Chloroplast thylakoid membrane-stabilised emulsions

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Research ArticleReceived: 13 April 2010 Revised: 5 August 2010 Accepted: 5 September 2010 Published online in Wiley Online Library: 19 October 2010

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4187

Chloroplast thylakoid membrane-stabilisedemulsionsMarilyn Rayner,a∗ Helena Ljusberg,a Sinan C Emek,b Emilie Sellman,a

Charlotte Erlanson-Albertssonc and Per-Åke Albertssonb

Abstract

BACKGROUND: Thylakoid-stabilised emulsions have been reported to possess satiety-promoting effects and inhibit pancreaticlipase–colipase activity in vitro, which prompted the investigation of their interfacial properties.

RESULTS: Thylakoid membranes isolated from spinach were used as an emulsifier/stabiliser in oil (triglyceride)-in-wateremulsions. Emulsions were characterised with respect to droplet size, interfacial tension, creaming, surface load and electronmicroscopy. The effects of pH and thylakoid concentration were also considered. Droplet size decreased with increasingthylakoid concentration, reaching a plateau around 15 µm beyond concentrations of 2 mg protein mL−1 oil. The resultingemulsions were stable against coalescence but were subject to creaming. The surface pressure (air/water interface) of thethylakoid isolate was 44 mN m−1 and the surface load 13 mg m−2 at 10 mg protein mL−1 oil. Electron micrographs showedthylakoids adsorbed as bunched vesicles on the drop surfaces. The stabilisation mechanism can be described as a combinedeffect of surface-active molecules, mainly membrane proteins but also membrane lipids, exposed on surfaces of thylakoidmembrane vesicles adsorbed as particles.

CONCLUSION: Thylakoid membranes effectively stabilise oil-in-water emulsions, which should facilitate their incorporationin food with satiety-promoting effects. To the authors’ knowledge, this is the first study on the emulsifying properties of anisolated biological membrane as a functional ingredient.c© 2010 Society of Chemical Industry

Keywords: thylakoids; biological membranes; oil-in-water emulsion; emulsion stability

INTRODUCTIONEmulsions are dispersions of two immiscible liquids. In this studythe emulsions were made with triglyceride oil, water and emulsifier,i.e. thylakoid membranes, using a lab-scale homogeniser. Earlierstudies using thylakoids isolated from green leaves as a functionalfood ingredient for appetite regulation1,2 also indicated that thiseffect was enhanced by incorporating them into an emulsifiedfood product and that they worked well as an emulsifier per se.3

Thus the aim of the present study was to characterise the abilityof thylakoids, the photosynthetic membranes of chloroplasts, tostabilise oil-in-water emulsions. This was quantified by measuringcreaming, droplet size, surface load and surface activity of theemulsifier under several processing conditions. The surface load isthe mass of emulsifier covering a unit area of droplet surface and isused to determine how much emulsifier is needed to produce anemulsion.4 This is an important issue when looking at the possibilityfor thylakoid membranes to become a functional food ingredient.

The chloroplast thylakoid membranes account for a largepart of the cells of green leaves. They are responsible for theconversion of solar energy into production of ATP and NADPH,which are used in the chloroplast for carbon dioxide fixation.The thylakoid membrane forms a physically continuous three-dimensional network of paired membranes enclosing the lumenand separating it from the surrounding stroma of the chloroplast(Fig. 1).5 – 8 The membrane contains more than 100 differentproteins involved in photosynthetic electron transport.6,7 Together

with their bound pigments such as chlorophyll, carotenes andxanthophylls, the membrane proteins account for approximately70% of the thylakoid mass.8 The membrane lipids, i.e. galactolipids(mono- and digalactodiglycerols), phospholipids and sulfolipids,9

account for the majority of the remaining 30%. Thylakoids exposehydrophilic, hydrophobic and charged groups and have anisoelectric point at 4.7.10

