Purification And Identification Of The Bacteriocin Isolated From Lactic Acid Bacteria

Lactic acid bacteria (LAB), a kind of probiotics, play an essentialrole in food fermentation processes. It can inhibit or kill variousfood-borne pathogenic and spoilage bacteria, so it is beneficial to humanhealth. The antimicrobial effect may be due to the production of organicacids, hydrogen peroxide and diacetyl, as well as bacteriocins, a family ofantimicrobial peptides, synthesized by the ribosome. These substances areproteinaceous, antimicrobial compounds produced by bacteria usuallyonly inhibit closely related species. There has been a recent explosion inthe interest of bacteriocins as natural biopreservatives in food systemsand they have been considered to be the most effective alternative toantibiotics.Since nisin was approved for use in foods as a 'direct food additive',studies on bacteriocins have become one of the most active fields in the microbiological researches. The studies mainly involve the screening andidentification of bacteriocin-producing strains; the purification,biochemical properties, mechanisms, fermentation conditions andapplications of bacteriocins, as well as the constructions of theengineering strains. In recent years, although the studies of bacteriocinshave made significant progress in molecular level and applicationresearches, the purification efficiency is still not ideal. Therefore, the aimof this study here is to screen out a broad spectrum bacteriocin, establishan easy purification method and then identify the structure of thebacteriocin.1,Bacteriocins were screened from the 13 lactic acid bacteria by theagar well diffusion method, using Micrococcus luteus 10209 as theindicator. Five strains showed the antibacterial activity, among which theNum.8 strain L.plantarum DSM20205 (isolated from baby-waste),identified by PCR, exhibited the highest antibacterial activity and stability.In addition, the biochemical properties of the L.plantarum DSM20205were also detected here, the gelatin hydrolysis and urea test and so on areall negative, while most of the sugar fermentation reactions are positive.According to the involved references, this bacteriocin found here shouldbe the first isolated from L.plantarum DSM20205 and was namedplantaricin DSM20205 in this paper.2,The results showed that plantaricin DSM20205 belonged to the broad-spectrum LAB bacteriocin. It showed inhibitory not only againstsome Gram-positive bacteria, but also some Gram-negative bacteria. Afew Lactobacillus bacteriocins with an extended spectrum of activityhave already been described. The spectrum of plantaricin DSM20205 waseven wider, including Staphylococcus citreus, Staphylococcus aureus,Escherichia coli, Salmonella, Listeria monocytogenes, Pseudomonasaeruginosa and Pseudomonas putida.3,The biochemical characteristics of plantaricin DSM20205 inculdethe effect of heat, pH, enzymes on its activity. Results indicated thatplantaricin DSM20205 had good heat stability, strong tolerance to acidpH and nice sensitivity to some proteases, such as trypsin, pepsin andproteinase K.4,Compared with other separation and purification methods,ammonium sulfate gradient precipitation was used here to recover thebacteriocin from culture supernatant, the saturation was from 45ï¼…to 60ï¼….The fractionation (60ï¼…saturation) was further purified to homogeneityby ultrafiltration and gel filtration chromatography under certainconditionns. HPLC analysis verified the sample's purity. The molecularsize of plantaricin DSM20205 was 2366.056Da, estimated onTricine-SDS-PAGE and MALDI-TOF-MS. The results of N-terminalsequence analysis showed that this bacteriocin contained serine, leucine,proline, glutamine, asparagine.