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Food Hydrocolloids xxx (2014) 1e9

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Food Hydrocolloids

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

Effect of barrier properties of zein colloidal particles and oil-in-wateremulsions on oxidative stability of encapsulated bioactive compounds

Yuanjie Pan a, Rohan V. Tikekar b, Min S. Wang a, Roberto J. Avena-Bustillos c,Nitin Nitin a,d,*

aDepartment of Food Science and Technology, University of California-Davis, Davis, CA 95616, United Statesb Program in Food Science, School of Technology and Professional Studies, Drexel University, Philadelphia, PA 19104, United StatescWestern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA 94710, United StatesdDepartment of Biological and Agricultural Engineering, University of California-Davis, Davis, CA 95616, United States

a r t i c l e i n f o

Article history:Received 2 February 2014Accepted 1 May 2014Available online xxx

Keywords:Free radical induced oxidationOxygenZein colloidal particleOil-in-water emulsionFluorescence spectroscopy

* Corresponding author. 2221 RMI South, Old DavisStates. Tel.: þ1 530 752 6208.

E-mail address: nnitin@ucdavis.edu (N. Nitin).

http://dx.doi.org/10.1016/j.foodhyd.2014.05.0020268-005X/� 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Pan, Y., etstability of encapsulated bioactive compoun

a b s t r a c t

Background: Oxidation of encapsulated bioactive compounds is a key challenge that limits shelf-life ofbioactive containing products. The objectives of this study were to compare differences between theoxidative barrier properties of biopolymer particle based encapsulation system (zein colloidal particles)and oil-in-water emulsions and evaluate the impact of these differences on oxidative stability ofencapsulated bioactives.Methods: Both zein colloidal particles and oil-in-water emulsions were stabilized by casein protein. Theoxidative barrier properties of the selected encapsulation systems were determined by measuring thepermeation rate of peroxyl radicals and oxygen across the interface. Peroxyl radical permeation rateswere correlated with stability of a model bioactive, curcumin and oxygen permeation rates werecorrelated with stability of another model bioactive, retinol.Results: Radical permeation rate was significantly higher in oil-in-water emulsions compared to zeincolloidal particles, indicating enhanced barrier property of zein colloidal particles against peroxyl radicalinduced oxidation. Consistent with these results, stability of curcumin encapsulated in zein colloidalparticles was significantly higher compared to that in oil-in-water emulsions. Oxygen permeationmeasurements showed no significant differences in the barrier properties of both encapsulation systemsagainst oxygen permeation. Consistent with these results, the oxidative stability of retinol was similar inboth encapsulation systems.Conclusions: The results of this study demonstrate the advantages of biopolymer particle based encap-sulation system in limiting free radical induced oxidation of encapsulated bioactives and also demon-strate the ineffectiveness of both encapsulation systems in limiting oxygen permeation.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Encapsulation of bioactives is a common approach used inpharmaceutical, food and cosmetic industries (Huang, Yu, & Ru,2010; Luo, Teng, & Wang, 2012). The overall goals of diverseencapsulation approaches are to enhance stability and improvedelivery of bioactives (Huang et al., 2010; Luo et al., 2012; Luo,Zhang, Whent, Yu, & Wang, 2011). To achieve these goals,

Road, Davis, CA 95616, United

al., Effect of barrier propertids, Food Hydrocolloids (2014)

diverse biopolymer particles and emulsions are commonly usedin various product formulations (Benjamin, Silcock, Leus, &Everett, 2012; Sant, Nadeau, & Hildgen, 2005). Among emul-sions, oil-in-water emulsions are commonly selected forencapsulation of hydrophobic bioactives such as antioxidantsand vitamins (Benjamin et al., 2012; Hambleton, Fabra,Debeaufort, Dury-Brun, & Voilley, 2009; Kohli, Chopra, Dhar,Arora, & Khar, 2010). For biopolymer based particles, the poly-mer materials selected for these formulations include syntheticpolymers such as poly(lactic-coglycolic acid) and natural bio-polymers including alginates and zein (Liu, Sun, Wang, Zhang, &Wang, 2005; Ogawa, Decker, & McClements, 2003; Parris, Cooke,& Hicks, 2005; Zhong et al., 2009). Natural edible biopolymers

es of zein colloidal particles and oil-in-water emulsions on oxidative, http://dx.doi.org/10.1016/j.foodhyd.2014.05.002

Y. Pan et al. / Food Hydrocolloids xxx (2014) 1e92

have many advantages compared to synthetic biopolymers asthese materials can be used for diverse applications includingfood.

Oxidation degradation limits the shelf-life of products contain-ing bioactive compounds such as vitamins, antioxidants and anti-inflammatory compounds (McClements, Decker, & Weiss, 2007;Tikekar, Johnson, & Nitin, 2011). One of the key goals of encapsu-lation systems is to limit oxidative degradation of encapsulatedbioactives (McClements et al., 2007). Currently, many of theencapsulation systems are empirically selected and bothbiopolymer particles and emulsion based formulations are used infood, pharmaceutical and cosmetic industries.

