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NON-THERMAL PROCESSING | Ultrasonication
Ultrasonication K Scho¨ssler, Technische Universität Berlin, Berlin, Germany H Ja¨ger, Technische Universität Berlin, Berlin, Germany; and Nestlé PTC Singen, Singen, Germany C Bu¨chner, S Struck, and D Knorr, Technische Universität Berlin, Berlin, Germany Ó 2014 Elsevier Ltd. All rights reserved.
Introduction Ultrasound is the sound energy emitted by sound waves with frequencies above the human hearing. In dependence of the intensity and the mechanisms of ultrasound treatments, two different approaches of ultrasonic processing are applied in food and bioprocessing. Low-intensity ultrasound with intensities <1 W cm2 is applied at high frequencies (MHz range) and with low amplitudes. Such ultrasound applications are nondestructive and can be applied for testing and imaging applications. In contrast, high-intensity ultrasound is characterized by low frequencies (20–100 kHz) and high amplitudes, and reaches intensities of 10–1000 W cm2. It is applied to alter material characteristics, increase processing rates, inactivate microorganisms and enzymes, and assist several processes in food and bioprocessing. With respect to food microbiology, the major uses of ultrasound are stimulating living cells, declumping, and cell disruption in analytical microbiology and ultrasound-assisted microbial inactivation for preservation purposes. Living cells are stimulated to increase the production of secondary metabolites as products or to improve the stress tolerance of the treated cells. At slightly higher intensities, ultrasonic waves can be applied to separate cell aggregates for exact cell enumeration, which is applied as a pretreatment in analytical microbiology. Ultrasound-induced cell disruption can be applied to improve detection for cell content. On the other hand, inactivation of microorganisms is a key processing step in the production of the majority of industrially processed food products. Conventional processes are based on heat inactivation, which often is related to product degradation. Heat-labile compounds, such as flavor compounds, color pigments, or vitamins, are easily destroyed at elevated temperatures and lead to a loss of the freshness characteristics of a treated product. Ultrasound was shown to act synergistically with a large variety of lethal stresses, such as heat, elevated pressure, chemicals, or cell permeabilization by pulsed electric fields (PEFs).
Mechanisms of Action in the Sound Field The sound wave as a pressure wave is a mechanical force acting on the transmission medium. The pressure alterations lead to cyclic compression and decompression of the treated product and associated mechanical, chemical, and thermal effects. Mechanical effects include acoustic streaming, mixing, and shear effects. Strong mechanical forces are attributed to ultrasound-induced cavitation, the formation, growth, oscillation, and eventual collapse of gas- and vapor-filled bubbles in liquid transmission media. The violent bubble collapse occurs when high pressure variations are achieved, which is limited to lower frequencies (<1 MHz). This so-called transient
Encyclopedia of Food Microbiology, Volume 2
cavitation is the main mechanism of action in most highintensity ultrasound applications. The associated effects include shear effects and microstreaming in the vicinity of oscillating bubbles, as well as high temperature and pressure peaks at the bubble collapse due to a sudden compression of the gas present in the bubble. Large numbers of cavitation bubbles may cause sound-wave absorption due to thermal, viscous, or acoustic damping and lead to reduced ultrasound effects. When bubbles collapse near solid surfaces, liquid jets and high-energy shockwaves can lead to strong mechanical impacts on the treated product, which often are cited to be responsible for the disruption of microbial cells during ultrasonic processing. The extreme conditions related to the violent bubble collapse can lead to chemical effects in the sound field. In water vapor–filled cavitation bubbles primary H$ and OH$ radicals are generated due to high local temperature and pressure peaks. The radicals and their reaction products, for example, hydrogen peroxide, are highly reactive and can cause oxidation reactions, which may affect microorganisms as well as product quality. In addition to the local hot spots occurring during the bubble collapse, thermal effects of ultrasound occur due to the absorption of the acoustic energy as well as due to thermal and viscous damping during the oscillation of cavitation bubbles. The temperature increase in a sonicated product depends on the treatment intensity and the specific heat capacity of the medium and is linear until the ambient temperature is exceeded. Heat convection, conduction, and radiation will then limit further temperature increase.
Stimulation of Living Cells Ultrasound treatment is generally associated with damage to cells, but evidence is emerging for beneficial effects of sonication on metabolic activity of living cells. Increasing the production of secondary metabolites by stimulating living cells is a concept with growing interest in food- and biotechnology. Enhanced metabolic productivity of microbial, plant, and animal cells in bioreactors can greatly improve the economics of the respective processes. The application of low-power ultrasound was shown to enhance the growth of algal cells in a liquid nutrient media and resulted in an increase in the production of protein. Applying ultrasound as a processing aid during yogurt production was shown to decrease necessary fermentation time with improved product texture and consistency. Fish eggs treated with 1 MHz ultrasound for 35 min three times a day showed reduced hatch time, which represents a benefit for fish-farming. Ultrasonication of seeds before sowing resulted in reductions in germination times for bean and rice as well as lotus
http://dx.doi.org/10.1016/B978-0-12-384730-0.00401-8
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seeds. Ultrasonication involves the treatment of seeds in water and is believed to assist in the breaking of dormancy. Sonication (20 kHz, 140 mm) applied under dry conditions several months before sowing improved sunflower germination. Ultrasound facilitates the disintegration of complex media, thereby exposing a much larger surface area to enzymes and microbial cells. Such disintegration may improve the accessibility of substrates for bioconversion, hence improving the metabolic activity of cells. By integrating an ultrasound pretreatment of a corn-meal slurry in bioethanol production, the overall ethanol yield resulting from fermentation with Saccharomyces cerevisiae could be significantly increased with a short processing time. The cavitation generated by ultrasound creates powerful hydromechanical shear forces as well as microstreaming, which improve the distribution of solutes and the mass transfer. The concept of using ultrasound for enhanced microbial productivity resulted in the development of sonobioreactors. Mass transfer and the exchange of nutrients and products between the growth media and the cells is a prerequisite for high reaction rates in biocatalysis. Microstreaming and the formation of microbubbles in a sonicated bioreactor reduce fluid boundary layers and enhance the mass transfer. In addition, ultrasound may enhance mass transfer within a cell due to intracellular microstreaming. A membrane permeation–enhancing effect as well as rotation of cell organelles and induced circulation within cells can be associated with ultrasound as well. These effects not only contribute to improved mass transfer but also may represent stressors to the biological system. Hence, an increase in productivity may be related to stimulation and an induced stress response resulting in an increased production of secondary metabolites. In addition, the enhancement of cell wall and membrane fluidity by ultrasound treatment was concluded to be one reason for the stimulation and was found to affect growth and proliferation of rice callus cells. Reversible permeabilization induced by ultrasound and resulting in minimal cell injury will allow the repeated harvest of cellular content that was shown for in vitro grown plant cells. The given examples show various effects of ultrasound on living cells that range from the promotion of enzyme activity to growth stimulation and the improvement of the penetrability of the cell membrane. Hence, ultrasound has a large potential for further application in cell and fermentation engineering.
