Antimicrobial Activities And Mechanisms Of Native Lactofarrin And Recombinant Lactoferrin In Vivo And In Vitro

The antimicrobial activities of native lactoferrin (LF), lactoferricin (Lfcin) and recombinant porcine lactoferrin (rPLF) including test Sf9 cells and silkworms were determined using broth microdilution method, agarose diffusion method and time-kill method in susceptibility test, with clinically used antibiotics controlled. Atomic force microscopy (AFM) was used to study the antimicrobial mechanism. Feeding experiment was conducted to evaluate the effect of rPLF on intestinal microflora and diarrhea rate of weanling pigs and approach to the mechanism. Ninety weanling pigs (Duroc LandracexYorkshine ternary crossed pigs) were allotted randomly to three groups by weight and sex, and each group included three replicates of ten pigs. The control group received the basal diet, and the treatment 2 and 3 received the diets supplemented with antibiotics and rPLF respectively. The pigs were fed experiment diets for 15 days after pretest for 5 days, which were provided with adequate feed and water. By the end of feeding experiment, all pigs were weighed, and three ones in each group were selected to slaughter. At the same time, the diarrhea rate of pigs was calculated. Then, the samples of intestinal contents were obtained for analysis.The results showed as follows:(1) Native BLF showed more potent inhibitory effect on the test bacteria than HLF. For example, the MIC of BLF to E.coli ATCC 25922 was 1500g/ml, but the one of HLF was 2000g/ml. Moreover, the hydrolysates of LF produced by protease were found to increase in antimicrobial activities and the effect of pepsin was better than trypsin. Like the result of intact LF, the proteolytic BLF exhibited strongeractivities than hydrolysates of HLF. In contrast to the control ones, test Sf9 cells and silkworms (rPLF) significantly decreased the viability of test bacterium and the hydrolysates had better effect than untreated ones. It was suggested that rPLF had been expressed in the test Sf9 cells and silkworms.(2) LF and Lfcin exhibited different antimicrobial activities against the test strains, and the latter's activity was much higher than the former, which was 500-fold greater at most. In addition to this effect, LF was mostly bacteriostatic, whereas Lfcin demonstrated consistent bactericidal activity against the bacterium, the MBC of Lfcin to E.coli ATCC 25922 was 16g/ml. A rapid loss of vialitity of test bacterium was found after exposure to Lfcin and 90% colonies were killed at 2h using time-kill method. Besides that, the zone of inhibition of Lfcin was clearer and the edge was sharper using agarose diffusion method. This result demonstrated that inhibitory effect of Lfcin was more thorough than LF and antibiotics, which maybe correlate with the different antimicrobial mechanism.(3) Both LF and the lactoferricin showed no growth inhibitory activities against B. bifidum in the presence of maximum concentration (MICLF>5000g/ml, MICLfcin >64g/ml) and much lower effects than the ones of antibiotics.Except for the MIC to S.aureus was lower than the one of Lfcin, the antimicrobial potency of demethylvancomycin was weaker than the peptide in the mass. However, doxycycline showed strong activity against several test strains. Especially, the antibiotics exerted significant antimicrobial activity against B. bifidum and L acidophilus.(4) The activity of Apo-LF was more potent than that of native protein having effectiveness against the test strains, which inhibited the growth of test bacterium. The native LF exhibited higher activities than holo-LF, and the inhibitory effect of the latter was very weak, which suggested that LF performed the antimicrobial activity not only depending on chelating iron but also other mechnism.(5) It was found that many bacteria were killed after one hour exposure to Lfcin scanned by AFM. The dead cells showed irregular shape and partial region of cellmembrane was sunken. The sphere structure of whole cell body changed into compressed, which suggested the vesicles content have leaked. After one hour mixin...