Nanodispersing thymol in whey protein isolate-maltodextrin conjugate capsules produced using the emulsion–evaporation technique Bhavini Shah 1 , Shinya Ikeda, P. Michael Davidson, Qixin Zhong ⇑ Department of Food Science and Technology, University of Tennessee, Knoxville, TN 37996, United States article info Article history: Received 18 March 2012 Received in revised form 9 May 2012 Accepted 15 May 2012 Available online 23 May 2012 Keywords: Thymol Nanoscale delivery system Whey protein isolate Maltodextrin Conjugate Emulsion–evaporation abstract This work presents simple processes to prepare transparent aqueous dispersions of thymol, a lipophilic antimicrobial compound. The emulsion–evaporation technique involved the preparation of capsules by spray-drying oil-in-water emulsions containing thymol dissolved in hexane emulsified using conjugates of whey protein isolate and maltodextrin. Hydration of spray-dried capsules resulted in transparent and heat stable nanodispersions containing thymol at concentrations well above its solubility limit, even at pH around the isoelectric points of whey proteins. The efficiency of encapsulation and the heat-stability of nanodispersions were affected by the emulsion composition. An encapsulation efficiency of 51.4% was obtained for one sample that corresponded to dispersions, adjusted to pH 3.0–7.0, with mean diameters of <90 nm after heating at 80 °C for 15 min. The present study demonstrates a promising technology to produce nanoscale systems for delivering lipophilic components in aqueous foods such as clear beverages. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Recently, nanoscale encapsulation systems have attracted much interest to deliver lipophilic antimicrobials in foods (Moraru et al., 2009; Weiss et al., 2009). Examples of these antimicrobials include essential oils distilled from plants or plant parts (Tiwari et al., 2009) and berry extracts (Nohynek et al., 2006). While the distillates and extracts have complex compositions, phenolic compounds are responsible for the broad spectrum antimicrobial activity (Davidson and Zivanovic, 2003). The limited water solubility of antimicrobial compounds reduces their effectiveness (Sofos et al., 1998) and the homogeneity of their distribution in food matrices is required to in- sure the inhibition of microbial growth throughout food products. The effectiveness of lipophilic antimicrobials in foods is further reduced because of the interaction with and/or solubilization by hydrophobic components of foods (Davidson and Taylor, 2007). Encapsulation of lipophilic antimicrobials in nanocapsules poten- tially increases antimicrobial effectiveness by increasing the surface area available for contacting bacteria and improving dispersibility and solubility of antimicrobials in water-rich phases or solid–liquid interfaces where target microorganisms are likely to be preferen- tially located (McClements et al., 2007; Weiss et al., 2009). Further, delivery systems are expected to reduce the possible binding of anti- microbials with food constituents, protect the encapsulated com- pound from degradation, control the release rate of the encapsulated compound, and mask undesirable aroma and taste (Alamilla-Beltran et al., 2005; Weiss et al., 2009). Nanocapsules also enable the incorporation of lipophilic compounds in transparent systems because of their inability to scatter visible light (Weiss et al., 2009). Several nanoscale particulate structures have been studied as delivery systems of lipophilic antimicrobials. Oil-in-water micro- emulsions, by dissolving lipophilic antimicrobials in surfactant micelles smaller than 100 nm, are thermodynamically stable and transparent and effectively improve the antimicrobial activity of essential oil components such as eugenol and carvacrol against the growth of Listeria monocytogenes and Escherichia coli O157:H7 (Gaysinsky et al., 2005a,b, 2008, 2007). Drawbacks of microemul- sions for food applications include: (1) costly surfactants, (2) large quantities of surfactants, causing high viscosities, and (3) the use of co-surfactants such as short-chain alcohols that are typically required to achieve a moderate volume of the dispersed phase. Nanoemulsions can also be used to disperse lipophilic antimicrobi- als (Donsì et al., 2011, in press; Ziani et al., 2011) but practical methods of producing nanoemulsions are currently lacking, espe- cially for those based on generally-recognized-as-safe (GRAS) ingre- dients. Emulsion–evaporation is one of the methods to prepare nanoemulsions (McClements, 2011a,b). This is commonly fulfilled by first dissolving a lipophilic component in a volatile organic 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.05.019 ⇑ Corresponding author. Address: Department of Food Science and Technology, The University of Tennessee, 2605 River Drive, 23 Food Safety and Processing Building, Knoxville, TN 37996, United States. Tel.: +1 865 974 6196; fax: +1 865 974 7332. E-mail address: [email protected](Q. Zhong). 1 Current address: Mead Johnson & Company, LLC, Evansville, IN, United States. Journal of Food Engineering 113 (2012) 79–86 Contents lists available at SciVerse ScienceDirect Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Transcript
