Synergistic bactericidal effects and mechanisms of low intensity ultrasound and antibiotics against bacteria: a review

Low intensity ultrasonic therapy is always an important research area of ultrasonic medicine. This review concentrates on low intensity ultrasound enhancing bactericidal action of antibiotics against bacteria in vitro and in vivo, including planktonic bacteria, bacterial biofilms, Chlamydia, and bacteria in implants. These literatures show that low intensity ultrasound alone is not effective in killing bacteria, while the combination of low intensity ultrasound and antibiotics is promising. Low intensity ultrasound facilitating antibiotic treatment is still in its infancy, and still requires a great deal of research in order to develop the technology on medical treatment scale.     

Low intensity pulsed ultrasound for fracture healing: a review of the clinical evidence and the associated biological mechanism of action

Low intensity pulsed ultrasound is used in the clinical treatment of fractures and other osseous defects. Level I clinical studies demonstrate the ability of a specific ultrasound signal (1.5 MHz ultrasound pulsed at 1 kHz, 20% duty cycle, 30 mW/cm² intensity (SATA)) to accelerate the healing time in fresh tibia, radius and scaphoid fractures by up to 40%. Additionally, the same ultrasound signal has been shown to be effective at resolving all types of nonunions of all ages, following a wide range of fracture types and primary fracture management techniques.Recently, significant efforts have resulted in a more comprehensive understanding of the biological mechanism of action that produces the documented clinical outcomes. Low intensity pulsed ultrasound has been demonstrated to accelerate in vivo all stages of the fracture repair process (inflammation, soft callus formation, hard callus formation). In particular, accelerated mineralisation has been demonstrated in vitro with increases in osteocalcin, alkaline phosphatase, VEGF and MMP-13 expression. Integrins, a family of mechanoreceptors present on a wide range of cells involved in the fracture healing process, have been shown to be activated by the ultrasound signal. Downstream of the integrin activation, focal adhesions occur on the surface of cells with the activation of multiple signalling pathways, including the ERK, NF-κβ, and PI3 kinase pathways. These pathways have been directly linked to the production of COX-2 and prostaglandin, which are key to the processes of mineralisation and endochondral ossification in fracture healing.     

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.     

High intensity ultrasound modified ovalbumin: Structure,interface and gelation properties

Influence of high intensity ultrasound (HIUS) on the structure and properties of ovalbumin (OVA) were investigated. It was found that the subunits and secondary structure of OVA did not change significantly with HIUS treatment from the electrophoretic patterns and circular dichroism (CD) spectrum. The amount of free sulfhydryl groups increased and intrinsic fluorescence spectra analysis indicated changes in the tertiary structure and partial unfold of OVA after sonication increased. Compared with the untreated OVA, HIUS treatment increased the emulsifying activity and foaming ability, and decreased interface tension (oil–water and air–water interface), which due to the increased surface hydrophobicity and decreased the surface net charge in OVA, while the emulsifying and foaming stability had no remarkable differences. The increased particle size may be attributed to formation of protein aggregates. Moreover, the gelation temperatures of HIUS-treated samples were higher than the untreated OVA according to the temperature sweep model rheology, and this effect was consistent with the increased in surface hydrophobicity for ultrasound treated OVA. These changes in functional properties of OVA would promote its application in food industry.     

Comparison between airborne ultrasound and contact ultrasound to intensify air drying of blackberry: Heat and mass transfer simulation,energy consumption and quality evaluation

This study aimed at investigating the performances of air drying of blackberries assisted by airborne ultrasound and contact ultrasound. The drying experiments were conducted in a self-designed dryer coupled with a 20-kHz ultrasound probe. A numerical model for unsteady heat and mass transfer considering temperature dependent diffusivity, shrinkage pattern and input ultrasonic energies were applied to explore the drying mechanism, while the energy consumption and quality were analyzed experimentally. Generally, both airborne ultrasound and contact ultrasound accelerated the drying process, reduced the energy consumption and enhanced the retentions of blackberry anthocyanins and organic acids in comparison to air drying alone. At the same input ultrasound intensity level, blackberries received more ultrasound energies under contact sonication (0.299 W) than airborne sonication (0.245 W), thus avoiding the attenuation of ultrasonic energies by air. The modeling results revealed that contact ultrasound was more capable than airborne ultrasound to intensify the inner moisture diffusion and heat conduction, as well as surface exchange of heat and moisture with air. During air drying, contact ultrasound treatment eliminated the gradients of temperature and moisture inside blackberry easier than airborne ultrasound, leading to more homogenous distributions. Moreover, the total energy consumption under air drying with contact ultrasound assistance was 27.0% lower than that with airborne ultrasound assistance. Besides, blackberries dehydrated by contact ultrasound contained more anthocyanins and organic acids than those dried by airborne ultrasound, implying a higher quality. Overall, direct contact sonication can well benefit blackberry drying in both energy and quality aspects.     

