Biosensing with Oleosin-Stabilized Liquid Crystal Droplets.

Liquid crystals (LCs) are emerging as novel platforms for chemical, physical, and biological sensing. They can be used to detect biological amphiphiles such as lipids, fatty acids, digestive surfactants, and bacterial endotoxins. However, designing LC-based sensors in a manner that preserves their sensitivity and responsiveness to these stimuli, and possibly improves biocompatibility, remains challenging. In this work, the stabilization of LC droplets by oleosins, plant-sourced and highly surface active proteins due to their extended amphipathic helix, is investigated. Purified oleosins, at sub-micromolar concentrations, are shown to readily stabilize nematic LC droplets without switching their alignment, allowing them to detect surfactants at micromolar concentrations. Direct evidence of localization of oleosins at the LC-water interface is provided with fluorescent labeling, and the stabilized droplets remain stable over months. Interestingly, chiral LC droplets readily switch in the presence of nanomolar oleosin concentrations, an unexpected behavior that is explained by accounting for the energy barriers required for switching the alignment between the two cases. This leads thus to a twofold conclusion: oleosin-stabilized nematic LC droplets present a biocompatible alternative for bioanalyte detection, while chiral LCs can be further investigated for use as highly sensitive sensors for detecting amphipathic helices in biological systems.

Efficiency of aqueous oleosome extraction from capsicum seeds compared to classical oil extraction.

The extraction of oil from oilseeds in intact oleosomes is one of the suggested processes that could replace the extraction of oil by pressing and solvent extraction, being milder, environmentally less impactful and potentially more efficient in its use of resources. This study assesses the latter using an exergy assessment of oleosome extraction for food emulsions. The contribution of each part of the process to the overall impact was investigated. Based on current lab-scale data, oleosome extraction has nearly twice the exergy loss compared to the industrial process of oil extraction and industrial assembly of emulsions. The exergy losses of the lab-scale oleosome extraction are currently dominated by the chemical exergy associated with product loss during the separation of oleosomes from the rest of the biomass. This loss is expected to significantly decrease when upscaled to industrial scale. When substituted with industrial material efficiencies, the total exergy loss decreased to nearly a quarter of the original loss, representing oleosome extraction as a potentially more effective and environment-friendly option.

The mechanism of encapsulating curcumin into oleosomes (Lipid Droplets).

Organisms have evolved intracellular micron-sized lipid droplets to carry and protect lipids and hydrophobic minor compounds in the hydrophilic environment of cells. These droplets can be utilized as carriers of hydrophobic therapeutics by taking advantage of their biological functions. Here, we focus on the potential of plant-derived lipid droplets, known as oleosomes, as carriers for hydrophobic therapeutics, such as curcumin. By spectroscopy and confocal microscopy, we demonstrate that the oleosome membrane is permeable to hydrophobic curcumin molecules. Fluorescence recovery after photobleaching shows rapid curcumin diffusion towards oleosomes, with a diffusion time in the range of seconds. Following this, quenching probes and dilatational rheology reveal that part of the loaded curcumin molecules can accumulate at the oleosome interface, and the rest settle in the inner core. Our findings shed light on the loading mechanism of the plant-derived lipid droplets and underscore the significance of molecular localization for understanding the mechanism. This work not only enhances the understanding of the loading process but also shows potential for oleosomes use as lipid carriers.

Oil-water interface and emulsion stabilising properties of rapeseed proteins napin and cruciferin studied by nonlinear surface rheology.