Journal of Food Engineering 113 (2012) 79–86
Contents lists available at SciVerse ScienceDirect
Nanodispersing thymol in whey protein isolate-maltodextrin conjugatecapsules produced using the emulsion–evaporation technique
Bhavini Shah 1, Shinya Ikeda, P. Michael Davidson, Qixin Zhong ⇑Department of Food Science and Technology, University of Tennessee, Knoxville, TN 37996, United States
a r t i c l e i n f o
Article history:Received 18 March 2012Received in revised form 9 May 2012Accepted 15 May 2012Available online 23 May 2012
Keywords:ThymolNanoscale delivery systemWhey protein isolateMaltodextrinConjugateEmulsion–evaporation
0260-8774/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.jfoodeng.2012.05.019
⇑ Corresponding author. Address: Department of FThe University of Tennessee, 2605 River Drive, 23Building, Knoxville, TN 37996, United States. Tel.: +1 87332.
E-mail address: [email protected] (Q. Zhong).1 Current address: Mead Johnson & Company, LLC, E
a b s t r a c t
This work presents simple processes to prepare transparent aqueous dispersions of thymol, a lipophilicantimicrobial compound. The emulsion–evaporation technique involved the preparation of capsules byspray-drying oil-in-water emulsions containing thymol dissolved in hexane emulsified using conjugatesof whey protein isolate and maltodextrin. Hydration of spray-dried capsules resulted in transparent andheat stable nanodispersions containing thymol at concentrations well above its solubility limit, even atpH around the isoelectric points of whey proteins. The efficiency of encapsulation and the heat-stabilityof nanodispersions were affected by the emulsion composition. An encapsulation efficiency of 51.4% wasobtained for one sample that corresponded to dispersions, adjusted to pH 3.0–7.0, with mean diametersof <90 nm after heating at 80 �C for 15 min. The present study demonstrates a promising technology toproduce nanoscale systems for delivering lipophilic components in aqueous foods such as clearbeverages.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, nanoscale encapsulation systems have attracted muchinterest to deliver lipophilic antimicrobials in foods (Moraru et al.,2009; Weiss et al., 2009). Examples of these antimicrobials includeessential oils distilled from plants or plant parts (Tiwari et al.,2009) and berry extracts (Nohynek et al., 2006). While the distillatesand extracts have complex compositions, phenolic compounds areresponsible for the broad spectrum antimicrobial activity (Davidsonand Zivanovic, 2003). The limited water solubility of antimicrobialcompounds reduces their effectiveness (Sofos et al., 1998) and thehomogeneity of their distribution in food matrices is required to in-sure the inhibition of microbial growth throughout food products.The effectiveness of lipophilic antimicrobials in foods is furtherreduced because of the interaction with and/or solubilization byhydrophobic components of foods (Davidson and Taylor, 2007).Encapsulation of lipophilic antimicrobials in nanocapsules poten-tially increases antimicrobial effectiveness by increasing the surfacearea available for contacting bacteria and improving dispersibilityand solubility of antimicrobials in water-rich phases or solid–liquidinterfaces where target microorganisms are likely to be preferen-
ll rights reserved.
ood Science and Technology,Food Safety and Processing
65 974 6196; fax: +1 865 974
vansville, IN, United States.
tially located (McClements et al., 2007; Weiss et al., 2009). Further,delivery systems are expected to reduce the possible binding of anti-microbials with food constituents, protect the encapsulated com-pound from degradation, control the release rate of theencapsulated compound, and mask undesirable aroma and taste(Alamilla-Beltran et al., 2005; Weiss et al., 2009). Nanocapsules alsoenable the incorporation of lipophilic compounds in transparentsystems because of their inability to scatter visible light (Weisset al., 2009).