The aims of the study were to: (a) comparatively evaluateoxidative barrier properties of biopolymer particles and emulsionsand (b) correlate oxidative barrier properties of the selectedencapsulation systems with oxidative stability of model bioactiveencapsulants. For this purpose, zein colloidal particles and oil-in-water emulsions were selected as model systems. Zein is theprolamine fraction of corn protein (Shukla & Cheryan, 2001) andhas long been recognized for encapsulation of hydrophobicbioactive compounds (Hurtado-Lopez & Murdan, 2006; Liu et al.,2005; Patel, Hu, Tiwari, & Velikov, 2010). In this study, zeincolloidal particles and oil-in-water emulsions have the sameinterfacial composition (i.e. casein protein molecules). The hy-pothesis of this study was that structural differences betweenbiopolymer particles (solid core) and oil-in-water emulsion (fluidcore) will result in significant differences in oxidative barrierproperties and hence influence the oxidative stability of encap-sulated bioactives.

Oxidative barrier properties of zein colloidal particles and oil-in-water emulsion were characterized based on quantitative real-time, in-situ measurements of permeation of oxygen and peroxylradicals into the hydrophobic core of the encapsulation systems.Free radical initiated oxidation reaction is one of the commonpathways for oxidative degradation of bioactives (Cercaci,Rodriguez-Estrada, Lercker, & Decker, 2007; Fukumoto & Mazza,2000). In addition, presence of oxygen in the encapsulation corecan itself induce oxidation of oxygen susceptible bioactives such asantioxidants (Coupland &McClements,1996). Curcumin and retinolwere selected as model food grade bioactives in this study. Cur-cumin has significant potential for prevention and treatment ofvarious diseases including cancer and inflammatory diseases.However, limited water solubility and poor oxidative stability limitbroad application of curcumin in diverse products (Anand,Kunnumakkara, Newman, & Aggarwal, 2007; Patel et al., 2010).Retinol is one of the bioactive forms of vitamin A and is essential formaintaining activity of immune system, function of epithelial tis-sues and health of visual function (Eskandar, Simovic, & Prestidge,2009). However, retinol can be readily oxidized upon exposure tooxygen, heat, and light and its bioavailability from natural sourcesis highly variable (Hwang, Oh, & Oh, 2005; Loveday & Singh, 2008;Yoshida, Sekine, Matsuzaki, Yanaki, & Yamaguchi, 1999). Therefore,there is a significant need to enhance stability and improve deliveryof curcumin and retinol using encapsulation strategies such asbiopolymer particles and emulsions.

In summary, this study will compare the oxidative barrierproperties of two widely used encapsulation approaches, i.e.emulsions and biopolymer particles. Quantitative analysis ofpermeation of oxygen and free radical induced oxidation in emul-sions and biopolymer particles will provide a comprehensiveapproach to determine the optimized encapsulation system foroxidation susceptible bioactives. To the best of our knowledge, thisis the first study that has quantitatively measured the oxidativebarrier properties of biopolymer based particles and compared theresults with oil-in-water emulsions.

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2. Materials and methods

2.1. Materials

Sodium caseinate from bovine milk, sodium azide, 2,20-azobis-2-methylpropanimidamide dihydrochloride (AAPH), organiccanola oil, tris (4,7-diphenyl-1,10-phenanthroline) ruthenium(II)bis (hexafluorophosphate) complex, retinol, curcumin from Cur-cuma longa (Turmeric) and zeinwere obtained from SigmaeAldrich(St Louis, MO, USA). Ethanol, dimethyl sulfoxide (DMSO) andacetone were purchased from Fisher Scientific (Pittsburgh, PA,USA). Ultrapure water (16 MU-cm) was obtained from an in-housewater filtration system. A peroxyl radical sensitive dye, BOD-IPY�665/676 dye was purchased from Invitrogen Inc. (Carlsbad, CA,USA).

2.2. Synthesis of zein colloidal particles for oxygen and peroxylradical permeation measurements

Casein (4 g) was dissolved in 200 mL of ultrapure water. Onegram of zein protein powder was solubilized in 50 mL of 80%ethanol. This zein ethanolic solution was mixed with either 200 mLof BODIPY 665/676 dye prepared in acetone (0.5 mg/mL) orruthenium based oxygen sensitive dye prepared in DMSO (1 mg/mL). The zein solution was added to the casein solution drop-wise(5 mL/min) under vigorous stirring (4000 rpm) and dispersed inaqueous solution using a hand-held disperser operating at9000 rpm for 2 min (Ultra-Turrax model T25, IKA Works, Wil-mington, NC, USA). The coarse dispersion was subsequently passedeight times through a single stage homogenizer (Niro Soavi, Parma,Italy) operating at 80 MPa. The resultant solution was heated at60 �C for 45 min to evaporate ethanol and facilitate particle for-mation. The particle solution was centrifuged at 10,000 g for 1 h toprecipitate coarse particles and agglomerates. Sodium azide wasadded to the particle solution at a concentration of 0.1% to preventmicrobial growth. The zein colloidal particles were stored at 4 �Cuntil further use.

2.3. Preparation of oil-in-water emulsions for oxygen and peroxylradical permeation measurements

Casein (4 g) was dissolved in 200 mL of ultrapure water. Canolaoil (8 g) was mixed with either 200 mL of BODIPY 665/676 dyeprepared in acetone (0.5 mg/mL) or ruthenium based oxygen sen-sitive dye prepared in DMSO (1 mg/mL). Emulsion premix wasformed using a hand-held disperser operating at 9000 rpm for2 min (Ultra-Turrax model T25, IKA Works, Wilmington, NC, USA).This premix was passed through a single stage homogenizer (NiroSoavi, Parma, Italy) operating at 80MPa five times to obtain a stableemulsion. The emulsion was incorporated with 0.1% sodium azideto prevent microbial growth and stored at 4 �C until further use.

