The extracts utilized to determine the phenolic compounds, flavonoids content (determined only in pig feed and not in meat samples), as well as the antioxidant assays were carried out following the method proposed by Santos et al. [35 (link)], with slight modifications. Succinctly, three grams of previously chopped and thawed sample were disrupted for 60 s using an IKA T25 digital Ultra-Turrax (IKA®-Werke GmbH & Co. KG, Staufen, Germany) in 20 mL of CH3OH: H2O (80:20, v/v). Then, homogenate was stirred at 50 rpm on a rocking shaker (SW-3D-E, OVAN, Barcelona, Spain) for 15 min, at room temperature. After stirring, samples were placed in an ultrasound water bath apparatus (Bransonic® 8510E-DTH, Danbury, CT, USA) for 15 min at 25 °C. Subsequently, they were centrifuged at 14,000× g and 4 °C during 15 min, and supernatant was separated and filtered through 0.45 µm pore filter (Acrodisc® LC PVDF syringe filter; Pall Gelman Laboratory, Montreal, QC, Canada). The methanolic extracts were stored protected from direct exposition to light at −80 °C prior to their corresponding analysis.
T25 digital ultra turrax
Manufactured by IKA Group
Sourced in Germany, United States
The T25 digital Ultra-Turrax is a high-speed disperser designed for homogenizing, emulsifying, and dispersing a wide range of materials in laboratory settings. It features a digital speed control and display, allowing for precise adjustment and monitoring of the rotational speed during operation.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Vibra cell sonicator, by Sonics (2 mentions)
Pal salt, by Atago (1 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions)
Bm chemiluminescence western blotting kit mouse rabbit, by Roche (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Taqman reverse transcription reagent kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Inlab solids, by Mettler Toledo (1 mentions)
Avanti j e centrifuge, by Beckman Coulter (1 mentions)
Whatman no 1 filter paper, by Cytiva (1 mentions)
Sensifast sybr lo rox kit, by Meridian Bioscience (1 mentions)
Whatman no 1 filter paper, by GE Healthcare (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Gel doc ez system, by Bio-Rad (1 mentions)
Seven2go, by Mettler Toledo (1 mentions)
Continent 512r, by Hanil (1 mentions)
Alphasoft, by Alpha MOS (1 mentions)
29 protocols using t25 digital ultra turrax
1
Extraction and Analysis of Phenolic Compounds and Antioxidants
2
Fabrication of Drug-Loaded Lipid-Based Nanoparticles
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The RZ-NLCs were created using the HPH (high-pressure homogenization) method. Briefly, we added RZ to a mixture of Precirol® ATO 5, stearyl amine, oleic acid, and Phospholipon® 90H that had been heated to a temperature of 10 °C over the soft argument of each lipid. Aqueous surfactant solutions (conductivity 0.058 S/cm) were obtained from triple-distilled water. The lipid mixture and the heated aqueous surfactant solution were blended using a “high-shear homogenizer” (IKA® T-25 digital Ultra-Turrax®, IKA-Werke, Staufen im Breisgau, Germany) at a speed of 13,000 rpm. The lipid liquid was heated to the same level as the surfactant solution in water. The RZ-NLCs were made by creating a pre-emulsion and then homogenizing the mixture under high pressure. The drug-loaded NLCs were cryoprotected with mannitol before being freeze-dried at −80 °C [27 (link)]. The lyophilization process took approximately an hour and thirty minutes at room temperature (25 °C). In contrast, the secondary drying took four hours at zero degrees Celsius. A final moisture content of around 0.2% was achieved via lyophilization at 1 Pa pressure and a −70 °C cold trap temperature.
3
Spectrophotometric Assay of Peroxidase Activity
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POD activity was assayed using the spectrophotometric method described by Tappi, et al. [25 (link)]. Hence, 5 g of sample were mixed for 2 min with 50 mL of 0.1 M potassium phosphate buffer (pH 6.5) with a T25 digital Ultraturrax (IKA®-Werke GmbH & Co. KG, Staufen, Germany). After filtration, the solution was centrifuged for 10 min at 4 °C and 10,000× g, and the supernatant was collected and considered as enzymatic extract.
A solution containing 99.8 mL of 0.1 M potassium phosphate buffer (pH 6.5), 0.1 mL of 99.5% guaiacol and 0.1 mL of 30% hydrogen peroxide was considered as POD substrate. Enzymatic activity was assessed mixing 150 μL of enzymatic extract to 3 mL of substrate solution in 10-mm pathlength glass cuvettes followed by monitoring the increase in absorbance at 470 nm at 25 °C for 3 min.