Several nanoscale particulate structures have been studied asdelivery systems of lipophilic antimicrobials. Oil-in-water micro-emulsions, by dissolving lipophilic antimicrobials in surfactantmicelles smaller than 100 nm, are thermodynamically stable andtransparent and effectively improve the antimicrobial activity ofessential oil components such as eugenol and carvacrol againstthe growth of Listeria monocytogenes and Escherichia coli O157:H7(Gaysinsky et al., 2005a,b, 2008, 2007). Drawbacks of microemul-sions for food applications include: (1) costly surfactants, (2) largequantities of surfactants, causing high viscosities, and (3) the useof co-surfactants such as short-chain alcohols that are typicallyrequired to achieve a moderate volume of the dispersed phase.Nanoemulsions can also be used to disperse lipophilic antimicrobi-als (Donsì et al., 2011, in press; Ziani et al., 2011) but practicalmethods of producing nanoemulsions are currently lacking, espe-cially for those based on generally-recognized-as-safe (GRAS) ingre-dients. Emulsion–evaporation is one of the methods to preparenanoemulsions (McClements, 2011a,b). This is commonly fulfilledby first dissolving a lipophilic component in a volatile organic
Fig. 1. Principle of emulsion–evaporation using spray drying to produce powdered samples for final preparation of nanodispersions of lipophilic antimicrobials.
80 B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86
solvent to prepare an emulsion, followed by evaporating the organicsolvent from the emulsion using a rotary evaporator, as demon-strated for b-carotene (Chu et al., 2007) and corn oil or fish oil(Lee et al., 2011). However, essential oil components are typicallyvolatile and the feasibility of using emulsion–evaporation to pre-pare nanocapsules has not been reported. Further, the practicalityof preparing nanocapsules is to be considered for realisticapplications.
In this work, the feasibility of adopting spray drying in theemulsion–evaporation technique for eventual preparation of nan-odispersions of volatile essential oil was studied, based on the prin-ciple illustrated in Fig. 1. Hexane was used as a volatile solvent, andthymol, the major essential oil component from thyme, as a vola-tile lipophilic antimicrobial. Conjugates of whey protein isolate(WPI) and maltodextrin (MD) were prepared by drying heatingWPI/MD mixtures (the Maillard reaction) and used as the shellmaterial for encapsulating thymol. The efficiency of encapsulationusing the adopted processes was evaluated. The spray-dried cap-sules containing thymol were hydrated for characterization of dis-persibility, particle properties, and heat stability. The proposedapproach thus has promise to produce nanodispersions using sim-ple processes and low-cost GRAS ingredients.
2. Materials and methods
2.1. Materials
Thymol (99%) was purchased from Acros Organics (MorrisPlains, NJ). WPI was a gift from Hilmar Cheese Company (Hilmar,CA). MD products with an average dextrose equivalent (DE) of 4,10, and 18 were donated by Grain Processing Corporation (Musca-tine, IA). Other chemicals such as hexane and methanol were ob-tained from Fisher Scientific (Pittsburgh, PA).
2.2. Preparation of WPI–MD conjugates
WPI–MD conjugates were prepared by dry heating using amethod described by Akhtar and Dickinson, (2007), with somemodifications including the heating temperature, drying method,chain length of MD, and mass ratio of WPI and MD. WPI and MDwere hydrated at a mass ratio of 1:2 in deionized water for 14 hand spray-dried using a B-290 mini spray dryer (BÜCHI Labortech-nik AG, Flawil, Switzerland). Spray drying conditions included aninlet temperature of 150 �C, a compressed air pressure of 600 kPa,an air flow rate of 35 m3/h, and a feed rate of 6.67 mL/min. Thespray-dried powders were dry-heated at 90 �C for 2 h to form‘conjugates’. The conjugate powders were collected and stored ina freezer at �18 �C.