HYPOTHESIS: Two major protein families are present in rapeseed, namely cruciferins and napins. The structural differences between the two protein families indicate that they might behave differently when their mixture stabilises oil-water interfaces. Therefore, this work focuses on elucidating the role of both proteins in interface and emulsion stabilisation. EXPERIMENTS: Protein molecular properties were evaluated, using SEC, DSC, CD, and hydrophobicity analysis. The oil-water interface mechanical properties were studied using LAOS and LAOD. General stress decomposition (GSD) was used as a novel method to characterise the nonlinear response. Additionally, to evaluate the emulsifying properties of the rapeseed proteins, emulsions were prepared using pure napins or cruciferin and also their mixtures at 1:3, 1:1 and 3:1 (w:w) ratios. FINDINGS: Cruciferins formed stiff viscoelastic solid-like interfacial layers (Gs' = 0.046 mN/m; Ed' = 30.1 mN/m), while napin formed weaker and more stretchable layers at the oil-water interface (Gs' = 0.010 mN/m; Ed' = 26.4 mN/m). As a result, cruciferin-formed oil droplets with much higher stability against coalescence (coalescence index, CI up to 10%) than napin-stabilised ones (CI up to 146%) during two months of storage. Both proteins have a different role in emulsions produced with napin-cruciferin mixtures, where cruciferin provides high coalescence stability, while napin induces flocculation. Our work showed the role of each rapeseed protein in liquid-liquid multiphase systems.

Insights into the emulsification mechanism of the surfactant-like protein oleosin.

Oleosins are proteins with a unique central hydrophobic hairpin designed to stabilize lipid droplets (oleosomes) in plant seeds. For efficient droplet stabilization, the hydrophobic hairpin with a strong affinity for the apolar droplet core is flanked by hydrophilic arms on each side. This gives oleosins a unique surfactant-like shape making them a very interesting protein. In this study, we tested if isolated oleosins retain their ability to stabilize oil-in-water emulsions, and investigated the underlying stabilization mechanism. Due to their surfactant-like shape, oleosins when dispersed in aqueous buffers associated to micelle-like nanoparticles with a size of ∼33 nm. These micelles, in turn, clustered into larger aggregates of up to 20 µm. Micelle aggregation was more extensive when oleosins lacked charge. During emulsification, oleosin micelles and micelle aggregates dissociated and mostly individual oleosins adsorbed on the oil droplet interface. Oleosins prevented the coalescence of the oil droplets and if sufficiently charged, droplet flocculation as well.

The role of membrane components on the oleosome lubrication properties.

HYPOTHESIS: Oleosomes are natural oil droplets with a unique phospholipid/protein membrane, abundant in plant seeds, from which they can be extracted and used in emulsion-based materials, such as foods, cosmetics and pharmaceutics. The lubrication properties of such materials are essential, on one hand, due to the importance of the in-mouth creaminess for the consumed products or the importance of spreading the topical creams. Therefore, here, we will evaluate the lubrication properties of oleosomes, and how these properties are affected by the components at the oleosome membrane. EXPERIMENT: Oleosomes were extracted, and their oral lubricating properties were evaluated using tribology. To understand the influence of the oil droplet membrane composition, reconstituted oleosomes were also studied, with membranes that differed in protein/lecithin ratio. Additionally, whey protein- and lecithin-stabilised emulsions were used as reference samples. Confocal laser scattering microscopy was used to study the samples visually before and after tribological analysis. FINDINGS: Oleosomes followed a ball-bearing mechanism, which was probably related to their high physical stability due to the presence of membrane proteins. When the membrane protein concentration at the surface was reduced, the droplet stability weakened, leading to plating-out lubrication. Following our results, we elucidated the oleosome lubrication mechanism and showed their possible control by changing the membrane composition.

The dilatable membrane of oleosomes (lipid droplets) allows their in vitro resizing and triggered release of lipids.

