Fabrication of bilayer emulsion by ultrasonic emulsification: Effects of chitosan on the interfacial stability of emulsion

In this study, the stable system of bilayer emulsion was fabricated by ultrasonic emulsification. The effect of chitosan (CS) addition (0.05 %-0.4 %, w/v) at pH 5.0 on the stability of rice bran protein hydrolysate-ferulic acid (RBPH-FA) monolayer emulsion was investigated. It was found that the addition of CS (0.3 %) could form a stable bilayer emulsion. The droplet size was 3.38 μm and the absolute ζ-potential value was 31.52 mV. The bilayer emulsion had better storage stability, oxidation stability and environmental stabilities than the monolayer emulsion. The results of in vitro simulations revealed the bilayer emulsion was able to deliver the β-carotene to the small intestine digestive stage stably and the bioaccessibility was increased from 22.34 % to 61.36 % compared with the monolayer emulsion. The research confirmed that the bilayer emulsion prepared by ultrasonic emulsification can be used for the delivery of hydrophobic functional component β-carotene.     

Effect of ultrasonic pretreatment on the emulsification properties of Clanis Bilineata Tingtauica Mell protein

Clanis Bilineata Tingtauica Mell Protein (CBTMP) is a naturally high-quality insect protein resource, while its poor emulsification has limited its application in food industry. In order to change the present situation, in this research, the ultrasonic pretreatment (0 W, 200 W, 400 W, 600 W, and 800 W) method was used to improve the emulsification properties of CBTMP. Results indicated that ultrasound treatment especially at 400 W could significantly change the particle sizes, further increase the content of sulfhydryl group and surface hydrophobicity. The emulsification properties of emulsions were enhanced (from 4.16 ± 1.07 m²/g to 27.62 ± 2.20 m²/g) by sonicated CBTMP solution. Moreover, the physical stability of the emulsions to salt stress and centrifugation treatment was also promoted. Additionally, rheology revealed that a stronger network was formed at 400 W and all samples exhibited frequency-dependent and amplitude-dependent properties. The experiment demonstrated that ultrasound pretreatment was an effective means to improve the emulsification properties of CBTMP and it could provide a promising perspective for the application of CBTMP in food industry.     

High-speed imaging of ultrasonic emulsification using a water-gallium system

Aiming at elucidating ultrasonic emulsification mechanisms, the interaction between a single or multiple acoustic cavitation bubbles and gallium droplet interface was investigated using an high-speed imaging technique. To our best knowledge, the moment of emulsification and formation of fine droplets during ultrasound irradiation were observed for the first time. It was found that the detachment of fine gallium droplets occurs from the water-gallium interface during collapse of big cavitation bubbles. The results suggest that the maximum size of cavitation bubble before collapsing is of prime importance for emulsification phenomena. Previous numerical simulation revealed that the collapse of big cavitation bubble is followed by generation of high-velocity liquid jet directed toward the water-gallium interface. Such a jet is assumed to be the prime cause of liquid emulsification. The distance between cavitation bubbles and water-gallium interface was found to slightly affect the emulsification onset. The droplet fragmentation conditions are also discussed in terms of the balance between (1) interfacial and kinetic energies and (2) dynamic and Laplace pressure during droplet formation.     

Process optimization and stability of D-limonene-in-water nanoemulsions prepared by ultrasonic emulsification using response surface methodology

d-limonene in water nanoemulsion was prepared by ultrasonic emulsification using mixed surfactants of sorbitane trioleate and polyoxyethylene (20) oleyl ether. Investigation using response surface methodology revealed that 10% d-limonene nanoemulsions formed at S₀ ratio (d-limonene concentration to mixed surfactant concentration) 0.6-0.7 and applied power 18 W for 120 s had droplet size below 100 nm. The zeta potential of the nanoemulsion was approximately −20 mV at original pH 6.4, closed to zero around pH 4.0, and around −30 mV at pH 12.0. The main destabilization mechanism of the systems is Ostwald ripening. The ripening rate at 25 °C (0.39 m³ s⁻¹ × 10²⁹) was lower than that at 4 °C (1.44 m³ s⁻¹ × 10²⁹), which was in agreement with the Lifshitz-Slezov-Wagner (LSW) theory. Despite of Ostwald ripening, the droplet size of d-limonene nanoemulsion remained stable after 8 weeks of storage.     

