ORIGINAL PAPER

Evaluating the Effectiveness of β-Carotene Extractionfrom Pulsed Electric Field-Treated Carrot Pomace UsingOil-in-Water Microemulsion

S. Roohinejad & I. Oey & D. W. Everett & B. E. Niven

Received: 2 January 2014 /Accepted: 8 May 2014 /Published online: 18 May 2014# Springer Science+Business Media New York 2014

Abstract Thermodynamically stable microemulsions wereused to extract β-carotene from pulsed electric field (PEF)-treated carrot pomace. In this study, a three-level Box–Behnken design was used to predict the effect of extractiontime (10–110 min), extraction temperature (30–70 °C) andcarrot/microemulsion ratio (1:30–1:90w/w) on the β-carotenecontent, polydispersity index (PDI) and particle size of themicroemulsions. The β-carotene extracted from PEF-treatedcarrot pomace using microemulsions was higher than untreat-ed carrot pomace. The extraction efficiency of β-caroteneusing microemulsions was higher compared to 100 % hexaneor 100 % glycerol monocaprylocaprate oil. A mathematicalmodel was developed to predict the optimal extraction condi-tions using transparent microemulsions with high loading ofβ-carotene, low PDI and small microemulsion particle size.The model predicted that an extraction time of 49.4 min,temperature of 52.2 °C and carrot/microemulsion ratio of1:70 (w/w) would result in microemulsions with β-caroteneloading of 19.6 μg/g, PDI of 0.27 and particle size of 74 nm.This study demonstrates the potential of using oil-in-watermicroemulsions as extraction media for β-carotene.

Keywords Microemulsion . Extraction . Polydispersityindex . Particle size . Optimization .β-Carotene

Introduction

Interest in the replacement of synthetic pigments bynatural colourants extracted from plant materials formaking transparent beverages has been and is increasingin recent years due to high consumer demand for ‘morenatural’ and healthy beverages (Cai et al. 2005; Lópezet al. 2009). β-Carotene is the most abundant caroten-oid, which is found in high concentrations in manyfresh fruits and vegetables, such as carrots. The impor-tance of β-carotene in food goes beyond their role asnatural colourants; there are well-documented biologicalactivities such as strengthening the immune system,decreasing the risk of cancer and reducing the risk ofcoronary heart diseases (Van Poppel and Goldbohm1995; Kritchevsky 1999).

The physical state and location of carotenoids inplants strongly affects their accessibility during diges-tion, which subsequently limits release and absorption.This can occur due to deposition of carotenoids incrystalline form in the chromoplasts, or carotene crys-tals being stabilized by other components (Reiter et al.2003). Various techniques can be used to break downplant cell structure and improve the release of caroten-oids, such as mechanical homogenization and heattreatment (Hedrén et al. 2002); however, these tech-niques may promote reactions that cause the degrada-tion of carotenoids (Fratianni et al. 2010; Talcott andHoward 1999).

Organic solvents are normally used to extract oil-solublecompounds from cellular structures after employing physicalmethods to improve extractability. Conventional carotenoidextraction methods require large amounts of organic solvents,which are costly, environmentally hazardous, and requireexpensive disposal procedures (Mustafa et al. 2012).Furthermore, the traditional extraction of carotenoids is no

S. Roohinejad : I. Oey (*) :D. W. EverettDepartment of Food Science, University of Otago, PO Box 56,Dunedin 9054, New Zealande-mail: indrawati.oey@otago.ac.nz

D. W. EverettRiddet Institute, Private Bag 11 222, Palmerston North 4442,New Zealand

B. E. NivenCentre for Application of Statistics and Mathematics, University ofOtago, PO Box 56, Dunedin 9054, New Zealand

Food Bioprocess Technol (2014) 7:3336–3348DOI 10.1007/s11947-014-1334-6

longer recommended because of the risk of organic solventresidues and loss of carotenoids as a consequence of solventevaporation (Illés et al. 1999). Recently, alternative non-thermal processing methods such as pulsed electric field(PEF) have emerged to improve the release of carotenoidsthrough increasing the permeabilization of cells (Grimi et al.2007; Roohinejad et al. 2014) without any significant effecton the stability of β-carotene or total carotenoid content(Sanchez-Moreno et al. 2005). Moreover, the use of inexpen-sive and efficient alternate extraction systems, such asmicroemulsions, might be suitable to replace the use of or-ganic solvents.

