Abstract
Industrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2′-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2′- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control.Graphical abstract
Highlights
Human milk oligosaccharides (HMOs) are the third main ingredients in breastmilk after lactose and lipids [1]
For limit of detection (LOD) and limit of quantitation (LOQ) analysis, the stock solutions were double diluted from 1× to 1024× fold
Two complementary gel-buffer systems were evaluated for the analysis of a mixture of 10 synthesized human milk oligosaccharides (Table 1)
Summary
Human milk oligosaccharides (HMOs) are the third main ingredients in breastmilk after lactose and lipids [1]. HMOs are unconjugated glycans typically found in concentrations of 5–20 g/L in mother’s milk [10] This high concentration and structural diversity of these short but complex carbohydrates are unique to humans. Lactose is considered as the simplest HMO core structure that can be further decorated with fucose and/or sialic acid residues. Within the group of neutral HMOs lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT) are considered as core structures, and the fucosylated forms of 2′-fucosyllactose (2′-FL), 3fucosyllactose (3-FL), 2′,3-difucosyllactose (DFL or LDFT), lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-Nfucopentaose V (LNFP-V), lacto-N-difucohexaose I (LNDFH-I), and lacto-N-difucohexaose II (LNDFH-II) are frequently occuring compositions. The most abundant sialylated HMOs are 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), sialyl-lacto-N-tetraose a (LST-a), sialyl-lacto-N-tetraose b (LST-b), and sialyl-lacto-N-neo-tetraose c (LST-c), just to mention the most important ones [17,18,19,20,21]
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