A. Tamayo
Tenorio
a,
E. W. M.
de Jong
a,
C. V.
Nikiforidis
ab,
R. M.
Boom
a and
A. J.
van der Goot
*a
aFood Process Engineering Group, Wageningen University, P.O. Box 17, 6700 AA Wageningen, The Netherlands. E-mail: Atzejan.vandergoot@wur.nl
bBiobased Chemistry and Technology Group, Wageningen University, P.O. Box 17, 6700 AA Wageningen, The Netherlands
First published on 5th December 2016
Thylakoids membranes are sophisticated, dynamic structures found in plant leaves, composed of protein complexes in a dynamic lipid matrix. The interfacial absorption dynamics and viscoelasticity of thylakoid membranes fragments were measured to assess the properties of the interfacial layer and to elucidate an emulsifying mechanism that includes the role of thylakoid's composition and 3D structure. Thylakoid membranes were extracted from sugar beet leaves by a series of buffer washing, filtration and centrifugation. The extract containing the intact thylakoid membranes was suspended in water through high-pressure homogenisation, which disrupted the structure into membrane fragments. Thylakoid fragments showed surface and interfacial behaviour similar to soft particles or Pickering stabilizers with slow adsorption kinetics. After adsorption, an elastic and stable thin film was formed, indicating formation of new interactions between adjacent thylakoid fragments. In an emulsion, thylakoid fragments stabilised oil droplets against coalescence, despite droplet aggregation occurring already during emulsification. Droplet aggregation occurred by steric and electrostatic bridging, which in turn forms a 3D network where the oil droplets are immobilised, preventing further droplet coalescence or aggregation. It was concluded that both composition and structure of thylakoid fragments determine their emulsifying properties, conferring potential for encapsulation systems, where the search for natural materials is gaining more attention.
Thylakoids are located in the chloroplasts of green leaves in the form of stacks (granal thylakoids) that are interconnected by non-stacked lamellae (stromal thylakoids).2 These two domains are organized in a complex 3D network that is facilitated by the special composition of thylakoid membranes.3 Thylakoids consist of 60–65 wt% proteins and 35–40 wt% lipids.4–6 The thylakoid proteins are protein complexes that are unevenly distributed within the membrane; their highly specialised functions explain their complex composition and 3D arrangement. Unlike most eukaryotic membranes, thylakoid lipids are poor in phospholipids (∼5–12 wt%), but composed of galactolipids like monogalactosyldiacylglycerol (MGDG; ∼50%), digalactosyldiacylglycerol (DGDG; ∼30%), and sulfoquinovosyldiacylglycerol (SQDG; ∼5–12%).7 Both proteins and lipids in the thylakoid membranes adopt an intricate but dynamic structure,1 showing transitions between conformations (e.g. bilayer to hexagonal phases) to enable enzyme activity7 or to respond to stress conditions.4
Their sophisticated and dynamic structure motivates the study of thylakoids as a biobased material for valuable applications, as a response to the current need for resource optimisation and demand for natural ingredients. The use of more intact parts present in biomass is a promising route to reach those goals. Thylakoids are known to stabilize oil droplets (emulsifiers) and exhibit satiation effect upon consumption,8,9 which has been attributed to slow digestion rate of the thylakoids and the subsequent release of satiety hormones, together with lipolysis inhibition.9 Both the emulsifying ability and slow digestibility make thylakoids an excellent candidate as stabilizers of oil capsules for targeted release of healthy compounds. A further understanding of their functional properties may lead to advanced applications in foods, cosmetics and pharmaceuticals.
Little is known about the emulsifying mechanism and interfacial properties of thylakoid membranes8,10 and no literature reports on how these aspects are linked to their composition and 3D structure. It can be easily hypothesized that both proteins and lipids play an important role in droplet stabilisation. The interfacial properties of the protein/lipid complexes can be analysed based on adsorption dynamics and dilatational rheology analysis. The adsorption dynamics provide a deeper understanding of the kinetics of molecules towards an interface, while compression/expansion measurements define the interfacial viscoelasticity of the adsorbed layer through the dilatational modulus.
