Article pubs.acs.org/JAFC
Adsorption of Bile Salts to Milk Phospholipid and Phospholipid− Protein Monolayers Sophie Gallier,*,† Ethan Shaw,§ Andrea Laubscher,⊗ Derek Gragson,§ Harjinder Singh,† and Rafael Jiménez-Flores⊗ †
Riddet Institute, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand Department of Chemistry and Biochemistry and ⊗Dairy Products Technology Center, California Polytechnic State University, San Luis Obispo, California 93407, United States
§
ABSTRACT: The adsorption of bile salts to milk phospholipid and phospholipid−protein monolayers at the air−water interface was studied under simulated intestinal conditions using a Langmuir trough, epifluorescence microscopy, and atomic force microscopy. Surface pressure changes were affected by temperature, initial surface pressure, and bile composition. The rate of addition of bile salts and the initial surface pressure of the monolayers had an impact on the microstructure of the mixed monolayers. The presence of proteins in monolayers at different ratios did not affect the surface pressure change upon addition of bile. However, at 20 °C, the addition of bile to phospholipid and phospholipid−protein monolayers led to different features with branching and clustering of liquid-ordered domains and possible formation of bile salt-rich areas within liquid-ordered domains. This study provides a basic understanding of the interfacial changes occurring at the surface of milk fat globules and milk phospholipid liposomes during their passage in the duodenum. KEYWORDS: bile salt, phospholipid monolayer, Langmuir trough, atomic force microscopy
■
INTRODUCTION The digestion of lipids is a complex interfacial process. Lipolysis starts in the stomach and is highly influenced by the presence of surfactants at the oil−water interface.1 Gastric lipase is able to penetrate the membrane of oil droplets to access the triglyceride core.2 whereas pepsin hydrolyzes some of the proteins present at the oil−water interface. The partially hydrolyzed oil droplets then reach the small intestine and are mixed with pancreatic and bile secretions. Pancreatic secretions contain enzymes to digest lipids, proteins, and carbohydrates, and bile secretions contain negatively charged biliary salts, bilirubin, cholesterol, phospholipids, and electrolytes.3 Bile salts compete with the surface-active molecules at the oil−water interface, and their ability to displace these surfactants will have an impact on the lipolysis of lipid droplets by pancreatic lipase.2 Due to their structure, bile salts lie flat at the oil−water interface.4 The interaction of bile salts and phospholipids is important in physiological processes such as the solubilization and transport of lipolytic products and lipid-soluble compounds, the stability of liposomes encapsulating drugs or bioactives (for oral administration), and the adsorption of lipases onto phospholipid-stabilized lipid droplets.5 The milk fat globule membrane (MFGM), surrounding the milk fat core, is composed of a phospholipid trilayer with proteins and cholesterol.6,7 As gastric lipase does not hydrolyze phospholipids and some MFGM glycoproteins are resistant to hydrolysis by pepsin, the milk fat globule and its MFGM retain their integrity in the stomach8 and reach the small intestine almost unchanged. Milk phospholipids have raised interest as emulsifiers for liposomal encapsulation of bioactives and drugs9,10 and their delivery in the gastrointestinal tract.11 The MFGM and milk phospholipids will then interact with bile salts. © 2014 American Chemical Society
We have previously studied the MFGM structure in its native state6 and using model systems12,13 as well as the in vitro14 and in vivo15 digestion of bovine milk fat globules. The use of model systems allows the understanding of the interfacial phenomena occurring at the surface of oil droplets during digestion, such as the adsorption of bile salts to phospholipid or galactolipid monolayers, the adsorption of the pancreatic lipase−colipase complex to bile salt−phospholipid or bile salt−galactolipid monolayers, or the orogenic displacement of protein by bile salts.16−18 Phospholipids can be present as monolayers (emulsions), bilayers (liposomes or cells), or multilayers (milk fat globules); monolayers are commonly used to study interfacial phenomena in these systems due to their experimental simplicity.16,18 Using a Langmuir trough, temperature, surface pressure, and surfactant composition can be easily controlled. Working with monolayers at the oil−water interface is difficult. For example, the solvents used to remove the oil phase before imaging of Langmuir−Blodgett (LB) films would also remove polar lipids from the monolayer. In addition, results obtained at the air−water interface are in agreement with those at the oil−water interface.16,19 Thus, here, experiments at the air−water interface were performed to obtain an understanding of how the lateral distribution of interfacial surfactants (phospholipids and proteins) affects the competitive adsorption of bile salts to the surface of the lipid droplets in the duodenum. In a previous work, we have characterized the hydrolysis of phospholipid and phospholiReceived: Revised: Accepted: Published: 1363
October 3, 2013 January 22, 2014 January 23, 2014 January 23, 2014 dx.doi.org/10.1021/jf404448d | J. Agric. Food Chem. 2014, 62, 1363−1372
Journal of Agricultural and Food Chemistry
Article
and β-lactoglobulin were prepared fresh daily and diluted to 1 mg mL−1 in PBS buffer (pH 7.45), and stock solutions (120 or 60 mM) of bile salts and bile extract were prepared with simulated intestinal fluid (SIF, 150 mM NaCl, 10 mM CaCl2, pH 7.0). SIF was used as the subphase simulating the ionic strength and the pH found in the small intestine. At pH 7, both proteins carry a negative net charge.13,17 The aqueous surface was cleaned before and between experiments until a surface pressure of
