Mixer

For loading of OVA into AEP-MSNs, OVA (0.5 mL, 0.5 mg/mL‚ 1 mM PB) and AEP-MSN (0.5 mL, 2 mg/mL, 1 mM PB) were mixed and incubated in Eppendorf mixer (400 rpm, 25°C, Nijmegen, the Netherlands) for different time periods (0, 0.5, 1, 2, 4, 8 and 24 h). After incubation, the suspensions were centrifuged and the encapsulation efficiency (EE%) of OVA was determined by measuring the difference in its intrinsic fluorescence intensity with a plate reader (Tecan M1000, Männedorf, Switzerland) (excitation wavelength = 280 nm and emission wavelength = 320 nm) in the supernatant before and after the encapsulation.
To determine the maximum loading capacity (LC%) of OVA in AEP-MSNs, the AEP-MSNs (2 mg/mL) were mixed with different initial concentrations of OVA (ranging from 0.25, 0.5, 1, 1.5, 2 to 3 mg/mL) and incubated in an Eppendorf mixer (400 rpm, 25°C) for 0.5 h. Next, the suspensions were centrifuged at 9000 g for 5 min. The EE% of OVA was determined by measuring the difference in their intrinsic fluorescence intensity in the supernatant before and after the encapsulation with a plate reader (Tecan M1000).
The EE% and LC% were calculated as below: $$\mathrm{EE}\%=\frac{t_{\mathit{ova}}-f_{\mathit{ova}}}{t_{\mathit{ova}}}\times 100\%$$
$$\mathrm{LC}\%=\frac{t_{\mathit{ova}}-f_{\mathit{ova}}}{\mathit{OVA}\mathit{\text{loaded}}\mathit{AEP}-\mathit{\text{MSNs}}}\times 100\%$$
Where t_ova represents the total content of OVA, and f_ova is the content of free OVA (OVA in the supernatant).

DNA was extracted from the frozen fecal samples using an extraction method previously described (16 (link)). Briefly, 50 mg of the samples were mixed with 1 ml of sterile PBS buffer and incubated for 10 min prior to a bead beating on an Eppendorf Mixer (model 5432, Eppendorf, Hamburg, Germany) at 4°C for 30 min. The samples were centrifuged at 13,000 rpm for 1 min and about, 200 ml of the supernatant was transferred to a clean 2.0 ml sample tube. DNA was extracted from the supernatant using the EZ1 DNA extraction robot (Qiagen bioinformatics, Aarhus, Denmark) and the DNA tissue kit (Qiagen bioinformatics, Aarhus, Denmark). The extracted DNA was frozen at −20°C until further analysis.