2.4. Structural analysis of zein colloidal particles

Surface appearance of zein colloidal particles was examinedusing a Transmission Electron Microscope (TEM) located in theDepartment of Medical Pathology and Laboratory Medicine at theUniversity of California, Davis. A drop (6 mL) of zein colloidal particlesamples was added onto a carbon coated TEM grid (400 mesh, TedPella, Redding, CA, USA) and incubated at room temperature for10 min. After 10 min, the excess liquid was wicked away using apiece of filter paper, and 8 mL of 1% sodium phosphotungstate wasadded to the TEM grid and wicked away immediately to negativestain the sample. The TEM grid was allowed to dry in air for anadditional 10 min prior to imaging. TEM images of the samples

es of zein colloidal particles and oil-in-water emulsions on oxidative, http://dx.doi.org/10.1016/j.foodhyd.2014.05.002

Y. Pan et al. / Food Hydrocolloids xxx (2014) 1e9 3

were viewed and acquired using a Philips CM120 Biotwin TEM, (FEICompany, Hillsboro, OR, USA) at 80 kV for low magnification, and100 kV at 200,000� magnification in order obtain image of oneparticle.

2.5. Size characterization of zein colloidal particles and oil-in-wateremulsion

The hydrodynamic sizes of zein colloidal particles and oil-in-water emulsion were measured using a particle size analyzer(Malvern Nano Series, Malvern Instruments, Inc., Westborough,MA, USA). The settings for the analyzer to measure the particle sizeof zein colloidal particles were - material type: protein, particlerefractive index¼ 1.45, dispersant type: water, dispersant refractiveindex ¼ 1.33, temperature: 25 �C. The settings for the analyzer tomeasure the droplet size of oil-in-water emulsions wereematerialtype: oil, particle refractive index ¼ 1.47, dispersant type: water,dispersant refractive index ¼ 1.33, temperature: 25 �C. Size mea-surements were analyzed based on the number average particledistribution for zein colloidal particles and number average dropletsize distribution for oil-in-water emulsion.

2.6. Fluorescence imaging of encapsulated BODIPY dye and oxygensensitive dye in zein colloidal particles and oil-in-water emulsion

Fluorescence images were obtained to demonstrate that theBODIPY and oxygen sensitive dyes were uniformly distributed inthe hydrophobic core of coarse zein colloidal particles and coarseoil-in-water emulsion, respectively. Fluorescence images of coarsezein colloidal particles and coarse emulsionwere obtained using anOlympus IX 71microscope (Olympus Inc., Center Valley, PA, USA) at40� magnification (100 ms exposure, excitation filter: 480/30 nm,emission filter: 570/60 nm for the oxygen sensitive dye; 100 msexposure, excitation filter: 540/30 nm, emission filter: 605/55 nmfor the BODIPY dye).

2.7. Measurement of permeation of peroxyl radicals

Peroxyl radicals were generated in the aqueous phase of zeincolloidal particles and oil-in-water emulsion using AAPH. AAPH(40 mM) was dissolved in ultrapure water. One milliliter of zeincolloidal particle or emulsion sample was then mixed with 1 mL of

Fig. 1. TEM images of (a) shape of zein colloidal particles (b) a close up view of a representatiand cracks (white arrows) (scale bars are 200 nm and 50 nm, respectively).

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AAPH solution to achieve a final concentration of 20 mM AAPH.Immediately after addition of AAPH, 200 mL of each sample wasplaced in a 96 well clear bottom black plate and fluorescence in-tensity was measured at a regular time interval of 20 min for a totaltime period of 20 h using a plate-reader (SpectramaxM5,MolecularDevices, Carlsbad, CA, USA). Zein colloidal particle and oil-in-wateremulsion samples mixed with equal volume of ultrapure waterwere used as controls. The excitation and emissionwavelengths forfluorescence measurements were 620 nm and 675 nm, respec-tively. The relative fluorescence intensity was calculated usingequation (1),

RFIradical ¼It AAPH=I0 AAPH

It control=I0 control� 100 (1)

where RFIradical ¼ Relative fluorescence intensity of BODIPY dye

It AAPH ¼ Fluorescence intensity of sample after ‘t’ min of expo-sure to AAPH,I0 AAPH ¼ Fluorescence intensity of sample immediately afteraddition of AAPH,It control ¼ Fluorescence intensity of control sample after time ‘t’min,I0 control ¼ Fluorescence intensity of control sample at timet ¼ 0 min.

The data was normalized with respect to the relative fluores-cence of control samples in order to account for changes in fluo-rescence intensity induced by evaporation during the course ofmeasurement (w20 h). The changes in fluorescence intensity werefit to equation (3).

