**3.3. Purification and characterization of bacteriocins**

Purification of bacteriocin peptides or small proteins into homogeneity is necessary in order to fully characterize them, particularly the determination of molecular mass, the primary structure or amino acid sequence and secondary structure. For pediocin, it was found that a simple and rapid method is effective for its purification. This method involves adsorption of pediocin onto the cell wall of the producer cell at pH 6 and 0.05 M NaCl and then subsequent desorption at pH 2.0 and 1 M NaCl (Elegado et al., 1997; Yang et al., 1992). This method seemed more applicable to pediocin but not with the lactococcin, nisin or plantaricin. The reason is not clear but it could be related to variation in cell wall properties. The pH-adsorption/desorption method was able to provide materials for pH and temperature tolerance assays, estimation of molecular mass through SDS-PAGE, residual activity determination after protease, amylase and other enzyme actions (Laxamana et al., 2011). Enough amount of semi-purified bacteriocin from pediococci using this method was obtained for further purification through preparative reverse phase HPLC for various characterization studies, including the determination of secondary structures by circular dichroism and confirmation of double bonds through trypsin digestion and electrospray mass spectrometry (Elegado and Kwon, 1998). Other preparative purification methods prior to reverse phase HPLC and spectrometry included ion exchange chromatography and gel filtration chromatography (Elegado et al., 2003), and hydrophobic interaction chromatography (Villarante et al., 2011). This method could also be applied with bacteriocins of pediococci and lactobacilli. The properties obtained from well characterized bacteriocinogenic LAB are shown in Table 3.


**Table 3.** List of purified and characterized bacteriocins from LAB isolated from Philippine indigenous fermented foods.
