β-Lactoglobulin-linoleate complexes: In vitro digestion and the role of protein in fatty acids uptake

peer-reviewedThe dairy protein β-lactoglobulin (BLG) is known to bind fatty acids such as the salt of the essential longchain fatty acid linoleic acid (cis,cis-9,12-octadecadienoic acid, n-6, 18:2). The aim of the current study was to investigate how bovine BLG-linoleate complexes, of various stoichiometry, affect the enzymatic digestion of BLG and the intracellular transport of linoleate into enterocyte-like monolayers. Duodenal and gastric digestions of the complexes indicated that BLG was hydrolyzed more rapidly when complexed with linoleate. Digested as well as undigested BLG-linoleate complexes reduced intracellular linoleate transport as compared with free linoleate. To investigate whether enteroendocrine cells perceive linoleate differently when part of a complex, the ability of linoleate to increase production or secretion of the enteroendocrine satiety hormone, cholecystokinin, was measured. Cholecystokinin mRNA levels were different when linoleate was presented to the cells alone or as part of a protein complex. In conclusion, understanding interactions between linoleate and BLG could help to formulate foods with targeted fatty acid bioaccessibility and, therefore, aid in the development of food matrices with optimal bioactive efficacyS. Le Maux is currently supported by a Teagasc Walsh Fellowship and the Department of Agriculture, Fisheries and Food (FIRM project 08/RD/TMFRC/650). We also acknowledge funding from IRCSET-Ulysses Travel Grant

β-lactoglobulin/linoleate complexes: binding properties and biological functions

Food structure can have a profound influence on delivering health benefits. Bioaccessibility of nutrients can be affected by their interaction with food components. The dairy protein β-lactoglobulin (βlg) is known to bind hydrophobic ligands such as fatty acids (FA). However, this protein is highly sensitive to the process conditions used in the dairy industry. Therefore βlg is often present in non-native or aggregated form in processed food. This structural change may modify the protein affinity for FA and the biological properties of the FA/protein complexes. The aim of this thesis was to investigate the interaction of bovine βlg in different structural forms (native, covalent dimer and nanoparticles) with linoleate (C18:2, cis,cis-9,12-octadecadienoic acid) and conjugated linoleic acids (CLA, C18:2), and the impact of those complexes on their biological activity in vitro. Two different sets of binding sites were determined for the interaction between linoleate and βlg, regardless of its state of aggregation, using intrinsic fluorescence spectroscopy and isothermal titration calorimetry. By increasing the level of βlg aggregation, the linoleate/βlg stoichiometry increased but the association constants remained similar for both sets of binding sites. In the presence of linoleate, the native protein was more sensitive to gastric in vitro digestion, due to the increased level of denaturation/aggregation of βlg. Transport of linoleate in Caco-2 cells was decreased in presence of the native βlg as observed by confocal microscopy and a monolayer that mimics the intestinal barrier. Cytotoxicity of linoleate on Caco-2 cells was reduced when the FA was bound to βlg compared to free FA. CLA, which is less water soluble than linoleate, is more cytotoxic when complexed by βlg than in its free form. Therefore, it is proposed that βlg can act as a molecular carrier and alter the bioaccessibility of FA depending on their solubility

Emulsion-Based Delivery Systems for Tributyrin, a Potential Colon Cancer Preventative Agent

Tributyrin, a short-chain triglyceride oil used as a food additive, has been reported to be a potential preventive agent against colon cancer. The purpose of this study was to develop tributyrin delivery systems based on food-grade oil-in-water emulsions that could potentially be incorporated into foods. Emulsions containing only tributyrin as the lipid phase were highly unstable to droplet growth due to Ostwald ripening (OR) because of the relatively high water solubility of this low molecular weight triacylglycerol. The stability of the emulsions to OR could be greatly improved by incorporating \u3eor=15-25% corn oil (a food-grade oil with a low water solubility) into the lipid phase. In addition, the tendency for droplet sedimentation to occur was reduced because the density contrast between the lipid and water phases was reduced in the mixed tributyrin/corn oil systems. The potential anticarcinogenic ability of the tributyrin emulsions was demonstrated using a cell culture model. Treatments with emulsions containing tributyrin significantly inhibited the viability of HT29 colon carcinoma cells. These results have important implications for the development and testing of nutraceuticals encapsulated in food-grade delivery systems as anticancer agents

β-lactoglobulin/Linoleic Acid Complexes – Formation, Binding Stoichiometry and Cytotoxic Capability

β-lactoglobulin (β-lg) is a major whey protein in bovine milk, which has the ability to bind hydrophobic ligands such as fatty acids. It is well known that the whey protein alpha-lactalbumin also forms a stable complexe with fatty acid, which is cytotoxic to a wide variety of tumour cells. The aim of this project was to investigate the binding stoichiometry of β-lg and linoleic acid (LA; cis,cis-9,12-octadecadienoic acid) and investigate the impact of this complex on viability of intestinal cancer cells in vitro. The interaction between β-lg and fatty acids was studied by HPLC and GC. Complexes with a binding stoichiometry of 1, 2 and 3 LA:β-lg were produced. The effect of the complexes on cell viability was measured by exposing the human colorectal adenocarcinoma epithelial cell line, Caco-2, to various concentrations of complexes from 0 to 72 hours. LA on its own exhibited a cell viability effect at 58 M as measured by MTS assays. A cytotoxic effect at 35 M was measured by an impedance-based biosensing microelectronic real-time cell analyzer system (XCELLigence RTCA system, Roche Diagnostics). Neither the β-lg (0 to 75 μM) nor β-lg/LA complexes (LA concentration from 0 to 200 μM) had an adverse effect on Caco-2 cells. In vivo it is assumed that β-lg gradually releases LA during gut transit which, in turn, may modulate fatty acid uptake by the gut and/or have a satiating effect. Future work will investigate fatty acid uptake and satiety signaling in the gut in response to β-lg/LA complexe