Modulation of luciferase activity using high intensity focused ultrasound in combination with bioluminescence imaging, magnetic resonance imaging and histological analysis in muscle tissue

This study investigates the effect of high intensity focused ultrasound (HIFU) to muscle tissue transfected with a luciferase reporter gene under the control of a CMV-promoter. HIFU was applied to the transfected muscle tissue using a dual HIFU system. In a first group four different intensities (802 W/cm², 1401 W/cm², 2117 W/cm², 3067 W/cm²) of continuous HIFU were applied 20 s every other week for four times. In a second group two different intensities (802 W/cm², 1401 W/cm²) were applied 20 s every fourth day for 20 times. The luciferase activity was determined by bioluminescence imaging. The effect of HIFU to the muscle tissue was assessed by T1-weighted ± Gd-DTPA, T2-weighted and a diffusion-weighted STEAM sequence obtained on a 1.5-T GE-MRI scanner. Histology of the treated tissue was done at the end. In the first group the photon emission was at 3067.6 W/cm² 1.28 × 10⁷ ± 3.1 × 10⁶ photon/s (5.5 ± 1.2-fold), of 2157.9 W/cm² 8.1 ± 2.7 × 10⁶ photon/s (3.2 ± 1.1-fold), of 1401.9 W/cm² 9.3 ± 1.3 × 10⁶ photon/s (4.9 ± 0.4-fold) and of 802.0 W/cm² 8.6x ± 1.2 × 10⁶ photon/s (4.5 ± 0.6-fold) compared to baseline. In the second group the photon emission was at 1401.9 W/cm² and 802.0 W/cm² 14.1 ± 3.6 × 10⁶ photon/s (6.1 ± 1.5-fold), respectively, 5.1 ± 4.7 × 10⁶ photon/s (6.5 ± 2.0-fold). HIFU can enhance the luciferase activity controlled by a CMV-promoter.     

Effect of ultrasound pretreatment on structural,physicochemical, rheological and gelation properties of transglutaminase cross-linked whey protein soluble aggregates

A solution (10%, w/v) of whey protein soluble aggregates (WPISA) was pretreated with high-intensity ultrasound (HUS, 20 kHz) for different durations (10–40 min) before incubation with transglutaminase (TGase) to investigate the effect of HUS on the structural, physicochemical, rheological, and gelation properties of TGase cross-linked WPISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that HUS increased the amounts of high-molecular-weight polymers/aggregates in WPISA after incubation with TGase. HUS significantly increased (P < 0.05) the degree of TGase-mediated cross-linking in WPISA, as demonstrated by a reduction in free amino group contents. HUS significantly increased (P < 0.05) the particle size, intrinsic fluorescence intensity, and surface hydrophobicity of TGase cross-linked WPISA, but had no significant impact (P > 0.05) on the zeta-potential or total free sulfhydryl group content of TGase cross-linked WPISA. The apparent viscosity and the consistency index of TGase cross-linked WPISA were significantly increased by HUS (P < 0.05), which indicated that HUS facilitated the formation of more high-molecular-weight polymers. HUS significantly increased (P < 0.05) the water holding capacity and gel strength of glucono-δ-lactone (GDL)-induced TGase cross-linked WPISA gels. The results indicated that HUS could be an efficient tool for modifying WPISA to improve its degree of TGase-mediated cross-linking, which would lead to improved rheological and gelation properties.     

