The effect of nanoparticles on the phase separation of waxy corn starch+locust bean gum or guar gum

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Food Hydrocolloids xxx (2014) 1e8

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Food Hydrocolloids

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The effect of nanoparticles on the phase separation of waxycorn starch þ locust bean gum or guar gum

Brent S. Murray*, Nataricha PhisarnchanananFood Colloids & Processing Group, School of Food Science and Nutrition, University of Leeds, Leeds, LS2 9JT, UK

a r t i c l e i n f o

Article history:Received 9 September 2013Accepted 6 January 2014

Keywords:Phase separationPolysaccharidesPickering stabilizationParticles

* Corresponding author.E-mail address: [email protected] (B.S. Murr

0268-005X/$ e see front matter � 2014 Published byhttp://dx.doi.org/10.1016/j.foodhyd.2014.01.004

Please cite this article in press as: Murracorn starch þ locust bean gum or guar gum

a b s t r a c t

Phase separation of mixtures of a gelatinized waxy corn starch (S) and locust bean gum (LBG) has beenstudied in detail at pH 7 and room temperature in the presence and absence of up to 1 wt.% silica nano-particles (primary particle size 20 nm diameter). A range of silica particles of varying surface hydropho-bicities was used, surfacemodified so as to have 100% (i.e., unmodified) to 65% of their surface SiOH groupsremaining. In the absence of particles the S þ LBG system separated via spinodal decomposition. In thepresence of particles the phase separationwas significantly curtailed. Confocalmicroscopy showed that theparticles had a strong preference for the starch microdomains that formed, rather than the gum phase,whilst there was an increasing tendency for particle aggregation to occur within the starch microdomainsand possibly at the W/W interface between the two phases as the particle hydrophobicity and particleconcentration was increased. Measurements on starch þ guar gum system showed that this behavedsimilarly to the S þ LBG system. Measurements of the bulk rheology of the gum and starch phases in thepresence and absence of the particles suggested that the inhibition of the phase separationwas not due tochanges in the viscoelasticity of the starch or gum microdomains themselves, but flocculation of theparticles in the gumphase, possibly via the depletionmechanism. It is suggested that the formation of largeaggregates of particles aids their accumulation at the W/W interface and subsequently slows down phaseseparation.

� 2014 Published by Elsevier Ltd.

1. Introduction

Mixtures of aqueous biopolymers have been widely studied formany years due to their important role in applications in foods,pharmaceuticals, nutraceuticals, etc. (Garnier, Schorsch, & Doublier,1995). Starch is the main storage carbohydrate in plant organismsand consists of amylose and amylopectin, whilst waxy starchesconsist of almost exclusively amylopectin, a highly branched, highmolecular weight molecule. Arabinogalactan or galactomannangums such as locust bean gum (LBG) and guar gum (GG) alsoconsist of very high molecular weight polymers, but, in contrast toamylopectin, branching is limited to attachment ofmonosaccharideside chains to a linear polysaccharide backbone. Thus LBG and GGform highly entangled, viscous but highly shear thinning solutionsat relatively low concentrations, whilst amylopectin forms veryweak gels (but is a good thickening agent) at relatively high con-centrations that also break down on shear in a similar manner. Thevery different conformation of amylopectin and LBG (or GG) means

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that they have difficulty forming simple mixtures even at relativelylow concentrations and this leads to their phase separation.

Phase separation of aqueous polysaccharides was first reportedby Albertsson (1962) and since then there have been numerousstudies of the thermodynamic incompatibility of starch and otherhydrocolloids (Alloncle & Doublier, 1991; Closs, Conde-Petit, Rob-erts, Tolstoguzov, & Escher, 1999; Conder-petit, Pfirter &Escher,1997; Frith, 2010; Kulicke, Eidam, Kath, Kix, & Kull, 1996; Tolsto-guzov, 1986; Tolstoguzov, 2006), since suchmixtures form the basisof the texture of numerous food products. It is important to havesome understanding and control of this phenomenon since exces-sive phase separation may cause unacceptable changes in the sen-sory properties or appearance (Firoozmand, Murray, & Dickinson,2012; Semenova & Dickinson, 2010), whilst at the same time suchsystems potentially provide a novel way of influencing desiredtexture and small molecule (flavours, drugs, etc.) release charac-teristics via dispersions of one type of aqueous phase in another, socalled water-in-water (W/W) emulsions. Studies (Frith, 2010;Williams et al., 2001) have shown how the detailed microstruc-ture ofW/Wdispersions of protein andpolysaccharide solutions canbe controlled bychanges in the composition and solution conditions

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B.S. Murray, N. Phisarnchananan / Food Hydrocolloids xxx (2014) 1e82

(salt, pH, temperature, shear rate, etc.). Filaments, droplets orbicontinuous structures can be produced.

