ORIGINAL CONTRIBUTION

Rheological investigation of pectin-based emulsion gelsfor pharmaceutical and cosmetic uses

Francesca R. Lupi & Domenico Gabriele & Lucia Seta &

Noemi Baldino & Bruno de Cindio & Rosamaria Marino

Received: 6 June 2014 /Revised: 19 September 2014 /Accepted: 15 October 2014 /Published online: 26 October 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Emulsion gels are structured emulsions suitable fordifferent uses for their specific behaviour, which is stronglydependent on the characteristics of the gelled dispersing phase.Therefore, it is important to adopt the specific gelling agent totune the final emulsion rheological behaviour properly. Pectinis extremely interesting among potential hydrophilic gellingagents owing to its specific characteristics. In the present work,four different low-methoxyl pectins were adopted to preparegels to be used as the dispersing phase in cosmetic or pharma-ceutical emulsion gels. The rheological characterisation of pec-tin gels, prepared at room temperature to avoid the damage topotential thermolabile components, was carried out with smallamplitude oscillations. The obtained gels were used, togetherwith a common non-ionic surfactant (Tween 60), to prepareolive oil emulsion gels suitable to design new cosmetic prod-ucts. A simple empirical model, proposed to relate the emulsioncomplex modulus to the oil fraction and properties of thedispersing phase, has shown itself to be a potentially usefultool to design formulations with desired properties.

Keywords Rheology . Structured emulsion . Pectin .

Filled gel . Cosmetics . Olive oil

Introduction

Structured emulsions (both O/WandW/O) are widely adoptedin industry, e.g. for food uses, cosmetic and pharmaceutical

creams production, and many others fields (Gabriele et al. 2009;Leal-Calderon et al. 2007; Lupi et al. 2011; McClements 2012).These systems, characterised by a “semisolid” behaviour of thecontinuous phase, are often described as “emulsion filled gels”,“composite gels” (Houzé et al. 2005; Lorenzo et al. 2013),“emulsion gels” (Dickinson 2012) or “emulgels” (Ajazuddinet al. 2013) with the aim of evidencing the relevant role playedby the structured dispersing phase in developing the emulsionmicrostructure. Among the different potential uses, emulsiongels (mainly the oil-in-water ones) have been finding relevantimportance in pharmaceutical and cosmetic applications for top-ical delivery because they possess the main advantage of bothemulsions (ability to penetrate the skin, control of rheologicalproperties, appearance and degree of greasiness) and hydrogels(greaseless, easily spreadable, easily removable, compatible withseveral excipients, and water-soluble or miscible) commonlyadopted for topical drug delivery (Ajazuddin et al. 2013).

Emulsion gels can be extremely useful in these fieldsbecause the structured dispersing phase (i.e. the gel) improvessignificantly the emulsion stability (Kokini and van Aken2006); moreover, the macroscopic properties (such as thetexture, spreadability and drug release rate) can be tuned bymodifying the characteristics of both gel and emulsion satis-fying some of the most important requirements for theseproducts (Aikens and Frieberg 1996; Gilbert et al. 2013;Masmoudi et al. 2005). Even if both oil-in-water and water-in-oil emulsion gels can be obtained, it seems that, for anumber of cosmetic and pharmaceutical uses, O/W systemscan have a higher customer acceptability since they are water-washable and non-greasy (Mohamed 2004; Yorgancioglu andBayramoglu 2013). Moreover, they are able to deliver bothlipophilic (entrapped in internal oil droplets) and hydrophilic(entrapped in the gel network) active components while con-trolling their release rate and stability (Ajazuddin et al. 2013).

A number of gelling agents have been adopted and testedfor emulsion gels production (Ajazuddin et al. 2013; Bais

F. R. Lupi :D. Gabriele (*) : L. Seta :N. Baldino :B. de CindioDepartment of Informatics, Modeling, Electronics and SystemEngineering (D.I.M.E.S.), University of Calabria, Via P. Bucci, Cubo39C, 87036 Rende, CS, Italye-mail: domenico.gabriele@unical.it

R. MarinoSilva Extracts Srl, Via Marco Polo 72/74, 87036 Rende, CS, Italy

Rheol Acta (2015) 54:41–52DOI 10.1007/s00397-014-0809-8

et al. 2005); nevertheless, the use of food biopolymers also inthe pharmaceutical field as potential stabilising or emulsifyingagents (Bouyer et al. 2012) is being receiving increasinginterest in the scientific community. Among them, pectin hasnot been deeply investigated yet, and it seems to be extremelypromising to be used in emulsion gels preparation owing to itsspecific characteristics.

Pectin is a polysaccharide extracted from different botani-cal sources (particularly apple or citrus peel), mainlyconsisting of α-(1–4)-D-galacturonic acid chain interruptedby 1,2-linked rhamnose units (Ovodov 2009). The carboxylgroups distributed along the chain are, in a certain amount,esterified with methanol and the degree of methyl esterifica-tion (DM) divides pectin into high-methoxyl pectins (HM),containing more than 50 % of esterified carboxyl groups, andlow-methoxyl pectins (LM) with a lower than 50 % degree ofesterification. Pectin is widely used for its thickening andgelling properties that are profoundly affected by its finestructure, which in turn, is determined by the specific sourceand by the adopted extraction conditions (Munarin et al. 2012;Willats et al. 2006). As a consequence a wide range of mac-roscopic properties can be obtained by selecting the suitableproduct as evidenced by the large number of current applica-tions (Liu et al. 2007; May 1990; Thakur et al. 1997).

