Probiotics are living bacteria which can improve the balance of microbiota. There are many recent studies on the effects of probiotics, including oral health promotion and the prevention of oral diseases. However, the mechanisms that underlie the activity of probiotic bacteria against periodontal pathogens have not been clearly elucidated. In order to clarify the antibacterial mechanisms of probiotics against periodontal pathogens, we have performed to identify antibacterial substances produced by Lactobacillus fermentum ALAL020 with the highest antibacterial activity against Porphyromonas gingivalis among the screened 50 strains of lactic acid bacteria.
The culture supernatant in MRS broth was neutralized and purified by gel filtration column chromatography and reverse-phase HPLC. The molecular weight and structure of purified substances was analyzed with LC-MS/MS, FT-IR and NMR.
The major antibacterial substance in culture supernatant produced by L. fermentum ALAL020 was purified as a single peak fraction and the molecular weight (m/z) was 226.131. An LC-MS analysis of this low molecular weight compound revealed that the composition formula was C11H18O3N2. The IR spectra of this compound exhibited amino group and carboxyl group was not contained. From the UV-VIS absorption spectrum with the PDA, it was not absorbed in the wave length area longer than 240nm. The results of structural analysis by NMR showed as a cyclic dipeptide, hexahydro-7-hydroxy-3-(2-methylproryl)pyrrolo[1,2-a]pyrazine-1,4-dione.
We purified and determined an antibacterial substance produced by L. fermentum ALAL020 which was main active constituent against P. gingivalis. This substance was suggested as a novel substance produced by a probiotic bacterium, and might have a possibility for prevention of periodontal disease.