Physiology of Consumption of Human Milk Oligosaccharides by Infant Gut-associated Bifidobacteria

Sadaki Asakuma,Emi Hatakeyama,Tadasu Urashima,Erina Yoshida,Takane Katayama,Kenji Yamamoto,Hidehiko Kumagai,Hisashi Ashida,Junko Hirose,Motomitsu Kitaoka

doi:10.1074/jbc.m111.248138

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.

Highlights

Human milk contains between 10 and 20 g/liter of oligosaccharides (the degree of polymerization (DP)2 Ն3) as the third most abundant solid component after lactose and lipids (1)
The results showed that Bifidobacterium longum subsp. infantis ATCC15697 can grow vigorously in the presence of human milk oligosaccharides (HMOs) acting as carbon sources, whereas Bifidobacterium bifidum ATCC29521 (JCM1255), Bifidobacterium breve ATCC15700 (JCM1192), and B. longum subsp. longum ATCC15707 (JCM1217) did not grow
The purified HMOs were added to the medium at a concentration of 1%, and their consumptions by bifidobacteria were monitored using normal phase HPLC

Summary

Introduction

Human milk contains between 10 and 20 g/liter of oligosaccharides (the degree of polymerization (DP) Ն3) as the third most abundant solid component after lactose and lipids (1). The use of the matrix may interfere with the measurement of low molecular compounds, and the approach may not be suitable for the detection of HMOs with DP ϭ 3 and the degradation products (mono- and disaccharides) of HMOs during cultivation This particular drawback is apparent in the recent reports where the presence of trisaccharides 2Ј-fucosyllactose (Fuc␣1– 2Gal␤1– 4Glc, 2Ј-FL) and 3-fucosyllactose (Gal␤1– 4(Fuc␣1– 3)Glc, 3-FL) are not described. We have conducted refined HPLC profiling of HMO consumption by the infant gut-associated bifidobacteria, in which the concentrations of the main fractions of HMOs (DP of 3– 6) and the degraded intermediates (mono- and disaccharides) were determined These results were interpreted with respect to the metabolic functionalities of the bifidobacteria. This is the first study that unequivocally reveals the fate of each tested HMO during bacterial fermentation and the results indicate that bifidobacterial species/subspecies share the degraded products

Methods

Results

Discussion

Conclusion