Synergistic antibacterial effects of ultrasound and thyme essential oils nanoemulsion against Escherichia coli O157:H7

Essential oil nanoemulsions have been proven to have stronger antimicrobial effects compared to the essential oil alone or coarse emulsion. Sonoporation could be the promising candidate to trigger a synergistic effect with thyme essential oil nanoemulsion (TEON) and produce a more effective antibacterial efficacy. Therefore, in this study, the bactericidal effects of ultrasound (US) in combination with TEON treatments against Escherichia coli (E. coli) O157:H7 were investigated. The remarkable synergistic effects of US (20 kHz, 255 W/cm², 9 min) and TEON (0.375 mg/mL) treatments at 22 °C reduced E. coli O157:H7 populations by 7.42 ± 0.27 log CFU/mL.The morphological changes of cells exposed to different treatments were observed by scanning electron microscopy and transmission electron microscopy. The results showed that the synergistic effects of the ultrasound and TEON treatments altered the morphology and interior microstructure of organism cells. Laser scanning confocal microscopy (LSCM) images revealed that the combination treatments of ultrasound and TEON altered the permeability of cell membranes, and this affected the integrity of E. coli O157:H7 cells. This was further indicated by the high amounts of nucleic acids and proteins released from these cells following treatment.The results from this study illustrated the mechanisms of the synergistic effects of sonoporation and TEON treatments and provided valuable information for their potential in food pasteurization.     

Nondestructive monitoring,kinetics and antimicrobial properties of ultrasound technology applied for surface decontamination of bacterial foodborne pathogen in pork

In this study, electronic nose (E-nose) and Hyperspectral Imaging (HSI) was employed for nondestructive monitoring of ultrasound efficiency (20KHZ) in the inactivation of Salmonella Typhimurium, and Escherichia coli in inoculated pork samples treated for 10, 20 and 30 min.Weibull, and Log-linear model fitted well (R² ≥ 0.9) for both Salmonella Typhimurium, and Escherichia coli inactivation kinetics. The study also revealed that ultrasound has antimicrobial effects on the pathogens. For qualitative analysis, unsupervised (PCA) and supervised (LDA) chemometric algorithms were applied. PCA was used for successful sample clustering and LDA approach was used to construct statistical models for the classification of ultrasound treated and untreated samples. LDA showed classification accuracies of 99.26%,99.63%,99.70%, 99.43% for E-nose - S. Typhimurium, E-nose -E. coli, HSI - S. Typhimurium and HSI -E. coli respectively. PLSR quantitative models showed robust models for S. Typhimurium- (E-nose Rp² = 0.9375, RMSEP = 0.2107 log CFU/g and RPD = 9.7240 and (HSI Rp² = 0.9687 RMSEP = 0.1985 log CFU/g and RPD = 10.3217) and E. coli -(E-nose -Rp² = 0.9531, RMSEP = 0.2057 log CFU/g and RPD = 9.9604) and (HIS- Rp² = 0.9687, RMSEP = 0.2014 log CFU/g and RPD = 10.1731).This novel study shows the overall effectiveness of applying E-nose and HSI for in-situ and nondestructive detection, discrimination and quantification of bacterial foodborne pathogens during the application of food processing technologies like ultrasound for pathogen inactivation.     

Experimental study on the influence of low-frequency and low-intensity ultrasound on the permeability of the Mycobacterium smegmatis cytoderm and potentiation with levofloxacin

Tuberculosis is an infectious disease caused by the bacterium M. tuberculosis. The aim of this study was to investigate the bactericidal effect and underlying mechanisms of low-frequency and low-intensity ultrasound combined with levofloxacin treatment against M. smegmatis (a surrogate of M. tuberculosis). As part of this study, M. smegmatis was continuously irradiated with low frequency ultrasound (42 kHz) using several different doses whereby both intensity (0.138, 0.190 and 0.329 W/cm²) and exposure time (5, 15 and 20 min) were varied. Flow cytometric analyses revealed that the permeability of M. smegmatis increased following ultrasound exposure. The survival rate, structure and morphology of bacteria in the lower-dose (I_SATA = 0.138 W/cm² for 5 min) ultrasound group displayed no significant differences upon comparison with the untreated group. However, the survival rate of bacteria was significantly reduced and the bacterial structure was damaged in the higher-dose (I_SATA = 0.329 W/cm² for 20 min) ultrasound group. Ultrasound irradiation (0.138 W/cm²) was subsequently applied to M. smegmatis in combination with levofloxacin treatment for 5 min. The results demonstrated that the bactericidal effect of ultrasonic irradiation combined with levofloxacin is higher compared to ultrasound alone or levofloxacin alone.     