Effect of ultrasonic power on the emulsion stability of rice bran protein-chlorogenic acid emulsion

In this study, rice bran protein–chlorogenic acid (RBP–CA) emulsion was subjected to an ultrasonic-assisted treatment technique. The encapsulation efficiency and loading capacity of chlorogenic acid (CA), and the morphology, particle size, zeta (ζ)-potential, atomic force microscopy image, viscosity, turbidity, and interfacial protein content of the emulsion under different ultrasonic power were investigated. The results revealed that the emulsion exhibited an encapsulation efficiency and loading capacity of 86.26 ± 0.11% and 17.25 ± 0.06 g/100 g, respectively, at an ultrasonic power of 400 W. In addition, the size of the emulsion droplets decreased and became more evenly distributed. Furthermore, the viscosity of the emulsion decreased significantly, and it exhibited a turbidity and interfacial protein content of 24,758 and9.34 mg/m², respectively. Next, the storage, oxidation, thermal, and salt ion stabilities of the emulsion were evaluated. The results revealed that the ultrasonic-assisted treatment considerably improved the stability of the emulsion.     

Effect of ultrasound on the structural characteristics and oxidative stability of walnut oil oleogel coated with soy protein isolate-phosphatidylserine

In this study, the three-dimensional network system formed by rice bran wax (RBW) was used as the internal structure, and the external structure formed by soybean protein isolate (SPI) and phosphatidylserine (PS) was added on the basis of the internal structure to prepare walnut oil oleogel (SPI-PS-WOG). Ultrasonic treatment was applied to the mixed solution to make SPI-PS-WOG, on the basis, the effects of ultrasonic treatment on SPI-PS-WOG were investigated. The results showed that both β and β’ crystalline forms were present in all SPI-PS-WOG samples. When the ultrasonic power was 450 W, the first weight loss peak in the thermogravimetric (TGA) curve appeared at 326 °C, which was shifted to the right compared to the peak that occurred when the ultrasonic power was 0 W, indicating that the thermal stability of the SPI-PS-WOG was improved by the ultrasonic treatment. Moreover, when the ultrasonic power was 450 W, the oil holding capacity (OHC) reached 95.3 %, which was the best compared with other groups. Both confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) showed that the ultrasonic treatment of appropriate power succeeded in making the SPI-PS-WOG samples more evenly dispersed in the internal structure and denser in the external structure. In terms of oxidative stability, it was found that the peroxide value of SPI-PS-WOG remained at 9.8 mmol/kg oil for 50 days under 450 W ultrasonic power treatment, which was significantly improved compared with liquid walnut oil (WO). These results provide a new idea for the preparation of oleogels, and also lay a theoretical foundation for the application of ultrasonic treatment in oleogels.     

Ultrasonic assisted water-in-oil emulsions encapsulating macro-molecular polysaccharide chitosan: Influence of molecular properties,emulsion viscosity and their stability

An ultrasonic technique was applied to formulation of two-phase water-in-paraffin oil emulsions loading a high-molecular polysaccharide chitosan (CS) and stabilized by an oil-soluble surfactant (Span80) at different operational conditions. The influence of chitosan molecular properties, phase volume ratio (φ_w), Span80 volume fraction (φ_s) and ultrasonic processing parameters were systemically investigated on the basis of mean droplet diameter (MDD) and polydispersity index (PDI) of emulsions. It was observed that the molecular weight (M_w) of CS was an important influential factor to MDD due to the non-Newtonian properties of CS solution varying with M_w. The minimum MDD of 198.5 nm with PDI of 0.326 was obtained with ultrasonic amplitude of 32% for 15 min at an optimum φ_w of 35%, φ_s of 8%, probe position of 2.2 cm to the top of emulsion, while CS with M_w of 400 kDa and deacetylation degree of 84.6% was used. The rise of emulsion viscosity and the reduction of negative zeta potential at φ_w increasing from 5% to 35% were beneficial to obtain finer droplets and more uniform distribution of emulsions, and emulsion viscosity could be represented as a monotonically-decreasing power function of MDD at the same φ_w. FTIR analysis indicated that the molecular structure of paraffin oil was unaffected during ultrasonication. Moreover, the emulsions exhibited a good stability at 4 °C with a slight phase separation at 25 °C after 24 h of storage. By analyzing the evolution of MDD, PDI and sedimentation index (SI) with time, coalescence model showed better fitting results as comparison to Ostwald ripening model, which demonstrated that the coalescence or flocculation was the dominant destabilizing mechanism for such W/O emulsions encapsulating CS. This study may provide a valuable contribution for the application of a non-Newtonian macromolecule solution as dispersed phase to generate nano-size W/O emulsions via ultrasound, and widen knowledge and interest of such emulsions in the functional biomaterial field.     

