type, transparency, depth of the ﬂuid 204 Chapter 11 column, and color) are critical process factors in the inactivation of microorganisms. The lethality of the light pulses is different at different wavelengths. Light pulses induce photochemical reactions in foods. UV-rich light causes photo chemical changes, while visual and infrared light cause photothermal changes. UV light inactivates pathogens and spoilage microorganisms. The antimicrobial effects of these wavelengths are primarily mediated through absorption by highly conjugated carbon–carbon double bond systems in proteins and nucleic acids. 11.7 Magnetic Fields Static magnetic ﬁeld (SMF) and oscillating magnetic ﬁeld (OMF) have the potential to be used as microbial inactivation methods. For SMF, the magnetic ﬁeld intensity is constant with time, while an OMF is applied in the form of constant amplitude or decaying amplitude sinusoidal waves. The magnetic ﬁeld may be homogeneous or heterogeneous. In a homogeneous magnetic ﬁeld, the ﬁeld intensity is uniform in the area close to the magnetic ﬁeld coil, while in a heterogeneous ﬁeld it is nonuniform, with the intensity decreasing as the distance from the center of the coil increases. OMF can be applied in the form of pulses reversing the charge for each pulse and the intensity of each pulse decreases with time to about 10% of the initial intensity. Processing of food with OMF involves sealing food in a plastic bag and subjecting it to 1–100 pulses in an OMF with a frequency between 5 and 500 kHz and intensity of 5–50 T at temperatures in the range of 0–50 °C for a total exposure time ranging from 25 to 100 ms. Frequencies higher than 500 kHz are less effective for microbial inactivation and tend to heat the food material. Magnetic ﬁeld treatments are carried out at atmospheric pressure and moderate temperatures. The temperature of the food increases 2–5 °C during the application of magnetic ﬁelds. The effects of magnetic ﬁelds on microbial growth and reproduction are inhibitory, stimulatory, or not observable. The inactivation of microorganisms is possible with OMF in milk, yogurt, orange juice, and bread dough. Only one pulse of OMF is adequate to reduce the bacterial population between 102 and 103 cfu g 1. The effect of magnetic ﬁelds on the microbial population of foods depends on electrical resistivity, magnetic ﬁeld intensity, microbial growth stage, number of pulses, frequency, and property of food (such as resistivity, electrical conductivity, and thickness of the food). A single pulse of intensity of 5–50 T and frequency of 5–500 kHz generally reduces the number of microorganisms by at least 2 log cycles. High-intensity magnetic ﬁelds can affect membrane ﬂuidity and other properties of cells. For inactivation of microorganisms by OMF, foods need to have a high electrical resistivity (greater than 10–25 Ω cm). Alternative Food Processing Techniques 205 Three theories are available to explain the inactivation mechanisms for micro organisms with SMF or OMF. The ﬁrst theory states that a “weak” OMF can loosen the bonds between ions and proteins. Many proteins vital to the cell metabolism contain ions. Energy is transferred selectively from the magnetic ﬁeld to the ions with a frequency of the magnetic ﬁeld. The interaction site of the magnetic ﬁeld is the ions in the cell, and they transmit the effects of magnetic ﬁelds from the interaction site to other cells, tissues, and organs. The second theory considers the effect of SMF and OMF on calcium ion bond in calcium-binding proteins. The calcium ions continually vibrate at an equilibrium position in the binding site. A steady magnetic ﬁeld causes the plane of vibration to rotate or proceed in the direction of magnetic ﬁeld at a frequency. Adding a magnetic ﬁeld disturbs or loosens the bonds. The third theory considers the inactivation of microorganisms depending on the coupling of energy into critical molecules, such as DNA. Within 5–50 T range, the amount of energy per oscillation coupling to one dipole in DNA is 10 2 to 10 3 eV. Several oscillations and collective assembly of enough local activation may result in the breakdown of covalent bonds in the DNA molecule and inhibition of the growth of microorganisms. 