Purification and Characterization of Plantaricin LPL-1, a Novel Class IIa Bacteriocin Produced by Lactobacillus plantarum LPL-1 Isolated From Fermented Fish

Abstract

Bacteriocins are ribosomally synthesized peptides or proteins possessing antibacterial activity against foodborne pathogens and spoilage bacteria. A novel bacteriocin, plantaricin LPL-1 was determined as a class IIa bacteriocin according to the YGNGV motif, and producer strain Lactobacillus plantarum LPL-1 was identified based on physio-biochemical characteristics and 16S rDNA sequence. The novel bacteriocin, plantaricin LPL-1 was purified by salt precipitation, cation exchange, gel filtration, and reverse phase high-performance liquid chromatography (RP-HPLC). The molecular mass of plantaricin LPL-1 was 4347.8467 Da by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis and entire amino acid sequence of plantaricin LPL-1 was VIADKYYGNGVSCGKHTCTVDWGEAFSCSVSHLANFGHGKC. Plantaricin LPL-1 possessed the merits of easy degradation by proteases, wide pH stability (2-10), high thermal stability (121°C, 20 min), surfactants stability and bactericidal activity against foodborne spoilage and pathogens bacteria. The mode action and membrane permeabilization of plantaricin was identified. The information of plantaricin LPL-1 indicated that it is not only a novel class IIa bacteriocin, but also a promising natural and safe biologic preservative for the food preservation industry.

Keywords: Lactobacillus plantarum; antibacterial activity; characteristics; mode of action; plantaricin LPL-1.

FIGURE 1

Dynamics of growth and production of plantaricin LPL-1 from L. plantarum LPL-1. Growth curve (); pH (); and Activity against indicator ().

FIGURE 2

Purification of plantaricin LPL-1 produced by L. plantarum LPL-1 by chromatography column. (A) Cation exchange column; (B) gel filtration chromatography; and (C): RP-HPLC process. (a) Process of purification; (b): antibacterial activity of absorbance peaks against indicator strain compared with CK (control test) by agar well diffusion assay; and (c): Tricine-SDS-PAGE of purified activity fraction.

FIGURE 3

Mass spectrum of plantaricin LPL-1 by MALDI-TOF-MS.

FIGURE 4

Alignment of reported class IIa bacteriocin, plantaricin 423, plantaricin C19, and plantaricin LPL-1 from L. plantarum. Alignments were obtained using Vector NTI 11.5 with default settings.

FIGURE 5

Effects of pH, temperature and surfactant on stability of plantaricin LPL-1. (A) pH; (B) temperature; and (C) surfactants.

FIGURE 6

Mode of action of plantaricin LPL-1 against the indicator cells of Listeria monocytogenes 54002. (): Viable cell count without bacteriocin; (Δ): viable cell count with bacteriocin; (): optical density at 600 nm without bacteriocin; and (□): optical density at 600 nm with bacteriocin.

FIGURE 7

Analysis of membrane permeabilization Listeria monocytogenes 54002 using RT-PCR instrument combined with SYTOX^TM Green Dead Cell Stain. (): Treatment with 128 μg/mL plantaricin; (•): treatment with 256 μg/mL plantaricin; and (): control test with sodium phosphate buffer.

FIGURE 8

CLSM images of dead Listeria monocytogenes 54002. (A) Control test with sodium phosphate buffer and treatment with 256 μg/mL plantaricin for 10 min (B), 20 min (C) and 30 min (D).