Calcium chloride 0.1 m

The L. salivarius spp. salivarius microorganisms were encapsulated by homogenization pressures using the methodology described by [16 (link)] with some modifications. Briefly, a mixture of 25 mL of microorganism solution containing 10¹⁰ CFU/mL of L. salivarius isolated by centrifugation at 7700× g for 15 min at 10 °C (Beckman Coulter AvantiTM J-25, Brea, CA, USA), 100 mL of sodium alginate (3%) (Sigma-Aldrich, Steinheim, Germany), 1 mL of tween 80 (Sharlau, Sentmenat, Spain), and 200 mL of commercial sunflower oil was homogenized at 70 MPa in two passes (Panda Plus Niro Soavi, Parma, Italy). Calcium chloride 0.1 M (Sigma-Aldrich, Steinheim, Germany) was used to break the emulsion and kept overnight at 4 °C to separate the phases. Microcapsules were isolated from the liquid phase by centrifugation at 7700× g for 15 min at 10 °C (Beckman Coulter AvantiTM J-25, Brea, CA, USA).