Milk foodome was measured as previously described using GC–MS and 1HNMR spectroscopy (16 (link)). Briefly, frozen milk samples were thawed at room temperature and vigorously vortexed until homogenization. After sonication, 200 μl milk was mixed with 300 μl of 80% methanol (containing 10 ppm internal standard, sorbitol) and 100 μl dichloromethane and vigorously vortexed followed by centrifugation at 13,572 g for 10 min at 4°C. Then 100 μl upper aqueous layer was transferred into a 200 μl glass insert and dried overnight using ScanVac (Labogene, Lynge, Denmark) at 40°C and 1,000 rpm. Immediately after drying, glass inserts were sealed with airtight magnetic lids into GC–MS vials, stored at 4°C,and analyzed by GC–MS within 24 h. For 1H NMR analysis, frozen milk samples were thawed at room temperature, vigorously vortexed until homogenization and 1.8 ml of milk samples were centrifuged at 13,572 g for 30 min at room temperature. Then, 600 μl of an aliquot from the clear solution was mixed with 135 μl of phosphate buffer in deuterated water and transferred into NMR SampleJet tubes of L = 103.5 mm and O.D. = 5.0 mm, kept at 5°C and analyzed within 24 h. GC–MS and 1H NMR datasets were subsequently processed using PARADASe (22 (link)) or SigMa (23 (link)) software, respectively, to convert the raw milk foodomics data into an informative metabolite table.
Scanvac
Manufactured by Labogene
Sourced in Denmark
The ScanVac is a laboratory vacuum concentrator designed for the efficient evaporation and concentration of samples. It features a compact and durable construction, providing a reliable solution for sample preparation in various research and analytical applications.
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Lab products found in correlation
Avanti j 26sxp, by Beckman Coulter (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Mira sem, by TESCAN (1 mentions)
208hr sputter coater, by Cressington (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
Lysing matrix b, by MP Biomedicals (1 mentions)
Snakeskin dialysis tubing, by Thermo Fisher Scientific (1 mentions)
Quantipro bca assay kit, by Merck Group (1 mentions)
Evo hd, by Zeiss (1 mentions)
Sephadex g 25 column, by Cytiva (1 mentions)
Cary 60 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
Trypsin gold ms grade, by Promega (1 mentions)
C18 cartridge, by Waters Corporation (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Pierce concentrator 7 ml 9 k, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Sb 1200, by Eyela (1 mentions)
22 protocols using scanvac
1
Milk Foodomics Analysis by GC-MS and NMR
2
Silk Scaffolds Characterization by SEM
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The silk scaffolds (± HSMMs) were fixed in 4% PFA/PBS for 30 min at RT and then dehydrated in ethanol solutions of increasing concentration (30–100%) for 20 min per concentration, freeze dried using a ScanVac (LaboGene, Lynge, Denmark), mounted on aluminium stubs and sputter coated with platinum (5 nm) using a 208HR sputter coater (Cressington, Redding, CA, USA). The data were collected using either a Neon 40EsB scanning electron microscope (SEM) (Carl Zeiss, Oberkochen, Germany) or MIRA SEM (TESCAN, Brno, Czech Republic).
3
Integrative Extraction of Proteins and Metabolites
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The integrative extraction of proteins and metabolites was performed as described before (27, 31). Approximately 0.5 g lysing matrix B (MP Biomedicals) and 750 µL ice-cold MCW (methanol: chloroform: water 2.5:1:0.5) were added to the frozen cell pellets. Homogenization was performed in a FastPrep 24 instrument (MP Biomedicals; 5 * 30 s, 6.5 ms−1; cooled on ice between circles), followed by 15 min incubation on ice. Samples were centrifuged (21000 g/4 min/4 °C) and the supernatants were transferred in new tubes for subsequent metabolite purification. A second extraction of the pellets was performed with 250 µL MCW. Samples were vortexed, 5 min incubated at room temperature (RT), centrifuged (21000 g/4 min/4 °C) and the supernatant was transferred to the tubes for metabolite purification. H2O (300 µL) was added to the supernatants to achieve a phase separation. After centrifugation (21000 g/4 min/4 °C), the upper polar phases were transferred to new tubes, carefully dried in a vacuum concentrator (ScanVac, Labogene) and stored at −20 °C until derivatization.
4
Serum Lipid and Fatty Acid Extraction
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All samples were kept on ice during the extraction of serum lipids and FA. In short, 0.5 ml frozen serum was thawed at 4 °C and centrifuged at 1000 × g for one minute. 100 μl serum was transferred to Eppendorf tubes and 10 μl of internal standard (10 ng/μl of triheptadecanoin in chloroform (CHCl3)) was added to each sample. Furthermore, a 750-μl mixture of methanol (MeOH) and CHCl3 (2:1 v:v) was added to each sample, vortexed for 10 s and sonicated for 10 min. Samples were spun down for 5 min at 2000 × g. The organic phase, containing lipids, was removed and saved in Eppendorf tubes. The sample was then re-extracted by the same procedures as described above. The second extract was combined with the first extract for each sample and evaporated in a SpeedVac (ScanVac, Labogene, Denmark). The lipids obtained were dissolved and vortexed in 1 ml MeOH/CHCl3 (2:1 v:v) and further divided into two portions each containing 500 μl and stored at − 80 °C until analysis of FA of total lipids and targeted lipidomics analysis, respectively.
