The extraction process for EPS was previously described (Kong et al., 2019) (link). Briefly, after removing protein (8,000 × g for 20 min, 4°C), the fermentation broth was concentrated to one-quarter of its original volume, and EPS was precipitated using triple volume of 95% (vol/vol) ethanol × 3. The EPS was collected by dialysis (molecular weight cut-off: 8,000-14,000 Da) and freeze-dried for physicochemical analysis. Both the molecular weight and intrinsic viscosity were analyzed using a high-performance size-exclusion chromatography system equipped with multiple detectors (Wyatt Technology), including a multiangle laser light scattering detector (DAWN HELEOS-II, MALLS), a differential pressure viscometer (ViscoStar III, DP), and a refractive index detector (Optilab T-Rex, RI). Samples were prepared using 0.1 mol/L NaNO 3 at a concentration of 2 mg/mL. Monosaccharide composition analysis was conducted using high-performance anion-exchange chromatography (Dionex ICS-5000). Samples were hydrolyzed at 60°C for 30 min and diluted in water, followed by hydrolysis at 100°C for 4 h. The hydrolysates were diluted 100-fold in pure water and filtered through a 0.22-μm membrane (ANPEL Co., Ltd.) for analysis.
0.22 μm membrane
Manufactured by ANPEL
Sourced in China
The 0.22-μm membrane is a filtration product designed to remove particles and microorganisms from liquids. It has a pore size of 0.22 micrometers, which is effective at trapping bacteria and other small particulates. This membrane is often used in various industries, such as water purification and pharmaceutical processing, to ensure the quality and safety of the final product.
Automatically generated - may contain errors
Lab products found in correlation
Ics 5000, by Thermo Fisher Scientific (1 mentions)
Vacuum freeze dryer, by Ningbo Scientz Biotechnology (1 mentions)
Shim pack uflc cbm30a, by Shimadzu (1 mentions)
Hp 5 capillary column, by Shimadzu (1 mentions)
Gc 2010 system, by Shimadzu (1 mentions)
Uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 1260, by Agilent Technologies (1 mentions)
3 protocols using 0.22 μm membrane
1
Extraction and Characterization of EPS
2
Untargeted Metabolome Analysis of Freeze-Dried Plant Samples
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Plant samples were freeze-dried using a vacuum freeze dryer (Scientz, Ningbo, China). The samples were then ground with a hybrid grinder (MM400, Retsch, Germany). To dissolve lyophilized powder, 1.2 ml of 70% aqueous methanol was added to the plant samples at 4°C overnight. The supernatant was obtained after centrifugation at 10,000 × g for 10 min, and filtered using a 0.22 μm membrane (ANPEL, Shanghai, China). The supernatant was subjected for LC–MS/MS analysis and conducted by Wuhan Metware Biotechnology Co., Ltd. following the standard procedure (Jang et al., 2018 (link); Wang et al., 2021 (link)). We used the ultra-performance liquid chromatography (Shim-pack UFLC CBM30A, Shimadzu, Japan) and tandem mass spectrometry (Applied biosystems, Framingham, United States) for metabolome analysis. Metabolites from all samples were subjected to orthographic projection of primary component analysis (PCA) and potential structure identification analysis (OPLS-DA). Metabolites with fold change ≥2 or fold change ≤0.5 and variable importance in project (VIP) ≥1, were defined as differentially changed metabolites (DCMs). Enrichment analysis of DCMs was performed using the KEGG database.
3
Bacterial Growth and Metabolite Analysis
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Bacterial growth was determined by measurement of the optical density (OD) at 600 nm using a UV-VIS Spectrophotometer (Thermo Scientific, Waltham, MA, USA). PAE concentrations were detected by GC-2010 system (SHIMADZU, Kyoto, Japan) equipped with a HP-5 capillary column (in radium 0.25 mm, length 30 m, membrane thickness 0.25 μm) and a flame ionization detector (FID) at 300 °C, with an injection volume of 5 μL. Ultrahigh purity nitrogen served as the carrier gas at a flow rate of 1.51 mL/min. The column temperature gradually increased from 160 to reach 280 °C at the rate of 10 °C/min and then was held for 4 min at 280 °C, carrier gas for FID-H2 (40 mL/min) and air (400 mL/min). Samples to be analyzed were extracted with the equal volume of hexane and filtered through 0.22 μm membrane (ANPEL, Shanghai, China). The metabolites of DEHP were analyzed using liquid chromatogram (LC, Agilent 1260) coupled with a triple quadrupole mass spectrometer (QQQ, Agilent G6400).
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