Modulation and Stabilization of Silk Fibroin-Coated Oil-in-Water ...

416 J.J. RAO et al.: Stabilization of Silk Fibroin-Coated O/W Emulsions, Food Technol. Biotechnol. 47 (4) 413–420 (2009)Creaming stability measurementA mass of 10 g of emulsions was transferred into aglass test tube (internal diameter 15 mm, height 125 mm),tightly sealed with a plastic cap, and then stored at roomtemperature for 7 days. After storage, some of the emulsionswere separated into a number of layers: a 'cream'layer at the top, a 'suspension' layer in the middle, and a'serum' layer at the bottom. These layers could be distinguishedby their visual appearance: the cream layer wasoptically opaque and whiter than the original emulsion,the suspension layer had the same appearance as the originalemulsion, and the serum layer was either slightlyturbid or transparent. The total height of the emulsion(H T ), the height of the serum layer (H SL ), and the heightof the cream layer (H CL ) were measured. The extent ofcreaming was characterized by creaming indices (CI),which were defined for the serum and cream layers respectively,as follows:CI SL =100·(H SL /H T ) /4/CI CL =100·(H CL /H T ) /5/The creaming indices provided indirect informationabout the extent of droplet aggregation in an emulsion,for example, the higher the creaming indices, the greaterthe particle aggregation.Statistical analysisExperiments were performed at least twice usingfreshly prepared samples. Average and standard deviationswere calculated from these duplicate measurements.Results and DiscussionOptimum conditions to form silkfibroin-coated dropletsThe purpose of these experiments was to establishthe optimum silk fibroin concentration required to formstable emulsions. The electrical charge (z-potential), particlesize distribution, creaming stability, microstructure,amount of destabilized oil and mean particle diameter(d 32 ) of emulsions (10 % corn oil, by mass; 10 mM phosphatebuffer, pH=7.0) containing different silk fibroinmass fractions (0.2–3 %) were measured 24 h after preparation.The electrical charge of the emulsion droplets wasnegative at pH=7.0 because this pH was above the pI ofthe absorbed silk fibroins. There was no significant changein z-potential within the range of silk fibroin mass fraction(0.2–3.0 %), with the average being (–30.7±1.4) mV.Since the emulsion droplets are coated with a chargedbiopolymer, electrostatic repulsion is expected to play animportant role to prevent aggregation (13,14).The mean particle diameter (d 32 ) of emulsion decreasedas the mass fraction of silk fibroin increasedfrom0.2to1%,itsminimumbeing(0.47±0.05) mm containing1 % (by mass) of silk fibroin (Fig. 1). The largeaggregates were observed under the microscope (d 32 >10mm), and rapid creaming (CI CL >40 %) was observed ind 32 / m m3.53.02.52.01.51.00.50.00.00.50.2% 0.5% 1% 1.5%1.0 1.5 2.0 2.5w(silk fibroin)/%Fig. 1. Influence of silk fibroin mass fraction on the mean particlediameter (d 32 ) of corn oil-in-water emulsions. Inserted isthe morphology of relevant emulsions under microscope 400´;more staining means worse dispersionthe emulsions containing 0.2 and 0.5 % (by mass) of silkfibroin, whereas no aggregation or creaming were observedat 1%(bymass)silk fibroin. These results can beattributed to the following reasons: (i) the total dropletsurface area that can be stabilized by the increased massfraction of silk fibroin; (ii) the rate at which the dropletsurfaces were covered with increased mass fraction ofsilk fibroin; (iii) the frequency of droplet collisions decreaseddue to the increase in aqueous phase viscosity.All of these factors should facilitate droplet disruptionand prevent droplet coalescence within the homogenizer,therefore leading to the formation of smaller droplet sizes.However, the mean particle diameter (d 32 ) of theemulsion increased again when the mass fraction of silkfibroin was over 1.5 %. Apart from this, the foam wasobserved after homogenizing. Therefore, the foam capacityof the silk fibroin was measured (Fig. 2). There wasno foam capacity when the silk fibroin mass fractionwas very low (i.e. 0.2 or 0.5 %), after which it increasedwith the increase of silk fibroin mass fraction. The highestfoam capacity was 136 % at the silk fibroin mass fractionof 3 %. This observation suggests that higher silkfibroin mass fraction enhances foaming capacity, whichcan be attributed to the above conclusion that the averageparticle diameter increased with the higher silk fibroinmass fraction. In addition, oil destabilization mea-Foam capacity/%160140120100806040203.03.500.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5w(silk fibroin)/%Fig. 2. Influence of silk fibroin mass fraction on the foam capacityof corn oil-in-water emulsions

