D fructose

Recombinant Bacillus subtilis 1A751/pUB-P43dpe-dal was constructed in reference to the method by reported He et al. [21 (link)] and the DPEase came from Clostridium scindens 35704. D-allulose (≥98.0% purity) was produced by our laboratory. D-fructose (≥99.0% purity) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). HFA was obtained from Resindion S.R.L (Mitsubishi Chem. Co., Milano, Italy).

The definitive endoderm cells were encapsulated in 2% (w/v) alginate (Sigma-Aldrich, Saint Louis, MO, USA). The encapsulation was performed using a system based on the syringe extrusion method [24 ]. Briefly, on Day 3 of cell differentiation, the adherent cells were digested with dispase, and a confluent aggregate of adherent cells was transferred to Knockout DMEM medium with the above added supplement (Knockout SR, GlutaMAX, NEAA,2-Mercaptoethanol, DMSO and P/S), then mixed with an equal amount of 4% (w/v) sodium alginate to form a 2% sodium alginate cell suspension of 3 × 10⁶ cells/mL. The mixture of cell aggregates in 2% purified sodium alginate was rapidly extruded through a 27 G needle injection syringe and the droplets were allowed to fall into a gelation bath (100 mM CaCl₂ (Sangon Biotech, Shanghai, China) + 20 mM D-fructose (Sangon Biotech, Shanghai, China) + 1 × HEPES (Gibco, Waltham, MA, USA), pH 7.4). The droplets produced were allowed to settle for 10 min in the gelation bath to ensure gel formation, after which the microbeads were washed twice with 0.9% saline, and then resuspended in differentiation medium as described above to continue differentiation for 15 days according to the above differentiation method.

Proline was measured according to the method of Bates et al. (1973) (link) using the ninhydrin reagent. Proline content was estimated using an UV-VIS spectrophotometer at 520 nm and determined from calibration curve using L-Proline (Sangon, Shanghai, China) as standard. Sugars contents were determined according to the method as described by Liu et al. (2008) (link). Briefly, sugars were extracted from dried plant tissues in 80% ethanol at 80°C for 40 min. Sucrose, glucose, and fructose contents were read at 480, 630, and 480 nm and determined from calibration curve using Sucrose, Glucose and D-Fructose (Sangon, Shanghai, China) as standard, respectively.