Ultrasound Pretreatment in Analytical Microbiology A number of microbiological methods are based on the reliable and reproducible detection and enumeration of microorganisms. High-power ultrasound may disrupt agglomerates of cells but also may inactivate cells by disruption of the cell structure. Declumping of cell clusters may be beneficial for the enumeration of cells, whereas the disintegration of the cells will become relevant when measuring substances in the cell content. For that purpose, a prior sample pretreatment will result in a facilitated release and detection of cell content.
The two phenomena, declumping and cell disintegration, and the scale of these effects were found to depend on sound intensity and frequency. Higher frequencies (580 kHz for Escherichia coli/Klebsiella pneumonia; 1146 kHz for cyanobacteria) and shorter treatment times were found to increase the detectable colony forming units indicating a deagglomeration. Reports indicate that ultrasonic pretreatment of milk causes increases in total numbers of recoverable bacteria by breaking up clumps of bacteria normally occurring in milk. Conventional microbiological methods such as the detection of microbial antigen in clinical specimens by agglutination may be improved by ultrasound application. Antigen detection by immune-agglutination of coated latex microparticles was found to be enhanced in rate and sensitivity by the application of a noncavitating ultrasonic standing wave. The physical forces promote the formation of agglutinates by increasing particle–particle contact. Particles suspended in a megahertz-frequency ultrasonic standing wave experience accumulation at the pressure nodes and are subjected to secondary acoustic attractive forces that bring the particle surfaces in proximity. This increases the rate of particle collision needed to enable antigen–antibody crosslinking and enhances the speed and sensitivity. Ultrasonically increased particle–particle interactions using standing waves can be realized in a controlled manner with some advantages compared with conventional effects such as Brownian motion, gravity, or microvortices produced by agitation. Standing waves have been shown to be useful in the separation and purification of solutions acting as a form of particle separator. Ultrasonic filters retain cells in an acoustic field allowing for the downstream purification of, for example, antibodies from fermentation filtrates containing cells. Disruptive cavitation was shown to enhance the sensitivity of enzyme immunoassays by increasing molecular diffusion across liquid–solid surfaces as occurring between immobilized antibodies and antigens present in a solvent. The recent developments in ultrasonic equipment, including the development of new and powerful devices, allow high-throughput applications. Sample treatments for protein identification, including the enzymatic digestion, can be boosted by ultrasound application, and the technology will find further applications in such analytical areas as metabolomics or genomics.
Inactivation of Microorganisms High-intensity ultrasound can affect the viability of microorganisms due to the mechanical, chemical, and thermal effects occurring in the sound field. The types of ultrasound-induced cell injury reach from radical-induced DNA changes to thermal and mechanical alteration of the cell membrane, weakening the cell structure. Several studies have shown that an ultrasound treatment alone is mostly insufficient to cause inactivation rates relevant for food processing. The ultrasound-induced changes at a cellular level have the potential to increase damage caused by other lethal factors, such as elevated temperature, pressure, the use of chemicals, or cell permeabilization. Combination treatments of such processes with ultrasound have led to additive and often even synergistic effects on the inactivation of
NON-THERMAL PROCESSING j Ultrasonication microorganisms. The following sections will summarize the knowledge on these combination treatments and the assumed underlying mechanisms of action.
Ultrasound and Temperature Thermal treatments represent the conventional way of food preservation targeting on microbiological safety, inactivation of enzymes, digestibility, and stability. The application of heat on food material for preservation could cause negative effects on texture, taste, and nutrients, such as natural antioxidants and bioactive compounds, which are undesirable and should be reduced as much as possible. Therefore, treatment temperature plays a critical role regarding the preservation process. The combination of ultrasound and heat treatment is known as thermosonication. Results of numerous studies showed that ultrasound and temperature had synergistic effects on inactivation of microorganisms. Microbiological cells became more sensitive to heat after being treated with ultrasound. It was also shown that above a critical treatment temperature, the additional use of ultrasound does not result in an increased inactivation of microorganisms in comparison to heat treatment only. The reason could be the cushioning effect of vapor in cavitation bubbles. During ultrasound treatments of liquid media, temperature affects vapor pressure, surface tension, and viscosity. With increasing temperature in the liquid medium, the equilibrium vapor pressure of the system and the formation of cavitation bubbles increase. The cavitation bubbles contain more vapor, which cushions the implosion of the bubbles during cavitation and therefore the cavitation-related effects. In contrast, increased temperature results in decreased viscosity of the liquid medium, which leads to easier bubble formation. Consequently, there is an optimum temperature for maximum cavitation in liquid media at which the strongest effects of ultrasound emerge. Ultrasonic effects in liquids, such as bubble implosion, high pressure, hot spots, microjets, microstreaming, and formation of free radicals, which occur during cavitation, can cause cellwall disruption and therefore lower the heat sensibility of bacterial cells. Electron microscopy scans of E. coli cells after ultrasonic treatments showed extensive damage on cell segments and intracellular constituents as well as breakage on cell membranes, shrinkage, and surface pitting. Recent studies of thermosonication with mild temperatures (35–45 C) have demonstrated the additional effect of the combination with ultrasound regarding the inactivation of Saccharomyces cereviseae, Staphylococcos aureus, Salmonella enterica, E. coli, and others. Ultrasound application during direct-steam injection heating was investigated aiming at maximizing cavitation effects and improving heat transfer. Studies have shown improved inactivation but also different sensitivities of Lactobacillus acidophilus and E. coli cells, which probably are attributed to differences in the cell-wall structure. The observed higher resistance of L. acidophilus cells could be of interest in pasteurization of probiotic fermented products where the selective retention of Lactobacilli might be of interest. Heat resistance of bacterial spores is reduced by ultrasound because cavitational effects, such as the high local pressure peaks during the bubble collapse, cause the release of
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dipicolonic acid (DPA), calcium, and other low-molecularweight substances from the spore into the surrounding medium. This release does not result in loss of viability, but increases the heat sensibility of spores because the permeability of the protoplast membrane simultaneously induces entrance of water into the cell. This hydration then can lead to a reduction of the heat resistance of bacterial spores. Thermosonication is a possibility to lower the treatment temperature in pasteurization treatments and to obtain the same results as conventional thermal treatment. This results in reduced thermal damage of the product, leading to better quality and improvement of taste, texture, and nutritional value. Combined treatments can result in higher energy consumptions than heat treatments alone, indicating the need to define the required pasteurization or sterilization effect and relate it to the improvements in food quality or food safety achievable for a certain product.