2.8. Curcumin stability in zein colloidal particles and oil-in-wateremulsion

Zein colloidal particles and oil-in-water emulsion encapsulatingcurcumin were prepared using the same experimental approach asdescribed for the samples encapsulating a radical sensitive dye.Curcumin was dissolved in oil for encapsulation in emulsion and inethanolic solution of zein protein for encapsulation in zein colloidalparticles. Curcumin was added to ethanol or oil to achieve a finalconcentration of 6.9 mg/mL ethanol or 200 mg/g oil. Concentration

ve zein colloidal particle highlighting structure of the particle core e surface roughness

es of zein colloidal particles and oil-in-water emulsions on oxidative, http://dx.doi.org/10.1016/j.foodhyd.2014.05.002

Fig. 2. Particle size distribution of zein colloidal particles and droplet size distributionof oil-in-water emulsion.

Fig. 3. Representative fluorescence microscopy images of BODIPY dye encapsulated in(a) coarse zein colloidal particles (b) coarse oil-in-water emulsion droplets. Ex/Em ¼ 480/570 nm, objective ¼ 40�, scale bar ¼ 10 mm.

Y. Pan et al. / Food Hydrocolloids xxx (2014) 1e94

of curcumin was selected based on the solubility limit of curcuminin ethanol and oil. Oxidative stability of encapsulated curcuminwasmeasured by characterizing changes in absorbance of curcumin as afunction of incubation time. Zein colloidal particles and oil-in-water emulsions containing encapsulated curcumin were incu-bated with and without 10 mM AAPH at 22 �C in dark for 24 h. Tomeasure curcumin concentration, emulsions were disrupted byadding 1 mL acetone to 200 mL zein colloidal particles or oil-in-water emulsion. The mixture was centrifuged at 14,000 g for10 min. One milliliter of the supernatant was placed in a cuvetteand the absorbance was measured at 425 nm using a UVeVisiblespectrophotometer (GENESYS 10S Series, Thermo Scientific, USA). Ablank sample was prepared using 1 mL acetone and 200 mL ultra-pure water. To account for any difference in the encapsulation ef-ficiency of curcumin in zein colloidal particles and oil-in-wateremulsion, the data was normalized with respect to the controls attime ¼ 0 for each formulation.

2.9. Measurement of permeation of oxygen

After homogenization, zein colloidal particle and oil-in-wateremulsion samples were transferred to three aluminum foil wrap-ped plastic tubes (5 mL each) and purged with nitrogen for 3 h toremove oxygen. Efficient removal of oxygen provides a large dy-namic range to measure changes in fluorescence upon exposure tooxygen. After purging, the samples were exposed to atmosphere bypipetting 200 mL of sample/well in a 96 well clear bottom blackplate optimized for fluorescence measurement. Zein colloidal par-ticle and oil-in-water emulsion samples were exposed to ambientair within a plate-reader. A loss in fluorescence intensity as a resultof exposure to atmospheric oxygen was measured using a plate-reader (Model: M5, Molecular Devices, Carlsbad, CA, USA). Theexcitation and emission settings for the plate-reader were 485 nmand 615 nm, respectively. The plate-reader was set at 23 �C.Changes in fluorescence intensity as a function of time wererecorded at a regular interval of 15 s for 30 min. The relative fluo-rescence intensity was calculated using the following equation,

RFIoxygen ¼ ðIt � INÞðI0 � INÞ � 100 (2)

where RFIoxygen ¼ Relative fluorescence intensity of oxygen sensi-tive dye

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It ¼ Fluorescence intensity at time ‘t’ s after exposure to atmo-spheric oxygen,IN ¼ Fluorescence intensity at equilibrium oxygenconcentration,I0 ¼ Fluorescence intensity in the absence of oxygen (nitrogenpurged sample at t ¼ 0 s).

The changes in fluorescence intensity were fit to equation (3).

2.10. Retinol stability in zein colloidal particles and oil-in-wateremulsion

Zein colloidal particles and oil-in-water emulsion encapsulatingretinol were prepared using the same experimental approach asdescribed for the samples encapsulating a radical sensitive dye.Retinol was dissolved in oil for encapsulation in emulsion and inethanolic solution of zein protein for encapsulation in zein colloidalparticles. Retinol was added to ethanol or oil to achieve a finalconcentration of 0.6 w/w %. Oxidative stability of encapsulatedretinol was determined by measuring changes in absorbance ofretinol as a function of incubation time. Zein colloidal particles andoil-in-water emulsion containing encapsulated retinol were

es of zein colloidal particles and oil-in-water emulsions on oxidative, http://dx.doi.org/10.1016/j.foodhyd.2014.05.002

Fig. 4. Permeation of peroxyl radicals from the aqueous phase to the core of zeincolloidal particles and oil-in-water emulsion. Radicals were generated using (a) 10 mMof AAPH and (b) 20 mM of AAPH in the aqueous phase of zein colloidal particles andoil-in-water emulsions. The radical permeation rate was measured based on loss influorescence of a peroxyl radical sensitive dye (C11-BODIPY 665/676) encapsulated inthe core of zein colloidal particles and emulsions. Each data point represents anaverage of three independent measurements � standard deviation.

