Calcium chloride

To evaluate the effect of fat on L. monocytogenes resistance to gastric fluid, the simulated gastric fluid (Czuprynski et al., 2002 (link)) was prepared by combining 8.30 g proteose- peptone (Sigma-Aldrich, USA), 3.50 g D-glucose (Samchun Pure Chemical Co. Ltd., Korea), 2.05 g sodium chloride (Duksan Pure Chemicals, Korea), 0.60 g potassium phosphate (Duksan Pure Chemicals), 0.11 g calcium chloride (Samchun Pure Chemical), 0.37 g potassium chloride (Duksan Pure Chemicals), 0.10 g lysozyme (Wako Pure Chemical Industries Ltd., Japan), 50 mg bile salt (Sigma-Aldrich), and 13.30 mg pepsin (Yakuri Pure Chemical Co. Ltd., Japan) per liter of distilled water. The simulated gastric fluid was adjusted to pH 2.0, using 1 N HCl. The frankfurters were transferred from vacuum packages to a filter bag (BagFilter^®, Interscience, France) containing 50 mL of simulated gastric fluid, and the samples were homogenized with a pummeler (BagMixer^®, Interscience, France) for 30 s. The homogenates were then placed in a water bath at 37℃, and samples were analyzed at 0, 30, 60, 90, and 120 min. The homogenates were diluted with BPW, and 0.1-mL portions of the diluents were then plated on TSAYE and Palcam agar to determine survivals of total bacteria and L. monocytogenes, respectively. The plates were incubated at 30℃ for 48 h, and colonies were manually counted.

The raw silk cocoons (Bombyx mori, Uljin silk farm, Korea) were cut into small pieces and degummed two times with 0.3 mM sodium oleate (Junsei, Japan) and 0.5 mM sodium carbonate (OCI, Korea) solution at 103°C for 40 min. Remnant salt was removed by boiling silk in distilled water twice at 100°C for 20 min [25 (link),26 (link)]. The obtained silk fibroin (SF) was rinsed thoroughly with distilled water and dried in a fume hood at room temperature for two days. Dried SF was dissolved in a ternary solvent consisting of calcium chloride (Samchun, Korea), distilled water, and ethyl alcohol (Duksan, Korea) with the molar ratio of 1:8:2. The reaction was continued at 91°C for 3 h. The dialysis was performed with a cellulose membrane tube (MWCO: 12–14 kDa) against distilled water. The distilled water was exchanged seven times every 5 h for 3 days at room temperature. The clear supernatant was collected after centrifugation (9000 rpm for 10 min) and lyophilized (FD8508, IlShinBioBase, Korea). The SF powder was vacuum-packed for long-term preservation.