In the future, we are going to spray our bio-stimulator into the environment. To make sure whether the bacteria contain anti-microbial peptide will not exist in the final product, we design the processing standards in the laboratory.
Bacteriocins are usually heat stable, we use high-temperature sterilization to double make sure our peptide solution does not contain any living E. coli. However, peptides may degrades after long time sterilization. To find out the best fitted time for sterilization, we boiled our bacteriocins for 0, 15, 30, and 45 minutes, and put them on LB Agar plate and cultured it at 37℃ for 16 hours.
These are the results of the plates, we can easily observe that the bacteria exists only in the sample that is not boiled. After fifteen minutes of sterilization, there are no alive bacteria exist.
We also ran SDS-PAGE to check the degradation of bacteriocins. The result showed that the peptide didn’t degrade after boiling in 45 minutes. We chose Enterocin 96 and Enterocin B as the representatives. From this, we deduced similar results from the other bacteriocins.
M: Protein Ladder 5–245 kDa; A: Sample without high-temperature sterilization; B: Sample sterilize at 100℃ for 15 mins; C: Sample sterilize at 100℃ for 30 mins; D: Sample sterilize at 100℃ for 45 mins.
Figure 4: Determine the stability of bacteriocins after high-temperature sterilization. The experimental samples are Enterocin 96+intein + CBD (35.9 kDa) and Enterocin B+ intein + CBD (35.5 kDa).