Proteins, both intrinsic and extrinsic, are the major and activecomponent of the thylakoid membrane. Proteins are the mostcommonly used surfactant in food, since they are edible, surface-active and provide superior resistance against coalescence.11

Protein emulsifiers derived from milk and milk-based emulsionshave been thoroughly studied, e.g by Walstra4 and Buchheimand Dejmek.12 Many vegetable proteins exhibit surface-active

∗ Correspondence to: Marilyn Rayner, Department of Food Technology, Engi-neering and Nutrition, Faculty of Engineering, Lund University, PO Box 124,SE-221 00 Lund, Sweden. E-mail: [email protected]

a Department of Food Technology, Engineering and Nutrition, Faculty ofEngineering, Lund University, Lund, Sweden

b Department of Biochemistry, Centre for Molecular Protein Science, ChemicalCentre, Lund University, Lund, Sweden

c Appetite Control Unit, Department of Experimental Medical Science, BMC, LundUniversity, Lund, Sweden

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fd

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a

e

b

Figure 1. Schematic diagram of thylakoid membrane showing its differentdomains: a, grana stack; b, stroma lamellae; c, end membranes; d, margins;e, lumen; f, plastoglobule.

properties and are thus used in the food and pharmaceuticalindustries to stabilise the air/water or oil/water interface of variousfoam and emulsion products. Much of the published work hasfocused on the interfacial or emulsifying properties of purifiedfractions such as globulins from soya,13 pea legumin and vicilin,14

field bean,15 lupin seed,16 faba bean,17 chickpea18 and sweetpotato.19 The interest in developing vegetable-based emulsifiersfrom the food industry has been motivated by several factors:they are non-animal-based (in contrast to many food emulsifiersderived from milk or egg), are isolated from a natural source, areless allergenic and have added health and/or nutritional benefits.20

Other plant extracts and isolates have also shown the ability toinhibit fat digestion. For example, galactolipids (fractionated oatoil), when incorporated in vegetable oil emulsions, significantlyreduced ad libitum energy intake (20–30%) by increasing satiety inhuman trials.21 Studies by Chu et al.22 showed that galactolipids,in particular digalactosyldiacylglycerol (DGDG) isolated fromspinach, also had an inhibitory effect on lipase. They reportedthat, compared with a lecithin-stabilised emulsion (from egg), theDGDG emulsion had an increased lag time for lipolysis (3.5-fold)and a decreased maximum rate (1.7-fold). Albertsson et al.1 foundthat thylakoid membranes inhibited pancreatic lipase–colipaseactivity in vitro in a dose-dependent way, reducing lipase activityby up to 80%. Furthermore, in contrast to the work of Chu et al.,22

the thylakoids’ inhibitory action lay in the membrane (includingproteins, pigments and lipids) rather than in the lipid fractionalone, which reduced lipase activity by only about 10%.1 Whenincluded in food, thylakoids induce satiety hormones such ascholecystokinin, leptin and enterostatin while reducing the hungerpeptide ghrelin, concomitant with reduced serum triglyceride andbody fat.1,2,23,24 This has been demonstrated in long-term studieson mice24 and rats1,23 and short-term studies on humans.2 Thesein vivo results have been interpreted as being due to a prolongationof lipid digestion inducing satiety.25,26

Two mechanisms have been suggested as the main cause ofthe inhibition of lipase–colipase activity and the resulting effectson appetite regulation and are illustrated in Fig. 2.

