Sonication as a pre-processing step to alter acid whey generation during Greek yoghurt manufacture.

We investigated sonication as a pre-processing step to reduce acid whey generation during Greek yoghurt manufacture. The generation of a large amount of acid whey during the production of Greek yoghurt is an ongoing problem in the dairy industry and many studies are currently focusing on reducing it. We focused on the use of ultrasonication as a novel approach to minimize the casein fraction in the acid whey stream and simultaneously improve the gel properties. Ultrasound applied before the fermentation modified the structural properties and bonding behaviours of milk proteins, and enhanced the retention of casein in the yoghurt gel after the fermentation and straining steps. Therefore, the use of low-frequency ultrasonication as a pre-processing step may have the potential to provide significant economic benefits to the Greek yoghurt manufacturing process. Moreover, it improved the nutritional and physicochemical properties compared to regular Greek yoghurts.

Immunogenicity in Oreochromis niloticus vaccinated with sonicated antigens against streptococcosis.

Streptococcosis causes great economic losses in intensive culture of tilapia. Vaccination is the most effective and safest way to tackle infectious diseases. Thus, this study sought the more effective and safer antigenic fraction after sonication of Streptococcus agalactiae to elaborate a vaccine against streptococcosis in Nile tilapia. For this, twenty-one days after vaccination with different fractions (soluble and insoluble) of S. agalactiae, the fish were challenged with the homologous strain (LD50). Then, samples were taken at zero, 14, 28, 60 and 90 days post-vaccination (DPV, n = 7). Blood and organs (cranial kidney, spleen and liver) were collected from vaccinated and unvaccinated fish. Finally, insoluble fraction vaccine presented the best effect, resulting in a 100% relative percent of survival (RPS) and without clinical manifestations. In view of the results, it was to evaluate the role of the insoluble fraction of the antigen in the protective immunity against streptococcosis. The results indicate that the spleen might be the main organ in the vaccine response in Nile tilapia due to the great morphological and immunological differences in vaccinated fish, evidenced by the greater of melanomacrophage centers (MMC) and IgM + lymphocytes in relation to the non-vaccinated fish. At 60 DPV, it was observed the peak of the protective immunity related to the maximum concentration of proteins, circulating leukocytes, antibody titers in the serum and tissue changes with greater expression of IgM + and MMC number in the spleen and kidney of Oreochromis niloticus. Vaccination with insoluble fraction of S. agalactiae was safe and provided effective protection against streptococcosis with maximum protective response at 60 DPV.

High-speed centrifugation of extender of freeze-thaw boar semen.

In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Much has been developed in the search for alternatives for this biological product. This work aimed to evaluate the processed egg yolk through ultracentrifugation and/or sonication in the cryopreservation of swine semen. Twenty-seven semen doses were purchased from a commercial boar stud and processed for cryopreservation using egg yolk lactose 11% (control) extender, processed using two different methods: high-speed centrifugation and sonication. Then, they were submitted to freeze-thawing protocol and were assessed for kinematic and cell structural parameters. Samples in which extenders underwent centrifugation had better results in velocity parameters, meanwhile those that only sonication was performed had poorest results in this parameter. The preservation of the membrane and mitochondria structure had better results when the diluent was only centrifuged in comparison with the other treatments. Therefore, centrifugation of extender containing egg yolk is important for better cryopreservation of swine semen.

Characterization of the physical, microbiological, and chemical properties of sonicated raw bovine milk.

