Inactivation of Enterobacter aerogenes in reconstituted skim milk by high- and low-frequency ultrasound

The inactivation of Enterobacter aerogenes in skim milk using low-frequency (20 kHz) and high-frequency (850 kHz) ultrasonication was investigated. It was found that low-frequency acoustic cavitation resulted in lethal damage to E. aerogenes. The bacteria were more sensitive to ultrasound in water than in reconstituted skim milk having different protein concentrations. However, high-frequency ultrasound was not able to inactivate E. aerogenes in milk even when powers as high as 50 W for 60 min were used. This study also showed that high-frequency ultrasonication had no influence on the viscosity and particle size of skim milk, whereas low-frequency ultrasonication resulted in the decrease in viscosity and particle size of milk. The decrease in particle size is believed to be due to the breakup of the fat globules, and possibly to the cleavage of the κ-casein present at the surface of the casein micelles. Whey proteins were also found to be slightly affected by low-frequency ultrasound, with the amounts of α-lactalbumin and β-lactoglobulin slightly decreasing.     

Inactivation of microorganisms by low-frequency high-power ultrasound: 1. Effect of growth phase and capsule properties of the bacteria

The aim of this study was to determine the effects of high-intensity low-frequency (20 kHz) ultrasound treatment on the viability of bacteria suspension. More specifically, we have investigated the relationship between the deactivation efficiency and the physical (size, hydrophobicity) and biological (gram-status, growth phase) properties of the microbes. Enterobacter aerogenes, Bacillus subtilis, Staphylococcus epidermidis, S. epidermidis SK and Staphylococcus pseudintermedius were chosen for this study owing to their varying physical and biological properties. The survival ratio of the bacteria suspension was measured as a function of the ultrasound power (up to 13 W) for a constant sonication time of 20 min. Transmission electron microscopy was used to evaluate the ultrasound-induced damages to the microbes. Ultrasound treatment resulted in lethal damage to E. aerogenes and B. subtilis (up to 4.5-log reduction), whereas Staphylococcus spp. were not affected noticeably. Further, E. aerogenes suspensions were more sensitive to ultrasonication in exponential growth phase than when they were in stationary phase. The results of this study demonstrate that the main reason for bacterial resistance to ultrasonic deactivation is due to the properties of the bacterial capsule. Microbes with a thicker and “soft” capsule are highly resistant to ultrasonic deactivation process.     

Effect of high intensity ultrasound on the fermentation profile of Lactobacillus sakei in a meat model system

The objective of this study was to investigate the efficacy of high intensity ultrasound on the fermentation profile of Lactobacillus sakei in a meat model system. Ultrasound power level (0–68.5 W) and sonication time (0–9 min) at 20 °C were assessed against the growth of L. sakei using a Microplate reader over a period of 24 h. The L. sakei growth data showed a good fit with the Gompertz model (R² > 0.90; SE < 0.042). Second order polynomial models demonstrated the effect of ultrasonic power and sonication time on the specific growth rate (SGR, μ, h⁻¹) and lag phase (λ, h). A higher SGR and a shorter lag phase were observed at low power (2.99 W for 5 min) compared to control. Conversely, a decrease (p < 0.05) in SGR with an increase in lag phase was observed with an increase in ultrasonic power level. Cell-free extracts obtained after 24 h fermentation of ultrasound treated samples showed antimicrobial activity against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium at lower concentrations compared to control. No significant difference (p < 0.05) among treatments was observed for lactic acid content after a 24 h fermentation period. This study showed that both stimulation and retardation of L. sakei is possible, depending on the ultrasonic power and sonication time employed. Hence, fermentation process involving probiotics to develop functional food products can be tailored by selection of ultrasound processing parameters.     

