Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria

Abstract

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

FIGURE 1.

HPLC profiles of the HMOs consumption by B. bifidum JCM1254 cells at different times. The sugars were derivatized by 2-anthranilic acid, solid phase-purified, and analyzed as described under the “Experimental Procedures.” The peaks (1–14) were identified by comparing their retention times with those of the standard sugars.

FIGURE 2.

Growth of the bifidobacteria in the basal media containing HMOs as carbon sources. Open squares, B. longum subsp. infantis JCM1222; closed diamonds, B. bifidum JCM1254; open triangles, B. longum subsp. longum JCM1217; closed circles, B. breve JCM1192.

FIGURE 3.

Changes in the concentrations of mono- and oligosaccharides in the culture of B. bifidum JCM1254 grown in HMO medium. The samples were taken at the indicated times and analyzed. A, monosaccharide concentrations: open squares, Fuc; closed circles, Gal; open triangles, Glc; closed diamonds, GlcNAc. B, di- and trisaccharide concentrations: open triangles, Lac; closed circles, LNB; open squares, 2′-FL; closed diamonds, 3-FL. C, tetrasaccharide concentrations: closed circles, LDFT; open triangles, LNT; open squares, LNnT. D, penta- and hexasaccharide concentrations: closed circles, LNFP I; open triangles, LNFP II + III; closed diamonds, LNDFH I; open squares, LNDFH II.

FIGURE 4.

Changes in the concentrations of mono- and oligosaccharides in the culture of B. longum subsp. infantis JCM1222 grown in HMO medium. The samples were taken at the indicated times and analyzed. The symbols used are the same as described in the legend of Fig. 3.

FIGURE 5.

Changes in the concentrations of mono- and oligosaccharides in the culture of B. longum subsp. longum JCM1217 grown in HMO medium. The samples were taken at the indicated times and analyzed. The symbols used are the same as described in the legend of Fig. 3.

FIGURE 6.

Changes in the concentrations of mono- and oligosaccharides in the culture of B. breve JCM1192 grown in HMO medium. The samples were taken at the indicated times and analyzed. The symbols used are the same as described in the legend of Fig. 3.