Simple and rapid purification of pediocin PA-1 from Pediococcus pentosaceous NCDC 273 suitable for industrial application

Abstract

The use of pediocins as food additives or drugs requires a simple and rapid method by which large quantities of homogeneous pediocin are produced at industrial level. Two centrifugation steps required during initial stages of purification i.e. separation of cells from fermentation broth and collection of precipitates after ammonium sulphate precipitation are the major bottlenecks for their large scale purification. In the present work, pediocin production by a new a dairy strain, Pediococcus pentosaceous NCDC 273 (identical to pediocin PA-1 at nucleotide sequence level), was found to be optimum at initial pH of 6.0 and 7.0 of basal MRS supplemented with 20g/l of glucose or lactose at 20 and 24h, respectively. Immobilization of cells through entrapment in alginate-xanthan gum gel beads with chitosan coating resulted in negligible cell release during fermentation. Thus, the cell free extract was directly collected through decantation, avoiding the need of centrifugation step at this stage. Subsequent ammonium sulphate precipitation at isoelectric point of pediocin PA-1 (8.85), using magnetic stirrer at high speed (approx. 1200rpm), resulted in forceful deposition of precipitates on the wall of precipitation beaker allowing their collection using a spatula, avoiding centrifugation step at this stage also. Further purification using cation-exchange chromatography resulted in yield of 134.4% with more than 320 fold purification with the specific activity of 19×105AU/mg. The collection of single peak of pediocin at 41.9min in RP-HPLC, overlapping with standard pediocin PA-1, resulted in yield of 1.15μg from 20μl of sample applied. The overlapping of RP-HPLC peak and SDS-PAGE band corresponding to 4.6kDa, confirmed the purity and identity of pediocin 273 as pediocin PA-1.