Columbia blood agar

The haemolysis of the LAB isolate was observed using the Columbia blood agar (Himedia, India) plates containing 1% blood (from human volunteers) and incubated at 37°C for 24 h and observed for haemolytic (γ-non-haemolytic, α and β-haemolytic) activity [28 (link)]. The bacterial DNase (Deoxyribonuclease) activity was analyzed by inoculating the culture of LAB isolate on the surface of the DNase test agar medium (HiMedia). A clear pink, purple zone around the bacterial colonies was considered positive for DNase activity [28 (link)].

Hemolytic activity of selected probiotic isolates was evaluated according to the method described previously [30 (link)]. Overnight grown probiotic cultures were inoculated on Columbia blood agar (M144, HiMedia, India) plates supplemented with 5% (v/v) sheep blood, and plates were incubated at 37°C for 48 h. Hemolytic activity was determined as α-hemolysis, β-hemolysis, and non-hemolysis based on the appearance of green zone, transparent zone and no halo around colonies, respectively.

Analytical-grade xylose was purchased from Loba Chemie (Mumbai, India). Food-grade XOSs for culture medium preparation were obtained from Triple Nine Solution Co. Ltd. (Bangkok, Thailand). Food-grade XOSs were confirmed to be free of xylose contamination before being used. Standard XOSs, including xylobiose (X2), xylotriose (X3), xylotetraose (X4), and xylopentaose (X5), were purchased from Megazyme (Wicklow, Ireland). The nutrient medium, trypticase soy broth, and Columbia blood agar were purchased from HiMedia (Nashik, India). The pepsin, bile salt, and pancreatin used in the preparation of simulated gastrointestinal tract (GIT) conditions were obtained from Sigma-Aldrich (St. Louis, MO, USA).