About 0.2 g of the cold extracted peanut oil was weighed into a 10 mL graduated flask, dissolved in 1-butanol, followed by 0.5 mL hydrochloric acid (0.1 M), and finally made up with 1-butanol. Pipette 3.0 mL of the prepared sample solution into a screw test tube and add 3.0 mL of TBA solution (0.2% in 1-butanol). In the same way, a blank experiment consisting of 2.85 mL 1-butanol, 0.15 mL hydrochloric acid (0.1 M), and 3.0 mL TBA solution (0.2% in 1-butanol) was carried along. The test tube was closed, shaken well, and incubated for 2 h in a water bath preheated to 95 °C. Finally, the sample was cooled down. After cooling, the sample was measured at a wavelength of 532 nm on the Ultrospec 1000 photometer (Pharmacia-Biotech, Uppsala, Germany) against the blank as a reference. For the previously performed calibration, 44.3 μmol of 1,1,3,3-tetramethoxypropane was dissolved in 6.0 mL hydrochloric acid (0.1 M) and then heated in a water bath at 40 °C for 45 min to release the malondialdehyde. After cooling down, the concentration of the stock solution was determined, and the calibration solutions with a concentration of 2 to 20 μM malondialdehyde were prepared and proceeded in the same way as for the sample solutions. Hydrochloric acid (0.1 M) was used as a blank [19] (link).
Ultrospec 1000
Manufactured by GE Healthcare
Sourced in Sweden
The Ultrospec 1000 is a UV/Vis spectrophotometer designed for general laboratory use. It is capable of measuring the absorbance of samples across a wavelength range of 320-800 nm. The instrument features a compact design and easy-to-use interface.
Automatically generated - may contain errors
Lab products found in correlation
Microbiome sample collection tube, by Transnetyx (1 mentions)
3 16pk centrifuge, by Merck Group (1 mentions)
Cto 6a oven, by Shimadzu (1 mentions)
High performance liquid chromatography (hplc), by Jasco (1 mentions)
Total starch assay procedure, by Megazyme (1 mentions)
Total starch assay kit, by Megazyme (1 mentions)
Waters 2489 uv vis detector, by Waters Corporation (1 mentions)
Gemini 300 mhz spectrometer, by Agilent Technologies (1 mentions)
1525 binary hplc pump, by Waters Corporation (1 mentions)
Hpx 87h, by Aminex (1 mentions)
Tryptone and yeast extract, by Merck Group (1 mentions)
Alcian blue, by Merck Group (1 mentions)
14 protocols using ultrospec 1000
1
Malondialdehyde Quantification in Peanut Oil
2
Microbial Characterization by Shotgun Sequencing
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After the ex vivo culture, the UV absorption of the suspension was measured at 600 nm by Pharmacia Biotech U.V./Visible Spectrophotometer (Ultrospec 1000), and 1 mL of the suspension (~1.00 absorbance unit) was centrifuged at 1000× g to get the pellet. Then, after removing the supernatant, the pellet was immediately suspended in the Transnetyx microbiome sample collection tube, and all the samples were submitted to the Transnetyx automatic whole-genome sequencing (WGS) platform. The platform employed shallow shotgun WGS with a minimum read depth of two million paired-end reads for the methodology and provides species/substrain-level taxonomic resolution on each sample. The analysis includes all microbe types, including bacteria, viruses, fungi, protists, and archaea. Data visualization of taxonomy, alpha- and beta-diversity, clustering, and PCoA were performed at the Onecodex (https://www.onecodex.com , accessed on 15 December 2021) cloud-computing website.
3
Quantifying Insulin Resistance in Rats
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This test was carried out to quantify insulin resistance in the study rats. According to previous reports, the euglycemic and hyperglycemic glucose clamp shows a good correlation when the first 15 minutes of the test are taken into account [30 (link),31 (link)]. Before the study began, all animals were in a fasting state for 12 hours and only had free access to water. To begin the measuring, the rats were anesthetized intraperitoneally with sodium pentobarbital (Anestesal) at a dose of 40 mg/kg. They then had a stabilization period of 15 minutes after which a blood sample(200–500μL) was taken from the tip of the tail to quantify the baseline glycemia level. Fast-acting crystalline insulin (Humolin, Eli Lilly Company) was administered intraperitoneally at a single dose of 0.75 IU/kg from a dilution with 0.5 IU/mL of isotonic saline solution (0.9% sodium chloride). Blood samples for quantifying the blood glucose levels were taken at 5 minutes, 10 minutes, and 15 minutes after insulin application. The serum of the blood sample was separated by centrifugation at 900g and frozen at −70°C for later glucose concentration determination, again employing the enzymatic-colorimetric method with glucose oxidase (Spinreact S.A.U.). In addition, absorbance was measured using a spectrophotometer at 505 nm(Ultrospec 1000, Pharmacia Biotech).