Microemulsions are defined as a system formed bythe dispersion of microdroplets of two immiscible liq-uids, stabilized by an interfacial membrane formed bysurfactants. They are thermodynamically stable, homo-geneous and optically isotropic solutions with an aver-age droplet size less than 100 nm. These delivery sys-tems are of great technological and scientific interestbecause of their potential to incorporate a wide rangeof bioactives (hydrophilic and hydrophobic) due to thepresence of both lipophilic and hydrophilic domains.These adaptable delivery systems provide protectionagainst oxidation, enzymatic hydrolysis and improvethe solubilization of lipophilic bioactives, hence enhancetheir bioavailability (Talegaonkar et al. 2008; Flanaganand Singh 2006).

Previous studies reported on the ability of microemulsionsto protect carotenoids against oxidation and increase solubili-zation (Arvanitoyannis 2009; Spernath et al. 2002). Moreover,microemulsions have attracted attention because of their ca-pability for selective extraction of biomolecules and metalions in liquid–liquid systems (Cortez et al. 2004; Dantaset al. 2003) and DNA condensation (Budker et al. 2002);however, there are very few reports on the potential ofmicroemulsions to extract food components from food com-plex mixtures (Paul and Moulik 2001). The utilization ofmicroemulsified systems for β-carotene extraction might bea preferable alternative to conventional solvent extraction dueto less hazardous solvents and lower energy consumption.

This study was designed to (1) evaluate the effectiveness ofoil-in-water (o/w) microemulsions as a potential alternative toreplace the use of organic solvents for extracting β-carotenefrom carrot pomace and (2) optimize the process conditions(extraction time, temperature and ratio) on dependent re-sponses (β-carotene content, polydispersity index (PDI) andparticle size) using a Box–Behnken design. The effect of PEFtreatment on the extractability of β-carotene from carrot pom-ace using microemulsions was initially compared to non-PEF-treated carrot pomace. The optimized extraction conditions toobtain the maximum β-carotene content, minimum PDI andsmall particle size were determined. To our knowledge, this isthe first study that has used microemulsions as an

environmentally friendly mixture for extraction of β-carotene from plant materials for use in making transparentbeverages.

Materials and Methods

Reagents

Methyl t-butyl ether and Tween 80 (polyoxyethylene sorbitanmonooleate) were purchased from Sigma-Aldrich (St. Louis,MO, USA) and n-hexane from J.T. Baker (Phillipsburg, NJ,USA). Glycerol monocaprylocaprate (Capmul MCM; CASno. 26402-26-6) was kindly donated by Abitec Corporation(Janesville, WI, USA). The fatty acid composition of CapmulMCM consists of caprylic acid (C8), capric acid (C10) andlauric acid (C12) in the ratio of 97.3:2.6:0.1. Ethanol (95 %),methanol (HPLC-grade) and acetone were purchased fromBiolab (Scoresby, Victoria, Australia). A β-carotene standardfor HPLC analysis was purchased from CaroteNature(Lupsingen, Switzerland). All solvents used in this experimentwere HPLC-grade, with the exception of ethanol.

Preparation of Pulsed Electric Field-Treated Carrot Pomace

Fresh carrots (Daucus carota cv. Nantes) harvested betweenJune and July 2013 were purchased from a supplier of locallygrown products (Kaan's Catering Supplies Ltd., Dunedin,New Zealand). Samples were washed, sliced and processedinto a purée by mixing with distilled water (1:1 ratio). PEFtreatment was carried out and compared with untreated sam-ples (control) of the corresponding batches. Carrot purée wastreated using an ELRACK-HVP 5 PEF unit (Quakenbrück,Germany) in a batch treatment configuration. For each PEFtreatment, carrot purée (100 g) was placed inside the PEFchamber of dimensions 80×100×50 mm with a sample ca-pacity of 400 mL, consisting of two stainless steel parallelplate electrodes with a gap of 80 mm. The treatment wasapplied according to the following optimum conditions: elec-tric field strength of 0.6 kV/cm with constant frequency of5 Hz, pulse width of 20 μs and treatment time of 3 ms, asdetermined from a previous study (Roohinejad et al. 2014).The temperature of the carrot purée was measured prior to andafter PEF treatment, using an electric thermometer (BAT-10thermometer, Physitemp, Clifton, NJ, USA). All samples weretreated using bipolar square pulses. PEF-treated carrot puréewas centrifuged twice at 15,300×g for 30 min at 4 °C, and thesolid phase (pomace) was separated and freeze-dried. Afterthis process, the dehydrated carrot pomace (carrot fibre) wasmilled, passed through a 14 mesh (1.19 mm) sieve and storedat −18 °C for later use. Untreated carrot pomace was alsofreeze-dried, milled, sieved and stored as for the PEF-treatedcarrot pomace.

