Quantification of amino acids, derived biogenic amines, ammonium, and GABA was carried out by UHPLC following a derivatization with diethyl ethoxymethylenemalonate (DEEMM, Sigma-Aldrich, St. Louis, MO, USA) in 100 µL of fecal water, by the procedure described in Redruello et al. [33 (link)], adapted and optimized to human fecal cell-free supernatants by Salazar et al. [34 (link)]. Samples were filtered through 0.22 µm pore diameter PTFE membranes (VWR International, Radnor, PA, USA) prior to injection of 1 µL into the UHPLC chromatographic system. Concentrations of the analyzed compounds in fecal supernatants were calculated in millimolar (mM) and referred as concentration in fecal samples.
Ptfe membrane
Manufactured by Avantor
Sourced in United States, Germany
The PTFE (Polytetrafluoroethylene) membrane is a thin, non-porous film that is chemically resistant and has a high thermal stability. It serves as a barrier material, providing protection and separation in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Precellys 24, by Bertin Technologies (2 mentions)
Cryolys temperature controller, by Bertin Technologies (2 mentions)
Deemm, by Merck Group (1 mentions)
Etoac, by Avantor (1 mentions)
Pestinorm, by Avantor (1 mentions)
Daidzein, by LC Laboratories (1 mentions)
Glass vial, by Avantor (1 mentions)
Microcentrifuge tube, by Avantor (1 mentions)
Hypersil ods c18 column, by Thermo Fisher Scientific (1 mentions)
11 protocols using ptfe membrane
1
Quantification of Fecal Metabolites
2
Beer De-carbonation and DEEMM Derivatization
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Containers of beer were left open for 2 h at room temperature to remove their carbon dioxide content. Samples of the de-carbonated beers were then centrifuged at 8000 g for 5 min to eliminate any particulate matter. DEEMM derivatization reactions were performed as described in Redruello et al. (2013) , using 100 l samples (or of standard solution when constructing calibration curves). When necessary, samples were diluted with 0.1 N HCl. After derivatization, samples were filtered through 0.22 m polytetrafluoroethylene (PTFE) membranes (VWR) into conical vials (VWR) prior to injection into the UHPLC system.
3
Lipid Extraction and Quantification
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A Precellys®24 bead homogenizer with a Cryolys temperature controller (all Bertin Technologies SAS, Montigny-le-Bretonneux, France), was employed for homogenization and a solvent system previously described by Blight and Dyer was applied for lipid extraction [47 (link)]. Twenty milligrams of dried pellet were accurately weighed and homogenized with zirconium oxide beads (0.5 ± 0.01 g, Ø 1.4 mm) in 1.0 mL of a cold mixture of chloroform:methanol (1:2, v/v). Three bead-beating cycles at 6000 rpm for 30 s with 15 s intermediate pause were applied to obtain a homogenous sample. An addition of 0.333 mL of a cold chloroform followed and tubes were vortexed for 20 s. Phase separation was induced by adding 0.333 mL of water. A phase separation was accelerated by a short and mild centrifugation cycle. A chloroform layer containing lipids was collected and cleared of cell debris with a syringe filter with PTFE membrane, 0.2 um, Ø 13 mm (VWR International). Final extracts were stored at −30 °C in glass vials until analyzed with ultra-performance convergence chromatography with high-resolution mass spectrometry (UPC2-ESI-QTOF). Dichloromethane was used as a sample diluent prior to injection. An aliquot of the lipid extract was transferred to a preweighted glass vial and evaporated on a heating block 40 °C while flushing with N2 gas for ca. 20 min to determine the amount of total lipids.