11.8 Ultrasound Industrial applications of ultrasound, such as cleaning surfaces, enhancement of dewatering, drying, and ﬁltration, inactivation of microorganisms and enzymes, disruption of cells, degassing of liquids, acceleration of heat transfer and extraction processes, and enhancement of any process, depend on the diffusion of sound. Ultrasound technology has a wide range of current and future applications in the food industry. Ultrasound has lethal effects on microbial cells, enzymes, and spores. Different species of microorganisms differ in their resistance to ultrasound: large cells appear to be more sensitive to ultrasound, coccal forms are more resistant than rod-shaped bacteria, and aerobic bacteria are more resistant than anaerobic bacteria. The use of ultrasound alone to lyse microbial cells is also a well- established laboratory method to extract intracellular components. Bacteria can be reduced by approximately 4 log cycles using a 10 min ultrasound treatment in broth, but in milk, a 30 min ultrasonic treatment can only reduce the number of Salmonella by 0.8 log cycle. The components of milk offer signiﬁcant protection against microbial cells. Ultrasound of 20 kHz/ 160 W using a cell disrupter with heating over a range of 5–62 °C can be used for the inactivation of bacteria. The combination of ultrasound and heating is signiﬁcantly more effective in inactivating bacteria. Ultrasound alone has no effect on bacterial spores, but thermoultrasonication can reduce the spore population. 206 Chapter 11 Figure 11.6 Mechanism of ultrasound-induced cell damage (US = ultrasound). Critical process factors are the amplitude of the ultrasonic waves, the exposure/ contact time with the microorganism, the type of microorganism, the volume of the food, the composition of the food, and the temperature of treatment. The inactivation effect of ultrasound can be attributed to the generation of intracellular cavitation and these mechanical shocks can disturb cellular structural and functional components up to the point of cell lyses (Figure 11.6). The percent of killed microorganisms can increase with an increase in exposure time and ultrasonic intensity. The bactericidal effect of ultrasound on vegetative bacteria is generally attributed to intracellular cavitations. Micromechanical shocks are created by making and breaking microscopic bubbles induced by ﬂuctuating pressures in the ultrasonication process. These shocks disrupt cellular structural and functional components up to the point of cell lysis. Ultrasound alone has no effect on spores. The other cotreatments have a main effect on any spore inactivation due to occurrence of ultrasound-inducing injury and repair. 11.9 Pulsed X-Rays Little is known about the inactivation of microorganisms with X-rays or pulsed X- rays. Electron beam, gamma rays, and X-rays are used to inactivate microorgan isms. There may be differences in the free radicals formed by these different processes. Electron beams have a limited penetration depth of about 5 cm in food, while X-rays have signiﬁcantly higher penetration depths (60–400 cm) depending on the energy used. Pulsed X-rays are a new technology that utilizes a solid-state opening switch to generate electron beam X-ray pulses of high intensity. Power pulses in the gigawatt range open at voltages of hundreds of kV and operate repetitively. Opening times range from 30 ns down to a few nanoseconds. Repetition rates have been demonstrated up to 1000 pulses per second in burst mode operation. Pulsed X-rays can reduce bacteria in foods, such as ground beef. The system consists of an X-ray accelerator with a thyristor-charging unit, a magnetic pulse compressor, a solid-state opening switch, an electron beam diode Alternative Food Processing Techniques 207 load, and an X-ray converter. The thyristor-charging unit converts three-phase, 240 V/440 V power to direct current. To inactivate surface and subsurface bacteria, fully packaged foods are sterilized by X-ray treatment. As a method of food preservation, X-ray treatment has low energy requirements. Microbial inactivation by all types of ionizing radiation involves two main mechanisms: direct interaction of the radiation with cell components and indirect action from radiolytic products, such as the water radicals (H+, OH , etc.). The primary target of ionizing radiation is chromosomal DNA, although effects on the cytoplasmic membrane may also play a role in the inactivation of microorganisms. Changes in chromosomal DNA and/or cytoplasmic membrane can cause microbial inactivation or growth inhibition. Ions, excited atoms, and molecules generated during irradiation have no toxic effect on humans. 