2.3. Preparation of capsules by spray-drying
Fig. 1 shows the processes of encapsulating thymol using theemulsion–evaporation. Emulsions were prepared by emulsifyingan oil phase containing thymol dissolved in hexane into an aque-ous phase containing WPI–MD conjugates, according to the formu-lations shown in Table 1. Emulsions with a total volume of 100 mLwere prepared using a Virtis-Sentry Cyclone I.Q.2 microprocessorhomogenizer operated at 15000 rpm for 3 min. The homogenizerwas equipped with a 20 mm diameter rotor–stator shaft assemblythat had a flow-through head with slotted orifices (width = 1 mm,height = 10 mm). The emulsions were then spray-dried using themini spray dryer and conditions as detailed previously.
Formulations in Table 1 were designed to study variables inemulsion preparation. The first set of emulsions (samples 1–6)was prepared using a fixed (10% v/v) volume fraction of the oilphase. The oil phase was formulated to 0.1–20% w/v thymol in hex-ane, and the aqueous phase contained a fixed amount (1% w/v) ofWPI–MD conjugates, corresponding to theoretical thymol loadinglevels of 1–69% estimated based on Eq. (1).
Theoretical loading ð%Þ ¼ ðMass of thymol in feed=Totalnon-solvent mass in feedÞ � 100 ð1Þ
The second set of emulsions (samples 7–12) also contained10% v/v volume fraction of the oil phase that was dissolved withvaried amounts (20–50% w/v) of thymol, and the aqueous phasecontained 11–28% w/v WPI–MD conjugates. The third set of emul-sions (samples 13–16) varied in volume fraction (15–30%) of the oilphase containing a fixed concentration of thymol (20% w/v), butthe aqueous phase contained a fixed amount (11% w/v) of WPI–MD conjugates. In all the preparations explained above, MD witha DE of 18 was used solely. To examine the effect of MD structure,a limited number of conjugates were prepared using MD with a DEof 4 (sample 15) and 10 (sample 16). Furthermore, controls to sam-ples 3 and 7 (samples C-3 and C-7) were prepared using mixturesof non-conjugated WPI and MD. The mixture was prepared byspray-drying a solution containing WPI and MD, but the spray-dried powder was not subjected to dry heating (at 90 �C for 2 h,as in the preparation of conjugates).
2.4. Characterization of spray-dried capsules
2.4.1. Mass yieldThe mass yield defined in Eq. (2) was used to calculate percent-
ages of the collected mass of spray-dried products with referenceto the non-solvent mass in the corresponding emulsion beforespray-drying (feed).
Table 1Formulations used to prepare emulsionsa for spray-drying and encapsulation performanceb.
a Emulsions were prepared with an oil phase of thymol in hexane and an aqueous phase of whey protein isolate (WPI)-maltodextrin (MD) conjugate (samples 1–16) ormixture (samples C-3 and C-7) in deionized water with a WPI:MD ratio of 1:2.
b Values are means ± standard errors of means from four measurements, two from each of two replicates.
B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86 81
Mass yield ð%Þ ¼ ðTotal mass of collected product=Total non-solvent mass in feedÞ � 100 ð2Þ
2.4.2. Thymol loading in spray dried powdersSpray-dried powders were dissolved at a concentration of 8 mg/
mL in 40% v/v aqueous methanol for quantification of the thymolcontent using high performance liquid chromatography (HPLC) inaccordance with a literature method (Ghosheh et al., 1999). The1200 series HPLC system from Agilent Technologies, Inc., (SantaClara, CA) was equipped with a 1200 series quaternary pump, adiode array detector, and a 1200 series vacuum degasser. A ZORBAXEclipse Plus-C18 column (Agilent Technologies, Inc., Santa Clara,CA) was used. HPLC grade water mixed with methanol at a volumeratio of 40:60 was used as the mobile phase in an isocratic mode ata flow rate of 1 mL/min. A 20 lL volume of sample was injected. UVspectra were acquired in the wavelength range between 190 and370 nm and the chromatogram at 254 nm was extracted and ana-lyzed using Chemstation Plus software (Agilent Technologies, Inc.,Santa Clara, CA). A calibration curve previously established usingstandard solutions with various thymol concentrations was usedto determine thymol concentration based on sample peak areas.The actual loading of thymol was defined in Eq. (3).