Results were expressed as residual enzymatic activity (%) considered as the ratio between PAW treated sample versus untreated one and measured on three independent extracts.
A solution containing 99.8 mL of 0.1 M potassium phosphate buffer (pH 6.5), 0.1 mL of 99.5% guaiacol and 0.1 mL of 30% hydrogen peroxide was considered as POD substrate. Enzymatic activity was assessed mixing 150 μL of enzymatic extract to 3 mL of substrate solution in 10-mm pathlength glass cuvettes followed by monitoring the increase in absorbance at 470 nm at 25 °C for 3 min.
Results were expressed as residual enzymatic activity (%) considered as the ratio between PAW treated sample versus untreated one and measured on three independent extracts.
4
Nanoemulsion Preparation by Phase Inversion
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Nanoemulsions (NEs) were prepared by emulsion phase inversion technique coupled with high stirring energy input as previously described [4 (link)]. Briefly, NEs composed of an oil core of medium chain triglycerides (MCT) were stabilized by a shell of surfactants, made of a mixture of polyoxyethylene (40) stearate (Myrj®52) and oleoyl polyoxyl-6 glycerides (Labrafil®M1944CS), hydrophilic and hydrophobic surfactants, respectively. To prepare the oil phase, the MCT oil core and surfactants were homogenized under magnetic stirring (750 rpm) using a thermostated bath at 80 °C. Then, the aqueous phase (PBS 5 mM pH 7.4), heated up to 80 °C, was added into the organic melt phase. The process of high stirring was then performed in two cycles of 10 min using a rotor–stator disperser (T25 digital Ultra-Turrax® equipped with an S25N10G shaft, IKA®-Werke GmbH & Co. KG, Staufen, Germany) rotating at 11,000 rpm at 80 °C. The resulting colloidal system was cooled to room temperature under magnetic stirring for 30 min. CCM was added to the oil phase during NEs preparation. Nanoemulsions were prepared with a lipid concentration in the final phase of 142.86 w/v with 1.33% (w/w) of curcumin.
All studies and measurements presented in this work were performed in triplicate.
All studies and measurements presented in this work were performed in triplicate.
5
Quantitative Analysis of Berries by GC-FID
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Fifteen grams of frozen
berries was crushed with a T25 digital Ultra-Turrax (IKA Werke GmbH
& Co. KG, Staufen im Breisgau, Germany) and extracted with 10
mL of Milli-Q water at room temperature. The extraction was assisted
with ultrasonication (15 min) and centrifugation (4420g for 15 min). After the supernatant was collected, the residue was
extracted with the same procedure three times. The supernatants from
the four times of extraction were combined and diluted with Milli-Q
water to a final volume of 50 mL. Sugars and simple organic acids
in the samples were analyzed as trimethylsilyl (TMS) derivatives by
Shimadzu GC-2010 equipped with a flame ionization detector (FID) (Shimadzu
Corp., Kyoto, Japan). The compounds were identified on the basis of
the retention time of reference standards. A mixed internal standard,
consisting of sorbitol (for sugars) and tartaric acid (for acids),
was used for quantification. The methods for preparation of samples
and standards, as well as gas chromatographic conditions, were the
same as described in the previous research.12 (link)
berries was crushed with a T25 digital Ultra-Turrax (IKA Werke GmbH
& Co. KG, Staufen im Breisgau, Germany) and extracted with 10
mL of Milli-Q water at room temperature. The extraction was assisted
with ultrasonication (15 min) and centrifugation (4420g for 15 min). After the supernatant was collected, the residue was
extracted with the same procedure three times. The supernatants from
the four times of extraction were combined and diluted with Milli-Q
water to a final volume of 50 mL. Sugars and simple organic acids
in the samples were analyzed as trimethylsilyl (TMS) derivatives by
Shimadzu GC-2010 equipped with a flame ionization detector (FID) (Shimadzu
Corp., Kyoto, Japan). The compounds were identified on the basis of
the retention time of reference standards. A mixed internal standard,
consisting of sorbitol (for sugars) and tartaric acid (for acids),
was used for quantification. The methods for preparation of samples
and standards, as well as gas chromatographic conditions, were the
same as described in the previous research.12 (link)
6
Microfluidization of Ivermectin Nanoparticles
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In accordance with the experimental design, IVM-NC were prepared using a top-down approach via the microfluidization technique (Rawas-Qalaji et al., 2015 (link); Verma et al., 2009 (link)). Increasing amounts of IVM (ranging from 0.0125 mg to 0.05 mg) and Food and Drug Administration (FDA) approved, biocompatible stabilizers such as Tween® 80, PVA, and SDS were added to 50 mL of purified water, to result in a drug concentration range of 0.025% to 0.1% (w/v) and a stabilizer-to-drug ratio of 1 to 4. The resulting mixture was then processed in a microfluidizer (M-110P V3, Microfluidics Corporation, USA) under a pressure of 30,000 psi for 1 to 5 cycles. In instances where homogenization was necessary as per the experimental design, the mixture was homogenized prior to microfluidization at 10,000 rpm for 10 min using a high-speed homogenizer (T-25 digital Ultra Turrax, IKA-Werke GmbH & Co., Germany).