It has been reported that lipid droplets (LDs), called oleosomes, have an inherent ability to inflate or shrink when absorbing or fueling lipids in the cells, showing that their phospholipid/protein membrane is dilatable. This property is not that common for membranes stabilizing oil droplets and when well understood, it could be exploited for the design of responsive and metastable droplets. To investigate the nature of the dilatable properties of the oleosomes, we extracted them from rapeseeds to obtain an oil-in-water emulsion. Initially, we added an excess of rapeseed oil in the dispersion and applied high-pressure homogenization, resulting in a stable oil-in-water emulsion, showing the ability of the molecules on the oleosome membrane to rearrange and reach a new equilibrium when more surface was available. To confirm the rearrangement of the phospholipids on the droplet surface, we used molecular dynamics simulations and showed that the fatty acids of the phospholipids are solubilized in the oil core and are homogeneously spread on the liquid-like membrane, avoiding clustering with neighbouring phospholipids. The weak lateral interactions on the oleosome membrane were also confirmed experimentally, using interfacial rheology. Finally, to investigate whether the weak lateral interactions on the oleosome membrane can be used to have a triggered change of conformation by an external force, we placed the oleosomes on a solid hydrophobic surface and found that they destabilise, allowing the oil to leak out, probably due to a reorganisation of the membrane phospholipids after their interaction with the hydrophobic surface. The weak lateral interactions on the LD membrane and their triggered destabilisation present a unique property that can be used for a targeted release in foods, pharmaceuticals and cosmetics.

The emulsifying ability of oleosomes and their interfacial molecules.

Oleosomes are natural oil droplets, present in all organisms and abundant in oilseeds. After their aqueous extraction from oilseeds, they can be directly utilized as oil droplets in food, cosmetics and all types of oil-in-water emulsion systems. However, to expand the potential uses of oleosomes as green ingredients and to valorize oilseeds as efficient as possible, we explored their emulsifying ability. Oleosomes were extracted from rapeseeds, and 10.0 wt% oil-in-water emulsions were created after homogenization with 0.5-6.0 wt% oleosomes, and the droplet size of the emulsions and their structure was measured by laser diffraction and confocal laser scanning microscopy (CLSM), respectively. The emulsion with an oleosome concentration lower than 1.0 wt% gave unstable emulsions with visible free oil. At oleosome concentrations at 1.5 wt% or higher, we obtained stable emulsions with droplet sizes between 2.0 and 12.0 µm. To investigate the role of the oleosome interfacial molecules in stabilizing emulsions we also studied their emulsifying and interfacial properties (using drop tensiometry) after isolating them from the oleosome structure. Both oleosomes and their isolated interfacial molecules exhibited a similar behavior on the oil-water interfaces, forming predominantly elastic interfacial films, and also showed a similar emulsifying ability. Our results show that oleosomes are not stabilizing the oil-in-water emulsions as intact particles, but they provide their interfacial molecules, which are enough to stabilize an oil-water surface up to about 2 times bigger than the initial oleosome surface. The understanding of the behavior of oleosomes as emulsifiers, opens many possibilities to use oleosomes as alternative to synthetic emulsifiers in food and pharma applications.

On the influence of protein aggregate sizes for the formation of solid and hollow protein microparticles.

Microparticles can function as carriers of e.g. pharmaceuticals and food ingredients. Hollow microparticles can enhance the capacitance due to their large interior void. For preparing microparticles, polymers have been assembled into spherical structures through the use of porous CaCO3 templates, followed by polymer cross-linking and selective template removal. However, this often results in the formation of microparticles with a solid core. Here we use proteins with different aggregate size distributions (<10 nm or >100 nm) to either form solid or hollow microparticles. Proteins were mixed with CaCl2 and Na2CO3 solutions, which from CaCO3 microcrystals (with 20-60 nm pores) with encapsulated proteins. Here it will be shown that small protein aggregates uniformly distributed into the CaCO3 templates. However, larger protein aggregates accumulated at the template edges. Au3+ ions were then added, which oxidize and cross-link proteins and are reduced to form gold nanoparticles (AuNPs). After removal of the templates, the small proteins formed solid microparticles and the larger protein aggregates hollow microparticles. This method of fabrication of solid and hollow protein microparticles, with embedded AuNPs, could be used for generating biomaterials with a broader range of applications, such as hosting molecules and multimodal imaging due to the presence of the AuNPs.

Hollow protein microparticles formed through cross-linking by an Au3+ initiated redox reaction.

