Use of pulsed nuclear magnetic resonance to predict emulsion stability

Pulsed nuclear magnetic resonance (NMR) was used to measure extent of oil solidification during cooling of oil-in-water emulsions. “Percent interaction,” derived from these measurements, was found to correlate well with actual resistance of the emulsion to creaming and phase separation during storage. Average oil droplet size gave a fair correlation with stability, but the correlation of required Hydrophile-Lipophile Balance (HLB) with stability was poor. Pulsed NMR cooling curve measurements on emulsions offer an improved method for prediction of emulsion stability. Presented at the AOCS Meeting in Chicago, September 1976.     

Interaction Between Polar Components and the Degree of Unsaturation of Fatty Acids on the Oxidative Stability of Emulsions

Minor components (polar components) and the degree of unsaturation of the fatty acids are the main factors responsible for the oxidative stability of bulk oils and emulsions. The isolated effects of these two factors and their interaction were evaluated in oil–in-water emulsions stored at 32 °C. Samples of coconut, olive, soybean, linseed and fish oils, both full and stripped of their polar components, were used to prepare the emulsions (1% w/w). The maximum concentration of hydroperoxide (LOOH_max) and the rate of formation of hydroperoxides (μmol L⁻¹ h⁻¹) were used to measure the primary products. Hexanal, propanal and malondialdehyde were used to determine the secondary products of the oxidized emulsions containing polyunsaturated fatty acids. LOOH_max varied from 0.16 to 12.75 mmol/kg among the samples. The interaction between the polar components and the degree of unsaturation of the fatty acids was significant (p < 0.001) when the hydroperoxides were evaluated. In general, the degree of unsaturation (β₁) and the absence of polar components (β₂), respectively, represented 30 and 20% of the contribution to increase the mean oxidation, with the interaction (β₁₂) contribution being more sensitive to the rate of formation of hydroperoxides (16%) than to the LOOH_max (5%). The significance of this interaction suggests that both strategies present synergism and should be applied to improve the oxidative stability of food emulsions.     

31P nuclear magnetic resonance studies on alkyl phosphate emulsifiers in cosmetic oil-in-water emulsions

Cosmetic oil-in-water emulsions with a stearyl phosphate emulsifier are examined by means of static and dynamic ³¹P nuclear magnetic resonance (NMR) techniques to characterize the molecular properties of the emulsifier in situ. The interfacially bound emulsifier can be deteced by high-resolution NMR spectroscopy, whereas the excess emulsifier exists as a solid lipid phase not detectable by this technique. The emulsions and the emulsifier raw material, consisting of monostearyl phosphate as well as distearly phosphate, are examined by solid state cross polarization magic angle spinning NMR spectroscopy to prove the existence of solid emulsifier phases in the emulsions. By applying dynamic ³¹P NMR methods to the interfacially bound emulsifier, information about the molecular dynamics at the interface is obtained. The results of the T ₁ and T ₂ relaxation time measurements indicate a restricted motion of the molecules that is dependent on the oil droplet size in the emulsions. This is verified by ³¹P NMR pulsed gradient spin echo self-diffusion measurements on emulsions with different droplet sizes. Only about 5 wt% of the total emulsifier used is bound at the interface; the excess forms solid lipid phases. The coverage of the interface seems to be independent of the emulsifier concentration. Only the monoester of the emulsifier raw material shows interfacial activity. Its mobility indicates the two-dimensional environment of the molecules on the surface of the oil droplets.     

Interdroplet heterogeneous nucleation of supercooled liquid droplets by solid droplets in oil-in-water emulsions

The crystallization of oil droplets inn-hexadecane oil-inwater emulsions was monitored by pulsed nuclear magnetic resonance (NMR). The emulsions initially contained an equal mixture of solid droplets and supercooled liquid droplets at either 6 or 8°C. The degree of crystallization in the droplets was determined by measuring the NMR signal 30 μs after a 90^o radio frequency pulse was applied to the sample. The signal from the solid droplets decayed rapidly after the radio frequency pulse, allowing the measured signal to be related to the fraction of liquid droplets. No crystallization was observed in a sample that contained only supercooled liquid droplets, but crystallization was observed when solid droplets of the same material were present. This indicated that crystallization was induced in the liquid droplets by the solid droplets and was most likely caused by interactions between solid and liquid droplets-that is, by interdroplet heterogeneous nucleation. The rate of induced crystallization decreased as the viscosity of the continuous phase was increased and the size of the droplets was increased, but was independent of droplet concentration (20–40%).     

