Initially, frozen LM were freeze-dried (FreeZone 6, Labconco, Kansas City, USA) at 0.2 mbar for 72 h. Afterwards, wings and legs were manually removed and LM were ground into coarse meal using a blender (C-Series 5200, Vitamix, Cleveland, USA). For fat extraction, n-hexane was added at a ratio of 1:5 w/v and continuously stirred for 27 h at room temperature. After 1 h of settling, the fat-containing hexane supernatant was separated via decantation and the solid residue was dried in a vacuum dryer (VD 23, Binder, Tuttlingen, Germany) at 35 °C and 200 mbar for 72 h. For protein extraction, the dried and defatted LM coarse meal was mixed with deionized water (1:2 w/v) and the pH was set to 9.0 by adding 4 M or 1 M NaOH for rough and fine adjustment, respectively. After 2 h of stirring, the LM slurry was filtered through a cheese-filtering cloth (pore size 0.2–0.5 mm, 100% cotton) for chitin separation. Finally, the filtrate was freeze-dried, ground in a mortar grinder and stored at 3 °C until further use.
Vd 23
Manufactured by Binder
Sourced in Germany
The VD 23 is a versatile laboratory equipment designed for a range of applications. It functions as a vacuum desiccator, providing a controlled environment for the storage, preservation, and drying of various materials and samples. The VD 23 utilizes a vacuum system to remove air and maintain a reduced pressure environment.
Automatically generated - may contain errors
Lab products found in correlation
Whatman, by Cytiva (3 mentions)
Freezone 6, by Labconco (1 mentions)
Lambda 35, by PerkinElmer (1 mentions)
N hydroxysuccinimide nhs, by Merck Group (1 mentions)
6770 freezer mill, by SPEX (1 mentions)
Freezone triad, by Labconco (1 mentions)
Whatman no 1 filter paper, by Cytiva (1 mentions)
No 1 filter paper, by Cytiva (1 mentions)
Basic hei vap ml, by Heidolph (1 mentions)
8 protocols using vd 23
1
Extraction and Purification of Locust Meal
2
DESIR Technique for Spiked Sample Extraction and Analysis
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Spiked samples
(10 g) were extracted using 10 mL of acetone solvent
(analytical grade, Merck, DE) in a glass vial. Then, each sample was
shaken for 1 min using a vortex mixer (model 232, Fisher Scientific,
UK), kept at 25 ± 2 °C for 10 min, and passed through a
filter paper (model no. 1, Whatman, UK). The DESIR technique was performed
by placing a glass microfiber filter paper (model GF/A, Whatman, UK)
on a 55 mm diameter glass Petri dish. The embedded 2 mL of extract
sample in the GF/A filter paper was then evaporated in a vacuum-drying
oven (model VD 23, BINDER GmbH, DE) at 35 ± 2 °C for 60
min. The DESIR samples were kept in a desiccator at 25 ± 2 °C
for 30 min before the NIR measurement.17 (link),21 (link) The FT-NIR
measurements and spectra collection were performed according to the
method described inSection 4.1.2 . above.