Low-intensity ultrasound enhances the antimicrobial activity of neutral peptide TGH2 against Escherichia coli

In recent years, foodborne diseases caused by Escherichia coli are a major threat to the food industry and consumers. Antimicrobial peptides (AMPs) and ultrasound both have good inhibitory effects on E. coli. In this work, the mechanism of action and synergistic effect of an in silico predicted AMP, designated as TGH2 (AEFLREKLGDKCTDRHV), from the C-terminal sequence of Tegillarca granosa hemoglobin, combined with low-intensity ultrasound was explored. The minimal inhibitory concentration (MIC) of TGH2 on E. coli decreased by 4-fold to 31.25 μg/mL under 0.3 W/cm² ultrasound treatment, while the time kill curve analysis showed that low-intensity ultrasound combined with peptide TGH2 had an enhanced synergistic bactericidal effect after 0.5 h. The permeability on E. coli cell membrane increased progressively during combined treatment with peptide TGH2 and low-intensity ultrasound, resulting in the leakage of intracellular solutes, as shown by transmission electron microscopy (TEM). Structural analysis using circular dichroism (CD) revealed that peptide TGH2 has an α-helical structure, showing a slight untwisting effect under 0.3 W/cm² ultrasound treatment for 0.5 h. The findings here provide new insight into the potential application of ultrasound and AMPs combination in food preservation.     

Effect of ultrasound and chlorine dioxide on Salmonella Typhimurium and Escherichia coli inactivation in poultry chiller tank water

This study evaluated the application of ultrasound alone or combined with chlorine dioxide (ClO₂) for Salmonella Typhimurium and Escherichia coli inactivation in poultry processing chiller tank water. A Full Factorial Design (FFD) 2² was conducted for each microorganism to evaluate the effect of ultrasound exposure time (x₁: 1 to 9 min; fixed: 37 kHz; 330 W; 25 °C) using a bath, and ClO₂ concentration (x₂: 1 to 17 mg L⁻¹) on microorganism count expressed in log CFU mL⁻¹ in distilled water. Variable x₂ had a negative effect on Salmonella Typhimurium (-5.09) and Escherichia coli (-2.00) count, improving the inactivation; while a x₁ increase present no inactivation improvement, explaining the use of x₁ lower level (1 min) and x₂ higher level (17 mg L⁻¹). The best condition for microorganism inactivation based on FFD was evaluated in chiller tank water (with organic matter) at 25, 16, and 4 °C; x₁ was kept (1 min), however x₂ was adjusted to obtain the same residual free chlorine (2.38 mg L⁻¹) considering the ClO₂ consumption by organic matter, achieving the value of 30 mg L⁻¹. An inactivation of 49% and 31% were observed for Salmonella Typhimurium and Escherichia coli. When ultrasound was replaced by a simple agitation in the presence of ClO₂, there was no inactivation for both microorganisms. Moreover, at poultry carcass pre-chilling (16 °C) and chilling (4 °C) conditions, the synergism of ultrasound combined with ClO₂ was more pronounced, with microorganisms’ reductions up to 100%.     