Correlation between iodide dosimetry and terephthalic acid dosimetry to evaluate the reactive radical production due to the acoustic cavitation activity

Acoustic cavitation plays an important role in sonochemical processes and the rate of sonochemical reaction is influenced by sonication parameters. There are several methods to evaluate cavitation activity such as chemical dosimetry. In this study, to comparison between iodide dosimetry and terephthalic acid dosimetry, efficacy of sonication parameters in reactive radical production has been considered by iodide and terephthalic acid dosimetries. For this purpose, efficacy of different exposure parameters on cavitations production by 1 MHz ultrasound has been studied. The absorbance of KI dosimeter was measured by spectrophotometer and the fluorescence of terephthalic acid dosimeter was measured using spectrofluorometer after sonication. The result of experiments related to sonication time and intensity showed that with increasing time of sonication or intensity, the absorbance is increased. It has been shown that the absorbance for continuous mode is remarkably higher than for pulsing mode (p-value < 0.05). Also results show that with increasing the duty cycles of pulsed field, the inertial cavitation activity is increased. With compensation of sonication time or intensity in different duty cycles, no significant absorbance difference were observed unless 20% duty cycle. A significant correlation between the absorbance and fluorescence intensities (count) at different intensity (R = 0.971), different sonication time (R = 0.999) and different duty cycle (R = 0.967) were observed (p-value < 0.05). It is concluded that the sonication parameters having important influences on reactive radical production. These results suggest that there is a correlation between iodide dosimetry and terephthalic acid dosimetry to examine the acoustic cavitation activity in ultrasound field.     

Inactivation of Escherichia coli and Streptococcus mutans by ultrasound at 500 kHz

This paper shows a systematic study of the 500 kHz frequency ultrasound efficiency on the microbial inactivation as a function of ultrasonic power delivered into the bacterial suspension. The inactivation of Escherichia coli IAM 12058, a Gram-negative bacterium and Streptococcus mutans JCM 5175, a Gram-positive bacterium is enhanced by increasing the ultrasonic power in the range of 1.7–12.4 W and the logarithm of survival ratio decreases linearly with irradiation time, except for E. coli sonicated with the highest power level. The rate constants were estimated in the linear region of the plots representing survival ratio logarithm vs. sonication time. A better understanding of the inactivation process at 500 kHz could be gained by suppressing the chemical effects with a radical scavenger. We find out that the rate constants increase with the ultrasonic power delivered into the solution and dramatically decrease by the addition of t-butanol as a radical scavenger to the bacterial suspension. For comparison, experiments were carried out at a low frequency level of 20 kHz. It was found out that for the same ultrasonic power delivered into the bacterial suspension, the inactivation was slightly enhanced at 500 kHz frequency. The examinations of bacterium performed with a TEM revealed lethal damages arising from the interaction of bacterial cells with the cavitational bubbles. A significant amount of empty cell envelopes as well as their cytoplasmatic content was detected. Thus, based on these new data, the mechanism of bacterial inactivation by ultrasounds at high frequency is discussed here.     

Ultrasound-enhanced coagulation for Microcystis aeruginosa removal

Source water eutrophication has caused serious problems in drinking water supplies, with enhanced coagulation widely used to remove the resulting algae. This paper investigates the use of sonication to improve the removal by coagulation of Microcystis aeruginosa, a common species of toxic algae. The results show that sonication significantly enhances the reduction of algae cells, solution UV254, and chlorophyll a without increasing the concentration of aqueous microcystins. The main mechanism involved the destruction during ultrasonic irradiation of gas vacuoles inside algae cells that acted as ‘nuclei’ for acoustic cavitation and collapse during the “bubble crush” period, resulting in the settlement of cyanobacteria. Coagulation efficiency depended strongly on the coagulant dose and sonication conditions. When the coagulant dose was 0.5 mg/l, 5 s of ultrasonic irradiation increased algae removal efficiency from 35% to 67%. As further sonication enhanced the coagulation efficiency only slightly due to better mixing, optimal sonication time was 5 s. The most effective sonication intensity was 47.2 W/cm², and the highest removal ratio of M. aeruginosa was 93.5% by the sonication–coagulation method. Experiments with reservoir water showed that this method could be successfully applied to natural water containing multiple species of algae.     

