Molecular Mass Estimation of Bacteriocin

The molecular mass of the purified bacteriocin was estimated by Tricine SDS PAGE 16.5% gel and Zymogram [17 (link),18 (link)]. The aliquot of purified bacteriocin and crude extract (CRE) were loaded in duplicate wells along with the Precision plus protein™ dual Xtra prestained protein standard (Bio-Rad Laboratories Inc., Hercules, CA, USA). The samples were electrophoresed in Tris-Tricine buffer solution at 10 mA for 4 h (Cleaver Scientific Ltd., Rugby, UK). The gel was divided so that each half contained both samples. One half-gel was stained with Coomassie blue, followed by a destaining step. Another half was used to detect the in-gel antimicrobial band by a Zymogram assay. It was fixed in a fixing solution (40% ethanol and 10% acetic acid) for 30 min, washed in water for 2 h, and placed onto a 7-mm layer of Mueller–Hinton agar (0.8% agar, 3% NaCl) inoculated with V. parahaemolyticus cells (~10⁶ CFU/mL). Another 7 mL of melting Mueller–Hinton ½ strength agar (0.8% agar, 3% NaCl) inoculated with V. parahaemolyticus cells was poured over the gel. After solidification, the plate was incubated for 8–12 h at 30 °C. An inhibition zone appeared in the gel if the samples contained bacteriocin.