Characterization and purification of a bacteriocin-like substance produced by Lactobacillus crispatus LBS 17-11 isolated from an oral cavity of human subject

Gaku Akimaru, DDS (1), Susumu Imai, PhD (2), Kaoru Sogabe, DMD, PhD (2), Ayako Okada, DDS, PhD (2), Tomohiko Terai, PhD (3), Takekazu Okumura, PhD (3), Nobuhiro Hanada, DDS, PhD (2), and Hiroshi Kawahara, DDS, PhD (1)

(1) Department of Dental Anesthesiology, (2) Department of Translational Research, Tsurumi University School of Dental Medicine, Yokohama, and (3) Yakult Central Institute, Microbiological Research Department, Kunitachi, Japan

Purpose: Lactobacillus crispatus LBS 17-11 was isolated from an oral cavity of human subject, and possess growth inhibition activity against Streptococcus pneumoniae. The objective of this study was to purify a bacteriocin-like substance, and to determine its characteristic properties.
Materials and Methods: Antibacterial activities against S. pneumoniae ATCC 33400 during purification steps and against several oral bacteria were determined by a radial diffusion assay. Purification procedure for bacteriocin-like substance was achieved with ammonium sulfate precipitation of culture supernatant, and subsequent extraction with chloroform. Minimum inhibitory concentration (MIC) was determined by liquid microdilution method.
Results: The culture supernatant of L. crispatus LBS 17-11 showed a bacteriostatic activity against S. pneumoniae. This substance was partially purified up to 2045-fold specific activity (unit/mg protein). The inhibitory activity was shown against only S. pneumoniae, but Gram negative species. The antimicrobial activity was heat stable and partially lost by the treatment with proteases (trypsin and proteinase K). The molecular mass of bacteriocin-like substance was estimated to be approximately 7 kDa based on tricine SDS-PAGE. Binary combination of 1/2-MIC level of sodium lactate (56 mM) and the bacteriocin-like substance showed lower MIC level compared to that of the addition of the bacteriocin-like substance alone.
Conclusion: Bacteriocin-like substance produced by L. crispatus LBS 17-11 showed bacteriostatic activity against S. pneumoniae. The partially purified bacteriocin-like substance was heat stable and degraded by proteases. Molecular mass was estimated to be approximately 7 kDa.
(Asian Pac J Dent 2017; 17: 9-14.)
Key Words: bacteriocin, Lactobacillus crispatus, probiotics, Streptococcus pneumoniae