Characterization of Cationic Polyelectrolytes Adsorption to an Anionic Emulsion via Zeta-Potential and Microcalorimetry

The adsorption of cationic polyelectrolytes (PEs) onto anionic silicone emulsion droplets, suspended in a sodium chloride solution is studied via electrophoretic mobility measurements and isothermal titration calorimetry. These model systems are studied to better understand the interactions governing PE adsorption-induced emulsion flocculation, which is relevant to many industrial applications. Electrophoretic mobility measurements provide critical information for rationalizing the effect of the PE charge density on the loss of stability of silicone emulsions. The interaction strength is calculated from a Langmuir adsorption isotherm determined by a ζ-potential titration measurement. Microcalorimetry measurements independently validate the adsorption free energy. Emulsion flocculation and coacervation are observed in the visual phase behavior as well as the ζ-potential titration measurements. The effect of PE charge density shows that PE-surfactant coacervation is the driving force in these PE-emulsion systems.     

Characterization of fish oil-in-water emulsions using light-scattering,nuclear magnetic resonance,and gas chromatography-headspace analyses

The effect of the molecular environment on the physical and oxidative properties of homogenized or microfluidized fish oil-in-water emulsions (5% w/w tuna oil in pH 7 phosphate buffer) stabilized by whey protein isolate (WPI, 1 or 5% w/w) or lecithin (2.5% w/w) was examined. Laser light-scattering measurements showed that WPI-stabilized emulsions had smaller particle sizes than lecithin-stabilized emulsions, and that higher pressures reduced the particle size. WPI afforded more protection against oil oxidation than did lecithin, as evidenced by the lower headspace propanal of emulsions as measured by GC-headspace analysis, despite the larger interface in WPI-stabilized emulsions. Reducing the concentration of WPI in emulsions from 5 to 1% decreased the oxidative stability of WPI-stabilized emulsions. The ¹H NMR transverse relaxation times (T ₂) of FA chains in emulsion droplets stabilized by the same surfactants made by homogenization or microfluidization were different and not always related to particle size. The higher mobility (i.e., longer T ₂) of the unsaturated parts of the FA chains within an oil droplet, compared with the saturated parts, suggests that the unsaturated components tended to stay in the core of the oil droplets. This experimental result supports the hypothesis reported in other literature that the more unsaturated FA are buried in the oil core of oil-in-water emulsions. The lack of a universal correlation between particle size and oxidation suggests that the mobility of particles in an emulsion has an influence on the rate of oxidation.     

Characterization of Dispersion Systems Prepared with Mutton Tallow/Hemp Seed Oil-Based Diacylglycerols Using Ultrasonic or Mechanical Homogenization

The objective of this work was to compare the physical stability and physicochemical properties of emulsions, containing enzymatically modified fatty base and homogenized mechanically or by ultrasounds. In the study, lipase-catalyzed interesterification of mutton tallow and hemp seed oil, in a ratio of 3:1, 3:3, and 1:3 w/w, was performed in order to produce fatty bases of the emulsions. Reaction conditions were selected to obtain increased amount of the by-products (MAG and DAG), which were applied as the only emulsifiers in dispersion systems. The higher ratio of animal fat in the interesterified fatty basis of an emulsion had an impact on the greater stability of these systems. The correlation between thickening agent concentration in the prepared emulsions and stability was not observed. Smaller particle size was found in emulsions manufactured by ultrasonic homogenization, although it did not contribute to greater long-term stability of these emulsions. It was concluded that emulsions containing enzymatically modified fats and homogenized mechanically revealed greater physical stability than their counterparts homogenized with ultrasounds.     