One method of potentially controlling phase separation is to useparticles that strongly adsorb at the interface. Thus, for example,Herzig,White, Schofield, Poon, andClegg (2007) andClegg andCates(2008) have shown that in an oilewater (lutidineewater) systemphase separation via spinodal decomposition can be completelyarrested by inclusion of 3 vol% or more of hydrophobic silica parti-cles, locking in or ‘jelling’ of the bicontinuous oilewater structure toforma so-called ’bijel. In an oilewater system, the interfacial tensioncan be sufficiently high for there to be a significant energy barrier toparticle desorption from the interface, of the orders of thousands ofkBT per particle (Binks &Horozov, 2006). Particle-stabilized systems(but largely oil/water or air/water systems) in foods have beenreviewed recently by a number of authors (Dickinson, 2010, 2012;Murray, 2007). However, with polysaccharideepolysaccharidephase separation, both phases are aqueous and theW/W interfacialtension can be extremely low (g z 0.001e0.01 mN m�1, Wolf,Scirocco, Frith, & Norton, 2000) so that the gain in free energy byparticles occupying the interface could be negligible. Nevertheless,Firoozmand, Murray, and Dickinson (2009) and Balakrishnan,Nicolai, Benyahia, and Durand (2012) have reported accumulationof latex particles at aW/W interface, for starchegelatin anddextran-polyethylene oxide phase-separating systems, respectively.Firoozmand et al. (2009) also observed the same effect with sub-micron oil droplets. Hanazawa and Murray (2014) recentlyextended the principle of stable oil droplets acting as Pickering‘stabilizers’ in W/W systems to a xanthanecaseinate system, andalso provided tentative evidence (Hanazawa & Murray, 2013) thatsuch particles mechanically strengthened the WeW interface.

In most of the WeW systems studied with particles so far, thephase separation can be significantly slowed down and themicrostructure can be significantly altered but phase separation isnot completely stopped. For complete arrest of phase separation,although there may be sufficient interfacial energy to cause particleaccumulation at the interface, it is probably necessary to formstrong cross-links between the particles. This appears to have beenachieved by Nguyen, Nicolai, and Benyahia (2013) using proteinaggregates as particles in a dextran e poly (ethylene oxide) system.Although protein particles are an attractive possibility, since theircross-linking e.g., via hydrogen bonds, salt bridges, etc., can becontrolled by the solution conditions, they are complex entities tocontrol in themselves and there is the added complication that theywill tend to interact strongly with polysaccharides in the bulkphase via the same sort of bonding as at the interface.

In this work the effects of model silica particles on the phaseseparation of model systems consisting of a waxy corn starch (S)plus LBG or GG were studied. Phase separation in these specificmixtures (in the absence of particles) has been demonstrated else-where (Achayuthakan & Suphantharika, 2008; Ptaszek et al., 2009;Simonet, Garnier & Doublier, 2000). A range of silica particles ofvarying surface hydrophobicities was studied to see if this had anyeffect on their ability to adsorb to and stabilize the WeW interface.Only non-modified silica in a certain size range is a permitted foodadditive, but here the aim is to demonstrate the possibility of controlof polysaccharideepolysaccharide phase separation via particles. Ifthis is successful the long-term aim is to extend this principle toother insoluble particles that are more compatible with food.

2. Materials and methods

2.1. Materials

Waxy corn starch (S), product code S9679, and locust bean gum(LBG), product code G0753, were purchased from SigmaeAldrich

Please cite this article in press as: Murray, B. S., & Phisarnchananan,corn starch þ locust bean gum or guar gum, Food Hydrocolloids (2014), h

(Gillingham, UK). All polysaccharide mixtures were made up ina pH 7 phosphate buffer consisting of 0.05 mol dm�3

KH2PO4 þ Na2HPO4 þ 0.05 mol dm�3 NaCl. Sodium azide(0.02 wt.%) was also added as a bactericide. The pH was adjusted byadding either 1 M NaOH or 1 M HCl. Rhodamine B (product code R-6626) and acridine orange hemi (zinc chloride) salt, (product code158550) were also obtained from SigmaeAldrich.Water purified bya Milli-Q apparatus (Millipore, Bedford, UK), with a resistivity notless than 18.2 MU cm, was used for the preparation of all solutions.Silicone oil AS4 was from Fluka (Gillingham, UK). Silica particleswith different hydrophobicity, characterized as having 100, 80, 70and 65% of the natural surface density of silica SiOH groupsremaining after treatment with dimethyldichloromethane, were agift from Professor Binks, University of Hull, previously obtainedfrom Wacker-Chemie GmbH (Munich, Germany). The nominalparticle size was 20 nm.