The use of pectin in food emulsions has already beenstudied by different authors (Littoz and McClements 2008;Neirynck et al. 2004; Thakur et al. 1997); more recently, thepotential interfacial activity of pectin was also investigated,and mainly for sugar beet pectin (SBP), interesting interfacialand/or emulsifying effects were observed (Fissore et al. 2013;Gülseren and Corredig 2014; Yapo et al. 2007). The potentialuse of pectin in cosmetic or pharmaceutical matrices hasbeen proposed by Awasthi (2011) and Munarin et al.(2012) for its gelling properties, and by Burapapadhet al. (2010) for the emulsifying properties for topicaldrug delivery systems. Moreover, it was observed thatsmall amounts of oligogalacturonides promote organiza-tion and stratification of the epidermal cells in vitro sug-gesting potential effects of these pectic components asanti-age active ingredients (Lebreton-decoster et al. 2011).

An interesting characteristic of low-methoxyl pectin, whichcould make it particularly suitable for cosmetic and pharma-ceutical systems, is a predisposition to gel, in suitable condi-tions, at room temperature thereby avoiding potential damageto thermolabile products. In fact, often, the controlled deliveryof thermolabile components, such as for example, propolis(Biscaia and Ferreira 2009), different components frommicroalgae (Mendes et al. 2003), deoxyArbutin (Yang et al.2010) and so on, has to be achieved and, therefore, lowtemperatures have to be used for carrier preparation. As aconsequence, structuring agents that gel as a result of heating(possibly followed by cooling) (e.g. carrageenans) cannot beused, whereas LM pectin gels as an effect of the ionic bonds

via calcium ions bridges and, therefore, gelation can occuralso in room condition (Thakur et al. 1997).

For this work, emulsion gels based on commercial citruspectins were prepared and their rheological properties werestudied and modelled according to the functionality of biphas-ic systems. It is known from the literature that rheologicalproperties of emulsion gels are strongly dependent on theproperties of the dispersing gel phase (Kokini and van Aken2006; Lorenzo et al. 2013); therefore, as a first step of thework, the rheological investigation of pectin gels was carriedout. In a further step, simple oil in water emulsions wasprepared and studied with the aim of relating macroscopicemulsion properties to the gel characteristics (i.e. pectin con-centration) and oil fraction.

Virgin olive oil was used as the oil phase because it is anatural product widely used, mainly in the past, in cosmeticand dermo-protective creams. Its similarity to sebum compo-sition, the presence of oleic acid that acts as a skin softener andthe presence of antioxidant components, such as vitamins Aand E, make it extremely active in skin protection and regen-eration (Varka and Karapantsios 2011; Viola and Viola 2009).

Finally, a common non-ionic surfactant, i.e. Tween 60, alsoused for cosmetic systems and emulsion gels (Ajazuddin et al.2013) was adopted in emulsion preparation.

The model systems produced in the present work arevirtually suitable as cosmetic creams for the controlled deliv-ery of thermolabile compounds in the aqueous phase, sincethey are all samples where the water phase was completelyhold at room temperature.

Materials and methods

Pectin characterisation

Four low-methoxyl citrus pectins, at different degrees ofmethoxylation, were kindly supplied by Silva Extracts srl(Italy). Degree of esterification for each sample was directlysupplied by pectin manufacturer and it is shown in Table 1.Pectin were characterised by determining their molecularweight and their potential interfacial activity.

Table 1 Characteristics of pectins used for gel preparation

Pectin DM (%) Mv (kDa) γeq (mN/m)

A 33.3 89±7 9.17±0.06

B 27.2 59±1 11.1±0.2

C 37.1 73±6 9.84±0.03

D 41 123±1 9.9±0.5

Degree of methoxylation (DM) value for each pectin was directly givenby the producer (Silva Extracts srl, Italy)

42 Rheol Acta (2015) 54:41–52

The average viscometric molecular weight (Mv) was deter-mined by using the Mark-Houwink equation (Lapasin andPricl 1995) which relates intrinsic viscosity, [η], and Mv:

η½ � ¼ KMav ð1Þ

K and a are empirical parameters depending on the partic-ular solvent-polymer pair and on the temperature. The intrin-sic viscosity was obtained processing the experimental data ofdiluted polymer solutions via an extrapolation procedurebased on the Huggins (Eq. 2) or Kraemer (Eq. 3) equations(Lapasin and Pricl 1995):

ηspc

¼ η½ � þ k 0 η½ �2c ð2Þ

ln ηrð Þc

¼ η½ � þ k 00 η½ �2c ð3Þ

where c is the polymer concentration and ηsp is the specificviscosity that can be obtained from the relative viscosity ηr(i.e. the solution, ηsol, to solvent, ηs, viscosity). The parametersk′ and k″ are known as Huggins and Kraemer constant, re-spectively. Viscosity of diluted solutions was measured byusing a Cannon-Fenske type capillary viscometer size 25(Cannon Instrument Company, USA) immersed in a thermo-static water bath (Julabo, USA) at 30.0 °C±0.1 °C. The flowtime was measured with an accuracy of 1 s. After the sampleloading into the viscometer, the solution was allowed toequilibrate at the bath temperature before starting the test.The relative viscosity was obtained as the ratio between theflow times of pectin solution and solvent (a 0.1 M NaClaqueous solution). The following values were assumed forMark–Houwink equation parameters (Migliori et al. 2010):K=9.55·10−4 (g/dl) and a=0.73.