4
Synthesis and Characterization of EmPEO1.9
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The complete end group modification reaction of mPEO1.9 to EmPEO1.9 was carried out at room temperature under argon atmosphere in a glovebox. NaOtBu (1.5 equiv, 9.32 mmol, 0.896 g) was dissolved in THF (60 mL) and subsequently added to a solution of mPEO1.9 (1.0 equiv, 6.29 mmol, 12.0 g) in THF (60 mL). After 72 h, epichlorohydrin (8.0 equiv, 50.3 mmol, 4.65 g, 3.94 mL) was added dropwise to the reaction within 15 min and stirred for six days. Subsequently, the volatile components were removed at 55 °C under vacuum and the solid residue was dissolved in THF. Undissolved components, mainly formed sodium chloride (NaCl), were removed by centrifugation (Sigma 3-18KS, Osterode am Harz, Germany, 10000 rpm for 10 min), followed by filtration with syringe filter (PTFE membrane, pore size = 0.2 µm, VWR) and drying at room temperature under vacuum. Afterwards, the product was dissolved in a little amount of toluene at 40 °C, precipitated into cold Et2O and collected by centrifugation as before. This process was repeated three times. The resulting EmPEO1.9 was dried at 30 °C under vacuum < 1 × 10−6 mbar (yield: 89–93%). The product was characterized by 1H-nuclear magnetic resonance (NMR) spectroscopy, 13C-NMR spectroscopy, gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA).
5
Time-course analysis of bacterial metabolites
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Metabolites such as daidzein, dihydrodaidzein, and equol were identified and quantified by UHPLC using a reversed-phase Acquity UPLC™ BEH C18 1.7 μm column [31 (link)]. Samples (0.2 mL) were harvested from bacterial cultures every 2 h for 24 h, filtered through a 0.2 µm PTFE membrane (VWR, Radnor, PA, USA), and used directly in UHPLC analyses. Metabolite concentrations were estimated based on calibration curves prepared with known quantities of the corresponding standard compounds (all from Toronto Research Chemicals). Measurements were obtained for four independent cultures.
6
Extraction and Quantification of Fungal Metabolites
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The entire solid cultures were sliced into small pieces with a flat spatula and transferred into 250 mL glass flasks. 100 mL of ethyl acetate (EtOAc, PESTINORM, VWR Chemicals, Leuven, Belgium) was added and the mixture was homogenized with an Ultra-Turrax at 19,000 rpm before filtering through a folded cellulose membrane filter (type 113P, Rotilabo®ROTH, Karlsruhe). The filtrate was collected into a glass separating funnel and washed with 100 mL of Milli-Q®water (Arium®Lab water systems, Sartorius) to remove salts and water-soluble compounds. A total of 100 mL of crude EtOAc extract was collected and evaporated to dryness by a rotary evaporator (150 rpm at 40°C). Dried extracts were re-dissolved in 3 mL of UPLC/MS grade methanol (MeOH) and pipetted into a pre-weighed 4 mL-amber glass vial through a 13 mm syringe filter w/0.2 μm PTFE membrane (VWR International, Darmstadt, Germany). After drying the extracts under a nitrogen atmosphere, vials were weighed to determine the extract amount. The extracts were stored at -20°C.
7
Mass Balance Evaluation of Tryptophan and Kynurenine
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A mass balance evaluation was performed to characterize how Trp and Kyn are distributed between the receptor chamber, the donor chamber, and the skin membrane. Hence, at the end of the experiment (after 45 h), the skin membrane was removed from the Franz cell and rinsed by immersing the membrane into 20 mL of PBS solution for about 10 s. After that, the skin membrane was placed in a glass vial containing 5 mL of 131 mM NaCl (pH of 12.0) and sonicated for 1 h for extraction of Trp and Kyn. For the calculations of the results from these experiments, only the skin area exposed to Trp and Kyn solution was taken into account, corresponding to 0.64 cm2. However, in the control experiments, where no Trp and Kyn were added, the total area of the skin membrane was used (2.0 cm2). Next, the solution was collected in a centrifuge tube, while the described extraction procedure was repeated two more times. Finally, the three extraction solutions were dried by evaporation by a Genevac system at 35 °C. The obtained samples were resuspended in 500 µL of MilliQ water and syringe filtered (13 mm, w/0.2 μm PTFE membrane, VWR International, USA) prior to the HPLC analysis. The amount of Trp and Kyn remaining in the lower donor chamber was estimated by diluting the sample 10 times and directly measuring it with HPLC–UV.