Synergistic bactericidal effect of ultrasound combined with citral nanoemulsion on Salmonella and its application in the preservation of purple kale

In this study, a novel citral nanoemulsion (CLNE) was prepared by ultrasonic emulsification. The synergistic antibacterial mechanism of ultrasound combined with CLNE against Salmonella Typhimurium and the effect on the physicochemical properties of purple kale were investigated. The results showed that the combined treatment showed obviously inactivate effect of S. Typhimurium. Treatment with 0.3 mg/mL CLNE combined with US (20 kHz, 253 W/cm²) for 8 min reduced S. Typhimurium populations in phosphate-buffered saline (PBS) by 9.05 log CFU/mL. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein and nucleic acid release assays showed that the US combination CLNE disrupt the integrity of S. Typhimurium membranes. Reactive oxygen species (ROS) and malondialdehyde (MDA) detection indicated that US+CLNE exacerbated oxidative stress and lipid peroxidation in cell membranes. The morphological changes of cells after different treatments by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) illustrated that the synergistic effect of US+CLNE treatment changed the morphology and internal microstructure of the bacteriophage cells. Application of US+CLNE on purple kale leaves for 6 min significantly (P < 0.05) reduced the number of S. Typhimurium, but no changes in the physicochemical properties of the leaves were detected. This study elucidates the synergistic antibacterial mechanism of ultrasound combined with CLNE and provides a theoretical basis for its application in food sterilization.     

In vitro and in silico approaches to investigate antimicrobial and biofilm removal efficacies of combined ultrasonic and mild thermal treatment against Pseudomonas fluorescens

A combined ultrasonic and thermal (US-TM) treatment was developed in this study to achieve a high efficacy of P. fluorescens biofilm control. The present study demonstrated that combined a moderate ultrasound treatment (power ≥ 80 W) and a mild heat (up to 50 °C) largely destroyed biofilm structure in 15 min and removed>65.63% of biofilm from a glass slide where cultivated the P. fluorescens biofilm. Meanwhile, the viable cell count was decreased from 10.72 to 6.48 log₁₀CUF/mL. Differences in biofilm removal and lethal modes of US-TM treatment were confirmed through microscopies analysis in vitro. The ultrasound first contributed to releasing the bacteria in the biofilm to the environment and simultaneously exposing inner bacteria at the deep layer of biofilm depending on shear force, shock waves, acoustic streaming, etc. When the biofilm structure was destroyed, US-TM treatment would synergistically inactivate P. fluorescens cells. In silico studies adopted COMSOL to simulate acoustic pressure and temperature distribution in the bioreactor; both of them were significantly influenced by various factors, such as input power, sonotrode position, materials and volume of container, etc. Facing the biofilm issue existing on the surface of container, boundary conditions were exported and thereby pointing out potential “dead ends” where the ultrasound may not be effectively transduced. Both in vitro and in silico results may inspire the food industry to adopt US-TM treatment to achieve biofilm control.     

Synergistic effect of ultrasonic pre-treatment combined with UV irradiation for secondary effluent disinfection

The ultraviolet (UV) disinfection efficiency is often affected by suspended solids (SS). Given their high concentration or large particle size, SS can scatter UV light and provide shielding for bacteria. Thus, ultrasound is often employed as a pre-treatment process to improve UV disinfection. This work investigated the synergistic effect of ultrasound combined with UV for secondary effluent disinfection. Bench-scale experiments were conducted in using samples obtained from secondary sedimentation tanks. These tanks belonged to three wastewater treatment plants in Beijing that use different kinds of biological treatment methods. Several parameters may contribute to the changes in the efficiency of ultrasound and UV disinfection. Thus, the frequency and energy density of ultrasound, as well as the SS, were investigated. Results demonstrated that samples which have relatively higher SS concentrations or higher percentages of larger particles have less disinfection efficiency using UV disinfection alone. However, the presence of ultrasound could improve the disinfection efficiency because it has synergistic effect. Changes in the particle size distribution and SS concentration notably affected the efficiency of UV disinfection. The efficiency of Escherichia coli elimination can be decreased by 1.2 log units as the SS concentration increases from 16.9 mg/l to 25.4 mg/l at a UV energy density of 40 mJ/cm². UV disinfection alone reduced the E. coli population by 3.4 log units. However, the synergistic disinfection of ultrasound and UV could reach 5.4 log units during the reduction of E. coli at a 40 kHz frequency and an energy density of 2.64 kJ/l. The additional synergistic effect is 1.1 log units.     