2.2. Preparation of solutions

Stock solutions of starch (0.2e4 wt.%) were prepared bydispersing the starch powder in phosphate buffer, followed byheating in an oil bath at 90 �C for 15 min with constant stirring, byhand. Stock solutions of gums were prepared by dispersing 1 wt.%LBG or GG in the buffer under the same conditions as for the starch.The gum solutions were then left to cool and centrifuged at11,000 rpm and 25 �C for 1 h in a high speed Beckman Coulter (J2-HS) centrifuge to remove insoluble materials. This contributed20� 2wt.% of the original powders. Panda (2004) has reported thatcommercial LBG may contain up to 27% impurities. Stock solutionswere stored at room temperature before use. The stock solutionswere diluted with buffer to the required concentrations based onthe soluble part remaining. To prepare mixtures, stock solutionswith or without added silica particles were heated separately at90 �C for 5 min before blending them immediately after removalfrom the oil bath by an Ultra Turrax T25 homogenizer (IKA-WerkeGmbH &Co., Staufen Germany) at 24,000 rpm for 1 min. Equalvolumes of starch and LBG solution were mixed and the tempera-ture of the samples was 70 � 5 �C after mixing. For samplesintended for confocal microscopy, rhodamine B or acridine orangewere added during blending.

2.3. Determination of phase separation and phase diagrams

The S þ gum mixtures with or without added silica particleswere observed visually after centrifugation. Phase diagrams wereconstructed by making a large number of samples at differentcompositions in centrifuge tubes. The samples were centrifuged at4200 rpm at 25 � 5 �C for 1 h in an MSE T606 bench-top centrifuge(MSE, London UK) and then the heights of the upper and lowerphases measured and converted to volumes from the known ge-ometry of the tubes.

2.4. Confocal laser scanning microscopy (CLSM)

Confocal microscopy was performed using a Leica TCS SP2confocal laser scanning microscope (Leica Microsystems, ManheimGermany) connected with a Leica Model DM RXE microscope base.The confocal was used with Ar/ArKr (488, 514 nm) and He/Ne(543,633 nm) laser sources. Laser excitation of the fluorescentsamples was at 543 nm (z29% intensity of laser) for RhodamineBlue (RB) and 488 nm (z49% intensity of laser) for Acridine Orange(AO). A 20� objective with numerical aperture 0.5 was used toobtain all images, at 1024�1024 pixel resolution. 1% wt.% of RB and0.5% (w/v) AO were dissolved in Millipore water and the solutionswere stored in the dark when not being used. For mixtures without


0.00

0.05

0.10

0.0 1.0 2.0

[LBG]/wt. %

[S]/ wt. %)

Fig. 1. Phase diagram showing the estimated binodal (d) between single-phase (P)and biphasic (C) mixtures of starch (S) and locust bean gum (LBG), as determined bythe centrifugation procedure described in the text.

[LBG]/ wt.%0.0 0.1 0.2 0.3

%volLBG

0

50

100

Fig. 2. Volume of upper LBG phase as a % of the overall volume (%vol LBG) versus wt.%of LBG [LBG] in the mixtures at different wt.% starch concentrations: 0.15 (P); 0.25(6); 0.5 (,); 1 (C); 1.5 (:); 2 (-).The dashed line at 50% indicates initial %volume ofLBG solution (since equal volumes of solutions were mixed).

B.S. Murray, N. Phisarnchananan / Food Hydrocolloids xxx (2014) 1e8 3

silica particles, 100 ml of the RB solution were added per 5 ml of thestarch solution before blending with LBG. For polysaccharidemixtures with silica particles, 100 ml of the AO solution were addedper 5 ml of either the S or gum phase before blending. Afterblending the mixtures via the Ultra Turrax samples were immedi-ately poured into a welled slide 30 mm diameter and 0.3 mm indepth. RB showed preferential staining of the starch whilst thecationic AO showed strong affinity for the silica particles. Unla-belled areas were therefore assumed to be gum-rich regions. Thefirst image was captured 5 min after blending the mixtures. Theappearance of samples was recorded again at 0.5, 3, 6 and 24 h.Image analysis was performed using Image J software.

2.5. Bulk rheology

Bulk shear rheology of the polysaccharide solutions wasmeasured with a Kinexus Rheometer (Malvern Instruments, Wor-cestershire UK) using the rSpace software to control the rheometer,measure and analyse the results. The environmental controllercartridge holds the lower geometry and controls the temperature ofsample as required. The temperature was set at 25 �C in everyexperiment. Two geometries and cartridges were used: cone withplate cartridge (CP2/60:PL65) for very viscous samples and doublegap and bob with cylinder cartridge (DG25:DO25/DI25) for lowerviscosity samples. After adding the sample to the geometry, it wasthen left to achieve steady state for 5 min. Viscosities weremeasured over a range of shear rates using the shear rate mode inrSpace software. The starting shear rate was 0.1 s�1 and the finalshear rate 100 s�1 the whole range taking 16 min in total. Inoscillatory mode, the elastic and viscous components G0 and G0’were measured at 1% strain, in the range 0.01e1 Hz, taking 26 minin total for each run. Silicone oil was layered around the edge of thesample to prevent sample evaporation and drying.

