Aims: To compare the physiological behaviour of Bi®dobacterium infantis ATCC 15697 growing on synthetic oligofructose or its components. Methods and Results: The studies were carried out in regulated or non-regulated batch cultures on semi-synthetic media. Differences between the carbohydrate utilization patterns with glucose, fructose, sucrose and fructo-oligosaccharides (FOS) were determined. Glucose was the preferred substrate for growth and biomass production, whereas fructose was the best for lactate and acetate production. With sucrose, biomass production reached the level obtained with glucose, whereas with FOS, more metabolites were produced, as with fructose. In a mixture of FOS, the shorter saccharides were used ®rst and fructose was released in the medium. Fructofuranosidase, an enzyme necessary to hydrolyse FOS, was inducible by fructose. Conclusions: Glucose contained in FOS and sucrose might sustain growth and cell production, while fructose might enable the production of major metabolites. Signi®cance and Impact of the Study: A better understanding of the bi®dogenic nature of oligofructose has been gained.

Aims: To characterize the b-fructofuranosidase of Bifidobacterium infantis ATCC 15697 and to compare it with other bacterial b-fructofuranosidases. Methods and Results: The b-fructofuranosidase of B. infantis ATCC 15697 was purified 46AE8 times over the crude extract by anion exchange chromatography, ultrafiltration and gel filtration. The sequence of 15 amino acid residues of the NH 2 terminal was determined. This enzyme was a monomeric protein (M r 70 kDa) with b-fructofuranosidase and invertase activities. The isoelectric point was pH 4AE3, the optimum pH 6AE0 and pKas (4AE5 and 7AE2) of two active groups were obtained. The activities were inhibited by Hg 2+ and p-chloromercuribenzoic acid (pCMB). The optimal temperature was 37°C and activities were unstable at 55°C. b-fructofuranosidase activity was more efficient than that of invertase with V m ⁄ K m ratios of 0AE65 and 0AE025 · 10 )3 l min )1 mg )1 , respectively. The enzyme catalyses the hydrolysis of fructooligosaccharides, sucrose and inulin at relative velocities of 100, 10 and 6, respectively. Conclusions: The enzyme of B. infantis ATCC 15697 is an exo-inulinase which has b-fructofuranosidase and invertase activities. This protein was different from the b-fructofuranosidase of another strain of B. infantis (B. infantis JCM no. 7007). Significance and Impact of the Study: A better knowledge of bacterial b-fructofuranosidases, especially from bifidobacteria, has been gained.

Resting cells and growing cells of bifidobacteria strains exhibited an ability to remove cholesterol in the presence of bile salts. In resting cell assays, the removed cholesterol was precipitated in the presence of cholic acid at pH values lower than 5.4. However, this precipitated cholesterol was redissolved when the pellets were washed with phosphate buffer, pH 7, and no cholesterol was found in the cells. It appears that this precipitation is a transient phenomenon. In the case of growing cells, the removed cholesterol was partially recovered when cells were washed with phosphate buffer, pH 7, while the remaining cholesterol was extracted from the cells. Cultured in the presence of radiolabeled free or esterified cholesterol, bifidobacteria strains were able to assimilate esterified cholesterol. It is concluded that the removal of cholesterol from the growth medium by bifidobacteria strains is due to both bacterial assimilation and precipitation of cholesterol.

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. 2000. Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N H -nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The speci®c growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDpH) was involved in Lactobacillus bile salt resistance.