Avantitmj 25

A 500 ml cultures of T. kivui were grown in complex or defined medium to late exponential growth phase (OD₆₀₀ of 1.7 to 2.3) and then harvested by centrifugation (Avanti^TMJ-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 7,000 × g and 4°C for 10 min. The harvested cells were washed with 30 ml of the respective medium by centrifugation at 8,500 rpm (5948 × g) and 4°C for 10 min (Avanti^TMJ-25 and JA-25.50 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States). Then, the cells were resuspended in 5 ml of the respective medium and kept in 16 ml Hungate tubes. Resuspended cells were distributed into in Hungate tubes to a final volume of 10 mL and a final protein concentration of 10 mg ml^–1. All the steps were performed under strictly oxygen free conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI, United States) filled with N₂/CO₂ (80/20; v/v) for carbonate medium or with 100% N₂ for carbonate free medium. As substrate, 25 mM glucose or 25 mM mannitol was added to the resting cells. The experiment started with incubation at 65°C in water bath with shaking (150 rpm). 0.8 ml subsamples were taken for determination of protein, substrate and product concentration. The total protein concentration in the cell suspension was measured using the method by Schmidt et al. (1963) (link).

Preparation of resting cells were performed under anoxic condition. Cells of T. kivui wild type and ∆cooS were grown on glucose or on glucose + 100% CO in the headspace, in 500 ml of complex media to mid exponential phase and were harvested by centrifugation (AvantiTMJ-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 11,500 × g, 4 °C for 10 min. The supernatant was discarded and the cells were washed three times with imidazole buffer (50 mM imidazole, 20 mM MgSO₄, 20 mM KCl, 20 mM NaCl, 4 mM DTE, 4 μM resazurin, pH 7). After centrifugation, cells were resuspended in 5 ml of same buffer and kept in 16 ml gas tight Hungate tubes. The headspace of the Hungate tubes were changed to 100% N₂. The protein concentration was measured according to (Schmidt et al. 1963 (link)).

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).