I. LIGHT IN FOOD PRESERVATION
A. Ultraviolet Radiation
Ultraviolet (UV) radiation has long been known to be the major factor in the bactericidal action of sunlight. It is mainly used in sterilizing air and thin liquid films due to low penetration depth. When used at high dosage there is a marked tendency toward flavor and odor deterioration before satisfactory sterilization is achieved. But low-level radiation at carefully applied doses can often usually extend the shelf life of foods without damaging quality [10].
The technique of using UV radiation to kill off bacteria in water is well known. UV irradiation is safe, environmentally friendly, and more cost effective to install and operate than conventional chlorination. It does not affect the taste of the water as does chlorine. High-intensity UV-C lamps have become available, which can increase the potential of destroying surface bacteria on food [22]. UV radiation has been used in dairy plants for many years. It is also being used in the ice cream industry and in meat and vegetable processing plants [52].
1. Ultraviolet Radiation in Food Preservation and Deterioration
Food-Preservation Enhancement by UV Radiation
Ultraviolet irradiation is being applied commercially in the use of bactericidal ultraviolet lamps in various food-processing applications: tenderizing or aging of meat, curing and wrapping of cheeses, prevention of surface mold growth on bakery products, and air purification in bottling and food-processing establishments and over pickle vats [10].
The lethal effect of ultraviolet light on microorganisms has been well documented. The practical application of this has been controversial because of the type and intensity of radiation, methods of estimating lethality, and other factors. A study of the germicidal powers of ultraviolet light shows that 383% of the yeast and 3372% of the molds were killed in apple cider through layers varying from 2 to 25 mm in thickness [10]. A greater part of the light was absorbed by coloring agents. Incident energy levels of 253.7 nm inhibited 90% of Bacillus megatherium at 1100 mWs/cm2 and 90% of Sarcina lutea at 19,800 mWs/cm2 [2]. There was a 90% reduction in the microbial count of apple juice. Coupled with effective refrigeration , this could be of commercial significance.
It is generally agreed that the wavelength for maximum germicidal effect is 2600 Å. Low-pressure mercury-vapor lamps have a maximum output at 2537 Å, a value close to the peak wavelength for bactericidal effectiveness. The lethal action varies with the time of exposure and intensity of light. Other influential factors include temperature, hydrogen ion concentration, and the number of organisms per unit area exposed. The relative humidity affects the death rate of bacteria suspended in air, this being most noticeable at relative humidities greater than 0.50, at which point an increase in relative humidity results in a decreased death rate [10].
Spores of bacteria are generally more resistant to ultraviolet light than vegetative bacteria; B. subtilis is reported to be 510 times more resistant than Escherichia coli. Molds are more resistant than vegetative bacteria, while yeasts differ less from bacteria in this respect. It has been suggested that some mold species may be protected by fatty or waxy secretions on the cell surface, which shield them from the rays. Pigments apparently also afford some protection; dark-pigmented spores are more resistant to UV irradiation than nonpigmented types [10].
Short exposures, even long enough to cover one or more life cycles of the organism, are more efficient than higher radiation intensities for brief periods. This presumably is due to the fact that during certain stages of the life cycle the susceptibility to ultraviolet radiation is increased.
The effect of UV irradiation on bacteria and fungi such as Penicillium and Aspergillus has been reported by Kleczkowski [62]. UV radiation has been reported to inhibit fungal development in grapes [23], kumquats, and oranges [99]. Moy et al. [87] combined UV and gamma radiations for the preservation of papaya. Combined methods can avoid high doses of gamma and UV radiations.
Lu et al. [75] studied the efficacy of gamma rays (0.13 kGy), electron beams (0.15.0 kGy), or UV radiation (4.473.3 kerg/mm2) to preserve Walla Walla onions up to 4 weeks at 2025°C. UV-radiated onions exhibited the greatest percentage of marketable product and reduction in postharvest rot. Sprouting was observed with control, UV, and electron beam-irradiated onions but not with gamma-irradiated onions. No significant total sugar, pH, moisture, ascorbic acid, color, texture, or sensory quality changes were observed in the onions irradiated with UV. The optimum UV doses were in the range of 35.873.3 kerg/mm2 for Walla Walla onions. In addition, UV irradiation is much more economical and safer to use than gamma or electron beam irradiation.
Ranganna et al. [97] studied the efficacy of ultraviolet radiation treatment in the control of both soft rot and dry rot diseases of potato tubers for short-term storage of 3 months. They used four UV radiation dose levels (75, 100, 125, and 150 kerg/mm2) and three incubation levels each for Fusarium solani fungi (0, 1, and 2 days) and the bacteria Erwinia carotovora var. carotovora (0, 6, and 12 hr). The highest UV dose level was found to be more effective than the other three in controlling infection by the above fungi and bacteria. Visual observations of potato quality found no significant changes in the tuber qualities such as firmness and color.
Maple sap is susceptible to microbial infection, which lowers the quality of the syrup. Schneider et al. [109] studied the reduction of living cells of bacteria (Pseudomonas-25 and Pseudomonas-11) and yeast (Cryptococcus albidus) strains suspended in maple sap when exposed to UV radiation of different intensities and for different lengths of time. The two bacterial strains were equally as sensitive as the yeast. Increase in exposure time had the same effect regardless of the method of UV irradiation employed.
