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Food Hydrocolloids 34 (2014) 208e216

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Food Hydrocolloids

journal homepage: www.elsevier .com/locate/ foodhyd

Effect of b-casein on surface activity of Quillaja bark saponin atfluid/fluid interfaces

Kamil Wojciechowski*, Aleksandra Kezwon, Joanna Lewandowska, Kuba MarcinkowskiDepartment of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland

a r t i c l e i n f o

Article history:Received 29 May 2012Accepted 19 September 2012

Keywords:SaponinsQBSSurface tensionb-Casein

* Corresponding author.E-mail address: kamil.wojciechowski@ch.pw.edu.p

0268-005X/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodhyd.2012.09.010

a b s t r a c t

Effect of a model bovine milk protein, b-casein, on surface activity of Quillaja bark saponin (QBS) fromSigma was studied at three fluid/fluid interfaces: air/water, tetradecane/water and olive oil/water. In allcases, the protein concentration was fixed at 10�6 mol L�1, and QBS concentration was varied between5$10�7 and 1$10�3 mol L�1. Dynamic interfacial tension on the timescale 5 se3600 s was measured usinga drop shape analysis technique. For the air/water system, they were complemented with short-term(50 mse5 s) measurements using a maximum bubble pressure technique. The dynamic resultstogether with the extrapolated equilibrium surface pressures are discussed from the point of view ofa complexation between b-casein and QBS, with the surface activity of the complex changing with itsstoichiometry. At low biosurfactant/protein ratios, the interfacial tension at all three interfaces passesthrough a maximum, corresponding to a transient decrease of both foam and emulsion formation ability.In addition, the effect of QBS on deterioration of b-casein’s surface activity upon ageing at roomtemperature is discussed.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Saponins, belonging to a large group of biosurfactants, are beingmore andmore often used in food and cosmetic industry because oftheir foaming and emulsifying properties (the name “saponin”derives from the Latin sapo, that means “soap”). Despite beinghighly toxic to the heat-labile animals, and showing strong fungi-cidal and insecticidal properties, saponins display low oral toxicityto mammals (Dini, Schettino, Simioli, & Dini, 2001). Saponins occurin about 500 varieties of plants and in some animal species. Inplants, they constitute an important part of the defence system andcan be found in parts such as root, tuber, bark, leaves, seeds andfruits (Wina, Muetzel, & Becker, 2005), i.e. the tissues mostsusceptible to attack by fungi, bacteria and insecticides.

A saponin molecule is composed of two parts connected bya glycosidic bond: sugar (glycone) and non-sugar (aglycone). Thesugar part consists of saccharides such as glucose, galactose,xylose, rhamnose, arabinose and glucuronic acid, while the non-sugar part is either triterpenoid or steroid (Güçlü-Üstünda�g &Mazza, 2007). Among several possible sources of saponins forindustrial applications, a Quillaja saponaria Molina tree, cultivated

l (K. Wojciechowski).

All rights reserved.

in warm climates, mainly in South America, seems to be the mostpromising and most widely exploited. Its bark contains at least 60different saponins identified using mass spectrometry (Nord &Kenne, 1999). The saponins present in the extract of Q. saponariaMolina’s bark contain mostly triterpene aglycones, and carrya general name “Quillaja Bark Saponin” (QBS) (San Martín &Briones, 1999). It should be stressed, however, that the commer-cially available QBS products vary greatly depending on both thesource and the extraction procedure. Consequently, also surfaceproperties of different QBS show great variations (Piotrowski,Lewandowska, & Wojciechowski, 2012; Stanimirova et al., 2011;Wojciechowski, Piotrowski, Popielarz, & Sosnowski, 2011). Despitethis variability and lack of clear definition, QBS is an approvedingredient for use in food and beverages under the United StatesFDA regulation 21 CFR 172.510 as a flavouring agent and bearsFEMA (Flavour and Extract Manufacturers’ Association) GRAS(Generally Recognized As Safe) status (number 2973). In theEuropean Union, it is an approved ingredient (E999) to water-based non-alcoholic drinks and ciders. In Japan, the Quillajaextract is allowed for human consumption (as emulsifier andfoaming agent) and for use in cosmetics.

Caseins are primary components of milk proteins. Among them,b-casein is certainly one of the most studied and understoodrandom coil proteins, especially in terms of its surface properties(Latnikova, Lin, Loglio, Miller, & Noskov, 2008). A characteristic

K. Wojciechowski et al. / Food Hydrocolloids 34 (2014) 208e216 209

feature of b-casein (b-cas) is the lack of tertiary structure and veryweak secondary structure in dilute solutions. However, the pres-ence of clearly distinct hydrophilic and hydrophobic domains,together with the accumulation of most of the negative chargeclose to the hydrophilic N-terminus, make b-cas a highly surfaceactive biopolymer, with adsorption properties similar to those ofblock polymers (Dickinson, 2006; Wilde, Mackie, Husband,Gunning, & Morris, 2004). Upon adsorption, an initially flat mole-cule starts to expel its hydrophilic parts, forming loops and trainsprotruding into the aqueous subphase (Cicuta & Hopkinson, 2001;Maldonado-Valderrama et al., 2005; Noskov, Latnikova, Lin, Loglio,& Miller, 2007). Thanks to its excellent surfactant properties andavailability in large quantities, b-cas is used not only in food andpharmaceutical industries, but also for production of plastics,paints, adhesives, paper, putty, lacquers, etc.