4
Quantification of Fermentation Metabolites
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The cell density was followed by measuring the optical density of the culture at 620 nm (OD620nm) using UV–Vis spectrophotometer (Ultrospec 1000 (Pharmacia Biotech, Sweden). The cell dry weight (CDW) was determined by centrifugation (Sigma 3-16PK centrifuge) of 5 mL fermentation broth at 4000 g for 20 min in a dried preweighed tube and drying the cell pellet for 12 h at 100 °C in an oven before weighing again and correlating with the volume.
Glycerol, propionic acid, acetic acid, succinic acid, and other minor metabolites were analyzed by HPLC (Jasco) equipped with Aminex HPX-87H organic acid analysis column (Bio-rad, Hercules, California, USA), CTO-6A oven (Shimadzu, Kyoto, Japan), Jasco AS 950–10 intelligent pump, PU 980 automatic intelligent injector (Jasco), and ERC 7515A refractive index detector (ERC, Saitama, Japan). Samples were diluted in MilliQ quality water, acidified with 20% (v/v) H2SO4 (20 μL per 1 mL of the sample), and then filtered through a 0.45 μm syringe filter prior to analysis. Chromatography was performed using 5 mM H2SO4 as mobile phase flowing at a rate of 0.6 mL/min, column temperature was maintained at 55 °C, and RI detector temperature at 30 °C.
Glycerol, propionic acid, acetic acid, succinic acid, and other minor metabolites were analyzed by HPLC (Jasco) equipped with Aminex HPX-87H organic acid analysis column (Bio-rad, Hercules, California, USA), CTO-6A oven (Shimadzu, Kyoto, Japan), Jasco AS 950–10 intelligent pump, PU 980 automatic intelligent injector (Jasco), and ERC 7515A refractive index detector (ERC, Saitama, Japan). Samples were diluted in MilliQ quality water, acidified with 20% (v/v) H2SO4 (20 μL per 1 mL of the sample), and then filtered through a 0.45 μm syringe filter prior to analysis. Chromatography was performed using 5 mM H2SO4 as mobile phase flowing at a rate of 0.6 mL/min, column temperature was maintained at 55 °C, and RI detector temperature at 30 °C.
5
Carotenoids Extraction from R. marinus
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The carotenoids were extracted from the R. marinus DSM 16675 strain during batch, fed-batch, and sequential batch cultivations. The extraction was performed according to the method described by Biehler et al. (Biehler et al. 2010 (link)). One milliliter of culture sample was pelleted down at 13,000 rpm for 5 min and the pellet was re-suspended in 1 mL of acetone (99.5%), shaken for 1 h on a rocking table at room temperature and then, separated from the supernatant by centrifugation at 13,000 rpm for 3 min. The absorbance of the cell-free supernatant was measured at 450 nm using a UV quartz cuvette (Hellma) in a UV/Visible spectrophotometer (Pharmacia biotech, Ultrospec 1000) and plotted against the cultivation time.
6
Intraperitoneal Glucose Tolerance Test
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Twelve hours before the study, the rats were put on fasting with only free access to water. To start the glucose level measurements, the animals were anesthetized intraperitoneally with a dose of 40 mg/kg of sodium pentobarbital (Anestesal) after which there was a 15-minute stabilization period. A blood sample was then drawn from the tip of the tail to quantify the blood glucose level in a control condition; a dose of 2 g/kg of glucose (Pisa) was administered intraperitoneally from a 40% solution [24 (link),32 (link)]. The glycemia levels were determined at 30 minutes, 60 minutes, and 120 minutes after the administration of the glucose solution. The blood samples (200 μL and 500 μL) were taken at the tip of the tail of each rat and were deposited in tubes for microsamples; once it coagulated, the serum was separated by centrifugation at 900g and stored at −70°C for later glucose measuring. Quantification was determined in triplicate by the enzymatic-colorimetric method using glucose oxidase (Spinreact S.A.U. Spain), and absorbance was measured using a spectrophotometer at 505 nm (Ultrospec 1000, Pharmacia Biotech).
7
Measuring Cell Growth via OD620 and CDW
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Cell growth was monitored by measuring OD at 620 nm using an Ultrospec 1000 spectrophotometer (Pharmacia Biotech, Uppsala, Sweden) and correlating it with cell dry weight (CDW). To determine CDW, 10 mL of the culture samples was centrifuged in preweighed Falcon tubes at 4000 x g for 15 min, the pellet was washed once with 10 mL distilled water, and then centrifuged and lyophilized until a constant weight was reached. The centrifuge tube was weighed again; a decrease in weight with respect to the original weight yielded the CDW.
