Difco lactobacilli mrs

Three hundred ninety three of the 453 assessed fecal samples (nearly 87%) were confirmed to contain B. longum subsp. longum DNA employing PCR and a subspecies-specific primer pair^{20 (link)}, of which 177 were selected to retrieve bifidobacteria by cultivation on TOS propionate agar (Eiken Chemical, Tokyo, Japan) supplemented with 50 mg/l mupirocin (Merck KGaA, Darmstadt, Germany). Following incubation at 37 °C for 48 hours under anaerobic conditions, ten colonies per sample were randomly selected and were examined by colony PCR using the above mentioned species-specific primer set. Each identified B. longum subsp. longum isolate was cultivated in Difco Lactobacilli MRS (Becton Dickinson, NJ) supplemented with 0.05% L-cysteine HCl (Kanto Chemical, Tokyo, Japan) at 37 °C for 16 hours under anaerobic conditions before DNA extraction. In this manner a total of 162 B. longum subsp. longum strains were isolated, of which a proportion (based on age representation) was selected for sequencing (see below).

The bacterial strains used in this study and general information about these strains are listed in Table 1. The bifidobacterial strains were obtained from stock cultures maintained in the Morinaga Culture Collection (MCC; Morinaga Milk Industry Co., Ltd., Zama, Japan) and the American Type Culture Collection (ATCC, VA). Each strain was cultivated in Difco Lactobacilli MRS (Becton Dickinson, NJ) supplemented with 0.05% L-cysteine·HCl (Kanto Chemical, Tokyo, Japan) at 37°C for 16 h under anaerobic conditions before DNA extraction. The microorganisms were collected by centrifugation, washed once with sterile saline, resuspended in an equivalent volume of sterile saline, and used as seed cultures for fermentation studies.

Thirty bacterial isolates from human feces were used in this study (Table1). Each bacterial isolate was identified based on a nearly full 16S rRNA sequence, which was deposited in DNA Data Bank of Japan (DDBJ). All bacterial strains were cultured in Difco Lactobacilli MRS (de Man-Rogosa Sharpe) broth (Becton Dickinson, Sparks, MD) at 37°C under anaerobic conditions. Heat-killed bacteria were prepared by heating bacteria at 95°C for 10 min.