Freshly pressed apple juice or fresh cider contains many microorganisms that cause deterioration within 2 days at room temperature unless they are inhibited or destroyed. The microbial population of fresh cider was greatly reduced and storage life prolonged without affecting the flavor by specially designed UV lamps [46]. Harrington and Hills [46] found that percentage reduction of microbial populations was affected by the clarity of the cider, the length of UV exposure, and the presence of potassium sorbate. This is very suitable where the initial microbial count is high and refrigeration is not adequate.
Meat becomes tender upon storage as a result of enzymic activity. This process is speeded up at relatively high temperatures, which favor the growth of surface microorganisms. By controlling such growth with ultraviolet light, the advantages of high-storage temperature can be better utilized and loss of meat is controlled. In this particular case, irradiation alone is the less likely active factor. The lamps employed emit rays not only in the germicidal 2537 Å range but also in the 1850 Årange. These shorter waves convert atmospheric oxygen to ozone; irregular and shaded areas of an irradiated surface are sterilized by the ozone. UV radiation is also used in storage vats and other tanks over both conveyers and for final treatment of both caps and stoppers [10].
Putrefaction of fresh meat can occur in a few hours as a result of the action of spoilage bacteria. UV radiation at a wavelength of 253.7 nm was effective in destroying surface bacteria on fresh meat by 2 log cycles (99% reduction) decrease on smooth surface beef after a radiation dose of 150 mWs/cm2. A further increase in dose level to 500 mWs/cm2 reduced bacteria 3 log cycles. Since UV radiation does not penetrate most opaque materials, it was less effective on rough-surface cuts of meat, such as round steak, because bacteria were partly shielded form the radiation. No deleterious effects on color (redness) or general appearance were observed, and UV irradiation of meat carcasses could also effectively increase the lag phase of bacterial growth until adequate cooling of the surface has occurred [115].
The physical appearance of a cut of meat in the display case is the most important factor determining consumer selection of a beef product. Reagan et al. [98] mentioned that significant increases in shelf life may be obtained by exposure of beef muscle and fat surface to UV light (maximum wavelengths 3660 Å for 2 min). Decreases in initial count and/or attenuation of the bacteria present on retail cuts via the use of UV light resulted in increased consumer acceptability, higher muscle color ratings, and increased shelf life beef [98].
Kaess and Weidemann [60] found that continuous UV (0.224 µW/cm2) irradiation of psychrophilic microorganisms growing on muscle slices at 0°C and 0.993 equilibrium relative humidity resulted in an extension of the lag phase of Pseudomonas and of the molds Thamnidium and Penicillium, but not of the yeast Candida scottii. A minimum intensity of 2 µW/cm2 at the meat surface is necessary to prolong storage life substantially. Lower-equilibrium relative humidity did not substantially increase UV effects. The relative extension of storage life at 10°C was comparable to that obtained at 0°C. Simultaneous use of UV radiation (0.2 µW/cm2) and ozone (0.5 mg/m3) produced synergistic effects with molds, but not with bacteria [60].
The use of UV radiation is effective in inhibiting the action of spoilage bacteria on fish and seafood [22]. UV radiation at 254 nm and doses of 300 mWs/cm2 from a photochemical reactor of 4.8 Ws/cm2 from a high-intensity UV-C lamp (40 sec at 120180 mW/cm2) reduced surface microbial counts on mackerel by 2 or 3 log cycles [52]. Huang and Toledo [52] found that Spanish fresh mackerel kept 7 days longer than untreated sample when the skin surface was treated with high-intensity UV light and stored in ice at -1°C. When UV irradiated and packed in 0°C ice, surface microbial counts on vacuum-packaged mackerel lagged 4 days behind those on mackerel wrapped in 1 mil polyethylene [52].
The use of UV radiation has some disadvantages, for example, it does not penetrate most opaque materials and it is less effective on rough surfaces [22]. Huang and Toledo [52] found that rough-surface fish such as croaker and mullet had little surface bacterial count reduction with a UV-C-13 lamp at doses 120180 mW/cm2 for up to 50 seconds. They found that spray-washing with water containing 10 ppm chlorine by itself or in combination with UV radiation was necessary to reduce surface bacterial counts on rough-surface fish to the same extent as on smooth-surface fish.
Quality Defects Caused by UV
Fat oxidation by photochemical action results in off-flavors such as rancidity, tallowiness, fishiness, cardboard flavor, and oxidized flavor [32]. Coe and Le Clerc [21] attributed rancidity to the ultraviolet light range of the spectrum. Packaging materials having the ability to screen ultraviolet light have been developed for food products. Ellickson and Hasenzahl [32] observed that processed cheese in normal cellophane wax-coated wrappers becomes oxidized within 12 hours, and within 48 hours the top slice became inedible. This process can be retarded by incorporating a substituted benzophenone within the wax coating normally applied to certain types of cheese wrappers. Hirsch [50] stated that the meaty portion of bacon is subject to fading when exposed to UV light. Fading can be reduced appreciably through vacuum-packaging and a UV barrier on the packaging material. A good vacuum-packaging operation will deliver a bacon package with no less than 28 inches of vacuum. By incorporating polyvinylidine chloride (PVDC) into the packaging material, both oxygen and UV light are screened and the product survives a considerably longer period of time without fade. Generally, retinoids are very susceptible to oxidation because of their alkyl chains with highly conjugated double bonds [110]. Shimoyamada et al. [110] found that retinol and retinoic acid bound to b-lactoglobulin were less susceptible to light-induced oxidation by UV light irradiation than those that were free or bound to bovin serum albumin. They found different mechanisms of protection against light-induced oxidation compared to enzymatic oxidation. Iwanami et al. [58] studied effect of UV radiation on a lemon flavor composed of lemon oil, water (pH 6 phosphate beffer), and ethanol. Three compounds of aldehyde were newly identified as photoreaction products of citral. Limonene, terpinolene, and nonanal decreased, while p-cymene increased after UV radiation. Other components, such as sesquiterpene hydrocarbons, citronellal, linalool, and terpineols, were slightly changed. These results suggested that citral is a UV-unstable component in lemon flavor and that the photolysis of citral could affect other components in lemon flavor during UV radiation [58].
