Large-scale preparation and glycan characterization of sialylglycopeptide from bovine milk glycomacropeptide and its bifidogenic properties

Abstract

Bovine glycomacropeptide (GMP) is a 7,000-Da glycopolypeptide released from κ-casein during cheese making. The O-glycan chains linked to GMP have many biological activities, but their utilization for nutraceutical products is limited due to their low content. To concentrate the functional glycan chains of GMP, we prepared sialylglycopeptide concentrate (SGC) from GMP-containing whey protein concentrate via proteolytic digestion of peptide chains and concentration of sialylglycopeptide by ultrafiltration using membranes with a molecular weight cut-off of 1,000 Da. The abundant saccharides detected in the prepared SGC were N-acetylneuraminic acid (Neu5Ac: 32.3% wt/wt), Nacetylgalactosamine (11.3%), and galactose (10.2%), which constitute O-glycans attached to GMP. The Neu5Ac content in SGC was found concentrated at approximately 4.8-fold of its content in GMP-containing whey protein concentrate (6.8%). Structural analysis of O-glycopeptides by liquid chromatography tandem mass spectrometry identified 88 O-glycopeptides. Moreover, O-acetylated or O-diacetylated Neu5Ac was detected in addition to the previously characterized O-glycans of GMP. Quantitative analysis of O-glycan in SGC by fluorescence labeling of chemically released O-glycan revealed that a disialylated tetrasaccharide was the most abundant glycan (76.6% of the total Oglycan). We further examined bifidogenic properties of SGC in vitro, which revealed that SGC served as a more potent carbon source than GMP and contributes to the growth-promoting effects on certain species of bifidobacteria. Overall, our study findings indicate that SGC contains abundant O-glycans and has a bifidogenic activity. Moreover, the protocol for the preparation of SGC described herein is relatively simple, providing a high yield of glycan, and can be used for large-scale preparation.

Potential Prebiotic Properties of Whey Protein and Glycomacropeptide in Gut Microbiome

“…Further, sialyl glycopeptide concentrate created through digestion and ultrafiltration of GMP-containing WPC (G-WPC) outperformed G-WPC as the sole carbon source for the growth of certain Bifidobacteria ( Fukudome et al, 2021 ). Although the glycan component of GMP is often hypothesized to be the basis for prebiotic activity, the peptide portion may be involved as well, as periodate-treated GMP did possess a small growth-promoting effect ( O’Riordan et al., 2018 ).…”

Glycomacropeptide: A comprehensive understanding of its major biological characteristics and purification methodologies

“…Additionally, they proposed that the sialic acid content in the hydrolysate is not directly linked to the promotion of bifidobacteria growth (Tian et al, 2015). Remarkably, the prebiotic activity was negated upon enzymatic digestion with proteases, underscoring the essential role of the amino acid sequence in facilitating the bifidobacterial growth (Fukudome et al, 2021;Morozumi et al, 2023). Furthermore, this effect was observed to be more pronounced with human-derived GMP compared to bovine GMP (Morozumi et al, 2023).…”