Effect of polyphenolic structure and mass ratio on the emulsifying performance and stability of emulsions stabilized by polyphenol-corn amylose complexes

O/W emulsions stabilized by polyphenol/amylose (AM) complexes with several polyphenol/AM mass ratios and different polyphenols (gallic acid (GA), epigallocatechin gallate (EGCG) and tannic acid (TA)) were prepared by a high-intensity ultrasound emulsification technique. The effect of the pyrogallol group number of polyphenols and the mass ratio of polyphenols/AM on polyphenol/AM complexes and emulsions was studied. The soluble and/or insoluble complexes gradually formed upon adding polyphenols into the AM system. However, insoluble complexes were not formed in the GA/AM systems because GA has only one pyrogallol group. In addition, the hydrophobicity of AM could also be improved by forming polyphenol/AM complexes. The emulsion size decreased with increasing pyrogallol group number on the polyphenol molecules at a fixed ratio, and the size could also be controlled by the polyphenol/AM ratio. Moreover, all emulsions presented various degrees of creaming, which was restrained by decreasing emulsion size or the formation of a thick complex network. The complex network was enhanced by increasing the ratio or pyrogallol group number on the polyphenol molecules, which was because the increasing number of complexes was adsorbed onto the interface. Altogether, compared to GA/AM and EGCG/AM, the TA/AM complex emulsifier had the best hydrophobicity and emulsifying properties, and the TA/AM emulsion had the best emulsion stability.     

Optimization of acoustic parameters for ultrasonic separation of emulsions with different physical properties

Ultrasound is an emerging and promising method for demulsification, which is highly affected by acoustic parameters and emulsion properties. Herein, a series of microscopic and dehydration experiments are carried out to investigate the parameter optimization of ultrasonic separation. The results show that the optimal acoustic parameters highly depend on the emulsion properties. For low frequency ultrasonic standing waves (USWs), mechanical vibrations not only facilitate droplet collision and coalescence, but also disperse the surfactant absorbed on the interface to decrease the interfacial strength. Therefore, low frequency ultrasound is suitable for separating emulsions with high viscosity and high interfacial strength. Increasing the energy density to produce moderate cavitation can increase demulsification efficiency. However, excessive cavitation results in secondary emulsification. In high frequency USWs, the droplets migrate directionally and form bandings, thereby promoting droplet coalescence. Therefore, high frequency ultrasound is favorable for separating emulsions with low dispersed phase content and small droplet size. Increasing the energy density can accelerate the aggregation of droplets, however, excessive energy density causes acoustic streaming that disturbs the aggregated droplets, resulting in reduced demulsification efficiency. This work presents rules for acoustic parameter optimization, further advancing industrial applications of ultrasonic separation.     

Fabrication of emulsions prepared by rice bran protein hydrolysate and ferulic acid covalent conjugate: Focus on ultrasonic emulsification

The aim of the paper was to investigate the effect of ultrasonic emulsification treatment on the fabrication mechanism and stability of the emulsion. The covalent conjugate made with rice bran protein hydrolysate (RBPH) and ferulic acid (FA) was used as the emulsifier. The effects of high intensity ultrasound (HIU) power with different level (0 W, 150 W, 300 W, 450 W and 600 W) on the stability of emulsion were evaluated. The results showed that ultrasonic emulsification can significantly improve the stability of the emulsions (p < 0.05). The emulsion gained better stability and emulsifying property at 300 W. It was able to fabricate emulsion with smaller particle size, more uniform distribution and higher interfacial protein content. It was confirmed by fluorescent microscopy and cryo-scanning electron microscopy (cryo-SEM) furtherly. And it was also proved that the emulsion treated by proper HIU treatment at 300 W had better storage stability. Excessive HIU treatment (450 W, 600 W) had negative effects on the stability of emulsion. The stability of emulsion (300 W) against different environmental stresses was further explored, which established a theoretical basis for the industrial application of emulsion in food industry.     