3. Results and discussion

3.1. Phase separation diagram and phase characteristics

After centrifugation of the mixtures, phase separation wasevident in the formation of an opaque lower phase and an upperclear phase. Since the original starch solution was also slightlyopaque, the lower phase was assumed to be the starch-rich phase,although independent I2/KI staining and subsequent confocal mi-croscopy of this phase confirmed this. The upper phase wastherefore assumed to be the gum-rich phase. The rate of phaseseparation depended on the composition and speed and time ofcentrifugation, but after 1 h at the speed used (4200 rpm) therewasno significant further change in the different phase volumes withincreasing centrifugation time. Independent measurements wheresamples were left for up to one month under normal gravityshowed that complete phase separation had still not taken place.The higher the concentration of gum and/or starch, the slower wasthe rate of separation, undoubtedly due to the increasing viscosityof the gum and starch phases at increasing bulk polymer concen-tration (Cp)e see later. From these observations the phase diagramswere constructed. The phase diagram for starch þ LBG is shown inFig. 1. It is seen that the binodal line lies close to starch concen-tration [S] axis and very close to the LBG concentration [LBG] axis atlow [S] but higher [LBG]. At higher concentrations of LBG (0.15, 0.20and 0.25 wt.%) the mixtures with starch were biphasic at all starchconcentrations down to the lowest studied (0.1 wt %). The phasediagramwas practically identical for GG and is therefore not shown.

Fig. 2 shows the volume of the upper LBG-rich phase as functionof the initial [LBG] mixed with different [S]. It is clear that as [S]increased, there was a reduction in the volume of the LBG-rich


phase. This phase must therefore become progressively enrichedin LBG, assuming that the majority of the LBG migrates to this re-gion. As a simple example, Fig. 3 shows the hypothetical maximumconcentration [LBG0] of LBG in this region, calculated from the datain Fig. 2 making the assumption that all the LBG ends up in thisphase. It is seen that for [S] > 1 wt.% it is possible for [LBG0] >>

[LBG] whilst for [S] < 1 wt.% [LBG0] can be <[LBG], i.e., there can bean effective dilution of the LBG because the volume of the starchphase contracts relative to the LBG-rich phase. It is important tounderstand these concentration effects at higher [LBG] where thephase separation is more strong (but more slow) because the [LBG]will strongly affect the viscosity of both the mixtures and themicroviscosity of the evolving LBG-rich domains. The bulk rheologyof the gum and starch solutions as a function of their concentra-tions was therefore measured.

3.2. Bulk rheology of different phases in the absence of particles

Fig. 4 shows the bulk shear viscosity (h) measured over theshear rate (g) range 0.1e100 s�1 for selected low and high con-centrations of starch, LBG and GG. As expected, all the solutions areshear thinning, particularly at the higher concentrations and amuch higher [S] is required to give h equivalent to those of thegums, reflecting the extremely high molecular weight and lowoverlap concentration of the latter. This is mademore clear in Fig. 5,where h at the lowest shear rate (0.1 s�1) is plotted as a function ofCp. It is also seen that LBG and GG gave relatively similar h, althoughLBG gave moderately higher h than GG above [Cp] >> 0.1 wt.%.Since g ¼ 0.1 s�1 is a relatively low shear rate, the above values of hmay be taken as an indication of the ’zero’ shear rate limiting h

characteristic of the gums solutions under rest, i.e., operatingwithin the solutions as the microdomains of gum evolve to formthe macro-phase separating regions. It is therefore instructive to


0.0 0.1 0.2 0.3 0.4 0.5

[LBG]'/ wt.%

0.0

0.1

0.2

0.3

0.4

0.5

[LBG]/ wt.%

Fig. 3. Hypothetical maximum concentration [LBG0] of LBG in the LBG phase separatedregions, calculated (see text) on the basis of Fig. 2, versus wt.% of LBG [LBG] in themixtures at different wt.% starch concentrations: 0.15 (P); 0.25 (6); 0.5 (,); 1 (C);1.5 (:); 2 (-). The dashed diagonal line indicates the case where [LBG0] ¼ [LBG].

Cp/ wt.%0.01 0.1 1

0.1s

/Pa s

0.01

0.1

1

10η

Fig. 5. Viscosity at g ¼ 0.1 s�1 versus wt.% concentration (Cp) of different biopolymers:starch (C); LBG (6); GG (,).

'0.1s

/ Pa s50

75η


take the hypothetical maximum [LBG0] from Fig. 3 and combinethemwith the h data in Fig. 5 to calculate a predicted h of the gum-rich regions that phase separate. This has been done for LBG and theresults are shown in Fig. 6 for varying [S]. Above [S] >> 1 wt.%, asexpected from Fig. 3, it is seen that h of the LBG-rich phase could bedifferent from the h of the original LBG solution mixed with thestarch solution, but also that the strong dependence of h on [Cp](Fig. 5) means that this LBG-rich phase h could be verymuch higherindeed. Increasing microviscosity is expected to slow down the rateof phase separation and in the extrememay inhibit it so greatly thatseparation is not evident over short time-scales, giving the illusionof complete mixing.

