Reaction sites and functionality of enzymatically cross-linked casein micelles

• Applicant: Professor Dr. Thomas Henle
• Subject Area: Food Chemistry
• Term: from 2015 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 271806234

Enzymatic cross-linking of casein by microbial transglutaminase (mTG) is widely used in the food industry in order to modify the functional properties of the milk proteins. In the current scientific literature, corresponding reports mainly focus on applications and on technological-phenomenological aspects. The structural consequences resulting from mTG-treatment as well as the molecular background of the enzymatic reaction are hardly studied until now. In milk, caseins are aggregated as micelles. Based on current studies of the applicant, this project is based on the hypothesis that the selectivity of enzymatic cross-linking of casein particularly depends on the structure of the casein micelle. As a consequence of intramicellar protein-protein-interactions, well-defined hot spots (i.e. lysine and arginine residues) for selective modification by mTG should exist. This, in turn, is the basis for the formation of casein networks with defined functional properties. The project will follow two general aims: (i) Reaction sites of enzymatic crosslinking of individual caseins within micelles It will be clarified, at which positions within the protein sequence crosslinking is induced by mTG and, thus, how the internal structure of the micelle affects the enzymatic reaction. For this, individual caseins (alpha s1-, alphaS2, beta- und kappa-casein), non-micellar sodium caseinate as well as casein micelles isolated from raw milk will be incubated with mTG and the hot spots attacked by mTG will be identified after enzymatic hydrolysis using LC/MS (peptide mapping). Two strategies, namely labelling of reactive glutamine or lysine residues with dansylcadaverine or triglycine, or direct analysis of isopeptide-containg peptides, respectively, will be followed. (ii) Functionality of mTG-cross-linked casein micelles. Size and stability of mTG-modified casein micelles as well as interactions of mTG-modified casein micelles with selected compounds will be studied. It will be clarified, how intramicellar cross-linking affects the stability of casein micelles in the presence of ethanol, EDTA and peptidases. Information concerning interactions with guest molecules (lysozyme, phoshoserine and beta-carotene) will provide information about the internal structure of the micelles and will show perspectives concerning the use of enzymatically cross-linked casein micelles as tools for encapsulation and delivery of bioactive food compounds. The project will contribute to the fundamental understanding of the course of mTG-induced cross-linking within the casein micelle and will deliver general information concerning the structure of the casein micelle before and after mTG-treament. Furthermore, perspectives for the preparation and use of enzymatically cross-inked casein micelles as nanocapsules for nutritionally relevant compound will be shown.

• DFG Programme: Research Grants