Study on the emulsification and oxidative stability of ovalbumin-pectin-pumpkin seed oil emulsions using ovalbumin solution prepared by ultrasound

Pumpkin seed oil (PSO), which is a valuable compound with high nutritional value used for the prevention of various chronic diseases, is prone to oxidation. In this work, small and uniform (su) ovalbumin (OVA) and pectin (PEC) were used to stabilize PSO in the form of an emulsion. The results showed that suOVA-PEC-PSO emulsion with a droplet size of 9.82 ± 0.05 μm was successfully self-assembled from PSO, PEC, and suOVA solution (with a droplet size of 230.13 ± 14.10 nm) treated with 300 W ultrasound, owing to the formation of a more stable interfacial film on the surface of droplets. The interfacial, rheological, emulsifying, and antioxidant properties of the suOVA-PES-PSO emulsions were excellent, owing to the synergistic effects between PEC and suOVA solution. Moreover, the physical stability of the suOVA-PEC-PSO emulsions to salt stress, a freeze-thaw cycle, and heat treatment was also increased and the oxidation of linolenic acid was notably delayed. These results have extended the food-related applications of OVA and PSO, and provide a promising foundation for further exploration of the self-assembly of composite emulsions by small and uniform proteins.     

Effect of ultrasonic power on the stability of low-molecular-weight oyster peptides functional-nutrition W1/O/W2 double emulsion

Ultrasonic-assisted treatment is an eco-friendly and cost-effective emulsification method, and the acoustic cavitation effect produced by ultrasonic equipment is conducive to the formation of stable emulsion. However, its effect on the underlying stability of low-molecular-weight oyster peptides (LOPs) functional-nutrition W₁/O/W₂ double emulsion has not been reported. The effects of different ultrasonic power (50, 75, 100, 125, and 150 W) on the stability of double emulsions and the ability to mask the fishy odor of LOPs were investigated. Low ultrasonic power (50 W and 75 W) treatment failed to form a well-stabilized double emulsion, and excessive ultrasound treatment (150 W) destroyed its structure. At an ultrasonic power of 125 W, smaller particle-sized double emulsion was formed with more uniform distribution, more whiteness, and a lower viscosity coefficient. Meanwhile, the cavitation effect generated by 125 W ultrasonic power improved storage, and oxidative stabilities, emulsifying properties of double emulsion by reducing the droplet size and improved sensorial acceptability by masking the undesirable flavor of LOPs. The structure of the double emulsion was further confirmed by optical microscopy and confocal laser scanning microscopy. The ultrasonic-assisted treatment is of potential value for the industrial application of double emulsion in functional-nutrition foods.     

Dual-frequency multi-angle ultrasonic processing technology and its real-time monitoring on physicochemical properties of raw soymilk and soybean protein

To improve the soybean protein content (SPC), flavor and quality of soymilk, the effects of dual-frequency ultrasound at different angles (40 + 20 kHz 0°, 40 + 20 kHz 30°, 40 + 20 kHz 45°) on physicochemical properties and soybean protein (SP) structure of raw soymilk were mainly studied and compared with the conventional single-frequency (40 kHz, 20 kHz) ultrasound. Furthermore, the intensity of the ultrasonic field in real-time was monitored via the oscilloscope and spectrum analyzer. The results showed that 40 + 20 kHz 45° treatment significantly increased SPC. The ultrasonic field intensity of 40 + 20 kHz 0° treatment was the largest (8.727 × 10⁴ W/m²) and its distribution was the most uniform. The emulsifying stability of SP reached the peak value (233.80 min), and SP also had the largest particle size and excellent thermal stability. The protein solubility of 40 + 20 kHz 30° treatment attained peak value of 87.09%. 20 kHz treatment significantly affected the flavor of okara. The whiteness and brightness of raw soymilk treated with 40 kHz were the highest and the system was stable. Hence, the action mode of ultrasonic technology can be deeply explored and the feasibility for improving the quality of soymilk can be achieved.     