Pectin interfacial properties were investigated measuringtransient interfacial tension (γ) with a Pendant Drop Tensiom-eter (FTA 200, First Ten Angstroms, USA) at room tempera-ture (22±1 °C) according to a procedure described by Setaet al. (2012, 2013). Solutions prepared with 1 % (w/w) ofpectin, corresponding to the saturation value (data not shown),were produced dissolving 0.3 g of each pectin in 30 ml ofbuffer solution (pH=4.2, prepared in bidistilled water) contin-uously stirred for 12 h. Larger concentrations were not inves-tigated because they did not change the interfacial tensionvalues of the final systems, and also because they yielded highconsistency samples, disturbing the correct performance of theanalysis. Tests were carried out allowing the formation of adroplet of pectin solution (about 18 μl) in a cuvette filled witholive oil (about 5 ml) and measuring interfacial tension for 3 h(about 10,800 s) until an apparent steady state value wasobtained (maximum variation of 3 % in 600 s; Seta et al.2012, 2013). According to Seta et al. (2012) non-purified oilwas used, with the aim of investigating the behaviour of

interfaces close to the adopted real systems where oil impuri-ties can affect the interface behaviour.

Pectin gels: materials and preparation

Pectin gels were produced, according to a standard proceduresuggested by Silva Extracts srl, by mixing pectin within acitrate buffer solution (constituted of distilled water, citricacid, and tribasic sodium citrate, Carlo Erba reagents, Italy)at pH=4.2.With the aim of better guaranteeing the dissolutionof powder and to avoid aggregate formation, pectin waspreliminary mixed with ethanol (Minimum assay (G.C.)99.8 % (v/v), Panreac, Spain) in a volume/weight ratio equalto 2 (ml of ethanol over grams of pectin) and then this mixturewas dropped to the buffer solution at 25 °C while stirring(ARE heating magnetic stirrer, Velp scientific, Italy). As al-ready discussed, LM pectin gels by effect of calcium bridgesbetween carboxyl groups (according to the so-called egg boxmodel (Thakur et al. 1997)), therefore, a calcium solutionmade of 2.2 % (w/v) of calcium chloride dihydrate (CarloErba reagents, Italy) in distilled water was used. Differentpectin concentrations were used for gel preparation, and thecalcium chloride/pectin ratio was kept constant for all samplesat 0.18 (w/w) by adding the proper volume of calcium solutionto the buffer containing pectin. In the case of samples pro-duced with more than 1.5 % (w/w) of pectin, the buffer wasadded to the ethanol/pectin mixture to favour the completedispersion of the system. The amount of buffer solution wasadjusted, for each sample, to maintain constant the totalamount of prepared gel at 100 g.

Final suspensions were continuously and gently stirred for1 h (ARE Velp scientific, Italia), and therefore, stored at 25 °Cfor 24 h, in order to allow the proper gel formation beforeexperimental tests. Gel samples were identified by the G letterfollowed by the pectin identifier (i.e. A, B, C, D); for eachpectin the following mass fractions were used to prepare gels:0.003, 0.004, 0.005, 0.01 and 0.015. Only for pectin B, anadditional gel was prepared at 0.02 mass fraction.

Emulsion gels: materials and preparation

A number of pectin gel formulations, selected according to theresults obtained by rheological characterisation, were used asthe aqueous dispersing phase of oil-in-water emulsions.

The oil phase was prepared by dissolving a constantamount (5 % w/w on a total emulsion basis) of a commonnon-ionic emulsifier, Tween 60 (polyoxyethylene sorbitanmonostearate; Sigma Aldrich Inc., Germany) in virgin oliveoil (De Santis, Italy), supplied from a local supermarket. Theoil was previously heated up to 70 °C on a heating magneticstirrer (ARE Velp Scientific, Italy), and the emulsifier wasadded at this temperature allowing it to be completely melted;even though the emulsifier was mainly hydrophilic (HLB=

Rheol Acta (2015) 54:41–52 43

14.9 (Gabriele et al. 2010)), it was dissolved in oil becauseaqueous phase was prepared at 25 °C. This temperature valuewas chosen with the aim of preparing systems potentiallyuseful for entrapping thermolabile hydrophilic active agents.It is worth noticing that if lipophilic agents have to be used, adifferent procedure, involving oil phase preparation at roomconditions, should be used and, therefore, the emulsifiershould be dissolved in aqueous phase.

The oily mixture obtained was then cooled down to 25 °Cunder a continuous stirring action and used for emulsionpreparation.

The aqueous phase was prepared by dispersing pectin inethanol in a continuously stirred beaker, then buffer solution(already described) was added to the mixture, stirred forfurther 2 min with an Ultra Turrax homogeniser (UT T50,IKA-Werke, Germany, speed of mixing 2,000 rpm).

The emulsion was prepared dropping the oil phase to theaqueous phase at 25 °C maintaining constant mixing condi-tions (UT T50, IKA-Werke, Germany, speed of mixing2,000 rpm) for 2 min, and after the emulsion formation,calcium chloride solution was added increasing the speed ofmixing up to 8,000 rpm for 30 s to promote the calciumdissolution in the whole system. According to the adoptedprocedure, emulsification was obtained before gelation ofdispersing phase (obtained by calcium addition), following aliterature procedure used for oil-in-water filled emulsion gelsbased on a different polysaccharide (Lorenzo et al. 2013): inthis way, the obtained gel is not damaged by the homogeni-sation process.

A preliminary rheological and microstructural analysis ofemulsified samples did not reveal any changes in rheologicalproperties after a minimum resting time of 36 h. Therefore, allsamples were completely analysed after 36 h from prepara-tion. Emulsions were stored at 4 °C for the whole restingperiod. Samples were identified by the E letter followed bythe identifier of the pectin (i.e. A, B, C, D) adopted in theaqueous phase; samples with different oil and pectin contentwere numbered consecutively (see Table 2).

