Lipid oxidation in oil-in-water emulsions: Iron complexation by buffer ions and transfer on the interface as a possible mechanism.

Lipid oxidation is the main hurdle for omega-3 fatty acid enrichment in food and beverages. Fat oxidation reduces the quality and safety of supplemented products. A tuna oil-in-water emulsion (20%v/v) was exposed to iron-induced oxidation. Emulsions with changing emulsifiers and buffers were analyzed under different storage conditions (argon purging, pH variation) using Conjugated Dienes and Thiobarbituric acid reactive substances assays. The results showed that free iron ions cannot interact with oxygen. However, buffers (Citrate and phosphate) chelate iron ions (Fe (II)). Depending on the pH value and the type of buffer-Fe (II) complex, its prooxidant activity and spatial distribution are influenced. The complex charge defines the interactions with the oil-water interface, i.e., positively charged interfaces repel positively charged complexes which keeps the prooxidant away. The mechanistic understanding of this work will help formulators and product developers to choose the right buffer and emulsifier combination for oxidation sensitive emulsions.

Detection of Lipid Oxidation in Infant Formulas: Application of Infrared Spectroscopy to Complex Food Systems.

 Fish- or algal oils have become a common component of infant formula products for their high docosahexaenoic acid (DHA) content. DHA is widely recognized to contribute to the normal development of the infant, and the European Commission recently regulated the DHA content in infant formulas. For many manufacturers of first-age early life nutrition products, a higher inclusion level of DHA poses various challenges. Long-chain polyunsaturated fatty acids (LC-PUFAs) such as DHA are very prone to oxidation, which can alter the organoleptic property and nutritional value of the final product. Traditional methods for the assessment of oxidation in complex systems require solvent extraction of the included fat, which can involve harmful reagents and may alter the oxidation status of the system. A rapid, efficient, non-toxic real-time method to monitor lipid oxidation in complex systems such as infant formula emulsions would be desirable. In this study, infrared spectroscopy was therefore chosen to monitor iron-induced oxidation in liquid infant formula, with conjugated dienes and headspace volatiles measured with GC-MS as reference methods. Infrared spectra of infant formula were recorded directly in mid- and near-infrared regions using attenuated total reflectance Fourier-transform (ATR-FTIR) and near-infrared (NIRS) spectrophotometers. Overall, good correlation coefficients (R2 > 0.9) were acquired between volatiles content and infrared spectroscopy. Despite the complex composition of infant formula containing proteins and sugars, infrared spectroscopy was still able to detect spectral changes unique to lipid oxidation. By comparison, near-infrared spectroscopy (NIRS) presented better results than ATR-FTIR: prediction error ATR-FTIR 18% > prediction error NIRS 9%. Consequently, NIRS demonstrates great potential to be adopted as an in-line or on-line, non-destructive, and sustainable method for dairy and especially infant formula manufacturers.

Fast, direct and in situ monitoring of lipid oxidation in an oil-in-water emulsion by near infrared spectroscopy.

Lipid oxidation has implications on food, cosmetics and other fat containing products. Fatty acid autoxidation alters both the quality and safety of these products. Efficient and fast methods are needed to track lipid oxidation in complex systems. In this study, an oil-in-water emulsion (20% v/v of fish oil stabilized with high oleic sunflower lecithin) was subjected to iron-initiated oxidation. Conjugated dienes (CDs) were measured after fat extraction using a standardized method. Near infrared spectroscopy (NIRS) has been used to record chemical changes occurring during oxidation directly in the emulsion. Variations were noticed in different spectral regions. Partial least squares regression (PLSR) revealed correlations between conjugated diene values and NIRS spectra. High coefficients of determination (R2 = 0.967 and 0.996) were found for calibration and prediction respectively. The CD value was predicted from NIRS spectra with an error of 7.26 mmol eq. LH kg-1 oil (7.8% error). Limits of detection (LOD) and quantification (LOQ) of 4.65 and 15.5 mmol eq. LH kg-1 oil were estimated. NIRS is a rapid and simple method for measuring lipid oxidation (CD value) in an emulsion without prior fat extraction. NIRS can replace the reference methods that use hazardous solvents and consume time. Therefore, NIRS enables in-line monitoring for process and quality control.

Fast and direct analysis of oxidation levels of oil-in-water emulsions using ATR-FTIR.

