Screening Of A Nisin-Producing Strain And Studying On Its Antibacterial Mechanism

In this thesis,screening and identification of a Nisin-producing strain,the optimal Nisin production condition,extraction and purification of Lactobacillin produced by the strain and antibacterial mechanism are studied systematically.21 strains of Lactic acid bacteria isolated from the leaf of Chinese cabbage have resistance to Nisin.By Oxford plate assay system,one strain with strong antibacterial activity designated as T0625 is screened from them.Based on the result of measurement,the antibacterial activity of the fermented broth is thermo-stable under the low pH,and insensitive to pepsin and trypsin as well.The fermented broth shows inhibition to some gram-positive bacteria,in contrast,little inhibition activity to gram-negative bacteria,yeast or mould.By morphology observation,physiological and biochemical identification and 16S rDNA sequencing,the strain of T0625 is finally identified as Lactococcus lactis subsp.Lactis and named Lactococcus lactis subsp.Lactis T0625.By single factor experiments and orthogonal experiments,the optimum culture medium and optimal fermentation conditions of producing Lactobacillin are obtained as follows:2% glucose,4%peptone,2%yeast extract,1%beef extract,0.003mol/L MgSO₄·7H₂O,0.05% Vitamin C;poured solution volume of 250mL(in 250mL triangle bottle),inoculated amount of 7%,initial pH of 6.5,culturing temperature at 35℃for 12 hours.By(NH₄)₂SO₄ salting-out,dialysis and Sphadex G-50 column chromatography, Lactobacillin produced by T0625 is purified.SDS-PAGE electrophoresis is used for determining the effect of purification.The electrophoretic band of Lactobacillin after purification is the same as the band of Standard of Nisin.This Lactobacillin is named Nisin T0625.The antibacterial mechanism of Nisin T0625 is studied preliminarily.The antibacterial tests are conducted after adding Nisin T0625 into Staphylococcus aureus broth.The results show that the content of protein and the conductivity of Staphylococcus aureus broth increase. The exponential phase of growth doesn't occur.Observation under the transmission electron microscope reveals that Nisin T0625 can make the cell distorted,cell plasmolysis.Thereafter the cell is broken and the cytoplasm flow out until it is decomposed even into irregular residues.SDS-PAGE shows that the content of protein decreases in Staphylococcus aureus under Nisin T0625 treatment,in contrast,the content of protein in the broth increases.It can be hypothesized that the action site of Nisin T0625 is cell membrane,whose integrity is damaged by Nisin T0625.The results prove that the antibacterial mechanism of Nisin T0625 is similar with that of Nisin,namely,the results support the viewpoint of "Pore Formation".