Ultrasound assisted modulation of yeast growth and inactivation kinetics

The yeast Saccharomyces cerevisiae is well known for its application in the food industry for the purpose of developing fermented food. The ultrasound (US) technology offer a wide range of applications for the food industry, including the enhancement of fermentation rates and inactivation of microbial cells. However, a better understanding and standardization of this technology is still required to ensure the scaling-up process. This study investigated the effect of the US technology on the growth of S. cerevisiae using frequencies of 20, 25, 45 and 130 kHz, treatment periods from 2 to 30 min. Furthermore, yeast kinetics subjected to US treatments were evaluated using modelling tools and scanning electron microscopy (SEM) analysis to explore the impact of sonication on yeast cells. Yeast growth was monitored after different US treatments plotting optical density (OD) at 660 nm for 24 h at 30 ⁰C. Growth curves were fitted using models of modified Gompertz and Scale-Free which showed good parameters of the fit. In particular, US frequencies of 45 and 130 kHz did not have a disruptive effect in lag phase and growth rate of the yeast populations, unlike the frequency of 20 kHz. Moreover, inactivation curves of yeast cells obtained after exposure to 20 and 25 kHz also observed the best fit using the Weibull model. US frequency of 20 kHz achieved significant reductions of 1.3 log cfu/mL in yeast concentration and also induced important cell damage on the external structures of S. cerevisiae. In conclusion, the present study demonstrated the significant effect of applying different US frequencies on the yeast growth for potential application in the food industry.     

Synergetic effects of ultrasound and slightly acidic electrolyzed water against Staphylococcus aureus evaluated by flow cytometry and electron microscopy

This study evaluated the synergetic effects of ultrasound and slightly acidic electrolyzed water (SAEW) on the inactivation of Staphylococcus aureus using flow cytometry and electron microscopy. The individual ultrasound treatment for 10 min only resulted in 0.36 log CFU/mL reductions of S. aureus, while the SAEW treatment alone for 10 min resulted in 3.06 log CFU/mL reductions. The log reductions caused by combined treatment were enhanced to 3.68 log CFU/mL, which were greater than the sum of individual treatments. This phenomenon was referred to as synergistic effects. FCM analysis distinguished live and dead cells as well as revealed dynamic changes in the physiological states of S. aureus after different treatments. The combined treatment greatly reduced the number of viable but nonculturable (VBNC) bacteria to 0.07%; in contrast, a single ultrasound treatment for 10 min induced the formation of VBNC cells to 45.75%. Scanning and transmission electron microscopy analysis revealed that greater damage to the appearance and ultrastructure of S. aureus were achieved after combined ultrasound-SAEW treatment compared to either treatment alone. These results indicated that combining ultrasound with SAEW is a promising sterilization technology with potential uses for environmental remediation and food preservation.     

Ultrasound assisted hydration of navy beans (Phaseolus vulgaris)

The use of ultrasound to enhance the transport phenomena in food processes has been well recognised in recent times. The objective of this study was to evaluate the effect of sonication on hydration rate and pasting profile of navy beans. The hydration kinetics for control and ultrasound assisted soaking was mathematically described using mechanistic (Fickian diffusion) and empirical (Peleg’s equation, Weibull model and First Order equation) models. Ultrasound enhanced the rate of hydration which was evident from the plot of kinetic data and model parameters. The effective diffusivities for water transport without and with ultrasound application were estimated to be 1.36 × 10⁻¹⁰ m²/s and 2.19 × 10⁻¹⁰ m²/s respectively, considering Fickian diffusion. The Weibull model was concluded to best predict the hydration kinetics of navy beans in an ultrasonic field. Significant increase in peak viscosity of sonicated bean powder was observed compared to control.     