11.10 Ozone Ozone (O3) is a triatomic naturally occurring form of oxygen. Ozone is generated naturally by ultraviolet irradiation from the sun and from lightning. It can be generated commercially by UV light (at 185 nm) or corona discharge. Ozone has a relatively short half-life (20 min in water at room temperature). It decomposes into simple oxygen with no safety concerns about residual ozone in the treated food product. Ozone is partially soluble in water and its solubility increases as the temperature decreases. Ozone is considered as a GRAS (generally regarded as safe) substance for food application by the U.S. Food and Drug Administration (FDA). Ozone has a very characteristic odor; it not only masks odors but also possesses a very marked deodorization property because of its oxidizing power in relation to odoriferous substances. Saturated hydrocarbons and organic compounds (functional groups: the carboxylic group, alcohol, or ester) do not react with ozone. It is a strong antimicrobial agent with numerous potential applications in food industry. Relatively low concentrations of ozone and short contact times are sufﬁcient to inactivate bacteria, molds, yeasts, parasites, and viruses. Ozone appli cations in food industry are mostly related to product surface and water treatment. Ozone can be used to inactivate microorganisms from water, meat, poultry, eggs, ﬁsh, fruits, vegetables, dry foods, cheese, and so on. It is also useful in detoxiﬁcation and elimination of mycotoxins and pesticide residues from agricultural products. Ozone does not produce signiﬁcant toxic residues in the environment after treat ment. There are many advantages of using ozone as a potent oxidizing agent in food and other industries. (i) Ozone is 52% more effective over a much wider spectrum of microorganisms than chlorine and other disinfectants. (ii) It is potentially useful in decreasing the microbial load, the level of toxic organic compounds, and the chemical and biological oxygen demand in the environment. (iii) Ozone converts many nonbiodegradable organic compounds into biodegradable forms. (iv) Ozone decomposes spontaneously to oxygen; thus, using ozone minimizes the 208 Chapter 11 accumulation of inorganic waste in the environment. (v) The higher oxidizing power and spontaneous decomposition also make ozone a viable disinfectant for ensuring the microbiological safety and quality of food products. (vi) Ozone is used for the preservation of foods to prevent microbial growth on surface of the walls of the cold store and the packaging materials. Ozone is an effective killing agent for microorganisms through oxidation of their cell membranes. Most of the pathogenic, foodborne microorganisms are susceptible to this oxidation effect. During food processing operations, surface disinfections of the product (raw or partially processed) are very important. Ozone more easily attacks the cells on the surface area. Penetration power of the ozone through tissue is very weak. A sufﬁcient concentration of ozone is therefore essential for the destruc tion of microorganisms and for delaying their multiplication on foods. The fungicidal action of ozone is generally greater than its bactericidal effect. Its bactericide power is problematic at low concentrations. Ozone at a concentration less than 0.2 mg m 3 of air does aid the growth of bacteria rather than inhibiting them. Ozone can be applied to reduce microorganisms, color, odors, turbidity, and organic compounds, and destroy chlorine by-products, pesticides, and toxic organic compounds present in water. Ozone is also used to remove iron, manga nese, and sulfur, and control taste and odor of fresh water. Practical applications of ozone to process water require concentrations ranging from 0.5 to 5.0 ppm (depending on the water source) with less than 5 min contact time. Ozone can destroy pesticides, chemical residues, and microorganisms on fruits and vegeta bles. Ozone is very effective in removing ethylene through chemical reaction to extend the storage life of many fruits and vegetables. Ozone application in the atmosphere of a cheese ripening room inactivates microorganisms including mold spores without