Effect of high hydrostatic pressure and high dynamic pressure on stability and rheological properties of model oil-in-water emulsions

Both static and dynamic high pressure applications provide interesting modifications in food structures which lead to new product formulations. In this study, the effects of two different treatments, high hydrostatic pressure (HHP) and high dynamic pressure (HDP), on oil-in-water emulsions were identified and compared. Microfluidization was selected from among the HDP homogenization techniques. The performance of each process was analyzed in terms of rheological modifications and emulsion stability improvements compared with the coarse emulsions. The stability of the emulsions was determined comparatively by using an analytical photo-centrifuge device employing novel analysis technology. Whey protein isolate (WPI) in combination with a food polysaccharide (xanthan gum, guar gum or locust bean gum) were used as emulsifying and stabilizing ingredients. The effective disruption of oil droplets and the degradation of polysaccharides by the shear forces under high pressure in HDP microfluidization yielded finer emulsions with lower viscosities, leading to distinctive improvements in emulsion stability. On the other hand, improvements in stability obtained with HHP treatment were due to the thickening of the emulsions mainly induced by protein unfolding. The corresponding increases in viscosity were intensified in emulsion formulations containing higher oil content. Apart from these, HHP treatment was found to be relatively more contributive to the enhancements in viscoelastic properties.     

Effect of exposure parameters on cavitation induced by low-level dual-frequency ultrasound

In order to quantify the effects of exposure parameters under therapeutic conditions such as sonodynamic therapy, it is necessary initially to evaluate the inertial cavitation activity in vitro. In this study, the dependence of cavitation activity induced by the low-level dual-frequency ultrasound irradiation on exposure parameters has been studied. Experiments were performed in the near 150 kHz and 1 MHz fields in the progressive wave mode. It has been shown that at constant ultrasound energy the fluorescence intensity for continuous sonication is higher than for pulsed mode. With increasing the duty cycle of pulsed field, the inertial cavitation activity is increased. The activity of cavitation produced by simultaneous combined sonication by two ultrasound fields is remarkably higher than the algebraic sum of effects produced by fields separately (p-value < 0.05). This study shows that simultaneous combined dual-frequency ultrasound sonication in continuous mode is more effective in producing inertial cavitation activity at low-level intensity. Therefore, it is concluded that investigations in this combined ultrasound sonication can be useful in sonodynamic therapy for superficial tumors.     

Effect of dual-frequency ultrasound on the formation of lysinoalanine and structural characterization of rice dreg protein isolates

The effect of dual-frequency ultrasound treatment with different working modes on the lysinoalanine (LAL) formation and structural characterization of rice dreg protein isolates (RDPI) was studied during alkaline exaction processing. Ultrasonic notably decreased the LAL amount of RDPI and enhanced the protein dissolution rate. The LAL content of RDPI, especially sequential dual frequency 20/40 kHz, decreased by 12.02% (P < 0.05), compared to non-sonicated samples. Herein, the protein dissolution rate was higher. The changes in sulfhydryl groups was positively correlated with the LAL formation. The amino acids (AA) such as threonine (Thr), lysine (Lys), and arginine (Arg) were reduced, resulting in a decrease in LAL content following sonication. Besides, ultrasonication altered protein secondary structure by reducing random coil and β-sheet contents, while α-helix and β-turn contents increased. Alterations in the surface hydrophobicity, particle size, particle size distribution, and microstructure indicated more irregular fragment with microparticles of RDPI by sonochemical treatment. Thus, ultrasound treatment may be a new and efficacious process for controlling the LAL generation in prepared-protein food(s) during alkali extraction.     

A study on the emulsification of oil by power ultrasound

A commercial ultrasonic probe was used to study emulsification of an o/w system (5 wt% soybean oil stabilised with 1 wt% Tween 80 in water). Two different sets of experiments were performed.

Firstly, we investigated the effect of power, duty cycle and ultrasound time on the production of an oil-in-water emulsion from a coarse pre-emulsion mix. The droplets reached a stable size (0.7 μm) within 5 min independent of the power and duty cycle used.

Secondly, the mechanism of emulsification was studied by observing the emulsification process at an oil–water interface (no pre-mix) with a high-speed camera. Transient cavitation is thought to be responsible for acoustic emulsification; however there have been no measurements to relate the transient cavitation zone to the production of an emulsion. It has already been shown that the transient cavitation in probe systems is directly under the probe tip. High-speed observations showed that an emulsion could only be obtained if the interface was within a few millimetres of the probe tip. These results strongly suggest that the transient cavitation zone is responsible for the acoustic emulsification of oil.     