(10 g) were extracted using 10 mL of acetone solvent
(analytical grade, Merck, DE) in a glass vial. Then, each sample was
shaken for 1 min using a vortex mixer (model 232, Fisher Scientific,
UK), kept at 25 ± 2 °C for 10 min, and passed through a
filter paper (model no. 1, Whatman, UK). The DESIR technique was performed
by placing a glass microfiber filter paper (model GF/A, Whatman, UK)
on a 55 mm diameter glass Petri dish. The embedded 2 mL of extract
sample in the GF/A filter paper was then evaporated in a vacuum-drying
oven (model VD 23, BINDER GmbH, DE) at 35 ± 2 °C for 60
min. The DESIR samples were kept in a desiccator at 25 ± 2 °C
for 30 min before the NIR measurement.17 (link),21 (link) The FT-NIR
measurements and spectra collection were performed according to the
method described in
3
Preparation and Characterization of Interpolymer Complexes
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EPO and S100 solutions were prepared by dissolving the copolymers in ethanol, tetrahydrofuran and isopropanol–acetone mixture (60:40, % v/v). These solutions were mixed at a constant temperature (25 °C) at different molar ratios (Table 1 ). Formation of the IPC occurred immediately upon mixing these solutions, which initially resulted in cloudy colloidal solution, followed by aggregation of primary polycomplex particles and their eventual precipitation. The IPC samples were prepared in a laboratory reactor system LR 1000 control under continuous and simultaneous agitation at 1000 rpm during 2 h using Eurostar 60 control overhead stirrer (IKA® Werke GmbH, Staufen, Germany). After isolation of the precipitates of IPC particles from solutions by centrifugation (10 min, 3000 rpm, centrifuge ELMI, Latvia), they were washed three times with ultrapure water (Smart2Pure UV/UF, Thermo Scientific, Waltham, MA, USA.), and subsequently vacuum-dried (vacuum oven VD 23, Binder, Germany) for 2 days at 40 °C until constant weight. The solid samples were ground with a grinder, ball milled (IKA® Ultra Turrax® Tube Drive P control Workstation, 10 min, 2000 rpm) and stored in tightly sealed containers at room temperature.
4
Vacuum Dried Fruit Powder
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The fruit puree was spread on a baking tray with an average thickness of 1.5 cm. The drying process was conducted in a vacuum oven (VD 23; Binder, Germany), with the temperature and pressure set at 70°C and 1 bar, respectively. After 24 h, the dried flakes were ground into powder (Gopinatha et al., 2020) .
5
ACE Inhibitory Activity Assay Protocol
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The ACE-inhibitory activity analysis was performed according to Sun et al. (2011) with some modification.
Hippuryl-Histidyl-Leucine (HHL) was dissolved in 50 mM sodium borate buffer (pH8.3) containing 0.5 M NaCl to achieve a final concentration of 8.3 mM. A reaction mixture containing 150 μL of HHL and 50 μL of sample (1 mg/mL) was pre-incubated at 37°C for 5 min. Subsequently, 50 μL ACE (25 mU/mL) was added to initiate the reaction and followed by incubation at 37°C for 60 min. The reaction was terminated by the addition of 250 μL of 1 N HCl. The released hippuric acid was extracted by the addition of 1.5 mL ethyl acetate followed by centrifugation at 2000 × g at 4°C for 10 min. The upper layer (1 mL) was transferred to a new test tube and evaporated at 40°C in a vacuum oven (Binder GmbH, VD 23, Germany). Then, 1 mL of deionised water was added to the test tube and the absorbance of the sample was measured at 228 nm using a UV-Vis spectrophotometer (Perkin Elmer, Lambda 35, USA). The ACE inhibition activity was calculated using the following equation: Inhibition activity (%) = [(A-B) / (A-C)] × 100%, where A is the absorbance of control (sample replaced with deionised water), B is the absorbance of the reaction mixture, C is the absorbance when the stop reaction solution was added before the reaction occurred (blank).
Hippuryl-Histidyl-Leucine (HHL) was dissolved in 50 mM sodium borate buffer (pH8.3) containing 0.5 M NaCl to achieve a final concentration of 8.3 mM. A reaction mixture containing 150 μL of HHL and 50 μL of sample (1 mg/mL) was pre-incubated at 37°C for 5 min. Subsequently, 50 μL ACE (25 mU/mL) was added to initiate the reaction and followed by incubation at 37°C for 60 min. The reaction was terminated by the addition of 250 μL of 1 N HCl. The released hippuric acid was extracted by the addition of 1.5 mL ethyl acetate followed by centrifugation at 2000 × g at 4°C for 10 min. The upper layer (1 mL) was transferred to a new test tube and evaporated at 40°C in a vacuum oven (Binder GmbH, VD 23, Germany). Then, 1 mL of deionised water was added to the test tube and the absorbance of the sample was measured at 228 nm using a UV-Vis spectrophotometer (Perkin Elmer, Lambda 35, USA). The ACE inhibition activity was calculated using the following equation: Inhibition activity (%) = [(A-B) / (A-C)] × 100%, where A is the absorbance of control (sample replaced with deionised water), B is the absorbance of the reaction mixture, C is the absorbance when the stop reaction solution was added before the reaction occurred (blank).