1. Adsorption of the lipase–colipase complex onto the thylakoidmembrane surface such that the substrate-binding site of theenzyme is hidden at the thylakoid surface. This mechanism issupported by binding experiments.1

2. Adsorption of the thylakoid membranes at the surface of thefat droplets, thereby sterically hindering the lipase–colipasecomplex from coming into contact with its substrate. Thismechanism is supported by electron microscopy, which showsthe attachment of thylakoids to the surface of oil droplets inwater.1

Figure 2. Trilogy mechanism for thylakoid effect on intestinal fat digestion.(A) When lipase (large circles) and colipase (small circles) form a 1 : 1complex, a conformational change takes place and a hydrophobic surfaceis exposed. Hence the lipase–colipase complex can adsorb both to the bilesalt-covered surface of the triglyceride oil droplet and to the hydrophobicregion of the thylakoids. At the same time the thylakoids can adsorb tothe oil droplet. (B) If we use thylakoids as an emulsifier, they are alreadypresent in the food matrix before ingestion. This allows for both coverageof the oil droplet and steric hindrance of the action of the lipase complexat the oil interface as well as direct interaction with the lipase complex byadsorption of the enzyme’s substrate-binding site to the thylakoids.

These two interactions together with the affinity of the li-pase–colipase complex for its oil substrate lead to the ‘trilogy’described in Fig. 2. The trilogy interactions stress the importanceof the surface properties of the thylakoid membranes. A larger ex-posed surface is expected to increase the effect of thylakoids, i.e.inhibition of lipase–colipase activity. This is achieved by premixingthe oil phase with the thylakoids, reducing the droplet size and in-creasing the interfacial area covered by the thylakoids. This in turnincreases the surface available for interaction and inhibition of theenzymes. It is therefore of great interest to study the emulsifyingproperties of thylakoids when included in the formulation of food.Since they are used to retard fat digestion, one needs to incorporatethem into a food product containing fat, e.g. an emulsion.

To our knowledge, this is the first time an intact biological mem-brane has been used as both an emulsifier and a functional ingredi-ent. As mentioned above, many other plant extracts have emulsify-ing and/or lipase-inhibiting properties, but they exist in the realmof molecules or hydrocolloids, whereas the thylakoids used in this

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study are in the form of intact thylakoids or sub-thylakoid mem-brane vesicles in the size range 1–5 µm. They are more accuratelydescribed as emulsion-stabilising particles. Some plant-based par-ticles that have been used to stabilise emulsions are starch granulesthat have been hydrophobically modified by octenyl succinyl an-hydride (OSA) treatment.27 Thylakoids are of practical interestowing to their natural abundance, as they are found in all greenleaves, their isolation is achieved by a mechanical solvent-free andreadily scalable process23 and they are naturally able to stabilisethe oil/water interface without chemical modification.

EXPERIMENTALPreparation of thylakoid isolateIn all experiments, frozen spinach leaves (a kind gift fromFindus AB, Bjuv, Sweden) were used. Thylakoids were preparedessentially as described by Albertsson et al.1 First, 100 g of leavesand 200 mL of water were homogenised in a 1 L blender (Braun4142, Braun AG, Kronberg, Germany) for about 5 min to obtain asmooth green slurry, which was then filtered through a Monodurpolyester filter (20 µm mesh). The filtrate was poured into 400 mLtubes and centrifuged (Sorvall Evolution RC SLA-3000, KendroLaboratory Products, Langenselbold, Germany) at 6084 × g for10 min. The supernatant was discarded and the thylakoids in thepellet were washed by resuspension in water and centrifugedat 16 893 × g for 15 min. The thylakoid pellet was resuspended,distributed into small test tubes, frozen in a −18 ◦C freezer andstored until use. The chlorophyll and protein concentrations ofthe thylakoid isolate were determined by the methods of Porraet al.28 and Bradford29 respectively

Preparation of emulsionsThe oil-in water emulsions consisted of 15 mL of rapeseed oil(Zeta, Fernando Di Luca, Stockholm, Sweden) and 135 mL of watercontaining varying amounts of thylakoids. The emulsions wereprepared using an in-house two-step lab-scale homogeniser30 ata set pressure of 10 MPa (100 bar) together with a cooler and amixer. The mixture of thylakoids and rapeseed oil was added to thehomogeniser, which was run for a determined number of loops, inthis case 20. Thereafter the emulsion was divided into test tubesand stored at 6 ◦C for further analysis.