Actual loading ð%Þ ¼ ðMass of thymol in collected product=Total mass of collected productÞ � 100 ð3Þ
2.4.3. Encapsulation efficiencyThymol retention was defined as the ratio of the actual loading
of thymol (Eq. (3)) to the theoretical loading (Eq. (1)).
The encapsulation efficiency (EE) was defined as in Eq. (5) tocompare total thymol mass in a spray-dried product and the corre-sponding thymol mass in the feed prior to spray-drying.
EE ð%Þ ¼ ðMass of thymol in collected product=Mass of thymolin feedÞ � 100 ð5Þ
By the present definitions, the EE equals to the thymol retention(Eq. (4)) times the mass yield (Eq. (2)).
2.4.4. Capsule morphology2.4.4.1. Scanning electron microscopy (SEM). Spray-dried capsuleswere coated onto a two-way adhesive tape mounted on a stainlesssteel stub. After sputter-coating with a gold layer of �5 nm, struc-tures of powders were imaged using a LEO 1525 surface scanningelectron microscope (LEO Electron Microscopy, Oberkochen,Germany).
2.4.4.2. Atomic force microscopy (AFM). Spray-dried capsulesprepared from formulation 7 were hydrated at a powder concen-tration of 5% w/v in deionized water at room temperature (20 �C)for 14 h and adjusted to pH 7.0 using 1 N NaOH and 1 N HCl. Theaqueous dispersion was heated at 80 �C for 15 min, diluted to adry matter concentration of ca. 10 ppm, drop-deposited on freshlycleaved mica sheets, air-dried, and imaged using an atomic forcemicroscope (XE-100, Park Systems Inc., Santa Clara, CA) at roomtemperature. A sample assembly was mounted on the closed-loopXY scanner, brought into contact with a beam-shaped nanoprobecantilever tip (Bruker, CA) oscillated at a preset amplitude and fre-quency around a resonance frequency, and scanned using an inde-pendently operated Z scanner. Topographical images weregenerated based on vertical movements of the Z scanner.
2.4.5. Thermal stabilitySpray-dried capsules were hydrated at a powder concentration
of 5% w/v in deionized water at room temperature (20 �C) for 14 hand adjusted to pH 3.0, 5.0, and 7.0 using 1 N NaOH or 1 N HCl andan NaCl concentration of 0 and 50 mM. Two mL volumes of theaqueous dispersions were placed in 4 mL glass vials and heatedfor 15 min in a water bath maintained at 80 �C, followed by coolingin a room temperature water bath immediately.
2.4.5.1. Visual observation. To evaluate the heat stability visually,the aqueous dispersions of spray-dried capsules were photo-graphed before and after heating at 80 �C for 15 min.