Mixtures of proteins and surfactants are widely used in almostevery real-life formulation, they can also be found in variousnatural biological systems, e.g. in blood serum, which consists ofa mixture of albumin and several kinds of low molecular weightsurfactants. Interactions between a protein and a low molecularweight surfactant component may result in both synergistic andantagonistic effects, and are of great concern for many industries.For this reason, several studies have been dedicated to studyingthem using mixtures of model protein and simple surfactantcomponents (Fainerman, Zholob, Leser, Michel, & Miller, 2004;Kotsmar et al., 2009; Miller, Fainerman, Makievski, Kragel, &Wustneck, 2000; Wilde et al., 2004). Typically, interfacial tensionand related techniques (e.g. surface rheology) are used as a conve-nient analytical tool for this purpose. Despite the numerous actualand even more potential applications of QBS in food, pharmaceu-tical and cosmetic industries, still not much is known about itsinterfacial interactions with proteins (Morton & Murray, 2001;Potter, Jimenez-Flores, Pollack, Lone, & Berber-Jimenez, 1993;Sarnthein-Graf & La Mesa, 2004; Shimoyamada, Ootsubo, Naruse, &Watanabe, 2000). With this paper we wish to contribute to fillingthis gap by studying interfacial properties of mixtures of a modelnon-globular protein (bovine milk b-casein, b-cas) and a bio-surfactant (Quillaja bark saponin from Sigma, QBS). The dynamicinterfacial tension results are complemented with foam andemulsion formation ability tests performed with a modifiedBikerman’s method, dynamic light scattering and confocalmicroscopy.

2. Experimental

The medium-term interfacial tension measurements were per-formed using a drop profile analysis tensiometer PAT-1 (SinterfaceTechnologies, Germany) as described in detail in Loglio et al. (2001),at constant temperature (21 �C) controlled with a thermostaticbath. A small (5e30 ml) drop of QBS/b-cas solution in phosphatebuffer (pH 7, I ¼ 0.02 mol L�1) was formed at the tip of a steelcapillary immersed in a glass cuvette (10 ml) filled with air, tetra-decane or olive oil. All experiments were performed during at least2700 s and repeated at least three times (typically during 3600 s,repeated six times). The equilibrium interfacial tension (geq) wascalculated as an average of the values extrapolated using t�1/2 / 0and t�1 / 0 asymptotic solutions of WardeTordai equation(Fainerman, Makievski, & Miller, 1994).

The short-term surface tension measurements were performedwith the aqueous solutions prepared as described above usinga maximum bubble pressure tensiometer BP-1 (Kruess, Germany)with a Teflon capillary. The latter was used to avoid wetting by thephosphate buffer, which was found to irreversibly adsorb on glasscapillaries and preventing any measurements (Wojciechowskiet al., 2011).

The foamability was assessed by a modified Bikerman’s methodusing a home-made device (Piotrowski et al., 2012). Nitrogen gaswas introduced at constant flow (50 dm3 h�1) into a 0.4 m columnfilled with the test QBS or QBS/b-cas solutions through a poroussintered glass. After 15 s of bubbling, the photo of the foam wastaken.

Olive oil-in-water emulsions (0.2% v/v) were prepared by soni-cation of the pre-mixed (600 rpm, 10 min) olive oil dispersions inphosphate buffered QBS or QBS/b-cas solutions (pH 7). Sonicationwas performed for 15 min with the power of 34 W at 20 kHz usingSonopuls HD 2070 (BANDELIN, Germany). For the fluorescencemicroscopy studies, a liposoluble dye (Nile Blue) was added to theemulsion after sonication and mixed mechanically (600 rpm) for1 min. Particle size of emulsions was measured with Zetasizer3000HS (MALVERN, UK) at 25 �C freshly after preparation and after1 h storage at room temperature. The results are presented as theSauter mean diameter, d32, defined as follows:

d32 ¼P

Ni$d3iPNi$d2i

(1)

where: Ni is a percentage of the total scattering intensity for a givenscattering droplet diameter (di, nm).