Food Bioprocess Technol (2014) 7:3336–3348 3337

Experimental Design

A preliminary study was carried out to determine the optimalβ-carotene extraction efficiency from untreated carrot pomace(UCP) and PEF-treated carrot pomace (PCP) using an o/wmicroemulsion (Capmul MCM/Tween 80/phosphate buffer(30:20:50, w/w)). The optimum extraction conditions weredetermined by means of a three-factor, three-level Box–Behnken experimental design combined with response sur-face modelling (RSM) and quadratic programming. RSMconsists of a group of empirical techniques which are appro-priate for the evaluation of relationships between controlledexperimental factors andmeasured responses according to oneor more selected criteria (Bayraktar 2001). Using a Box–Behnken design instead of a complete factorial design hasmany advantages, including (i) they are all spherical designsand factors with only three levels required to be run, (ii) someof these designs can provide orthogonal blocking, (iii) thereare no runs where all factors are at either the maximum orminimum levels and (iv) additional experiments and timeconsuming laboratory work can be eliminated (Yetilmezsoyet al. 2009).

The conditions for optimized extraction were time of ex-traction (X1), temperature of extraction (X2) and carrot pomaceto microemulsion ratio (X3), at three equidistant levels. Theresponse variables were the β-carotene content (Y1), PDI (Y2)and particle size (Y3). A regression model containing tencoefficients including linear and quadratic effect of factorsand linear effect of interactions was assumed to describerelationships between experimental factors (X1, X2 and X3)and the responses (Y1, Y2 and Y3) as follows:

Yk ¼ β0 þX

i¼1

3

βiX i þX

i¼1

3

βiiX2i þ

X

i¼1

2 X

j¼iþ1

3

βijX i:X j

ð1Þ

where β0, βi, βii and βij are the intercept, regression coeffi-cients of the linear, quadratic and interaction terms of themodel, respectively, and Xi and Xj are the independent vari-ables and Y is the dependent variable (β-carotene content, PDIand particle size). The combination of factors at the centre oflevel was run in triplicate. The numbers of experiments (N)were calculated as follows:

N ¼ 2 k k−1ð Þ þ cp ð2Þ

where k is the number of factors and cp is the number ofreplicates at the central point. In total, 15 combinations ofthree factors were used. The extraction of carotenoids anddetermination of β-carotene was carried out using HPLC.

All experiments were carried out in a randomized order toeliminate bias. The factors, their levels and codes for the levelsare shown in Table 1. The 3D response graph and profile forpredicted values and desirability level for factors were plottedusing Minitab software (version 16, Minitab Inc., State Col-lege, PA, USA).

β-Carotene Extraction Using a Microemulsion

Pseudo-ternary phase diagrams containing oil–surfactant–wa-ter mixtures were constructed to define the microemulsionregion. Briefly, samples were prepared by mixing appropriateamounts of medium chain triglycerides (Capmul MCM),phosphate buffer (0.01 M, pH 6.8) and surfactant (Tween80) in vials at room temperature and mixed well using a vortexmixer. Buffers were prepared using NaH2PO4/Na2HPO4.NaOH (0.1 M) or HCl (0.1 M) were used to maintain the pHof the solution. The pseudo-ternary phase diagrams wereconstructed by preparing 100 samples of differing composi-tions to define the phase boundaries in each phase diagram,using SigmaPlot software (Version 12.3, Systat Software Inc.,Chicago, IL, USA). A clear, transparent o/w microemulsionliquid region with a maximum oil loading and minimumparticle size (30 % Capmul MCM and 20 % Tween 80 in50 % 0.01 M, pH 6.8 phosphate buffer) was selected for thisexperiment. Freeze-dried carrot pomace (UCP and PCP) weremixed with microemulsions, flushed with nitrogen for 1 min,placed in a thermostatic orbital shaker (Model OM 11, RatekInstrument Ltd., Boronia, Victoria, Australia) and shaken at300 rpm continuously at different temperatures (30, 40, 50, 60and 70 °C), times (10, 20, 30, 60, 90, 110 min), and carrotpomace to microemulsion ratios (1:30, 1:40, 1:60, 1:70 and1:90). Freeze-dried carrot pomace was used instead of carrotpomace due to the reason that water present in the carrotpomace will change the percentage of water in the formulationwhich consequently affects microemulsion transparency.After completing the extraction process, the suspensions were

Table 1 Levels of independent and dependent variables in Box-Behnkendesign

Independent variables Level

Low (−1) Middle (0) High (+1)

X1=time of extraction (min) 10 60 110

X2=temperature of extraction (°C) 30 50 70

X3=PCP/microemulsion ratio (w/w) 1:30 1:60 1:90

Dependent variables Goal

Y1=β-carotene Maximize

Y2=PDI Minimize

Y3=particle size Minimize

PCP pulsed electric field-treated carrot pomace, PDI polydispersity index

3338 Food Bioprocess Technol (2014) 7:3336–3348

transferred to 50 mL Polyallomer centrifuge bottle (Beckman,Fullerton, CA, USA) and centrifuged at 12,100×g for 15 minat 4 °C and the clear supernatant liquid was decanted. The β-carotene content, PDI and droplet size were determined usingthe methods described below.

