Ultrasonic emulsification of food-grade nanoemulsion formulation and evaluation of its bactericidal activity

Basil oil (Ocimum basilicum) nanoemulsion was formulated using non-ionic surfactant Tween80 and water by ultrasonic emulsification method. Process of nanoemulsion development was optimized for parameters such as surfactant concentration and emulsification time to achieve minimum droplet diameter with high physical stability. Surfactant concentration was found to have a negative correlation with droplet diameter, whereas emulsification time had a positive correlation with droplet diameter and also with intrinsic stability of the emulsion. Stable basil oil nanoemulsion with droplet diameter 29.3 nm was formulated by ultrasonic emulsification for 15 min. Formulated nanoemulsion was evaluated for antibacterial activity against Escherichia coli by kinetics of killing experiment. Fluorescence microscopy and FT-IR results showed that nanoemulsion treatment resulted alteration in permeability and surface features of bacterial cell membrane.     

Ultrasonic emulsification of eucalyptus oil nanoemulsion: Antibacterial activity against Staphylococcus aureus and wound healing activity in Wistar rats

The plant derived essential oil nanoemulsion was prepared using a mixture of components containing eucalyptus oil as organic phase, water as continuous phase, and non ionic surfactant, Tween 80, as emulsifier at a particular proportion of 1:1 v/v%. The ultrasonication was applied for varied processing time from 0 to 30 min to study the effect of time on the formation of nanoemulsion and physical stability of formulation by this method. The transparency and stability of emulsion was enhanced when the sonication time was increased compared to hand blender emulsion. The most stable nanoemulsion was obtained in 30 min sonication having the mean droplet diameter of 3.8 nm. The antibacterial studies of nanoemulsion against Staphylococcus aureus by time kill analysis showed complete loss of viability within 15 min of interaction. Observations from scanning electron microscopy of treated bacterial cells confirmed the membrane damage compared to control bacteria. Furthermore, the wound healing potential and skin irritation activity of the formulated nanoemulsion in Wistar rats, suggested non-irritant and higher wound contraction rate with respect to control and neomycin treated rats. These results proposed that the formulated system could be favourable for topical application in pharmaceutical industries.     

Process optimization of ultrasound-assisted curcumin nanoemulsions stabilized by OSA-modified starch

This study reports on the process optimization of ultrasound-assisted, food-grade oil–water nanoemulsions stabilized by modified starches. In this work, effects of major emulsification process variables including applied power in terms of power density and sonication time, and formulation parameters, that is, surfactant type and concentration, bioactive concentration and dispersed-phase volume fraction were investigated on the mean droplet diameter, polydispersity index and charge on the emulsion droplets. Emulsifying properties of octenyl succinic anhydride modified starches, that is, Purity Gum 2000, Hi-Cap 100 and Purity Gum Ultra, and the size stability of corresponding emulsion droplets during the 1 month storage period were also investigated. Results revealed that the smallest and more stable nanoemulsion droplets were obtained when coarse emulsions treated at 40% of applied power (power density: 1.36 W/mL) for 7 min, stabilized by 1.5% (w/v) Purity Gum Ultra. Optimum volume fraction of oil (medium chain triglycerides) and the concentration of bioactive compound (curcumin) dispersed were 0.05 and 6 mg/mL oil, respectively. These results indicated that the ultrasound-assisted emulsification could be successfully used for the preparation of starch-stabilized nanoemulsions at lower temperatures (40–45 °C) and reduced energy consumption.     

Physiochemical and cytotoxicity study of TPGS stabilized nanoemulsion designed by ultrasonication method

The main aim of the present work was to prepare TPGS stabilized D-α-Tocopherol, lemon oil, tween-80, and water nanoemulsion by low cost and highly effective sonication method. The prepared nanoemulsion showed good stability for 60 days at variable temperature conditions i.e. 4, 25 and 37 °C. The tolerance of the prepared nanoemulsion to salt (50 mM–500 mM) and pH (pH 2–pH 7.4) was also studied. The morphology and droplet size of pure and quinine loaded nanoemulsion was characterized with transmission electron microscopy. The prepared formulation was transparent and the obtained average particle size ranged between 25 nm and 35 nm. The nanoemulsion was found to be non toxic. The cell viability study of pure nanoemulsion carried out on Hep G2 cells revealed that the cell viability was 100%. The formulation further exhibited high quinine loading and release capacity with cumulative release up to 76 ± 2% and 65 ± 2% at pH 7.4 and pH 5.5 respectively. The interaction between quinine and vitamins (riboflavin, thiamine and biotin) was also carried out (aqueous medium). The study revealed that riboflavin had strong interaction with quinine and vitamins vis-à-vis thiamine and biotin.     