Fluorescence confocal microscope Olympus Fluoview FV10i wasemployed to visualise the emulsions after placing a drop of a freshlyprepared sample between two glass slides. Two predefined detec-tion channels were used: lex ¼ 506 nm, lem ¼ 527 nm (fluo-3) andlex ¼ 548 nm, lem ¼ 559 nm (mKusabira-Orange).

All the glassware was cleaned with acetone and Hellmanex IIsolution (Hellma Worldwide) and rinsed with copious amounts ofMilli-Q water. Tetradecane (Aldrich 172456, �99%) and olive oil(Sigma O1514, highly refined, low acidity) were purchased fromSigmaeAldrich, andwerepurified by shakingwith Florisil� followedby centrifugation at 6000 rpm during 30 min and filtering throughWhatman’s filter paper (No. 1). For olive oil a single shaking/centrifugation/filtering cycle using Florisil 30e60 mesh (SigmaeAldrich, 288691) was sufficient, while for tetradecane e fourcycles were necessary: two with Florisil 30e60 mesh, and two with60e100 mesh (Fluka, 46385, for chromatography). The criterion forthe surface purity of the oil phase was a constancy of the corre-sponding interfacial tension during at least 3600 s (the timescale ofthe typical experiment) against pure water (whose surface purityhad been confirmed prior to contacting with the oil phase) andagainst the phosphate buffer used as the aqueous phase. FreshMilli-Q (Millipore)water (18.2$106U cm)was used for themeasurements.NaH2PO4$2H2O (71505), Na2HPO4$2H2O (71645), Nile blue(222550), Quillaja bark saponin, QBS (84510, Mw ¼ 1650 g mol�1)and b-casein from bovinemilk, b-cas (C6905,Mw ¼ 23,800 gmol�1)were purchased from SigmaeAldrich, and used as received.

3. Results

3.1. Dynamic interfacial tension

Dynamic interfacial tension decays (g(t)) for QBS/b-cas mixturesat three fluidefluid interfaces were measured using the Axisym-metric Drop Shape Analysis (ADSA)method on amedium timescale5e3600 s. In all cases, the protein concentration was fixed at10�6mol L�1, which is sufficiently high to quickly attain equilibriumsurface pressures, but still below the b-casein critical micelleconcentration (Portnaya et al., 2006). Since b-cas has been shown toadsorb with near-bulk values of an effective diffusion coefficients(Fainerman & Miller, 2005; Mellema, Clark, Husband, & Mackie,1998; Miller, Fainerman, Aksenenko, Leser, & Michel, 2004), thedynamic curves obtained under these conditions will subsequently

K. Wojciechowski et al. / Food Hydrocolloids 34 (2014) 208e216210

be extrapolated using an asymptotic solutions of a diffusion-basedWardeTordai equation. The dynamic decays are shown in Fig.1 (leftpanel), together with the corresponding results for pure QBS (rightpanel). For comparison, also the dynamic interfacial tension decaysfor pure b-cas solutions are shown as solid lines in panels on the leftside of Fig. 1. The close-to-equilibrium values of dynamic interfacialtension are reached at all interfaces within first 15 min of adsorp-tion. The surface tension decays, g(t), are the slowest at the air/water interface, while the tetradecane/water interface seems topromote the fastest decays of g(t), closely followed by the olive oil/water one. It is worth noting that the same ordering applies to thekinetics of interfacial tension decays for pure b-cas solutions, and isprobably related to solvation of the protein molecules by the non-aqueous phase. For all three interfaces, addition of low amounts ofQBS at bulk concentrations comparable to that of b-cas, does notalter significantly the rate of interfacial tension decays. AbovecQBS ¼ 5$10�7 mol L�1, however, the decays start to differentiatedepending on the nature of the non-aqueous phase. At the air/water interface they slightly deepen until the QBS concentrationreaches 1$10�5 mol L�1, where a significant slowing down of thesurface tension decay is observed. Above cQBS ¼ 1$10�5 mol L�1, therate of QBS/b-cas adsorption increases again, although still remainsslower than in the corresponding systemwithout the protein. Uponfurther increase of the biosurfactant concentration, above the QBS/b-cas ratios exceeding 102 (i.e., above cQBS ¼ 1$10�4 mol L�1), theadsorption kinetics of QBS recovers and the dynamic curves start to

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/mN

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/mN

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γγ

γ

Fig. 1. Dynamic interfacial tensions for QBS/b-cas mixtures (left panel), compared with the(middle), and olive oil/water (down). QBS concentrations in the aqueous phase: 5$10�7 mol(=), 1$10�4 mol L�1 (<), 4$10�4 mol L�1 ( ), 1$10�3 mol L�1 ( ). In all mixed solutions b-cpure b-cas (1$10�6 mol L�1).

