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The non-protein nitrogen fraction of infant formula: composition, physiochemical properties, analytical chemistry and in vitro bioactivity
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Date
2025
Authors
Gowen, Niamh
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
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Abstract
The non-protein nitrogen (NPN) fraction of milk is comprised of several nitrogenous components that are non-proteinaceous in nature and accounts for 20-25% of the total nitrogen in human milk, compared to just 3-5% of total nitrogen in bovine milk, and 5-16% of total nitrogen in infant formulas, depending on the ingredients used. These differences in NPN concentration among different milk sources highlight the need for a deeper understanding of the NPN fraction in infant formula to better replicate the NPN profile of human milk. This thesis aims to provide a comprehensive examination of the NPN fraction of milk and dairy ingredients, with a specific focus on urea and polyamines as selected major and minor NPN components, respectively.
One of the challenges in advancing understanding of the NPN fraction is the lack of data available on the NPN content of modern dairy ingredients used in infant formula manufacturing. This challenge was addressed by characterizing and quantifying the NPN content and profiles of thirteen dairy ingredients. The influence of NPN fractionation methods on the NPN profile of a stage 1 infant formula was also investigated, in which the choice of fractionation method was shown to qualitatively influence the NPN profile.
The effect of urea, a major NPN component, on the techno-functional properties of a model infant formula protein system representing a stage 1 (0-6 months) infant formula was assessed across a pH range of 6.4-7.4. The study aimed to understand the role, if any, that urea plays in these functions, so if removed or altered, the implications on the physical stability and functionality of such products could be predicted. Increased concentrations of urea influenced the foaming, viscosity and heat stability properties of the systems, while negligible differences in zeta-potential and solubility of the systems were observed.
The subsequent chapters focused on polyamines which are minor bioactive NPN components. A fit-for-purpose analytical method was developed and validated, the optimal environmental conditions for maximum polyamine recovery in an infant formula matrix were established, and the effect of infant formula matrix on recovery was evaluated. The polyamine profiles and content of human milk and thirteen dairy ingredients commonly used in infant formula manufacturing were characterised and quantified, in which an association between polyamines and total protein content was identified and the impact of ingredient selection on the total polyamine content of an infant formula demonstrated. Finally, potential biological functions of polyamines were investigated. Specifically, the differences, if any, in the immune responses of THP-1 macrophages following treatment with two distinct spermine to spermidine ratios: 0.75:0.25 and 0.25:0.75 was evaluated. These ratios were chosen to reflect polyamine profiles often found in human milk and infant formula. Both ratios generally led to an increased production of pro-inflammatory cytokines; however, the 0.75:0.25 spermine to spermidine
ratio exhibited a more pronounced effect compared to the 0.25:0.75 treatment.
The findings of this research provide valuable insights into the significance of the NPN fraction as a whole, and the importance of selected NPN components, in the nutritional profile in infant formula. The findings will also help to support the development of infant formulas that more
closely resemble the NPN profiles of human milk.
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Keywords
Non-protein nitrogen , Infant formula , Urea , Polyamines , Physiochemical properties , Analytical chemistry , Bioactivity
Citation
Gowen, N. 2025. The non-protein nitrogen fraction of infant formula: composition, physiochemical properties, analytical chemistry and in vitro bioactivity. PhD Thesis, University College Cork.