In this study, we extracted thylakoid membranes from sugar beet leaves based on protocols developed for photosynthesis analysis, rendering a rather pure thylakoid isolate. The characterisation of the isolated thylakoids focused on their technological functionality as emulsifiers. Their interfacial absorption and viscoelasticity were measured to assess the properties of the interfacial film. These fundamental measurements were further used to build a hypothesis on the emulsification mechanism with thylakoids as emulsifying structures. The discussion describes the possible relations of the interfacial behaviour with the thylakoid's composition and native 3D arrangements. Moreover, extraction of thylakoids from side streams like sugar beet leaves, constitutes a high value application for leafy biomass that takes advantage of functional structures present in nature.11
![]() | ||
Fig. 1 Extraction protocol of thylakoid membranes from leaves. At each centrifugation step, only the thylakoid-containing layer was recovered to continue the protocol. Thy = thylakoids. |
![]() | (1) |
Ed = Ed′ + Ed′′ | (2) |
The real part (Ed′) is called the storage modulus and represents the elastic energy stored in the interface (i.e. dilatational elasticity). The imaginary part (Ed′′) is called the loss modulus and accounts for the energy dissipation in the relaxation process (i.e. dilatational viscosity).
![]() | (3) |
Thylakoid emulsions were also observed with a light microscope (Axiovert-Zeiss, Gottingen, Germany) fitted with a digital camera (Axio Cam MRc 5, Gottingen, Germany). The samples were observed with and without polarised light.
Composition | wt% ± sd |
---|---|
Protein | 50.5 ± 6.5 |
Dietary fibre | 14.2 ± 3.2 |
Lipids | 17.1 ± 2.3 |
Ash | 4.6 ± 1.9 |
To visualise the thylakoids in their native form and after extraction, a leaf cross section and the thylakoid extract were analysed with the aid of TEM. Thylakoid structures were identified inside the chloroplast of the fresh leaf (Fig. 2a, arrows) as well as in the thylakoid extract (Fig. 2b). The thylakoid extract and the fresh leaf contained additional cell structures such as plastoglubules. The latter are thylakoid microdomains responsible for storage and synthesis of lipids2 and they are identified as dark spherical structures next to the membrane structures.
![]() | ||
Fig. 2 TEM micrographs. (a) Leaf cross section focused on a chloroplast portion, thylakoid membranes (Thy) and plastoglobules (P) are identified with the arrows. (b) Thylakoid extract from sugar beet leaves, focused on a grana structure. (c) 3D schematic model of thylakoid grana structure, adapted from Ruban & Johnson.1 The plane denotes a transversal cut, which is normally observed with TEM micrographs. (d) A cartoon of the transversal view of the thylakoid grana. |
The extract was a very dense material (semisolid) rich in stacked thylakoid membranes (grana). The stacked thylakoids are 3D structures as depicted in Fig. 2c, and the interconnected supramolecular structures observed with TEM are actually a transversal view of the cylindrical structure. The cylindrical grana stacks are made of membranous discs piled one on top of the other, surrounded by unstacked membranes that are helically bound around the grana.18 A cartoon of the transversal view of the grana is shown in Fig. 2d, showing the crowded distribution of protein complexes throughout the lipid bilayer. The protein complexes in adjacent membranes are bridged through electrostatic interactions mediated by Mg2+ ions. Thylakoids are negatively charged due to protein phosphorylation and Mg2+ ions can balance the electrostatic repulsion by forming bridges between membranes, leading to membrane stacking.19,20 The membrane lipids (i.e. DGDG) also play a role in stacking by screening the negative charges and through hydrogen bonding between polar heads of adjacent membranes.21 Thus thylakoid membranes have the capacity to stack (or aggregate).
![]() | ||
Fig. 3 ζ-Potential of thylakoid extract upon pH change. Thylakoid membranes were extracted from sugar beet leaves. n = 3. |
In this study, the thylakoid extract was suspended in water by high-pressure homogenisation before any analysis. The thylakoid extract, rich in intact membrane structures (Fig. 2b), was initially not soluble in water and appeared as large clusters (∼100 μm) under the optical microscope (Fig. 4a). After homogenisation, these clusters were broken into smaller fragments (<5 μm) (Fig. 4b), resulting in a light-green, almost transparent suspension. The mechanical treatment is expected to supply enough energy to the system to break the membrane structures at the weaker domains and even alter the ultrastructure (staked, non-stacked conformation). According to the size distribution analysis of the homogenized thylakoid dispersion (Fig. 4b), the resulting thylakoid fragments include membrane domains and protein clusters surrounded by lipids (Fig. 4c).