Ultrasonic processing of dairy systems in large scale reactors

High intensity low frequency ultrasound was used to process dairy ingredients to improve functional properties. Based on a number of lab-scale experiments, several experimental parameters were optimised for processing large volumes of whey and casein-based dairy systems in pilot scale ultrasonic reactors. A continuous sonication process at 20 kHz capable of delivering up to 4 kW of power with a flow-through reactor design was used to treat dairy ingredients at flow rates ranging from 200 to 6000 mL/min. Dairy ingredients treated by ultrasound included reconstituted whey protein concentrate (WPC), whey protein and milk protein retentates and calcium caseinate. The sonication of solutions with a contact time of less than 1 min and up to 2.4 min led to a significant reduction in the viscosity of materials containing 18% to 54% (w/w) solids. The viscosity of aqueous dairy ingredients treated with ultrasound was reduced by between 6% and 50% depending greatly on the composition, processing history, acoustic power and contact time. A notable improvement in the gel strength of sonicated and heat coagulated dairy systems was also observed. When sonication was combined with a pre-heat treatment of 80 °C for 1 min or 85 °C for 30 s, the heat stability of the dairy ingredients containing whey proteins was significantly improved. The effect of sonication was attributed mainly to physical forces generated through acoustic cavitation as supported by particle size reduction in response to sonication. As a result, the gelling properties and heat stability aspects of sonicated dairy ingredients were maintained after spray drying and reconstitution. Overall, the sonication procedure for processing dairy systems may be used to improve process efficiency, improve throughput and develop value added ingredients with the potential to deliver economical benefits to the dairy industry.     

Dual frequency cavitation event sensor with iodide dosimeter

The inertial cavitation activity depends on the sonication parameters. The purpose of this work is development of dual frequency inertial cavitation meter for therapeutic applications of ultrasound waves. In this study, the chemical effects of sonication parameters in dual frequency sonication (40 kHz and 1 MHz) were investigated in the progressive wave mode using iodide dosimetry. For this purpose, efficacy of different exposure parameters such as intensity, sonication duration, sonication mode, duty factor and net ultrasound energy on the inertial cavitation activity have been studied. To quantify cavitational effects, the KI dosimeter solution was sonicated and its absorbance at a wavelength of 350 nm was measured. The absorbance values in continuous sonication mode was significantly higher than the absorbance corresponding to the pulsed mode having duty factors of 20–80% (p < 0.05). Among different combination modes (1 MHz_100% + 40 kHz_100%, 1 MHz_100% + 40 kHz_80%, 1 MHz_80% + 40 kHz_100%, 1 MHz_80% + 40 kHz_80%), the continuous mode for dual frequency sonication is more effective than other combinations (p < 0.05). The absorbance for this combined dual frequency mode was about 1.8 times higher than that obtained from the algebraic summation of single frequency sonications. It is believed that the optimization of dual frequency sonication parameters at low-level intensity (<3 W/cm²) by optically assisted cavitation event sensor can be useful for ultrasonic treatments.     

Combination of supercritical CO2 and high-power ultrasound for the inactivation of fungal and bacterial spores in lipid emulsions

For the first time, this study addresses the intensification of supercritical carbon dioxide (SC-CO₂) treatments using high-power ultrasound (HPU) for the inactivation of fungal (Aspergillus niger) and bacterial (Clostridium butyricum) spores in oil-in-water emulsions. The inactivation kinetics were analyzed at different pressures (100, 350 and 550 bar) and temperatures (50, 60, 70, 80, 85 °C), depending on the microorganism, and compared to the conventional thermal treatment. The inactivation kinetics were satisfactorily described using the Weibull model.Experimental results showed that SC-CO₂ enhanced the inactivation level of both spores when compared to thermal treatments. Bacterial spores (C. butyricum) were found to be more resistant to SC-CO₂ + HPU, than fungal (A. niger) ones, as also observed in the thermal and SC-CO₂ treatments. The application of HPU intensified the SC-CO₂ inactivation of C. butyricum spores, e.g. shortening the total inactivation time from 10 to 3 min at 85 °C. However, HPU did not affect the SC-CO₂ inactivation of A. niger spores. The study into the effect of a combined SC-CO₂ + HPU treatment has to be necessarily extended to other fungal and bacterial spores, and future studies should elucidate the impact of HPU application on the emulsion’s stability.     