Fish oil based vitamin D nanoencapsulation by ultrasonication and bioaccessibility analysis in simulated gastro-intestinal tract

Recently, nanoemulsions have been employed for different applications including food and drug industries for efficient nutrient delivery system. In this study, vitamin D (a lipophilic molecule) was encapsulated in fish oil for higher oral bioavailability. The oil-in-water nanoemulsion was formulated by ultrasonication technique with a droplet size range of 300–450 nm and a shelf life of more than 90 days. The influence of oil, water and surfactant concentration was investigated by phase diagram. The formulated nanoemulsion had encapsulation efficiency in the range of 95.7–98.2%. Further, nanoemulsion passed through simulated gastro-intestinal tract revealed an increased bioavailability than non-encapsulated vitamin. Thus, the formulation can be used as a drug delivery vehicle for various lipophilic compounds. Till date, no one have fabricated an efficient nano-vehicle for the delivery of vitamin D as well as analyzed the efficient delivery system in simulated GI-tract, this is first of its kind study in this regard. This can be scaled up further after analyzing the safety aspects.     

Skin permeation of d-limonene-based nanoemulsions as a transdermal carrier prepared by ultrasonic emulsification

Nanoemulsions can be used for transporting pharmaceutical phytochemicals in skin-care products because of their stability and rapid permeation properties. However, droplet size may be a critical factor aiding permeation through skin and transdermal delivery efficiency. We prepared d-limonene nanoemulsions with various droplet sizes by ultrasonic emulsification using mixed surfactants of sorbitane trioleate and polyoxyethylene (20) oleyl ether under different hydrophilic–lipophilic balance (HLB) values. Droplet size decreased with increasing HLB value. With HLB 12, the droplet size was 23 nm, and the encapsulated ratio peaked at 92.3%. Transmission electron microscopy revealed spherical droplets and the gray parts were d-limonene precipitation incorporated in spherical droplets of the emulsion system. Franz diffusion cell was used to evaluate the permeation of d-limonene nanoemulsion through rat abdominal skin; the permeation rate depended on droplet size. The emulsion with the lowest droplet size (54 nm) achieved the maximum permeation rate. The concentration of d-limonene in the skin was 40.11 μL/cm² at the end of 360 min. Histopathology revealed no distinct voids or empty spaces in the epidermal region of permeated rat skin, so the d-limonene nanoemulsion may be a safe carrier for transdermal drug delivery.     

Optimization of ultrasound induced emulsification on the formulation of palm-olein based nanoemulsions for the incorporation of antioxidant β-d-glucan polysaccharides

Polysaccharides of β-d-glucan configuration have well-known antioxidant activity against reactive free radicals generated from the oxidation of metabolic processes. In this study, β-d-glucan-polysaccharides extracted from Ganoderma lucidum were incorporated in palm olein based nanoemulsions which act as carrier systems to enhance the delivery and bioactivity of these polysaccharides and could be potentially useful for skin care applications. Initially response surface statistical design (Central Composite Design – CCD) was subjected to optimize the formulation variables of oil-in-water (O/W) nanoemulsions induced by ultrasound. The optimal formulation variables as predicted by CCD resulted in considerably improving the physical characteristics of ultrasonically formulated nanoemulsions by minimizing their droplet size, polydispersity index and viscosity. Moreover, the β-d-glucan-loaded nanoemulsions exhibited good stability over 90 days under different storage conditions (4 °C and 25 °C). The studies using palm olein based β-d-glucan-loaded nanoemulsion generated using ultrasound confirm higher antioxidant activity as compared to free β-d-glucan.     

Physical and chemical characteristics of ultrasonically-prepared water-in-diesel fuel: Effects of ultrasonic horn position and water content

An ultrasonic technique was applied to preparation of two-phase water-in-oil (W/O) emulsified fuel of water/diesel oil/surfactant. In this study, an ultrasonic apparatus with a 28 kHz rod horn was used. The influence of the horn tip position during ultrasonic treatment, sonication time and water content (5 or 10 vol%) on the emulsion stability, viscosity, water droplet size and water surface area of emulsion fuels prepared by ultrasonication was investigated. The emulsion stability of ultrasonically-prepared fuel significantly depended on the horn tip position during ultrasonic irradiation. It was found that the change in the stability with the horn tip position was partly related to that in the ultrasonic power estimated by calorimetry. Emulsion stability, viscosity and sum of water droplets surface area increased and water droplet size decreased with an increase in sonication time, and they approached each limiting value in the longer time. The maximum values of the viscosity and water surface area increased with water content, while the limiting values of the emulsion stability and water droplet size were almost independent of water content. During ultrasonication of water/diesel oil mixture, the hydrogen and methane were identified and the cracking of hydrocarbon components in the diesel oil occurred. The combustion characteristics of ultrasonically-prepared emulsion fuel were studied and compared with those of diesel oil. The soot and NOx emissions during combustion of the emulsified fuel with higher water contents were significantly reduced compared with those during combustion of diesel oil.     

