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.

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.