2. Disinfection Effects of UV Radiation
UV rays, which are nonionizing radiation, have been used extensively in the industrial disinfection of equipment, glassware, and air for many years [39]. The bactericidal effect of ultraviolet light is widely used for sanitation purposes. It is particularly effective in destroying airborne organisms and consequently becomes an important sanitary aid to in-plant installations. It may eliminate detrimental contamination and keep away objectionable invaders. Cerny [17] found that high-intensity UV irradiation may be used in the sterilization of packing materials for aseptic packaging. The penetrating power of ultraviolet rays is very low, so that lethal action is confined to organisms on or near the surface of irradiated materials. Aerial disinfection is severely limited by the presence of dust particles in the atmosphere. Several different UV lamps are available commercially for food industry applications for processing or disinfection.
3. UV Mode of Action
A number of conflicting theories have been proposed with regard to the mode of action of ultraviolet light. These include indirect lethal action resulting from the production of hydrogen peroxide, and various chemical and physiochemical changes in the constituents of the cell. The production of hydrogen peroxide is not generally considered to be the mechanism by which ultraviolet light induces its effect, although organic peroxides may be involved. It has been suggested that substances in the cell nucleus are involved in the destructive action by ultraviolet light. UV wavelengths of 200290 nm penetrate cell membranes to disrupt DNA molecules, preventing cell replication [10]. Also, the degradation of the cell walls destroys bacteria and causes a germicidal effect [2,120].
B. Visible Light Radiation
The germicidal effect of sunlight is due largely to the ultraviolet radiation received at the earth's surface. The wavelength is 290300 µm. Altitude and latitude and clarity of the atmosphere affect its effectiveness. Visible light having electromagnetic radiation of wavelength 400750 µm is absorbed by relatively few of the compounds present in nonphotosynthetic organisms. Light that is not absorbed has little or no effect. This is also true for the longer ultraviolet wavelengths of 300400 µm. Ultraviolet radiation with a wavelength of less than 300 µm, on the other hand, is strongly absorbed by proteins and nucleic acids. Relatively small doses of such radiation will cause chromosome breakage, genetic mutation, inactivation of enzymes, or death [10].
Cool white fluorescent illumination (14.5 W/m2 for 72 hr) of apples at 2°C enhances red color without hampering fruit quality and storability potential [102]. Saks et al. [101] studied the Dort and Ofra cultivars of strawberry illuminated at 14.5 and 17.5 W/m2 in white fluorescent light at 2°C. A 2-hour treatment was sufficient to overcome the genetic limitation of white shoulders in Dorit and poor red color in Ofra. Illumination enhanced both external and internal fruit color with no effect on quality attributes (e.g., freshness of calyx, fruit firmness, and fruit decay) when treated and kept in storage simulating air or sea transport followed by shelf life (18°C). The treatment reduced fruit rot in both cultivars. In fruit inoculated with Botrytis cinerea, the most common storage pathogen of strawberry, the appearance of disease symptoms was delayed.
C. Photoreactivation
If microorganisms are treated with dyes (e.g., erythrosin), they may become sensitive to damage by visible light. This effect is known as photoreactivation. Some food ingredients coud induce the same reaction. Such dyes are said to possess photodynamic action [10]. Spores may occasionally fail to show photoreactivation when inactivated with ultraviolet light, whereas the corresponding vegetative cells sometimes show photoreactivation. The simplest explanation for this is that radiation damages genetic material in the spore and that certain bacteria may produce diploid spores as a result of specific disruptions. Such bacterial spores exhibit two types of radiation inactivation curves: B. subtilis, B. brevis, and B. mesentericus are inactivated in a single-hit fashion, whereas B. megaterium, B. cereus, and B. mycoides are affected by a multiple hit. In all cases there is no effect on spore survival if the postirradiation medium is changed from yeast extract to a purely chemically defined medium [10].
When surface microbial contamination is the major cause of spoilage in certain seafood, the application of intense, short pulses of incoherent, continuous, broad-spectrum light can be used to increase the shelf life. The extension is achieved through two processes: by the destruction of spoilage-causing microorganisms and by the inactivation of enzymes. These effects are obtained through a complex photothermal and photochemical mechanism mediated by the use of wavelengths of less than 300 nm. The pulsed light waves transfer thermal energy to a thin surface layer without raising the interior temperature of the product [22]. Colby and Flick [22] concluded that increased efficiency is possible by the use of dyes or other chemical compounds that selectively bind to either microorganisms or enzymes, thereby increasing their susceptibility to the pulsed electromagnetic waves.