The structure of the membrane fragments is probably dominated by the hydrophobic interactions between the lipid's hydrophobic tail and hydrophobic protein domains or by lipid/lipid and protein/lipid ionic interactions and hydrogen bonding, similar to the interactions occurring in the native thylakoid membranes.7,23 These strong interactions prevent the release of lipids from the proteins upon membrane disruption and yield protein/lipid complexes. Nevertheless, lipids that were indeed detached can still bind to membrane proteins, which offer a complementary amphipathic surface. These types of interactions are described for protein-surfactant mixtures and are thermodynamically more favourable than micelles.24 Thus, complexes between proteins and lipids are expected to account for the majority of the thylakoid fragments obtained through homogenisation. The proteins in these fragments can include large protein clusters, since the densely packed protein complexes in thylakoid membranes can arrange in super and megacomplexes mediated by pigments and small proteins.25
In addition to the particle size, the micrographs facilitated the identification of the liquid-crystalline structure of the membrane lipids, which were observed as bright spots under polarised light (Fig. 4a and b). Following mechanical rupture, crystalline structures were still observed, although at smaller sizes. Lipid crystal structures in thylakoid membranes include lamellar bilayers, together with hexagonal and cubic phases. The bilayer is a planar structure whereas the hexagonal phase consists of cylindrical inverted micelles packed on an hexagonal lattice.26 The cubic phase is a bicontinuous structure also formed by inverted micelles with fatty acyl chains pointing toward the outside of tubules and the polar head groups toward the centre.27,28 The type and amount of membrane lipids and protein complexes determine the crystal conformation adopted by the lipids.26,27 These crystal structures are expected to be formed by the lipids surrounding the protein complexes upon thylakoid homogenisation, given the crystals observed in the homogenised material.
All tested concentrations (0.01, 0.06, 0.13 and 0.25 w/v%) showed a similar pattern of rapid initial decline which then slows down, reaching a semi-equilibrium at the end of the measurement. This is expected when larger entities, such as the thylakoid fragments, adsorb to an interface. Due to the mechanical homogenisation of the thylakoids, the proteins are part of small membrane fragments consisting of both proteins and lipids. These lipids can surround the proteins and cover the hydrophobic domains,28 producing stable thylakoid fragments that behave like soft particles at the air–water interface. As soft particles, the thylakoid fragments lower the surface tension due to capillary forces rather than conformational changes at the interface. In particle-stabilised interfaces, lateral attractive capillary forces occur due to deformation of the fluid interface around the particles. Such interactions contribute to the mechanical stability of the interfacial layer.29
![]() | ||
Fig. 6 Dynamic surface tension (γ) measurements of 0.001 w/v% thylakoid solution as a function of time. (a) Time expressed using a time logarithmic scale. I, II and III indicate the three phases of diffusion, adsorption and finally rearrangement and equilibrium, as defined for single proteins. And (b) time in linear scale. In red: theoretical curve using eqn (1). |
Similar adsorption kinetics have been observed for globular proteins (i.e. lipases, glutamate dehydrogenase),30 β-casein31,32 and for oleosins.33 In all cases, the equilibrium interfacial tension is reached after ∼20 h, except for oleosins (∼7 h). Long times for adsorption are common for complex proteins,34 but also for soft particles that are surface active.35,36 As indicated earlier, the thylakoid fragments as expected to consist of membrane proteins covered by lipids, which can provide a stable conformation to the protein/lipid complexes and render a soft particle that adsorbs to the interface.