Innovative processing technologies, such as ultrasonication, can change the properties of milk, allowing for the improvement or development of dairy foods. Yet taking bench-scale equipment to pilot plant scale has been challenging. Raw milk, standardized to 3% fat and warmed to inlet temperatures of 42 or 54°C, was exposed to continuous, high-intensity, low-frequency ultrasonication (16/20 kHz, 1.36 kW/pass) at flow rates of 0.15, 0.30, and 0.45 L/min that resulted in resident times within the reaction cell of 6, 3, and 2 min per pass, respectively. Multiple passes (3, 5, and 7, respectively) were required to obtain a total exposure time of 14 to 18 min. Evaluation of fat droplet sizes, enzyme coagulation properties, and microstructure of milk and milk gels, as well as determining compositional and lipid properties, were conducted to determine the potential of the ultrasound system to effectively modify milk. Laser scanning particle sizing and confocal microscopy showed that the largest droplets (2.26 ± 0.13 µm) found in raw milk were selectively reduced in size with a concomitant increase in the number of submicron droplets (0.37 ± 0.06 µm), which occurred sooner when exposed to shorter bursts of ultrasonication (0.45 L/min flow rates) and at an inlet temperature of 54°C. Ultrasound processing with milk entering at 42°C resulted in faster gelling times and firmer curds at 30 min; however, extended processing at inlet temperature of 54°C reduced curd firmness and lengthened coagulation time. This showed that ultrasonication altered protein-protein and protein-lipid interactions, thus the strength of the enzyme-set curds. Scanning electron microscopy revealed a denser curd matrix with less continuous and more irregular shaped and clustered strands, whereas transmission electron microscopy showed submicron lipid droplets embedded within the protein strands of the curd matrix. Processing at inlet temperature of 54°C with flow rates of 0.30 and 0.45 L/min also reduced the total aerobic bacterial count by more than 1 log cfu/mL, and the number of psychrophiles below the limit of detection (10 cfu/mL) for this study. Ultrasonication exposures of 14 to 18 min had minimal effect on the milk composition, fatty acid profiles, and lipid heat capacity and enthalpy. The findings show that this continuous ultrasound system, which is conducive to commercial scale-up, modifies the physical and functional properties of milk under the parameters used in this study and has potential use in dairy processing.

Pretreatment with Ultrasonication Reduces the Size of Azelaic Acid-Chitosan Nanoparticles Prepared by Electrospray.

Nowadays, electrospray is becoming a favourable approach for preparing monodispersed nanoparticles. However, this approach is quite recent and requires further works to optimize control over physicochemical properties of its products. This study aimed to determine the possible effects of sonication as a pretreatment to reduce the size of azelaic acid-chitosan particle before using electrospray. The results showed that sonication treatment can produce submicron particles of azelaic acid-chitosan. By diluting the solution and increasing sonication time and amplitude, smaller particles were obtained with the smallest one at 516 nm, sized by dynamic light scattering. The pretreated solution was then electrosprayed to reduce the size of nanoparticles to 80 nm, indicating that sonication may play an important role in reducing the size of electrosprayed nanoparticles. The electrosprayed nanoparticles were nearly monodispersed and almost spherical in shape.

Bacterial recovery using sonication versus swabbing of titanium and stainless steel implants inoculated with Staphylococcus pseudintermedius or Pseudomonas aeruginosa.

OBJECTIVE: To evaluate the use of sonication to improve recovery of bacteria from metal discs infected with bacteria commonly associated with implant infections in veterinary medicine. METHODS: In vitro study in which sterile titanium (Ti6Al4V) and stainless steel (AIS1316-L) discs were incubated with either Staphylococcus pseudintermedius or Pseudomonas aeruginosa for 24 hours. The following three groups were compared: 1) the sonication group involved immersing the discs in sterile saline and sonicating for five minutes; 2) the sham group was considered a negative control in which the discs were immersed in saline for five minutes without sonication; and 3) the swab group involved systematically swabbing the implant with a sterile culturette. All samples were plated on blood agar and incubated for 24 hours. Colonies were then counted and compared. RESULTS: For both species of bacteria, there was a significant increase in bacterial colonies isolated using sonication compared to the other two study groups (p = 0.0001). No differences in bacterial growth were found between the two types of metal implants. There was a significant increase in bacterial colony counts for S. pseudintermedius when comparing the swab group versus the sham group, but this was not significant for P. aeruginosa. CLINICAL SIGNIFICANCE: Sonication significantly improves recovery of bacteria commonly associated with veterinary implant-associated surgical site infections compared to swabbing of implants in vitro. A prospective clinical evaluation is indicated to determine the in vivo efficacy of sonication in veterinary patients.