Effects of ultrasound-assisted low-concentration chlorine washing on ready-to-eat winter jujube (Zizyphus jujuba Mill. cv. Dongzao): Cross-contamination prevention,decontamination efficacy,and fruit quality

Wash water is circulated for use in the minimal processing industry, and inefficient disinfection methods can lead to pathogen cross-contamination. Moreover, few disinfection strategies are available for ready-to-eat fruits that do not need to be cut. In this study, the use of chlorine and ultrasound, two low-cost disinfection methods, were evaluated to disinfect winter jujube, a delicious, nutritious, and widely sold fruit in China. Ultrasound treatment (28 kHz) alone could not decrease the cross-contamination incidence of Escherichia coli O157:H7, non-O157 E. coli, and Salmonella Typhimurium, and free chlorine treatment at 10 ppm decreased the incidence from 55.00% to 5.00% for E. coli O157:H7, 65.00% to 6.67% for non-157 E. coli, and 70.00% to 6.67% for S. Typhimurium. The cross-contamination incidence was completely reduced (pathogens were not detected in sample) when the treatments were combined. The counts of aerobic mesophiles, aerobic psychrophiles, molds, yeasts, and three pathogens in the group subjected to combination treatment (28 kHz ultrasound + 10 ppm free chlorine) were significantly lower than those in the control, chlorine-treated, and ultrasound-treated groups during storage (0–7 d at 4 °C). Analysis of weight loss, sensory quality (crispness, color, and flavor), instrument color (a*/b*), soluble matter contents (total soluble solids, reducing sugar, total soluble sugar, and titratable acid), and nutritional properties (ascorbic acid and polyphenolic contents) indicated that treatment with ultrasound, chlorine, and their combination did not lead to additional quality loss compared with properties of the control. Additionally, the activities of phenylalanine ammonia-lyase and polyphenol oxidase were not significantly increased in the treatment group, consistent with the quality analysis results. These findings provide insights into disinfection of uncut ready-to-eat fruits using a minimum dose of disinfectant for cross-contamination prevention under ultrasonication. The use of ultrasound alone to decontaminate fresh produce is accompanied by a high risk of pathogen contamination, and the use of sanitizers to decrease cross-contamination incidence is recommended.     

Synergism between ultrasonic pretreatment and white rot fungal enzymes on biodegradation of wheat chaff

Lignocellulosic biomass samples (wheat chaff) were pretreated by ultrasound (US) (40 kHz/0.5 W cm⁻²/10 min and 400 kHz/0.5 W cm⁻²/10 min applied sequentially) prior to digestion by enzyme extracts obtained from fermentation of the biomass with white rot fungi (Phanerochaete chrysosporium or Trametes sp.). The accessibility of the cellulosic components in wheat chaff was increased, as demonstrated by the increased concentration of sugars produced by exposure to the ultrasound treatment prior to enzyme addition. Pretreatment with ultrasound increased the concentration of lignin degradation products (guaiacol and syringol) obtained from wheat chaff after enzyme addition. In vitro digestibility of wheat chaff was also enhanced by the ultrasonics pretreatment in combination with treatment with enzyme extracts. Degradation was enhanced with the use of a mixture of the enzyme extracts compared to that for a single enzyme extract.     

Ultrasound-mediated drug delivery using liposomes modified with a thermosensitive polymer

Ultrasound-mediated drug delivery was established using liposomes that were modified with the thermosensitive polymer (TSP) poly(NIPMAM-co-NIPAM), which sensitized the liposomes to high temperatures. TSP-modified liposomes (TSP liposomes) released encapsulated calcein under 1 MHz ultrasound irradiation at 0.5 W/cm² for 120 s as well as the case under incubation at 42 °C for 15 min. In addition, uptake of the drug released from TSP liposomes by cancer cells was enhanced by ultrasound irradiation. In a cell injury assay using doxorubicin (DOX)-loaded TSP liposomes and ultrasound irradiation, cell viability of HepG2 cells at 6 h after ultrasound irradiation (1 MHz, 0.5 W/cm² for 30 s) with DOX-loaded TSP liposomes (TSP/lipid ratio = 1) was 60%, which was significantly lower than that of the control conditions such as DOX-loaded TSP liposomes alone and DOX-loaded intact liposomes under ultrasound irradiation.     