Hollow microparticles (MPs) are of great relevance in the materials industry for a wide range of applications, such as catalysis, coatings, and delivery of theranostics. Here, we report the formation of hollow MPs through the assembly of lipoproteins in CaCO3 templates. Proteins interact in the pores of CaCO3 templates through attractive hydrophobic forces and form dense edges of hollow MPs. To further cross-link the proteins, Au3+ was added to initiate a redox reaction, where proteins were oxidized forming inter- and intramolecular covalent bonds, while Au3+ was reduced and gold nanoparticles (AuNPs) were formed. The obtained protein-based hollow MPs have a diameter of 6 µm and the AuNPs are embedded on their surface. Through this research, we suggest a new route to design biobased Au-protein hollow MPs in simple steps, which can allow new possibilities for carrying functional molecules and bioimaging.

Au3+-Induced gel network formation of proteins.

The formation of protein gel networks in aqueous systems is a result of protein intermolecular interactions after an energy input, like heating. In this research, we report that a redox reaction between Au3+ ions and proteins can also lead to the formation of a protein gel network. Amino acids, like cysteine and tyrosine, get oxidized and form covalent bonds with neighboring protein molecules, while Au3+ ions get reduced to Au+ and Au0, nucleate and form gold nanoparticles. The protein gel network formation occurs within 2 h at room temperature and can be tuned by varying Au3+/protein ratio and accelerated by increasing the incubation temperature. The proposed Au3+-induced gel network formation was applied to different proteins, like egg yolk high-density lipoprotein, bovine serum albumin and whey protein. This research opens new insights for the investigation of the metal-protein interactions and may aid in the design of novel hybrid-soft nanocomposite materials.

Air-water interfacial behaviour of whey protein and rapeseed oleosome mixtures.

HYPOTHESIS: Plant seeds store lipids in oleosomes, which are storage organelles with a triacylglycerol (TAG) core surrounded by a phospholipid monolayer and proteins. Due to their membrane components, oleosomes have an affinity for the air/oil-water interface. Therefore, it is expected that oleosomes can stabilise interfaces, and also compete with proteins for the air-water interface. EXPERIMENTS: We mixed rapeseed oleosomes with whey protein isolate (WPI), and evaluated their air-water interfacial properties by interfacial rheology and microstructure imaging. To understand the contribution of the oleosome components to the interfacial properties, oleosome membrane components (phospholipids and membrane proteins) or rapeseed lecithin (phospholipids) were also mixed with WPI. FINDINGS: Oleosomes were found to disrupt after adsorption, and formed TAG/phospholipid-rich regions with membrane fragments at the interface, forming a weak and mobile interfacial layer. Mixing oleosomes with WPI resulted in an interface with TAG/phospholipid-rich regions surrounded by whey protein clusters. Membrane components or lecithin mixed with proteins also resulted in an interface where WPI molecules aggregated into small WPI domains, surrounded by a continuous phase of membrane components or phospholipids. We also observed an increase in stiffness of the interfacial layer, due to the presence of oleosome membrane proteins at the interface.

Adsorption of rapeseed proteins at oil/water interfaces. Janus-like napins dominate the interface.

Plants offer a vast variety of protein extracts, typically containing multiple species of proteins that can serve as building blocks of soft materials, like emulsions. However, the role of each protein species concerning the formation of emulsions and interfaces with diverse rheological properties is still unknown. Therefore, deciphering the role of the individual proteins in an extract is highly relevant, since it determines the optimal level of purification, and hence the sustainability aspects of the extract. Here, we will show that when oil/water emulsions were prepared with a rapeseed protein extract containing napins and cruciferins (in a mass ratio of 1:1), only napins adsorbed at the interface exhibiting a soft solid-like rheological behavior. The dominance of napins at the interface was ascribed to their small size (radius r = 1.7 nm) and its unique Janus-like structure, as 45% of the amino acids are hydrophobic and primarily located at one side of the protein. Cruciferins with a bigger size (r = 4.4 nm) and a more homogeneous distribution of the hydrophobic domains couldn't reach the interface, but they appear to just weakly interact with the adsorbed layer of napins.