D. Pulsed Light
Pulsed light with an intensity 20,000 times that of normal sunlight is applied at rates up to 20 flashes per second. The flashes of this high-intensity light are a very quick way to transfer large amounts of thermal energy to the surface of a material, raising a thin surface layer to a high enough temperature to affect vegetative cells on that surface. This method inactivates microorganisms through a combination of photothermal and photochemical reactions. The UV light needs to be filtered out from the pulsed light when it is used to treat UV-sensitive foods, thus most of the remaining energy is in the visual and IR spectrum and the inactivation mode is photothermal [77].
II. Sound in Food Preservation
Ultrasound is sound energy with a frequency range that covers the region from the upper limit of human hearing, which is generally considered to be 20 kHz, to beyond hundreds of MHz [122]. Ultrasonic technology was first developed as a means of submarine detection in World War I, and developments in this area have continued to the present in different fields of technology [65]. The two applications of ultrasound in foods are (a) characterizing a food material or process, such as estimation of chemical composition, measurements of physical properties, nondestructive testing of quality attributes, and monitoring food processing; and (b) directly affecting food preservation or processing. Low-power ultrasound is used in a variety of capacities as a sensing medium. Only preservation and processing aspects will be considered in this chapter. Applications of sound to directly improve processes and products is less popular in food manufacturing [40]. High-intensity sound is mainly used for such applications with a frequency in either the sonic (<18 kHz) or the ultrasonic (>18 kHz) range, depending on the application [40].
A. Generation and Propagation of Sound
1. Generators
The first ultrasonic apparatuses were piezoelectric generators of quartz submerged in oil that generated ultrasonic waves of a very high frequency but low intensity (10 W/cm2). Modern ultrasonic instruments consist essentially of a piezoelectric generator having a crystal of zirconate titanate that changes its shape under the effect of an electric field of 100 V and a frequency of 20 kHz, supplied by a standard 5060 Hz, 120220 V converter. The high-intensity equipments are characterized by relatively low frequencies, up to about 100 kHz, by continuous (as distinct from pulsed) operation, and by power levels from 10 kW upward [45]. This electric energy, transformed into mechanical energy of the same frequency, is transmitted to a titanium alloy disruptor horn. The horn transmits and amplifies this energy onto its tip, which is submerged into the menstruum being ultrasonicated. Amplification depends on the volume and shape of the disruptor [103]. Berlan and Mason [8] reviewed the available generators. The geometry of the chamber and sonicating horn is also important, and the effect of any given ultrasonication treatment is inversely related to the volume of the chamber [25].
2. Propagation and Attenuation into the Medium
Sound involves the propagation and transmission of vibrational energy above the upper limits of audible sound. Ultrasonic vibrations pass through a body as a system of pulsating energy waves propagated by alternating compression-expansion zones. As a wave propagates through a relaxing medium, its amplitude decreases or attenuates and sound energy is lost. Ultrasonic attenuation is a measure of the relative amplitudes of a wave at two locations in space [45]. When moving through a liquid they cause the phenomenon known as cavitation. This involves formation of tiny vapor bubbles or voids within the liquid. The collapse of these cavities is responsible for the creation of pressures up to several hundred atmospheres. This cavitation occurs at high frequency or at very low amplitude [53,80,108].
Three causes of attenuation are diffraction, scattering, and absorption. Diffraction and scattering are properties of the shape and macroscopic structure of the material. The diffraction effects are constant if the sound source is inside the Fresnel zone, i.e., near the field. Scattering contributes less than 1% to absorption, thus is negligible [49]. Ultrasonic waves absorbed in liquid depend on viscosity, thermal conductivity, and thermal relaxation. Absorption due to viscosity and thermal conduction takes place in liquids as well as in gases, but the thermal conduction effects are usually negligible compared with those due to viscosity [9,45].
As a result of the specific absorption of acoustic energy by materials, particularly at their interfaces, a selective temperature increase may take place. Arkhangel'skii and Statnikov [1] theoretically found that the temperature change due to absorption at a solid wall, under given conditions, was 0.1°C for water and about 1°C for air. These results were also verified experimentally [40]. Floros and Liang [40] indicated that a significant temperature increase (560°C) may occur in sugar solutions as a result of ultrasonication, particularly at high sugar concentrations. High-intensity acoustic energy passing through a solid medium generates a rapid series of alternating constructions and expansions, much like when a sponge is squeezed and released repeatedly. This mechanism is known as rectified diffusion [40]. Sound travels by sine waves that have a node and an antinode. At the node position, the velocity is zero and the pressure is maximum. At the antinode position, the velocity is maximal and the pressure is zero [40].
The cavitation threshold (minimum oscillation of pressure for cavitation to occur: amplitude of pressure) depends on (a) dissolved gas content (liquids saturated with gases have a very low threshold, which increases linearly with the vapor pressure of the liquid); (a) hydrostatic pressure; (c) specific heat of the gas bubble; (d) tensile strength of the liquid, and (e) temperature [103].
Sala et al. [103] summarized many different parameters that influence the efficacy of ultra-sound, all of which should be at their optimum for achieving maximum cavitation. Thus, ultrasonication conditions should be carefully chosen and controlled for maximum benefit [8].
B. Consequences of Ultrasound
The beneficial or deteriorative use of sound depends on its chemical, mechanical, or physical effects on the process or products [40]. In this section, the applications of sound in food processing are summarized from selected reviewed papers.