To test the emulsion at different pH values, 1 mL of rapeseedoil was added to 9 mL of an aqueous suspension of thylakoids(1 mg of chlorophyll equivalent to about 10 mg of thylakoids). Themixture was blended using an Ultra-Turrax mixer (170W, IKA Werk,Staufen, Germany).

Particle size measurementThe particle size (mean particle diameter d4,3) was measured bylaser diffraction (Coulter LS 130, optical mode: Mie, refractiveindex 1.470, Coulter Electronics Ltd, Luton Beds, England) ina flow-through cell. A small volume of emulsion was addedto the flow system and pumped through the optical chamberfor measurements. Light scattering was performed directly afteremulsification and 24 h after homogenisation.

To inspect the microstructure of the emulsions, a drop from amixed sample was placed on a covered microscope slide. An opticallight microscope (Olympus BX50, Tokyo, Japan) with a digital videocamera (Sony Hyper Had, Sony, Tokyo, Japan) connected to a PCrunning image analysis software (Matrox Inspector 2.1, MatroxElectronic Systems Ltd., Dorval, QC, Canada) was used to confirm

that the size distributions obtained from light scattering reflectedthe size ranges of individual droplets and did not include largeaggregates of droplets or other structures.

Surface tension measurementThe surface tension was measured with a dynamic drop tensiome-ter (IT Concept, Longessaigne, France) at the air/water interface.The thylakoid isolate consisted of 32.6 mg thylakoid mL−1 wa-ter (calculated from chlorophyll determination). The data weresubjected to axis-symmetrical drop shape analysis by computer.

CreamingThe phase separation of water and emulsion layer was monitoredin the following way. The emulsions were left in the fridge for 4 h,after which the heights of the emulsion layer and water phasewere marked on the glass test tubes. The volumes of the phaseswere then determined in a measuring glass. The emulsificationindex (E)31 was calculated as

E(%) = (volume of emulsion layer/total volume of emulsion)

× 100 (1)

Surface loadAnalyses were carried out to determine the concentrations ofchlorophyll and protein in the water phase of the producedemulsions and to estimate the amounts in the emulsion layer bytaking the difference between the known amounts of thylakoidsadded to the emulsion and the measured concentrations in thewater phase. Subsequently the surface load (�, mg m−2) could becalculated based on the total surface area of the droplets foundduring particle size measurement and the estimated amountfound in the oil phase, assuming that the thylakoids not presentin the water phase were adsorbed to the oil/water interface.

The oil and water phases of the produced emulsions wereseparated in a separation funnel for 5 h at room temperature. Thewater phase was collected and its volume was measured. Thethylakoids were isolated by ultracentrifugation (Beckman L7-65,Palo Alto CA, USA) at 106 551×g for 1 h and the pellet was retainedfor chlorophyll and protein analyses (as described above).

Adjustment of pHAdjustments of pH were made using 12 mol L−1 HCl or 5 mol L−1

NaOH while measuring the pH with a pH meter (PHM 82,Radiometer, Copenhagen, Denmark) calibrated with pH 4 and7 solutions. The initial pH of the emulsions was in the range 6–6.5,with pH decreasing with increasing thylakoid concentration.

Electron microscopyEmulsion samples were fixed with 25 g L−1 glutaraldehyde in0.15 mol L−1 kakodylate buffer, embedded in Epon epoxy resinand stained with 30 g L−1 uranyl acetate and lead citrate prior toexamination by transmission electron microsopy (JOEL 1200 EX,JOEL, Tokyo, Japan).

RESULTS AND DISCUSSIONDroplet size, coalescence and surface loadThe droplet size was measured immediately and 24 h afterhomogenisation. The initial mean droplet diameter d4,3 decreased

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Figure 3. Droplet size as a function of added thylakoids, measuredimmediately and after 24 h. Note that there is very little change in meandroplet diameter after 24 h of storage, indicating that the emulsion isstable against coalescence even though it creams.