Ultrasound and Pressure The combination of ultrasound and pressure at sublethal temperatures is known as manosonication and provides an alternative way to enhance effects of cavitation in liquid media. Increasing external pressure results in a higher cavitation threshold and greater intensity of bubble collapse as the pressure in the cavitation bubble during collapse can be considered approximately as the sum of acoustic and hydrostatic pressure. Therefore an increase of external pressure increases the pressure inside the cavitation bubble leading to a more violent and rapid bubble collapse. During manosonication a backpressure from 1 to 5 bar is applied with a constant pressure pump, such as centrifugal pumps or gear pumps. This is the result of numerous studies that additional application of static pressure during an ultrasound treatment increases the inactivation of vegetative cells. For instance, a pressure of 100–500 kPa in addition to sonication at sublethal temperatures (40–54 C) could implement a reduction of 5-log E. coli cells in 2 min. Sonication without pressure at the same temperatures on the other hand could inactivate 4-log in 4 min. The lethal effect of ultrasound is increased with applied external static pressure. For example the D-value of Y. enterocolitica at 30 C, 600 kPa and sonication (150 mm) was determined with 0.22 min. Without application of pressure sonication at sublethal temperatures lead to a D-value of 1.5 min. Ultrasonic treatments with increased pressure from 0 to 200 kPa of Listeria monocytogenes at sublethal temperatures (40 C) could reduce the D-value from 4.3 to 1.5 min. Grampositive and coccal bacteria forms are the most resistant microorganism for manosonication treatment. The D-value of different vegetative microorganisms decreased drastically with combined pressure. Observation on the influence of amplitude on the lethality of manosonication (200 kPa, 40 C) demonstrated that the D-values of all species examined decreased with increasing amplitude between 62 and 150 mm. The same influence of amplitude on lethality was observed for Bacillus subtilis spores. Manosonication treatment could inactivate 99.9% of a Bacillus subtilis spore population. An upper-pressure level between 400 and 600 kPa has been reported for manosonication in different studies, which
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depends on intensity of the ultrasonic field, microorganism sensitivity to ultrasound, and medium properties. Above the upper-pressure limit an additional increase in static pressure does not result in increased inactivation of bacterial cells. The reason for such an upper-pressure limit is probably the inhibition of cavitation at high static pressures, as combined forces of overpressure and cohesive forces of liquid molecules constrain the ultrasonic field. This results in a reduced number of imploding bubbles, reduced cavitation, and consequently diminished effects of manosonication. The literature states that an optimum operating pressure exists.
Manothermosonication The combination of ultrasound and heat under pressure is known as manothermosonication. The advantage of this combination is that the loss of the cavitational effect at temperatures near the boiling point due to the high–water vapor pressure in the liquid medium can be overcome by applying an external pressure. The result is that cavitation is possible above the water boiling point, which leads to an increased lethal effect of the treatment. Several studies have shown the influence of manothermosonication on the inactivation of microorganisms. Observations demonstrated that the D-value of Y. enterocolitica decreased rapidly between 50 and 58 C throughout manothermosonication with 200 kPa, 117 mm, and 20 kHz compared with sole heat treatment. Below 50 C no influence of temperature on the inactivation rate was observed. Temperatures over 58 C resulted in equal D-values of heat and manosonication treatment. Two mechanisms are responsible for the inactivation of microorganisms during manothermosonication: the heat and the manosonication treatment. The lethal effects of both mechanisms add up to the inactivation rate, which depends on treatment temperature. A synergistic effect between manothermosonication and a reduced aW value of the treatment media was observed. With decreasing water activity, the synergistic effect on inactivation of Salmonella enterica serovar Enteritidis increased. The reason for this effect could be the sensitizing effect of heat during treatment. Manothermosonication between 70 and 90 C increased the lethality of the treatment on Bacillus subtilis spores due to the synergistic effect of heat and ultrasound.
Ultrasound and Pulsed Electric Fields Microbial inactivation of vegetative cells by PEFs involves the application of short pulses of high electric field intensity, which leads to an irreversible perforation of the cell membrane and eventually to cell death. This technology offers pasteurization with low energy input and higher retention of heat-sensitive food compounds. In some cases, however, intense PEF treatments have to be applied to achieve substantial microbial inactivation. Since technical as well as quality aspects limit PEF treatment intensity, combining PEF and ultrasound according to the hurdle concept is a promising approach. This holds especially true, because ultrasound and PEF inactivate by different mechanisms, which could potentially lead to synergistic effects. Few studies investigate the combination of ultrasound with PEF, and contradicting experimental results have been
published. The effect of combined TS (thermosonication) and PEF treatment on the inactivation of Listera innocua in milk yielded a degree of inactivation, which was comparable to conventional pasteurization, whereas individual treatments with TS and PEF resulted only in moderate bacterial inactivation. Compared with the conventional pasteurization process the TS/PEF treatment featured a shorter treatment time and less exposure to temperature. It was reported that TS/PEF-treated orange juice had comparable microbial shelf life stability and similar overall consumer acceptability as the high-temperature, short-time (HTST) -pasteurized juice. Synergistic effects of PEFultrasound were reported for the inactivation of Streptococcus thermophilus in Ringer solution. In both configurations (PEF followed by ultrasound and ultrasound followed by PEF) the combined treatment caused higher inactivation than the additive sum of single ultrasound and PEF treatments. Contradictory results were published on the inactivation of Salmonella enteritidis in liquid whole egg. In this application, both combinations (PEF/ultrasound and ultrasound/PEF) only exhibited additive effects and no synergy was observed. Treatments were performed in batch mode instead of a continuous flow through operation, which may be one reason for differences in experimental results. The insights gained so far indicate that the combined treatment with PEF and ultrasound might be a promising alternative to conventional pasteurization. Yet, further studies are required to confirm the proposed mechanisms.