affecting the sensory qualities of cheese. Ozone treatment improves the storage quality and decreases mesophilic aerobes and sulﬁte-reduc ing anaerobes on meat and in the meat transport vehicles. The poultry processing industry uses a large volume of water. The potential for reuse of poultry processing water represents an attractive economic beneﬁt to the industry. Ozonation yields the highest quality of processing water and reduces micro organisms (such as coliforms, E. coli, and Salmonella) by 99%. There would be no signiﬁcant difference in measured carcass quality including skin color, taste, or shelf life using recycled, ozone-treated water as opposed to fresh makeup water. Eggs can be treated with ozone gas. All quality parameters (acid, peroxide, and thiobarbituric acid values) would have better values in the ozone-treated eggs. Treatment of ozone-washed eggs with heat (59.4 °C) in an ozone vacuum chamber is called hyperpasteurization. Treatment of the skin of the ﬁsh with ozone decreases the viable bacteria. Ozone treatment improves the sensory quality by decreasing the formation of trimethylamine. Disinfections of air are an important part of clean room technology in the food industry. Ozone is effective on airborne microorganisms and air disinfection systems. The bacteri cidal action of ozone reduces organisms on food preparation surfaces and inhibits Alternative Food Processing Techniques 209 cold-tolerant bacteria on foods. Most of the color molecules contain aromatic rings and/or a series of carbon–carbon double bounds. Ozone reacts very rapidly with these bound molecules and consequently removes the color. All odoriferous compounds in fruits and vegetables are destroyed by ozone by oxidation. Ozone is very effective when the taste-producing compounds are chemically unsaturated compounds. A disadvantage of ozone as a disinfectant is that unlike chlorine, it is extremely unstable. Surface oxidation of food may result from excessive use of ozone. Discoloration and undesirable odors in ozone-treated meat can occur. Ozone changes the surface color of the fruits and vegetables (such as peaches and carrots). Ozone decreases ascorbic acid in fruits (such as broccoli) and content in products (such as wheat ﬂour). Ozone had a negative effect on the sensory quality of grains, ground spices, milk powder, and ﬁsh cake due to the lipid oxidation. Ozone releases a strong odor, which may be detected at very low concentrations. The perceptional threshold is 0.04 mg O3 m 3 of air. When ozone is breathed at higher concentra tions, the odor becomes disagreeable. Ozone at concentrations higher than 0.04 mg O3 m 3 of air causes loss of the sense of smell. At concentrations above 1 mg O3 m 3 of air, a sensation of dryness of the throat and nose is felt, followed by coughing and headache. A concentration of 4 mg O3 m 3 of air disturbs the sense of taste, ﬁnally causing vomiting and an irritation of the bronchial tubes. In practical applications of ozone in the food industry, safe use is an important point. Good manufacturing practice and hazard analysis and critical control point systems are also needed to control high-ozone-demand materials in food processing. Ozone can be used as a safe and effective antimicrobial agent in many food applications. The most important thing is that the use of ozone for drinking water allows solving many different problems in food industry. When compared with chlorine and other disinfectants, lower concentrations of ozone and shorter contact times are sufﬁcient in controlling or reducing microbial population and chemical residues. Ozone has an effect on the metabolism of living cells (such as fruits and vegetables). All oxidizable compounds are attacked by ozone. Ozone can only act on the surface of vegetable foodstuffs and there are no compounds on the surface liable to be subject to rapid oxidation. Ozone produces greater lethality rates for microorganisms (such as bacteria, molds, yeasts, amoebic cysts, and viruses) than chlorine or other chemical sanitizing agents. Use of ozone during processing or storage extends the shelf life of foods. However, the use of chlorine in food industry requires regulations due to toxicity issues. Ozone does not produce signiﬁcant toxic residues in the environment after the treatment. 