During the pendant drop analysis, the protein hydrophobic groups are partly covered by the surrounding lipids and partly exposed to the oil–water interface. Once at the interface, new non-covalent bonds are formed, and possibly new bridges and interactions. This process is often irreversible and results in closely-packed, cross-linked, gel-like structures at the interface.37 During adsorption of the thylakoid fragments, the membrane lipids play an important role. As single amphiphiles, lipids can displace proteins from the interface because they are effective surface-active substances and lower the interfacial tension more than proteins do.34,38 This displacement will depend on the concentration of both lipids and proteins. A lipid-controlled adsorption occurs much faster than with proteins and should result in a quick lowering of the interfacial tension. However, no decrease of interfacial tension was observed during the first seconds of analysis with the thylakoid fragments (Fig. 6a), suggesting that the thylakoids lipids are somehow not available to freely adsorb to the oil–water interface. This observation supports the idea that thylakoid fragments behave as intact particles and most of lipids remain complexed within this system.
To further analyse the interfacial behaviour of the thylakoid fragments, the interfacial tension was plotted against a linear time scale (Fig. 6b). Similar to the surface tension measurements, the interfacial tension decreased before reaching a plateau value. The thylakoids lowered the interfacial tension to ∼10 mN m−1, similar to values obtained by large proteins like ovalbumin,39 β-lactoglobulin, β-caseine and human serum albumin.40
Finally, the experimental data in Fig. 6b was fitted well by (1) as depicted by the red line. The relaxation times t1 and t2 were equal to 13250 s and 3058 s, respectively, with a coefficient of determination (r2) of 0.9916. These parameters describe the interfacial tension decay with time and they give an indication of the contributions of adsorption and rearrangement to the dynamic surface tension. In this case, t1 was four times larger than t2, suggesting a relatively slow adsorption compared to fast arrangements of fragments at the interface. Certainly, the large sizes of the thylakoid fragments determined a slow diffusion and adsorption, typical of large molecules or soft particles, resembling Pickering emulsifiers.
Before oscillations started, the elastic modulus was ∼10 mN m−1 and increased with the drop deformation rate up to 23.3 mN m−1. Above a frequency of 0.02 s−1, the elastic modulus reached a plateau at ∼23 mN m−1. This behaviour suggests that the adsorbed layer formed a mechanically stable structure.
The dynamic viscoelastic behaviour can be analysed more intuitively with the loss tangent (tanδ), which is equal to Ed′′/Ed′. When tan
δ > 1, Ed′′ is dominant and the interface mainly exhibits viscous properties. In contrast, when tan
δ < 1, the interface mainly exhibits elastic properties. In our system, the tan
δ was below 1 at all frequencies and from 0.01 s−1, with low frequency dependence (Fig. 7b). Thus, the surface elasticity had a greater contribution to the dilatational modulus than the surface viscosity. Between the thylakoid fragments, the protein/protein, protein/lipid and lipid/lipid interactions will take place at the interface and result in an elastic interfacial film or 2D network, which can show viscoelastic response to mechanical disturbances.42,43
![]() | ||
Fig. 8 Visual appearance of oil-in-water emulsions stabilised by thylakoid extract at different concentrations. Pictures were taken right after emulsions preparation. |
To further characterise the colloidal stability of the emulsions, the ζ-potential was measured as a function of pH (Fig. 10). The IEP was found at pH 5.1, higher than for the pure thylakoid solution (pH 4.7), but the charge range was similar (±30 mV). The change of the IEP suggests re-arrangement of the proteins and lipids in the thylakoid fragments. Upon homogenisation, the resulting protein/lipid complexes had a different distribution of charges around protein complexes that induced an IEP shift.
![]() | ||
Fig. 10 ζ-Potential of thylakoid stabilised emulsion upon pH change. 0.05 w/v% thylakoid solution. n = 3. |
At the emulsion conditions (pH 6.8), the oil droplets covered with thylakoids are expected to repel each other due to the negative charge that is exposed at this pH. However, having droplet aggregates at this pH suggests that the attractive forces are stronger than the electrostatic repulsion and aggregation occurs, probably in a similar manner as thylakoid membranes stack on top of each other in the native structure. In the emulsion system, the exposed negative charges on the thylakoid fragments, together with the available multivalent cations, can balance the electrostatic repulsion and facilitate the inter droplet interactions. Additionally, emulsions prepared at different pH conditions (pH 3.0, 4.7, and 7.0) showed similar properties and stability (data not shown) as the emulsions at pH 6.8. This observation confirmed that electrostatic interactions played no major role on the stabilisation mechanism. Instead, stabilising forces on the interface and bulk phase might be ruled by hydrophobic and van der Waals’ interactions, which can contribute to stability of the interfacial layer as well as bridging of droplets. Moreover, the attractive interactions between droplets might be enhanced by small thylakoid fragments that can induce depletion flocculation and contribute to phase separation of the emulsions.
