4 m naoh

For the spray drying procedure previously described, methods were used with slight modifications (29 (link), 35 (link)). Initially, excipients and phages at titers of 5 × 10¹⁰ PFU/mL (1% phage added to final volume) were dissolved in ultrapure water to make up a 200 mL volume solution. The excipients tested were trehalose (Glentham Life Sciences Ltd, UK), leucine (Glentham Life Sciences Ltd, UK), mannitol (Fisher Scientific, UK) and Eudragit S100 (Evonik, Germany). Eudragit S100 was dissolved in ultrapurified water (5% wt/vol) by the addition of 4 M NaOH (Fisher Scientific, UK) drop by drop, until the solution turned clear, which indicated polymer dissolution (26 (link)).
The excipient-phage solution was spray dried using a laboratory scale LabPlant Spray Dryer (UK) with a two-fluid nozzle for atomization, and the nozzle had a diameter of 0.5 mm (Fig. 1a). Air speed of 4.3 ms⁻¹ and liquid flow rate of 280 mlh⁻¹ were used. The drying temperature was controlled by changing the inlet temperature from 80°C to 100°C, and the corresponding outlet temperatures varied from 40°C to 60°C. Dried powder phages were passed through the cyclone, collected in 100 mL glass bottles, and stored at 4°C until use. To determine phage titer, 0.10 g of dried powder phage was suspended in 900 μL phage suspension buffer, diluted 10-fold, and titered by plaque assays (Kropinski et al., 2009).

Anaerobic (N₂-sparged) batch fermentations were performed in triplicate using 10% fecal slurry (1% fecal inoculum) under controlled conditions [water-jacket vessels (Soham Scientific, Soham, United Kingdom), pH 6.8, temperature 37°C]. Basal medium contained per liter: 2 g peptone (Oxoid, Basingstoke, United Kingdom), 2 g yeast extract (Oxoid), 0.1 g NaCl (Fisher Scientific, Fair Lawn, NJ, United States), 0.04 g K₂HPO₄ (BDH, Toronto, ON, Canada), 0.04 g KH₂PO₄ (BDH), 0.01 g MgSO₄7H₂O (BDH), 0.01 g CaCl₂6H₂O (Honeywell, Morris Plains, NY, United States), 2 g NaHCO₃ (Oxoid), 2 mL Tween 80 (Sigma–Aldrich, Oakville, ON, Canada), 0.05 g Hemin (Sigma–Aldrich) dissolved in 1 mL of 4 M NaOH (Fisher Scientific), 10 μL Vitamin K (Sigma–Aldrich), 0.5 g l-Cysteine HCL (Sigma–Aldrich), 0.5 g Bile Salts (Oxoid), and 4 mL of Resazurin (Sigma–Aldrich) (0.025 g/100 mL). Vessels were dosed with the substrates (1% wt/vol) after simulated in vitro upper gastrointestinal digestion and dialysis (Mandalari et al., 2008 (link)) and inoculated with 10% fecal slurry. The final volume of each culture was 200 mL. Samples were harvested at time points 0 (immediately after incubation) and 24 h.

For the spray drying procedure previously described, methods were used with slight modifications (29 (link), 35 (link)). Initially, excipients and phages at titers of 5 × 10¹⁰ PFU/mL (1% phage added to final volume) were dissolved in ultrapure water to make up a 200 mL volume solution. The excipients tested were trehalose (Glentham Life Sciences Ltd, UK), leucine (Glentham Life Sciences Ltd, UK), mannitol (Fisher Scientific, UK) and Eudragit S100 (Evonik, Germany). Eudragit S100 was dissolved in ultrapurified water (5% wt/vol) by the addition of 4 M NaOH (Fisher Scientific, UK) drop by drop, until the solution turned clear, which indicated polymer dissolution (26 (link)).
The excipient-phage solution was spray dried using a laboratory scale LabPlant Spray Dryer (UK) with a two-fluid nozzle for atomization, and the nozzle had a diameter of 0.5 mm (Fig. 1a). Air speed of 4.3 ms⁻¹ and liquid flow rate of 280 mlh⁻¹ were used. The drying temperature was controlled by changing the inlet temperature from 80°C to 100°C, and the corresponding outlet temperatures varied from 40°C to 60°C. Dried powder phages were passed through the cyclone, collected in 100 mL glass bottles, and stored at 4°C until use. To determine phage titer, 0.10 g of dried powder phage was suspended in 900 μL phage suspension buffer, diluted 10-fold, and titered by plaque assays (Kropinski et al., 2009).