Production And Physicochemical Characterization Of Bacteriocin Lactobacillin XH1Produced By Lactobacillus Acidophilus

Lactic acid bacteria bacteriocin is a kind of polypeptide, protein or protein compound withantibacterial activity which is produced by the mechanism of ribosome synthesis in the process ofmetabolism. It can inhibit other Gram-positive bacteria, such as Lactococcus lactis, grapes bacteria,Listeria monocytogenes, et al, but not the majority of Gram-negative bacteria, fungi. Bacteriocin producingspecies have now been identified in all genera of LAB including Lactococcus, Streptococcus, Lactobacillus,Leuconostoc, Pediococcus and Enterococcus. These probiotics can improve the intestinal ecology, and is akind of potential natural food preservatives, has become a hot to research and development applications.This paper studied isolation and purification of La-XH1bacteriocins produced by Lactobacillusacidophilus which was domesticated and preserved by our laboratory, and then analyzed thephysicochemical properties and inhibitory mechanism.1. After ruling out the influence of organic acids and hydrogen peroxide with flat-dug wells agardiffusion method, some biological characteristics of Lactobacillin XH1bacteriocin produced byLactobacillus acidophilus, such as antibacterial activities, thermal stability, the optimum pH and thesensitivity of protease, were researched. The results showed Lactobacillin XH1, a kind of broad spectrumantimicrobial substance, could inhibit Escherichia coli, Staphylococcus aureus and Bacillus anthraci. Itwas active under pH1.0-5.0and the optimum pH was2.0. Its activity nearly unchanged after being treatedfor20min at121℃and had certain thermal stability. It was sensitive to trypsin, pepsin and papain, butinsensitive to proteinase K and neutral protease.2. Lactobacillin XH1bacteriocin produced by Lactobacillus acidophilus was extracted by twomethods, chloroform extraction and ammonium sulfate gradient precipitation (the saturation was from60%to80%, the precipitate was collected, dissolved in water, and concentrated). Two kinds of crude extractsshowed significant inhibitory effect. The fractionation was further purified to homogeneity by cellufineA-500anion-exchange chromatography. The anti-microbial activity was determined. The active fractionswere pooled and dialyzed. Activity was estimated by the critical dilution method. The bacteriostasispotency and specific activity of the two crude extracts were significantly increased. Tricine-SDS-PAGEshowed that its molecular weight was between14.4～20.1kDa. 3. Antibacterial mechanism of Lactobacillin XH1bacteriocin on Escherichia coli was investigatedthrough the cell membrane damaged and changes of intracellular material leaked in Escherichia coli cells.E. coli cells produced holes, and cell walls were partially damaged. It could be due to the bacteriocinmolecules embedded in the cell membrane to enhance the membrane permeability. The leakage ofultraviolet substances, lactate dehydrogenase macromolecules leaded to disruption of cellular matter andenergy metabolism. The concentration of intracellular K+of E. coli decreased rapidly, which resulted in theimbalance of membrane potential. And to maintain the membrane potential, the intracellular ATP wasconsumed. Which coule be causing the cells to die eventually. This research laid a foundation forapplication of Lactobacillin XH1bacteriocin and studying on its structure-activity relationship.