Rheological characterisation

Gel samples were investigated with Nova rheometer(Rheologica AB, Sweden) equipped with a plate-plate geom-etry (φ=25 or 40 mm according to the specific sample con-sistency); temperature was controlled with a Peltier systemacting under the lower plate and a constant gap of 1.5±0.1 mmwas adopted for all samples during the characterisation carriedout using small amplitude oscillation tests. Frequency sweeptests were performed at a constant stress within the linearviscoelastic region, in the frequency range 0.1–10 Hz (previ-ous stress sweep tests were carried out in order to evaluate thelinear viscoelastic domain) and at a fixed temperature value of25 °C; a narrower frequency range (up to 1 Hz) was

investigated for low pectin concentrations (lower than 0.01w/w) owing to instrument limitations related to head inertiaissues (Franck 2003) more evident when low consistencysamples were investigated. Dynamic temperature ramp tests(time cure tests) were carried out by heating the sample from25 °C to 70 °C at 1 °C/min, always guaranteeing the linearviscoelastic regime: in fact, the applied stress was modifiedduring the test according to the temperature changes.

Emulsion gels characteristics were studied with a DSR-500(Rheometric Scientific, USA) also equipped with a plate-plategeometry of the same diameter used for gels; emulsionssamples were analysed with the same conditions describedfor gels, but a gap of 2.5±0.1 mm was adopted. All tests wererepeated three times and shown results are averaged values.

A cosmetic O/Wemulsion for topical uses, kindly suppliedby SA.TE.CA (Italy), and enriched with thermal water, wasalso characterised with a controlled strain rheometer (ARES-RFS, TA Instruments, USA) equipped with a plate-plate ge-ometry (φ=25 mm, gap=1.5±0.1 mm); a frequency sweeptest was carried out within the same frequency range adoptedfor model emulsions and obtained data were used as a bench-mark to be compared to the rheological behaviour of thepectin emulsion gels.

Contrast phase microscopy

With the aim of studying the effect of the investigated param-eters (pectin amount in gels, process condition, oil phasevolume fraction) on the internal microstructure of the emul-sions, an optical microscopy analysis was also performed toevaluate the droplet size distribution (DSD). The microphoto-graphs were taken using a phase contrast microscopy(MX5300H, MEIJI, Japan) equipped with phase contrast

Table 2 Emulsion gels characteristics: pectin type and their fractions inthe aqueous phases (gels) are listed

Sample ID Pectintype

Pectin fraction ingels, xp (w/w)

Oil phase fraction inemulsion gels, xo (w/w)

EA1 A 0.015 0.240

EB1 B 0.015 0.240

EC1 C 0.015 0.240

ED1 D 0.015 0.240

EB2 B 0.020 0.240

EB3 B 0.026 0.240

EB4 B 0.03 0.240

EB5 B 0.02 0.144

EB6 B 0.02 0.335

EB7 B 0.02 0.430

EB8 B 0.02 0.525

EB9 B 0.02 0.097

44 Rheol Acta (2015) 54:41–52

objective 40×. Samples were diluted and gently stirred (1:10,volume fraction) in distilled water (Seta et al. 2013) with theaim of reducing both the potential presence of droplets aggre-gates (and therefore, making photomicrographs more easy tobe analysed) and the sample opacity; in fact, being the externalphase a gel, it has not been possible to observe undilutedsamples owing to their opacity. After that, all samples wereplaced onto a glass slide inside a cover-imaging chamber(Sigma Aldrich, Germany) on which a cover slide was put.The cover chamber forms a sealed volume for thick and free-floating specimens.

An image database software was used to detect the differ-ent particle sizes of droplets (dhs image database, Germany)by greyscale detection. The software measures the number-based surface equivalent diameter. A statistic software analy-sis was used to interpret the DSD raw data acquired from eachemulsion batch (Statgraphics Centurion XV, version 15.2.13,USA). According to the procedure adopted by Seta et al.(2013), a lognormal model was used to interpret experimentaldata, and droplet size distribution was completelycharacterised with two parameters: the mean diameter d andthe standard deviation σ values (from the lognormal modelparameters). In particular, the width of the distribution, relatedto its polydispersity, can be evaluated from the standard devi-ation value (σ) in the case of Gaussian distributions (Seta et al.2013).

Results and discussion

Pectin characteristics

The preliminary determination of pectin molecular weightevidenced that Mv changes significantly among the samples(see Table 1) and it seems to decrease with decreasing DM;this is probably caused by the extraction and demethoxylationprocedures, industrially adopted and based on a non-selectiveacid hydrolysis which simultaneously reduces both DM andchain length. Moreover, it is worth recalling that, sinceadopted pectins are commercial products, differences in rawmaterials could be present and could affect the final molecularweight.

With the aim of investigating the potential emulsifyingactivity of pectins, interfacial tension (γ) between the twophases adopted for emulsion preparation was measured. Ex-perimental results evidence a transient behaviour (data notshown) from an initial value, equal to 17.1±0.3 mN/m, downto a steady value reached approximately after 8,000 s from thebeginning of the test. Equilibrium values (γeq) are shown inTable 1 and evidence that pectins exert an action on theinterface, confirming a potential interfacial activity of thesebiopolymers. Anyway, the reduction in interfacial values

seems smaller than those observed for typical commercialemulsifiers; for example Tween 60, at sunflower oil/waterinterface, exhibits equilibrium interfacial tensions approxi-mately equal to 4.6 mN/m (Seta et al. 2012) (starting frominitial values close to those observed in the present work forolive oil/water interface). Therefore it seems that investigatedpectins are not so effective in reducing interfacial tension ascommercial surfactants are, even though their interfacial ac-tivity is interesting.

