Acetic acid

Alginate (300 kDa), gelatin (bovine skin), phosphate-buffered saline (PBS), quercetin, calcium chloride, and acetic acid were obtained from Hi-Media, India Limited. The samples were prepared and characterized in PBS and double-distilled water.

Lipopolysaccharide (LPS) was purified from S. Typhimurium, using the hot phenol method as reported earlier (31 (link)). Briefly, Salmonella Typhimurium LT2 strain was cultured in Luria Broth (1L) at 37°C for 10h (OD 1.8). Formalin-inactivated cells were collected by centrifugation at 5000 g and washed twice with PBS. After resuspension in PBS (30 ml), 90% phenol (HiMedia) was used for 30 min at 68°C to extract crude LPS from the cell pellet. The suspension was centrifuged at 7,300 × g at 10°C for 1 h. The aqueous layer (60 ml) was precipitated with ethanol (final concentration 75%), and the precipitate was treated with DNAse (1 μg/mL), RNase (1 μg/mL) and Proteinase K (100 μg/mL) (all purchased from Roche), followed by dialysis overnight against PBS (pH 7.4) at 4°C. Extracted LPS was used to further extract O-specific polysaccharide (OSP). to this end, LPS was incubated with 1% acetic acid (HiMedia), pH 3.0, and 100°C for 1.5 hours, followed by ultracentrifugation at 64,000 ×g for 5h at 10°C, using a WX+ Ultra series centrifuge (Thermo Scientific). The supernatant was dialyzed for 48h against PBS (pH 7.4) and OSP was purified by size exclusion chromatography.