On the Emulsifying Properties of Self-Assembled Pea Protein Particles.

Pea proteins are promising oil-in-water emulsifying agents at both neutral and acidic conditions. In an acidic environment, pea proteins associate to form submicrometer-sized particles. Previous studies suggested that the emulsions at acidic pH were stabilized due to a Pickering mechanism. However, protein particles can be in equilibrium with protein molecules, which could play a significant role in the stabilization of emulsion droplets. Therefore, we revisited the emulsion stabilization mechanism of pea proteins at pH 3 and investigated whether the protein particles or the protein molecules are the major emulsifying agent. The theoretical and experimental surface load of dispersed oil droplets were compared, and we found that protein particles can cover only 3.2% of the total oil droplet surface, which is not enough to stabilize the droplets, whereas protein molecules can cover 47% of the total oil droplet surface. Moreover, through removing protein particles from the mixture and emulsifying with only protein molecules, the contributions of pea protein molecules to the emulsifying properties of pea proteins at pH 3 were evaluated. The results proved that the protein molecules were the primary stabilizers of the oil droplets at pH 3.

Assessment of rapeseed oil body (oleosome) lipolytic activity as an effective predictor of emulsion purity and stability.

The lipolytic activity in oil body creams as affected by recovery and washing protocols was investigated. The effect of thermal treatment on the hydrolytic activity and physical stability of fresh and aged (up to 30 days) oil body emulsions was studied. The use of alkaline pH solutions (9.5) to soak and grind rapeseeds were more effective reducing the contamination of oil body material from seed proteins/enzymes, compared with neutral pHs. Soaking and grinding seeds with a NaHCO3 solution (0.1 M, pH 9.5) yielded oil bodies with a similar composition to those prepared in urea (9 M); however, the physical stability over storage was compromised due to the presence of hydrolytic enzymes. Heating a dispersion of oil bodies for 6 mins at 95 °C did not alter the physical properties of oil bodies and significantly reduced lipolytic activity (>90% enzyme inactivation), resulting in a stable emulsion.

The effect of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations on rapeseed oleosome (oil body) extraction and stability at pH 7.

Oleosomes are storage vehicles of TAGs in plant seeds. They are protected with a phospholipid-protein monolayer and extracted with alkaline aqueous media; however, pH adjustment intensifies the extraction process. Therefore, the aim of this work was to investigate the extraction mechanism of rapeseed oleosomes at pH 7 and at the presence of monovalent and divalent cations (Na+, K+, Mg2+, and Ca+2). The oleosome yield at pH 9.5 was 64 wt%, while the yield at pH 7 with H2O was just 43 wt.%. The presence of cations at pH 7, significantly enhanced the yield, with K+ giving the highest yield (64 wt.%). The cations affected the oleosome interface and their interactions. The presence of monovalent cations resulted in aggregation and minor coalescence, while divalent cations resulted in extensive coalescence. These results help to understand the interactions of oleosomes in their native matrix and design simple extraction processes at neutral conditions.

Structure and functions of oleosomes (oil bodies).

Oleosomes are natural oil droplets, abundant in plants and more specifically in seeds, composing 20-50 wt% of their mass. The structure of oleosomes is the mechanism that seeds developed to safely store energy in the form of triacylglycerols and use it during germination. For this, the phospholipid/protein membrane that covers and protects the triacylglycerols has been wisely developed during evolution to grant them extreme stability against physical and chemical stresses. The remarkable property-performance relationships of oleosomes have generated a lot of interest to incorporate them in oil-in-water emulsions and take advantage of their sophisticated membrane. However, the structure-function relationship of the molecular components in the oleosome membrane is still not well understood and requires more attention in order to take complete advantage of their potential functions. The aim of this review is to give insights into the architecture of the oleosomes and to discuss the exploitation of their properties in advanced and broad applications, from carrying and protecting sensitive molecules to bio-catalysis.