Enumeration of clostridia in goat milk using an optimized membrane filtration technique.

A membrane filtration technique developed for counting butyric acid bacteria in cow milk was further developed for analysis of goat milk. Reduction of the sample volume, prolongation of incubation time after addition of proteolytic enzyme and detergent, and a novel step of ultrasonic treatment during incubation allowed filtration of goat milk even in the case of somatic cell counts (SCC) exceeding 10(6)/mL. However, filterability was impaired in milk from goats in late lactation. In total, spore counts were assessed in 329 farm bulk goat milk samples. Membrane filtration technique counts were lower than numbers revealed by the classic most probable number technique. Thus, method-specific thresholds for milk to evaluate the risk of late blowing have to be set. As expected, the spore counts of milk samples from suppliers not feeding silage were significantly lower than the spore counts of milk samples from suppliers using silage feeds. Not only were counts different, the clostridial spore population also varied significantly. By using 16S rRNA gene PCR and gene sequencing, 342 strains from 15 clostridial species were identified. The most common Clostridium species were Clostridium tyrobutyricum (40.4%), Clostridium sporogenes (38.3%), Clostridium bifermentans (7.6%), and Clostridium perfringens (5.3%). The 2 most frequently occurring species C. tyrobutyricum and C. sporogenes accounted for 84.7% of the isolates derived from samples of suppliers feeding silage (n=288). In contrast, in samples from suppliers without silage feeding (n=55), these species were detected in only 45.5% of the isolates.

Evaluation of high-intensity ultrasonication for the inactivation of endospores of 3 bacillus species in nonfat milk.

Endospores of Bacillus licheniformis [American Type Culture Collection (ATCC) 6634], Bacillus coagulans (ATCC 12245), and Geobacillus stearothermophilus (ATCC 15952) were spiked in sterile nonfat milk, and subjected to high intensity batch ultrasonication treatment at different amplitudes (80 or 100%) and durations (1 to 10 min). Increasing the amplitude from 80 to 100% did not result in enhanced inactivation of G. stearothermophilus endospores. However, an increase in the duration of ultrasonication from 1 to 10 min significantly increased the inactivation of endospores of all 3 species. About 48.96% of the G. stearothermophilus endospores were inactivated by ultrasonication alone, whereas ultrasonication and pasteurization combined increased the inactivation to 65.74%. Inactivation of endospores could be further enhanced to 75.32% by ultrasonication and higher heat (80 °C/1 min) combination. Endospores of B. licheniformis and B. coagulans were inactivated to a lesser extent compared with G. stearothermophilus spores. Ultrasonicated B. licheniformis endospores germinated in higher numbers when compared with untreated endospores resulting in their greater inactivation during the combined treatment. During microstructure imaging of ultrasonicated endospores, although no structural damage was noticed, they showed irregular shrinkage and wrinkles with surface coarseness. This may also have contributed to their reduced thermal resistance, in addition to sporulation.

Ultrasonicated Enterococcus faecium SF68 enhances neutrophil free radical production and udder innate immunity of drying-off dairy cows.