1. Effect on Microorganisms
Process Applications
Harvey and Loomis [47] first reported the lethal effect of ultrasound in microorganisms [103]. Most investigations in this field refer to the degree of killing attained in microbial cultures when they are submitted to defined ultrasonic treatment [10]. Bacteria, especially spores, are very resistant and require hours of ultrasonication [80,107].
Ultrasonics has been reported to pasteurize milk [18,47] and destroy cells at high frequencies [112]. An increase in total counts at low frequencies was reported by Huhtanen [54,55] and Stone and Fryer [116]. This is probably due to the breaking up of clumps and bacteria, which normally occur in milk. A decrease in Salmonella typhimurium counts were time dependent, with the greatest decrease occurring after a 30-minute ultrasonication treatment at 50°C. Stone and Fryer [116] also showed 30-minute ultrasonic treatments to be more effective than shorter treatments. Lee et al. [66] showed a 4 log reduction in Salmonellae with a 10-minute ultrasonic treatment in peptone water and a 0.78 log reduction in chocolate milk treated for 30 minutes. Thus, treatment duration needed for microbial reduction depends on the substrate or medium.
Ultrasound is used to free bacteria adhered to surfaces to facilitate the removal of contaminated flora [26]. Salmonella and Campylobacter have long been associated with poultry and poultry products. Lillard [70] showed that bacteria are firmly attached to poultry skin, and although bactericides are lethal to samonella in processing water, they do not seem to access bacteria that are firmly attached to or entrapped in poultry skin [59,71]. Daufin and Saincliviert [24] reported that most bacteria from milk films on metal surfaces can be affected by ultrasonic waves of 80 kHz. Sams and Feria [104] studied ultrasonic treatment (47 kHz) of broiler drumsticks by dipping in deionized water at 25 or 40°C for 15 or 30 minutes in the presence of lactic acid with pH adjusted to 2 or 4. They found no significant effect of treatment and offered the explanation that the irregular broiler skin surface may provide some level of physical protection for bacteria against cavitation. Stumpf et al. [117] and Miller [85] observed that ultrasound waves are transmitted most efficiently over flat surfaces, whereas irregular surfaces reflected or refracted the waves, creating stationary waves, which greatly reduced cavitation. Salmonellae that were attached to broiler skin were reduced 11.5 log by sonication in peptone at 20 kHz for 30 minutes, by less than 1 log by chlorine alone, but by 2.54 log by sonicating skin in chlorine solution [72]. The difference in the results from Sams and Feria [104] may be due to the use of skin pieces rather than whole drumsticks, the use of peptone, or the greater synergistic effect of chlorine and sonication compared to sonication and heat [73].
Mode of Action
The mechanical disruption of cells by the very intense currents generated by ultrasound is the main lethal effect on microorganisms. Dorothy [29] concluded from electron micrographs that ultrasonic energy destroyed microorganisms by physical forces rather than chemical ones. Later Elliott and Winder [33] attributed bacterial destruction to thermal effects due to high-temperature hot spots. Since the volume of the high-temperature region is small, only a very small number of cells were affected [103]. Suslick [113] found that highly reactive chemical radicals and reaction products, such as hydrogen peroxide, are also lethal. Now most authors agree that, among other factors, cavitation is the mechanical effect due to extreme variations in pressure, which are responsible for the destruction of bacterial cells [73,103,108,111]. It has been mentioned that microorganisms can withstand high pressure but are incapable of withstanding the rapid alternating pressures produced during cavitation [103].
2. Effect on Enzymes
El'Piner [34] reviewed the effect of ultrasound on enzymes and other food components. The effect of ultrasound on enzymes depends on [103] (a) the ultrasonic field, (b) the molecular structure of enzymes, and (c) the nature of the sonicating medium, especially the nature of the dissolved gas. Inactivation effects generally require long irradiation periods and the presence of oxygen and are reduced if hydrogen replaces oxygen in the medium or when antioxidants are present [20,43,105]. Dunn and Macleod [31] found that the formation of free radicals by cavitation affects enzyme inactivation, which is related to the dissolved gas.
At low temperature, catalases are resistant to ultrasound [103] and yeast invertase is fairly resistant at low concentration [1,93], while pepsin is resistant at low concentration [90]. Ribonuclease is not inactivated in the presence of either oxygen or hydrogen, and in some cases serum aminopeptidase shows a similar nature [30]. However, lysozyme, alcohol dehydrogenase, hyalurodinase, lactate dehydrogenase, malate dehydrogenase, polyphenoloxidase, and other oxidases are much more sensitive [20,43,61,83,105].
3. Process and Quality Enhancement
Ultrasound is used in processes such as liquid degasification, homogenization, mixing, emulsification, crystallization, and the aging of meat, liquors, and wines. Ultrasonic-assisted cutters are also used in the food industry to enhance cutting performance and achieve a faster, cleaner, sharper cut with minimal waste due to self-cleaning blades.
Microwave Ultrasonics
Shukla [111] stated that the active force in microwave ultrasonics (above 16 kHz) is mechanical in nature due to cavitation, unlike heat in microwave heating.
Emulsification
Processes and apparatus utilizing the principle of ultrasound have found extensive use in emulsification and are potentially useful for cleaning operations. Ultrasonic treatment of wine induces cavitation. Dissolved bases such as carbon dioxide and sulfur dioxide are driven away in a rather drastic manner. Ultrasonic preservation of wine is therefore not advisable [10]. Ultrasonic homogenization can produce uniform solutions with reduced particle size. Martinez et al. [78] found ultrasonic homogenization of expressed human milk prevented fat loss during tube feeding.