Effect of ultrasonic frequency on size distributions of nanosized mist generated by ultrasonic atomization

Ultrasonic atomization is used to produce fine liquid mists with diameter ranges below 100 nm. We investigated the effect of the frequency on the size distribution of ultrasonic mist. A bimodal distribution was obtained for the mist generated by ultrasonic atomization with a wide-range particle spectrometer. The peak diameter decreased with increasing frequency, and the number concentration of the mist increased in the smaller range. We determined the relation between the size distribution of the mist and the ultrasonic frequency, and we proposed a generation mechanism for the ultrasonic nanosized mist based on the amount of water vapor around the liquid column. Increasing the power intensity and density by changing the surface diameter of the ultrasonic oscillator affected the number concentration and size distribution of the nanosized mist. Using this technique, the diameter of the mist can be controlled by changing the frequency of the ultrasonic transducer.     

Effects of homogeneous and ultrasonic treatment on casein/phosphatidylcholine complex-emulsions: Stability and bioactivity insights

Casein (CAS), a typical protein emulsifier, has functional properties limited by its chemical structure in practical production applications. This study aimed to combine phosphatidylcholine (PC) and casein to form a stable complex (CAS/PC) and improve its functional properties through physical modification (homogeneous and ultrasonic treatment). To date, few studies have explored the effects of physical modification on the stability and biological activity of CAS/PC. Interface behavior analysis showed that compared to homogeneous treatment, PC addition and ultrasonic treatment could decrease the mean particle size (130.20 ± 3.96 nm) and increase the zeta potential (−40.13 ± 1.12 mV), indicating the emulsion is more stable. The chemical structural analysis of CAS showed that PC addition and ultrasonic treatment promoted changes in its sulfhydryl content and surface hydrophobicity, exposing more free sulfhydryl groups and hydrophobic binding sites, thereby enhancing its solubility and improving the stability of the emulsion. Additionally, storage stability analysis revealed that the incorporation of PC with ultrasonic treatment could improve the root mean square deviation value and radius of gyration value of CAS. These modifications resulted in an increase the binding free energy between CAS and PC (−238.786 kJ/mol) at 50 °C, leading to an improvement in the thermal stability of the system. Furthermore, digestive behavior analysis indicated that PC addition and ultrasonic treatment could increase the total FFA release from 667.44 ± 22.33 μmol to 1250.33 ± 21.56 μmol. In conclusion, the study underscores the effectiveness of PC addition and ultrasonic treatment in enhancing the stability and bioactivity of CAS, offering novel ideas for designing stable and healthy emulsifiers.     

A fundamental study on the degradation of paracetamol under single- and dual-frequency ultrasound

The degradation of paracetamol, a widely found emerging pharmaceutical contaminant, was investigated under a wide range of single-frequency and dual-frequency ultrasonic irradiations. For single-frequency ultrasonic irradiation, plate transducers of 22, 98, 200, 300, 400, 500, 760, 850, 1000, and 2000 kHz were employed and for dual-frequency ultrasonic irradiation, the plate transducers were coupled with a 20 kHz ultrasonic horn in opposing configuration. The sonochemical activity was quantified using two dosimetry methods to measure the yield of HO• and H₂O₂ separately, as well as sonochemiluminescence measurement. Moreover, the severity of the bubble collapses as well as the spatial and size distribution of the cavitation bubbles were evaluated via sonoluminescence measurement. The paracetamol degradation rate was maximised at 850 kHz, in both single and dual-frequency ultrasonic irradiation. A synergistic index higher than 1 was observed for all degrading frequencies (200 – 1000 kHz) under dual-frequency ultrasound irradiation, showing the capability of dual-frequency system for enhancing pollutant degradation. A comparison of the results of degradation, dosimetry, and sonoluminescence intensity measurement revealed the stronger dependency of the degradation on the yield of HO• for both single and dual-frequency systems, which confirms degradation by HO• as the main removal mechanism. However, an enhanced degradation for frequencies higher than 500 kHz was observed despite a lower HO• yield, which could be attributed to the improved mass transfer of hydrophilic compounds at higher frequencies. The sonoluminescence intensity measurements showed that applying dual-frequency ultrasonic irradiation for 200 and 400 kHz made the bubbles larger and less uniform in size, with a portion of which not contributing to the yield of reactive oxidant species, whereas for the rest of the frequencies, dual-frequency ultrasound irradiation made the cavitation bubbles smaller and more uniform, resulting in a linear correlation between the overall sonoluminescence intensity and the yield of reactive oxidant species.