Effect of the lupin protein/surfactant ratio on linear viscoelastic properties of oil-in-water emulsions

This work studies the influence of a low-molecular-weight emulsifier/lupin protein ratio on the linear viscoelastic properties of oil-in-water emulsions. With this aim, dynamic viscoelasticity tests, as well as droplet-size distribution measurements, were done. Four types of low-molecular-weight emulsifiers were used. From the results, we conclude that emulsions stabilized by emulsifier blends show, in general, lower values of both the linear viscoelasticity functions and Sauter diameter than those found when lupin protein was used as the only emulsifier. Protein-free emulsions may have similar parameter values when the surfactant induces a structured gel-like continuous phase. Evolution of the linear viscoelastic functions with the composition of the mixed emulsifier has been explained on the basis of changes in interdroplet interactions, rheological behavior of the continuous phase, and surface viscoelasticity of the adsorbed emulsifier layer. However, the above-mentioned behavior cannot be explained from changes in the emulsion droplet-size distribution.     

Label-Free Detection of Protein interactions with peptide aptamers by open circuit potential measurement

Label-free electrical detection of protein interactions has been achieved by direct measurement of variations in open circuit potential (OCP) using an accurate differential voltage measurement. Our model system involves a panel of peptide aptamers that recognise specific protein partners of the cyclin-dependent kinase (CDK) family.Different peptide aptamers immobilized on gold electrodes were used for the detection of human CDK2 and CDK4. The formation of the peptide aptamer recognition layer and the efficiency of its interaction with CDK proteins in yeast cell lysates were characterized by quartz crystal microbalance measurements. The interaction of the peptide aptamers with CDK proteins was successfully detected by direct OCP measurements. The OCP dependence on the pH of the measurement buffer confirms that the effects observed are due to the change in charge upon protein interaction. Variations in charge transfer resistance and in protein/double-layer capacitance were investigated by means of electrochemical impedance spectroscopy with charged redox markers in solution.The present work shows that electrical detection of protein interactions can be achieved by direct measurement of OCP variations using suitable differential voltage instrumentation. The results indicate that label-free detection of protein interactions is possible with potentiometric transducers such as field-effect transistors.     

Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study

Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM–SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM–SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM–SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM–SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM–SAM interactions.     

High-resolution solid-state NMR studies on uniformly [13C,15N]-labeled ubiquitin

Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of 13C', 13Calpha, and 13Cbeta resonance frequencies suggests that 13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR.     

Orientation-dependent coarse-grained potentials derived by statistical analysis of molecular structural databases

We present results obtained for anisotropic potentials for protein simulations extracted from the continually growing databases of protein structures. This work is based on the assumption that the detailed information on molecular conformations can be used to derive statistical (a.k.a. ‘knowledge-based’) potentials that can describe on a coarse-grained level the side chain-side chain interactions in peptides and proteins. The complexity of inter-residue interactions is reflected in a high degree of orientational anisotropy for the twenty amino acids. By including in this coarse-grained interaction model the possibility of quantifying the backbone-backbone and backbone-side chain interactions, important improvements are obtained in characterizing the native protein states. Results obtained from tests that involve the identification of native-like conformations from large sets of decoy structures are presented. The method for deriving orientation-dependent statistical potentials is also applied to obtain water-water interactions. Monte Carlo simulations using the new coarse-grained water model show that the locations of the minima and maxima of the oxygen-oxygen radial distribution function correspond well with experimental measurements.     

Antioxidant Activity of Hybrid Grape Pomace Extracts Derived from Midwestern Grapes in Bulk Oil and Oil‐in‐Water Emulsions

Natural antioxidants to inhibit oxidation in edible oils are in high demand. Grape pomace is an abundant, inexpensive source of polyphenolic antioxidants, which are responsible for numerous health benefits. We examined pomace from eight varieties of Midwestern hybrid grapes for phenolic content and antioxidant activity. Ethanolic extracts produced from the pomace of each grape variety were added to two model systems, bulk soybean oil and oil‐in‐water emulsions, to determine antioxidant activity. Oxidation was monitored in each model system at a temperature appropriate to that particular system. While the extracts had relatively little effect in bulk oil, we observed dose‐dependent antioxidant effects of some extracts in oil‐in‐water emulsions. Oxidation in bulk oils was assessed via total polar compounds and polymerized triacylglycerols. Oxidation in emulsions was assessed by peroxide value, headspace oxygen measurements, gas chromatography of headspace volatiles, and fatty acid analysis. Pomace extracts derived from red grapes generally outperformed those from white grapes, with the Marechal Foch variety showing high antioxidant activity at intermediate concentrations. At higher concentrations, Marechal Foch, Corot Noir, Frontenac, and Norton extracts showed promising antioxidant activity. This is the first report on antioxidant activity in an oil and emulsion setting for many of these grape varieties.     