8
Lipid Extraction from Dried Cell Pellets
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Lipid were extracted as described earlier (Bartosova et al., 2021a (link)) with modifications. The cell pellets were snap-freezed by submerging them in liquid nitrogen for about 10 s and lyophilized overnight. Five milligram(s) of dried cell pellets were mixed with zirconium oxide beads (0.5 ± 0.01 g, Ø 1.4 mm) in 2 ml vials with 1 ml of a cold mixture of chloroform:methanol (1:2, v/v). The mixtures were homogenized with three bead-beating cycles at 6,500 rpm for 30 s with 15 s intermediate pause by a Precellys® 24 bead homogenizer with a Cryolys temperature controller (all Bertin Technologies SAS, Montigny-le-Bretonneux, France). Cold chloroform 333 μl were then added, followed by vortexing for 20 s. Phase separation was induced by adding 333 μl of water, followed by vortexing for 20 s. The phase separation was accelerated by centrifuging at 14,000 rpm for 5 min at 15°C. A lower chloroform layer containing lipids was collected and cleared of cell debris with a syringe filter with PTFE membrane, 0.2 um, Ø 13 mm (VWR, United States). For determination of total lipids, 300 μl of the lipid extracts were transferred to a pre-weighed glass vial, left in fume hood for evaporation and weighed after 2 days.
The remaining extracts were flushed with a stream of nitrogen and stored at −80°C in dark glass vials for analyses of lipid classes and species.
The remaining extracts were flushed with a stream of nitrogen and stored at −80°C in dark glass vials for analyses of lipid classes and species.
9
Quantitative Analysis of Isoflavone Metabolites
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daidzein, genistein, and their derived metabolites dihydrodaidzein, dihydrogenistein, O-DMA, and equol were detected and quantified by UHPLC based on the method for isoflavone determination in urine samples reported by Redruello et al. [37 (link)]. Briefly, the control strains were independently cultured in GAM-Arg medium supplemented with 12.5–100 µM daidzein or genistein (LC Laboratories, Woburn, MA, USA). Furthermore, the selected strains were inoculated in pairs, triads, and tetrads and cultured with 100 µM of each isoflavone as above. After overnight incubation, cultures were centrifuged at 16,000× g for 2 min, and then filtered through a 0.2 µm polytetrafluoroethylene (PTFE) membrane (VWR, Radnor, PA, USA). The culture supernatants were used directly in UHPLC analyses. Quantification was performed against calibration curves for isoflavone and isoflavone-derived standards obtained from a commercial source (LC Laboratories). In this work, the limit of quantification (LoQ) for the different compounds analyzed were, in µM, 6.25 for daidzein, genistein, and dihydrogenistein, 5.62 for O-DMA, 3.13 for dihydrodaidzein, and 3.12 for equol.
10
Bread and Sourdough Sample Analysis
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Breads and sourdough samples were stored at -20°C before analysis. Samples (10 g) were homogenized in a glass beaker with 60 mL of 4.0 mM H 2 SO 4 solution (Sulphuric acid 97 %, AnalaR Normapur, VWR) using an Ultra-Turrax homogenizer (Yellowline DI25 Basic Homogenizer). Further, the bread suspension was transferred to a 100 mL volumetric flask and supplemented with 4.0 mM H 2 SO 4 . The suspension was centrifuged for 10 minutes at 25000 G. The supernatant was transferred to 1.7 mL micro centrifuge tubes (VWR) and centrifuged for a second time at 25000 G. Further, the supernatant was filtered over a 0.45 µm filter (PTFE-membrane, 13 mm diameter; VWR) directly into 1.5 mL glass vials (VWR). Three samples of each baking tests were analyzed.
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