Sorvall lynx 4000 centrifuge

Microparticles containing VEGF was prepared by double emulsion solvent evaporation method (w/o/w) as described by in the literature with slight modifications (Anugraha et al., 2015; Rui et al., 2012). Briefly, 5 µg of rVEGF121 was dissolved separately in 0.25 mL of 10% polyvinyl alcohol (w/v) (PVA) (M.W. 30 to 70 kDa; 87%–90% hydrolyzed) (Sigma-Aldrich, Bangalore, India) and 100 mg of polylactide (M.W. 75–120 kDa (Sigma-Aldrich, Bangalore, India) in 2 mL of dichloromethane (50 mg/mL) was separately prepared. Both mixtures pooled together and sonicated for 2 min at a burst speed of 40% amplitude with a repetitive on/off cycle for 9 s. The emulsion was slowly added into 75 mL of 5% PVA under vigorous agitation in a magnetic stirrer. The microparticles formed through overnight solvent evaporation were centrifuged (Thermo scientific SORVALL LYNX 4000 centrifuge) at 10,000 × g/20 min/4 °C. The centrifugation was repeated twice with sterile distilled water to remove any surfactants. The pellet was lyophilized (Bench TopPro with Omnitronics Sp Scientific) overnight to form a free-flowing powder. The same way 200 µL of PBS was encapsulated with PLA to be used as a control.

Predator stock lysates were made by coculturing the predator and the prey (E. coli LF82, 10⁸ CFU/mL) inoculating pieces of YPSC medium double-layered plate in Diluted Nutrient Broth 2× (DNB2×) (Bacto Nutrient Broth 1.6 g/L, yeast extract 0.1 g/L, casaminoacids 0.5 g/L, CaCl₂ × 2H₂O 0.3 g/L, MgCl₂ × 6H₂O 0.6 g/L) [28 (link)]. The coculture was then incubated at 30 °C on a rotary shaker for at least 72 h, until the cultures cleared. The fresh co-culture was filtered three times with 0.45-μm pore-size filters (Millex^®, Merck KGaA, Darmstadt, Germany) to eliminate the prey cells. In order to ensure the effective elimination of the LF82, aliquots (10 µL) of the filtered co-culture were plated onto BHI agar plates. Further, the filtrated co-culture was washed three times at 29,000× g for 45 min (Sorvall LYNX 4000 centrifuge, Thermo Fisher Scientific Inc), re-suspending the pellet in 10 mL of phosphate-buffered saline (PBS) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for two cycles, and then in 2 mL of PBS after the last step. The predator concentration was then evaluated by counting the plaque-forming units (PFU) and seeding the B. bacteriovorus preparation onto a double-layered plate of YPSC medium as described above. We obtained a PFU count of between 5 × 10⁸ and 5 × 10⁹ PFU/mL. The predator stock was prepared fresh for each experiment.

Before measuring SSC, TA, and pH, homogenized fruit samples were thawed at 4°C and 35 g of each fruit sample were put in a 50-ml centrifuge tube and centrifuged (Sorvall Lynx 4000 Centrifuge; Thermo Fisher Scientific, Waltham, MA, United States) at 12,000 × g for 20 min at 4°C. The resultant supernatant was filtered through two-layer cheesecloth and used for the measurements. A few drops of the filtered supernatant were placed on the prism of a digital refractometer (model r2i300; Reichert Analytical Instruments, Depew, NY, United States) to measure SSC in Brix. A 3.0-ml aliquot of the supernatant was mixed with 50 mL DI water for measuring TA and pH, using a 905 Titrando automatic titration system (Metrohm USA Inc., Riverview, FL, United States) with TA measurements expressed as percent citric acid.