Oil and water determination in emulsions by pulsed low-resolution NMR

A pulsed, low-resolution nuclear magnetic resonance (NMR) technique has been used for the oil and water determination in O/W emulsions. The method is based on the analysis of the longitudinal magnetization decay curve, that, due to the different relaxation times of oil and water, consists of 2 recognizable components. Correlation of NMR results with fat content is described. A good correlation between the NMR response and fat content by weight has been found. The rapidity and accuracy of the measurements are comparable to those of other techniques.     

PFG‐NMR on W1/O/W2‐emulsions: Evidence for molecular exchange between water phases

A major question in the investigation of multiple emulsions of WOW‐type is that of stability and ripening, which is often reflected in the droplet‐size distribution (DSD). The DSD is an important parameter for judging product properties. Instability in multiple emulsions is often linked to molecular exchange between inner and outer water phases (W₁, W₂), which has to be taken into account. For example, coalescence phenomena and Ostwald ripening affect microscopic and/or macroscopic stability and therefore the multi‐dispersity of an emulsion. This is especially important as the inner compartment can act for encapsulating active agents. NMR investigations on emulsions are mainly based on the analysis of diffusion properties of molecules in the different compartments. For determination of the DSD of the inner and outer droplets, it is crucial to know the physical effects influencing the NMR‐signal. Depending on these effects, the NMR data are analyzed by different models, depending for example on the occurrence and time scale of molecular exchange between the phases. They are shown to yield correct DSDs. Evidence for the effect of diffusion between phases is given by using the phenomenon of driven diffusion, which allows a direct detection of the exchange by NMR. The exchange is confirmed by confocal laser scanning microscopy.     

Flow Alignment of Extracellular Vesicles: Structure and Orientation of Membrane‐Associated Bio‐macromolecules Studied with Polarized Light

Extracellular vesicles (EVs) are currently in scientific focus, as they have great potential to revolutionize the diagnosis and therapy of various diseases. However, numerous aspects of these species are still poorly understood, and thus, additional insight into their molecular‐level properties, membrane–protein interactions, and membrane rigidity is still needed. We here demonstrate the use of red‐blood‐cell‐derived EVs (REVs) that polarized light spectroscopy techniques, linear and circular dichroism, can provide molecular‐level structural information on these systems. Flow‐linear dichroism (flow‐LD) measurements show that EVs can be oriented by shear force and indicate that hemoglobin molecules are associated to the lipid bilayer in freshly released REVs. During storage, this interaction ceases; this is coupled to major protein conformational changes relative to the initial state. Further on, the degree of orientation gives insight into vesicle rigidity, which decreases in time parallel to changes in protein conformation. Overall, we propose that both linear dichroism and circular dichroism spectroscopy can provide simple, rapid, yet efficient ways to track changes in the membrane–protein interactions of EV components at the molecular level, which may also give insight into processes occurring during vesiculation.     

Polar Interfacial Interactions in RPLC

The three principal forces that govern the interaction between dissolved proteins and solid (flat or particulate) surfaces immersed in a polar liquid are: A. Lifshitz-van der Waals forces (LW interactions) B. Polar (hydrogen bond, or Lewis acid-base) forces (AB interactions, which include “hydrophobic” interactions) C. Electrostatic forces (EL interactions) EL interaction energies can be determined via electrokinetic measurements. LW and AB interaction energies are derived from the LW component and the electron-acceptor (γ⊕) and electron-donor (γ⊖) parameters of the AB component of the surface tension, all three of which can be determined by contact angle (Θ) measurements on (a) hydrated layers of protein and (b) flat layers of particulate surface, using at least 3 polar liquids in each case. By this approach, the optimal conditions for attachment as well as for detachment of a given protein to a well-characterized solid surface or particle can be determined quantitatively. Reversed phase liquid chromatography of immunoglobulin G (1) on phenyl Sepharose (2) shows that the elution of the protein, by an increasing concentration of ethylene glycol (EG) in the aqueous medium (3), occurs at the EG concentration where the value of ΔG₁₃₂^TOT changes from negative to positive.     