Ultrasound processed nanoemulsion: A comparative approach between resveratrol and resveratrol cyclodextrin inclusion complex to study its binding interactions,antioxidant activity and UV light stability

Resveratrol is a naturally occurring therapeutic molecule used for treatment of diseases caused by oxidative stress. This investigation elucidates the advantages of fabrication of size controlled resveratrol inclusion complex. This has been done by encapsulating resveratrol-cyclodextrin inclusion complex in a phospholipid stabilized nanoemulsion formulated by ultrasonication emulsification method. The prepared nanoemulsion has been compared with resveratrol encapsulated nanoemulsion system. The morphology of the resveratrol nanoemulsion and inclusion complex nanoemulsion have been observed using transmission electron microscopy with average size 20.41 ± 3.41 and 24.48 ± 5.70 nm respectively. The nanoemulsion showed good loading and release efficiency. The radical diminishing potential of resveratrol and its inclusion complex has been compared in nanoemulsion. The effect of UV irradiation (365 nm) on resveratrol in different solvent systems (ethanol, water and nanoemulsion) indicated that nanoemulsion prevents degradation of resveratrol. Efforts have also been made to explore the interactions between bovine serum albumin and resveratrol in nanoemulsion.     

Impact of process parameters in the generation of novel aspirin nanoemulsions – Comparative studies between ultrasound cavitation and microfluidizer

In the present investigation, the operating efficiency of a bench-top air-driven microfluidizer has been compared to that of a bench-top high power ultrasound horn in the production of pharmaceutical grade nanoemulsions using aspirin as a model drug. The influence of important process variables as well as the pre-homogenization and drug loading on the resultant mean droplet diameter and size distribution of emulsion droplets was studied in an oil-in-water nanoemulsion incorporated with a model drug aspirin. Results obtained show that both the emulsification methods were capable of producing very fine nanoemulsions containing aspirin with the minimum droplet size ranging from 150 to 170 nm. In case of using the microfluidizer, it has been observed that the size of the emulsion droplets obtained was almost independent of the applied microfluidization pressure (200–600 bar) and the number of passes (up to 10 passes) while the pre-homogenization and drug loading had a marginal effect in increasing the droplet size. Whereas, in the case of ultrasound emulsification, the droplet size was generally decreased with an increase in sonication amplitude (50–70%) and period of sonication but the resultant emulsion was found to be dependent on the pre-homogenization and drug loading. The STEM microscopic observations illustrated that the optimized formulations obtained using ultrasound cavitation technique are comparable to microfluidized emulsions. These comparative results demonstrated that ultrasound cavitation is a relatively energy-efficient yet promising method of pharmaceutical nanoemulsions as compared to microfluidizer although the means used to generate the nanoemulsions are different.     

An experimental study on the motion of water droplets in oil under ultrasonic irradiation

The motion of a single water droplet in oil under ultrasonic irradiation is investigated with high-speed photography in this paper. First, we described the trajectory of water droplet in oil under ultrasonic irradiation. Results indicate that in acoustic field the motion of water droplet subjected to intermittent positive and negative ultrasonic pressure shows obvious quasi-sinusoidal oscillation. Afterwards, the influence of major parameters on the motion characteristics of water droplet was studied, such as acoustic intensity, ultrasonic frequency, continuous phase viscosity, interfacial tension, and droplet diameter, etc. It is found that when the acoustic intensity and frequency are 4.89 W cm⁻² and 20 kHz respectively, which are the critical conditions, the droplet varying from 250 to 300 μm in lower viscous oil has the largest oscillation amplitude and highest oscillation frequency.     

