Vice Lactobacillus Casei Prime Antibacterial Activity And Antibacterial Mechanism

Antibacterial substance that was produced from fermentation of Lactobacillus paracasei HD1.7 by extraction, separation, purification. The antibacterial substance was bacteriocin that was studied systematically at antibacterial activity and antibacterial mechanism in this work. The results offered a theoretical base for the development and ultilization of bacteriocin as a kind of natural food preservative of antibacterial bacteriocin. It is believed that the application of bacteriocin of Lactobacillus paracasei HD1.7 as a kind of natural food preservative will be very broadly in the future.The bacteriocin of Lb.parcaseiHD1.7produced by Lb.parcaseiHD1.7 is a small heat-stable peptide which can inhibit spoilage microorganisms in food. Recently, antibacterial mechanisms of Lactobacillus paracasei bacteriocin was that it can make sensitive cells death by affecting the cytoplasmic membrane. In this work, antibacterial activity and mode of action of Lactobacillus paracasei bacteriocin against spoilage microorganisms were studied and major findings were listed as follows:The results showed bacteriocin of Lb.parcaseiHD1.7 had obvious action in antibacterial upon Gram-negative bacteria and Gram-positive bacteria, and antibacterial spectrum was wide. The micro-dilution broth method was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the bacteriocin. To Staphylococcus aureus of Gram-positive bacteria, the MIC and MBC of bacteriocin were 77.5IU/mL; To Escherichia coli, the MIC and MBC of bacteriocin were 155 IU/mL and 310 IU/mL.In this experiment, the effects of pH value, temperature, and metal ion on bacteriocin of Lb.parcaseiHD1.7 antibacterial activity were investigated. The results showed that antibacterial activity of the bacteriocin was the best when pH values was 3.0; while pH values was adjusted to 6.0 ,antibacterial activity of the bacteriocin was greatly reduced. The bacteriocin had a good endurance to temperature. When the Staphylococcus aureus and Escherichia coli were treated by bacteriocin at 121℃for 30min, the antibacterial ratio was 78%. The effect of Na⁺, K⁺on activity was a little along with ion concentration. The bacteriocin was not only promoted but also inhibited; the effect of Mg²⁺,Ca²⁺ on activity were inhibited very strongly;the effect of Fe³⁺,Al³⁺ on activity promoted growth at first, and then inhibited growth at last.Lactobacillus paracasei bacteriocin can increase the cell membrane permeability of Escherichia coli and Staphylococcus aureus by electrical conductivity method. When Escherichia coli were treated by Lactobacillus paracasei bacteriocin (155IU/mL) for 6 h, the relative seepagevity ratio was 64.45%, and at the same time the permeability change was 52.51%. When Staphylococcus aureus were treated by bacteriocin (77.5IU/mL) for 6 h, the relative seepagevity ratio was 75.32%, and at the same time the permeability change was 72.19%. The result showed the membrane permeability was affected by the bacteriocin. The relative seepagevity ratio of membrane obviously ascends which resulted in the dissipation of the proton motive force (PMF) and induced the cells death.The results indicate that fluorescent probe is feasible in the determination of the alteration of Escherichia coli and Staphylococcus aureus. When Escherichia coli were treated by Lactobacillus paracasei bacteriocin for 4.5h, flurescent intensity of FDA declined gradually with the increase of time. The number of living Escherichia coli was less than the control about 1.83% from 0.5h to 1h. The relative increasing ratio was 41.55% from 1h to 5h. The results showed that Lactobacillus paracasei bacteriocin can increase the cell membrane permeability of Escherichia coli, break the cell membrane and inhibit the growth of Escherichia coli. When Staphylococcus aureus were treated by Lactobacillus paracasei bacteriocin for 0.5h, flurescent intensity of FDA declined strongly. When Staphylococcus aureus were treated by Lactobacillus paracasei bacteriocin from 1h to 5h, flurescent intensity of FDA declined gradually with the increase of time. The number of living Staphylococcus aureus was less than the control about 14.1% from 0.5h to 1h. The relative increasing ratio was 60.67% from 1h to 5h. The results showed that Lactobacillus paracasei bacteriocin can quickly kill Staphylococcus aureus by break the cell membrane with the fluorescent probe penetrating to the outside.The leakage of nucleic acid and big protein from cells of determination that turned out a large nucleic acid obviously ascended when Escherichia coli were treated by Lactobacillus paracasei bacteriocin for 3h, and then big protein ascended after 6h. The result showed Escherichia coli were damaged by bacteriocin for the leakage of nucleic acid and big protein partly. It was increasing of the leakage of nucleic acid and big protein from Staphylococcus aureus treated by bacteriocin at 3 h later. The result showed Staphylococcus aureus were broken strongly by bacteriocin. There would be a pore on the cell membrane of Staphylococcus aureus, which ensured killing of target bacteria.