Alginate

The encapsulation of a phage cocktail—S4lw and D5lw—was conducted as previously described with minor modification^{29 (link)}. First, phages S4lw and D5lw at approximately 10⁹ PFU/mL were mixed together at a 1:1 ratio as a phage cocktail. A total of 100 mL of phage cocktail in SM buffer was added with 0.02 M Na₂CO₃ (Sigma-Aldrich, Oakville, Ontario, Canada) and around 1.2—1.8% sodium alginate (Buchi, Switzerland), depending on nozzle sizes used for the encapsulation. After alginate was dissolved, the phage-alginate mixture was subject to encapsulation using a Buchi B-390 encapsulator (Buchi, Switzerland) with different nozzle sizes (200, 300, 450, 750, and 1000 μm) accordingly. At the same time, a clean beaker containing 200 mL of 1.8% CaCl₂ (Sigma-Aldrich, Oakville, Ontario, Canada) with a low-rate spinning stir bar was used to catch and harden the resulting phage-encapsulated beads from the encapsulator. After solidifying, the beads were separated from the solution and washed with SM buffer (three times) to remove any free phages. The beads were stored in the SM buffer at 4 °C for further experiments.

Previously it has been shown that of the three LAB strains, L. plantarum strain 22F gave the best in vitro performance^{19 (link)}. Hence, this strain was selected to use in the microencapsulation procedure. Alginate (1% w/v) (Sigma-Aldrich, Missouri, USA) and chitosan (0.4% w/v) (Union Chemical 1986, Bangkok, Thailand) were used as inner and outer wall materials. A total of 10⁹ CFU/mL of L. plantarum strain 22F was added at a ratio of 1:5 (v/v) to Alginate solution. The mixture was atomized through a spray dryer (Mini Spray Dryer B-290, Buchi, Flawil, Switzerland) with the inlet temperature set at 130 °C, and then the Alginate powder was collected. One gram of this powder was added to 100 mL of chitosan solution before atomizing through the spray dryer under the same conditions as previously described. These double-coated powders containing L. plantarum strain 22F were recovered from the collecting vessel and stored at room temperature for 6 months before use⁵⁹ .

Initially, 10 g of sodium alginate (Buchi, France) was dissolved in 400 mL of an aqueous 4% (w/v) glucose solution to produce 500 mL of sodium alginate solution at 2%. ASCs from each donor were dispersed in 20 mL of this 2% alginate solution, at 4 × 10⁶ cells/mL. Then, cells were encapsulated by the prilling vibration technique using the B-390 Encapsulator (Buchi, France) fitted with a 150-µm nozzle. The laminar jet of the suspension through the 150-µm nozzle was adjusted to a flowrate of 3.1 mL/min. The vibration frequency, applied to split the laminar jet into fine droplets, was set to 366 Hz. Finally, the voltage of the electrode forming an “umbrella” at the nozzle outlet (to avoid microparticles sticking together) was adjusted to 2000 V. The droplets of sodium alginate containing encapsulated cells then encountered the aqueous jellification solution, containing 76 mM calcium chloride (Calbiochem, France), 85 mM glucose (Macopharma, France), and 6 mM HEPES (Sigma Aldrich, St. Louis, MO, USA) at physiological pH and osmolarity, where the alginate microparticles were formed.
Microparticles were rinsed with lactated Ringer’s solution (Macopharma, France) and stored in ASC culture medium.