3.3. Microscopic observations of the effect of particles on phaseseparation

The viscosities of the bulk phases become an importantconsideration when trying to explain the effect of particles on themacro- and microscopic aspects of phase separation, as seen in thefollowing. For the sake of brevity and because the GG and LBGappeared to behave so similarly, only microscopic images for theLBG-starch system are presented below. Fig. 7 shows typical ex-amples of confocal micrographs from the starch þ LBG system, inthis case for 2 wt.% starchþ0.3 wt.% LBG. Fig. 7(a) shows the system5� 2 min after mixing in absence of any added particles. Even afterjust this short ageing time, microscopically the system showedsignificant phase separation, possessing a morphology typical of aspinodal decomposition mechanism. In real time the system wasquite dynamic, with movement and fusion of the bright (starch-rich) domains and parallel growth of the dark background (LBG-

/ s-10.1 1 10 100

/ Pa s

0.01

0.1

1

10

γ

η

Fig. 4. Viscosity (h) versus shear rate (g) for solutions of 4 wt.% starch (C); 1 wt.%starch (P); 0.5 wt.% GG (-); 0.2 wt.% GG (,); 0.5 wt.% LBG (:); 0.2 wt.% LBG (6).


rich) domains. Fig. 7(b) shows that within the same system 24 hlater no such regions were evident, just an ill-defined backgroundof faint ’blobs’ of a wide size range but all below ca. 10 mm. Withinthis ageing time and the small height (0.3 mm) of the sample wellon the microscope slide almost complete stratification of the LBGand starch occurs. The small background blobs may represent thesmall fraction of starch that still remains within the bulk LBG phase.

Fig. 7(c) shows the dramatic effect on the microstructure ofadding just 0.5 wt.% of silica particles to the system. Even after 24 hageing, plenty of the early (<1 h ageing)microstructurewas evidentthroughout the sample, although not as many starch-rich domainscould be found as immediately after mixing (e.g., as in Fig. 7(a)). InFig. 7(c) the silica particles added were the 100%SiOH particles, i.e.,where the silica surface had been unmodified and should possess100% of it natural surface density of SiOH groups. Such particlesshould be quite hydrophilic andmight be expected to be compatiblewith either the gum or starch phase and dispersewithin each phaseequally. Nevertheless, the very bright dense regions in the micro-graphs suggested formation of quite large aggregates of the silicaparticles. It should be recalled that the primary particle size of thesilica is approximately 20 nm and individual particles of this sizewould not be visible at this resolution. Some of the aggregates inFig. 7(c) are at least 20 mmacross. Furthermore, most of the particlesand their aggregates appeared to reside largely within the starch-rich domains. Experiments were repeated many times where theparticles were deliberately dispersed within the gum phase beforemixing this with the starch phase, but this seemed to have no effecton the distribution of particles on ageing: after 3 h or more 99% ofthe particles and their aggregates visible appeared to reside withinthe starch-rich phase. This propensity for particles topreferone typeof phase, for no obvious reason, is a feature that has beennoted byus

0.1s / Pa s0 25 50 75

0

25

η

Fig. 6. Hypothetical maximum viscosity (h0) at 0.1 s�1 of the LBG phase separatedregions, calculated (see text) on the basis of Figs. 2 and 5, versus viscosity (h) of theoriginal LBG phase after mixing with different wt.% starch concentrations: 0.15 (P);0.25 (6); 0.5 (,); 1 (C); 1.5 (:); 2 (-). The dashed diagonal line indicates the casewhere h0 ¼ h.


Fig. 7. Representative confocal micrographs of mixtures of 2 wt.% starch þ0.3 wt.% LBG in the absence and presence of a final particle silica particle concentration of 0.5 wt.% ofdifferent surface characteristics: (a) no particles, age 5 min; (b) no particles, age 24 h; (c) 100%SiOH (i.e., unmodified) particles, age 24 h; (d) 80%SiOH particles, age 24 h; (e) 70%SiOHparticles, age 24 h; 65%SiOH particles, age 24 h. Dark regions are gum-rich, brighter regions are starch-rich and/or particle-rich, very bright regions are clusters of particles. The sizebar ¼ 75 mm.


previously on completely different systems, for example sodium-caseinate þ xanthan þ emulsion droplet ‘particles’ (Hanazawa &Murray, 2014); sodium caseinate þ xanthan þ latex particles(Moschakis, Murray, & Dickinson, 2006) and alsostarch þ gelatin þ latex particles or emulsions droplet particles(Firoozmand et al., 2009). However, in Section 3.5 belowweproposea possible explanation.