82 B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86
2.4.5.2. Particle size distribution. Particle size distributions in theheated and unheated aqueous dispersions of spray-dried capsuleswere determined using Delsa™ Nano-Zeta Potential and SubmicronParticle Size Analyzer (Beckman Coulter, Inc., Brea, CA). The volume–length mean particle diameter (d4,3) was calculated using Eq. (6) inwhich ni is the number of particles corresponding to diameter di:
d4;3 ¼
X
i¼1
nid4i
X
i¼1
nid3i
ð6Þ
3. Results and discussion
3.1. Encapsulation performance
The encapsulation performance is summarized in Table 1. Thepreparations with an oil phase volume fraction of 10% v/v (samples1–10, 15, 16, C-3, and C-7) resulted in similar mass yields rangingfrom 73.8% to 82.8%. The mass yield decreased from 81.4% to 67.6%when the volume fraction of the oil phase increased from 10% v/v(sample 7) to 30% v/v (sample 14). The actual loading of thymol inspray-dried powders was much lower than the theoretical loading,corresponding to a retention of 9.2–63.4% and EE of 7.1–51.4%.From comparisons between samples 6 and 7, it is suggested thatan increase in the content of WPI–MD conjugates from 1.0% w/vto 11.1% w/v in the aqueous phase of emulsion significantly im-proved the retention of thymol in spray-dried capsules. The thymolretention decreased from 63.4% to 26.5% and EE from 51.4% to18.9% when the thymol content in the oil phase increased from20% w/v (sample 7) to 50% w/v (sample 10), regardless of the con-jugate level (16.7–27.8% w/v). The thymol retention and EE alsodecreased from 63.4% to 21.6% and from 51.4% to 14.7%, respec-tively, when the oil phase volume fraction increased from 10% v/
Fig. 2. Scanning electron micrographs of thymol-containing capsules prepar
v (sample 7) to 30% v/v (sample 14). The vapor pressure of thymolis 8.0 kPa at 150 �C (Syracuse Research Corporation, 2011), whichwas the inlet temperature during spray-drying, which causes theloss of thymol during spray-drying. In terms of the retention ofthymol, the best formulation was found to consist of 20.0% w/vthymol in hexane, 11.1% w/v conjugates in the aqueous phase,and 10% v/v oil phase (i.e., sample 7). The mass yield of this formu-lation (81.4%) was the second best among all examinedformulations.
Effects of DE of MD on thymol retention can be deduced fromcomparisons between samples 7, 15, and 16. The DE is a measureof the reducing power of MD relative to that of glucose with anidentical mass. The DE is inversely correlated with the averagemolecular weight of MD as MD consists of 1 ? 4 and 1 ? 6 linkeda-d-glucose. From the DE values of 4, 10, and 18, theoretical num-ber average molecular weights of the MD used in this study are ex-pected to be 4000, 2500, and 1000, respectively. In Table 1, boththe thymol retention and EE are shown to increase with increasingDE or decreasing molecular weight of MD. It was confirmed thatsample 7, the DE of which was the largest, had an advantage oversamples 15 and 16 in terms of the retention of thymol.
3.2. Morphology of spray-dried capsules
SEM images of spray-dried capsules show structural characteris-tics such as shape, size, and surface defects (Fig. 2). Most samplesdemonstrated spherical shell structures varying from 1 to 5 lm insize. Extensively agglomerated capsules were observed for samples1–6 that contained a relatively small amount (1% w/v) of WPI–MDconjugates in the aqueous phase during emulsion preparation. It ispossible that these capsule shells were mechanically weaker thanthose prepared from higher contents of WPI–MD conjugates andforced to aggregate upon evaporation of hexane and water during
ed from formulations in Table 1. The scanned dimension is 30 � 40 lm.
B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86 83
spray-drying. Defects such as holes and cracks were detected on thesurface of several samples (e.g. samples 9 and 14), presumably dueto evaporation of hexane. Many capsules also showed partially col-lapsed spherical structures or dents on shell structures (e.g. samples10 and 16). Ruptured capsules were also observed (e.g., samples 7and 8). Ruptures may have occurred upon rapid evaporation of vol-atile components within emulsion droplets during spray-drying ormanual scraping and collection of capsules using a spatula.
The observation based on SEM was verified using AFM. For SEMimaging, spray-dried capsules were sputter-coated with gold,while those for AFM imaging were first hydrated in water, air-dried, and then imaged without metal coating. Most particles weresmaller than 100 nm but there were several bigger structures com-posed of loosely-aggregated smaller particles (Fig. 3) assembledaround a void space similar to the shell-type structure observedin SEM (Fig. 2). This likely resulted from spray-dried capsules thatwere not fully dissociated into individual capsules upon rehydra-tion and heating at 80 �C. The presence of ruptured capsules wasevident (Fig. 3), confirming that rupture occurred during spray-drying, not during sample preparation for SEM. Furthermore, indi-vidual particles in the flocculated structure are much bigger thanindividual whey protein molecules that are �2–3 nm based onAFM (not shown) and have hydrodynamic radii of 2.6–4.9 nm forb-lactoglobulin (Parker et al., 2005), 2.0 nm for _a-lactalbumin(Molek and Zydney, 2007), and 3.7 nm for bovine serum albumin(Brownsey et al., 2003) at neutral pH. This indicates that moleculesin the shell of spray-dried capsules underwent self-assemblingprocess during hydration and possibly heating.