Sustained release formulations of citronella oil nanoemulsion using cavitational techniques

Nanoemulsion synthesis has proven to be an effective way for transportation of immobile, insoluble bioactive compounds. Citronella Oil (lemongrass oil), a natural plant extract, can be used as a mosquito repellent and has less harmful effects compared to its available market counterpart DEET (N, N-Diethyl-meta-toluamide). Nanoemulsion of citronella oil in water was prepared using cavitation-assisted techniques while investigating the effect of system parameters like HLB (Hydrophilic Lipophilic Balance), surfactant concentration, input energy density and mode of power input on emulsion quality. The present work also examines the effect of emulsification on release rate to understand the relationship between droplet size and the release rate. Minimum droplet size (60 nm) of the emulsion was obtained at HLB of 14, S/O¹ ratio of 1.0, ultrasound amplitude of 50% and irradiation time of 5 min. This study revealed that hydrodynamic cavitation-assisted emulsification is more energy efficient compared to ultrasonic emulsification. It was also found that the release rate of nanoemulsion enhanced as the droplet size of emulsion reduced.     

Fabrication of polymeric nanocapsules from curcumin-loaded nanoemulsion templates by self-assembly

In this study, biodegradable polymeric nanocapsules were prepared by sequential deposition of food-grade polyelectrolytes through the self-assembling process onto the oil (medium chain triglycerides) droplets enriched with curcumin (lipophilic bioactive compound). Optimum conditions were used to prepare ultrasound-assisted nanoemulsions stabilized by octenyl-succinic-anhydride (OSA)-modified starch. Negatively charged droplets (−39.4 ± 1.84 mV) of these nanoemulsions, having a diameter of 142.7 ± 0.85 nm were used as templates for the fabrication of nanocapsules. Concentrations of layer-forming cationic (chitosan) and anionic (carboxymethylcellulose) biopolymers were optimized based on the mean droplet/particle diameter (MDD/MPD), polydispersity index (PDI) and net charge on the droplets/capsules. Prepared core–shell structures or nanocapsules, having MPD of 159.85 ± 0.92 nm, were characterized by laser diffraction (DLS), ζ-potential (ZP), atomic force microscopy (AFM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Furthermore, physical stability of curcumin-loaded nanocapsules in suspension was determined and compared at different storage temperatures. This study may provide information regarding the formation of ultrasound-assisted polymeric nanocapsules from the nanoemulsion templates which could be helpful in the development of delivery systems for lipophilic food bioactives.     

Pretreatment of defatted wheat germ proteins (by-products of flour mill industry) using ultrasonic horn and bath reactors: Effect on structure and preparation of ACE-inhibitory peptides

The ultrasonic horn and bath reactors were compared based on production of angiotensin-converting-enzyme (ACE) inhibitory peptides from defatted wheat germ proteins (DWGP). The DWGP was sonicated before hydrolysis by Alcalase. Degree of hydrolysis, ACE-inhibitory activity, surface hydrophobicity, fluorescence intensity, free sulfhydryl (SH), and disulfide bond (SS) were determined. The highest ACE-inhibitory activity of DWGP hydrolysate was obtained at power intensity of 191.1 W/cm² for 10 min in the ultrasonic horn reactor. The fixed frequency of 33 kHz and the sweep frequency of 40 ± 2 kHz resulted in the maximum ACE-inhibitory activity. The combined irradiation of dual fixed frequency (24/68 kHz) produced significant increase in ACE-inhibitory activity compared with single frequency (33 kHz). The ultrasonic probe resulted in significant higher ACE-inhibitory activity compared with ultrasonic bath operating at single or dual fixed and sweep frequencies. The changes in conformation of the DWGP due to sonication were confirmed by the changes in fluorescence intensity, surface hydrophobicity, SH_f and SS contents and they were found in conformity with the ACE-inhibitory activity in case of the ultrasonic horn reactor but not in bath reactor.     

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.     

Physicochemical characterization of black seed oil-milk emulsions through ultrasonication

The ultrasonic formation of stable emulsions of a bioactive material, black seed oil, in skim milk was investigated. The incorporation of 7% of black seed oil in pasteurised homogenized skim milk (PHSM) using 20 kHz high intensity ultrasound was successfully achieved. The effect of sonication time and acoustic power on the emulsion stability was studied. A minimum process time of 8 min at an applied acoustic power of 100 W was sufficient to produce emulsion droplets stable for at least 8 days upon storage at 4 ± 2 °C, which was confirmed through creaming stability, particle size, rheology and color analysis. Partially denatured whey proteins may provide stability to the emulsion droplets and in addition to the cavitation effects of ultrasound are responsible for the production of smaller sized emulsion droplets.     