Figure 4. Droplet size and surface load as a function of added thylakoids.

rapidly with increasing thylakoid concentration but approacheda plateau value of about 10 µm between 2 and 10 mg thylakoidprotein mL−1 oil (Figs 3 and 4). This may have been limited by theintensity of the lab-scale homogeniser. The measured droplet sizeremained about the same for 24 h, showing that the emulsion wasstable at least to initial coalescence; even though it creamed aftera few hours, it was easily redispersed. This indicates that, althoughthe emulsion separated owing to gravity, the droplets coveredby the thylakoids did not coalesce or strongly aggregate and theinterfacial area remained intact.

The surface load � (mg m−2) is a variable used for measuring theemulsifying efficiency of an ampiphilic molecule and correspondsto the mass of emulsifier covering a unit area of droplet surface.If the emulsifier concentration is low, some proteins unfold atthe interface (e.g. sodium caseinate) and form a polypeptidemonolayer with a � of about 1 mg m−2. Highly soluble proteinsgive a plateau value of 3 mg m−2, while aggregated and globularproteins (e.g. soy protein isolates) can yield values greater than5 mg m−2.32 Protein aggregates tend to be preferentially adsorbedduring emulsification, thereby increasing� even more. The surfacelayer will become substantially stronger if additional proteinsaccumulate beyond the interfacial layers.4

In the experiments, surface load was found to be a function ofthylakoid concentration. However, fluctuations in the surface loadvalues at the two or three lowest concentrations in Fig. 4 may bedue to experimental error, as the amount of protein remaining inthe water phase was very low and close to the detection limit ofthe method used. Furthermore, the large and unstable emulsiondroplets (>80 µm) also increase the uncertainty of these datapoints at low concentrations. The surface load reached a value ofabout 13 mg protein m−2 when 10 mg thylakoid protein mL−1 oilwas used (Fig. 4). The thylakoids’ protein/chlorophyll ratio is about3–4 mg mg−1.23 The thylakoid membrane has an area of about2 m2 mg−1 chlorophyll.1,33 Combining these last three statementsresults in an estimated surface load of 7–9 m2 thylakoid m−2 dropsurface. This indicates that the thylakoids should be bunched up inexcess on the oil surface, which can be seen in the micrographs inFig. 5. In the intact chloroplast the thylakoid membrane is foldedinto stacked regions, called grana, connected via single pairedregions, called stroma lamellae (Fig. 1).5,34 The stacked structuresare preserved during isolation if the suspension medium containsa salt such as 0.15 mol L−1 NaCl or 10 mmol L−1 MgCl2. Whenthe thylakoids are suspended in a solution of low ionic strength,they reversibly unstack.35 During preparation of the thylakoidsin water, as used here, the thylakoid membrane unfolds andthereby exposes a much larger surface than in the intact stackedstate. In the unfolded state there is a partial randomisation ofthe membrane proteins. If the pH of a suspension of unfoldedthylakoids is reduced to 4.7, stacking is induced all over themembrane and will change the exposed thylakoid surface.35 Thusthe surface load may depend on both the ionic strength and pH.

Ability of thylakoids to stabilise oil-in-water emulsionsA plateau value of surface load may already have been reached atlow concentrations of thylakoids, and the increase in intensityof the green colour of the water phase could then dependon an excess of thylakoids (Fig. 6). However, the creaming rate

Figure 5. Electron micrographs of oil droplets covered with thylakoid membranes at pH 7 (left) and 4.7 (right).