Fig. 3. Topographical AFM image of thymol-containing capsules prepared from sample
Fig. 4. Photographs of aqueous dispersions of thymol-containing capsules before and aft(labeled on vials) were hydrated at a content of 5% w/v in deionized water and adjusted
3.3. Thermal stability of nanodispersions
Photographs of heated and unheated aqueous dispersions ofspray-dried capsules prepared from samples 3 and 7 are presentedin Fig. 4. At all examined pH (3.0, 5.0, and 7.0) and NaCl conditions(0 and 50 mM), all dispersions remained clear after heating at 80 �Cfor 15 min, with noticeable increases in visual clarity after heating.Such observation was consistent with the results from particle sizeanalysis. The d4,3 determined from particle size distribution data(Fig. 5) for selected samples are summarized in Table 2. Disper-sions of sample 3 or 7 demonstrated d4,3 less than 250 nm beforeheating and less than 100 nm after heating (Table 2). The reducedparticle sizes after heat treatment indicate improved hydration ofspray-dried powders at an elevated temperature, which is charac-teristic of disruption of inter-particle hydrogen bonding. The d4,3 ofsample 7 increased slightly after addition of 50 mM NaCl. Theinsignificant difference between samples containing 0 and50 mM NaCl indicates the dominance of steric repulsion, thestrength of which is independent on the ionic environment fornon-ionizable MD, over electrostatic repulsion that is weakenedby the addition of NaCl.
Fig. 6 shows effects of DE of MD on the heat stability. At pH 5.0,dispersions containing sample 7 remained transparent, while thosecontaining samples 15 and 16 became turbid after heating. It iswell known that as pH approaches isoelectric point of wheyproteins (pH 5.2 for b-lactoglobulin, the most abundant whey pro-tein), electrostatic repulsion between protein molecules is remark-ably reduced, due to reduced surface net charges, and protein
7 in Table 1. The scanned dimension is 8.4 � 8.4 lm in (A) and 3.8� 3.8 lm in (B).
er heating at 80 �C for 15 min. Spray-dried capsules prepared from samples 3 and 7to pH 3.0, 5.0, and 7.0 and 0 and 50 mM NaCl before heating.
84 B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86
aggregation is facilitated (Tziboula and Donald Muir, 1993). Thepresent results suggest that steric repulsion provided by MD con-jugated with whey proteins increased with increasing DE.
3.4. Significance of conjugates to nanodispersion properties
When whey proteins are conjugated with carbohydrates, theirdenaturation properties, solubility, emulsification and emulsionstabilization properties can be significantly affected. Akhtar andDickinson, (2007) demonstrated improved solubility of WPIupon conjugation with MD even at pH 5.0, where the net charge
Fig. 5. Particle size distributions of aqueous dispersions of thymol-containing capsulessamples 3 (A), 7 (B), C-3 (C) and C-7 (D) were hydrated at a content of 5% w/v in deioni
Table 2d4,3 of thymol-containing dispersions adjusted to pH 3.0, 5.0 and 7.0 before and after hea
Sample ID NaCl (mM) d4,3 before heating (nm)
pH 3.0 pH 5.0
3 0 194 ± 4 298 ± 950 245 ± 8 317 ± 5
7 0 67 ± 1 100 ± 150 70 ± 2 113 ± 6
15 0 203 ± 2 420 ± 250 227 ± 1 367 ± 2
16 0 130 ± 2 393 ± 150 155 ± 1 451 ± 2
C-3 0 353 ± 5 1444 ± 2150 298 ± 6 3631 ± 46
C-7 0 325 ± 4 1267 ± 1950 343 ± 15 1335 ± 50
a Values are means ± standard errors of means from four measurements, two from ea
of b-lactoglobulin is close to zero and the electrostatic repulsionis not strong enough to prevent protein aggregation (Kulmyrzaevand Schubert, 2004). To illustrate the significance of conjugateson improvement of dispersion properties, conjugated (samples3 and 7) and non-conjugated (samples C-3 and C-7) WPI andMD were compared for encapsulation performance and heatstability.