Post-mortem ultrasound and freezing of rabbit meat: Effects on the physicochemical quality and weight loss

High intensity ultrasound (HIU) is a technique with the potential to improve meat quality, however, more research is needed on its application within the chain of cold storage and freezing. This study evaluates the effect of HIU (40 kHz, 9.6 W/cm², 20 and 40 min) and post-mortem development on the yield and physicochemical quality of rabbit meat in samples treated with HIU pre- and post-storage in a freezer (120 h at −20 °C). Twenty rabbit carcasses were vacuum packed 12 h post-mortem, placed in a fridge at 4 °C for 24 h, and divided in two groups (HIU application before or after freezing), before assigning the treatments. The results show that HIU before freezing produced intense and bright orange-yellow colours, whereas its application after freezing resulted in pale red tones. HIU application accelerates rigor mortis resolution when it is applied before freezing and causes a significant decrease in pH immediately following the HIU treatment. Post-freezing application of HIU is not recommended because it considerably increased weight loss and toughening of the meat when long exposure times were used (40 min). In contrast, a short treatment duration with HIU mitigated the effects of freezing and produced significant increases in water-holding capacity (WHC) after cold storage. The yield (weight loss) of the rabbit meat was not affected when HIU was applied pre-freezing. The application of HIU pre-freezing constitutes a promising technology because it increased the tenderness and the WHC of rabbit meat. However, more research is needed to improve the appearance before scaling up to industrial levels.     

Enhancing carrot convective drying by combining ethanol and ultrasound as pre-treatments: Effect on product structure,quality, energy consumption,drying and rehydration kinetics

Ultrasound was combined with ethanol to improve different aspects of carrot convective drying, evaluating both processing and product quality. The ultrasound in water treatment resulted in cellular swelling and small impact on texture. Differently, the ultrasound in ethanol and ethanol treatments modified both carrot microstructure (cell wall modifications of parenchymatic tissue) and macrostructure (shrinkage and resistance to perforation). Pre-treatments with ultrasound in ethanol and ethanol improved the drying kinetics, reducing the processing time (~50%) and the energy consumption (42–62%). These pre-treatments also enhanced rehydration, whose initial rate and water retention were higher than the control. In addition, the carotenoid content was preserved after drying, for all the treatments. Any impact on shrinkage was observed. A mechanistic discussion, based on structural modification (microstructure and macrostructure) and physical properties of water and ethanol, was provided. As conclusion, this work not only described positive aspects of combining the technologies of ultrasound and ethanol as pre-treatments to convective drying, but also proposed mechanisms to explain the phenomena.     

Synergistic inactivation of bacteria based on a combination of low frequency,low-intensity ultrasound and a food grade antioxidant

This study evaluated a synergistic antimicrobial treatment using a combination of low frequency and a low-intensity ultrasound (LFU) and a food-grade antioxidant, propyl gallate (PG), against a model gram-positive (Listeria innocua) and the gram-negative bacteria (Escherichia coli O157:H7). Bacterial inactivation kinetic measurements were complemented by characterization of biophysical changes in liposomes, changes in bacterial membrane permeability, morphological changes in bacterial cells, and intracellular oxidative stress upon treatment with PG, LFU, and a combination of PG + LFU. Combination of PG + LFU significantly (>4 log CFU/mL, P < 0.05) enhanced the inactivation of both L. innocua and E. coli O157:H7 compared to PG or LFU treatment. As expected, L. innocua had a significantly higher resistance to inactivation compared to E. coli using a combination of PG + LFU. Biophysical measurements in liposomes, bacterial permeability measurements, and scanning electron microscope (SEM)-based morphological measurements show rapid interactions of PG with membranes. Upon extended treatment of cells with PG + LFU, a significant increase in membrane damage was observed compared to PG or LFU alone. A lack of change in the intracellular thiol content following the combined treatment and limited effectiveness of exogenously added antioxidants in attenuating the synergistic antimicrobial action demonstrated that oxidative stress was not a leading mechanism responsible for the synergistic inactivation by PG + LFU. Overall, the study illustrates synergistic inactivation of bacteria using a combination of PG + LFU based on enhanced membrane damage and its potential for applications in the food and environmental systems.     