6
Fabrication of Cartilage-Derived ECM Scaffolds
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Cartilage-derived ECM scaffolds were fabricated in aseptic conditions as previously reported 6b . First, cartilage (native and engineered) was cut with a sharp blade, each group separately. Cartilage slurry was created by pulverizing cartilage using a cryogenic mill (6770 Freezer/Mill, SPEX, UK). Pulverized cartilage was then homogenized in UPW by a homogenizer (IKAT10, IKA Works, USA) to generate a fine slurry. Scaffolds were fabricated using a previously used know slurry concentration (250 mg ml -1 ) 6b . Cartilage slurry was poured into moulds (60 μl) and lyophilized (FreeZone Triad, Labconco) 6b, 6c . The scaffolds were crosslinked by dehydrothermal (DHT) and 1-Ethyl-3-3-dimethyl aminopropyl carbodiimide (EDAC) 6b, 13 . DHT method was carried out in an oven (VD23, Binder, Germany), at 115˚C and 2 mbar (24 hours). EDAC (Sigma-Aldrich) crosslinking was performed (2 hours; 6 mM) with N-Hydroxysuccinimide (NHS; Sigma-Aldrich). It was used a molar ratio of 2.5 M EDAC/M N-Hydroxysuccinimide 13b . Identical process of fabrication of the scaffolds was performed for the three different types of ECM: derived from porcine ECM (Native), from human engineered sheets (Eng) and from human engineered sheets with microspheres (Eng-MS) (Figure 1). human infrapatellar fat pad-derived stem cells.
7
Extraction and Analysis of Miang Polyphenols
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Miang samples were dried at 50°C for 24 h in a vacuum drier (Binder VD 23, Germany) and the dried sample was powdered and sieved through 60 mesh (250 μm). A weight of 5 g of dry powdered Miang was extracted in 100 ml of 80% (v/v) acetone on an incubator shaker at 30°C with 250 rpm for 1 h. After extraction, the extract was filtered through Whatman filter paper (No. 1) to remove any suspended material. The solvent was completely removed from the filtrate at 40°C for 20 min by an EYELA N-1000 rotary evaporator (Tokyo Rika-kikai Co. Ltd., Japan). Dried Miang extract was dissolved with 20 ml of 80% (v/v) acetone, and the supernatant was used for the determination of total polyphenol content, total tannin, total flavonoid, and antioxidant activity based on DPPH free radical scavenging activity.
8
Ginger Rhizome Extraction and Purification
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Initially, 400 g of Z. officinale rhizomes underwent individual extraction using a 50:50 mixture of ethanol and ethyl acetate. The pH of the extraction solution was adjusted to fall within the range of 5.0 to 7.0 by adding 1.0 M NaOH or HCl as needed. The extraction process employed the maceration technique and lasted for a period of three days. Subsequently, the resulting liquid was meticulously filtered through Whatman No. 1 filter paper, and the collected filtrate was then subjected to evaporation using a rotary evaporator (Heidolph Basic Hei-VAP ML, Schwabach, Germany), resulting in the formation of a highly viscous extract dissolved in cosolvent. To ensure optimal extraction efficiency, the maceration process was repeated two more times following the same protocol. The residual ethanol present in the herbal material was eliminated by subjecting it to further evaporation in a vacuum drying chamber (Binder VD 23, Tuttlingen, Germany). Evaporation continued until a consistent weight was attained, indicating the complete removal of all remaining cosolvent.
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