5
Quantifying Extracellular Algal Biomolecules
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Hundred mL aliquots from Control, +ASTM and +Infochemicals algal cultures were centrifuged at 4500 × g for 15 min at 4°C to extract sEPS. Supernatant was first passed through a 0.22 μm pore-size filter and dialyzed against distilled water using a SnakeSkin Dialysis Tubing (3.5 kDa MWCO, Thermo Scientific). After dialysis the sEPS were freeze-dried (CoolSafe, ScanVac, LaboGene) and re-suspended in 1200 μl of HPLC grade water for further quantification assays. Carbohydrates were measured using the anthrone method using glucose as a reference standard (Le and Stuckey, 2016 (link)). Proteins were measured using the BCA assay kit (QuantiProTM BCA Assay Kit, Sigma Aldrich) using bovine serum albumin (BSA) as a reference standard (Georgiou et al., 2008 (link)). Fatty acids were measured using the method reported by Kapoore (2014) . The concentrations of each fraction were normalized by the algal cells’ concentration.
6
Lipid Extraction from RBC Supernatant
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Lipid extraction from stored RBCs supernatant was performed by gently mixing the samples with 0.5 mL of EtOAc containing 0.13% acetic acid (v/v). All samples were spiked with 5 µl of the IS. After 10 min of vigorous shaking (1500 rpm), samples were centrifuged (10,000 rpm, 10 min, 4 °C), and 450 µl of the organic layer was transferred to fresh tubes and evaporated in speed vac (Scan Vac, Labogene, Demark) at 4 °C. The dry residue was reconstituted in 25 µL of nitrogen-purged EtOH and then directly injected (2 µl) into the LC–MS/MS system.
7
Freeze-drying Effects on Rice Microstructure
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The cooked white, brown, and germinated brown rice samples were frozen at −80 °C for 20 h using a dry cube (F570, Eppendorf, UK) freezer before being subjected to a freeze dryer chamber (Scanvac, cool safe 90–80 A, Labogene, Denmark) at 0.1 hPa from −15–25 °C for 2 h. No significant change in the sample structure was observed from this procedure. The microstructure of the freeze dried samples were observed under a scanning electron microscope (SEM) (EVO® HD, Carl Zeiss, Germany) at an accelerating voltage of 10 kV and a working distance of 7–8.5 mm in secondary electron mode. Magnification of 1Kx.was applied for observation.
8
Plant Biomass and Water Content Analysis
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At the end of a two-week exposure, the plants were harvested, washed with tap and distilled water, and separated into roots and shoots. The separate plant organs were weighed (fresh weight, FW) and frozen in liquid nitrogen. The material was freeze-dried for 3 days (0.001 mbar, − 95 °C, ScanVac, Labo-Gene, Denmark), weighed (dry weight, DW) and used for photosynthetic pigment content analysis. Water content (WC) was calculated as .
9
Protein Separation via Size-Exclusion Chromatography
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For SEC separation, 5 g of hydrolyzed protein was mixed with 35 mL of HCl (0.01 N). Then, three volumes of ethanol were added to this solution and stored at 4 °C during the night for deproteinization. The mixture was centrifuged at 12,000 rpm for 20 min at 4 °C to remove the precipitated proteins, and ethanol was evaporated using a rotary evaporator. Samples were lyophilized overnight (SCANVAC, Labogene ApS, Denmark). Before injection into the SEC column, 1 g of the dried sample was resuspended in 10 mL of 0.01 N HCl and filtered through a 0.45 μm syringe filter to remove non-soluble impurities. A total of 5 mL of the sample was injected on the Sephadex G25 column (2.5 × 65 cm) (Amersham Biosciences, Uppsala, Sweden). The separation process was performed using a flow rate of 15 mL per hour of degassed 0.01 N HCl, and 5 mL peptide fractions were collected using an automatic collector. Finally, the absorbance was measured at 254 and 280 nm in a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Palo Alto, CA, USA), and the biological activities were also assayed in all fractions [24 (link),25 (link)].
10
Protein Extraction from Orange Seeds
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The defatted orange seed flour (containing 22.5% protein) was mixed with bidistilled water (at a ratio of 1:20 at room temperature). Then, NaOH at a concentration of 1 N was used to get a pH of 10 and the mixture was homogenized during 1 h at room temperature and later centrifuged (15 min, 12,000 rpm) using an Avanti J-26S XP centrifuge (Beckman Coulter Inc., Indianapolis, IN, USA). Later, HCl at a concentration of 1 N was used to decrease the pH to 3 and centrifuge to obtain a clean pellet (15 min, 12,000 rpm), which was washed with bidistilled water and freeze dried (SCANVAC, Labogene ApS, Lillerød, Denmark) [19 (link)]. The protein concentrates contained 75.1% protein and were used for the hydrolysis process.
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