8
Quantitative Starch Analysis in Wheat Bran
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Starch determination in wheat bran was performed using a total starch assay kit (Megazyme) based on the use of two enzymes: thermostable α-amylase and amyloglucosidase. Briefly, the starch was hydrolyzed to soluble branched and unbranched maltodextrin using thermostable α-amylase at pH 5, 100°C, 6 min, using a heating block (Techne Dri-block). Then maltodextrin was hydrolyzed to D-glucose using amyloglucosidase at 50°C, 30 min in the heating block. Finally, the glucose was oxidized to D-gluconate and the released hydrogen peroxide was converted to quinone-imine dye using peroxidase (GOPOD reagent enzymes) and then the absorbance of the dye was measured at 510 nm by a UV/ Visible spectrophotometer (Ultrospec 1000, Pharmacia biotech) with D-glucose as standard (total starch assay procedure, Megazyme, Ireland).
9
Synthesis and Characterization of Ara-A
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All reagents were purchased from Merck Sigma-Aldrich (Milan, Italy). Agarose gel 6B-CL was activated to glyoxyl–agarose as previously reported [24 (link)]. TLC analyses were performed using commercial silica gel 60 F254 aluminum sheets. HPLC analyses were performed using a Waters 1525 Binary HPLC Pump, equipped with a Waters 2489 UV–vis detector (Waters, Milford, MA, USA) and an Ascenti C18 column (25 cm × 4 mm, 4 μm particle size). The Bradford assay and CpUP activity assay were performed using the spectrophotometer Ultrospec 1000 (Pharmacia Biotech, Cologno Monzese, Italy). 1H-NMR of ara-A (11 ) in DMSOd6 was recorded on a Varian Gemini 300 MHz spectrometer (Varian, Palo Alto, CA, USA). The continuous flow reactions were performed using either a R2+/R4 series or an E series flow reactor, commercially available from Vapourtec (Cambridge, UK) equipped with Omnifit glass columns with one fixed and one adjustable endfits. Recombinant CpUP and AhPNP were prepared as previously reported [17 (link)]. One international unit (IU) corresponds to the amount of enzyme that transforms 1 μmol of substrate per minute under specific temperature and pH values, while the specific activity is defined as units of enzyme activity per milligram of protein.
10
Quantification of Cell Growth and Metabolite Analysis
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Cell density: The cell density was followed by measuring the optical density at 620 nm (OD620nm) using a spectrophotometer (Ultrospec 1000, Pharmacia Biotech, Uppsala, Sweden) after proper dilution of the sample.
Cell dry weight: For measuring the cell dry weight (CDW - g/L), 10 mL of the fermentation broth was collected in a 15-mL Falcon tube, centrifuged at 5000×g for 10 min at room temperature. The supernatant was discarded, and the cell pellet was dried overnight at 105 °C.
Finally, the optical density was correlated with the cell dry weight (g/L) where 1 OD620nm unit was equivalent to 0.366 gCDW/L.
Analytes: The concentration of the substrates (glucose and glycerol) and products (PA, AA, and SA) was determined using JASCO HPLC (Tokyo, Japan) as described elsewhere [61 (link)]. Briefly, 50 µL of properly diluted and acidified samples were injected into the mobile phase (0.5 mM sulfuric acid), flowing at a rate of 0.4 mL/min. Separation of the different components was done at 55 °C using the Biorad column, Aminex HPX-87H (Richmond, CA, USA). The detection was done using an ERC refractive index (RI) detector (Kawaguchi, Japan).
Cell dry weight: For measuring the cell dry weight (CDW - g/L), 10 mL of the fermentation broth was collected in a 15-mL Falcon tube, centrifuged at 5000×g for 10 min at room temperature. The supernatant was discarded, and the cell pellet was dried overnight at 105 °C.
Finally, the optical density was correlated with the cell dry weight (g/L) where 1 OD620nm unit was equivalent to 0.366 gCDW/L.
Analytes: The concentration of the substrates (glucose and glycerol) and products (PA, AA, and SA) was determined using JASCO HPLC (Tokyo, Japan) as described elsewhere [61 (link)]. Briefly, 50 µL of properly diluted and acidified samples were injected into the mobile phase (0.5 mM sulfuric acid), flowing at a rate of 0.4 mL/min. Separation of the different components was done at 55 °C using the Biorad column, Aminex HPX-87H (Richmond, CA, USA). The detection was done using an ERC refractive index (RI) detector (Kawaguchi, Japan).
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