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Auftrennung und Analyse von Oligosacchariden aus Humanmilch


Kottler,  R.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Kottler, R. (2010). Auftrennung und Analyse von Oligosacchariden aus Humanmilch. Diploma Thesis, Otto-von-Guericke-Universität, Magdeburg.

During the last decade an enormous step regarding knowledge about the composition and properties of human milk has been made. The three main components of human milk are pro-teins, fats and carbohydrates. As they are composed in human milk, they combine a large va-riety of properties. These were closely surveyed in a multiplicity of studies. Accordingly, it could be shown that during breastfeeding antibodies pass from the mother to the child. Furthermore human milk contains several anti-infective factors such as lactoferrin (Kunz et al., 1999; Rodriguez-Palmero et al., 1999) and immunoglobulin A protecting against microorganisms (Glass et al., 1983). This all supports the development of a good immune system of the newborn, e.g. lowering the risk of infections like otitis media (Dewey et al., 1995) and urinary tract infections (Mårild et al., 2004). Also the risk of developing allergies is reduced (Greer et al., 2008). Another really interesting fact was investigated by diverse studies, that breastfeeding of infants is associated with higher intelligence later in life (Kramer et al., 2008). Within another study there is an even more interesting fact that dietary sialic acids improve the learn-ing and memory abilities of piglets (Wang et al., 2007). Most studies concerning human milk and breastfeeding were focused on the effect of whole human milk. Therefore, this work is intended to establish or to improve analytical techniques, which allow the characterization of individual milk components, namely human milk oligosaccharides (HMOS). The characterization of HMOS is essential to understand the relationship between their structures and biological effects (Erney et al., 2000). The role and significance of HMOS is still not fully understood, which leads to the strong necessity to find out which structures do exist and how they can be identified. Such distinguished information can be used to improve the quality of infant nutrition. This is done in order to provide the best possible start of the infant into life (Hoffman, 2003). In the following diploma thesis a method for analyzing the oligosaccharide pattern of human milk is presented. It comprises protein-precipitation, oligosaccharide purification, clean-up steps, as well as the “fingerprint” analysis of oligosaccharide pools by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF) using a ABI PRISM 3100 capillary DNA-sequencer (Applied Biosystems, USA). It turned out, that CGE-LIF is a valuable alternative to time consuming chromatographic techniques, as well as mass spectrometric or nuclear magnetic resonance spectroscopic based methods. The aim of this diploma thesis was to investigate the ability of the CGE-LIF to analyze the oligosaccharide composition of human milk. During this work the reliability of this technique has been proven by analyzing the oligosaccharide fingerprints of different human milk samples. Another goal was the validation of the capillary DNA-sequencer as an analysis tool for HMOS. With respect to the reproducibility, it could be shown that migration times of the APTS (8-aminopyrene-1,3,6-trisulfonic acid) labeled human milk oligosaccharides were stable within the 6 month measuring time span. During this study the deviation within the migra-tion times of the measured HMOS were for Lacto-N-fuco-pentaose I a typical HMOS 2,2 %. The procedure used allows a quick, unequivocal and sensitive method for the detection of Secretor gene (Se) secondary products and consequently reveals the secretor or nonsecretor status of the milk donor. Additionally it is possible to observe distribution trends of the Se gene products. Furthermore it was possible to see the differences between the secondary gene products from the Lewis gene (Le) within different individual human milks (Thurl et al., 1997; Erney et al., 2000, Thurl et al., 2010). It could be shown within this thesis, that the variety of different HMOS is very high. In sum, CGE-LIF delivers a powerful tool to efficiently and reliably characterize even complex mixtures of HMOS and thereby, it helps to improve the knowledge about their structures and biological effects. This new insides will be valuable to further improve infant nutrition, as well as a variety of other products.