Continuous counter-current electrophoretic separation of oleosomes and proteins from oilseeds

Kübra Ayan

This research summary is based on the article “Continuous counter-current electrophoretic separation of oleosomes and proteins from oilseeds,” recently published in Food Hydrocolloids. It explains how Kübra Ayan and colleagues developed a simpler electrophoretic separation system for extracting oleosomes and proteins from oilseeds. The Food Process Engineering Group & Biobased, Chemistry and Technology Group performed the work at Wageningen University in the Netherlands.

Abstract

Oleosomes and proteins are two functional compounds that can be extracted from oilseeds following a mild aqueous extraction procedure. A centrifugation step can achieve their ultimate separation; however, it requires a large amount of water, capital investment, and intensive maintenance. Therefore, we proposed a simpler electrophoretic separation system that relies on a balance between electrophoresis and counter-current fluid flow. In the developed system, compounds with high electrophoretic mobility (electrophoresis rate) are captured by an electric field, and compounds with lower electrophoretic mobility are dragged by the solvent flow. As a result, the compounds migrate in opposite directions, and the separation is performed. To observe this behavior in the case of oleosome and protein separation, a single-channel PDMS microfluidic device was fabricated, and the movement of oleosomes and proteins was tracked under changing electric field strengths and fluid flow velocity. The results indicated that oleosomes have higher electrophoretic mobility than proteins, and they can be separated when a counter-current solvent flow rate is set between their electrophoresis rates.

Introduction | Electrophoretic separation and pressure-driven flow

Oilseed components like oils and proteins can be utilized as functional food ingredients. Oils are stored in special organelles named oleosomes, which are natural oil-in-water emulsions with great stability¹. Furthermore, oilseed proteins are excellent sources of plant-based proteins with functionalities comparable to animal-based proteins².

To obtain these compounds, however, a proper extraction strategy is needed. Currently, an aqueous extraction is used, but there is still some room for improvement and alternative techniques are being investigated to develop a more sustainable route. Therefore, a simpler electrophoretic separation approach has been introduced for oilseed oleosomes and protein fractionation.

Electrophoretic separation can be useful as both oleosomes and proteins are charged compounds. In the electrophoretic separation, compounds are separated according to their electrophoretic mobility, migration rate of a compound under a unit electric field strength (1 V/cm).

Oleosomes and proteins obtained from rapeseed have distinct electrophoretic mobility values at pH ≥ 5.0, yet both are negatively charged. To achieve a separation between the two, an additional force like counter-current flow that causes opposite migration of the compounds is needed. When an electric field and pressure-driven solvent flow are combined, oleosomes (high electrophoretic mobility) are retained by the electric field, and proteins (low electrophoretic mobility) go along with the flow. Figure 1 illustrates the designed separation technique.

Aims

• Designing of a continuous electrophoretic separation technique for oleosome and protein fractionation.
• Observation of migration pattern of oleosomes and proteins under different electric field strength and fluid flow velocity values using a PDMS – based microfluidic device.

Experiment setup | Balancing electrophoresis and counter-current fluid flow

Oleosomes and proteins are extracted from rapeseed and dispersed in 1.0 mM potassium phosphate buffer at pH 8.0 to achieve 0.1 mg/mL final concentration. Oleosomes are dyed by curcumin (λ_ex = 430 nm), and proteins are dyed by fast green dye (λ_ex = 633 nm) for visualization under an inverted fluorescent microscope.

• A single channel PDMS – based microfluidic device (L: 2 cm, W: 400 mm, D: 30 mm) was fabricated to miniaturize the electrophoretic separation of oleosomes and proteins.
• Two stainless steel tubing were inserted into the inlet and outlet parts of the microfluidic device to serve as electrodes. Then, they are connected to a power supplier using crocodile clips.
• The sample dispersion (mixture of oleosomes and proteins) was introduced into the channel using an OB1 MK3+ flow controller connected to 0.5 mm ID silicone tubing and a 25 μm ID flow resistor.
• Migration of only oleosomes, only proteins, and their mixture was recorded and investigated under increasing electric field strength and/or pressure (flow rate).

Materials

• 76 mm silicon wafer
• SU-8 25 photoresist (Kayaku, MA, USA)
• Micro-writer device (MicroWriter ML3, Durham Magneto Optics Ltd., Germany)
• Propylene glycol monomethyl ether acetate (PGMEA)
• PDMS and a curing agent (SYLGARD™ 184 Silicone Elastomer)
• Plasma Cleaner (PDC–32 G, Harrick Plasma, NY, USA)
• Polyvinyl alcohol (PVA)
• Pressure controller (OB1 MK3+, Elveflow, France)
• Power supplier
• Fluorescent microscope (Nikon- Ti2-Eclipse)

Key findings | Electrophoretic separation pattern is limited by pH alteration

• Migration of oleosomes and protein particles is governed by the combination of electrophoresis and pressure-driven flow.
• Proteins are taken up by the hydrodynamic flow before oleosomes under constant electric field strength due to their lower electrophoretic mobility (Figure 3).

• When the electric field and pressure difference are balanced, oleosomes and proteins move in opposite directions, and the separation is observed (Figure 4).

• The separation pattern is limited by pH alteration due to the production of electrolysis products, like H⁺ and OH^– ions, as it affects the electrophoretic mobility of the compounds. Therefore, it has to be controlled for an upscaled system.

Conclusions | Electrophoretic separation can be used for upscaling extraction processes

A continuous electrophoretic separation process can be utilized to separate oleosomes and proteins from oilseeds and any other compounds that differ in their electrophoretic mobility. The developed system is also suitable for upscaling as it is based on a simple balance between two anti-parallel forces (electrophoresis and solvent flow).