Y. Pan et al. / Food Hydrocolloids xxx (2014) 1e9 5

incubated at 22 �C in dark for 24 h. To measure retinol concentra-tion, zein colloidal particles or emulsions were disrupted by adding1 mL methanol to 50 mL zein colloidal particles or oil-in-wateremulsion. The mixture was centrifuged at 14,000 g for 10 min.One milliliter of the supernatant was placed in a cuvette and theabsorbance was measured at 325 nm using a UVeVisible spectro-photometer (GENESYS 10S Series, Thermo Scientific, USA). A blanksample was prepared using 1 mL methanol and 50 mL ultrapurewater. To account for any difference in the encapsulation efficiencyof retinol in zein colloidal particles and oil-in-water emulsion, thedata was normalized with respect to the controls at time ¼ 0 foreach formulation.

2.11. Curve fitting

The changes in fluorescence intensity were fit to an exponentialmodel

DI ¼ I0e�ðt=sÞa (3)

using Matlab CFTool 2012 (MathWorks, Torrance, CA, USA), whereI0 is the initial intensity of fluorescence, s is the time required for anexponential decay (i.e. the time for the initial fluorescence to reach1/e), and a is the shape parameter. Shape parameter indicates thetime lag before fluorescence intensity starts to decrease. As time lagincreases, the shape parameter increases as well and indicatesresistance of the formulation to oxidative stress. A nonlinearregressionmodel with “Trust Region” algorithmwas used for fittingthe data.

2.12. Statistical analysis

Statistical analysis was carried out using Microsoft Excel 2007(Microsoft Inc., Bellevue, WA, USA). Analysis of Variance (ANOVA)and Student’s t-test were used to identify significant differencesbetween the treatments (a ¼ 0.05).

3. Results and discussion

3.1. Structural analysis of zein colloidal particles

For structural analysis, zein colloidal particles were imaged us-ing transmission electronmicroscopy (TEM). TEM results in Fig.1(a)show that zein colloidal particles prepared using homogenizationfollowed by solvent evaporation approach were spherical in shapeand did not form aggregates even after drying on a TEM coppergrid. The lack of aggregation of zein colloidal particles can beattributed to the presence of interfacial coating (Anton, Benoit, &Saulnier, 2008). In this case, casein molecules at the interfaceprovided an effective interfacial coating to limit aggregation of zeincolloidal particles. A higher magnification (200,000�) TEM imageof the selected zein colloidal particle (Fig. 1(b)) shows nanoscalecracks and roughness near the interface of the zein colloidal par-ticle. The cracks and roughness observed on the surface of the zeincolloidal particles could be attributed to both variation in surfacedensity of casein protein on the zein colloidal particle and porosityof the zein core. Since oil-in-water emulsions have a uniform fluidcore, the structural details of the lipid core were not evaluatedusing TEM.

3.2. Particle and droplet size measurements

Fig. 2 shows the particle size distribution for zein colloidalparticles and the droplet size distribution for oil-in-water emulsionmeasured using dynamic light scattering. The results show a partial

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overlap in the size distribution between zein colloidal particles andoil-in-water emulsion. The results also show that the average par-ticle diameter for zein colloidal particles (104.9 � 0.1 nm) wassmaller than that for oil-in-water emulsion (204 � 7 nm). The PDI(polydispersity index) values associated with the size distributionof zein colloidal particles and oil-in-water emulsionwere 0.241, and0.226, respectively. Storage stability of zein colloidal particles andoil-in-water emulsion was also measured based on size measure-ments. The results show no significant (p > 0.05) changes in size ofboth oil-in-water emulsion droplets and zein colloidal particleswithin 2e3 weeks of refrigerated storage (data not shown).

3.3. Peroxyl radical permeation across zein colloidal particles andoil-in-water emulsion

Efficacy of zein colloidal particles and oil-in-water emulsion inlimiting free radical mediated oxidation processes was determinedby measuring permeation of free radicals from the aqueous phaseto the hydrophobic core of zein colloidal particles and oil-in-water

es of zein colloidal particles and oil-in-water emulsions on oxidative, http://dx.doi.org/10.1016/j.foodhyd.2014.05.002

Table 1Nonlinear regression fit parameters for the changes in fluorescence intensity of a peroxyl radical sensitive BODIPY dye encapsulated in zein colloidal particles and oil-in-wateremulsion as a function of time. Values in the parentheses represent 95% confidence interval.

Samples I0 s a R2

Oil-in-water emulsion 102.5 (100.1e104.9) 8.242 (8.063e8.422) 2.315 (2.151e2.480) 0.99Zein colloidal particles 98.77 (97.69e99.85) 18.44 (18.26e18.62) 4.618 (4.301e4.936) 0.99

Y. Pan et al. / Food Hydrocolloids xxx (2014) 1e96

emulsion. Permeation of peroxyl radicals was measured based onchanges in fluorescence intensity of a peroxyl radical sensitive dye(BODIPY) encapsulated in the core of zein colloidal particles andoil-in-water emulsion. The selected BODIPY dye is highly hydro-phobic and has high specificity to react with peroxyl radicals (Pan,Tikekar, & Nitin, 2013; Tikekar & Nitin, 2011). This approach hasbeen successfully used in our prior studies to measure the influenceof lipid core design and interfacial composition on the rate ofpermeation of peroxyl radicals across emulsions, lipid nano-particles and lipid bilayers (Bricarello, Prada, & Nitin, 2012; Panet al., 2013; Tikekar & Nitin, 2011, 2012).

