0.22 μm syringe filter

We placed each group of mice on an RD, or an HFD containing 60% fat for 12 weeks and collected stool samples at 12 weeks. Stool samples were dissolved in Phosphate Buffered Saline (PBS) and centrifuged at 5, 20, and 340 g for 5 min each. Supernatant fractions were pelleted once at 10,000 g for 30 min and then filtered through a 0.45-μm syringe filter (Sartorius Stedim Biotech, Goettingen, Germany) followed filtration through a 0.22-μm syringe filter (Sartorius Stedim Biotech, Goettingen, Germany). The filtrates were then subjected to density-gradient centrifugation in a Beckman ultracentrifuge (Beckman Coulter, Fullerton, USA) at 100,000 g for 2 h at 4 °C. Fraction between 10% and 40% OptiPrep solution (Sigma, St. Louis, USA) was taken, and EVs were prepared by centrifugation at 150,000 g for 2 h at 4 °C using a Beckman ultracentrifuge. EVs were diluted in PBS and stored at –80 °C. The protein concentration of EVs was assessed by a BCA assay (Thermo Fisher Scientific, Waltham, USA).

HIV-1_NL4-3 stocks were made by transfection of HEK293T cells with a proviral DNA plasmid (39 (link), 40 (link)). The medium was replaced at 24 h posttransfection, and HIV-1 was harvested at 72 h posttransfection. All virus stocks were quantified by a CAp24 enzyme-linked immunosorbent assay (ELISA) and stored in small aliquots at −80°C as previously described (18 (link)).
VLPs that convey NB-ZSG or ZSG were produced as previously described (21 (link)). Briefly, 10 μg of the pSRS11-SF-γC vector (25 (link)) expressing NB-ZSG or ZSG and 2 μg of a plasmid expressing Gag-Pol were transfected into Phoenix-Ampho cells (41 (link)) with X-tremeGENE HP DNA transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer’s protocol. The cell medium was replaced at 24 h posttransfection. VLPs were harvested by filtration with a sterile 0.22-μm syringe filter (Sartorius Stedim Biotech GmbH, Göttingen, Germany) at 48 h and 72 h posttransfection.

Nanoparticles were prepared by the desolvation technique as described by Langer et al.^{11 (link)} modified by Sánchez-Segura et al.^{14 (link)} and readjusted from Sánchez-Arreguin et al.^{10 (link)}. Briefly, 200 mg of BSA powder were dissolved in 2 mL of 10 mmol NaCl solution, pH 9.4, and filtered through a 0.22 μm syringe filter (Sartorius, Goettingen, Germany). The solution was maintained under agitation at 200 rpm using a stirrer (Eurostar 20, IKA, Wilmington, NC, USA) for 30 min at room temperature. The formulations of increasing concentration of capsaicin were generated by the addition of 4 mL of an ethanol-capsaicin solution at 0, 812, 1625, 2437, and 3250 µg mL⁻¹ for each drying treatment. The rate of addition was 1.0 mL min⁻¹ at 200 rpm of stirring speed. The crosslinking process was carried out by the addition of 5 mL of 4% glutaraldehyde in a 10 mmol NaCl solution in agitation at 1000 rpm for 30 min in dark conditions.

Enzymatic saccharification of pretreated biomass was evaluated using the commercial cellulase enzyme solution Cellic® CTec2 (Novozymes A/S, Bagsværd, Denmark) at an enzyme load of 20 FPU g_solids⁻¹. The reactions were performed in duplicate inside 2 mL microcentrifuge tubes containing 1.0 mL solutions comprising 3% w/w dry solids in 50 mM citrate buffer (pH 5). The mixture was incubated in a thermomixer at 50 °C and 800 rpm for 72 h, with samples taken at 8, 24, 48, and 72 h. After collection, the samples were placed in a water bath at 100 °C for 5 min to denature the enzyme and then centrifuged at 12 000 × g for 10 min at room temperature. The supernatant was removed from the solids, filtered through a 0.22 μm syringe filter (Sartorius, Göttingen, Germany) and the sugars were quantified by HPLC as previously described (see section “Organosolv fractionation”).

The total flavonoid content was extracted according to Liu et al. (2019) (link), with a minor modification. Sample powder (0.05 g FW) was sonicated in 1 mL of 100% methanol for 30 min at room temperature. After centrifugation at 12,000 rpm for 10 min, the sample supernatant was filtered through a 0.22 μm syringe filter (Sartorius, Gottingen, Germany) into an UPLC vial. Target flavonoids were detected using an Acquity UPLC I-class/VION IMS QTOF (Waters Corp., Milford, MA, United States) instrument, equipped with a photodiode array detector (PDA). The separation of the target flavonoids was achieved on a C₁₈ BEH (1.7 μm, 2.1 × 100 mm) column equipped with a C₁₈ BEH (1.7 μm) precolumn (Waters) at 45°C, and the mobile phases consisted of eluent A (0.1% formic acid in Milli-Q water) and eluent B (0.1% formic acid in MeCN, Dingguo Corp., Beijing, China) with a flow rate of 0.4 mL min^–1. The procedures were as follows: 5–20% B (0–3 min), 20–100% B (3–10 min), 100% B (10–12 min), 100–95% B (12–15 min), 95% B (15–19 min). Finally, 1 μL of the sample was injected into the liquid chromatograph-mass spectrometer (LC-MS) system and the UV wavelength was set at 254 and 335 nm for the detection of genistein and scutellarin. The mass spectrometric analysis and construction of the standard curve were performed according to Xia et al. (2014) (link).

The TSB used in the study was obtained from plants aged at least two years, whereas the TSF was collected from a seven-year-old plant. The collected materials were washed and boiled twice with reverse osmosis water for 60 min. Then, the crude extracts were collected to freeze and dry to form powder. TSB and TSF extracts were dissolved in sterile phosphate-buffered saline (PBS; pH 7.4) and filtered using a 0.22-μm syringe filter (Sartorius Stedim Biotech Inc., Göttingen, Germany).