For the isolation of
Bifidobacteria, fecal samples were collected from healthy Koreans (aged 20 to 30 years old) by BD BBLanaerobic sample collection and transport system (Becton Dickinson and Co, USA) to maintain anaerobic conditions. Fecal samples were serially diluted tenfold from 10
-1 to 10
-8, and 100 μl were spread into selective blood liver agar (Nissui Pharm, Japan) containing 5% sheep blood. After 48 h of incubation in anaerobic conditions (90% N
2, 5% H
2, 5% CO
2) (Bactron Anaerobic Chamber, USA) at 37°C, brown or reddish-brown colonies 2 mm to 3 mm in diameter were selected for further identification [
18]. A fructose-6-phosphate phosphoketolase (F6PPK) test was performed to ensure that the colonies selected were
Bifidobacteria [
19]. To identify the isolated
Bifidobacterium spp. at the species level, 16S rRNA sequencing was performed by Bio leaders (Daejeon, Korea). We established an
N-methyl-
N'-nitro-
N-nitrosoguanidine (MNNG)-induced mutant of
B. adolescentis SPM1005, which we named SPM1005-A.
B. adolescentis SPM1005-A, was cultured at 37°C for 48 h on general anaerobic medium (GAM, Nissui Pharm, Japan) under anaerobic conditions and then centrifuged at 1,200 rpm for 15 minutes. The supernatant was separated from the bacterial cell pellet and filtered using an 0.2-μm syringe filter (Sartorius Stedim Biotech, Germany). The purified supernatant was used for further experiments. Written informed consents were obtained from all volunteer who provided samples and the protocol was approved by the Institution Review Board of Office of Research Development, Sahmyook University.