Adherence interactions between milk proteins and human intestinal surface layer components : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Palmerston North, New Zealand

Abstract

Recent research suggests a number of food-derived proteins may be used as orally delivered functional components. The native structure is often vital to their activity and requires protection during the digestive process. Nutrient vehicles are used as protective envelopes and as a mechanism for targeting specific sites of activity, e.g. the small intestine. This study evaluated molecules which adhere to one or more in vitro models of three human intestinal surface layers. Successful candidates could then be incorporated into nutrient vehicles, promoting adhesion to the surface layers and resulting in prolonged retention of the active ingredient at the site of action or absorption. To identify molecules that adhere to models of the intestinal surface, an adhesion protocol was developed to screen the proteome of whole milk, skim milk and whey for candidate proteins. Molecules adhering to model layers of the human gastrointestinal tract (intestinal epithelial cells, mucin or bacteria with the propensity to form a biofilm) were screened by SDS-PAGE analysis and identified by mass spectrometry and Western blot. The binding behaviour of selected proteins was further investigated by flow cytometry. The combined results showed that milk and whey proteins exhibit different binding affinities to the models of individual surface layers. a-Lactalbumin was found to adhere to a model of the intestinal epithelial cells, while ß- lactoglobulin showed binding to the protective mucin layer. Lactoferrin and various components of immunoglobulins showed highest binding affinity to bacteria. Finally, IgM appeared to adhere to all three tested model layers of the human gastrointestinal surface. Least binding was observed to the intestinal epithelial cells in culture. The validity of the developed adhesion protocol was demonstrated by replicating adhesion of immune-related proteins, lactoferrin and immunoglobulins, to bacterial cells. This work reveals new important characteristics of milk-derived proteins in their ability to adhere to models of the gastrointestinal surface. These may be further utilised in site-specific targeting of functional foods.