Influence of sodium alginate pretreated by ultrasound on papain properties: Activity,structure, conformation and molecular weight and distribution

The aim of the study was to investigate the impact of sodium alginate (ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and molecular weight and distribution of papain. ALG solutions were pretreated with ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45 W/cm²), 135 kHz, 50 °C for 20 min. The maximum relative activity of papain increased by 10.53% when mixed with ALG pretreated by ultrasound at 0.25 W/cm², compared with the untreated ALG. The influence of ultrasound pretreated ALG on the conformation and secondary structure of papain were assessed by fluorescence spectroscopy and circular dichroism spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG increased the number of tryptophan on papain surface, especially at 0.25 W/cm². It indicated that ultrasound pretreatment induced molecular unfolding, causing the exposure of more hydrophobic groups and regions from inside to the outside of the papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes in the secondary structure of the papain. The content of α-helix was slightly increased after ultrasound pretreatment and no significant change was observed at different ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the secondary structure of papain, especially at 0.25 W/cm². The free sulfhydryl (SH) content of papain was slightly increased and then decreased with the increase of ultrasonic power. The maximum content of free SH was observed at 0.25 W/cm², under which the content of the free SH increased by 6.36% compared with the untreated ALG. Dynamic light scattering showed that the effect of ultrasound treatment was mainly the homogenization of the ALG particles in the mixed dispersion. The gel permeation chromatography coupled with the multi-angle laser light scattering photometer analysis showed that the molecular weight (M_w) of papain/ALG was decreased and then increased with the ultrasonic pretreatment. Results demonstrated that the activity of immobilized papain improved by ultrasonic pretreatment was mainly caused by the variation of the conformation of papain and the effect of interactions between papain and ALG. This study is important to explain the intermolecular interactions of biopolymers and the mechanism of enzyme immobilization treated by ultrasound in improving the enzymatic activity. As expected, ALG pretreated by appropriate ultrasound is promising as a bioactive compound carrier in the field of immobilized enzyme.     

Kinetic modeling of inactivation of natural microbiota and Escherichia coli on cherry tomato treated with fixed multi-frequency sonication

The suitability of some non-linear kinetic models (Weibull {with or without tail}, Log-linear, Log-linear shoulder {with or without tail}, Biphasic linear, Logistic, Multi-target and Single-target models) were evaluated to determine the inactivation kinetics of inoculated E. coli, and natural microbiota (i.e. mesophilic aerobic bacteria, and mold and yeast) on cherry tomato treated with fixed multi-frequency ultrasound. Almost all the studied model fitted well (R² ≥ 0.9) for the inactivation kinetics; however, the Weibull, Log-linear shoulder, and Biphasic linear model showed the highest statistical parameters (0.9 ≤ adj. R² ≤ 0.99 and smallest RMSE and SSE values). All the three models could be used to compare the kinetic behavior of E. coli and the first two models for the kinetic behavior of mesophilic aerobic bacteria and mold and yeast during sonication treatment. Two distinctive inactivation curves were obtained for the mono-frequency and the multi-frequency (dual and tri-frequency) for all the microbial inactivation. The remarkable results obtained for dual and tri-frequency sonication shows to be an effective and promising alternative to the traditional microbial inactivation techniques and the common practice of using ultrasound with other sanitizing methods.     

Targeted and ultrasound-triggered drug delivery using liposomes co-modified with cancer cell-targeting aptamers and a thermosensitive polymer

