Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

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 intactN -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 releasedN -glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-releasedN -glycans supported the rapid growth ofBifidobacterium longum subsp.infantis (B. infantis ), a species that grows well on human milk oligosaccharides, but did not support growth ofBifidobacterium 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 thatB. infantis consumed 73% of neutral and 92% of sialylatedN -glycans, whileB. lactis degraded only 11% of neutral and virtually no (<1%) sialylatedN -glycans. These results provide mechanistic support thatN -linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, releasedN -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 ofN -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 conjugatedN -glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth ofB. infantis , which is a key infant gut microbe. The selectivity of consumption of individualN -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 thatB. infantis can consume the range of glycan structures released from whey protein concentrate.