Ultrasound and Chemicals Combining ultrasound with stress factors like a low pH, natural antimicrobials, or chemicals is expected to give synergistic effects and increase the efficiency of the microbial deactivation. Several authors investigated the combination of ultrasound with acidic conditions, and contradicting results have been published. Most authors report no significant influence of the pH when ultrasound is applied at nonlethal temperatures. Thus, it appears that the resistance to ultrasound is not affected by acidic conditions. Thus far, ultrasound resistance differs from heat resistance, which is generally decreased at a low pH value. In these premises, a low pH can be beneficial for ultrasound, because the ultrasound treatment is usually connected to a temperature rise at high treatment intensities. If this generated heat is not removed or ultrasound is applied at elevated temperatures (Thermosonication), synergistic effects can be expected due to the interaction of heat and pH. Many researchers successfully applied ultrasound in combination with sanitizing agents for the decontamination fresh fruit and vegetables. Synergistic effects occurred for the combination of ultrasound with commercial sanitizers like sodium dichloroisocyanurate, chlorine dioxide, or peracetic acid on cherry tomatoes. The bactericidal properties of chlorine dioxide were enhanced by ultrasound for the treatment of apples and lettuce. Ultrasound in conjunction with chlorine, acidified sodium chlorite, peroxyacetic acid, or acidic electrolyzed water increased the reduction of E. coli on spinach. Salicylic acid combined with ultrasound was more effective than the salicylic acid treatment alone for reducing Penicillium expansum in peach fruit. The functional principle is probably that cavitation induced by ultrasound detaches cells from the
NON-THERMAL PROCESSING j Ultrasonication surface of the fruit or vegetable. Thus the susceptibility of the microorganism to the sanitizer is increased. The combination of ultrasound with natural antimicrobials also seems to be a promising approach. For example, an enhanced inactivation of Listeria innocua was reported for the vanillin, when applied in conjunction with heat and ultrasound. Chitosan combined with ultrasound led to an enhanced inactivation of S. cerevisiae.
Conclusion and Future Trends The three large areas of ultrasound application in food microbiology, namely, cell stimulation, pretreatments for improved analytics, and microbial inactivation, highlight the high diversity of this technology and its large potential in future process development. The underlying mechanisms are not yet fully understood, however, and make target-oriented processing difficult. Hence, future research work should focus on the identification of the mechanisms of action, especially during the combination treatments applied for preservation purposes. A better understanding of the interaction of the different lethal factors and the specific response of different kinds of microorganisms, and the role of the medium composition will improve process development. It is the inevitable precondition for the transfer of this promising technology to industrial-level food preservation.
See also: Minimal Methods of Processing: Manothermosonication; Potential Use of Phages and Lysins; Nonthermal Processing: Pulsed Electric Field; Nonthermal Processing: Pulsed UV Light; Nonthermal Processing: Irradiation; Nonthermal Processing: Microwave; Nonthermal Processing: Cold Plasma for Bioefficient Food Processing; Nonthermal Processing: Steam Vacuuming; Thermal Processes: Pasteurization; Thermal Processes, Commercial Sterility (Retort).
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Further Reading Chisti, Y., 2003. Sonobioreactors: using ultrasound for enhanced microbial productivity. Trends in Biotechnology 21 (2), 89–93. Earnshaw, R.G., Appleyard, J., Hurst, R.M., 1995. Understanding physical inactivation processes: combined preservation opportunities using heat, ultrasound and pressure. International Journal of Food Microbiology 28, 197–219. Ellis, R.W., Sobanski, M.A., 2000. Diagnostic particle agglutination using ultrasound: a new technology to rejuvenate old microbiological methods. Journal of Medical Microbiology 49, 853–859. Feng, H., Barbosa-Cánovas, G.V., Weiss, J. (Eds.), 2011. Ultrasound Technologies for Food and Bioprocessing. Food Engineering Series, Barbosa-Cánovas, G. V. series (Ed.), Springer ScienceþBuisness Media, LLC. Gastélum, G.G., Avila-Sosa, R., López-Malo, A., Palou, E., 2010. Listeria innocua multi-target inactivation by thermo-sonication and vanillin. Food and Bioprocess Technology 5, 665–671. Huang, T.-S., Xu, C., Walker, K., West, P., Zhang, S., Weese, J., 2006. Decontamination efficacy of combined chlorine dioxide with ultrasonication on apples and lettuce. Journal of Food Science 71, M134–M139. Humphrey, V.F., 2007. Ultrasound and matter – physical interactions. Progress in Biophysics and Molecular Biology 93, 195–211. Knorr, D., Zenker, M., Heinz, V., Lee, D.-U., 2004. Applications and potential of ultrasonics in food processing. Trends in Food Science and Technology 15, 261–266. Lee, H., Zhou, B., Feng, H., Martin, S.E., 2009. Effect of pH on Inactivation of Escherichia coli K12 by sonication, manosonication, thermosonication, and manothermosonication. Journal of Food Science 74, E191–E198. Lee, H., Zhou, B., Liang, W., Feng, H., Martin, S.E., 2009. Inactivation of Escherichia coli cells with sonication, manosonication, thermosonication, and manothermosonication: microbial responses and kinetics modeling. Journal of Food Engineering 93 (3), 354–364. Lorimer, J.P., Mason, T.J., 1987. Sonochemistry part 1-the physical aspects. Chemical Society Reviews 16, 239–274. Mason, T.J., Paniwnyk, L., Lorimer, J.P., 1996. The uses of ultrasound in food technology. Ultrasonics Sonochemistry 3, 253–260. Raso, J., Pagán, R., Condón, S., Sala, F.J., 1998. Influence of temperature and pressure on the lethality of ultrasound. Applied and Environmental Microbiology 64 (2), 465–471. Ross, A.I.V., Griffiths, M.W., Mittal, G.S., Deeth, H.C., 2003. Combining nonthermal technologies to control foodborne microorganisms. International Journal of Food Microbiology 89, 125–138. Santos, H.M., Capelo, J.L., 2007. Trends in ultrasonic-based equipment for analytical sample treatment. Talanta 73, 795–802. Thompson, L.H., Doraiswamy, L.K., 1999. sonochemistry: science and engineering. Industrial & Engineering Chemistry Research 38, 1215–1249.
