Spectra por molecularporous membrane tubing

The obtained mixture was incubated for 2 h at 37 °C on a magnetic stirrer. Then, the mixture was heated to 90 °C for 10 min to inactivate the enzymes used in the digestion process. At the end, the samples were centrifuged for 20 min at 7000 rpm. The pellet was incubated with ethanol (96%) at room temperature for 24 h with agitation. The solution was centrifuged for 20 min at 7000 rpm, obtaining a precipitate (TE) that was discharged, and the ethanol in the digested ethanolic extract (TE-dig) was removed using a rotavapor before the lyophilization of the residue. The supernatant obtained from the digestion process was divided into two parts. One part was lyophilized, obtaining the crude digested aqueous extract (TW-dig); the other one was dialyzed with membrane cut-off 3.5 kDa (Spectra/Por molecularporous membrane tubing, Thermo Fisher Scientific, Milan, Italy) against 250 mL of water for 24 h at 4 °C to separate the low molecular weight (mw) fraction (<3.5 kDa) and the high mw fraction (>3.5 kDa). The solutions inside the dialysis tube (>3.5 kDa) and out of the tube (<3.5 kDa) were lyophilized.
To prepare the undigested aqueous and ethanolic extracts (TW and TE, respectively), the same procedure was followed for extraction without the first part of enzymatic digestion. The detailed scheme of in vitro digestion and the obtained extracts and fractions is illustrated in Figure 1.

The protocol for synthesizing GelMA polymers with the medium substitute degree followed our previous studies [28 (link),29 (link)]. Briefly, 10% type A gelatin powders from porcine skin were added into Dulbecco’s phosphate-buffered saline (DPBS) at 50 °C and then stirred at 240 rpm until the gelatin powder dissolved completely. Subsequently, MA was gently added into the gelatin solution drop by drop, and the final concentration of MA was 1.25 v/v%. After 2 h of reaction at 50 °C under a low-speed stirring process, an equal volume of pre-warm DPBS was added into the gelatin/MA solution, followed by another stirring process for 10 min. Afterward, the mixture was moved to the dialysis membrane (Spectra/Por molecular porous membrane tubing, MWCO 12,000–14,000, Thermo Fisher Scientific Inc., Waltham, MA, USA) to execute a purification process. In the purification process, the GelMA mixture in the dialysis membrane was placed in distilled water (DI water) at 40 °C at 500 rpm, and the DI water was changed twice daily. After 5 days of dialysis, the purified GelMA polymers were moved to the centrifugal tubes and further stored at −80 °C. The GelMA polymers were obtained via a 4-day freeze-dried process to remove the DI water before experiments.