Drying and Acoustically Assisted Diffusion
Mechanisms: A number of mechanisms may enhance water removal during drying or other mass transfer unit operations. These mechanisms include temperature increase at the interfaces, pressure fluctuation due to cavitation, developing microchannels by fracture, turbulence at the boundary, and structure change of the medium. The heat effect was assumed to be responsible for the significant increase in diffusion of sodium ions through living frog skin under ultrasound [67].
The contraction caused by acoustic energy releases a minute quantitity of water, and enhanced migration takes place during acoustic drying and dewatering [36]. In more dense materials, the alternating acoustic stress facilitates dewatering by either maintaining existing channels for water movement or creating new ones. Microscopic channels are created in directions normal to wave propagation during rarefaction or parallel to wave propagation during compression [89]. This mechanism also affects pressure gradient at gas/liquid interfaces, which enhance evaporation. Although the pressure variation introduced by the sound wave is very low, its effect is strong because of the rapid rate of pressure oscillation [12].
Acoustic waves cause extreme turbulence known as acoustic streaming or microstreaming at the interfaces [92]. This increases convective mass transfer by reducing the diffusion boundary layer. Furthermore, this has significance where ordinary mixing is not possible [1].
The mechanism that prevents binding or bridge formation may also affect the process, such as filtration and cleaning surfaces [40]. In the drying of foods, for example, sound may reduce the water- binding energy [123]. This was supported by the results of Huxsoll and Hall [56] for sonic drying of wheat and corn.
Acoustic drying may solve some problems faced when drying heat-sensitive materials, and it may be useful in removing the bound water from certain foods [40]. The effect of acoustics on the drying rate and permeation through membranes are shown in Figure 1 [40]. Acoustic radiation increases the efficiency of drying by extending the constant rate period [11,44]. Floros and Liang [40] reviewed the estimated acceleration due to sound and found that it varied from 1.00 to 4.75 factors. This indicates that in several cases the improvement is not large enough to justify commercialization, but in gelatin, yeast, and orange powder the rates are doubled or tripled, thus acoustic drying is beneficial.
Diffusion of a number of substances through membranes by acoustic is also reviewed by Floros and Liang [40], and the increment varied from 1.2 to 6.0 fold. Thus, ultrasound may result in significant improvements in the processes involving membranes, such as filtration, ultrafiltration, reverse osmosos, and dialysis [40].
Factors affecting accoustically enhanced diffusion: The factors affecting acoustically enhanced diffusion, reviewed by Floros and Liang [40], are as follows:
1. Temperature: Huxsoll and Hall [56] found an approximate 20% net increase in drying rate for whole wheat dried at low temperature (21°C). At higher temperature (94°C), the positive effect of sonication diminished to about 6%. Similarly, for green rice at 20.5°C, sound caused a 15% net increase, but no improvement at 40°C [88].
Conflicting results were found for drying yeast cake, when moisture migration increased by 80% at 25°C and 200% at 37°C [13]. Thus, Floros and Liang [40] concluded that temperature can make the effect of sound either more or less pronounced, depending on the specific products or processes.
2. Acoustic Intensity: Intensity is a measure of the amount of acoustic energy traveling through a given area [40]. The effect of acoustic intensity on diffusion is shown in Figure 2 [40]. The experimental results are summarized by Floros and Liang [40] as follows: acceleration of diffusion by sound is a function of intensity [86], the function is nonlinear [37,68], and cavitation produced by high intensity sound negatively affects diffusion through membranes [37,51]. As shown in Figure 2, there is a threshold intensity value below which the effect of acoustic on diffusion cannot be observed. For acoustic drying the threshold value is about 130 dB [38] or 145 dB [1,13]. Above the threshold, an optimum intensity is observed where the effect of acoustic energy on diffusion is maximal [37]. Above the optimum, diffusion may be retarded due to the extreme turbulence at interfaces or vapor locks in porous media by violent cavitation.
3. Acoustic Frequency: Theoretically the diffusion coefficient is a function of sound frequency [3]. Howkins [51] observed a slight increase in diffusion as the frequency increased from 20 kHz to 1 MHz. In acoustic drying mostly frequencies in the audible (sonic) range are used [40]. An optimum value of 8.1 kHz was found for potatoes [6] and of 610 kHz for other materials [12]. Ultrasonic energy (<18 kHz) is used in case of liquid-liquid extraction and membrane separation, such as osmosis. Floros and Liang [40] concluded that the effect of frequency is not clearly identified in the literature.
4. Direction of Acoustic Wave: The effect of sound on diffusion is maximal when it propagates in the same direction as the diffusion flow, and minimal when propagation is opposite to the direction of diffusion flow. When the direction is to diffusion flow, then the results are between the two extremes [68]. The theoretical analysis and discussion of Arkhangel'skii and Statnikov [1] support the above observations [40].
5. Pulsation of Acoustic Wave: Sound waves may be applied in a continuous or a pulsed (on-off) mode [40]. Lehmann and Krusen [67] and Mortimer et al. [86] found that both continuous and pulsed acoustic energy of the same average intensity resulted the same effect on diffusion, whereas Dinno et al. [27] stated that pulsation causes a significantly larger effect.