To determine the distribution of BODIPY dye in zein colloidalparticles and oil-in-water emulsion, coarse zein colloidal particlesand coarse oil-in-water emulsion droplets containing BODIPY dyewere imaged using fluorescence microscopy (Fig. 3). Coarse zeincolloidal particles and coarse oil-in-water emulsion droplets wereprepared with the same proportions of organic and aqueous phasesas in the case of homogenized zein colloidal particles and oil-in-water emulsions respectively using a hand-disperser. The imaging

Fig. 5. Oxidative stability of encapsulated curcumin in (a) oil-in-water emulsion and(b) zein colloidal particles with and without 10 mM AAPH treatment. Both the controland treatment emulsion samples were stored in dark at room temperature for 20 h.N ¼ 3, **p < 0.01, compare to control. Error bars represent standard deviations.

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measurements illustrate that the BODIPY dye was uniformlydistributed in the core of zein colloidal particles (Fig. 3(a)) and oil-in-water emulsion (Fig. 3(b)). This imaging based observation is incontrast to the result obtained in our prior study using solid lipidnanoparticles (solid core), where extensive exclusion of theencapsulated BODIPY dye to the interface of solid lipid particles wasobserved (Tikekar & Nitin, 2011). Thus, in contrast to solid lipidparticles, the solid core of the biopolymer particles such as zein canretain encapsulated material in the core.

To measure the interactions of peroxyl radicals with theencapsulation systems, peroxyl radicals were generated in theaqueous phase using AAPH. Due to a relatively long half-life(approximately 175 h) of APPH in aqueous solution (Zimowskaet al., 1997), the rate of generation of peroxyl radicals can bemaintained constant for an extended duration of time. Fig. 4 (a) and

Fig. 6. Representative fluorescence microscopy images of (a) coarse zein colloidalparticles (b) coarse oil-in-water emulsion droplets encapsulating an oxygen sensitivedye. Ex/Em ¼ 540/605 nm, objective ¼ 40�, scale bar ¼ 10 mm.

es of zein colloidal particles and oil-in-water emulsions on oxidative, http://dx.doi.org/10.1016/j.foodhyd.2014.05.002

Fig. 7. Permeation of oxygen to the encapsulation core of zein colloidal particles andoil-in-water emulsion. The oxygen permeation rate was measured based on loss influorescence of a hydrophobic oxygen sensitive dye encapsulated in the core of zeincolloidal particles and emulsion. Each data point represents an average of three in-dependent measurements � standard deviation.

Y. Pan et al. / Food Hydrocolloids xxx (2014) 1e9 7

(b) show changes in fluorescence intensity of encapsulated BODIPYdye (peroxyl radical sensitive dye) as a function of time uponexposure to 10mM (Fig. 4(a)) and 20mM (Fig. 4(b)) AAPH dissolvedin the aqueous phase. At 10 mM AAPH, there was a significantdifference (p < 0.01) in the rate of fluorescence decay between zeincolloidal particles and oil-in-water emulsion. After 20 h of incu-bation, the relative fluorescence intensity for zein colloidal particleswas close to 100%, while the relative fluorescence intensity for oil-in-water emulsion decreased to approximately 35% (Fig. 4(a)). Tovalidate whether these differences in susceptibility of encapsulatedBODIPY dye to react with peroxyl radicals in the two encapsulationsystems remain consistent at higher AAPH concentration, mea-surements were also performed using 20 mM AAPH. At this level,the relative fluorescence intensity of zein colloidal particlesremained close to 100% for 9 h, after which the fluorescence in-tensity decreased steadily to 31% after additional 11 h of incubation(Fig. 4(b)). However, oil-in-water emulsion showed a steady declinein relative fluorescence intensity of the encapsulated peroxylradical sensitive dye and the relative fluorescence intensity wasapproximately 2% after 20 h of incubation with 20 mM AAPH(Fig. 4(b)). To quantify the differences in the decay of relativefluorescence intensity between zein colloidal particles and oil-in-water emulsion, the experimental data for the 20 mM AAPHtreatment was fitted to equation (3). The results of the curve fittinganalyses are shown in Table 1. Quantitative comparison of the ki-netic measurements shows that the time required for an expo-nential decay (i.e. s) in fluorescence intensity is approximately 2-fold higher in zein colloidal particles (w18 h) compared to oil-in-water emulsion (w8 h). Together, these results show that at agiven rate of peroxyl radical generation, the rate of fluorescenceloss in zein colloidal particles was significantly (p < 0.01) lowerthan that in oil-in-water emulsion.

These results demonstrate that zein colloidal particles havesignificantly (p < 0.01) higher oxidative barrier properties than oil-

Table 2Nonlinear regression fit parameters for the changes in fluorescence intensity of an oxygenfunction of time. Values in the parentheses represent 95% confidence interval.