resemble those for the pure biosurfactant solutions. Similardiscussion holds for the two other interfaces, although in differentranges of concentrations. At the tetradecane/water interface addi-tion of as little as 1.5$10�6 mol L�1 QBS slows down the rate ofadsorption for the mixture at early stages of adsorption. However,by the end of the dynamic measurement the correspondingdynamic curve merges with those for other concentrations below1$10�5 mol L�1. Above cQBS ¼ 4$10�5 mol L�1 the rate of interfacialtension decays seems to follow that for solutions without b-cas,although the surface pressures attained are systematically smaller(more shallow decays are observed). For the olive oil/water case,despite an initial deepening of the decay at 1.5$10�6 mol L�1 of QBS,between 1$10�5 mol L�1 and 4$10�5 mol L�1, the curves resemblethose for bare b-cas. Only above the QBS/b-cas ratios exceeding 102

the decays start to follow those for bare QBS.The drop shape analysis based methods allow for dynamic

measurements only on timescales longer than a few seconds.However, despite being a relatively large molecule (Mw ¼ 24 kDa),b-cas adsorbs at fluidefluid interfaces quickly, following a diffu-sion-controlled model with the effective diffusion coefficient, Deff,in the range 5$10�11e2$10�10 m2 s�1 (Mellema et al., 1998; Milleret al., 2004). The short-term dynamic surface tension data ob-tained with a maximum bubble pressure (MBP) technique areshown in Fig. 2, combined with the medium-term data obtainedfrom the ADSA method to show the mutual consistency of the twomethods.

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corresponding pure QBS solutions (right panel) at: air/water (top), tetradecane/waterL�1 (-), 1.5$10�6 mol L�1 (C), 4$10�6 mol L�1 (:), 1$10�5 mol L�1 (;), 4$10�5 mol L�1

as concentration was fixed at 10�6 mol L�1. The solid black lines represent the data for

0,1 1 10 100 100035

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75 /m

N.m

-1

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γ

Fig. 2. Combined short- and medium-term dynamic interfacial tensions for QBS/b-casmixtures at air/water interface. b-cas concentration was fixed at 1$10�6 mol L�1, QBSconcentrations: 5$10�7 mol L�1 (-), 1.5$10�6 mol L�1 (C), 4$10�6 mol L�1 (:),1$10�5 mol L�1 (;), 4$10�5 mol L�1 (=), 1$10�4 mol L�1 (<), 4$10�4 mol L�1 ( ),1$10�3 mol L�1 ( ). Data for pure b-cas (1$10�6 mol L�1) is added for comparison ( ).

K. Wojciechowski et al. / Food Hydrocolloids 34 (2014) 208e216 211

3.2. Aging of b-casein in the presence of QBS

Miller et al. (2004) have observed that the surface activity of b-casein aqueous solutions deteriorates with time upon storage atroom temperature. For the same concentration as employed in thepresent study (1$10�6 mol L�1), the noticeable changes have startedabout 10 h after the solution had been prepared. In order to assessthe effect of QBS on b-cas ageing, the dynamic surface tensionmeasurements for each QBS/b-cas mixturewere repeated after 24 hstorage at room temperature. The data are collected in Fig. 3, pre-sented as transient differences between the surface tension decaysmeasured after 24 h and those for fresh solutions (Ds(t)). They canbe interpreted as differential dynamic surface tensions. In pureb-cas solutions, in full agreement with Miller et al. (2004), thesurface tension decrease after 24 h storage is slower and the valuesattained at the end of the 1-h experiment are slightly smaller than

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mN

.m-1

t /s

Δσ ⁄

Fig. 3. Transient differences between the surface tension decays for QBS/b-casmixtures measured after storage for 24 h at room temperature and as-prepared. b-casconcentration was fixed at 1$10�6 mol L�1, QBS concentrations: 5$10�7 mol L�1 (-),1.5$10�6 mol L�1 (C), 4$10�6 mol L�1 (:), 1$10�5 mol L�1 (;), 4$10�5 mol L�1 (=),1$10�4 mol L�1 (<), 4$10�4 mol L�1 ( ), 1$10�3 mol L�1 ( ). Data for pure b-cas(1$10�6 mol L�1) is added for comparison ( ).

for fresh the solution. Addition of as low as 5$10�7 mol L�1 of QBS(corresponding to QBS/b-cas molar ratio of 0.5) practicallycompletely cancels the protein’s surface activity within 24 h.Increasing this molar ratio up to 10 does not significantly changethe situation. Only when it is further increased above 102

(cQBS ¼ 1$10�4 mol L�1), the trend reverses, and the aged QBS/b-casmixtures start to acquire additional surface activity upon storage atroom temperature.

