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 20 g/l of glucose or lactose at 20 and 24 h, 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. 1200 rpm), 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 19×10⁵ AU/mg. The collection of single peak of pediocin at 41.9min in RP-HPLC, overlapping with standard pediocin PA-1, resulted in yield of 1.15 μg from 20 μl of sample applied. The overlapping of RP-HPLC peak and SDS-PAGE band corresponding to 4.6 kDa, confirmed the purity and identity of pediocin 273 as pediocin PA-1.

Optimization of Culture Conditions for Bacteriocin Production by Soil Isolates Pediococcus pentosaceus LB44 and Weissella confusa LM85

“…The optimum temperatures for the production of pediocin by P. pentosaceus ST18 and P. pentosaceus ACCEL were 30 and 37°C, respectively (21,22). The maximum bacteriocin production by P. acidilactici 13 and P. pentosaceous NCDC 273 was reported at 37°C (8,13). Optimum bacteriocin production by L. casei LA-1 was reported at 26 to 37°C (4).…”

“…The effect of different carbon sources on growth and bacteriocin production was studied as described by Vijay Simha et al (8). MRS medium containing different concentrations of glucose and lactose (10,20,30 and 40 g/L) were prepared in different sets and filter-sterilized.…”

“…Optimum growth and bacteriocin production depends on various factors such as pH, temperature, NaCl concentrations, and carbohydrate source (4,(6)(7)(8)(9)(10). These parameters are generally specific to producer strains (4).…”

Produção de bacteriocina por Bifidobacterium lactis a partir de soro de leite

“…Estas bacteriocinas podem ser adicionadas tanto parcialmente purificadas quanto purificadas em concentrados, o que exige a aprovação específica como conservantes, do ponto de vista legal. Até agora, a nisina e a pediocina PA-1 são as únicas bacteriocinas licenciadas como conservantes de alimentos (SIMHA et al, 2012). Muitos estudos preliminares sobre a atividade de bacteriocinas in vitro ou em sistemas alimentares estão sendo realizados com preparações parcialmente purificadas, obtidas a partir de caldos de fermentações de culturas, mas, na maioria dos casos, uma baixa concentração de bacteriocinas é recuperada HOLZAPFEL, 1996;STILES, 1996), o que demonstra a importância de se abordar esforços nas técnicas de purificação.…”

“…Bacteriocins can be added as partially purified or purified concentrates, which would require specific approval as preservatives from the legislative viewpoint. So far, nisin and pediocin PA-1 are bacteriocins licensed as food preservatives (Simha, Sood, Kumariya, & Garsa, 2012). Many preliminary studies on the activity of bacteriocins in vitro or in food systems are carried out with partially-purified preparations obtained from culture broths, but in the most cases a low concentration of bacteriocin is often recovered (Schillinger et al, 1996;Stiles, 1996), which demonstrates the significance to address many efforts in this direction.…”