Samples I0 s

Oil-in-water emulsion 98.67 (96.90e100.4) 4.283Zein colloidal particles 101.0 (99.60e102.3) 6.005

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in-water emulsion. Since both oil-in-water emulsion and zeincolloidal particles are stabilized by casein molecules at the inter-face, the higher resistance to permeation of peroxyl radicals in zeincolloidal particles can be attributed to differences in the corestructure between zein colloidal particles and oil-in-water emul-sion droplets. Zein colloidal particle core is formed by precipitationof zein proteins and has no liquid phase in it. Solvent evaporationprocess used for preparation of zein colloidal particles results inprecipitation of zein proteins to form a particle with a porous core.Porosity in the core of the zein colloidal particle results fromevaporation of a solvent as demonstrated in the previous studywith polymer nanoparticles (Sant et al., 2005). In contrast, the oilcore of emulsion is in liquid phase. Differences in the oxidativebarrier properties of zein colloidal particles and emulsion could beattributed to the differences in physical state of the core in thesecolloidal systems. At the lower concentration levels of AAPH (at10 mM), the peroxyl radicals were quenched upon reacting withthe zein matrix, thus no significant reduction in fluorescence ofperoxyl radical sensitive dye was observed. At the higher concen-tration levels of AAPH (at 20mM), the zeinmatrix wasmore rapidlyoxidized resulting in increased interactions of the peroxyl radicalswith encapsulated peroxyl radical sensitive dye. It is important tonote that other experimental parameters such as pH, temperatureand concentration of emulsifier weremaintained constant betweenzein colloidal particles and oil-in-water emulsion and there was nosignificant (p > 0.05) change in the size of zein colloidal particlesand oil-in-water emulsion after incubationwith 10 mM and 20mMAAPH for 24 h (data not shown).

3.4. Stability of curcumin in zein colloidal particles and oil-in-wateremulsion

Impact of structural differences between biopolymer particlesand emulsions on stability of encapsulated curcumin (a modelbioactive susceptible to oxidation) was evaluated. Curcumin is anantioxidant and has the ability to scavenge reactive free radicals(Chauhan, Kandadai, Jain, & Kumar, 2003; Priyadarsini et al., 2003).Therefore, it was selected as a model bioactive compound to eval-uate radical mediated oxidation. Fig. 5(a) compares the stability ofcurcumin in 10 mM AAPH treated and control oil-in-water emul-sions. The results show that about 78% of the encapsulated curcu-minwas degraded in AAPH treated oil-in-water emulsion after 20 hof incubation. In contrast, no significant (p > 0.05) degradation ofcurcumin was observed in the control oil-in-water emulsion(without AAPH treatment) over the same incubation time. Fig. 5(b)compares the stability of curcumin in 10 mM AAPH treated andcontrol zein colloidal particles. During the 20 h of incubation, nosignificant (p > 0.05) degradation of encapsulated curcumin wasobserved in AAPH treated zein colloidal particles. These differencesin degradation of curcumin between zein colloidal particles and oil-in-water emulsion followed a similar trend as the differences in therate of permeation of peroxyl radicals in zein colloidal particles andoil-in-water emulsion (Fig. 4(a)). These results demonstrate thatzein colloidal particles were more effective in limiting oxidativedegradation of encapsulated curcumin as compared to oil-in-wateremulsion upon exposure to AAPH. It is important to note that AAPH

sensitive dye encapsulated in zein colloidal particles and oil-in-water emulsion as a

a R2

(4.178e4.388) 1.141 (1.103e1.179) 0.99(5.881e6.128) 0.9556 (0.9346e0.9764) 0.99

es of zein colloidal particles and oil-in-water emulsions on oxidative, http://dx.doi.org/10.1016/j.foodhyd.2014.05.002

Fig. 8. Stability of encapsulated retinol in zein colloidal particles and oil-in-wateremulsion during 24 h of storage. Both zein colloidal particle and emulsion sampleswere stored in dark at room temperature for 24 h. N ¼ 3, *p< 0.05. Error bars representstandard deviations.

Y. Pan et al. / Food Hydrocolloids xxx (2014) 1e98

treatment of samples simulates an accelerated oxidative stabilityanalysis of encapsulated bioactives (Watanabe et al., 2010) and doesnot represent the shelf-life of encapsulated curcumin under normalstorage and processing conditions. These results validate thatstructural differences between biopolymer particles and emulsionscan significantly influence oxidative degradation of encapsulatedbioactives.

3.5. Permeation of oxygen across zein colloidal particles and oil-in-water emulsion

Permeation of oxygen into the encapsulation core of zeincolloidal particles and oil-in-water emulsion was measured basedon changes in fluorescence intensity of an oxygen sensitive dyeencapsulated in the core of zein colloidal particles and oil-in-wateremulsion. The fluorescence intensity of this hydrophobic oxygensensitive dye is inversely proportional to the local oxygen con-centration present in the encapsulation matrix. This approach hasbeen successfully used in our prior studies to measure diffusion ofoxygen in emulsions (Tikekar et al., 2011; Tikekar & Nitin, 2011).

Spatial distribution of the oxygen sensitive dye encapsulatedwithin the core of zein colloidal particles and oil-in-water emulsionwas also characterized using fluorescence imaging of coarse zeincolloidal particles (Fig. 6(a)) and coarse oil-in-water emulsiondroplets (Fig. 6(b)). The results show that the dye was uniformlydistributed in the core of zein colloidal particles and oil-in-wateremulsions and no exclusion of the dye was observed from thecore of zein colloidal particles and oil-in-water emulsions. Thisimaging data is in agreement with our prior measurements ofencapsulation of the oxygen sensitive dye in oil-in-water emulsions(Tikekar et al., 2011). Fig. 7 shows the rate of loss of fluorescence foroxygen sensitive dye encapsulated within nitrogen purged zeincolloidal particles and oil-in-water emulsion upon exposure to at-mospheric oxygen. It is apparent that in both samples, the fluo-rescence intensity decreased rapidly and after 15 min of exposureto atmospheric oxygen, the relative fluorescence reached 0%, indi-cating that oxygen concentration inside the encapsulation systemswas in equilibriumwith that in the aqueous phase and surroundingair (Fig. 7). To quantify differences in the decay of relative fluores-cence intensity between zein colloidal particles and oil-in-wateremulsion, the experimental data was fitted to equation (3). Theresults of the curve fitting analyses are shown in Table 2. Quanti-tative comparison of the kinetic measurements shows that the timerequired for an exponential decay (i.e. s) in fluorescence intensity is