Proper dry cow management is critical not only for subsequent milk production and fertility but also for mastitis control. A phenomenon of immunosuppression was commonly observed in transition cows, an example being the high susceptibility of the mammary gland during early the dry period to new infectious agents. Polymorphonuclear neutrophils (PMN) play important defence roles in the mammary gland of newly dried cows. One of the bactericidal mechanisms of PMN is through producing reactive oxygen species (ROS), which can be efficiently quantified by chemiluminescence (CL) assay. In the current study, the potential of intramammary application of a commercial Enterococcus faecium SF68 (SF68) product to enhance the local innate immunity of newly dried mammary glands was evaluated based on the CL assay. The preliminary experiments in vitro indicated virtual dose-responsiveness of ROS generation from three different cell preparations, bovine blood PMN, bovine blood PMN pre-conditioned with cow milk, and the post-diapedesis model somatic cells from cow milk, on their exposure to phorbol 12-myristate 13-acetate (PMA), viable SF68, and ultrasonicated SF68, but not dry-heated SF68. Because ultrasonication treatment was found to profoundly enhance the immunogenicity of SF68 in vitro, in the following animal trial, single infusion of either 5 or 10×107 original cfu of ultrasonicated SF68 was randomly applied to the front quarters and phosphate-bufferedsaline (PBS) applied to the rear quarters of each of the four experimental cows on the first day of milk stasis. The results showed that within the first post-infusion week, ultrasonicated SF68 induced a faster and greater (P<0·05) recruitment of PMN into mammary lumen with no apparent local or systemic inflammatory sign. Meanwhile, ultrasonicated SF68 also induced a greater (P<0·05) ROS production in response to PMA challenge by in situ somatic cells of mammary secretion. Taken together, ultrasonicated SF68 modulated ROS generation of bovine neutrophils, and would be a potential enhancer of udder innate immunity in drying-off dairy cows. More thorough work is warranted.

Pre-treatment of sperm reduces success of ICSI in the pig.

In pigs, although ICSI is a feasible fertilization technique, its efficiency is low. In general, injected pig sperm are insufficient to induce oocyte activation and embryonic development. Pretreatments for disrupting sperm membranes have been applied to improve the fertility of ICSI oocytes; however, we hypothesize that such pretreatment(s) may reduce the ability of the sperm to induce oocyte activation. We first evaluated the effects of sperm pretreatments (sonication (SO) to isolate the sperm heads from the tails, Triton X-100 (TX), and three cycles of repeated freezing/thawing (3×-FT) for disrupting sperm membranes) on the rate of pronucleus (PN) formation after ICSI. We found that oocytes injected with control (whole) sperm had higher rates of PN formation than those obtained after subjecting the sperm to SO, TX, and 3×-FT. The amounts of phospholipase Cζ (PLCζ), which is thought to be the oocyte-activating factor in mammalian sperm, in sperm treated by each method was significantly lower than that in whole untreated sperm. Furthermore, using immunofluorescence, it was found that in pig sperm, PLCζ was localized to both the post-acrosomal region and the tail area. Thus we demonstrated for the first time that sperm pretreatment leads to a reduction of oocyte-activating capacity. Our data also show that in addition to its expected localization to the sperm head, PLCζ is also localized in the tail of pig sperm, thus raising the possibility that injection of whole sperm may be required to attain successful activation in pigs.

A unified approach to combine temperature estimation and elastography for thermal lesion determination in focused ultrasound thermal therapy.

Sonogram-based temperature estimation and elastography have both shown promise as methods of monitoring focused ultrasound (FUS) treatments to induce thermal ablation in tissue. However, each method has important limitations. Temperature estimates based on echo delays become invalid when the relationship between sound speed and temperature is nonlinear, and are further complicated by thermal expansion and other changes in tissue. Elastography can track thermal lesion formation over a wider range of elasticity, but with low specificity and high noise. Furthermore, this method is poor at small lesion detection. This study proposes integrating the two estimates to improve the quality of monitoring FUS-induced thermal lesions. Our unified computational kernel is tested on three types of phantoms. Experiments with type I and type II phantoms were conducted to calibrate the thermal mapping and elastography methods, respectively. The optimal settings were then used in experiments with the type III phantom, which contains ex vivo swine liver tissue. Three different spatial-peak temporal-average intensities (I(spta); 35, 133 and 240 W cm(-2)) were delivered with a sonication time of 60 s. The new procedure can closely monitor heating while identifying the dimensions of the thermal lesion, and is significantly better at the latter task than either approach alone. This work may help improve the current clinical practice, which employs sonograms to guide the FUS-induced thermal ablation procedure.