For particles to inhibit phase separation significantly one ex-pects that a preference for interfacial accumulation of particles and/or their aggregation at the interface to be an important require-ment, as explained in the Introduction. From Fig. 7(c), or any otherof the large number of images we have obtained on this system, itvery difficult to say with certainty that particles definitely accu-mulate at the interface, partly because of the presence of particleaggregates in the microdomains, which may or may not beanchored at the interface (althoughmany aggregates are clearly notat the interface). On the other hand, it has frequently been observedin Pickering stabilized systems that many parts of the interfaceappear denuded of particles although the emulsion droplets or gasbubbles appear perfectly stable. This may be because completecoverage is not necessary if the particles form a strong network atthe interface, or again it could be because at the resolution of theconfocal microscope a layer of particles just a few tens of nm thickis hard to detect, particularly if there are much larger particle ag-gregates around which will tend to dominate the fluorescencesignal. Certainly for emulsions stabilized by fragments of hydro-phobic starch granules (Yusoff & Murray, 2010) very little coveragecould be observed for stable oil droplets.

More hydrophobic silica particles have a greater tendency toaggregate, which could lead to them forming a stronger interfacialnetwork, and for this reason the same starch-LBG mixtures wereprepared with such particles. Fig. 7(d) shows a typical micrographobtained after 24 h with 0.5 wt.% of the 80%SiOH particles andindeed there seemed to be a greater fraction of the interfacial zonedecorated with particle aggregates and even greater persistence ofthe early spinodal decomposition structure than with the 100%


SiOH particles. Unfortunately, there was also a greater density ofparticle aggregates within the starch-rich domains and indeedthere is no obvious reason why more hydrophobic particles wouldhave a greater tendency to aggregate at the interface than in thebulk, unless the particles somehow get trapped at the interfaceduring migration from the gum domains to the starch domains.Again, however, deliberately mixing the particles in the starchphase before mixing with the gum phase had no substantial effecton themicrostructures observed. Experiments were also conductedwith 70%SiOH and 65%SiOH particles and representative micro-graphs for 24 h old systems are shown in Fig. 7(e) and (f), respec-tively. For some reason the 70%SiOH particles were not as effectiveas the 100%SiOH or 80%SiOH particles in preserving the spinodalmicrostructure whereas the 65%SiOH particles gave the most welldefined structures with very clear decoration of the interfacial re-gions with particles but also even larger aggregates within thestarch-rich domains, which appeared more coarse then with the100%SiOH or 80%SiOH particles. Being the most hydrophobic, the65%SiOH particles were the most difficult to disperse: eventuallygross bulk aggregation of particles must begin to negate any effectsthat particles might have on biopolymer phase separation at all.

3.4. Image analysis of phase separating microdomains

An attempt has been made to quantify the effects of particletype and concentration on the phase separation kinetics of the2 wt.% starch þ0.3 wt.% LBG system by performing image analysison different series of images. Fig. 8(a)e(c) show the extractedcharacteristic length scale (L) as function of time for 0.5, 0.7 and1.0 wt.% particles, respectively. L, the largest dominant dimensionin any direction on the image, was determined from the two-dimensional fast-Fourier transform of the captured micrographs,as described previously (Firoozmand et al., 2012), using Image Jsoftware.Without particles, Lwas approximately 60 mm after 5 min(the shortest ageing time inwhich it was possible to obtain images).In less than 25 min after this time the domain size grew so rapidly


time/h0 10 20

L/ μm

50

100

150

XX

X

X X

time/h0 10 20

L/ μm

50

100

150

XXX X X

time/h0 10 20

L/ μm

50

100

150

XX X XX

(a)

(b)

(c)

Fig. 8. Characteristic length scale, L, versus time since mixing for mixtures of 2 wt.%starch þ0.3 wt.% LBG plus different final concentrations of added silica particles: (a)0.5 wt.% (b) 0.7 wt.%; (c) 1 wt.%. Data and representative confocal micrographs areshown for systems with silica particles of different surface characteristics: (>) 100%SiOH (i.e., unmodified); (,) (80%SiOH); (6) 70%SiOH; (�) 65%SiOH.


that discrete domains were not visible because separate layersstarted to form in the well of the slide. At 0.5 wt.% particles, the 80%SiOH particles are effective in keeping L< 100 mm for 24 h, whereaswith the 100%SiOH or 70%SiOH particles the domain size is over200 mm in >2 h (data points not shown except for 70%SiOH at0.5 h). These trends are generally in agreement with the qualitativeassessment of the effects at 0.5 wt.% particles in Fig. 7, discussedabove. Also in agreement is the fact that, using the most hydro-phobic 65%SiOH particles recovers some increase in domain sizestability, although L is approximately 1.8� larger at 24 h than withthe 80%SiOH particles. Fig. 8(b) shows the effect of 0.7 wt.% parti-cles and it is seen that this relatively small increase in particleconcentration gave a dramatic increase in stability for all 4 particletypes. Now the 80%SiOH, 70%SiOH and 65%SiOH particles were themost effective, giving approximately the same small increase in Lfrom 60 to 70� 5 mm in the first 5 h, and Lwas the same after 24 h,within experimental error. The 100%SiOH particles gave approxi-mately the same stability in the first 5 h, but L then increased to