Effects of low-intensity ultrasound on the growth,cell membrane permeability and ethanol tolerance of Saccharomyces cerevisiae

Effects of low-intensity ultrasound (at different frequency, treatment time and power) on Saccharomyces cerevisiae in different growth phase were evaluated by the biomass in the paper. In addition, the cell membrane permeability and ethanol tolerance of sonicated Saccharomyces cerevisiae were also researched. The results revealed that the biomass of Saccharomyces cerevisiae increased by 127.03% under the optimum ultrasonic conditions such as frequency 28 kHz, power 140 W/L and ultrasonic time 1 h when Saccharomyces cerevisiae cultured to the latent anaphase. And the membrane permeability of Saccharomyces cerevisiae in latent anaphase enhanced by ultrasound, resulting in the augment of extracellular protein, nucleic acid and fructose-1,6-diphosphate (FDP) contents. In addition, sonication could accelerate the damage of high concentration alcohol to Saccharomyces cerevisiae although the ethanol tolerance of Saccharomyces cerevisiae was not affected significantly by ultrasound.     

Ascorbic acid stability in fruit juices during thermosonication

Thermosonication is an emerging technology useful for inactivating microorganisms and enzymes in fruit juices. However, the effect of the ultrasound processing on the ascorbic acid content is not clear and the results reported in the literature are contradictory. In this work, the impact of sonication and thermosonication on the ascorbic acid content was first evaluated in model systems. Degassed model solutions at four different pH values (3–6) were processed with or without sonication for 60 min at two different conditions of temperature (25 and 55 °C). In all cases, the ascorbic acid was stable under the treatment. After that, two commercial deaerated fruit juices were processed with and without US at 55 °C. The ascorbic acid was also retained in these juices after the ultrasound processing under the most severe conditions studied. In conclusion, previous degassing/deaerating of fruit juices is recommended to prevent ascorbic acid degradation when thermosonication is applied.     

Effect of non-acoustic parameters on heterogeneous sonoporation mediated by single-pulse ultrasound and microbubbles

Sonoporation—transient plasma membrane perforation elicited by the interaction of ultrasound waves with microbubbles—has shown great potential for drug delivery and gene therapy. However, the heterogeneity of sonoporation introduces complexities and challenges in the realization of controllable and predictable drug delivery. The aim of this investigation was to understand how non-acoustic parameters (bubble related and bubble-cell interaction parameters) affect sonoporation. Using a customized ultrasound-exposure and fluorescence-imaging platform, we observed sonoporation dynamics at the single-cell level and quantified exogenous molecular uptake levels to characterize the degree of sonoporation. Sonovue microbubbles were introduced to passively regulate microbubble-to-cell distance and number, and bubble size. 1 MHz ultrasound with 10-cycle pulse duration and 0.6 MPa peak negative pressure were applied to trigger the inertial collapse of microbubbles. Our data revealed the impact of non-acoustic parameters on the heterogeneity of sonoporation. (i) The localized collapse of relatively small bubbles (diameter, D < 5.5 μm) led to predictable sonoporation, the degree of which depended on the bubble-to-cell distance (d). No sonoporation was observed when d/D > 1, whereas reversible sonoporation occurred when d/D < 1. (ii) Large bubbles (D > 5.5 μm) exhibited translational movement over large distances, resulting in unpredictable sonoporation. Translation towards the cell surface led to variable reversible sonoporation or irreversible sonoporation, and translation away from the cell caused either no or reversible sonoporation. (iii) The number of bubbles correlated positively with the degree of sonoporation when D < 5.5 μm and d/D < 1. Localized collapse of two to three bubbles mainly resulted in reversible sonoporation, whereas irreversible sonoporation was more likely following the collapse of four or more bubbles. These findings offer useful insight into the relationship between non-acoustic parameters and the degree of sonoporation.     