Moreover, it is worth noting that equilibrium values, forinvestigated pectins, are included in a narrow range of valuesfor all the investigated pectin and relevant differences, thatcould make one biopolymer preferable with respect to theother ones, do not seem present.

Gel rheology: the effect of pectin content

As already discussed, it is well known that the rheologicalbehaviour of structured emulsions, or emulsion gels, is mainlydue to the mechanic properties of the solid-like continuousphase (Kokini and van Aken 2006; Rodríguez-Abreu andLazzari 2008). Therefore, first of all, the rheological investi-gation of the hydrogel, which is the aqueous phase of emul-sions, was carried out with the aim of investigating potentialdifferences among discussed pectins and, if possible, ofselecting the most proper polysaccharide for the preparationof final emulsion gels.

All the investigated gel samples, as obviously expected,showed a typical solid-like behaviour, with both moduli al-most linear in a log-log scale and a storage modulus G′ muchhigher than G″ (loss modulus) in frequency sweep tests,within the investigated frequency range. A simple way tosummarise and compare the rheological characteristics of allinvestigated gels is to evaluate the complex modulus G* andthe phase angle δ. Data obtained for samples GB at differentweight fractions are shown in Fig. 1 as function of adoptedfrequency: they evidence a non-linear monotonous increase inG*, with increasing concentration, whereas a more complexnon monotonous trend is observed for δ.

Complex modulus and phase angle, at 1 Hz, for all theinvestigated gel samples are shown in Fig. 2a, b, respectively.Samples GB and GD at very low pectin fraction (xp) (i.e.approximately 0.003) are characterised by a phase angle valuesignificantly larger than those observed at higher concentra-tion. According to the data shown in Fig. 2b, samples GB andGD with the lowest pectin fraction are less structured thanmore concentrated systems. Samples GA and GC do notexhibit, at the lowest concentration, phase angle values sig-nificantly larger than the others, suggesting that the potentialthreshold could be lower than the lowest investigated fraction.The observed differences, among tested samples, cannot besimply attributed to differences in DM or molecular weight,whereas they are probably due to the specific characteristics of

Rheol Acta (2015) 54:41–52 45

pectin structure such as the length of non-methylated regions(often named as degree of blockiness (Fraeye et al. 2009)) orthe partial replacement of hydrogen ions, on carboxyl groups,with monovalent cations which, according to the industrialempirical knowledge, could make the pectin less reactive to-wards calcium ions. When larger concentrations are used, adecrease in phase angle is observed, followed by a more or lesspronounced increase. Therefore, for all samples, an apparentminimum in phase angle is present for concentrations rangingbetween 0.004 and 0.005, according to the specific pectin. Itcan be speculated that for these concentrations all the potentiallinks between pectin chains, promoted by the adopted calciumconcentration, were accomplished, and an “optimal” structureextension is obtained. On the other hand, increasing the pectincontent the gelation rate could increase making the calciumdistribution within the sample more difficult and less uniformand yielding, and as a final result, a less structured gel. Thisexperimental evidence is in agreement with the literature inwhich optimal gel strength for intermediate calcium concentra-tions is reported, whereas weaker systems are obtained at bothlow and high calcium levels (May 1990).

When complex modulus G* is considered, a monotonousnon-linear increase with pectin amount is observed and, for

each pectin, when plotting complex modulus as concentrationfunction on log-log scale, two linear regions can be identified.The slope change can be approximately identified at the pectinconcentration corresponding to the minimum in phase anglecurve, i.e. between 0.004 and 0.005. This power law behav-iour was already observed in the literature for storage modulus(that is approximately equal toG* for the considered systems)as a function of pectin content (Cardoso et al. 2003; Fraeyeet al. 2009; MacDougall et al. 1996), even though no slopechanges have so far been observed for G′. This evidence isprobably due to the different range of investigated pectinfractions, in fact, in the present work, lower pectin concentra-tions were used with respect to the systems discussed in thecited literature.

On the other hand, it is worth noticing that a similar slopechange was observed when plotting G′ as a function of calci-um content (in a double log plot) for commercial citrus pectins(Cardoso et al. 2003), suggesting a change in material behav-iour according to the pectin to calcium ratio.

It can be highlighted that at a high pectin content a slowerslope is observed evidencing a lower dependence on polymer

Fig. 1 Frequency sweep tests at 25 °C in linear conditions for gels basedon pectin B at different weight fractions. Complex modulus (a) and phaseangle (b)

Fig. 2 Rheological parameters of gel samples evaluated at 1 Hz in fre-quency sweep tests as a function of pectin fraction. Complex modulus a:symbols are experimental values and lines represent fitting Eq. 4; phaseangle b: symbols are experimental values and lines are only a guide forthe eye

46 Rheol Acta (2015) 54:41–52

concentration of complex modulus, probably because in theseconditions pectin yields mainly a thickening effect whereas atlower fractions a significant change in structure extension isalso present (also evidenced by the phase angle trend).

For all samples with a pectin fraction higher than the valuecorresponding to the minimum in phase angle, complex mod-uli data were fitted with a power-law model and results ob-tained are compared to experimental values in Fig. 2a; where-as, Table 3 shows the values of the constants C1 and C2,computed according to Eq. (4) that proposes the linearizationof the classical power-law equation:

ln G� 1Hz; xp� �� � ¼ C1þ C2⋅ln xp

� � ð4Þ

Constant C1 is the pre-exponential factor, equal to ln(G*)evaluated at xp=1, and as a consequence, proportional to thecomplex modulus value, while C2 is the slope of the curve:the lower the slope, the lower the effect of pectin on theincrease of G* modulus. It is worth noticing that C2 rangesbetween 2.3 and 4, values slightly larger than those observedin the literature (Cardoso et al. 2003) for citrus commercialpectin (close to 2) or olive pectic extract (close to 3). Highervalues of the curve slope could be ascribed to a lower numberof interconnections between polymer chains, as suggested bythe work of Lapasin and Pricl (1995) and Vriesmann andPetkowicz (2013) on similar gels, even if based on high-methoxyl pectin.