11.11 Antimicrobial Edible Films The new generation of edible ﬁlms is being especially designed to increase their functionalities by incorporating natural or chemical antimicrobial agents, 210 Chapter 11 antioxidants, enzymes, ingredients (such as probiotics, minerals, and vitamins), and plasticizers. Films have advantages over direct applications of the antimicro bial or antioxidant agents because they can be designed to slow down the diffusion of the active compounds from the surface of the food. Edible ﬁlms can enhance the nutritional value of foods by carrying basic nutrients in its matrix. The sensory quality of coated products can also be improved if in the matrix ﬂavors and pigments are added. 11.11.1 Antimicrobial Food Additives Food additives used to prevent biological deterioration are termed antimicrobials or preservatives. A wide variety of antimicrobials can be added to edible ﬁlms to control microbiological growth and extend product shelf life. Antimicrobials used for the formulation of edible ﬁlms must be classiﬁed as food-grade additives or GRAS by the relevant regulations. Some types of antimicrobial food additives allowed by international regulatory agencies (European Union and Food and Drug Act) are given in Table 11.2. The lactoperoxidase system (LPS) consists of three components: lactoperox idase, thiocyanate, and hydrogen peroxide. The last compound serves as a substrate for lactoperoxidase in oxidizing thiocyanate, resulting in the generation of highly reactive oxidizing agents. LPS shows bactericidal effects on Gram- negative bacteria and bacteriostatic effects on Gram-positive bacteria. LPS also shows antifungal and antiviral activities. Killing of cells is usually greater at low pH and low temperatures. Lysozyme exhibits antimicrobial activity against vegetative cells of different microorganisms. Gram-negative bacteria are generally less sensitive than Gram-positive bacteria to lysozyme, mainly as a result of protection of the peptidoglycan layer of the cell wall by the outer membrane. The rate of cell Table 11.2 Antimicrobial compounds used in edible ﬁlms. Chemical compounds Natural compounds Organic acids: acetic, benzoic, citric, lactic, malic, Polysaccharides: starch, cellulose, pectin, propionic, sorbic, and tartaric acids. chitosan, and gums. Organic acid salts: sodium acetate, sodium Plant extracts, essential oils, and spices: diacetate, sodium benzoate, sodium citrate, cinnamon, capsicum, lemongrass, oregano, sodium formate, calcium formate, sodium rosemary, garlic, vanilla, carvacrol, citral, lactate, sodium propionate, calcium propionate, cinnamaldehyde, vanillin, and grape seed extract. potassium sorbate, and sodium tartrate. Parabens: methylparaben, ethylparaben, Polypeptides: lysozyme, peroxidase, propylparaben, sodium salt of methylparaben, lactoperoxidase, lactoferrin, nisin, and natamycin. sodium salt of ethylparaben, and sodium salt of propylparaben. Inorganics: sodium bicarbonate, ammonium bicarbonate, and sodium carbonate. Others: EDTA–CaNa2. Alternative Food Processing Techniques 211 catalysis by lysozyme depends on the pH of the medium, showing a bell shape with a maximum at pH 5.0. Plants, herbs, and spices, as well as their derived essential oils and substances isolated from different extracts, contain a large number of compounds that inhibit the metabolic activity of bacteria, yeasts, and molds. Essential oils of angelica, anise, carrot, cardamom, cinnamon, cloves, coriander, dill weed, lemongrass, fennel, garlic, nutmeg, oregano, parsley, rosemary, sage, and thymol are inhibi tory to various spoilage and pathogenic bacteria, molds, and yeasts. Common chemical antimicrobial agents used in food systems may be incor porated into edible ﬁlms and coatings to inhibit the outgrowth of both bacterial and fungal cells. In fact, the antimicrobial compounds inhibit the growth of microorganisms present on the surface of foods. Due to the health concerns of consumers related to chemical preservatives, edible ﬁlms can be prepared using natural biopreservatives. Chitosan has antifungal and antibacterial properties. The most frequently used biopreservatives for antimicrobials are lysozyme and nisin. These biopreservatives show bactericidal effects on Gram-positive bacteria but they can also become effective on Gram-negative bacteria if they are combined with chelating agents, such as EDTA. Common other biopreservatives that may be used in