115 � 5 mm after 24 h. Increasing the particle concentration furtherto 1.0 wt.% (Fig. 8(c)) gave a further increase in the stability of L,which decreased for all particle types, hardly increasing at all from60� 5 mm between 0.1 and 24 h for the 80%SiOH, 70%SiOH and 65%SiOH particle, whereas for the 100%SiOH (unmodified) silica par-ticles there was an increase to 70 � 5 mm. Representative confocalmicrographs of some of the compositions have been included onFig. 8 to give the reader some visual idea of the microstructuraldifferences. (These are the thresholded images that were analysedfor L and thus have very clear boundaries). Higher particles con-centrations were not studied because the aim was to keep theeffective particle concentration within limits that might have apractical application. Unmodified silica can be added to foods butregulatory limits are around a few % (depending on the particletype) whilst it is hoped in the long term to mimic these effects ofsynthetic particles with food grade materials that we have usedelsewhere (Murray, Durga, Yusoff, & Stoyanov, 2011).

It is useful to estimate the minimum domain size that the par-ticles could be expected to stabilize. We have done this calculationpreviously (Luo et al., 2011; Yusoff & Murray, 2010) for other sys-tems, making various assumptions, the most important of whichare that particle contact angle at the interface ¼ 90� and that acertain minimum fraction of coverage, fcrit, of particles at theinterface is required. An adsorbed particle will then occupy an areaof pr2 at the W/W interface and, assuming all the particles arepresent at the interface, it is easy to show that the ratio, K, of theinterfacial area occupied by the particles to the total interfacial areais given by:

K ¼ M:R4$r$r$f$Bcrit

(1)

where R ¼ the radius of the domain size (i.e., domains assumedspherical); M ¼ the mass per unit volume of particles added;r ¼ the particle density; r ¼ the particle radius; f ¼ the volumefraction of the domains. For the latter, since equal volumes of the 2polysaccharides phases are mixed, f was assumed ¼ 0.5. In theabsence of other information, fcrit was taken as 0.5, although thisseems a reasonable value based on various experimental observa-tions (Luo et al., 2011; Yusoff & Murray, 2010). The primary particlesize of the silica was 20 nm and so setting r ¼ 10 nm the minimumW/W droplet size that the silica particles could cover (i.e., K¼ 1), ascalculated from Eq. (1), is 2.5 mm. This is close to the lateral reso-lution of the confocal system under the conditions used, given thedynamic state of the system, so that regions around this size orsmaller would not be clearly visible. For the smallest mean Lcaptured, around 60 mm, equating this to R ¼ 60/2 ¼ 30 mm, K ¼ 12,so that the particles present could easily stabilize domains of thissize. Clearly the domains quickly grow larger than this, so that theinterfacial area required to be stabilized is substantially less, whilston the other hand many of the particles are present as aggregates,which reduces the interfacial area that they can cover. In theabsence of more detailed information, all that can be said is that thesize of the domains observed is broadly consistent with the avail-ability of particles to stabilize the corresponding area of the W/Winterface. In addition, this point also highlights that alternatives tosilica nanoparticles must be found if it is desired to stabilize do-mains of the sizes seen, since only silica particles much larger than20 nm are permitted for food use.

3.5. Bulk rheology of the different phases in the presence of silicaparticles

It is clear that all the particles were capable of significantlyslowing down microscopic domain growth and hence macroscopic



phase separation, although themore hydrophobic particles seemedmore effective at the higher particle concentrations. There alsoseemed to be a threshold concentration of around 0.7 wt.% abovewhich there was a significant increase in stability. At the same timesignificant aggregation of particles occurred in the starch micro-domains as well as possibly at the starch-gum W/W interface. It iswell known (Iler,1979) that nanoparticle silica can undergo gelationat bulk concentrations as low as those studied here (i.e., 1 wt.% orless) so that an important question iswhether the change in domainstability is simply due to gelation (or a very significant increase inviscosity) of the starch-rich domains via the formation of a silicaparticle network within them. To test this hypothesis, the mosthydrophilic particles, i.e., the untreated 100%SiOH particles, weredispersed in the separate bulk phases at the different particle con-centrations and the bulk rheologymeasured. Fig. 9 shows the resultsof the 3 types of measurement: in Fig. 9(a) h at g¼ 0.1 s�1 (i.e., as inFigs. 5 and 6) plus oscillatory shear rheology at low frequencies and1% strain: in Fig. 9(b) the loss modulus (G00) at 0.1 Hz and in Fig. 9(c)the storage modulus (G0) at 0.01 Hz. These low shear conditionswere selected so as to be as close as possible to the ‘zero’ shearconditions extant during the spontaneous phase separating condi-tions. The oscillatory values were obtained from frequency sweepsbetween 0.01 and 1 Hz and values averaged for different runs at thefrequencies given. (Below0.1HzG00 valueswere toonoisy to give anymeaningful distinction between the different systems).