Pectins B–D exhibit a similar behaviour as shown by C1and C2 values having not relevant differences; whereas, sam-ple A shows values of both constants lower than those of othersamples.

The observed behaviour of C1 and C2 could be explainedconsidering the potential interactions among polymer chainsin LM pectin gels. According to the literature (Kastner et al.2012), three possible kinds of links can be formed in LMpectin: a weak interaction such as hydrophobic interactionsbetween methoxyl ester groups, hydrophilic interactions be-tween undissociated carboxyl groups and hydroxyl groups viahydrogen bonds, and stronger interactions like ionic linkagesvia calcium bridges (egg-boxmodel). LM pectin characterisedby a very lowDM, has a higher number of free carboxylic acidgroups in their alkyl chains, which can interact with divalentions (Ca2+) forming strong links between the pectin chains.

Therefore, the continuous structured network proves strongerwith respect to gels prepared with higher DM pectin (Fraeyeet al. 2010a). In the light of this, a lower DM gives moreconsistent gels. Increasing the DM (andMv as a consequence),the primary interactions with calcium ions decrease, and gelstrength decreases as a consequence. On the other hand whenDM is further increased, the possible primary interactions areclearly less, but the secondary interactions between methoxylgroups could become significant, giving again a substantialincrease in network structure and strength thanks to hydrophobicinteractions and hydrogen bonds (Löfgren et al. 2005). There-fore it could be speculated that in samples GC and GD second-ary interactions are enough relevant to give a final behaviourquite close to that of sample GB; on the other hand in sampleGA, having intermediate DM, both primary and secondaryinteractions are probably lower than those present in otherpectins and as a consequence a less structured gel is obtained.

Even though sample B is characterised by the highestmoduli at higher pectin concentrations and, therefore, itshould be considered the most promising material formanufacturing structured and very consistent emulsion gels,differences with the other gel samples were not so relevant tojustify the choice of one pectin as particularly suitable for thedesired production. As a consequence emulsion gels, in apreliminary phase, were prepared using all pectins to investi-gate their behaviour in biphasic systems.

Emulsion gels samples

As a preliminary step, a benchmark sample (i.e. a commercialcosmetic cream kindly supplied by a local producer, namedBS in the following discussion) was investigated with fre-quency sweep tests. A linear trend in a log-log plot wasobserved for both G′ and G″ as frequency function with thestorage modulus greater than the loss one, evidencing a prev-alent solid-like behaviour. According to the data analysisalready described for gels, the benchmark sample was de-scribed by using complex modulus and phase angle at 1 Hzand the following values were computed G* (1 Hz)=2,900±100 Pa and δ (1 Hz)=10.0±0.3°. Following the study carriedout on gel samples, emulsions gels, in which the dispersedphase is unstructured olive oil, were produced.

It is worth noting that, according to the previous discussionconcerning interfacial tension values, the investigated pectindoes not seem extremely effective in reducing interfacialtension at oil/water interface; therefore, with the aim ofguaranteeing the formation of stable emulsion, a commonnon-ionic emulsifier, i.e. Tween 60, was adopted in allformulations.

Four samples were obtained using the different pectinslisted in Table 1, with a mass fraction 0.015 (see Table 2).

The results obtained in terms of complex modulus G* andphase angle both evaluated at 1 Hz are shown in Table 4. It is

Table 3 Equation (4) parameters and coefficient of determination R2

values for linear regression

Sample ID—gel Pectin type C1 (−) C2 (−) R2

GA A 17±1 2.3±0.3 0.98

GB B 24.7±0.6 4.0±0.1 0.99

GC C 22±2 3.4±0.3 0.99

GD D 23.3±0.6 3.8±0.1 0.99

Rheol Acta (2015) 54:41–52 47

worth noting that when emulsion gels are compared to pectingels having the same pectin content (i.e. 0.015), a relevantdecrease in complex modulus is observed: this behaviour isknown and expected for emulsion gels where no interactionbetween the gel matrix and the dispersed particles is present(Pal 2002; Van Vliet 1988); therefore it can be speculated thatin the investigated systems the oil is an inactive filler.

A narrow range of phase angle values, quite close to that ofBS sample, can be observed, whereas larger variations areshown by G*, which is significantly lower than the valuemeasured for the benchmark. As a consequence, it was con-sidered more relevant to set the optimisation of the properrheological characteristics on the complex modulus value.With the aim of investigating, a potential relationship betweenmacroscopic properties and emulsion characteristics the DSDof all samples was investigated. In fact, it is worth recallingthat for unstructured and concentrated emulsions (with a dis-persed phase volume fraction higher than 0.5), or for weaklystructured but less concentrated emulsions (Gabriele et al.2009), the DSD, characterising emulsified systems or suspen-sions, exerts a significant influence on the bulk rheology of thesystem. According to the literature (Barnes 2000; Ford et al.1997), an increase in mean diameter and polydispersity (stan-dard deviation) of these emulsions yields to less viscoussystems, in general.

The droplets size distribution parameters, mean diameter dand standard deviation σ, for each emulsions evaluated 36 hafter the preparation, are shown in Table 4 and they do notevidence relevant differences. According to these data thesamples showing the lowest value of both mean diameterand standard deviation are EB1 and ED1; if the only variableacting on the rheology of such samples was the droplet sizedistribution, EB1 and ED1 should be characterised by higherconsistency with respect to the other samples. On the contrary,ED1 is the less consistent sample; therefore, it is clear from theobtained results that the bulk rheology of structured emul-sions, or filled gels, is not significantly influenced by DSDbut, prevalently, by the structuration of the continuous phase(gel) and by the volume (or mass) fraction of the dispersedphase.