Please cite this article in press as: Pan, Y., et al., Effect of barrier propertistability of encapsulated bioactive compounds, Food Hydrocolloids (2014)

approximately 1.4-fold longer in zein colloidal particles (w6 min)as compared to oil-in-water emulsion (w4 min). However, thisdifference may not have a meaningful impact since in both theencapsulation systems oxygen permeation was very rapid andequilibrium conditions were reached in approximately 15 min.

Based on these results, we can conclude that the rate ofpermeation of oxygen was similar in both zein colloidal particlesand oil-in-water emulsion, but the rate of permeation of peroxylradicals was significantly different between zein colloidal particlesand oil-in-water emulsion. These differences in the rates ofpermeation of oxygen and radicals indicate the inherent differencesin the chemical nature of the oxidative molecules. Since oxygen is asmall molecule and has no significant reactivity with the zeinprotein matrix, its diffusion in the core of the matrix is notrestricted by the structure of zein colloidal particle core, whileperoxyl radicals that are highly reactive can be quenched uponinteractions with the zein matrix. These results also indicate thatthe zein colloidal particle has a porous core that allows for a rapiddiffusion of oxygen. These results are in agreement with our priorstudy comparing barrier properties of solid lipid particles andnano-structured lipid carriers (NLCs) (Tikekar & Nitin, 2012). Theresults of this prior study demonstrated that the rate of permeationof peroxyl radicals was significantly influenced by the solid lipidcore fraction of NLCs, but the rate of permeation of oxygen was notsignificantly impacted.

3.6. Stability of retinol in zein colloidal particles and oil-in-wateremulsion

Fig. 8 compares the stability of encapsulated retinol in zeincolloidal particles and oil-in-water emulsion. Retinol is highlysusceptible to oxidation. Among various pathways for oxidation ofretinol, oxygen induced oxidative degradation is one of the keypathways. In this oxidation reaction, retinol reacts with oxygenresulting in the formation of a retinoid-derived peroxyl radical(Tesoriere, DArpa, Re, & Livrea, 1997). Therefore, retinol wasselected as a model bioactive compound to correlate oxygenpermeation rate within encapsulation systems with the oxidativestability of encapsulant. After 24 h of storage, approximately 50%and 60% of encapsulated retinol was degraded in zein colloidalparticle and oil-in-water emulsion samples, respectively (Fig. 8).Comparison of the rate of degradation of retinol between zeincolloidal particles and oil-in-water emulsion suggests that the rateof oxidation of retinol was marginally but significantly (p < 0.05)lower for zein colloidal particles than oil-in-water emulsion.However, more than 50% retinol was degraded at 24 h underambient conditions in both encapsulation systems (Fig. 8). Theresults indicate that both zein colloidal particles and oil-in-wateremulsion were not effective in limiting oxygen induced oxidativedegradation of encapsulated retinol. These results are in agreementwith the oxygen permeation measurements in zein colloidal par-ticles and oil-in-water emulsion (Fig. 7). To further validate that theoxidative degradation of encapsulated retinol was indeed inducedby oxygen, zein colloidal particle and oil-in-water emulsion sam-ples were purged with nitrogen for 30 min, sealed and stored indark for 24 h. After 24 h, approximately 89% and 77% retinol wasretained in zein colloidal particle and oil-in-water emulsion sam-ples, respectively (results not shown). These results validate thatpresence of oxygen is the dominant mechanism for the oxidativedegradation of encapsulated retinol.

4. Conclusions

This study compared the performance of biopolymer particlebased formulations with oil-in-water emulsions for maintaining

es of zein colloidal particles and oil-in-water emulsions on oxidative, http://dx.doi.org/10.1016/j.foodhyd.2014.05.002

Y. Pan et al. / Food Hydrocolloids xxx (2014) 1e9 9

the oxidative stability of encapsulated bioactives. The results of thisstudy demonstrated that zein colloidal particles have significantlyhigher barrier properties than oil-in-water emulsion in limitingperoxyl radical induced oxidation processes. Consequently, the rateof permeation of peroxyl radicals was significantly lower in zeincolloidal particles compared to oil-in-water emulsion. Encapsu-lated curcumin was more stable in zein colloidal particlescompared to curcumin in oil-in-water emulsions. However, neitherzein colloidal particles nor oil-in-water emulsions were capable ofreducing the rate of permeation of oxygen into the encapsulationcore. Consistent with this observation, oxygen induced oxidation ofretinol was similar in zein colloidal particles and oil-in-wateremulsion. Overall, the results of this study enable rational designand engineering of nanoscale encapsulation systems for extendedoxidative stability of bioactives.

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