Isolation, Characterization Of Lactobacillus Plantrum163 And Its Bacteriocins Study

Bacteriocin of Lactic acid bacteria (LAB) is an antimicrobial peptide ribosomally synthesized by LAB against other bacteria, either within the same species, or across different genera. They have been considered "Generally recognized as safe" (GRAS) and can be used as food additives, feed additives and also in pharmaceutical preparations.The study of bacteriocins produced by LAB has become a worldwide focus of scientific research. Many bacteriocin producers have been identified and investigated from fermented food, so the discovery of new bacteriocin producers by LAB with broad-spectrum antibacterial activity from fermented foods has received great attention. In order to obtain lactic acid bacteria strain with application prospect, we collected and isolated LAB from traditional fermented foods of Yungui Plateau in the southwest China, and investigated the bacteriocin characteristics such as the primary structure, antibacterial mode, antibacterial spectrum and its stability. The main results are as follows:1. Screening and identification of Bacteriocin Producers430 strains of presumptive LAB isolated from traditional fermented foods produced in Guizhou province were screened for bacteriocin-producing strains. After agar-spot-test about 110 presumptive strains of LAB were exhibited antibacterial activity against S. aureus and E. Coli. However, by using the agar well diffusion assay method, the five strains were cultivated in MRS broth and their cell-free supernatants showed the excellent antibacterial activity after eliminating the effect of low pH and catalase. We selected the strain 163 as a candidate for this study. The strain 163 was identified as Lb. plantarum on the basis of its physiobiochemical characteristics and 16S rDNA sequencing (GenBank access number is JX524228), and named as Lb. plantarum 1632. Purification, structure identification and properties of Plantaricin 163 and Plantaricin 163-1Lb. plantarum 163 entered to logarithmic growth phase after inoculation for 4 h. The OD600 of fermentation broth increased and pH decrease rapidly, antibacterial activity begined to appear. Lb. plantarum 163 was in stationary phase after 10 h, The OD600 of fermentation broth OD600 and pH change smoothly, but antibacterial activity was exponentially increase. Lb. plantarum 163 reached to decreasing phase after 30 h, the OD600 and pH, antibacterial activity increased smoothly.Through the salt precipitation, gel filtration and reversed-phase chromatography (RP-HPLC), Plantaricin 163 and Plantaricin 163-1 were obtained. The complete amino acid sequence of plantaricin 163 was speculated as to VFHAYSARGNYYGNCPANWPSCRNNYKSAGGK and Plantaricin 163-1 as to YVCASPW based on MALDI-TOF-MS/MS, N-terminal sequence analysis and amino acid composition analysis. Plantaricin 163 and Plantaricin 163-1 were identified to be novel bacteriocins by comparison with the literature published and BLAST searches. Plantaricin 163 was highly stable in the high temperature and broad pH range, but sensitive to protease and exhibited a wide range of antimicrobial activity not only against LAB but also other foodborne pathogens, including Gram-positive and Gram-negative bacteria. The isoelectric point of plantaricin 163 was 9.66, the average hydrophilicity was -0.2, hydrophilic residue ratio was 38%, the net charge of pH 7 was 4, and the probability to form the loop structure was 87.5%. Similarly, Plantaricin 163-1 was also highly stable in the high temperature and broad pH range, but sensitive to protease K and pepsin, and exhibited a wide range of antimicrobial activity against not only LAB but also other foodborne pathogens including Gram-positive and Gram-negative bacteria. The isoelectric point of Plantaricin 163-1 was 6.2, hydrophilic residue ratio was 38%, the net charge of pH 7 was 4.3. Screening and optimization of food grade medium of Lb. plantarum 163Food-based medium for fermentation of the bacteriocin with Lb. plantarum 163 was screened (cabbage juice 200 mL/L, tomato juice 50 mL/L, glucose 10 g/L, K2HPO4 2 g/L and distilled water 1L). The key factors of the fermentation with Lb. plantarum 163 were determined as to be the inoculum concentration, K2HPO4, pH and cabbage juice by using Plackett-Burman Design. The quadratic polynomial mode for food-based medium of Lb. plantarum 163 was obtained by Box-Behnken Design. The best fermentation conditions were K2HPO4 1.89 g/L, cabbage juice 341.5 mL/L, inoculum concentration 3.56% (v/v) and pH 6.95. Under these conditions, the maximum antimicrobial activity was obtained, that was increased by 30% comparison with that befor the optimizing.5. Antibacterial mode of Plantaricin 163-1We also studied the antibacterial mode of Plantaricin 163-1 against S. aureus ATCC 25923 and E. Coli ATCC 25922 from macro, cellular and subcellular level. At the macro level the plantaricin 163-1 could kill S. aureus and E. coli, but the minimum inhibited concentration was different between of S. aureus, and E. coli.12.8 Au/mL of the plantaricin 163-1 could fully inhibite the growth of S. aureus but not did E. coli. The cell contents (nucleic acid, protein and lactate dehydrogenase) of S. aureus and E. coli, was leaked out by the treatment of the plantaricin 163-1, showed that the cell membrane of the bacteria was disrupted. By the observation of electronic microscope, it was found the formation of hole in cell wall with the plantaricin 163-1, indicated that the antibacterial mode of the plantaricin 163-1 could be hole model.