Rheological properties of emulsions containing modified soy protein isolates

The feasibility of replacing common emulsifiers with soy protein isolates (SPI) in low-calorie salad dressings was evaluated. Structural modifications of SPI were obtained by thermal-acidic treatment with or without neutralization (TH1.6N and TH1.6, respectively). Modification of flow properties of TH1.6 and TH1.6N emulsions by thermal treatment and different protein concentrations was evaluated through shear stress vs. shear rate measurements in a rotational viscometer. TH1.6N isolates generated emulsions with higher shear stress and apparent viscosity than those prepared with TH1.6. Heated TH1.6N emulsions at 10% protein gave the highest values of shear stress and plastic flow behavior. These emulsions had high consistency, viscosity, and elasticity. TH1.6N isolates had lower emulsifying capacity than TH1.6, probably due to the higher protein aggregation produced during neutralization, which prevented protein unfolding. These isolates would be suitable for the preparation of stable emulsions with adequate consistency and elasticity.     

Nanodisc‐Targeted STD NMR Spectroscopy Reveals Atomic Details of Ligand Binding to Lipid Environments

Saturation transfer difference (STD) NMR spectroscopy is one of the most popular ligand‐based NMR techniques for the study of protein–ligand interactions. This is due to its robustness and the fact that it is focused on the signals of the ligand, without any need for NMR information on the macromolecular target. This technique is most commonly applied to systems involving different types of ligands (e.g., small organic molecules, carbohydrates or lipids) and a protein as the target, in which the latter is selectively saturated. However, only a few examples have been reported where membrane mimetics are the macromolecular binding partners. Here, we have employed STD NMR spectroscopy to investigate the interactions of the neurotransmitter dopamine with mimetics of lipid bilayers, such as nanodiscs, by saturation of the latter. In particular, the interactions between dopamine and model lipid nanodiscs formed either from charged or zwitterionic lipids have been resolved at the atomic level. The results, in agreement with previous isothermal titration calorimetry studies, show that dopamine preferentially binds to negatively charged model membranes, but also provide detailed atomic insights into the mode of interaction of dopamine with membrane mimetics. Our findings provide relevant structural information for the design of lipid‐based drug carriers of dopamine and its structural analogues and are of general applicability to other systems.     

Influence of Fatty Acid Profile of Total Parenteral Nutrition Emulsions on the Fatty Acid Composition of Different Tissues of Piglets

Total parenteral nutrition (TPN) studies in human babies of very-low-birth-weight suggest that the lipid emulsions currently available are not optimum for neonatal nutrition. Since fatty acid metabolism in human and pigs is very similar, the present study examines how lipid emulsions used in clinical TPN (i.e. ClinOleic, Intralipid, Lipofundin or Omegaven), with different fatty acid compositions, administered to neonatal piglets for 7 days, influenced their tissue fatty acid composition as compared to those enterally fed with a sow milk replacer. A positive linear relationship was found between the proportion of all individual fatty acids in the lipid emulsions or in the milk replacer versus those in plasma, skeletal muscle, subcutaneous fat, liver, heart, pancreas, stomach or intestine total lipids or in brain phospholipids, the latter showing the lowest correlation coefficient. With the exception of brain, the proportion of either oleic acid or alpha-linolenic acid in the individual tissues was correlated with those present in the corresponding lipid emulsion or milk replacer, whereas the proportion of linoleic acid correlated significantly with all the tissues studied. With the exception of brain phospholipids, both eicosapentaenoic and docosahexaenoic acids were higher in the tissues of piglets receiving Omegaven than in all other groups. In conclusion, with the exception of the brain, fatty acid composition of plasma and different tissues in piglets are strongly influenced by the fatty acid profile of TPN emulsions. Fatty acid composition of brain phospholipids are, however, much less influenced by dietary composition, indicating an active and efficient metabolism that ensures its appropriate composition at this key stage of development.