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.

Giant unilamellar vesicles (GUVs) were constructed using an electroformation technique to mimic the morphology of the native milk fat globule membrane (MFGM) for the purpose of structural investigation. Bovine milk derived phospholipids were selected to manufacture GUVs which were characterized by confocal laser scanning microscopy after fluorescent staining. Circular nonfluorescent dark regions were observed in a 3/7 (mol/mol) surface mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine. Linear shaped dark lipid domains were found in GUVs containing sphingomyelin (SM) in the absence of cholesterol. The dark regions were interpreted as a gel phase formed by a high gel-liquid phase transition temperature (Tm) of DPPC and SM. This study provides a strategy for investigating the lipid structural organization within the native MFGM using a model lipid bilayer system and reveals that a SM and cholesterol association network is not the only requirement for nonfluorescent lipid domain formation and that PE is preferably located in the inner leaflet of the phospholipid bilayer.

Recently, phospholipids have been used for the liposomal encapsulation of drugs and bioactive compounds. Pancreatic phospholipase A2 is responsible for the hydrolysis of phospholipids in the intestines. Using fluorescence microscopy, Langmuir monolayers at the air–water interface and atomic force microscopy, the aim of this work was to obtain preliminary data on the effect of the liquid-ordered domains and proteins on the hydrolysis of phospholipids by pancreatic phospholipase A2. Increasing the enzyme:substrate ratio (1:40 to 1:2) and the temperature (30°C to 37°C) increased phospholipid hydrolysis as seen by a decrease in surface pressure and change in the surface area corresponding to the formation and desorption of hydrolytic products. Hydrolysis occurred rapidly in the phase coexistence region and led to the clustering of the liquid-ordered domains likely through reduction of electrostatic forces. Atomic force microscopic images of the monolayers under simulated intestinal conditions revealed the presence of packing defects in the liquid-ordered domains where phospholipase A2 could adsorb. Hydrolysis took place starting in the center of the liquid-ordered domains and proceeded towards their edges. It did not lead to any measurable height changes by atomic force microscopy within the domains but it was evident that mechanical changes took place during hydrolysis. The presence of β-casein appeared to increase the rate of hydrolysis, possibly through interaction with phospholipase A2 at the interface.

Phospholipid–protein monolayer films were studied as model systems to mimic the structure of the native bovine milk fat globule membrane (MFGM) and to understand lipid–protein interactions at the surface of the globule. Phospholipids extracted from bovine raw milk, raw cream, processed milk and buttermilk powder were spread onto the air–water interface of a Langmuir trough, β-casein was then added to the sub-phase, and Langmuir–Blodgett films were studied by epifluorescence microscopy and atomic force microscopy. In all films, β-casein was responsible for clustering of the sphingomyelin- and cholesterol-rich microdomains into larger platforms. This suggests that the same phenomenon may happen at the surface of the milk fat globule, where specific MFGM proteins may cause aggregation of microdomains.

Monolayers were prepared from phospholipids extracted from bovine milk and used as a model system to mimic the native milk fat globule membrane (MFGM) surface structure in various microscopic experiments. The natural complex mixtures of phospholipids were isolated from bovine raw milk, raw cream, processed whole milk, and buttermilk powder by total lipid extraction and solid-phase extraction. A Langmuir film balance mounted on an epifluorescence microscope was used to analyze the physical behavior of the monolayer films and the phase coexistence resulting from the formation of phospholipid microdomains within these films. Atomic force microscopy was used for nanometer-scale topographic resolution of the microdomains. This study allowed comparison of the behavior of phospholipid monolayers from dairy products at different stages of processing, analysis of the formation of microdomains, and the study of the effect of milk processing on lipid-lipid interactions and phase coexistence. It was observed that milk processing changes the physical behavior of phospholipid monolayers by altering the phospholipid profile and the fatty acid distribution.

The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied, but its structure, especially the lateral organization of its components, still remains unclear. We have used confocal laser scanning microscopy (CLSM) to investigate the surface structure of the MFGM in globules with different degrees of processing using two types of fluorescently labeled phospholipid probes and a protein dye. Using this technique, we have observed heterogeneities in the distribution of MFGM lipids and proteins relating to the processing and size of the globules. The effect of pretreating the milk (centrifugation, pasteurization-homogenization and churning) was studied by double-staining the surface of the milk fat globules, followed by observation using CLSM, and by determining the phospholipid profile of raw milk, raw cream, processed milk and buttermilk powder. Our findings agree with other techniques by showing that the composition of the MFGM changes with processing through the loss of phospholipids and the adsorption of caseins and whey proteins onto the surface.

Writing formal "journal-style" lab reports is often one of the requirements chemistry and biochemistry students encounter in the physical chemistry laboratory. Helping students improve their technical writing skills is the primary reason this type of writing is a requirement in the physical chemistry laboratory. Developing these skills is an important goal, yet sometimes students are not given enough time to review and revise their writing. Further, they seldom are taught how to review, and thus critical steps in the writing process are omitted. We have developed an approach with the goal of helping students improve their technical writing skills that employs the peer-review process and includes detailed guidance for writing reports in a progressive manner. We present these assignments, how student progress is assessed, and how the components fit to improve the technical writing abilities of the students.