6. Medium Properties: Concentration, viscosity, and porosity may alter the effect of sound on diffusion [40]. At a low sugar concentration of 10°Brix, sound accelerated the diffusion of water into the apple tissue 5- to 10-fold after 6 hours. At a medium concentration of 30°Brix, sound had no effect, while at 60°Brix some acceleration of water migration from the tissue was observed [69]. Thus, effect of sonication on osmosis is concentration dependent.
7. Other Factors: The positioning of sample in a sound field at the node or antinode may affect the outcome, but conclusive evidence for this does not exist. Other factors, such as gravitational forces, pressure, and acoustic impedance compared to the medium impedance may also affect diffusion by acoustic forces [40].
Biotechnological Processes
Ultrasound has also been used to break cell walls to investigate cellular components [84]. The application of ultrasound to biotechnological processes has recently gained attention because treatment caused by ultrasounic waves may release many useful components from living cells, such as intracellular enzymes. For example, glucose oxidase was continuously released from Aspergillus sp. under mild ultrasound [57] and vacuole-located pigment from Beta vulgaris cells were enhanced for repeated harvesting by the use of 1.02 MHz ultrasound [64,64]. This is due to the change in cell membranes and walls of microorganisms or plants that permitted intracellular substances such as enzymes or metabolites to be released [100].
Lactose-hydrolyzed milk products are thought to have therapeutic value for people who cannot tolerate the lactose that remains in normal milk products. Usually lactose-hydrolyzed yoghurt produced by fermentation of lactose-hydrolyzed milk or by the simultaneous addition of b-glactosidase and lactic acid bacteria [119]. It is well known that lactic acid bacteria have b-galactosidase activity, which can be used to hydrolyze lactose in fermented milk. Sakakibara et al. [100] studied milk fermentation with Lactobacillus delbrueckii under ultrasonic irradiation in a 450 cm3 bioreactor with a polyethylene film bottom. Ultrasonic treatment increased the hydrolysis of lactose in milk but decreased cell viability. However, the viable cell count increased again when the ultrasound was stopped, because ultrasound did not destroy the cell propagation ability of surving cells. When the sonication power was 17.2 kW/m2 and the sonication period 3 hours, 4.9 × 108 cfu/cm3 of the viable cell count and 55% lactose hydrolysis were attained. In contrast, the viable cell count was 2 × 109 cfu/cm3 and 35.6% of lactose was hydrolyzed during control fermentation. Sakakibara et al. [100] found that both aspects could be enhanced if sonication were carried out under optimum conditions for b-glactosidase activity and lactic acid bacteria viability, e.g., at suitable pH and temperature. Matsuura et al. [82] found that fermentation of wine, beer, and sake can be accelerated 5065% by ultrasonic treatment.
Functional Properties
Sala et al. [103] observed the following effects of ultrasound: (a) the reversible reduction of viscosity of aqueous solutions of starch, gum arabic, gelatin, and other macromolecules, (b) the depolymerization of starch and polymerization of dextrans to high molecular weight, and (c) the breakdown of DNA to fragments retaining the native configuration.
High-intensity sound affects the structural properties of fluids, particularly their viscosity [40]. Usually Newtonian fluids maintain their Newtonian characteristics, but dilatent and thixotropic fluids tend to either stiffen or become less viscous [36,89]. High intensity sound also permits protein breakdown and hydrolysis, simple cell lysis, and protein particulation and may help retain vitamins and other heat-sensitive ingredients [111].
Ultrasound showed potential for improving the mechanical strength of milk protein films. Banerjee et al. [4] studied the effects of ultrasound frequency (168 kHz and 520 kHz), acoustic power (low, medium, and high), and exposure time (0.5 and 1 hr) on the functional properties of whey protein concentrate and sodium caseinate films. The average tensile strength of the ultrasound-treated caseinate films was 224% higher than that of controls. The treatment was more effective on sodium caseinate than on whey protein concentrate film. Resistance to puncture was improved for both types of film treated at an acoustic power of 5.22 W. Stronger films can be formed by increasing exposure time. The improvement by ultrasound could be due to the reduction of particle size in a film-forming solution, which results in increased molecular interaction and products a film with greater rigidity and compactness. Elongation at break, water vapor permeability, and moisture content of films were not affected by the ultrasound treatment [4].
The application of ultrasound treatment can produce a coating with improved physical and mechanical properties, e.g., brightness, hardness, compactness, and adhesion strength [124]. Thus, food preservation by edible coating can be enhanced by forming improved functional coating on the surface. Sonication has been used to alter the resistance of proteins from cow's milk and hen's eggs to proteolysis [91].
Chen et al. [19] studied the effects of ultrasonic conditions and storage in acidic solution on changes in molecular weight and polydispersity of treated chitosan. The results showed that chitosan was degraded faster in dilute solutions and in lower-temperature solutions. The degradation increased with prolonged ultrasonic time, and chitosan was degraded during storage in an acidic solution at ambient temperatures [19].
Synthesis of Active Functional Organic Compounds
Low [74] clearly identified the value of ultrasound to organic synthesis as its ability not only to accelerate known reactions, particularly those that are heterogeneous, in solvent systems, but also to generate new chemistry that is not available using existing methodologies. He discussed two distinct areas: generation of a number of synthetically useful reagents and the effects of adding electron-transfer agents to sonochemical reactions, and the chemistry of p-allyltricarbonyliron lactone and lactam complexes, which have been shown to be useful intermediates for the synthesis of a variety of biologically active or functionalized compounds. Bremner [14] also reviewed recent applications of ultrasound in organic synthesis. Thus, ultrasound is a tool that is valuable in synthetic development.