Oxidation of omega-3 fatty acids is a major limitation on its enrichment in food and beverages. An efficient and simple method to monitor lipid oxidation in complex systems is essential to limit lipid oxidation during formulation and processing. Fish oil-in-water emulsions (20% v/v) were exposed to iron or free radical initiated oxidation. Conjugated dienes (CDs) were rapidly measured using a previously developed fat extraction method. Fourier transform infrared (FTIR) spectroscopy has been used to directly record chemical changes occurring during oxidation. Variations were noticed in different spectral regions despite the presence of broad water bands near 3400 and 1640 cm-1. Partial least squares regression (PLSR) revealed correlations between CD values and full FTIR spectra (4000-600 cm-1), and different spectral regions (e.g., 1800-1500 cm-1, 1500-900 cm-1). These correlations confirm that FTIR spectroscopy is a rapid and simple method for measuring lipid oxidation in complex foods without prior fat extraction.

Soybean oleosomes studied by small angle neutron scattering (SANS).

HYPOTHESIS: Oleosomes are stabilized by a complex outer phospholipid-protein-layer. To improve understanding of its structure and stabilization mechanism, this shell has to be studied in extracellular native conditions. This should be possible by SANS using contrast variation. Oleosomes are expected to be highly temperature stable, with molecular changes occurring first in the protein shell. Direct measurements of changes in the shell structure are also important for processing methods, e.g. encapsulation. EXPERIMENTS: Extracted soybean oleosomes were studied directly and after encapsulation with pectin by SANS using contrast variation. In order to determine structure and size, a shell model of oleosomes was developed. The method was tested against a simple phospholipid-stabilized emulsion. The oleosomes' temperature stability was investigated by performing SANS at elevated temperatures. FINDINGS: Size (Rg = 1380 Å) and shell thickness of native and encapsulated oleosomes have been determined. This is the first report measuring the shell thickness of oleosomes directly. For native oleosomes, a shell of 9 nm thickness surrounds the oil core, corresponding to a layer of phospholipids and proteins. Up to 90 °C, no structural change was observed, confirming the oleosomes' high temperature stability. Successful coavervation of oleosomes was shown by an increase in shell thickness of 10 nm after electrostatic deposition of pectin.

Label-free in situ imaging of oil body dynamics and chemistry in germination.

Plant oleosomes are uniquely emulsified lipid reservoirs that serve as the primary energy source during seed germination. These oil bodies undergo significant changes regarding their size, composition and structure during normal seedling development; however, a detailed characterization of these oil body dynamics, which critically affect oil body extractability and nutritional value, has remained challenging because of a limited ability to monitor oil body location and composition during germination in situ Here, we demonstrate via in situ, label-free imaging that oil bodies are highly dynamic intracellular organelles that are morphologically and biochemically remodelled extensively during germination. Label-free, coherent Raman microscopy (CRM) combined with bulk biochemical measurements revealed the temporal and spatial regulation of oil bodies in native soya bean cotyledons during the first eight days of germination. Oil bodies undergo a cycle of growth and shrinkage that is paralleled by lipid and protein compositional changes. Specifically, the total protein concentration associated with oil bodies increases in the first phase of germination and subsequently decreases. Lipids contained within the oil bodies change in saturation and chain length during germination. Our results show that CRM is a well-suited platform to monitor in situ lipid dynamics and local chemistry and that oil bodies are actively remodelled during germination. This underscores the dynamic role of lipid reservoirs in plant development.

The role of intact oleosin for stabilization and function of oleosomes.

Lipid storage in plants is achieved among all plant species by formation of oleosomes, enclosing oil (triacylglycerides) in small subcellular droplets. Seeds are rich in this pre-emulsified oil to provide a sufficient energy reservoir for growing. The triacylglyceride core of the oleosomes is surrounded by a phospholipid monolayer containing densely packed proteins called oleosins. They are anchored in the triacylglycerides core with a hydrophobic domain, while the hydrophilic termini remain on the surface. These specialized proteins are expressed during seed development and maturation. Particularly, they play a major role in the stabilization and function of oleosomes. To better understand the importance of oleosins for oleosome stabilization, enzymatic digestion of oleosins was performed. This made it possible to compare and correlate changes in the molecular structure of oleosins and changing macroscopic properties of oleosomes. Tryptic digestion cleaves the hydrophilic part of the oleosins, which is accompanied by a loss of secondary structures as evidenced by Fourier-transform infrared and sum frequency generation spectra. After digestion, the ability of oleosins to stabilize oil-water or air-water interfaces was lost. The surface charge and the associated aggregation behavior of oleosomes are governed by interactions typical of proteins before digestion and by interactions typical of phospholipids after digestion.

