Model xl

A 1.0-ml aliquot of the 16-h culture of each LAB strain was centrifuged at 9,000g for 10 min at 4°C. The resultant extracellular supernatants and bacterial cells were harvested separately. The bacterial cells were washed twice with sterile PBS, resuspended in 1.0 ml of sterile PBS, and sonicated for 10 min with an ultrasonicator (Model XL, Misonix, Farmingdale, NY, USA). The sonicated bacterial cells were fractioned into intracellular extracts and cell-wall pellet fractions by subsequent centrifugation at 13,000g for 20 min at 4°C. The extracellular supernatants, intracellular extracts, and cell-wall fractions of each bacterial strain were harvested, and the anti-PEDV activities were evaluated as described above.

A 5-mL aliquot of the overnight cultures of the strains LN and ATCC 23350 was centrifuged at 9,000×g for 10 min at 4°C, and the pellets were washed twice with sterile PBS (0.1 M, pH 7.0). Then, the LN cells or ATCC 23350 cells were added to 5 mL of PBS (0.1 M, pH 7.0) containing 5 ppm of ZEN, to yield a final bacterial concentration of 1×10¹⁰ colony forming units (CFU) mL^-1. The mixtures were incubated at 37°C for 24 h on an orbital shaker at 120 rpm. During the incubation period, 1.0-mL aliquots were taken at 0, 4, 8, 12, 24, 36, and 48 h, and centrifuged at 17,000 ×g for 10 min at 4°C to harvest the cell pellets. The resultant supernatants were analyzed for ZEN concentration. The collected cells were resuspended in 1 mL of 0.1 M of phosphate-buffered saline (PBS; pH 7.4), sonicated for 10 min with an ultrasonicator (Model XL, Misonix, Farmingdale, NY), and fractioned into intracellular supernatant and cell-wall pellet fractions by subsequent centrifugation at 17,000×g for 10 min at 4°C. The cell-wall pellet was extracted by acetonitrile-water (84:16, v/v) and centrifuged at 13000×g for 20 min at 4°C. The supernatant was analyzed for ZEN concentration.