99D-17

Probiotic bacteria have been frequently used as the active food ingredient attributed to their health benefits. Outstanding performance of probiotics mainly depends on their high culture viability. The objectives of this work were (1) to evaluate the effects of variables, including medium components and environmental factors, on the growth of one probiotic strain of Clostridium butyricum; and (2) to optimize the key factors that significantly affected the growth of the probiotic strain. A fractional factorial design was initially applied to investigate the main growth factors. Among the variables, glucose, yeast extract and medium initial pH were identified to have significant effects. Central composite experimental design and response surface methodology were subsequently adopted to derive a statistical model for optimizing the medium composition. The optimal values of the medium were 2.44%(w/v) glucose, 2.08% yeast extract (w/v), 1% tryptone (w/v), 0.1% (NH₄)₂SO₄ (w/v), 0.1% NaHCO₃ (w/v), 0.02% MnSO₄•H₂O (w/v), 0.02% MgSO₄•7H₂O (w/v), 0.002% CaCl₂ (w/v) and 2% agar (w/v) (if necessary) at pH 8.55. The subsequent verification experiments with 24 hrs incubation confirmed the validity of the model with the populations of the viable organisms at 10⁹ cfu/mL, which was a hundred times higher than that incubated in the initial medium. In addition, this tested strain was able to survive at low pH and relatively high bile concentrations compared favorably in these respects to other six probiotic Lactobacillus and Bifidobacterium strains (P<0.01). Further investigation also demonstrated that the tested probiotic strain not only did not show acute oral toxicity in the animal test, also it had exhibited a capacity to improve intestinal microbial balance by inhibiting the growth of pathogens such as Staphylococcus aureus, Salmonella typhimurium, Escherichia coli and Enterobacter spp.