Soy milk oleosome behaviour at the air-water interface.

Soy milk is a highly stable emulsion mainly due to the presence of oleosomes, which are oil bodies and function as lipid storage organelles in plants, e.g., in seeds. Oleosomes are micelle-like structures with an outer phospholipid monolayer, an interior filled with triacylglycerides (TAGs), and oleosins anchored hairpin-like into the structure with their hydrophilic parts remaining outside the oleosomes, completely covering their surface (K. Hsieh and A. H. C. Huang, Plant Physiol., 2004, 136, 3427-3434). Oleosins are alkaline proteins of 15-26 kDa (K. Hsieh and A. H. C. Huang, Plant Physiol., 2004, 136, 3427-3434) which are expressed during seed development and maturation and play a major role in the stability of oil bodies. Additionally, the oil bodies of seeds seem to have the highest impact on coalescence, probably due to the required protection against environmental stress during dormancy and germination compared to, e.g., vertebrates' lipoproteins. Surface pressure investigations and Brewster angle microscopy of oleosomes purified from raw soy milk were executed to reveal their diffusion to the air-water interface, rupture, adsorption and structural modification over time at different subphase conditions. Destroying the surface portions of the oleosins by tryptic digestion induced coalescence of oleosomes (J. Tzen and A. Huang, J. Cell. Biol., 1992, 117, 327-335) and revealed severe changes in their adsorption kinetics. Such investigations will help to determine the effects behind oleosome stability and are necessary for a better understanding of the principal function of oleosins and their interactions with phospholipids.

Soybean oleosomes behavior at the air-water interface.

Soy milk is a highly stable emulsion, the stability being mainly due to the presence of oleosomes or oil bodies, spherical structures filled with triacylglycerides (TAGs) and surrounded by a monolayer of phospholipids and proteins called oleosins. For oleosomes purified from raw soymilk, surface pressure investigations and Brewster angle microscopy have been performed to unveil their adsorption, rupture and structural changes over time at different subphase conditions (pH, ionic strength). Such investigations are important for (industrial) food applications of oleosomes, but are also useful for the understanding of the general behavior of proteins and phospholipids at interfaces. In addition a better comprehension of the highly stable oleosomes can lead to advancements in liposome manufacturing, e.g., for storage and transport applications. Although oleosomes have their origin in food systems, their unique stability and physical behavior show transferable characteristics which lead to a much better understanding of the description of any kind of emulsion. This study is one of the first steps toward the comparison of natural emulsification concepts based on different physical structures: e.g., the animals' low density lipoproteins, where apolipoproteins with phospholipids are located only at the interface and plant oleosomes with its oleosins, which are embedded in a phospholipid monolayer and reach deep inside the oil phase.

Photo-DHEA--a functional photoreactive dehydroepiandrosterone (DHEA) analog.

The steroid hormone dehydroepiandrosterone (DHEA) has beneficial effects on vascular function, survival of neurons, and fatty acid metabolism. However, a specific receptor for DHEA has not been identified to date. Here, we describe the synthesis of a photoreactive DHEA derivative (Photo-DHEA). In Photo-DHEA, typical characteristics of DHEA are conserved: (i) a "planar" tetracyclic ring system with a Δ(5) double bond, (ii) a 3ß-hydroxyl group, and (iii) a keto group at C17. In cell-based assays, Photo-DHEA showed the same properties as DHEA. We conclude that Photo-DHEA is suitable for radioiodination to yield a tool for the identification of the elusive DHEA receptor.

DHEA-Bodipy--a functional fluorescent DHEA analog for live cell imaging.

The androgen dehydroepiandrosterone (DHEA) has been reported to protect neuronal cells against dysfunction and apoptosis. Several signaling pathways involved in these effects have been described but little is known about the intracellular trafficking of DHEA. We describe design, synthesis and characterization of DHEA-Bodipy, a novel fluorescent DHEA analog. DHEA-Bodipy proved to be a functional DHEA derivative: DHEA-Bodipy (i) induced estrogen receptor alpha-mediated gene activation, (ii) protected PC12 rat pheochromocytoma cells against serum-deprivation-induced apoptosis, and (iii) induced stress fibers and focal adhesion contacts in SH-SY5Y human neuroblastoma cells. DHEA-Bodipy bound rapidly and specifically to plasma membranes of living PC12 cells. We analyzed metabolism and trafficking of DHEA-Bodipy in human neuroblastoma cells. DHEA-Bodipy is the first functional fluorescent DHEA derivative suitable for live cell imaging of intracellular DHEA transport and localization.