3.3. Interfacial tension isotherms

The decays of dynamic interfacial tension for QBS/b-casmixtures are much faster than e.g. for globular protein/QBSmixtures (Piotrowski et al., 2012; Wojciechowski et al., 2011). Thisprompted us to attempt construction of interfacial tensionisotherms by extrapolating our s(t) to t/N. Even though for mostproteins 1 h is considered too short, the high values of the effectivediffusion coefficient for b-cas and the shape of the dynamic curvesin our opinion justify extrapolation from this medium-term data.The equilibrium interfacial tension values were obtained from botht�1/2 / 0 and t�1 / 0 asymptotic solutions of WardeTordaiequation, as it is typically done for the surfactant and proteinsolutions, respectively (Fainerman et al., 1994; Maldonado-Valderrama, Martin-Molina, Galvez-Ruiz, Martin-Rodriguez, &Cabrerizo-Vilchez, 2004). The differences between the twoapproaches never exceeded 0.6 mN m�1, and the average valueswere used for construction of the interfacial tensions isotherms forthe air/water, tetradecane/water and olive oil/water interfaces(Fig. 4). For comparison, the respective isotherms for the corre-sponding pure QBS solutions at the same interfaces are also pre-sented. At all interfaces, the same type of behaviour can be noticed:a decrease of equilibrium interfacial tension upon addition of smallamounts of QBS, followed by a maximum, after which the isothermfollows more or less closely that for pure QBS.

3.4. Foams stabilised with QBS/b-cas

The foams stabilised with QBS and QBS/b-cas mixtures werecompared using the modified Bikerman’s method, as described inthe experimental part. The photographs of selected foams arecollected in Fig. 5. In general, the foams do not form in pure QBS atconcentrations below 1$10�5 mol L�1, while those stabilised solelyby b-cas at 1$10�6 mol L�1 are rather loose and of poor quality.Addition of small amounts of QBS (cQBS < 1$10�5 mol L�1) to theb-cas solution only slightly improves the foam quality. Interest-ingly, in mixed solutions, around the QBS concentration of1.5$10�6 mol L�1 a slight decrease of foam quality can be observed,in line with the deterioration of the surface activity noted indynamic surface tension measurements (Fig. 1). For the QBSconcentration of 4$10�5 mol L�1 the QBS-only foams become denseand wet, but the presence of low amounts of b-cas (1$10�6 mol L�1,i.e., at QBS/b-cas ratios exceeding 40), surprisingly, can still improvethem. At even higher QBS concentrations, cQBS¼ 1$10�3 mol L�1, thepresence of b-cas has no significant effect on foamability.

3.5. Emulsions stabilised with QBS/b-cas

The emulsion formation ability of themixed QBS/b-cas solutionswas fist assessed with dynamic light scattering (DLS). For thispurpose, diluted optically transparent emulsions (0.2 wt%) olive oil-in-water emulsions were produced by sonication. The results forfresh emulsions and after 1 h storage at room temperature shownin Fig. 6 point to a rather weak effect of the presence of b-cas onemulsion formation ability (t ¼ 0). Surprisingly, however, after 1 hstorage the emulsions stabilised with the mixtures seem to be

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γ

Fig. 4. Interfacial tension isotherms for: (,) mixtures of b-cas with QBS (cb-cas was fixed at 1$10�6 mol L�1) and for pure QBS (B) at three interfaces (from left to right): air/water,tetradecane/water and olive oil/water. The dotted lines represent the equilibrium interfacial tension for pure b-cas at 1$10�6 mol L�1.

K. Wojciechowski et al. / Food Hydrocolloids 34 (2014) 208e216212

significantly more prone to droplet growth than those stabilisedsolely with QBS, hence will probably be less stable. Since DLScannot provide a detailed information on the real drop size distri-bution, any subtle effects, e.g., those related to the transient dete-rioration of interfacial activity around cQBS ¼ 1$10�5 mol L�1 are notdetected.

The QBS and QBS/b-casein stabilised olive oil-in-water emul-sions were also analysed using a fluorescence confocal microscope.The QBS concentrations were selected to represent the character-istic points on the interfacial tension isotherms, in the range ofsmall (1.5$10�6, 4$10�6 mol L�1), intermediate (1$10�5 mol L�1) andhigh (1$10�3 mol L�1) concentrations. For emulsions stabilised withthe QBS/b-cas mixtures, the same set of QBS concentrations wasused, while b-cas concentration was fixed at 1$10�6 mol L�1, as forall other experiments in this study. The non-aqueous phase wasstained with a lipophilic Nile Blue dye in order to highlight theemulsion droplets. For each sample, several images at differentlocations were recorded, and the most representative ones wereused as the basis for semi-quantitative evaluation of the dropletsizes, monodispersity and abundance. In agreement with theinterfacial tension results for the olive oil/water interface, anincrease of QBS concentration in most samples resulted inimproved emulsion quality (increased number of more mono-disperse drops, cf Fig. 7A and B). The only exception, depicted inFig. 7C and D, was observed for the case of the mixture withcQBS ¼ 1$10�5 mol L�1, where both the number and monodispersityof the droplets slightly decreased as compared with emulsionsstabilised with pure QBS at the same concentration. It is worthnoting that the maxima in the interfacial tension isotherms (Fig. 4)were observed in the same range of QBS concentrations. Furtherincrease of cQBS in themixture improves the quality of emulsions, inline with the improvement observed in the corresponding pureQBS-stabilised emulsions.