Fig. 9 clearly shows that up to 3 wt.% untreated silica particlesadded to the 4 wt.% starch system there was no significant increasein h, G0 or G00. In fact if anything there was a slight decrease in thesemeasures of the phase viscoelasticity. In contrast, addition of par-ticles gave a significant increase in h, G0 and G00 of the 0.6 wt.% LBG.(Also shown are results for 0.5 wt.% guar gum, which once againgave similar results to those for 0.6 wt.% LBG). Because of the usual

Fig. 9. (a) Viscosity (h) at g ¼ 0.1 s�1; (b) loss modulus (G00) measured at 0.1 Hz;(c) storage modulus (G0) at 0.01 Hz for varying wt% of 100%SiOH (i.e., unmodified) silicaparticles added to individual solutions of 4 wt.% starch (C); 0.6 wt.% LBG (6); 0.5 wt.%GG (,).


migration of biopolymer into the different phase regions discussedearlier (e.g., see Fig. 3), plus migration of particles into the starchphase, plus the fact that a fair proportion of this silica seems to endup as insoluble lumps or aggregates, it is impossible, at present, tobe certain of the exact free particle and starch concentrations in themicrodomains observed. However, the results in Fig. 9 do notsuggest that a combination of starch concentration and nano-particle concentration could be reached within the starch domainssuch that these regions would form strong silica particle gels. Moreinteresting is the effect on gum phases, where the well-knownphenomenon of depletion flocculation of particles by the gum isthe likely cause of the significant increase in viscoelasticity of thisphase. Furthermore, this would provide a mechanism for the for-mation of particle aggregates in the first place, which possibly gettrapped to some extent at the W/W interface, since the larger theparticles the larger the desorption energy, for particles of the samesurface characteristics. Preliminary CLSM images that we hope topublish in the future seem to confirm greater tendency for particleaggregation in the gum phase than in the starch phase. Depletionflocculation of particles by the starch probably does not occurbecause amylopectin is a much more compact molecule than LBGor GG, with a much smaller hydrodynamic radius for a similarmolecular weight. The persistence of aggregates at the interface orwithin the starch microdomains may simply be a kinetic effect,since as both polymer domains becamemore viscous any aggregatere-arrangement or dissociation will be slowed down.

At present we have no further direct evidence that a silica par-ticle network forms at the W/W interface that strengthens this re-gion so as to explain the inhibition of phase separation. However, ina sodium caseinate þ xanthan systemwe have recently (Hanazawa&Murray, 2013) measured a significant increase in interfacial shearviscosity of the W/W interface in the presence of a low volumefraction of oil droplets covered inprotein that similarly seem to slowdown phase separation. Clearly, if cross-linking between silicaparticles at the interface could be turned on in someway this mightgive complete cessation of domain growth. Chemicals exist for this(Iler, 1979), but the methodology would not be permissible in food-stuffs. Particle cross-linking at the W/W interface might be easilyachieved with protein-coated particles, or with protein particles, asshown by Nguyen et al. (2013) in a dextran e poly (ethylene oxide)system. However, the addition of protein introduces furthercomplexity because protein molecules are quite surface active andthey will also interact strongly with polysaccharides. At present weare investigating other non-protein food-grade particles to see ifthey can act in the sameway as the synthetic silica particles in thesepolysaccharideepolysaccharide systems.

4. Conclusions

Phase separation in polysaccharideepolysaccharide systems canbe strongly influenced by the addition of a low volume fraction ofsilica nanoparticles. The nanoparticles show a strong preference forthe starch domains that form rather than the gum phase, whilstthere is an increasing tendency for particle aggregation to occurwithin the starch domains and possibly at the W/W interface be-tween the two phases as the particle hydrophobicity and particleconcentration is increased. Measurements of the bulk rheology ofthe gum and starch phases in the presence of particles suggests thatthe rheology of the interior of the starch domains is hardly affectedat all by the particles, whilst the gum phase viscoelasticity issignificantly increased by the addition of particles. It is suggestedthat depletion flocculation of the particles in the gum phase may bethe driving force behind the formation of particle aggregates at theW/W interface and within the starch domains that somehow slowsdown phase separation.



Acknowledgements

The authors would like thank Prof. Bernard P. Binks of theUniversity of Hull (UK) for the gift of some of the silica particlesused in this work and for useful discussions regarding particle-stabilized systems in general. Most of all, BM would like to thankProfessor Eric Dickinson, to whom this volume is dedicated, for hiswonderful support, advice and inspiration, past and present.

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The effect of nanoparticles on the phase separation of waxy corn starch+locust bean gum or guar gum