Evaluation of Prebiotic Glycan Composition in Human Milk and Infant Formula: Profile of Galacto-Oligosaccharides and Absolute Quantification of Major Milk Oligosaccharides by UPLC-Cyclic IM-MS and UPLC-MS/MS.

Prebiotic oligosaccharides have attracted immense interest in the infant formula (IF) industry due to their unique health benefits for infants. There is a need for the reasonable supplementation of prebiotics in premium IF products. Herein, we characterized the profile of galacto-oligosaccharides (GOS) in human milk (HM) and IF using ultrahigh-performance liquid chromatography-cyclic ion mobility-mass spectrometry (UPLC-cIM-MS) technique. Additionally, we further performed a targeted quantitative analysis of five essential HM oligosaccharides (HMOs) in HM (n = 196), IF (n = 50), and raw milk of IF (n = 10) by the high-sensitivity UPLC-MS/MS method. HM exhibited a more abundant and variable HMO composition (1183.19 to 2892.91 mg/L) than IF (32.91 to 56.31 mg/L), whereas IF contained extra GOS species and non-negligible endogenous 3'-sialyllactose. This also facilitated the discovery of secretor features within the Chinese population. Our study illustrated the real disparity in the prebiotic glycome between HM and IF and provided crucial reference for formula improvement.

Selective extraction of phospholipids from human milk using glass fabric modified with zirconium-based metal organic framework.

Phospholipids (PLs) are important and complex trace lipids in milk, which have positive effects on the infants' nervous and immune system development. Herein, a new method for selective extraction of PLs using glass fabric @ MOF-808 was proposed. Based on Lewis acid-base interaction, MOF-808 containing abundant Zr-OH groups was selected as the adsorption body, and glass fabric was used as a substrate to make the adsorbent easy to remove and reuse. The influencing factors such as loading solution, extraction time, eluent and elution time were further investigated. The adsorbent showed high adsorption capacity (3.31-6.54 mg/g for PLs) and good reusability (reused at least five times). The method showed low detection limits (1.61 µg/L - 10.24 µg/L) and quantification limits (5.24 µg/L-51.21 µg/L) for eight classes of PLs. The analysis of PLs in human milk at different lactation stages by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry could obtain up to 206 PLs, indicating that the method has extremely high extraction and anti-interference capabilities. This work is the first time to introduce MOF materials to selectively extract PLs and use glass fabric as a substrate for MOF-808, which has the advantages of easy recovery and high sensitivity. It provides technical support for the discovery of more PL species and has potential applications in phospholipidomics.

Development of a high-throughput method for the comprehensive lipid analysis in milk using ultra-high performance supercritical fluid chromatography combined with quadrupole time-of-flight mass spectrometry.

Milk lipids are one of the most complex materials in nature and are associated with many physiological functions, hence it is important to comprehensively characterize lipids profiles to evaluate the nutritional value of milk. A quick method was developed by ultra-high performance supercritical fluid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (UHPSFC-ESI-QTOF-MS) to analyze the non-polar and polar lipids profiles of cow, goat, buffalo, human milk, and infant formulas in 7 min. All chromatographic conditions were carefully optimized and their effect on the chromatographic behavior of lipid classes and species was discussed. Under optimized conditions, 12 lipid classes (triacylglycerols, diacylglycerols, monoglyceride, fatty acids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, sphingomyelin, lyso-phosphatidylcholine, and lyso-phosphatidylethanolamine) were separated and each class was further separated in single analysis to facilitate the identification. 250 lipid species in real samples were characterized and quantified. This result demonstrates the applicability of the UHPSFC-ESI-QTOF-MS method in the high-throughput and comprehensive lipid analysis of milk, and will hopefully help to provide nutritionists with the lipid distribution in different types of milk, as well as help in the design of more suitable infant formula for babies.

Investigation of alterations in phospholipids during the production chain of infant formulas via HILIC-QTOF-MS and multivariate data analysis.

Phospholipids play a key role in infant nutrition and cognitive function. In this study, hydrophilic interaction liquid chromatography coupled to quadrupole time-of-flight mass spectrometry method was firstly developed to analyze the composition of phospholipids. Then we characterized and quantified phospholipids extracted from raw, pasteurized, homogenized, and spray-dried milk to investigate the effect of the technological process on the composition of the phospholipids. Results indicate that the composition of the phospholipids underwent minor changes after pasteurization, while the concentration of phospholipids was significantly affected by the spray-drying process, especially phosphatidylethanolamine and phosphatidylinositol. Multivariate data analysis further verified the results and indicated that phospholipids containing polyunsaturated fatty acids had undergone significant changes during the production chain, especially in spray-drying. This work reveals the changes of phospholipids composition during the production chain of infant formulas and serve as a reference for the subsequent optimization of infant formulas to meet nutritional need of infants.

Biochemical characterization and mutational studies of a thermostable uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

Uracil DNA glycosylases (UDGs) play an important role in removing uracil from DNA to initiate DNA base excision repair. Here, we characterized biochemically a thermostable UDG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba UDG), and probed its mechanism by mutational analysis. The recombinant Tba UDG cleaves exclusively uracil-containing ssDNA and dsDNA at 65°C. The enzyme displays an optimal cleavage activity at 70-75°C. Tba UDG cleaves DNA over a wide pH spectrum ranging from 4.0 to 11.0 with an optimal pH of 7.0-9.0. In addition, Tba UDG activity is independent on a divalent metal ion; however, both Zn2+ and Cu2+ completely inhibit the enzyme activity. Tba UDG activity is also inhibited by high NaCl concentration. Tba UDG removes uracil from DNA with the following preference: U≈U/G>U/T≈U/C>U/A. Kinetic results showed that Tba UDG cleaves uracil-containing ssDNA and dsDNA at a similar rate. The mutational studies showed that the E118A, N159A and H216A mutants completely abolish cleavage activity and retain compromised binding activity while the Y127A mutant displays similar cleavage and binding activities with the wild-type protein, suggesting that residues E118, N159 and H216 are essential for uracil removal and necessary for uracil recognition.