Exploring the Treasure of Plant Molecules With Integrated Biorefineries.

Despite significant progress toward the commercialization of biobased products, today's biorefineries are far from achieving their intended goal of total biomass valorization and effective product diversification. The problem is conceptual. Modern biorefineries were built around well-optimized, cost-effective chemical synthesis routes, like those used in petroleum refineries for the synthesis of fuels, plastics, and solvents. However, these were designed for the conversion of fossil resources and are far from optimal for the processing of biomass, which has unique chemical characteristics. Accordingly, existing biomass commodities were never intended for modern biorefineries as they were bred to meet the needs of conventional agriculture. In this perspective paper, we propose a new path toward the design of efficient biorefineries, which capitalizes on a cross-disciplinary synergy between plant, physical, and catalysis science. In our view, the best opportunity to advance profitable and sustainable biorefineries requires the parallel development of novel feedstocks, conversion protocols and synthesis routes specifically tailored for total biomass valorization. Above all, we believe that plant biologists and process technologists can jointly explore the natural diversity of plants to synchronously develop both, biobased crops with designer chemistries and compatible conversion protocols that enable maximal biomass valorization with minimum input utilization. By building biorefineries from the bottom-up (i.e., starting with the crop), the envisioned partnership promises to develop cost-effective, biomass-dedicated routes which can be effectively scaled-up to deliver profitable and resource-use efficient biorefineries.

The behaviour of sunflower oleosomes at the interfaces.

Oleosomes are particles equipped with a sophisticated membrane, comprising a continuous monolayer of phospholipids and hydrophobic proteins, which covers the triglyceride core and grants them extreme physical and chemical stability. The noteworthy qualities of oleosomes have attracted strong interest for their incorporation in emulsion formulations; however, little is known about their emulsifying properties and their behaviour on interfaces. For these reasons, oleosomes were isolated from sunflower seeds (96.2 wt% oil, 3.1 wt% protein) and used as an emulsifier for the stabilization of O/W and W/O interfaces. In both cases, oleosomes showed high interfacial and emulsifying activity. Individual oleosome particles had a broad size distribution from 0.4 to 10.0 µm and it was observed that the membrane of the larger oleosomes (>1-5 µm) was disrupted and its fractions participated in the newly formed interface. Oleosomes with a smaller diameter (<1 µm) seemed to have survived the applied mild emulsification step as a great number of them could be observed both in the bulk of the emulsions and on the interface of the emulsion droplets. This phenomenon was more pronounced for the W/O interface where oleosomes were absorbed intact in a manner similar to a Pickering mechanism. However, when the triglycerides were removed from the core of oleosomes in order to focus more on the effect of the membrane, the remaining material formed sub-micron spherical particles, which clearly acted as Pickering stabilisers. These findings showcase the intriguing behaviour of oleosomes upon emulsification, especially the crucial role of their membrane. The study demonstrates relevance for applications where immiscible liquid phases are present.

In vitro lipid digestion in raw and roasted hazelnut particles and oil bodies.

Previous studies have proved that the physical encapsulation of nutrients by the cell walls of plant foods modulates macronutrient bioaccessibility during human digestion. In this study, we investigated structural factors that modulate lipid hydrolysis during in vitro digestion of raw and roasted hazelnut particles and isolated oil bodies. Isolated oil bodies exhibited a significantly higher lipid hydrolysis compared to hazelnut particles. Moreover, roasting had an impact on the structure of hazelnut cell walls implying a more efficient diffusion of digestive fluids and enzymes into the hazelnut cells. Heat treatment also caused destabilization of oil body interfacial protein membranes, facilitating their proteolysis under gastric conditions, altering the emulsion properties and enhancing fatty acid release during intestinal digestion. This study underlined the barrier role played by the plant cell wall as well as the impact of heat processing on lipid bioaccessibility in hazelnuts.