C. Thermo-Sonication
Sonication in conjunction with higher temperatures was shown to inhibit lipolytic activity and completely eliminate bacterial contaminants in human milk [79]. Ordonez et al. [94,95] reported that thermoduric streptococci as well as a strain of Staphylococcus aureus were rendered much more susceptible to damage by the combined effect of ultrasonic (20 kHz) and heat treatment than by either treatment separately. Sanz et al. [106] found a marked decrease in heat resistance (without killing) of Bacillus stearothermophilus spores by ultrasound at 20 kHz regardless of the heating temperature and the storage time between both treatments. Its heat resistance was reduced from one half to one third of original value. The heat resistance at 105°C of Bacillus cereus can Bacillus licheniformis decreased after a previous ultrasound treatment at 20 kHz [15]. Garcia et al. [41] showed that ultrasonic treatment (20 kHz, 150 W) of Bacillus subtilis spores in distilled water or milk resulted in little or no decrease in heat resistance, but simultaneous ultrasonic and heat treatments were effective. This effect became smaller at the higher-temperature treatment. This may be due to the increased vapor pressure, decreased viscosity and, as a consequence, reduction in the intensity of cavitation [103].
D. Mano-Thermo-Sonication
Mano-thermo-sonication decreased heat resistance of Bacillus subtilis to about one-tenth that of a simply heated control in the range 100112°C. It was also effective with other microorganisms, such as spore-formers, vegetative cells, and yeast. The lethality of mano-thermo-sonication was 630 times greater than that of the corresponding heat treatment at the same temperature, depending on the microorganism, when Sala et al. [103] studied the survival curves of Aeromonas hydrophila, S. cerevisiae, B. coagulans, and B. stearothermophilus. Sala et al. [103] found that with combined heat-ultrasound under pressure, the sensitizing effect could be retained even at temperatures above the boiling point of medium. The efficacy of mano-thermo-sonication depended on the intensity of the ultrasound (sonication time, amplitude, and instrument output) and the extent of the pressure [103]. An ultrasonication treatment at 45°C under pressure failed to show any lethal effect on spores of B. subtilis var. niger, but heat resistance was reduced from 0.1 to 0.015 minutes at 112°C under pressure (20 kHz, 117 µm, 300 kPa) [103]. Thus, the effect is not additive, but synergistic.
1. Mode of Action in Mano-Thermo Sonication
The physical effects of ultrasound could sensitize target molecules and/or structures. Thus, heat plus pressure is more effective [103]. Scherba et al. [108] mentioned that extremely high-temperature hot spots developed by cavitation cause heat inactivation. Extreme pressure changes and shock waves may cause physical damage to the cell. Ultrasonication disrupted the spore exosporium and released dipicolinic acid and low molecular weight polypeptides from the cortex of some bacterial spores [7,96]. This cortex degradation would lead to rehydration of the protoplast, resulting in a loss of heat resistance [42].
2. Effects of Mano-Thermo-Sonication on Enzymes
The effects of mano-thermo-sonication on lipoxygenase, peroxidase, polyphenoloxidase, and Pseudomonas fluorescens extracellular protease and lipase are summarized by Sala et al. [103]. The enzyme-inactivation efficacy of heat is increased by a factor that depends on the nature of the enzyme and the treatment conditions. The role of the different bonds and interactions involved in protein structure stabilization are not equally important in maintaining the native structure of the catalytic center of each of the enzymes, and they are not equally affected by heat and ultrasound.
Enzyme inactivation by a combined treatment of heat and ultrasound under pressure is not additive but a synergistic. The magnitude of the synergistic effect depends on the bonds and interactions of the active sites for catalysis, the nature of the groups participating in the catalysis, and the molecular weight of the protein [103].
The synergistic effect at constant pressure decreased with an increase in temperature in lipoxygenase and P. fluorescens protease [103]. This may be due to less effective collapse by an increase of the water vapor pressure in the cavitation bubble and the lower number of cavitation collapses available to release energy [103].
Lu and Whitaker [76] suggested a mechanism involving the release of the heme moiety of the enzyme previous to the denaturation of the liberated apoprotein during heat inactivation of peroxidase. A similar mechanism is also involved in mano-thermo-sonication [103]. Sala et al. [103] mentioned that it must be clarified whether the potentiating effects of ultrasound on peroxidase destruction are related to an increase in the dissociation rate or to the hindering of heme binding to the apoprotein. This could be due to an increase in denaturation rate or in the rate of heme destruction [103].
Sala et al. [103] summarized other factors effecting mano-thermo-sonication efficiency for enzyme inactivation: the effect (a) is almost independent of the ionic strength in the range 01, (b) increases as pH changes from 5 to 8, and the level of increment also depends on temperature and types of enzymes, (c) diminishes with increasing enzyme concentration, and (d) increases with soluble solids concentration [103]. The concentration effect is due to the increasing intensity of cavitation [28,35].
Sala et al. [103] concluded, based on the data in the literature, that the resistance of most microorganisms and enzymes to ultrasound is so high that it would probably produce extensive undesirable quality changes. Thus, a combination of ultrasound with other treatments, such as heat and pressure, should have a much better chance for practical use in the future.
Written by M. Shafiur Rahman in "Handbook of Food Preservation", Marcel Dekker, USA, 1999, excerpts pp.669-686. Digitized, adapted and illustrated to be posted by Leopoldo Costa.