Microemulsion Droplet Size, PDI and Zeta Potential Analysis

The particle size and PDI of the blank and carotenoid-loadedmicroemulsions were determined without any dilution by adynamic light scattering technique. Light scattering measure-ments were carried out using a high-performance particle sizer(HPPS; model HPP5001, Malvern Instruments Ltd.,Worcestershire, UK). The mean particle size was reported asa Z-average, which is the mean hydrodynamic diameter of theparticle derived from the cumulative analysis of the intensityof the scattered light (Schmidts et al. 2011). The zeta potentialof the microemulsions was determined using a zeta potentialanalyser (Zetasizer 3000, Malvern Instruments Ltd.) (Fanet al. 2014).

Microemulsion Stability

The stability of the optimized microemulsion prepared by theBox–Behnken mathematical model used in this study wasevaluated by analysing the β-carotene content, droplet sizeand PDI. After preparation, this microemulsion was stored at25 °C for 4 weeks and its stability was evaluated after storagefor 0, 2 and 4 weeks and compared with the predicted valuegiven by the model. If the microemulsions are not stable,separation or turbidity will be observed to increase duringthe storage time.

Encapsulation Efficiency

The β-carotene encapsulation efficiency of optimizedmicroemulsion was measured according to a method de-scribed by Qu et al. (2014). The microemulsion was pouredonto a 0.22 μm cellulose nitrate membrane (Sartorius,Göttingen, Germany) to remove the nonencapsulated β-carotene. The free β-carotene was retained in the membranewhile the filtrate containing the homogeneous suspension ofβ-carotene-loaded microemulsion was collected. The β-carotene encapsulation efficiency (DEE) was calculated asfollows:

DEE ¼ C1C2

� 100 ð3Þ

whereC1 represents the measured amount ofβ-carotene in themicroemulsion after filtration, and C2 is the total amount ofβ-carotene in the microemulsion before filtration.

Determination of Carotenoid Content in the Microemulsion

The extraction of carotenoids using a microemulsion wascarried out by solvent extraction (Wright et al. 2008).Briefly, samples were subjected to solvent extraction byadding 0.5, 3 and 1 mL of ethanol, acetone and deionizedwater, respectively, with 5 s of vortexing after the addition ofeach liquid. Hexane (2 mL) was added, the vials inverted tentimes and the organic layer was removed after 5 min using aglass transfer pipette. The hexane extraction was carried out intriplicate for each sample. The hexane extraction (6 mL) waspooled and the organic phase was dried under nitrogen at37 °C to the point of dryness. HPLC-grade methanol (1 mL)was added and vortexed for 5 s. The β-carotene content wasanalysed by HPLC after extraction.

The HPLC assay for the identification and quantification ofβ-carotene was based on the method of Lemmens et al.(2009). The separation of the β-carotene was carried out witha reversed phase C18-column (5 μm×250 mm×4.6 mm). Forthis purpose, an HPLC system equipped with a diode arraydetector (Agilent Technologies 1200 Series, Diegem,Belgium) was used. A linear gradient elution was applied toseparate the carotenoids. The gradient, starting from the initialcondition (81 % methanol, 15 % methyl t-butyl ether, 4 %reagent grade water) was built up in 20 min to the endcondition (41 % methanol, 55 % methyl t-butyl ether, 4 %reagent-grade water) at a flow rate of 1 mL/min. Identificationand quantification was carried out at 450 nm, at which pointthe maximal absorbance of β-carotene was found. The col-umn temperature and the autosampler temperature were set at25 and 4 °C, respectively. The mean value of β-carotene wasobtained from at least three independent extractions using aβ-carotene external standard curve.