Thermosonication as a potential quality enhancement technique of apple juice

Enzymatic browning and microbial growth lead to quality losses in apple products. In the present study, fresh apple juice was thermosonicated using ultrasound in-bath (25 kHz, 30 min, 0.06 W cm⁻³) and ultrasound with-probe sonicator (20 kHz, 5 and 10 min, 0.30 W cm⁻³) at 20, 40 and 60 °C for inactivation of enzymes (polyphenolase, peroxidase and pectinmethylesterase) and microflora (total plate count, yeast and mold). Additionally, ascorbic acid, total phenolics, flavonoids, flavonols, pH, titratable acidity, ^°Brix and color values influenced by thermosonication were investigated. The highest inactivation of enzymes was obtained in ultrasound with-probe at 60 °C for 10 min, and the microbial population was completely inactivated at 60 °C. The retention of ascorbic acid, total phenolics, flavonoids and flavonols were significantly higher in ultrasound with-probe than ultrasound in-bath at 60 °C. These results indicated the usefulness of thermosonication for apple juice processing at low temperature, for enhanced inactivation of enzymes and microorganisms.     

Variation of dissolved organic nitrogen concentration during the ultrasonic pretreatment to Microcystis aeruginosa

Algae cells were the main sources of dissolved organic nitrogen (DON) in raw water with plenty of algae, and ultrasonic pretreatment was one of the algae-controlling methods through the damage of algae cells. However, the variation of DON concentration during the ultrasonic treatment process was not confirmed. Variation of DON concentration during the processes of low frequency ultrasound treatment of Microcystis aeruginosa was investigated. In addition, the effect of sonication on the metabolite concentration, algae cellar activity and the subsequent coagulation performance were discussed. The results showed that after a long duration of ultrasonic (60 s), nearly 90% of the algal cells were damaged and the maximum concentration of DON attained more than 3 mg/L. In order to control the leakage extent of DON, the sonication time should be less than 30 s with power intensity of more than 1.0 W/cm³. In the mean time, ultrasonic treatment could inhibit the reactivation and the proliferation of algal, keep the algae cell wall integrity and enhance coagulation effectively under the same condition. However, ultrasound frequency had little effect on DON at the frequency range used in this study (20–150 kHz).     

Power ultrasound as a pretreatment to convective drying of mulberry (Morus alba L.) leaves: Impact on drying kinetics and selected quality properties

The effect of ultrasound pretreatment prior to convective drying on drying kinetics and selected quality properties of mulberry leaves was investigated in this study. Ultrasound pretreatment was carried out at 25.2–117.6 W/L for 5–15 min in a continuous mode. After sonication, mulberry leaves were dried in a hot-air convective dryer at 60 °C. The results revealed that ultrasound pretreatment not only affected the weight of mulberry leaves, it also enhanced the convective drying kinetics and reduced total energy consumption. The drying kinetics was modeled using a diffusion model considering external resistance and effective diffusion coefficient D_e and mass transfer coefficient h_m were identified. Both D_e and h_m during convective drying increased with the increase of acoustic energy density (AED) and ultrasound duration. However, D_e and h_m increased slowly at high AED levels. Furthermore, ultrasound pretreatment had a more profound influence on internal mass transfer resistance than on external mass transfer resistance during drying according to Sherwood numbers. Regarding the quality properties, the color, antioxidant activity and contents of several bioactive compounds of dried mulberry leaves pretreated by ultrasound at 63.0 W/L for 10 min were similar to that of mulberry leaves without any pretreatments. Overall, ultrasound pretreatment is effective to shorten the subsequent drying time of mulberry leaves without damaging the quality of final product.     