J.J. RAO et al.: Stabilization of Silk Fibroin-Coated O/W Emulsions, Food Technol. Biotechnol. 47 (4) 413–420 (2009)417surements (Fig. 3) confirmed that there was appreciable'oiling-off' in the emulsion with higher silk fibroin massfraction. At high silk fibroin mass fraction (³1.5 %), therewas more than 15 % of destabilized oil in the emulsions.All these results indicate that O/W emulsions can be preparedusing silk fibroin concentration. However, in orderto avoid the formation of foam during homogenizing,1%(bymass)ofsilkfibroin was used to preparethe emulsion (i.e. silk fibroin-to-oil mass ratio of 1:10) inall subsequent experiments, because this mass fractionenabled the production of emulsions containing relativelysmall droplets that were stable to creaming.Destabilized oil/%4030201000.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5w(silk fibroin)/%Fig. 3. Influence of silk fibroin mass fraction on the amount ofdestabilized oil in 10 % (by mass) corn oil-in-water emulsionsSurface activity of silk fibroinThe ability of protein to form and stabilize emulsionsis dependent on their ability to adsorb to interfacesand on the amount of protein required to saturatethe interface (15). The foam capacity is also dependenton it. In an attempt to explain the emulsifying abilityand foam capacity of silk fibroin, the surface activity ofsilk fibroin was measured.The dependence of surface tension (g) of silk fibroinmass fraction in 10 mM phosphate buffer solution wastested (Fig. 4). As the silk fibroin mass fraction increased,the surface tension decreased from 71.5 mN/min the absence of silk fibroin to a constant value of 58.8g/(mN /m )807570656055500.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5w(silk fibroin)/%Fig. 4. Influence of silk fibroin mass fraction on the surface tensionof silk fibroin solutions at 30 °CmN/m when the interface became saturated with silkfibroin of 1.5 %, then sharply dropped to 54.9 mN/mwith the continued increase in silk fibroin mass fractionto 3 %. The surface activity of silk fibroin was relativelylow when concentrations were lower. This can be attributedto its relatively high hydrophobic properties.Generation of foam involves the development of aprotein film surrounding a gas bubble and the packingof gas bubbles into an overall structure. The spontaneousadsorption of proteins from the solution to theair/aqueous interface is of central importance for theirfoaming performance. This phenomenon is thermodynamicallyfavourable due to the simultaneous dehydrationof the hydrophobic interface and hydrophobic portionsof the protein. Hydrophobic patches on the surface of aprotein initially drive this process, and surface hydrophobicityhas been correlated with improved foamingproperties (16). Once contacts are made with the interface,flexibility within the molecules can expose previouslyburied hydrophobic portions into the interface,potentially leading to interfacial denaturation of the moleculesand subsequent reduction in surface tension (17).This has been correlated with improved foamability. Inorder to avoid its foamability during emulsion processing,silk fibroin mass fraction of 1%intheemulsionsystem was selected.Influence of pH on the emulsion stabilityThe influence of storage pH (2–8) on the droplet charge(z-potential), mean particle diameter (d 32 ), and creamingstability of 10 % (by mass) of corn oil-in-water emulsionsstabilized with silk fibroin was measured (Figs. 5–7).z-potential/mV4020012 3 4 5 6 7 8 9-20-40-60Fig. 5. Influence of pH on z-potential of corn oil-in-water emulsionsstabilized with silk fibroin (1 %, by mass)d 32 / m m2.52.01.51.00.5pH0.01 2 3 4 5 6 7 8 9pHFig. 6. Influence of pH on the mean particle size of corn oil-in--water emulsions stabilized with silk fibroin (1 %, by mass)

418 J.J. RAO et al.: Stabilization of Silk Fibroin-Coated O/W Emulsions, Food Technol. Biotechnol. 47 (4) 413–420 (2009)8060CI SLCI CL1.00.8pH=3pH=7CI/%4020d 32/ m m0.60.40.202 3 4 5pH6 7 8Fig. 7. Influence of pH on the cream stability of corn oil-in--water emulsions stabilized with silk fibroin (1 %, by mass). Insertedis the cream stability of emulsions at various pH, separationsignifies unstability0.0unheated60 90t/°CFig. 8. Influence of thermal processing on the mean particle sizeof corn oil-in-water emulsions stabilized with silk fibroin (1 %,by mass)The z-potential was relatively high (25.7 mV) atpH=2.0, but it became less positive with increasing pHuntil it reached a value of zero at around pH=4.0, andthen became increasingly negative as the pH increasedfurther, until it reached a value of –35.4 mV at pH=8.0.The droplets in protein-stabilized emulsions have zeronet charge around the isoelectric point (IEP) of the adsorbedproteins. Our z-potential vs. pHmeasurementssuggested that the pI values of the silk fibroin werearound pH=4.0. This value is quite similar to the pIvalue reported by the other researchers (18).The mean particle diameter (d 32 ) remained relativelysmall (