Transmission Electron Microscopy

The morphology of the optimized microemulsions (blank andβ-carotene-loaded microemulsion) was examined using trans-mission electron microscopy (TEM). Samples (10 μL) wereplaced on plasma-glowed, carbon-coated, 300 mesh coppergrids (ProSciTech Pty Ltd, Thuringowa, Queensland,Australia). The carbon coating and plasma glowing steps werecarried out using an Edwards E306A coating system(Edwards Vacuum Ltd, Crawley, England). Plasma glowingrenders the carbon film hydrophilic immediately before spec-imen application to create an optimal specimen film. After60 s, the excess specimen was blotted from the grid. Thesample residue was contrasted using 1 % phosphotungsticacid at pH 6.8 before being immediately blotted again.Images were captured using a Philips CM100 BioTWINtransmission electron microscope (Philips/FEI Corporation,Eindhoven, The Netherlands) with a Mega View III digitalcamera (Olympus Soft Imaging Solutions GmbH, Münster,

Food Bioprocess Technol (2014) 7:3336–3348 3339

Germany). The TEM was operated at 100 kV acceleratingvoltage, and typically viewed at a magnification of×13,500.

Statistical Analysis

Three replications of independent experiment were carried outand triplicate samples of each experiment were analysed. Atwo-sample t test was used to compare the β-carotene con-centrations of the microemulsions obtained from untreatedand PEF-treated carrot pomace. The significance of indepen-dent variables and their interactions were analysed usingANOVA. The adequacy of the quadratic model was verifiedby ANOVA, determination coefficient (R2), adjusted determi-nation coefficient (Ra

2), correlation coefficient (R) and chi-square (χ2) tests. All statistical analyses were carried out usingMinitab software (version 16, Minitab Inc., State College, PA,USA).

Results and Discussion

Evaluation of Method

Effect of Pulsed Electric Field on Release of β-Carotene

To compare the extraction efficiency of carotenoids from PCPand UCP, a direct carotenoid extraction was carried out. Atwo-sample t test was used to compare with the extraction ofβ-carotene from PCP and UCP. In comparison to the untreatedsample, PEF treatment led to an insignificant (P>0.05) in-crease in temperature when the final temperature was below25 °C. The results showed that the β-carotene extracted fromPCPwas significantly (P

100% hexane and Capmul oil (the oil phase component of themicroemulsion). The microemulsion used in this study onlycontained 30 % oil and was able to maximally extract (up to12.75 %)β-carotene at 22.80±0.63μg/g for UCP and 24.79±0.62 μg/g for PCP (Fig. 1). The β-carotene content of PCPand UCP was also measured after hexane extraction underextraction conditions of 10 min, 30 °C and a ratio of 1:30 (w/w). The effective yield of β-carotene extracted using 100 %hexane was higher than using the microemulsion, i.e. 41.16±0.12 μg/g for UCP and 47.32±0.10 μg/g for PCP. The β-carotene content of PCP and UCP were also extracted with100 % Capmul oil at a fixed extraction time, temperature andratio of 60min, 60 °C and 1:30 (w/w), respectively. The valuesobtained from the oil extraction were 25.10±0.11 μg/g forUCP and 29.38±0.13 μg/g for PCP. The extraction efficien-cies using hexane and pure oil were lower than for themicroemulsion containing only 30 % oil, despite containing>99 % hydrophobic compounds which is more favourable todissolve carotenoids. The reasons for this may be due to (i)rehydration of carrot cells taking place during extraction withmicroemulsions which expanded the cells and increased theintracellular pressure, consequently improving the extractionefficiency; (ii) the microemulsion having a very small particlesize (0.05) when the temperature was increased to70 °C. It was previously reported that temperatures higherthan 65 °C might cause an increase in β-carotene degradation(Baysal et al. 2000). Thus, the temperature range of 50–70 °Cwas selected for further study and setting of the boundaries forthe Box–Behnken experimental design.

The effects of extraction time (10, 20, 30, 60, 90 and110 min) on β-carotene content in the microemulsions were

studied while keeping the extraction temperature and ratioconstant at 60 °C and 1:30 w/w, respectively. The resultsshowed that the concentration of β-carotene was significantlyhigher (P

27 (33) for a single replicate of a full factorial design whichreduces to 15 using a Box–Behnken experimental design. Theresults of this limited number of experiments provided astatistical model that was used to identify trends in highconcentration of β-carotene and low PDI and particle sizefor the extraction process. The data obtained in the Box–Behnken experiment was used to fit a second-order polyno-mial surface equation using three independent variables (Xvalues) for each of the three response variables (Yvalues) asdescribed by Eqs. (4) to (6):