4. Discussion

The surface activity of b-cas gets clearly enhanced in thepresence of small amounts of QBS, as shown by both increase ofthe rate of the medium-term interfacial tension decays (Fig. 1),and decrease of the equilibrium values (Fig. 4). For all threeinterfaces, this effect is most pronounced around a QBS/b-cas ratioof 1:1, which points to the formation of a complex with thesurface activity higher than that of the individual components,possibly of 1:1 stoichiometry. Even though the hydrophilicN-terminus of the b-cas molecule is predominantly negativelycharged, the first two segments are actually positively charged(see Fig. 3 in Dickinson (2006)). We can then speculate thatbinding of one or two negatively charged QBS molecules to thepositively charged segments of the hydrophilic N-terminus mightfurther increase its hydrophilic character, thus enhancing theprotein’s block-copolymer character. The fact that in the mixedsolutions containing low QBS concentrations the dynamic inter-facial tensions stabilise more quickly than in the correspondingpure QBS solutions suggests that the adsorption barrier of QBS isreduced in the presence of b-cas (cf left and right panels of Fig. 1).It could be then speculated that complexation of QBS by b-castakes place already in solution, and that the complexes thusformed adsorb in a way similar to the pure protein. Taking intoaccount the molar masses of the biosurfactant (1.6 kDa) and theprotein (23.5 kDa), as well as the random coil conformation ofb-cas in solution, complexation of a few QBS molecules should notchange much of its bulk diffusion behaviour. The adsorptionkinetics of QBS/b-cas complex would then still be diffusion-controlled, and similar to that of pure b-cas. As a result of thiscomplexation, an effective adsorption kinetics of QBS wouldincrease, since the faster-diffusing b-cas molecule would enable itto circumvent the adsorption barrier.

Fig. 5. Photographs of foams stabilised with QBS (upper row: cQBS ¼ 5$10�7, 1.5$10�6,1$10�5, 4$10�5, 1$10�3 mol L�1) and QBS/b-cas mixtures (lower row: cQBS ¼ 0, 5$10�7,1.5$10�6, 1$10�5, 4$10�5, 1$10�3 mol L�1) for b-cas concentration fixed at1$10�6 mol L�1.

05x10-71,5x10-64x10-61x10-54x10-51x10-44x10-41x10-3

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QBS, t=0QBS+b_cas, t=0

QBS, t=1 hQBS+b_cas, t=1 h

d 32 /n

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CQBS /M

Fig. 6. Average size (Sauter mean diameter, d32) of olive oil-in-water emulsion dropletsstabilized by QBS alone and a mixture of b-cas (cb-cas ¼ 1$10�6 mol L�1) with QBS, asa function of QBS concentration for freshly prepared emulsion (t ¼ 0), and after 1 hstorage at room temperature (t ¼ 1 h).

K. Wojciechowski et al. / Food Hydrocolloids 34 (2014) 208e216 213

Further evidence in favour of the biosurfactanteproteincomplexation taking place in solution comes from the existenceof local maxima in interfacial tension isotherms observed atintermediate QBS concentrations at all three interfaces. Similarmaxima have been observed in mixtures of polymers withanionic low molecular-weight surfactant, sodium alkyl sulfates(Mukherjee, Dan, Bhattacharya, Panda, & Moulik, 2011; Penfoldet al., 2007), and their appearance is usually explained by a tran-sient formation of a complex with reduced surface activity. Danet al. (2012) when studying the effect of b-cas on adsorption ofSDS, have observed that the local maxima in the interfacial tensionisotherms appear when the surfactant and the protein are intro-duced simultaneously, but not e when sequentially. In the formercase, the SDS/b-cas complex would be formed in the bulk, whereasin the latter e at the interface (i.e., the surfactant would interactwith a pre-adsorbed protein).

The maxima in the present system appear at QBS/b-cas ratiosexceeding one, when the stoichiometry of the complex changes,leading to a decrease of its surface activity. This might suggest thatthese additional QBS molecules probably attach to other availablepositive segments of b-cas, which are this time located in

hydrophobic parts of the molecule (see Fig. 3 in Dickinson (2006)).This seems likely, since most of the positively charged segments(13 out of 21) are located in hydrophobic segment of b-casein, aspointed out by Dickinson (2006). Despite the fact that the QBSmolecules do not possess clearly defined hydrophobic and hydro-philic regions, one can envisage that pairing of these positivesegments with QBS should increase the overall hydrophilicity ofthese segments. As a result, at the QBS/b-cas molar ratios slightlyexceeding one, the block copolymer-like character becomes lesspronounced, leading to a decrease of the surface activity of thecomplex. This is also reflected in the low value of the effectivediffusion coefficient obtained from the short-term surface tensiondata.