Response surface methodology for optimization of cinnamon essential oil nanoemulsion with improved stability and antifungal activity

The optimum formulation and ultrasonic condition for fabrication of cinnamon essential oil (CEO) nanoemulsion were determined using Response Surface Methodology (RSM). The CEO nanoemulsions were formed using an ultrasonic bath (43 kHz at power output of 210 W) and an ultrasonic probe (24 kHz at power of 400 W). Probe ultrasonication outperformed bath ultrasonication since it produced nanoemulsions with smaller droplet size, narrower size distribution as measured using polydispersity index (PDI), and higher viscosity. The influences of sonication time of 180.23–351.77 s, temperature of 4.82–45.18 °C, and Tween® 80 concentration of 1–3% w/w on the droplet size, PDI, and viscosity were investigated using RSM based on Box-Behnken design (BBD). The RSM revealed that the sonication time of 266 s, temperature of 4.82 °C, and Tween® 80 of 3% w/w produced the optimum CEO nanoemulsion with droplet size of 65.98 nm, PDI of 0.15, and viscosity of 1.67 mPa.s. Moreover, the optimum nanoemulsion had good stability in terms of droplet size and PDI when storing at 4, 30, and 45 °C for 90 days. The antifungal activity of the optimum CEO nanoemulsion was then investigated against Aspergillus niger, Rhizopus arrhizus, Penicillium sp., and Colletotrichum gloeosporioides in comparison to CEO coarse emulsion. The results showed that the CEO nanoemulsion had better antifungal activity than coarse emulsion of CEO.     

Ultrasonic pretreatment of corn slurry for saccharification: A comparison of batch and continuous systems

The effects of ultrasound on corn slurry saccharification yield and particle size distribution was studied in both batch and continuous-flow ultrasonic systems operating at a frequency of 20 kHz. Ground corn slurry (28% w/v) was prepared and sonicated in batches at various amplitudes (192–320 μm_{peak-to-peak (p–p)}) for 20 or 40 s using a catenoidal horn. Continuous flow experiments were conducted by pumping corn slurry at various flow rates (10–28 l/min) through an ultrasonic reactor at constant amplitude of 12 μm_p–p. The reactor was equipped with a donut shaped horn. After ultrasonic treatment, commercial alpha- and gluco-amylases (STARGEN^TM 001) were added to the samples, and liquefaction and saccharification proceeded for 3 h. The sonicated samples were found to yield 2–3 times more reducing sugars than unsonicated controls. Although the continuous flow treatments released less reducing sugar compared to the batch systems, the continuous flow process was more energy efficient. The reduction of particle size due to sonication was approximately proportional to the dissipated ultrasonic energy regardless of the type of system used. Scanning electron microscopy (SEM) images were also used to observe the disruption of corn particles after sonication. Overall, the study suggests that both batch and continuous ultrasonication enhanced saccharification yields and reduced the particle size of corn slurry. However, due to the large volume involve in full scale processes, an ultrasonic continuous system is recommended.     

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.     

Ultrasonic energy input influence οn the production of sub-micron o/w emulsions containing whey protein and common stabilizers

Ultrasonication may be a cost-effective emulsion formation technique, but its impact on emulsion final structure and droplet size needs to be further investigated. Olive oil emulsions (20 wt%) were formulated (pH ～ 7) using whey protein (3 wt%), three kinds of hydrocolloids (0.1–0.5 wt%) and two different emulsification energy inputs (single- and two-stage, methods A and B, respectively). Formula and energy input effects on emulsion performance are discussed. Emulsions stability was evaluated over a 10-day storage period at 5 °C recording the turbidity profiles of the emulsions. Optical micrographs, droplet size and viscosity values were also obtained. A differential scanning calorimetric (DSC) multiple cool–heat cyclic method (40 to ?40 °C) was performed to examine stability via crystallization phenomena of the dispersed phase.Ultrasonication energy input duplication from 11 kJ to 25 kJ (method B) resulted in stable emulsions production (reduction of back scattering values, dBS ～ 1% after 10 days of storage) at 0.5 wt% concentration of any of the stabilizers used. At lower gum amount samples became unstable due to depletion flocculation phenomena, regardless of emulsification energy input used. High energy input during ultrasonic emulsification also resulted in sub-micron oil-droplets emulsions (D₅₀ = 0.615 μm compared to D₅₀ = 1.3 μm using method A) with narrower particle size distribution and in viscosity reduction.DSC experiments revealed no presence of bulk oil formation, suggesting stability for XG 0.5 wt% emulsions prepared by both methods. Reduced enthalpy values found when method B was applied suggesting structural modifications produced by extensive ultrasonication. Change of ultrasonication conditions results in significant changes of oil droplet size and stability of the produced emulsions.