Y 1 ¼ 21:413þ 2:267X 1 þ 2:412X 2−3:685X 3−2:545X 12−4:765X 22−2:225X 32−0:727X 1X 2−2:927X 1X 3−4:012X 2X 3

ð4Þ

Y 2 ¼ 0:320þ 0:006X 1−0:0125X 2−0:196X 3 þ 0:017X 12 þ 0:035X 22þ0:187X 32 þ 0:072X 1X 2 þ 0:020X 1X 3−0:0175X 2X 3

ð5Þ

Y 3 ¼ 152:9þ 96:04X 1 þ 10:45X 2−278:42X 3 þ 59:38X 12 þ 65:88X 22þ206:13X 32 þ 61:58X 1X 2−81:17X 1X 3 þ 64:31X 2X 3

ð6Þ

Table 2 shows that the models can adequately predict themicroemulsion properties (i.e. the predicted values of β-carotene content, PDI and particle size) compared to theresulting characteristics of the experimentally preparedmicroemulsion (i.e. the experimental values), for thepredefined extraction time, extraction temperature and PCP/microemulsion ratio combinations.

Sen and Swaminathan (2004) reported that analysis ofvariance is essential to test the significance of the model. Byapplying ANOVA to the three regression Eqs. (4) to (6), themodels were found to be significant (P0.1) if the model fits the dataadequately (Cho et al. 2013). The P values of the lack-of-fitfor the models were 0.62, 0.31 and 0.36 for β-carotene, PDIand particle size, respectively. Insignificant lack of fit, togetherwith the high values of R2, Ra

2 and R indicated that thequadratic equation was capable of representing the systemunder the given experimental domain.

The significance of the model and each coefficient wasdetermined using F test values and corresponding P valuesthat are listed in Table 3. Larger F values and smaller P valuesindicate that the parameters in the regression model are moreimportant (Yetilmezsoy et al. 2009; Khajeh 2011). In thisstudy, taking the F values (58.07, 94.78 and 4,308.26 for β-carotene, PDI and particle size, respectively) into consider-ation, all models were significant (Table 3). For β-carotenecontent, the results showed that the linear-term and quadratic-term coefficient effects of time, temperature and ratio weresignificant, as was evident from their respective F value and Pvalues (Table 3). Based on the mean squares obtained from theANOVA, the linear-term coefficient of extraction ratio (X3),quadratic-term coefficient of temperature (X2

2) and the inter-action effect of temperature and ratio (X2X3) of β-caroteneshowed the very high level of significance compared to other

3342 Food Bioprocess Technol (2014) 7:3336–3348

terms. However, no significant difference (P>0.05) was ob-served on the interaction between time and temperature(X1X2).

For PDI, the linear-term (X3), quadratic-term (X22, X3

2)coefficients and interaction of time and temperature (X1X2)were significant. On other hand, all other terms were notsignificant. Thus, the extraction ratio could be considered asthe most significant single parameter. The particle size modelshows that all linear-term coefficients (X1, X2, X3), quadratic-term coefficients (X1

2, X22, X3

2) and interaction parameters(X1X2, X1X3, X2X3) were significant (P

pomace absorbing the o/w microemulsion containing 50 %water by simple diffusion and osmosis through the porescreated during PEF treatment. This would lead to expansionof the cells and consequently β-carotene could be solubilizedin the oil matrix and released by increasing the extraction time.The maximum β-carotene content of 27.2 μg/g was obtained

at 107.0 min, 62.0 °C and 1:30 (w/w) for X1, X2 and X3,respectively.

PDI indicates the size distribution of the microemulsiondiscrete phase, which was significantly affected by the time,temperature and ratio of the extraction. The response surfacegraph (Fig. 2d–f) showed that PDI decreased by increasing the