The influence of combined low-strength ultrasonics and micro-aerobic pretreatment process on methane generation and sludge digestion: Lipase enzyme,microbial activation,and energy yield

Low-frequency ultrasonics is a potential technology to reduce the hydrolysis phase period in anaerobic digestion process. In this study, the influence of combined low frequency ultrasonics and micro-aerobic (MA) pretreatment on sewage sludge solubilization, enzyme activity and anaerobic digestion were assessed. Initially, the effect of ultrasonic density (0.012, 0.014, 0.016, 0.018, 0.1, 0.12 and 0.14 W/mL) and irradiation time (1, 3, 5, 8, 9, 10 and 12 min) of 20 kHz frequency waves were investigated. Accordingly, the effect of micro-aerobic pretreatment (Air flow rate (AFR) = 0.1, 0.2, 0.3 and 0.5 VVM) within 20, 30, 40.48 and 60 h were examined. In addition, the effect of combined pretreatment on COD solubilization, lipase enzyme activation, ATP, percentage of live bacteria and methane gas production during the anaerobic process were examined. The results showed that the highest lipase activity (14.9 Umol/mL) was obtained under the effect of ultrasonic density of 0.1 W/ml within 9 min. The highest solubilization (65%) was observed under optimal micro-aerobic conditions: AFR = 0.2 (VVM) and micro-aerobic time: 40 h. Combined ultrasonic and micro-aerobic (US + MA) pretreatment increases the solubilization (70%), microbial activity (2080%) and lipase enzymatic activity (129%) compared to individual pretreatment. The Biogas production during anaerobic digestion pretreated with combined methods increased by 193% compared to the control, while the elevated values of biogas production in reactors pretreated by ultrasonic and micro-aerobic pretreatment alone were observed to be 101% and 165%, respectively. The net energy in reactor with the combined pre-treatment methods was calculated to be 1.26 kWh, while this value for control, pretreated ultrasonic and micro-aerobic reactors were obtained to be 0.56, 0.67 and 1.2 kWh, respectively.     

Fermentation of Saccharomyces cerevisiae in a 7.5 L ultrasound-enhanced fermenter: Effect of sonication conditions on ethanol production,intracellular Ca2+ concentration and key regulating enzyme activity in glycolysis

In this study, the effect of sonication on the fermentation process of a single-celled fungus was examined. During the experiment, Saccharomyces cerevisiae (S. cerevisiae) was used as the starting strain for ethanol fermentation (batch fermentation) in a 7.5 L automated fermentation tank. The fermentation tank connected with a six-frequency ultrasonic equipment. Non-sonication treatment was set up as the control. Sonication treatment with power density of 280 W/L and 48 h of treatment time were set up as trial groups for investigating the influence of different ultrasound frequency including 20, 23, 25, 28, 33 and 40 kHz on the changes in dry cell-weight, glucose consumption rate, and ethanol yield. The results showed that the dry cell-weight, glucose consumption rate, and ethanol content reached the best results under the ultrasonic condition of 28 kHz ultrasound frequency in comparison with other ultrasound frequency. The dry cell-weight and ethanol content of the 28 kHz ultrasonic treatment group increased by 17.30% and 30.79%, respectively in comparison with the control group The residual sugar content dropped to a lower level within 24 h, which was consistent with the change in ethanol production. Besides, the results found that the glucose consumption rate increased compared to the control. It indicated that ultrasound accelerated glucose consumption contributed to increase the rate of ethanol output. In order to explore the mechanism of sonication enhanced the content of ethanol output by S. cerevisiae, the morphology, permeability of S. cerevisiae and key enzyme activities of ethanol synthesis were investigated before and after sonication treatment. The results showed that after sonication treatment, the extracellular nucleic acid protein content and intracellular Ca²⁺ concentration increased significantly. The morphology of S. cerevisiae was observed by SEM and found that the surface of the strain had wrinkles and depressions after ultrasonic treatment. furthermore after sonication treatment, the activities of three key enzymes which catalyze three irreversible reactions in glycolysis metabolism, namely, hexokinase, phosphofructokinase and pyruvate kinase increased by 59.02%, 109.05% and 87.27%, respectively. In a word, low-intensity ultrasound enhance the rate of ethanol output by S. cerevisiae might due to enhancing the growth and cell permeability of strains, and increasing the activities of three key enzymes of ethanol biosynthesis.