Lactobacillus strains

The PP mixture comprised various Lactobacillus strains supplied by the American Type Culture Collection (ATCC) (Manassas, VA, USA). The strains included Lactobacillus gasseri (ATCC 33323), Lactobacillus plantarum (ATCC BAA-793), Lactobacillus reuteri (ATCC 23272), Lactobacillus helveticus (ATCC BAA-2840), Lactobacillus fermentum (ATCC 23271), Lactobacillus rhamnosus (ATCC BAA-2836), and Lactobacillus casei (ATCC BAA-2843). Following the supplier’s protocol, we cultured the strains in MRS broth (Beckton Dickinson, Sparks, MD), and preserved them as glycerol stocks at −80°C. To initiate culture growth, we prepared 5 mL of pre-warmed MRS broth with the glycerol stocks and then incubated these starter cultures at 37°C in a 5% CO₂ environment for 2 h. The bacteria were cultured overnight in 1 L of MRS broth under identical conditions until they reached the logarithmic growth phase, confirmed by measuring optical density at 600 nm (OD600). After growth, the bacterial cells were collected through several centrifugation steps at 3000×g and 4°C, followed by a wash in cold PBS. We then resuspended the bacterial pellets in 10% glycerol in PBS and promptly froze them in 1 mL aliquots for future administration to mice. The viability and concentration of the bacteria were verified by serial dilution and colony-forming unit (CFU) enumeration on agar plates.

L. johnsonii NCK88, L. acidophilus NCFM and L. plantarum WCFS1 were generously provided by Dr. Todd Klaenhammer, L. johnsonii LB1 was isolated directly from mouse cecal contents, and all other Lactobacillus strains were purchased from the American Type Culture Collection (ATCC). Cultures were inoculated in sterile de Man, Rogosa, Sharp (MRS) [41 ] broth from 10% frozen glycerol stocks and incubated anaerobically in an atmosphere composed of 85% N, 10% CO₂, and 5% H₂ at 37°C for two consecutive passages. E. coli C600 and E. coli DH5α were inoculated from 10% frozen glycerol stocks in sterile Lysogeny Broth (LB) and incubated aerobically at 37°C with 300 RPM shaking agitation. Transgenic E. coli C600 strains harboring pSF-OXB12:BSH plasmids were inoculated in sterile LB supplemented with 50 μg/mL kanamycin and incubated at 37°C with 300 RPM shaking agitation for two consecutive passages.