Despite the droplet aggregation, the thylakoid emulsions were stable against coalescence over time. The average diameter d43 did not increase over 7 days of storage (Fig. 11) and no free oil was observed on the emulsion's surface. The aggregation of droplets occurred already during emulsion preparation and the droplet clusters remained stable. This immediate aggregation might be caused by insufficient thylakoid fragments to saturate the droplet surface. In that case, the insufficient adsorbed molecules are shared between droplets and steric bridging occurs. Such bridging flocculation during emulsification has been described for single proteins (i.e. lactoglobulin) and an increase on protein concentration reduced droplet aggregation.45 Nevertheless, the increase of thylakoid concentration resulted in even more aggregation. Most likely, multiple layers of thylakoids were formed at the interface due to the excess of material and the electrostatic bridging between droplets was probably promoted. Since the thylakoids have a natural tendency to stack, one may expect that this also happens on an interface. A stack of thylakoids can then easily form a bridge towards neighbouring droplets, resulting in aggregation.
![]() | ||
Fig. 11 Average droplet size diameter d43 of emulsions stabilised by thylakoid solutions at different concentrations and after 7 days of storage at 4 °C. n = 3. |
After droplet aggregation, the adsorbed thylakoid fragments might form lateral interactions as part of the structural consolidation of the adsorbed layer. These interactions result in a 2D network around the oil droplets that ultimately stabilises the emulsion against coalescence over time.
A thylakoid stabilised emulsion was analysed with the aid of TEM (Fig. 12) to visualise the emulsion droplets and the thylakoid structures after emulsification. The size polydispersity of oil droplets and droplet aggregates are depicted in Fig. 12a and b, respectively. Aggregates contained small droplets with local flattening of the surface (see arrows) due to the elasticity of the adsorbed thylakoid layer, which deforms upon attraction. The local flattening may indicate a very strong bridging; strong enough to deform the droplets and hence enlarge their surface area. The elastic properties of the adsorbed layer were described during the dilatational rheology analysis of the thylakoids, suggesting the formation of a 2D network with viscoelastic response. The resulting surface flattening upon droplet aggregation can enhance the attractive forces between droplets.38 However, no coalescence was observed after droplet aggregation, confirming the mechanical stability of the adsorbed layer.
Moreover, no intact thylakoid structures were observed when using higher magnification level (Fig. 12c), which was the same magnification level used to visualise the thylakoid extract in Fig. 2b. Interestingly, no thylakoid vesicles stuck out of the droplets as previously described by Rayner, et al.8 when thylakoid-stabilised emulsions where tested. The different thylakoid isolation method and emulsion preparation might account for this major difference. In particular, the homogenisation step was done at higher pressure in our case (150 bar compared to 100 bar), and this high pressure can lead to more breakage of the thylakoid ultrastructure. Instead of having intact thylakoid domains reaching the oil–water interface, small thylakoid fragments adsorbed at the interface and formed a thin covering layer. This covering layer can be the cause for the smeared edges of the oil droplets in the close-up image (Fig. 12c). The smeared layer had a thickness of 30–50 nm, suggesting the adsorption of multiple layers since the thickness of lamellar thylakoids is ∼10 nm. Those observations confirm the tendency of the thylakoids to stack. The presence of multiple adsorbed layers promotes bridging between droplets either by steric or electrostatic bridging, which was confirmed by the immediate aggregation of droplets during emulsification even at higher thylakoid concentrations.
Additionally, the black dots or small droplets (10–80 nm) observed in the emulsions had comparable sizes to the plastoglobules identified in the thylakoid extract and in the homogenised material. These thylakoid microdomains have a diameter ranging between 30–100 nm and are assumed to remain in suspension after emulsion preparation due to their stable conformation and due to interface crowding by thylakoid fragments.
This journal is © The Royal Society of Chemistry 2017 |