J.J. RAO et al.: Stabilization of Silk Fibroin-Coated O/W Emulsions, Food Technol. Biotechnol. 47 (4) 413–420 (2009)419z-potential/mV201000 50 100 150 200 250-10-20-30-40pH=3pH=7c(NaCl)/mMFig. 10. Influence of NaCl on z-potential of corn oil-in-wateremulsions stabilized with silk fibroin (1 %, by mass)d 32 / m m54321pH=3pH=700 50 100 150 200 250c(NaCl)/mMFig. 11. Influence of NaCl on the mean particle size of corn oil--in-water emulsions stabilized with silk fibroin (1 %, by mass)with increasing NaCl concentration at pH=7.0 than atpH=3.0. The most likely reason for this difference is thatthe monovalent Na + ions are counterions for the anionicdroplets in the emulsion, whereas the monovalent Cl –ions are counterions for the cationic droplets in the emulsion.Also, the Na + ions are more sensitive to binding tothe droplets coated with silk fibroin at pH=3.0. This wasalso confirmed by the mean droplet diameter (d 32 )measurements,which showed that the magnitude of d 32 increaseat pH=7.0 was smaller than that at pH=3.0 withincreasing NaCl concentration, presumably because electrostaticscreening and ion binding effects reduced moreelectrostatic repulsion at pH=3.0 than at pH=7.0 betweenthe oil droplets. It was also found that the meanparticle diameters (d 32 ) at pH=3.0 and 7.0 sharply increasedeven when 50 mM of sodium chloride were added.This would account for the fact that lower amountof NaCl was needed to promote droplet aggregation inthe emulsion. The creaming stability (data not shown) ofemulsion was relatively high (CI SL >50 %) even at lowerconcentration of salt. All these results suggest that silkfibroin-stabilized emulsions were unstable against dropletaggregation even at lower ionic strength, when electrostaticinteractions would be screened. Hence, it can beconcluded that polymeric steric repulsion plays a lessimportant role than electrostatic repulsion in preventingthe droplets from aggregating.ConclusionsThis study has shown that silk fibroin, which can beisolated from silkworm cocoon, is very effective in stabilizingoil-in-water emulsion by absorbing to the surfaceof lipid droplets. It has also helped to identify optimummass ratio of corn oil and silk fibroin (corn oil/silk fibroin=10:1)for the preparation of O/W emulsions. Theelectrical properties and aggregation stability of the silkfibroin-coated O/W emulsions were determined as a functionof pH, ionic strength, and thermal processing becausethese environmental stresses are commonly encounteredin the food industry. The silk fibroin-coatedO/W emulsions proved to be stable to aggregation at pHvalues sufficiently far from their isoelectric point (pH=4.0)at relatively low salt concentrations (

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FTB 47 (4) 413-420.(FTB-2183)Moduliranje i stabilizacija emulzija ulja u vodi pomoću fibroina svileSažetakSvrha je ovoga rada bila pripremiti i okarakterizirati stabilnu emulziju ulja u vodi,koja sadrži kapljice obložene fibroinom svile. Fibroin svile, prirodni jestivi vlaknasti proteindobiven iz čahura dudova svilca, upotrijebljen je za pripremu emulzije kukuruznog ulja uvodi (masenog udjela od 10 %) na sobnoj temperaturi (pH=7, 10 mM fosfatni pufer).Primjenom fibroina svile masene koncentracije od 1 % dobivene su emulzije relativno malogpromjera čestica (d 32 =0,47 µm) i vrlo dobre stabilnosti (dulje od 7 dana). Ispitan je utjecajpH-vrijednosti (pH=2–8), toplinske obrade (60–90 °C, 20 min) i koncentracije soli(c(NaCl)=0–250 mM) na svojstva i stabilnost emulzije (ζ-potencijal, veličina čestica iraslojavanje emulzije). Izoelektrična točka kapljica stabiliziranih pomoću fibroina svilepostignuta je pri pH~4. Emulzije skladištene na sobnoj temperaturi bile su stabilne pri svimpH-vrijednostima, osim pri srednjoj (pH=4,0) kada su imale relativno nizak ζ-potencijal.Povećanjem pH-vrijednosti s 2 na 8 povećao se ζ-potencijal emulzija (s 25 na -35 mV).Uočena je stabilnost emulzija i nakon toplinske obrade (tijekom 20 minuta na 60 i 90 °C, pripH=3 i 7), a pri temperaturama višim od 60 °C ζ-potencijal se neznatno smanjio. Zbog učinkaelektrostatskog odbijanja emulzije su bile nestabilne čak i pri neznatnim koncentracijama soli(c(NaCl)=0-250 mM, pH=3 i 7). Rezultati upućuju na to da stabilizacija emulzija pomoćufibroina svile omogućuje proizvodnju funkcionalnih proizvoda na bazi ulja.Ključne riječi: fibroin svile, kukuruzno ulje, emulzija, stabilnost, pH