Isolation And Identification Of Bacteriocingenic Strains From Porcine Intestine

This study presented the anti-pathogenic bacteria isolation from the gut flora of healthy finishing pigs. Strains with clear inhibitory activity to pathogenic Escherichia coli indicator were collected using a seeding method. All the active strains were checked their inhibitory performance after the treatments of their supernatant by adjusting pH to neutral or incubating with catalase to eliminate their inhibitory activity from producing acidic products or hydrogen peroxide. Thereafter, both trypsin and papain were used to hydrolyze their peptides in the supernatant of each candidate strain cultures to confirm the production of anti-pathogenic peptides or proteins. The prime strain designated to Mangchang2 was identified as Bacillus subtilis according to its colony status and the results of Gram’s staining, metabolic test and 16 S rDNA sequencing. Further study was conducted to optimize the fermentation conditions of the Mangchang2 strain for producing the highest inhibitory supernatant against its indicator. There are three parts in this study.1. Serial dilution method was used to isolate the commensal bacteria in different intestinal parts(duodenum, jejunum, ileum, caecum, colon and rectum) of healthy pigs. A number of 2317 strains with clearly unique colony morphological characteristics were selected. A clinic isolated pathogenic Escherichia coli strain was used as the indicator in this study. Fifty strains with a clear inhibitory zone individually were collected as the active strains after twice anti indicator cultivation with a seeding method. Fortunately, there were 2 strains with the production of protein or multi-peptide like substance after the proteolysis test and the exemption of producing acid product or hydogen peroxide.2. The prime strain, Mangchang2 was identified as Bacillus according the results of its colonial morphology, Gram’s staining and several metabolic tests. Further 16 S rDNA sequencing confirmed with over 99% similarity that Mangchang2 was a Bacillus Subtilis.3. Further study was investigated into the optimization of the fermentation conditions of Mangchang2 producing bacteriocin like substance. The results showed the optimal condition for Mangchang2 producing inhibitory substance was PCA broth with a 7.0±0.1 initial pH, 3% inoculum concentration, and incubated at 37℃ for 6 hours.