Isolation And Application Of The Antimicrobial Protein Produced By Bacillus Subtilis R21-4

As an important type of natural preservative, antimicrobial peptide produced by microorganisms are safe , efficient and degradable by protease in the gut. And thus people pay more and more attention for the study and application of the antimicrobial protein produced by microorganisms. Bacillus species were popular in the nature with various metabolic modes. Most antimicrobials produced by Bacillus have broad spectrums of activity against microorganisms with good thermo-stabilization, broad pH ranges and their gene is convenient to clone and modify. All of these are all beneficial to the study of novel food preservatives and biocontrol agents. An antagonistic Bacillus sp. R21-4 and its antimicrobial protein were studied in this paper in order to provide useful data for future application of the natural antimicrobials to food and biocontrol. The results were showed as the following aspects.Bacillus sp. R21-4 was identified as Bacillus subtilis by the main biochemical indexes and genetic sequence analysis. R21-4 antimicrobial demonstrated a broad spectrum of activity against some Gram-negative bacteria, Gram-positive bacteria, as well as some fungi.The antibiotic titer of the culture supernatant to the sensitive indicator (B.cererus) was 320AU/mL under the optimal culture conditions. Most antimicrobial protein can be precipipitated by 60% saturated ammonium sulphate. And two active peaks were observed through the elution by Sephadex G75 gel filtration. The former peak was only active against B.cererus, while the latter one had greater antimicrobial activity against both B.cererus and E. coli. Direct detection of antimicrobial activity on Tricine-SDS—PAGE suggested a molecular mass of the protein about 6400D-7200D.The antimicrobial peptide displayed bactericidal action on B.cererus and E. coli by the means of liquid incubation. Observation showed that R21-4 antimicrobial peptide restrained the spores to germinate and made hyphas swelled and distorted. And the cytoplasm in the studied hyphas was concentrated after Verticillium dahliae Kleb.V991 was treated with the antimicrobial.Bacillus subtilis R21-4 was treated with spontaneous screening and then by UV mutagenesis. A stable strain was obtained with the antimicrobial titer increased by 58.49% than the parent strain.The antimicrobial effects of R21-4 protein on fresh pork and pock mince were studied. The results indicated that 3%sodium lactate had a better preservative activity than R21-4 antimicrobial protein with the titer of 640AU/mL, which was better than 0.1% potassium sorbate. R21-4 antimicrobial protein with the titer of 64AU/mL and 128AU/mL both presented obvious antimicrobial activity in the study on the inhibitory activity against microorganisms in the mince. The bacteria counts of the sample treated with 128AU/mL antimicrobial protein was about 6 orders less than those of control group.