Introduction Ultrasound is the sound energy emitted by sound waves with frequencies above the human hearing. In dependence of the intensity and the mechanisms of ultrasound treatments, two different approaches of ultrasonic processing are applied in food and bioprocessing. Low-intensity ultrasound with intensities <1 W cm2 is applied at high frequencies (MHz range) and with low amplitudes. Such ultrasound applications are nondestructive and can be applied for testing and imaging applications. In contrast, high-intensity ultrasound is characterized by low frequencies (20–100 kHz) and high amplitudes, and reaches intensities of 10–1000 W cm2. It is applied to alter material characteristics, increase processing rates, inactivate microorganisms and enzymes, and assist several processes in food and bioprocessing. With respect to food microbiology, the major uses of ultrasound are stimulating living cells, declumping, and cell disruption in analytical microbiology and ultrasound-assisted microbial inactivation for preservation purposes. Living cells are stimulated to increase the production of secondary metabolites as products or to improve the stress tolerance of the treated cells. At slightly higher intensities, ultrasonic waves can be applied to separate cell aggregates for exact cell enumeration, which is applied as a pretreatment in analytical microbiology. Ultrasound-induced cell disruption can be applied to improve detection for cell content. On the other hand, inactivation of microorganisms is a key processing step in the production of the majority of industrially processed food products. Conventional processes are based on heat inactivation, which often is related to product degradation. Heat-labile compounds, such as flavor compounds, color pigments, or vitamins, are easily destroyed at elevated temperatures and lead to a loss of the freshness characteristics of a treated product. Ultrasound was shown to act synergistically with a large variety of lethal stresses, such as heat, elevated pressure, chemicals, or cell permeabilization by pulsed electric fields (PEFs).
Mechanisms of Action in the Sound Field The sound wave as a pressure wave is a mechanical force acting on the transmission medium. The pressure alterations lead to cyclic compression and decompression of the treated product and associated mechanical, chemical, and thermal effects. Mechanical effects include acoustic streaming, mixing, and shear effects. Strong mechanical forces are attributed to ultrasound-induced cavitation, the formation, growth, oscillation, and eventual collapse of gas- and vapor-filled bubbles in liquid transmission media. The violent bubble collapse occurs when high pressure variations are achieved, which is limited to lower frequencies (<1 MHz). This so-called transient
Encyclopedia of Food Microbiology, Volume 2
cavitation is the main mechanism of action in most highintensity ultrasound applications. The associated effects include shear effects and microstreaming in the vicinity of oscillating bubbles, as well as high temperature and pressure peaks at the bubble collapse due to a sudden compression of the gas present in the bubble. Large numbers of cavitation bubbles may cause sound-wave absorption due to thermal, viscous, or acoustic damping and lead to reduced ultrasound effects. When bubbles collapse near solid surfaces, liquid jets and high-energy shockwaves can lead to strong mechanical impacts on the treated product, which often are cited to be responsible for the disruption of microbial cells during ultrasonic processing. The extreme conditions related to the violent bubble collapse can lead to chemical effects in the sound field. In water vapor–filled cavitation bubbles primary H$ and OH$ radicals are generated due to high local temperature and pressure peaks. The radicals and their reaction products, for example, hydrogen peroxide, are highly reactive and can cause oxidation reactions, which may affect microorganisms as well as product quality. In addition to the local hot spots occurring during the bubble collapse, thermal effects of ultrasound occur due to the absorption of the acoustic energy as well as due to thermal and viscous damping during the oscillation of cavitation bubbles. The temperature increase in a sonicated product depends on the treatment intensity and the specific heat capacity of the medium and is linear until the ambient temperature is exceeded. Heat convection, conduction, and radiation will then limit further temperature increase.
Stimulation of Living Cells Ultrasound treatment is generally associated with damage to cells, but evidence is emerging for beneficial effects of sonication on metabolic activity of living cells. Increasing the production of secondary metabolites by stimulating living cells is a concept with growing interest in food- and biotechnology. Enhanced metabolic productivity of microbial, plant, and animal cells in bioreactors can greatly improve the economics of the respective processes. The application of low-power ultrasound was shown to enhance the growth of algal cells in a liquid nutrient media and resulted in an increase in the production of protein. Applying ultrasound as a processing aid during yogurt production was shown to decrease necessary fermentation time with improved product texture and consistency. Fish eggs treated with 1 MHz ultrasound for 35 min three times a day showed reduced hatch time, which represents a benefit for fish-farming. Ultrasonication of seeds before sowing resulted in reductions in germination times for bean and rice as well as lotus
http://dx.doi.org/10.1016/B978-0-12-384730-0.00401-8
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seeds. Ultrasonication involves the treatment of seeds in water and is believed to assist in the breaking of dormancy. Sonication (20 kHz, 140 mm) applied under dry conditions several months before sowing improved sunflower germination. Ultrasound facilitates the disintegration of complex media, thereby exposing a much larger surface area to enzymes and microbial cells. Such disintegration may improve the accessibility of substrates for bioconversion, hence improving the metabolic activity of cells. By integrating an ultrasound pretreatment of a corn-meal slurry in bioethanol production, the overall ethanol yield resulting from fermentation with Saccharomyces cerevisiae could be significantly increased with a short processing time. The cavitation generated by ultrasound creates powerful hydromechanical shear forces as well as microstreaming, which improve the distribution of solutes and the mass transfer. The concept of using ultrasound for enhanced microbial productivity resulted in the development of sonobioreactors. Mass transfer and the exchange of nutrients and products between the growth media and the cells is a prerequisite for high reaction rates in biocatalysis. Microstreaming and the formation of microbubbles in a sonicated bioreactor reduce fluid boundary layers and enhance the mass transfer. In addition, ultrasound may enhance mass transfer within a cell due to intracellular microstreaming. A membrane permeation–enhancing effect as well as rotation of cell organelles and induced circulation within cells can be associated with ultrasound as well. These effects not only contribute to improved mass transfer but also may represent stressors to the biological system. Hence, an increase in productivity may be related to stimulation and an induced stress response resulting in an increased production of secondary metabolites. In addition, the enhancement of cell wall and membrane fluidity by ultrasound treatment was concluded to be one reason for the stimulation and was found to affect growth and proliferation of rice callus cells. Reversible permeabilization induced by ultrasound and resulting in minimal cell injury will allow the repeated harvest of cellular content that was shown for in vitro grown plant cells. The given examples show various effects of ultrasound on living cells that range from the promotion of enzyme activity to growth stimulation and the improvement of the penetrability of the cell membrane. Hence, ultrasound has a large potential for further application in cell and fermentation engineering.