It is worth noticing that for dilute emulsions, DSD issignificantly affected by both interfacial tension values (with

smaller droplets obtained for lower interfacial tension values)and by water phase viscosity (Seta et al. 2013). In the presentwork a correlation with dispersing phase consistency seemspresent, in fact droplet diameter seems to decrease with de-creasing G* (at 1 Hz) of pectin gels having the same concen-tration: probably the higher the aqueous phase consistency themore difficult the formation of small drops.

When emulsion samples are compared to the commercialbenchmark it results that phase angle values of all products arequite close each other whereas complex modulus of BS issignificantly larger than those of pectin emulsion gels.

Therefore, among the studied emulsions, thosecharacterised by the highest values of complex modulus at1 Hz (i.e. EB1) can be chosen as the starting point for design-ing samples having rheological characteristics as close aspossible to the benchmark sample. A further relevant charac-teristic of emulsions, potentially suitable for cosmetic (orpharmaceutical) use, is the long-term stability: assessmentmethods, in this area, are often based on accelerated condi-tions able to shorten the time over which the tests are carriedout and, among the potential procedures, aging tests at hightemperature, normally 40 °C, for a fixed period of time areprobably the most used (Semenzato et al. 2012; Zografi 1982).Recently, rheology based procedures are being proposed andit seems that dynamic temperature ramp tests can be consid-ered as valid methods to simulate the forced accelerateddestabilisation of cosmetic emulsions, if compared with thecommon accelerated aging tests (Semenzato et al. 2012). As aconsequence, time cure tests were carried out on all samplesand a typical behaviour is shown in Fig. 3 for sample EB1: itwas observed, as a general trend, an initial region wheremoduli are almost constant followed by a more or less sharpdecrease after a critical temperature. The observed behaviourcan be probably attributed to the rupture of the emulsionowing to the melting of the dispersing phase (i.e. the pectingel); in fact, according to the literature (Cárdenas et al. 2008;Fraeye et al. 2010b; Vithanage et al. 2010), it is known that by

Table 4 DSD parameters (mean diameter d and standard deviation σ)and rheological properties (complex modulus G* and phase angle δ at1 Hz) of emulsion gel samples

Sample G* at 1 Hz (Pa) δ at 1 Hz (°) d (μm) σ (μm)

EA1 530±20 11.8±0.2 1.40±0.02 0.31±0.03

EB1 650±40 10.9±0.2 1.26±0.02 0.21±0.02

EC1 431±9 11.2±0.4 1.33±0.04 0.28±0.04

ED1 340±20 10.6±0.3 1.24±0.05 0.20±0.02

Fig. 3 Time cure test of sample EB1. Full circles represent G′ modulus,empty circles G″ modulus, and diamonds represent phase angle δ

48 Rheol Acta (2015) 54:41–52

increasing the temperature LM pectin gels melt and a signif-icant decrease in dynamic moduli can be observed, eventhough specific effects depend on the adopted pectin. It isworth reminding that investigated emulsions are mainlystabilised by the structured external network and, therefore,when it melts the rupture of the emulsion occurs as evidencedby a rheological behaviour, with temperature, similar to thatalready observed in the literature for the rupture of egg yolkstabilised O/W emulsions induced by heating on rheometerplate (Moros et al. 2003).

Sample EB1 resulted to be more stable with respect to theother tested emulsions, being both the dynamic moduli (andthe loss tangent as a consequence) constant for a wider rangeof temperature. Therefore, pectin B seems to be the mostsuitable to produce stable structured emulsion gels, being ableto yield the highest values of dynamic moduli (at the sameconcentration) and the most stable sample, when compared toother pectins.

As a consequence, all other samples investigated in thepresent work were produced with the pectin B and increasingbiopolymer content in order to modify the rheological charac-teristics according to those of the benchmark sample. As spec-ified in Table 3, samples EB2–EB4 were produced keepingconstant the amount of oil and the type of pectin, varying itsfraction up to 0.03, while samples EB5–EB9 were made vary-ing the content of oil and leaving constant all the others vari-ables. The effect of increasing pectin content on the rheologicalcharacteristics of the emulsions was to increase the complexmodulus value in a non-linear manner while, obviously, theincrease of the oil phase content lowered the G* value.

According to the literature (Dickinson 2012), the emulsiongel modulus depends on the relation between the stiffness ofthe gel matrix and the stiffness of the filler particles and on oilfraction. Even though correlation models, based on compositegels behaviour, were proposed in the literature mainly forstorage modulus (Dickinson 2012), they are based on someassumptions such as homogenous droplet distribution, non-interacting particles, low filler volume fraction, etc. In thepresent work, the model proposed by van Vliet (1988) waspreliminary used to describe the emulsion-gel rheologicalbehaviour (comparison not shown) and an overestimate ofexperimental values was found. This could be due to thenon-homogenous distribution of particles, moreover the sam-ples investigated in the present work are moderately concen-trated and this could be a further deviation from the theoreticalmodel. As a consequence, owing to the difficulties in describ-ing the rheological behaviour of these complex systems byusing theoretical models and aiming at describing the ob-served behaviour, an empirical fitting model was investigated,trying to relate emulsion complex modulus to oil fraction andgel phase properties.