In this study, we demonstrated the feasibility of targeted and ultrasound-triggered drug delivery using liposomes co-modified with single stranded DNA aptamers that recognized platelet-derived growth factor receptors (PDGFRs) as targeting ligands for breast cancer cells and poly(NIPMAM-co-NIPAM) as the thermosensitive polymer (TSP) to sensitize these liposomes to high temperature. TSP-modified liposomes (TSP liposomes) released encapsulated calcein under 1 MHz ultrasound irradiation for 30 s at 0.5 W/cm² as well as the case under incubation for 5 min at 42 °C. Ultrasound-triggered calcein release from TSP liposomes was due to an increased local temperature, resulting from cavitation bubble collapse induced by ultrasound, and not due to an increase in the bulk medium temperature. Liposomes modified with PDGFR aptamers (APT liposomes) bound to MDA-MB-231 human breast cancer cells through PDGFR aptamers; however, they did not bind to primary human mammary epithelial cells (HMECs). The binding of APT liposomes was greatest for MDA-MB-231 cells, followed by MCF-7, WiDr, and HepG2 cancer cells. In a cell injury assay using doxorubicin (DOX)-loaded APT/TSP liposomes and ultrasound irradiation, cell viability of MDA-MB-231 at 24 h after ultrasound irradiation (1 MHz for 30 s at 0.5 W/cm²) with DOX-loaded APT/TSP liposomes was 60%, which was lower than that with ultrasound irradiation and DOX-loaded TSP liposomes or with DOX-loaded APT/TSP liposomes alone.     

The tumor affinity of chlorin e6 and its sonodynamic effects on non-small cell lung cancer

ObjectiveSonodynamic therapy (SDT) is a promising new approach for cancer therapy. The aim of this study was to investigate the tumor affinity of chlorin e6, a photosensitizer, and its sonodynamic effects on NSCLC.MethodsHuman lung adenocarcinoma cells SPCA-1 and mice bearing SPCA-1 tumor xenograft were exposed to ultrasound in the presence or absence of chlorin e6. Chlorin e6 distribution was detected by laser scan confocal microscope. Cell apoptosis and necrosis were studied by flow cytometry analysis. Tumor size and weight were measured after different treatments.ResultsThe concentration of chlorin e6 in tumor tissue was remarkably higher than that in normal muscle near tumor, and the difference was greatest at 18 h (the fluorescence intensity was 5.38-fold higher in tumor than in muscle, P < 0.05). In vivo, ultrasound (0.4–1.6 W/cm²) or chlorin e6 (10–40 mg/kg) alone had no remarkable anti-tumor effects, but the combination of ultrasound (1.6 W/cm²) with chlorin e6 (SDT) hampered tumor growth significantly (P < 0.05). Intraperitoneal injection of 40 mg/kg chlorin e6 exerted no notable side effect on blood, liver and kidney function. Flow cytometry analysis showed that chlorin e6-mediated sonodynamic effect was mainly through the induction of cell necrosis.ConclusionChlorin e6 is a promising sonosensitizer and chlorin e6-mediated SDT may provide a new approach for NSCLC therapy.     

Ultrasound-assisted alkaline proteinase extraction enhances the yield of pecan protein and modifies its functional properties

To enhance the extraction yield of pecan protein and modify its functional properties, this study investigated whether both ultrasound and enzyme have a synergistic impact on the extraction of pecan (Carya illinoinensis (Wangenh.) K. Koch) protein. The highest protein extraction rate (25.51%) was obtained under the conditions of 1415.43 W^.cm⁻², 15 min, pH 10.0, 50 °C, and 1% (w/w) alkaline proteinase. Owing to its high shear, mechanical energy and cavitation, the ultrasound process increased the solubility of the substrate making it readily accessible to the enzyme, thereby accelerating the chemical reaction and improving the yield of the protein. The optimized ultrasound-assisted enzymatic method (400 W, 20 kHz, 5 s/3s) effectively changed the secondary and tertiary structure of the pecan protein. The results of surface hydrophobicity, intrinsic fluorescence spectra, sulfhydryl content and scanning electron microscopy all indicated the unfolding of protein and exposure of hydrophobic groups and sulfhydryl groups. Moreover, the protein obtained by this method showed higher solubility (70.77%), higher emulsifying activity (120.56 m²/g), smaller particle size (326.7 nm), and better dispersion (0.305) than single ultrasound and non-ultrasound methods (p < 0.05). To conclude, ultrasound-assisted enzymatic method could be an appropriate technique to improve the yield and quality of the pecan protein. The study also provides a theoretical basis for the application of pecan protein in food processing.