Ultrasound Pretreatment in Analytical Microbiology A number of microbiological methods are based on the reliable and reproducible detection and enumeration of microorganisms. High-power ultrasound may disrupt agglomerates of cells but also may inactivate cells by disruption of the cell structure. Declumping of cell clusters may be beneficial for the enumeration of cells, whereas the disintegration of the cells will become relevant when measuring substances in the cell content. For that purpose, a prior sample pretreatment will result in a facilitated release and detection of cell content.
The two phenomena, declumping and cell disintegration, and the scale of these effects were found to depend on sound intensity and frequency. Higher frequencies (580 kHz for Escherichia coli/Klebsiella pneumonia; 1146 kHz for cyanobacteria) and shorter treatment times were found to increase the detectable colony forming units indicating a deagglomeration. Reports indicate that ultrasonic pretreatment of milk causes increases in total numbers of recoverable bacteria by breaking up clumps of bacteria normally occurring in milk. Conventional microbiological methods such as the detection of microbial antigen in clinical specimens by agglutination may be improved by ultrasound application. Antigen detection by immune-agglutination of coated latex microparticles was found to be enhanced in rate and sensitivity by the application of a noncavitating ultrasonic standing wave. The physical forces promote the formation of agglutinates by increasing particle–particle contact. Particles suspended in a megahertz-frequency ultrasonic standing wave experience accumulation at the pressure nodes and are subjected to secondary acoustic attractive forces that bring the particle surfaces in proximity. This increases the rate of particle collision needed to enable antigen–antibody crosslinking and enhances the speed and sensitivity. Ultrasonically increased particle–particle interactions using standing waves can be realized in a controlled manner with some advantages compared with conventional effects such as Brownian motion, gravity, or microvortices produced by agitation. Standing waves have been shown to be useful in the separation and purification of solutions acting as a form of particle separator. Ultrasonic filters retain cells in an acoustic field allowing for the downstream purification of, for example, antibodies from fermentation filtrates containing cells. Disruptive cavitation was shown to enhance the sensitivity of enzyme immunoassays by increasing molecular diffusion across liquid–solid surfaces as occurring between immobilized antibodies and antigens present in a solvent. The recent developments in ultrasonic equipment, including the development of new and powerful devices, allow high-throughput applications. Sample treatments for protein identification, including the enzymatic digestion, can be boosted by ultrasound application, and the technology will find further applications in such analytical areas as metabolomics or genomics.
Inactivation of Microorganisms High-intensity ultrasound can affect the viability of microorganisms due to the mechanical, chemical, and thermal effects occurring in the sound field. The types of ultrasound-induced cell injury reach from radical-induced DNA changes to thermal and mechanical alteration of the cell membrane, weakening the cell structure. Several studies have shown that an ultrasound treatment alone is mostly insufficient to cause inactivation rates relevant for food processing. The ultrasound-induced changes at a cellular level have the potential to increase damage caused by other lethal factors, such as elevated temperature, pressure, the use of chemicals, or cell permeabilization. Combination treatments of such processes with ultrasound have led to additive and often even synergistic effects on the inactivation of
NON-THERMAL PROCESSING j Ultrasonication microorganisms. The following sections will summarize the knowledge on these combination treatments and the assumed underlying mechanisms of action.
Ultrasound and Temperature Thermal treatments represent the conventional way of food preservation targeting on microbiological safety, inactivation of enzymes, digestibility, and stability. The application of heat on food material for preservation could cause negative effects on texture, taste, and nutrients, such as natural antioxidants and bioactive compounds, which are undesirable and should be reduced as much as possible. Therefore, treatment temperature plays a critical role regarding the preservation process. The combination of ultrasound and heat treatment is known as thermosonication. Results of numerous studies showed that ultrasound and temperature had synergistic effects on inactivation of microorganisms. Microbiological cells became more sensitive to heat after being treated with ultrasound. It was also shown that above a critical treatment temperature, the additional use of ultrasound does not result in an increased inactivation of microorganisms in comparison to heat treatment only. The reason could be the cushioning effect of vapor in cavitation bubbles. During ultrasound treatments of liquid media, temperature affects vapor pressure, surface tension, and viscosity. With increasing temperature in the liquid medium, the equilibrium vapor pressure of the system and the formation of cavitation bubbles increase. The cavitation bubbles contain more vapor, which cushions the implosion of the bubbles during cavitation and therefore the cavitation-related effects. In contrast, increased temperature results in decreased viscosity of the liquid medium, which leads to easier bubble formation. Consequently, there is an optimum temperature for maximum cavitation in liquid media at which the strongest effects of ultrasound emerge. Ultrasonic effects in liquids, such as bubble implosion, high pressure, hot spots, microjets, microstreaming, and formation of free radicals, which occur during cavitation, can cause cellwall disruption and therefore lower the heat sensibility of bacterial cells. Electron microscopy scans of E. coli cells after ultrasonic treatments showed extensive damage on cell segments and intracellular constituents as well as breakage on cell membranes, shrinkage, and surface pitting. Recent studies of thermosonication with mild temperatures (35–45 C) have demonstrated the additional effect of the combination with ultrasound regarding the inactivation of Saccharomyces cereviseae, Staphylococcos aureus, Salmonella enterica, E. coli, and others. Ultrasound application during direct-steam injection heating was investigated aiming at maximizing cavitation effects and improving heat transfer. Studies have shown improved inactivation but also different sensitivities of Lactobacillus acidophilus and E. coli cells, which probably are attributed to differences in the cell-wall structure. The observed higher resistance of L. acidophilus cells could be of interest in pasteurization of probiotic fermented products where the selective retention of Lactobacilli might be of interest. Heat resistance of bacterial spores is reduced by ultrasound because cavitational effects, such as the high local pressure peaks during the bubble collapse, cause the release of
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dipicolonic acid (DPA), calcium, and other low-molecularweight substances from the spore into the surrounding medium. This release does not result in loss of viability, but increases the heat sensibility of spores because the permeability of the protoplast membrane simultaneously induces entrance of water into the cell. This hydration then can lead to a reduction of the heat resistance of bacterial spores. Thermosonication is a possibility to lower the treatment temperature in pasteurization treatments and to obtain the same results as conventional thermal treatment. This results in reduced thermal damage of the product, leading to better quality and improvement of taste, texture, and nutritional value. Combined treatments can result in higher energy consumptions than heat treatments alone, indicating the need to define the required pasteurization or sterilization effect and relate it to the improvements in food quality or food safety achievable for a certain product.