At the lower level of conjugates (1% w/v) in emulsion prepara-tion (samples 3 and C-3), the retention of thymol and EE were bothrelatively low (<15%) (Table 1), presumably because the lowamount of solids resulted in the formation of thin and/or coarse
Fig. 6. Photographs of aqueous dispersions of thymol-containing capsules after heating at 80 �C for 15 min. Spray-dried capsules prepared from samples 7, 15, and 16 werehydrated at a content of 5% w/v in deionized water and adjusted to pH 3.0, 5.0, and 7.0 before heating.
Fig. 7. Photographs of aqueous dispersions of thymol-containing capsules before and after heating at 80 �C for 15 min. Spray-dried capsules prepared from samples C-3, andC-7 were hydrated at a content of 5% w/v in deionized water and adjusted to pH 3.0, 5.0, and 7.0, and 0 and 50 mM NaCl before heating.
B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86 85
shell structures that were more permeable to volatile thymol dur-ing spray-drying. For samples 7 and C-7 that contained 11.1% ofWPI–MD conjugates in the aqueous phase, the thymol retention(63.3% vs. 33.0%) and EE (51.3% vs. 25.1%) were higher for sample7. The positive impact of conjugation on the retention of thymolis consistent with improved emulsifying properties of whey pro-tein after conjugation with MD reported in the literature (Akhtarand Dickinson, 2007, Choi et al., 2010).
Spray-dried capsules prepared from samples C-3 and C-7 werehydrated at 5% w/v in deionized water, adjusted for pH and the NaClconcentration, and subjected to heat stability analyses. Visualappearance of dispersions before and after heating is presented inFig. 7 Dispersions were slightly turbid at pH 3.0 and 7.0 prior tothe heat treatment which corresponded to larger d4,3 than those ofsamples 3 and 7 (Table 2). At pH 5.0, d4,3 of samples C-3 and C-7 weregreater than 1 lm even before heating and both samples formedopaque gels after heating. No steric repulsion between capsules pre-pared from samples C-3 and C-7 is expected because MD moleculesare not covalently attached to whey proteins. Due to the absence ofsteric repulsion and remarkably reduced electrostatic repulsion, thecapsules aggregate readily at pH 5.0, eventually forming gels afterheating. The enhanced dispersibility and thermal stability of disper-sions prepared from WPI–MD conjugates supports the proposedmechanism of stabilization of nanocapsules in which MD moleculescovalently attached to whey protein molecules provide steric hin-drance against aggregation between nanocapsules even at pH 5.0.
4. Conclusions
The present study demonstrated the success of a spray drying-based emulsion–evaporation method for encapsulating volatilethymol. Upon hydration of the spray-dried powder, transparent
and heat stable nanodispersions were formed at thymol concentra-tions well above its solubility limit. The advantage of conjugationof WPI with MD over non-conjugated mixtures was demonstratedin improved dispersibility, transparency, and thermal stability. Theencapsulation performance of thymol and properties of nanodi-spersions were impacted by the content of WPI–MD conjugatesin the aqueous phase, the content of thymol in the oil phase, andthe volume fraction of the oil phase in emulsion preparation aswell as the DE of MD. The presented technology is applicable todisperse various lipophilic compounds such as antimicrobials, fla-vor oils, pigments, nutraceuticals, and fat-soluble vitamins likevitamin D in transparent beverages like clear fruit juices.
Acknowledgements
This work was supported by the USDA National Institute ofFood and Agriculture under the Project Number TEN02010-03476and The University of Tennessee Institute of Agriculture. We thankHilmar Cheese Company and Grain Processing Corporation fordonating materials.
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