The aim of this work was to assess the accuracy of different extraction methods of phospholipids and to measure the effect that processing has on phospholipid composition. Four methods of extracting phospholipids from buttermilk powder were compared to optimize recovery of sphingomyelin. Using the optimal method, the phospholipid profile of four dairy products (raw milk, raw cream, homogenized and pasteurized milk, and buttermilk powder) was determined. A total lipid extraction by the Folch method followed by a solid-phase extraction using the Bitman method was the most efficient technique to recover milk sphingomyelin. Milk processing (churning, centrifuging, homogenization, spray-drying) affected the profile of milk phospholipids, leading to a loss of sphingomyelin and phosphatidylcholine after centrifugation for cream separation. A corresponding decrease in the saturation content of the raw cream phospholipids and a loss of phosphatidylethanolamine after spray-drying to produce buttermilk powder were also observed.

Two experiments are described in which students explore phase transitions and critical phenomena by obtaining compression isotherms of phospholipid monolayers using a Langmuir trough. Through relatively simple analysis of their data students gain a better understanding of compression isotherms, the application of the Clapeyron equation, the balance between enthalpic and entropic contributions to the chemical potential, critical phenomena, and phase diagrams in general. Students use their data to determine the latent heat of transition, the entropy of transition, and the critical temperature for the liquid condensed to liquid expanded phase transition of a monolayer. Students also gain a general understanding of how molecular level changes in the structure of phospholipids, such as changes in chain length affect the structure and function of biological membranes.

The structure and function of the milk fat globule membrane (MFGM) from bovine milk was analyzed using a Langmuir-Blodgett trough and atomic force microscopy (AFM). Isotherms of bovine buttermilk powder were obtained at temperatures ranging from 17–37 degrees C. A collapse was observed at 28 + 1 mN/m at 19.7 + 0.2 degrees C. MFGM monolayers were deposited on cleaved mica at various surface pressures. Domains were observed. Funding: California State University Agricultural Research Initiative (ARI)

Nonlithographic techniques for patterning structures on the nanometer Scale can provide methods for direct control of particle spacing at surfaces. By using diblock copolymers, the surface density of a film can be established by the properties and area of the anchoring block, and the feature sizes can be set through the choice of free block dimensions. By depositing poly(styrene)-poly(ethylene oxide) (PS-PEO) diblock copolymers of different fractional composition of PEO on a surface by a Langmuir-Blodgett technique at different pressures, we show that the surface density of poly(styrene) aggregates can be controlled. The separation of PS aggregates on the surface is ensured by selection of the PEO composition so that its projected area is greater than that of the PS for all pressures less than that of the transition from a 2-dimensional to 3-dimensional film. The areal density of these resultant PS surface micelles can be tuned for a particular polymer composition and is linearly dependent on the deposition pressure which defines the region chosen on the phase diagram.

Vibrational sum frequency spectroscopy has been used in conjunction with dynamic surface tension measurements to study formation of a 1,2-dilauroyl-sn-phosphatidylcholine (DLPC) monolayer at a water–carbon tetrachloride interface. Surface tension measurements show that an aqueous solution of liquid crystalline phosphocholine vesicles (4.5 μM DLPC) requires several hours to form a tightly packed, fully equilibrated monolayer of DLPC monomers. Vibrational spectra of the interfacial region at different stages in the monolayer formation process indicate that the solvent structure undergoes dramatic re-organization as the monolayer forms. Initial adsorption of DLPC monomers severely disrupts the interfacial hydrogen bonding. Intensity in the OH stretching region oscillates in a systematic fashion during the first 2 h of monolayer formation before finally settling to a level characteristic of the fully equilibrated monolayer. Frequency shifts of the OH stretching vibration show that water molecules with their C2 axes aligned parallel to the interface experience a markedly different environment than those water molecules aligned perpendicular to the interface. This difference is attributed to the effect of the adsorbed, zwitterionic DLPC head-groups which, if aligned parallel to the interface, can stabilize in-plane water molecules.

Work from our laboratory on vibrational sum frequency spectroscopic investigations of molecular ordering at the carbon tetrachloride-water interface is reviewed. Simple charged surfactants adsorbed at the liquid-liquid interface are seen to induce alignment of interfacial water molecules to a degree which is dependent on the induced surface potential. Saturation of water molecule alignment occurs at a surfactant surface concentration corresponding to a calculated surface potential of approximately 160 mV. In complementary studies, the relative degree of hydrocarbon chain ordering within monolayers of symmetric phosphatidylcholines of different chain lengths is inferred by the relative signal contributions of the methyl and methylene symmetric stretch modes. The degree of hydrocarbon chain disorder observed depends strongly on the method of monolayer preparation. By one method, a decrease in hydrocarbon chain order is seen with increasing chain length. Another method of monolayer formation yielded very well ordered hydrocarbon chains for the longest chain phosphatidylcholine studied, and showed much greater disorder in shorter chain species which was comparable to the other preparation method. These studies are a foundation for further work with this technique geared towards understanding molecular-level structural features in membrane-like assemblies and surface biochemical interactions of relevance to biomedical research.