Ultrasound pretreatment of wheat dried distiller’s grain (DDG) for extraction of phenolic compounds

Wheat Dried distiller’s grain (DDG), a coproduct from the ethanol production process, is rich in potentially health-promoting phenolic compounds. In the extraction of phenolic compounds from DDG, the DDG cell wall is an important barrier for mass transfer from the inside to the outside of the cell. The effect of high-power ultrasound pretreatment on destruction of DDG cell walls and extraction yield and rate was investigated. Direct sonication by an ultrasound probe horn at 24 kHz was applied and factors such as ultrasound power and treatment time were investigated. The method of nitrogen (N₂) adsorption at 77 K was used as a means to determine and compare the changes in physical properties (specific surface area, pore volume and pore size) of the treated samples at different levels of ultrasound power and treatment time. Increasing specific surface area, pore volume and pore size caused by ultrasonic treatment implied development of new or larger pores and damaged cell walls. Also, it was observed that the ultrasound pretreatment of DDG particles increased the extraction yield and rate of phenolic compounds from DDG by 14.29%. Among tested ultrasound conditions, 100% ultrasound power for 30 s was evaluated as the best pretreatment condition.     

The inactivation kinetics of polyphenol oxidase in mushroom (Agaricus bisporus) during thermal and thermosonic treatments

The effect of thermal and thermosonic treatments on the inactivation kinetics of polyphenol oxidase (PPO) in mushroom (Agaricus bisporus) was studied in 55–75 °C temperature range. In both the processes, the inactivation kinetics of PPO followed a first-order kinetics (R² = 0.941–0.989). The D values during thermal inactivation varied from 112 ± 8.4 min to 1.2 ± 0.07 min while they varied from 57.8 ± 6.1 min to 0.88 ± 0.05 min during thermosonic inactivation at the same temperature range. The activation energy during thermal inactivation was found to be 214 ± 17 kJ/mol, while it was 183 ± 32 kJ/mol during thermosonic inactivation. The inactivating effect of combined ultrasound and heat was found to synergistically enhance the inactivation kinetics of PPO. The D values of PPO decreased by 1.3–3 times during thermosonic inactivation compared to the D values of PPO during thermal inactivation at the temperature range. Therefore, thermosonication can be further developed as an alternative to “hot break” process of mushroom.     

Ultrasound-assisted extraction of Na and K from swine feed and its application in a digestibility assay: A green analytical procedure

The study is aimed to evaluate the efficiency of ultrasound-assisted extraction (UAE) as a simple strategy focused on sample preparation for metal determination in biological samples. The extraction of sodium and potassium extraction was carried out from swine feed followed by determination of the concentration of these metals by flame atomic emission spectrometry (FAES). The experiment was performed to cover the study of the variables influencing the extraction process and its optimal conditions (sample mass, particle size, acid concentration, sonication time and ultrasound power); the determination of these analytical characteristics and method validation using certified reference material; and the analysis of pre-starter diets. The optimal conditions established conditions were as follows: mass: 100 mg, particle size:<60 μm, acid concentration: 0.10 mol L^?1 HCl, sonication time: 50 s and ultrasound power: 102 W. The proposed method (UAE) was applied in digestibility assays of those nutrients present in different piglet pre-starter feeds and their results proved to be compatible with those obtained from mineralized samples (P < 0.05). The ultrasound extraction method was demonstrated to be an excellent alternative for handless sampling and operational costs and the method also has the advantage of does not generating toxic residues that may negatively affect human health and contaminate the environment.     

Enhancement of artificial promoter activity by ultrasound-induced oxidative stress

We previously developed artificial promoters that were activated in response to X-ray irradiation. Sonication with 1.0 MHz ultrasound that causes intracellular oxidative stress was found to activate some of these promoters though to lesser degrees. The most sensitive one among these promoters showed intensity- and duration-dependent activations by sonication. In addition, its activation by sonication was attenuated when N-acetyl cysteine was present, suggesting the involvement of intracellular oxidative stress in the activation mechanism. Improved promoters for sensitivity to X-ray irradiation were also found more sensitive to sonication. The most improved one showed 6.0 fold enhancement after sonication with 1.0 MHz ultrasound at 1.0 W/cm² for 60 s. This enhancement was also attenuated with the presence of N-acetyl cysteine. When stably transfected HeLa cells with the most sensitive promoter were transplanted on to mice and sonicated, luciferase activity by the promoter increased to 1.35 fold in average though it was not statistically significant compared to control. Although gene regulation in vivo by sonication was not clear, this is the first report on artificially constructed promoters responsive to ultrasound.