A relative complex modulus (Gr*) was computed as the

ratio of the value of the emulsion gels (Ge*) to the value of

the pectin gel with the same concentration adopted in emul-sion preparation (i.e. the dispersing phase) (Gg

*):

G�r 1Hzð Þ ¼ G�

e 1Hzð ÞG�

g 1Hzð Þ ð5Þ

The oil volume fraction, ϕ, was estimated from the weightfraction of each component and an empirical model, having adependence on ϕ similar to literature equations (Pal 2002; VanVliet 1988), was proposed (Eq. 6):

G�r 1Hzð Þ ¼ 1þ a⋅ϕ

1−b⋅ϕð6Þ

where parameters a and b should depend on the ratio ofcomplex modulus of filler (i.e. oil phase) to the matrix (i.e.the pectin gel). The experimental data obtained for samplesEB5-EB8 were studied to evaluate the relationship betweenemulsion complex modulus evaluated at 1 Hz and the oilfraction. Moreover the non-emulsified gel having the samepectin content (i.e. 0.02 and named GB_2) was used as a limitsample of emulsion with an oil phase fraction equal to zero.

Fig. 4 ComplexmodulusG*, evaluated at 1 Hz in frequency sweep tests,versus oil volume fraction (a) and pectin mass fraction (b). Equation (7)model (fitting lines) and experimental values (symbols). GB_2 is the non-emulsified sample (i.e., the simple gel) containing a pectin fraction 0.02

Rheol Acta (2015) 54:41–52 49

Data obtained for samples EB1-EB4 were, then, used toinvestigate the dependence between emulsion rheology andproperties of the gelled aqueous phase. It was observed that aconstant value (equal to unity) can be assumed for parametera, whereas for parameter b the dependence on the ratio ofcomplex modulus of filler to the matrix should be considered;anyway, in the present case, since the composition (and there-fore the rheology) of oil phase was not changed, a simplerdependence only on matrix rheology was proposed and thefollowing final empirical model was obtained:

G�r 1Hzð Þ ¼ 1þ ϕ

1− b1 þ b2⋅ln G�g 1Hzð Þ

� �h i⋅ϕ

ð7Þ

where b1= 62±5 (−) and b2=−10.2±0.5 (−); the gel moduluscan be computed, according to Eq. 4 as

G�g 1Hzð Þ ¼ eC1xC2p ð8Þ

where C1 and C2 for pectin B are given in Table 3. It is worthreminding that Eqs. 7 and 8, being empirical fitting models,are only valid within the investigated range of experimentalconditions, i.e. 0≤ϕ≤0.5 and 0.015≤xP≤0.03.

Analogously to the models proposed by the review ofDickinson (2012), the dependence of the rheological parame-ters of oil-in-water emulsions-filled gels with oil phase vol-ume fraction and the mechanical characteristics of the gelledmatrix is extremely non-linear. Figure 4a, b shows the exper-imental data and the model proposed in Eq. (7) in terms ofG*versus xp and ϕ. It can be seen that, in agreement with thebehaviour of emulsified oil non-interacting with gel matrix(Dickinson 2012), complex modulus of emulsion gels de-creases with increasing oil fraction. Therefore, Eq. (7) canbe considered as an interesting empirical model suitable topredict the rheological characteristics of the considered sys-tems, within the investigated ranges, without carrying outother experimental investigations (that, in turn, would benecessary only if the raw materials would be changed). Thus,an additional sample was prepared and studied in order tofurther verify the model validity: sample EB9 was preparedlowering the oil fraction down to 0.05 with respect to the othersamples produced at a constant value of pectin fraction, andthe resulting G* value plotted in Fig. 4a reveals a goodpredictability of the model described by Eq. (7) (differencebetween experimental value and model prediction is lowerthan 13 %). Therefore, this empirical model could be used todesign emulsion gel formulations having the desired rheolog-ical properties (in terms of G*).

Finally, it is worth noticing that sample EB3, prepared byusing a pectin B fraction of about 0.026 and oil volumefraction 0.23 (corresponding to oil mass fraction 0.24) ischaracterised by rheological values very close to those of the

proposed benchmark sample (difference in complex modulusapproximately 1 %) and could be a valid replacement for it.

It was also tested the ability of the proposed model todescribe the rheological behaviour of emulsion gels basedon different pectins, i.e. samples EA1, EC1, and ED1, byusing the values of the constants C1 and C2 previously deter-mined for pectins A, C and D (see Table 3); a good agreementbetween model predictions and experimental complex modu-lus values was observed for samples EC1 and ED1 (differ-ences lower than 16 %) whereas a larger error (approximately37 %) was found for sample EA1; this suggests that furtherinvestigations on interactions between pectin gel and oil phasecould make more general the proposed empirical model.

Conclusions

This paper investigated pectin-based emulsion gels, or filledgels, simulating model systems for potential cosmetic appli-cations. These systems can be categorised as structured oil-in-water emulsions based on LM pectin hydrogel dispersingolive oil droplet. The rheological optimisation of emulsiongels was carried out by preliminarily investigating the charac-teristics of non-emulsified hydrogels. TheG*modulus of gelswas found to vary with pectin content with a power-lawbehaviour and two different regions were observed, accordingto the biopolymer concentration range. Among four differentLM pectins, the one producing the most consistent gel waschosen to produce structured emulsions. G* at 1 Hz for eachemulsion sample was evaluated varying the pectin fraction inthe gel phase and the oil content in the emulsion. Finally, anempirical predictive model, describing G* versus pectin andoil fraction, was obtained and it has shown itself suitable todescribe rheological behaviour of considered emulsion gelswithin the investigated range of concentrations.

Acknowledgments The authors are grateful for its support to PONR&C(Programma Operativo Nazionale Ricerca e Competitività 2007–2013)project PON01_00293 “Spread Bio Oil”.

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