Ultrasound and Pressure The combination of ultrasound and pressure at sublethal temperatures is known as manosonication and provides an alternative way to enhance effects of cavitation in liquid media. Increasing external pressure results in a higher cavitation threshold and greater intensity of bubble collapse as the pressure in the cavitation bubble during collapse can be considered approximately as the sum of acoustic and hydrostatic pressure. Therefore an increase of external pressure increases the pressure inside the cavitation bubble leading to a more violent and rapid bubble collapse. During manosonication a backpressure from 1 to 5 bar is applied with a constant pressure pump, such as centrifugal pumps or gear pumps. This is the result of numerous studies that additional application of static pressure during an ultrasound treatment increases the inactivation of vegetative cells. For instance, a pressure of 100–500 kPa in addition to sonication at sublethal temperatures (40–54 C) could implement a reduction of 5-log E. coli cells in 2 min. Sonication without pressure at the same temperatures on the other hand could inactivate 4-log in 4 min. The lethal effect of ultrasound is increased with applied external static pressure. For example the D-value of Y. enterocolitica at 30 C, 600 kPa and sonication (150 mm) was determined with 0.22 min. Without application of pressure sonication at sublethal temperatures lead to a D-value of 1.5 min. Ultrasonic treatments with increased pressure from 0 to 200 kPa of Listeria monocytogenes at sublethal temperatures (40 C) could reduce the D-value from 4.3 to 1.5 min. Grampositive and coccal bacteria forms are the most resistant microorganism for manosonication treatment. The D-value of different vegetative microorganisms decreased drastically with combined pressure. Observation on the influence of amplitude on the lethality of manosonication (200 kPa, 40 C) demonstrated that the D-values of all species examined decreased with increasing amplitude between 62 and 150 mm. The same influence of amplitude on lethality was observed for Bacillus subtilis spores. Manosonication treatment could inactivate 99.9% of a Bacillus subtilis spore population. An upper-pressure level between 400 and 600 kPa has been reported for manosonication in different studies, which
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depends on intensity of the ultrasonic field, microorganism sensitivity to ultrasound, and medium properties. Above the upper-pressure limit an additional increase in static pressure does not result in increased inactivation of bacterial cells. The reason for such an upper-pressure limit is probably the inhibition of cavitation at high static pressures, as combined forces of overpressure and cohesive forces of liquid molecules constrain the ultrasonic field. This results in a reduced number of imploding bubbles, reduced cavitation, and consequently diminished effects of manosonication. The literature states that an optimum operating pressure exists.
Manothermosonication The combination of ultrasound and heat under pressure is known as manothermosonication. The advantage of this combination is that the loss of the cavitational effect at temperatures near the boiling point due to the high–water vapor pressure in the liquid medium can be overcome by applying an external pressure. The result is that cavitation is possible above the water boiling point, which leads to an increased lethal effect of the treatment. Several studies have shown the influence of manothermosonication on the inactivation of microorganisms. Observations demonstrated that the D-value of Y. enterocolitica decreased rapidly between 50 and 58 C throughout manothermosonication with 200 kPa, 117 mm, and 20 kHz compared with sole heat treatment. Below 50 C no influence of temperature on the inactivation rate was observed. Temperatures over 58 C resulted in equal D-values of heat and manosonication treatment. Two mechanisms are responsible for the inactivation of microorganisms during manothermosonication: the heat and the manosonication treatment. The lethal effects of both mechanisms add up to the inactivation rate, which depends on treatment temperature. A synergistic effect between manothermosonication and a reduced aW value of the treatment media was observed. With decreasing water activity, the synergistic effect on inactivation of Salmonella enterica serovar Enteritidis increased. The reason for this effect could be the sensitizing effect of heat during treatment. Manothermosonication between 70 and 90 C increased the lethality of the treatment on Bacillus subtilis spores due to the synergistic effect of heat and ultrasound.
Ultrasound and Pulsed Electric Fields Microbial inactivation of vegetative cells by PEFs involves the application of short pulses of high electric field intensity, which leads to an irreversible perforation of the cell membrane and eventually to cell death. This technology offers pasteurization with low energy input and higher retention of heat-sensitive food compounds. In some cases, however, intense PEF treatments have to be applied to achieve substantial microbial inactivation. Since technical as well as quality aspects limit PEF treatment intensity, combining PEF and ultrasound according to the hurdle concept is a promising approach. This holds especially true, because ultrasound and PEF inactivate by different mechanisms, which could potentially lead to synergistic effects. Few studies investigate the combination of ultrasound with PEF, and contradicting experimental results have been
published. The effect of combined TS (thermosonication) and PEF treatment on the inactivation of Listera innocua in milk yielded a degree of inactivation, which was comparable to conventional pasteurization, whereas individual treatments with TS and PEF resulted only in moderate bacterial inactivation. Compared with the conventional pasteurization process the TS/PEF treatment featured a shorter treatment time and less exposure to temperature. It was reported that TS/PEF-treated orange juice had comparable microbial shelf life stability and similar overall consumer acceptability as the high-temperature, short-time (HTST) -pasteurized juice. Synergistic effects of PEFultrasound were reported for the inactivation of Streptococcus thermophilus in Ringer solution. In both configurations (PEF followed by ultrasound and ultrasound followed by PEF) the combined treatment caused higher inactivation than the additive sum of single ultrasound and PEF treatments. Contradictory results were published on the inactivation of Salmonella enteritidis in liquid whole egg. In this application, both combinations (PEF/ultrasound and ultrasound/PEF) only exhibited additive effects and no synergy was observed. Treatments were performed in batch mode instead of a continuous flow through operation, which may be one reason for differences in experimental results. The insights gained so far indicate that the combined treatment with PEF and ultrasound might be a promising alternative to conventional pasteurization. Yet, further studies are required to confirm the proposed mechanisms.