7
Moisture and Salt Content Determination
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The determination of moisture content was based on the AOAC method [15 ]. One gram of the crushed sample was homogenized with 9 mL of distilled water in a disperser (T 25 digital ULTRA-TURRAX, IKA-Werke GmbH & Co., KG., Staufen, Germany), and the upper filtrate was collected. The salt content of the filtrate was measured with a hand-held salinometer (PAL-SALT, Atago Co., Ltd., Tokyo, Japan). The weights of the samples before (W0) and after (W1) salting were measured, and the yields were calculated with Equation (1):
8
Emulsion Stabilization with SPAH and XG
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All emulsions prepared in this study contained 30% of oil phase (w/w) and various concentrations of SPAH and XG. Firstly, emulsions containing only SPAH, i.e., emulsions without XG, were prepared. These emulsions had the following concentrations of SPAH: 3, 5, or 7% (w/w) with respect to the total mass of the emulsion. Secondly, emulsions containing both SPAH and XG were prepared. The obtained emulsions had 3 or 5% of SPAH (w/w) with respect to the total mass of the emulsion and the following concentrations of XG in the continuous phase: 0.1, 0.2, 0.3, 0.4, or 0.5% (w/w). Sodium azide was used as a preservative in all emulsions. The concentration of salt in the aqueous phase of all emulsions was set to 0.01%. A rotor-stator homogenizer (T25 digital ULTRA-TURRAX, IKA®-Werke GmbH & Co., Staufen, Germany) was used for the preparation of all investigated emulsions. In order to prepare the disperse systems, the homogenizer operated at 15,000 rpm for 20 min in a water bath with a constant temperature of 25 °C.
9
Brassica carinata Sprout Preparation
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For the human pilot intervention study, seeds of Brassica carinata (provided by the World Vegetable Center (AVRDC)) were sown on a wet fleece, and put in a perlite/water mixture filled into an aluminum tray. Sprouts were grown in a greenhouse for 8 days and watered when needed. Sprouts (27 g) were harvested and mixed with 27 mL of water and homogenized using a T25 digital ULTRA-TURRAX® (IKA®-Werke GmbH & CO. KG, Staufen, Germany) leading to a beverage with a smoothie-like consistence. After 30 min of incubation, three replicates of 500 mg samples were analyzed for their glucosinolate hydrolysis product composition and content and the remaining beverage was immediately used for the intervention.
10
Preparation of MXF-Loaded PLGA Microspheres
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MXF-loaded PLGA microspheres (MXF-PLGA-MSs) were prepared using solvent vaporization method [32 (link)]. MXF and PLGA polymer were dissolved in an organic solvent composing of mixture of dichloromethane and methanol (1:1 ratio). Briefly, 20 mg of MXF and 20 mg of PLGA were dissolved in 1mL of organic internal solvent. The organic phase was then slowly dispersed in 4 mL of an aqueous phase containing 2% poly vinyl alcohol with continuous stirring. The resultant emulsion was homogenized for 2 h at a rotational speed of 3500 rpm using high shear homogenizer (T 25 digital ULTRA-TURRAX®, IKA®-Werke GmbH, Humburg, Germany). Further vaporization of organic solvent was assisted by mechanical stirring at 3500 rpm overnight. The resultant microspheres were collected by centrifugation at 3000 rpm for 30 min and purified by washing with double distilled sterile water for 3 times. The obtained microspheres were freeze dried until subsequent use.
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