edible ﬁlms are bacteriocins, such as lacticin and pediocin, and antimicrobial enzymes (such as chitinase and glucose oxidase). The use of calcium alginate ﬁlms with antimicrobials (such as PS) results in a signiﬁcant delay in microbial growth on the potato and extends the shelf life during the storage at 5 °C. 11.11.2 Applications of Edible Films on Foods Antimicrobial edible ﬁlms are used for improving the shelf life of food products without impairing consumer acceptability. They are used on foods in order to prevent surface contamination while gradually releasing the active substance. Antimicrobials alone never provide complete protection for food spoilage and poisoning, and combination of preservatives with another stress factor is a way to improve food safety. According to this trend, application of edible ﬁlms containing antimicrobials is usually made together with other preservation factors in order to improve the quality of food products, such as fruits, vegetables, meats, seafood, and cheese. 11.11.2.1 Application on Fruits and Vegetables Nowadays, many commercial edible coatings for use on fresh and fresh-cut fruits and vegetables are available on the market to reduce weight loss and physiological disorders, and maintain product quality. Most of them are used mainly to maintain the quality of citrus fruits and apples, and to a lesser extent mangoes, papayas, pomegranates, avocados, cucumbers, and tomatoes. Fruits or vegetables are usually coated by dipping in or spraying with a range of edible materials. A semipermeable membrane is formed on the surface for suppressing respiration, 212 Chapter 11 controlling moisture loss, retaining stable quality, market safety, nutritional value, and economic production cost, and for providing other functions. A variety of edible materials, including lipids, polysaccharides, and proteins, alone or in combination can be formulated to produce edible ﬁlms. Lipid-based coatings made of acetylated monoglycerides (AMs), waxes (such as beeswax, carnauba, candelilla, parafﬁn, and rice bran), and surfactants can be used on whole fruits and vegetables to reduce surface damage during handling and serve as moisture barriers. Colloidal suspensions of oils or waxes dispersed in water are typical early fruit-coating formulations. Chitosan-based coatings reduce weight loss from fresh strawberries during storage and show an important antimicrobial activity against Rhizopus and Cla dosporium. The addition of oleic acid to chitosan coatings enhances the antimicro bial activity, improves water resistance, and reduces the respiration rate of cold- storage fruits. However, chitosan–oleic acid coatings decrease aroma and ﬂavor of fruits. Starch, carrageenan, and chitosan can be used to optimize coating compo sition and properties of fresh fruits. Addition of calcium chloride to the chitosan coatings decreases the microbial growth rate and is also effective in reducing weight loss and ﬁrmness loss of coated fruits. Chitosan ﬁlms inhibit the growth of yeasts, and mesophilic and psychrotrophic bacteria on fresh-cut fruits, particularly when the samples are packaged in a modiﬁed atmosphere with low O2 level. Some antimicrobial ﬁlms can be applied to peach, cherry, and tomato for their antimicrobial activity and to retain product quality. Chitosan-based ﬁlms reduce the respiration rate and ethylene production, and delay ripening of tomatoes. Chitosan-coated fruits also show less postharvest decay and greatly reduced postharvest black rot caused by the plant pathogen Alternaria alternata, and reduced formation of gray and blue colors by Botrytis cinerea and Penicillium expansum, respectively. HPMC-based coatings containing sorbic acid (0.4%) enhance the inactivation of Salmonella enterica subsp. enterica ser. Montevideo on the surface of tomatoes. These coatings can cause a chalky appearance of the fruit surface, limiting its potential for commercial application. Film-forming solutions of sodium caseinate, chitosan, or carboxymethyl cellulose containing 1% of essential oil of spices (such as olive, rosemary, onion, capsicum, garlic, or oregano) can show antimicrobial activity against L. monocytogenes. Fruit and vegetable tissues may remain biologically active, suffering many physiological changes from postharvest, during storage, and until they are consumed or processed. In addition, operations (such as washing, sorting, trim ming, peeling, slicing, and coring) are usually carried out on these products, promoting cell tissue disruption and membrane collapse. Because of their char acteristics, fresh or minimally processed fruits and vegetables are very perishable and require some combination techniques to extend the shelf life. Edible ﬁlms protect perishable fruits and vegetables from deterioration by retarding dehydra tion, suppressing respiration, improving textural quality, helping to retain volatile ﬂavor compounds, and reducing microbial growth. In addition, they can be used Alternative Food Processing Techniques 213 as a vehicle for incorporating functional ingredients, such as antioxidants, ﬂavor, colors, and nutrients. The application of a starch–chitosan coating on minimally processed carrots inhibits total viable count, lactic acid bacteria, coliforms, yeasts, and molds. Edible ﬁlms have effects on the organoleptic and nutritional preser vation of fresh vegetables. Methylcellulose coatings containing ascorbic, citric, and stearic acids can lower the browning rate and reduce vitamin C in mushrooms and cauliﬂower. With regard to the fresh fruit and vegetable industry, the potential beneﬁts of using edible ﬁlms include the following: 1 To provide moisture barrier on the surface of product for preventing moisture loss. Moisture loss during postharvest storage of fresh product leads to weight loss and changes in texture, ﬂavor, and appearance. 2 To provide sufﬁcient gas barrier for controlling gas exchange between the fresh product and its surrounding atmosphere; this would slow down respiration and delay deterioration. The gas barrier function can in turn retard the enzymatic oxidation and protect the fresh product from browning discoloration and texture softening during storage. 3 To restrict the exchange of volatile compounds between the fresh product and its surrounding environment by providing gas barriers, which prevents the loss of natural volatile ﬂavor and color components from fresh product and the acquisition of foreign odors. 4 To protect from physical damage of product caused by mechanical impact, pressure, vibrations, and other mechanical factors. 5 To act as carriers of other functional ingredients, such as antimicrobial and antioxidant agents, nutrients, and color and ﬂavor ingredients for reducing microbial loads, delaying oxidation and discoloration, and improving quality. 11.11.2.2 Application on Meat and Meat Products Minimally processed ready-to-eat meats are a potential source of foodborne pathogens such as S. Typhimurium, L. monocytogenes, and E. coli O157:H7. Con tamination with pathogens may occur during further processing or packaging. Most outbreaks of contamination are associated with the consumption of meat products. Edible ﬁlms carrying antimicrobials are a promising tool for decreasing the risk of pathogenic bacteria and also for extending the product shelf life. In meat products, application of a ﬁlm not only is useful as a carrier of the antimicrobial but can also prevent moisture loss during storage of fresh or frozen meats, hold juices of fresh meat cuts when packed in plastic trays, reduce the rate of rancidity, and restrict volatile ﬂavor loss and the uptake of foreign odors. 11.11.2.3 Application on Seafood and Seafood Products The quality of seafood is quickly reduced during storage by chemical and enzymatic reactions leading to the initial loss of freshness, while microbial spoilage produces the end of the shelf life. Using edible ﬁlms in seafood prevents the 214 Chapter 11 contamination with microorganisms. Edible ﬁlms inhibit L. monocytogenes growth, which constitutes the major risk in freshly processed cold smoked salmon. Edible ﬁlms can also avoid oxidative spoilage, in the fat-containing seafood, by the use of antioxidant agents and prevent moisture loss. 11.11.2.4 Application on Cheese Cheese is a complex food product that contains mainly casein, fat, and water. In the case of fresh and semi-hard cheese, microbial stability must be controlled. Edible ﬁlms are used mainly to control microbial growth on the surfaces and to reduce the risk of postprocessing contamination with L. monocytogenes. Also, the ﬁlms must allow gas exchange with the environment in order to maintain cheese quality.

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