At the air/water and tetradecane/water interfaces, QBS behavessimilarly to its low molecular-weight ionic counterparts (e.g., SDS),which do not simply displace b-cas from the interface, but modifyits hydrophilic/hydrophobic character by attaching to it throughelectrostatic and hydrophobic interactions (Kotsmar et al., 2009).This hypothesis is supported by the fact that even at very high QBS/b-cas ratios ([10) for the tetradecane/water and air/water inter-faces both dynamic and equilibrium interfacial tensions still differfrom those obtained for pure QBS solutions. If b-cas were simplydisplaced by QBS, like it is observed for non-ionic low molecularweight surfactants, e.g. Tween or triblock polymeric surfactants(Latnikova et al., 2008; Maldonado-Valderrama et al., 2007;Woodward, Gunning, Mackie, Wilde, &Morris, 2009), the datawithand without b-cas should merge at high QBS concentrations. Thefoamability tests fully confirm the presence of this interaction, evenat cQBS ¼ 4$10�5 mol L�1.

The interaction between the QBS and b-cas molecules in theinterfacial region depends significantly on the type of the interface.For the air/water and tetradecane/water, besides the differences inthe isotherms described above, the critical micelle concentrations(cmc) shift to higher concentrations, suggesting that the complexeshave lower tendency to self-aggregate than the QBS alone. Inthe case of tetradecane/water interface, the slope of the isotherm

Fig. 7. Representative images of olive oil-in-water emulsions stabilised with: (A) pure QBS at 4$10�6 mol L�1, (B) the corresponding QBS/b-cas mixture with cQBS ¼ 4$10�6 mol L�1

and cb-cas ¼ 1$10�6 mol L�1, (C) pure QBS at 1$10�5 mol L�1, (D) the corresponding QBS/b-cas mixture with cQBS ¼ 1$10�5 mol L�1 and cb-cas ¼ 1$10�6 mol L�1.

K. Wojciechowski et al. / Food Hydrocolloids 34 (2014) 208e216214

(ds/dlogc) is also smaller for the mixture than for pure QBS, whichaccording to the Gibbs equation indicates a higher area occupied byeach QBS molecule in the mixture, as compared to the adsorbedlayers of pure QBS. This is a consequence of the incorporation of theprotein into the QBS layer, as a result of which some space at theinterface is effectively taken up by b-cas. The fact that this effect ismost pronounced at the tetradecane/water interface might belinked to the highest compatibility between the non-polar hydro-carbon chains of tetradecane and the hydrophobic segments ofb-cas and QBS.

As pointed by Maldonado-Valderrama, the highest surfacepressures and possibly the highest degrees of unfolding are reachedat non-polar organic solvent/water interfaces with high initialinterfacial tensions (i.e. those for bare interfaces) (Maldonado-Valderrama et al., 2004). Although the polarity of the air phase iseven smaller than that of tetradecane, at the air/water interface thehydrophobic segments of b-cas cannot be solvated, and the extentof unfolding is smaller. On the other hand, the olive oil, despitehaving the ability to solvate the hydrophobic parts of b-cas, is muchmore polar, mostly due to the presence of triglycerides and fattyacids. Since the main driving force for the proteins to adsorb atfluid/water interfaces is the possibility to expose their hydrophobicparts to the fluid phase, the increased polarity of the non-aqueousphase (e.g. olive oil) renders this interface less attractive for anadsorbing hydrophobic molecule. Consequently, b-cas does notstrongly attach to the olive oil/water interface, and gets easilydesorbed by QBS, as evidenced by the overlap of the interfacial

tension isotherms obtained for the pure QBS solutions and for itsmixtures with b-cas above cQBS ¼ 1$10�5 mol L�1 at the olive oil/water interface. This is also reflected in a rather subtle effect of thepresence of the protein on the olive oil-in-water emulsions. In theDLS results, which are averaged over the size distributions, prac-tically no effect could be seen, while the confocal images showsome tiny differences between the samples with and without theprotein in the range of concentrations where the maximum in theinterfacial tension isotherm is observed. At high QBS concentra-tions, in contrast to the other two interfaces, at the olive oil/water,QBS behaves similarly to non-ionic surfactants, e.g., Tweens or alkylphosphine oxides, which have been shown to remove proteins fromthe interfaces, either by competitive adsorption or a so-calledorogenic displacement mechanism (Kotsmar et al., 2009;Woodward et al., 2009). In the present case, however, the depletionis more due to the poor affinity of b-cas to the given interface, thanto the extent of modification of the protein’s hydrophobic characterby complexation with the surfactant.