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Figure 6. Appearance of emulsions with increasing thylakoid concentra-tion (1, 1.5, 2, 2.5 and 3 mg protein mL−1 oil).

decreased and the emulsification index increased with increasingconcentration, which points to a more stable emulsion. Thisis supported by our observation that, although the dropletsreadily creamed (owing to their large size), they were readilyredispersed and had not coalesced during the storage time.A probable stabilising mechanism could thus be some kind ofparticle stabilisation. Images from electron microscopy (Fig. 5) ofan oil and thylakoid emulsion show that the thylakoid membraneadsorbs to the interface as vesicles. Thylakoid membranes alwaysturn into vesicles when broken in media of low ionic strengthand never remain linear.36 The vesicles stick out from the dropletsurface, thereby preventing the droplets from coming into closecontact with each other and coalescing. Most probably it is thehydrophobic part of the membrane proteins that adsorbs to theinterface.

With higher thylakoid concentrations the emulsion layerbecame smoother and more creamy in appearance, with ashimmering surface, and more densely packed, with the dropsbeing impossible to distinguish from each other (Fig. 6). Emulsionsmade with the highest concentration of thylakoids, 10 mg mL−1

oil, seemed to have an intermediate phase between the emulsionlayer and the water phase, indicating that smaller droplets mayexist in the emulsion. As the thylakoid concentration increased, thewater phase became more and more green owing to increasingconcentrations of chlorophyll.

CreamingCreaming results give an indication of emulsion activity andstability, whereby a stable emulsion shows no separation betweenphases and the emulsion layer stays constant over time. Theobserved creaming rate was rapid at low thylakoid concentrations,where the formation of an emulsion layer took only a few seconds,probably owing to the presence of large droplets. The size ofthe creaming layer is related to the droplet size: as the dropletsize increases, the emulsion layer becomes smaller because ofless steric and electrostatic repulsion between oil droplets.37

With increasing concentration of thylakoids the creaming ratedecreased; based on droplet size measurements, this is to beexpected owing to the smaller droplet sizes as the concentrationincreases. Creaming was measured 4 h after homogenisation,though further observations several days later showed that theemulsion layer had not or only slightly decreased from its initialwidth and that the measured droplet size distribution remainedunchanged (Fig. 3). The results of the creaming experimentsin Fig. 7 show that the emulsification index E increased with

Figure 7. Emulsification index as a function of added thylakoids. Notethat the emulsification index increases with increasing concentration ofthylakoids, indicating higher stability at higher protein concentrations.

increasing thylakoid concentration. The large size of the thylakoidmembrane vesicles generates an increase in droplet sizes duringthe emulsification process. This size dependence can be similarto that of particle-stabilised emulsions.38 Recently, Yusoff andMurray,27 using starch particles (∼1 µm) to stabilise oil-in-wateremulsions, found that, although the droplet size they obtained waslarge (10–20 µm) and the emulsions creamed almost immediately,they were completely stable to coalescence for up to at least3 months, this being an interesting feature of particle-stabilisedemulsions.

Surface tensionThe surface tension of a clean air/water interface is 72 mN m−1.Surface-active molecules act to reduce this value. The differencebetween the measured surface tension of a sample and that ofthe clean air/water interface defines the surface pressure (π ).4 Thehigher the surface pressure, the better is the surfactant. Threemeasurements of surface tension were made at the air/waterinterface, resulting in a mean surface pressure of 28 mN m−1. Thisvalue is comparable to the surface pressure for other proteinsmeasured at the air/water interface, e.g. β-casein (26.7 mN m−1),bovine serum albumin (16.7 mN m−1) and lysosyme (21 mNm−1)39 and egg lecithin (31 mN m−1) and cholesterol (29 mNm−1).40 A comparison reveals that thylakoid membranes reducethe surface tension of water and do so to a higher extent thanmany commonly used highly surface-active protein emulsifiers.In summary, thylakoids, being biological membranes, exposehydrophobic groups or surfaces in a similar way to proteins,and we suggest that this is the main reason for their affinity to theinterface.