Ultrasound and Chemicals Combining ultrasound with stress factors like a low pH, natural antimicrobials, or chemicals is expected to give synergistic effects and increase the efficiency of the microbial deactivation. Several authors investigated the combination of ultrasound with acidic conditions, and contradicting results have been published. Most authors report no significant influence of the pH when ultrasound is applied at nonlethal temperatures. Thus, it appears that the resistance to ultrasound is not affected by acidic conditions. Thus far, ultrasound resistance differs from heat resistance, which is generally decreased at a low pH value. In these premises, a low pH can be beneficial for ultrasound, because the ultrasound treatment is usually connected to a temperature rise at high treatment intensities. If this generated heat is not removed or ultrasound is applied at elevated temperatures (Thermosonication), synergistic effects can be expected due to the interaction of heat and pH. Many researchers successfully applied ultrasound in combination with sanitizing agents for the decontamination fresh fruit and vegetables. Synergistic effects occurred for the combination of ultrasound with commercial sanitizers like sodium dichloroisocyanurate, chlorine dioxide, or peracetic acid on cherry tomatoes. The bactericidal properties of chlorine dioxide were enhanced by ultrasound for the treatment of apples and lettuce. Ultrasound in conjunction with chlorine, acidified sodium chlorite, peroxyacetic acid, or acidic electrolyzed water increased the reduction of E. coli on spinach. Salicylic acid combined with ultrasound was more effective than the salicylic acid treatment alone for reducing Penicillium expansum in peach fruit. The functional principle is probably that cavitation induced by ultrasound detaches cells from the
NON-THERMAL PROCESSING j Ultrasonication surface of the fruit or vegetable. Thus the susceptibility of the microorganism to the sanitizer is increased. The combination of ultrasound with natural antimicrobials also seems to be a promising approach. For example, an enhanced inactivation of Listeria innocua was reported for the vanillin, when applied in conjunction with heat and ultrasound. Chitosan combined with ultrasound led to an enhanced inactivation of S. cerevisiae.
Conclusion and Future Trends The three large areas of ultrasound application in food microbiology, namely, cell stimulation, pretreatments for improved analytics, and microbial inactivation, highlight the high diversity of this technology and its large potential in future process development. The underlying mechanisms are not yet fully understood, however, and make target-oriented processing difficult. Hence, future research work should focus on the identification of the mechanisms of action, especially during the combination treatments applied for preservation purposes. A better understanding of the interaction of the different lethal factors and the specific response of different kinds of microorganisms, and the role of the medium composition will improve process development. It is the inevitable precondition for the transfer of this promising technology to industrial-level food preservation.
See also: Minimal Methods of Processing: Manothermosonication; Potential Use of Phages and Lysins; Nonthermal Processing: Pulsed Electric Field; Nonthermal Processing: Pulsed UV Light; Nonthermal Processing: Irradiation; Nonthermal Processing: Microwave; Nonthermal Processing: Cold Plasma for Bioefficient Food Processing; Nonthermal Processing: Steam Vacuuming; Thermal Processes: Pasteurization; Thermal Processes, Commercial Sterility (Retort).
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Further Reading Chisti, Y., 2003. Sonobioreactors: using ultrasound for enhanced microbial productivity. Trends in Biotechnology 21 (2), 89–93. Earnshaw, R.G., Appleyard, J., Hurst, R.M., 1995. Understanding physical inactivation processes: combined preservation opportunities using heat, ultrasound and pressure. International Journal of Food Microbiology 28, 197–219. Ellis, R.W., Sobanski, M.A., 2000. Diagnostic particle agglutination using ultrasound: a new technology to rejuvenate old microbiological methods. Journal of Medical Microbiology 49, 853–859. Feng, H., Barbosa-Cánovas, G.V., Weiss, J. (Eds.), 2011. Ultrasound Technologies for Food and Bioprocessing. Food Engineering Series, Barbosa-Cánovas, G. V. series (Ed.), Springer ScienceþBuisness Media, LLC. Gastélum, G.G., Avila-Sosa, R., López-Malo, A., Palou, E., 2010. Listeria innocua multi-target inactivation by thermo-sonication and vanillin. Food and Bioprocess Technology 5, 665–671. Huang, T.-S., Xu, C., Walker, K., West, P., Zhang, S., Weese, J., 2006. Decontamination efficacy of combined chlorine dioxide with ultrasonication on apples and lettuce. Journal of Food Science 71, M134–M139. Humphrey, V.F., 2007. Ultrasound and matter – physical interactions. Progress in Biophysics and Molecular Biology 93, 195–211. Knorr, D., Zenker, M., Heinz, V., Lee, D.-U., 2004. Applications and potential of ultrasonics in food processing. Trends in Food Science and Technology 15, 261–266. Lee, H., Zhou, B., Feng, H., Martin, S.E., 2009. Effect of pH on Inactivation of Escherichia coli K12 by sonication, manosonication, thermosonication, and manothermosonication. Journal of Food Science 74, E191–E198. Lee, H., Zhou, B., Liang, W., Feng, H., Martin, S.E., 2009. Inactivation of Escherichia coli cells with sonication, manosonication, thermosonication, and manothermosonication: microbial responses and kinetics modeling. Journal of Food Engineering 93 (3), 354–364. Lorimer, J.P., Mason, T.J., 1987. Sonochemistry part 1-the physical aspects. Chemical Society Reviews 16, 239–274. Mason, T.J., Paniwnyk, L., Lorimer, J.P., 1996. The uses of ultrasound in food technology. Ultrasonics Sonochemistry 3, 253–260. Raso, J., Pagán, R., Condón, S., Sala, F.J., 1998. Influence of temperature and pressure on the lethality of ultrasound. Applied and Environmental Microbiology 64 (2), 465–471. Ross, A.I.V., Griffiths, M.W., Mittal, G.S., Deeth, H.C., 2003. Combining nonthermal technologies to control foodborne microorganisms. International Journal of Food Microbiology 89, 125–138. Santos, H.M., Capelo, J.L., 2007. Trends in ultrasonic-based equipment for analytical sample treatment. Talanta 73, 795–802. Thompson, L.H., Doraiswamy, L.K., 1999. sonochemistry: science and engineering. Industrial & Engineering Chemistry Research 38, 1215–1249.