Table 3 Regression coefficientsof the predicted quadratic poly-nomial models

df degrees of freedom, R2 deter-mination coefficient, Ra

2 adjusteddetermination coefficient, PDIpolydispersity index

Source Sum of squares df Mean square F value P value R2 Ra2

For β-carotene

Model 409.44 9 45.49 58.07 0.00 99.05 % 97.35 %

X1 41.13 1 41.13 52.50 0.00

X2 46.56 1 46.56 59.43 0.00

X3 108.63 1 108.63 138.66 0.00

X12 15.63 1 23.92 30.53 0.00

X22 78.40 1 83.85 107.02 0.00

X32 18.29 1 18.29 23.34 0.01

X1X2 2.12 1 2.12 2.70 0.16

X1X3 34.28 1 34.28 43.76 0.00

X2X3 64.40 1 64.40 82.20 0.00

Residual 3.92 5 0.78

Lack-of-fit 2.07 3 0.69 0.75 0.62

Pure error 1.84 2 0.92

For PDI

Model 0.465 9 0.052 94.78 0.000 99.42 % 98.37 %

X1 0.000 1 0.000 0.570 0.483

X2 0.001 1 0.001 2.290 0.190

X3 0.308 1 0.308 565.34 0.000

X12 0.000 1 0.001 2.07 0.209

X22 0.002 1 0.005 8.30 0.035

X32 0.130 1 0.130 238.18 0.000

X1X2 0.021 1 0.021 38.58 0.002

X1X3 0.002 1 0.002 2.94 0.147

X2X3 0.001 1 0.001 2.25 0.194

Residual 0.003 5 0.001

Lack-of-fit 0.002 3 0.001 2.360 0.31

Pure error 0.001 2 0.000

For particle size

Model 925,007 9 102,779 4,308.26 0.000 99.99 % 99.96 %

X1 73,793 1 73,793 3,093.26 0.000

X2 873 1 873 36.59 0.002

X3 620,147 1 620,147 25,995.27 0.000

X12 5,960 1 13,021 545.8 0.000

X22 9,293 1 16,023 671.67 0.000

X32 156,879 1 156,879 6,576.03 0.000

X1X2 15,168 1 15,168 635.83 0.000

X1X3 26,351 1 26,351 1,104.58 0.000

X2X3 16,542 1 16,542 693.4 0.000

Residual 119 5 24

Lack-of-fit 89 3 30 1.93 0.36

Pure error 31 2 15

3344 Food Bioprocess Technol (2014) 7:3336–3348

extraction temperature and ratio whereas it increased by in-creasing the extraction time. The minimum predicted PDI of0.20 was obtained at 10 min, 70 °C and 1:79 (w/w) for X1, X2and X3, respectively. In addition to PDI, particle size is also acrucial characteristic of microemulsion because it influencesthe bioactive release rate and absorption (Cho et al. 2013).Similar to PDI, the response surfaces (Fig. 2g–i) show thatparticle size increases with increasing the extraction tempera-ture and time, whereas it decreases when increasing the ex-traction ratio. The results clearly show that appropriate lowerextraction temperatures are important in controlling particlesize. The increase in particle size after increasing the extrac-tion temperature and time might be due to the agglomerationof particles during extraction. The minimum predicted particlesize of 46.8 nm could be obtained at 47.4 min, 44.5 °C and1:80 (w/w) for X1, X2 and X3, respectively.

All of the three responses should be evaluated in theoptimization of β-carotene microemulsion. However, it isalmost impossible to optimize all the objectives at thesame time because they do not coincide with each otherand conflict may occur between them. The optimumcondition obtained in one response may have contraryeffect on another response (Cho et al. 2013). Therefore,in order to find the best compromising formulation forall responses, the optimized formulation was achievedusing a desirability function to satisfy the desired goalssimultaneously: maximized β-carotene content and min-imized PDI and particle size. Preliminary investigationshowed that the optimum points for Y1, Y2 and Y3(Fig. 2) did not fall exactly in the same region. Thus,the multi-response optimization process was transformedinto a single response using mathematical calculation

A B C

D F

G HI

E

Fig. 2 Response surface graph for β-carotene (a–c), PDI (d–f) and particle size (g–i) from pulsed electric field-treated carrot pomace in microemulsionsas a function of temperature, time and ratio of carrot pomace to microemulsion. Variables not shown in each plot were kept constant at the centre levels

Table 4 Droplet size, polydis-persity index and zeta potential ofoptimized condition after storagefor 4 weeks at room temperature(25 °C)

Predicted value Experimental results

0 weeks 2 weeks 4 weeks

β-carotene content (μg/g) 19.62 19.81±0.13 19.79±0.11 19.76±0.11

Particle size (nm) 74.02 74.90±3.60 73.80±4.0 73.80±3.10

Polydispersity index 0.27 0.27±0.01 0.27±0.01 0.27±0.02

Zeta potential (mV) – −38±7 −37±6 −38±5

Food Bioprocess Technol (2014) 7:3336–3348 3345

(Cho et al. 2013). The results show that the optimizedlevels of X1, X2 and X3 were 49.4 min, 52.2 °C and 1:70w/w, respectively, and the predicted values of Y1, Y2 andY3 were 19.6 μg/g, 0.27 and 74.0 nm, respectively. Theindividual desirability of β-carotene, PDI and particlesize was 0.91247, 1 and 1, and the combined desirabilitywas 0.96993. To verify the precision of the model, trip-licate confirmatory samples were prepared under opti-mum conditions and the results were compared with theoptimized model.