Effect of pHOur results show that thylakoids form emulsions over the entirepH range 3–7, with the clearest water phase in the pH range3–5. In Fig. 8, at higher pH a small number of droplets stay in thelower phase, resulting in the darker green colour of the right-handsamples, whereas the water phase is clearer at low pH in thesamples shown to the left. This can be explained in the followingway. The isoelectric point of thylakoids is 4.7. This means thatat pH 3 and 4 (Fig. 8) the thylakoids have a net positive chargeand, if the electrical potential between the oil and water phases


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Figure 8. Appearance of thylakoid emulsions at different pH values (3, 4,5, 6 and 7).

A

B

C 2 µm

Figure 9. (A) Electron micrograph of thylakoid native membranes innormal stacked chloroplasts; (B) the chloroplast structure can be destackedby reducing the ionic strength of the buffer (dilution at pH 7.4); (C) thestacked structure can be reformed by lowering the pH from 7.4 to 4.7(adapted from previous work by Andersson et al.35).

is such that the water phase is slightly more positive than the oilphase, their adsorption to the interface will be facilitated. At pH6 and 7 the interfacial electrical potential will force the thylakoidstowards the water phase. However, this force is weak comparedwith the hydrophobic effects, so the majority of the thylakoids areattached to the interface and facilitate emulsificaton. However,the differences between the bottom phases as seen in Fig. 8may simply be due to different speeds of flotation of the oildroplets towards the upper oil phase. If the droplets are heavilyloaded by thylakoids, their density increases, which slows down oreven prevents this flotation. At the isoelectric point the thylakoids

precipitate. However, the forces between the precipitated particlesare rather weak, since they can be easily broken by mechanicalstirring of the precipitate. Thus the precipitation does not interferewith the emulsification process; however, once this is stopped,the precipitation tendency of the thylakoids will result in a weakthree-dimensional network that may reduce the mobility of theemulsion droplets.

Thylakoids can undergo significant and, in some cases, reversiblestructural changes during typical food-processing steps such asdilution and pH adjustment (Fig. 9). This can be important notonly for their function in lipase–colipase inhibition but also fortheir function in a food matrix. The stacked structure is stablein situ and requires an ionic strength above a certain value, i.e.0.1 mol L−1 NaCl or 10 mmol L−1 MgCl2, or a high concentrationof a water-soluble polymer such as 100–200 g kg−1 dextran. Ifthese conditions are not fulfilled, the thylakoids unstack, unfoldand form an irregular ‘blown-up’ structure of the membrane.Fragmentation of the thylakoid membrane by shearing forces(e.g. during emulsification), pressure treatments or sonicationresults in closed sub-thylakoid membrane vesicles, some of theminside-out.35 This increases the exposed surface of the thylakoidmembrane and perhaps leads to its adsorption to the oil/waterinterface.

CONCLUSIONSA basic study has been made to provide information about theemulsion-stabilising characteristics of thylakoid membranes fromchloroplasts. To our knowledge, this is the first time an isolatedbiological membrane has been used as both an emulsifier anda functional ingredient. This is interesting from a practical pointof view, as it is not necessary to further isolate protein or lipidfractions, since the entire biological membrane can be used as it is.

We found that thylakoids provide excellent stability againstcoalescence. The stability against creaming was poorer and seemsto be dependent on the thylakoid concentration, but it may alsobe a result of the homogenisation conditions, as our droplets wererelatively large (10 µm) and thus subject to high buoyancy effects.The stabilisation probably occurs because of vesicles attaching tothe oil surface to create a particle-stabilised emulsion in whichboth proteins and lipids are part of the active material. Thylakoidswill almost certainly have a future as a functional food ingredientowing to their appetite-regulating abilities, but more experimentsneed to be done to establish the stabilising mechanism and therole of thylakoids in food application as well as their physiologicaleffect in the human body.

ACKNOWLEDGEMENTSThis work was funded by the Carl Trygger Foundation (P-ÅAlbertsson) and the Royal Physiographic Society, Lund (P-ÅAlbertsson). We thank Rita Wallen (Department of Cell andOrganism Biology, Lund University) for taking the electronmicrographs.

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Chloroplast thylakoid membrane-stabilised emulsions