Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria
Authors: Karav Sercan , Le Parc Annabelle , Leite Nobrega de Moura Bell Juliana Maria , Frese Steven A. , Kirmiz Nina , Block David E. , Barile Daniela , Mills David A. 
Published: April 2016

Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-
N
-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact
N
-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released
N
-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released
N
-glycans supported the rapid growth of
Bifidobacterium longum
subsp.
infantis
(
B. infantis
), a species that grows well on human milk oligosaccharides, but did not support growth of
Bifidobacterium animalis
subsp.
lactis
(
B. lactis
), a species which does not. Conversely,
B. infantis
ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that
B. infantis
consumed 73% of neutral and 92% of sialylated
N
-glycans, while
B. lactis
degraded only 11% of neutral and virtually no (<1%) sialylated
N
-glycans. These results provide mechanistic support that
N
-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released
N
-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of
N
-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides.

IMPORTANCE
It has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated
N
-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of
B. infantis
, which is a key infant gut microbe. The selectivity of consumption of individual
N
-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip–quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that
B. infantis
can consume the range of glycan structures released from whey protein concentrate.