Characterization of Microemulsion Formulation AfterExtraction Under Optimal Conditions

To validate the adequacy of the model, additional experimentswere carried out to verify the predicted values. The stability ofmicroemulsion after extraction was also monitored after 2 and4 weeks storage as shown in Table 4. Further examinationsincluding measuring the particle size, PDI and zeta potential,were carried out to anticipate the stability of the β-carotene-loaded o/w microemulsion (Table 4). The results indicate thatthe predicted values were very similar to the experimentalresults, demonstrating that coupling with a desirability func-tion could be an efficient approach to optimize the extractionof β-carotene. In the period of the stability study (2 and4 weeks), no phase separation and no considerable change inβ-carotene content, PDI, particle size or zeta potential wereobserved, indicating that the microemulsion was stable duringstorage. The zeta potential under the optimized conditions was−37.8±6.5 mV which confirmed the previous reports that±30 mVof potential would be sufficient to prevent dropletcoalescence (Cho et al. 2013).

The encapsulation efficiency of β-carotene-loadedmicroemulsions prepared under optimized condition wasevaluated by determining the difference between the totalamount of β-carotene before and after filtration. The recoveryobtained was 98.2±0.5 %, indicating high loading efficiency.This demonstrates that most of the β-carotene was entrappedwithin droplets of the microemulsion. This result was inagreement with other studies reporting the advantage of usinga microemulsion technique for encapsulation of drug andbioactive compounds to achieve a high encapsulation efficien-cy (Qu et al. 2014; Yi et al. 2012).

The appearance of the blank and β-carotene-loaded o/wwas assessed by TEM using a negative-staining technique.TEM images are presented in Fig. 3 and shows that thesubmicron-sized droplets were well dispersed without anyaggregation or cluster formation, spherical in shape and ap-preciably homogeneous in size. The sizes are in good agree-ment with the light scattering measurements; a significantincrease in droplet size was observed in the absence (64.7±1.1, PDI of 0.30±0.02) and presence (74.9±4.6, PDI of 0.27±0.01) of β-carotene.

Conclusions

This is the first reported work on the use of oil-in-watermicroemulsions as media for the effective extraction of β-carotene from carrot pomace. PEF treatment improved theextractability of β-carotene compared to untreated carrotpomace. The effects of three independent variables (extractiontime, temperature and carrot pomace to microemulsion ratio)on β-carotene content, PDI and particle size were evaluatedby a Box–Behnken design, and optimized using a desirabilityfunction. The models showed that the extraction ratio was themost significant single parameter which influenced the extrac-tion of β-carotene, PDI and particle size, followed by extrac-tion temperature and extraction time. Under optimum condi-tions, the desirable observed responses were close to thepredicted values. The suitable extraction conditions were ob-tained at extraction time 49.4 min, extraction temperature

Fig. 3 Transmission electron micrographs of blank microemulsion drop-lets (a) and β-carotene-loaded microemulsion prepared under optimizedconditions (b). Scale bar 1,000 nm and magnification ×13,500

3346 Food Bioprocess Technol (2014) 7:3336–3348

52.2 °C and extraction ratio 1:70 (w/w). Under these condi-tions, the response variables were predicted to be 19.6 μg/g,0.27 and 74 nm for β-carotene content, PDI and particle size,respectively. This transparent β-carotene-loadedmicroemulsion could be used as a promising vehicle forfortification of transparent food, beverage and pharmaceuticalproducts.

Acknowledgments This research was part of the New Zealand-Koreajoint grant programme funded by the Ministry of Business, Innovation,and Employment (MBIE). The authors gratefully acknowledge RichardEasingwood for his help with the electron microscopy techniques andUniversity of Otago for PhD Scholarship for Shahin Roohinejad.

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http://dx.doi.org/10.1111/ijfs.12510http://dx.doi.org/10.1111/ijfs.12510

Evaluating...AbstractIntroductionMaterials and MethodsReagentsPreparation of Pulsed Electric Field-Treated Carrot PomaceExperimental Designβ-Carotene Extraction Using a MicroemulsionMicroemulsion Droplet Size, PDI and Zeta Potential AnalysisMicroemulsion StabilityEncapsulation EfficiencyDetermination of Carotenoid Content in the MicroemulsionTransmission Electron MicroscopyStatistical Analysis

Results and DiscussionEvaluation of MethodEffect of Pulsed Electric Field on Release of β-CaroteneSelection of Extraction Conditions

Optimization of MethodStatistical Evaluation of the Developed Model for β-Carotene ExtractionEffects of Extraction Parameters on ResponsesCharacterization of Microemulsion Formulation After Extraction Under Optimal Conditions

ConclusionsReferences