Proteinase k

EPS was isolated from JW15, as described in previous study (40) , Briefly, single colony was inoculated in sucrose broth (1% tryptone, 0.5% yeast extract, 0.5% dipotassium phosphate, 0.5% diammonium citrate, 5% sucrose, pH 7.0) and incubated at 37℃ in a shaking incubator for 48 h. Afterward, the sucrose broth was added with 4% (w/v) trichloroacetic acid (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 4℃ for 2 h. After centrifugation (10,000 × g, 4℃, 25 min), the supernatant was collected and precipitated with two volumes of cold 95% ethanol at 4℃ overnight, then centrifugated (10,000 × g, 4℃, 25 min). The resultant pellet was resuspended in triple distilled water. It was named as the crude EPS solution and treated with 50 μg/mL of DNase Ⅰ (Sigma-Aldrich) and RNase A (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37℃ for 6 h, followed by 200 μg/mL of proteinase K (GeneAll, Seoul, Republic of Korea) under the same condition. The solution was mixed with two volumes of chilled 95% ethanol and precipitated at 4℃ overnight. The EPS pellet was collected by centrifugation (10,000 × g, 4℃, 25 min), washed with 70% ethanol, and re-centrifuged. After the supernatant was removed, the purified EPS pellet was dissolved in triple distilled water and lyophilized.