Pure b-CAS solutions in phosphate buffer are quite stable ona short timescale under ambient conditions, probably due to thelack of highly developed secondary and tertiary structure. Never-theless, Miller et al. (2004) observed changes in dynamic surfacetension curves for b-cas at 1$10�6 mol L�1 after about 10 h storageat room temperature. With time, the decays were becoming lessand less pronounced, leading to smaller equilibrium surface pres-sures. The authors found out that both filtration and addition ofsodium azide hinders this process, and attributed the changes to

K. Wojciechowski et al. / Food Hydrocolloids 34 (2014) 208e216 215

bacterial degradation of the protein. A similar effect can be noticedin our results for pure b-cas (1$10�6 mol L�1), Fig. 3. When the b-cassolutions are stored in the presence of QBS, the changes in dynamicinterfacial tension are getting even more pronounced and reacha maximum around cQBS ¼ 4$10�5 mol L�1. This would suggest thatQBS accelerates the bacterial degradation, although itself does notundergo such a degradation (no degradation effects were observedin pure QBS solutions). However, with further increase of QBSconcentration, the surface activity of the complex starts to increaseupon storage. The change in behaviour between 4$10�5 and1$10�4mol L�1 is very sharp and is rather unlikely to be related onlyto the bacterial degradation. The sudden increase of surface activityafter 24 h might be due to the release of QBS molecules initiallybound to b-cas, when the latter degrades. With the progress ofbacterial degradation, the QBS-binding capacity of b-cas mightdecrease leading to the increased concentration of unbound QBS,and consequently e enhanced decays of surface tension. Accordingto another hypothesis, the enhancement of surface activity mightbe caused by slow dissociation of QBS micelles by b-cas. Althoughnormally micelle dissociation is a relatively fast process, for thepresent system this hypothesis is supported by the fact that thesudden increase of surface activity upon storage commences atconcentrations approaching the QBS’ cmc (see Fig. 4). The thirdalternative explanation for the strange behaviour depicted in Fig. 3would be the enhancement of the protein’s surface activity due tosome slow complexation processes. Initially, and at low surfactant-to-protein ratios, the less surface active complexes would beformed. However, at high surfactant-to-protein ratios, on thetimescale of several hours (i.e. upon slowly approaching thecomplex formation equilibrium) the QBS molecules would even-tually attach to the b-cas polymer chain preferentially at locationswhere their presence would enhance the surface activity. To thebest of our knowledge no such effect has been observed for smallmolecular weight synthetic surfactants (Kotsmar et al., 2009),although given the complex structure of QBS, with its hydrophobicand hydrophilic regions not being clearly defined, this result is notvery surprising.

5. Conclusions

The dynamic and equilibrium interfacial tension studies provedthat Quillaja bark saponin (QBS) from Sigma (84510) interacts at allthree fluidefluid interfaces (air/water, tetradecane/water and oliveoil/water) with a random coil milk protein, b-casein (b-cas). At verylow QBS/b-cas ratios (<1.5) a clear synergistic effect is observed inboth the kinetics and the equilibrium interfacial tensions of themixtures. Also the foam and emulsion quality seem to increase inthis region of QBS concentrations. However, binding of additionalQBS molecules to b-cas (1.5 < QBS/b-cas < 10) apparently reducessurface activity of the proteinesurfactant complex, resulting in localmaxima in the interfacial tensions isotherms. This effect can benoticed at each studied interface, but is definitely most pronouncedfor the air/water system, where the surface tension of the complexformed at 1$10�6 mol L�1 concentration of b-cas and 1$10�5 mol L�1

QBS is higher than that of the corresponding pure b-cas solution byw3 mN m�1. The reduction of surface activity at intermediate QBS/b-cas ratios is reflected in a slightly reduced foamability of themixtures. Similarly, the solutions where themaximum in interfacialtension isotherm can be noticed, show reduced ability to formmonodisperse olive oil-in-water emulsions. At even higher QBSconcentrations, the biosurfactant does not completely remove theprotein from the interface, except for the olive oil/water system,where b-cas is apparentlyweakly adsorbed, possibly due to the highpolarity of the olive oil. At the other two interfaces, with less polartetradecane and air, QBS continues to interact with b-casein even at

the molar ratios as high as 103. The results presented in this papershow that QBS displays interesting and complex surface properties,not only when present alone, but also in mixtures with otherpotential components of real-life foam/emulsion formulations (e.g.,bovine milk b-casein). Interaction of QBS with b-cas is morecomplex than that of its low molecular weight counterparts(synthetic surfactants), what opens several possibilities to employ itas a more sustainable alternative to the latter.

Acknowledgements

This work was financially supported by the PolishNational Science Centre, grant no. DEC-2011/03/B/ST4/00780. Prof.T. Sosnowski is acknowledged for help in measurements with BP-1.

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