Nanodispersing thymol in whey protein isolate-maltodextrin conjugate capsules produced using the emulsion–evaporation technique

Journal of Food Engineering 113 (2012) 79–86

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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

a r t i c l e

i n f o

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

a b s t r a c t 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 insure 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 potentially 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-

⇑ 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. 0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2012.05.019

tially located (McClements et al., 2007; Weiss et al., 2009). Further, delivery systems are expected to reduce the possible binding of antimicrobials with food constituents, protect the encapsulated compound 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 microemulsions, 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 microemulsions 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 antimicrobials (Donsì et al., 2011, in press; Ziani et al., 2011) but practical methods of producing nanoemulsions are currently lacking, especially for those based on generally-recognized-as-safe (GRAS) ingredients. 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

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B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86

Fig. 1. Principle of emulsion–evaporation using spray drying to produce powdered samples for final preparation of nanodispersions of lipophilic antimicrobials.

solvent to prepare an emulsion, followed by evaporating the organic solvent from the emulsion using a rotary evaporator, as demonstrated for b-carotene (Chu et al., 2007) and corn oil or fish oil (Lee et al., 2011). However, essential oil components are typically volatile and the feasibility of using emulsion–evaporation to prepare nanocapsules has not been reported. Further, the practicality of preparing nanocapsules is to be considered for realistic applications. In this work, the feasibility of adopting spray drying in the emulsion–evaporation technique for eventual preparation of nanodispersions of volatile essential oil was studied, based on the principle illustrated in Fig. 1. Hexane was used as a volatile solvent, and thymol, the major essential oil component from thyme, as a volatile lipophilic antimicrobial. Conjugates of whey protein isolate (WPI) and maltodextrin (MD) were prepared by drying heating WPI/MD mixtures (the Maillard reaction) and used as the shell material for encapsulating thymol. The efficiency of encapsulation using the adopted processes was evaluated. The spray-dried capsules containing thymol were hydrated for characterization of dispersibility, particle properties, and heat stability. The proposed approach thus has promise to produce nanodispersions using simple processes and low-cost GRAS ingredients.

2.3. Preparation of capsules by spray-drying Fig. 1 shows the processes of encapsulating thymol using the emulsion–evaporation. Emulsions were prepared by emulsifying an oil phase containing thymol dissolved in hexane into an aqueous phase containing WPI–MD conjugates, according to the formulations shown in Table 1. Emulsions with a total volume of 100 mL were prepared using a Virtis-Sentry Cyclone I.Q.2 microprocessor homogenizer operated at 15000 rpm for 3 min. The homogenizer was equipped with a 20 mm diameter rotor–stator shaft assembly that had a flow-through head with slotted orifices (width = 1 mm, height = 10 mm). The emulsions were then spray-dried using the mini spray dryer and conditions as detailed previously. Formulations in Table 1 were designed to study variables in emulsion preparation. The first set of emulsions (samples 1–6) was prepared using a fixed (10% v/v) volume fraction of the oil phase. The oil phase was formulated to 0.1–20% w/v thymol in hexane, and the aqueous phase contained a fixed amount (1% w/v) of WPI–MD conjugates, corresponding to theoretical thymol loading levels of 1–69% estimated based on Eq. (1).

Theoretical loading ð%Þ ¼ ðMass of thymol in feed=Total non-solvent mass in feedÞ  100 ð1Þ

2. Materials and methods 2.1. Materials Thymol (99%) was purchased from Acros Organics (Morris Plains, 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 (Muscatine, IA). Other chemicals such as hexane and methanol were obtained from Fisher Scientific (Pittsburgh, PA). 2.2. Preparation of WPI–MD conjugates WPI–MD conjugates were prepared by dry heating using a method described by Akhtar and Dickinson, (2007), with some modifications including the heating temperature, drying method, chain length of MD, and mass ratio of WPI and MD. WPI and MD were hydrated at a mass ratio of 1:2 in deionized water for 14 h and spray-dried using a B-290 mini spray dryer (BÜCHI Labortechnik AG, Flawil, Switzerland). Spray drying conditions included an inlet 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. The spray-dried powders were dry-heated at 90 °C for 2 h to form ‘conjugates’. The conjugate powders were collected and stored in a freezer at 18 °C.

The second set of emulsions (samples 7–12) also contained 10% v/v volume fraction of the oil phase that was dissolved with varied amounts (20–50% w/v) of thymol, and the aqueous phase contained 11–28% w/v WPI–MD conjugates. The third set of emulsions (samples 13–16) varied in volume fraction (15–30%) of the oil phase containing a fixed concentration of thymol (20% w/v), but the aqueous phase contained a fixed amount (11% w/v) of WPI– MD conjugates. In all the preparations explained above, MD with a DE of 18 was used solely. To examine the effect of MD structure, a limited number of conjugates were prepared using MD with a DE of 4 (sample 15) and 10 (sample 16). Furthermore, controls to samples 3 and 7 (samples C-3 and C-7) were prepared using mixtures of non-conjugated WPI and MD. The mixture was prepared by spray-drying a solution containing WPI and MD, but the spraydried 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 yield The mass yield defined in Eq. (2) was used to calculate percentages of the collected mass of spray-dried products with reference to the non-solvent mass in the corresponding emulsion before spray-drying (feed).

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B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86 Table 1 Formulations used to prepare emulsionsa for spray-drying and encapsulation performanceb. Sample ID

DE of MD

Thymol in hexane (% w/v)

Conjugate in water (% w/v)

Volume fraction of oil phase (% v/v)

Theoretical thymol loading (% w/w)

Mass yield (%)

Actual thymol loading (% w/w)

Thymol retention (%)

Encapsulation efficiency (EE) (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 C-3 C-7

18 18 18 18 18 18 18 18 18 18 18 18 18 18 4 10 18 18

0.1 1.3 1.8 5.0 10.0 20.0 20.0 30.0 40.0 50.0 20.0 20.0 20.0 20.0 20.0 20.0 1.8 20.0

1.0 1.0 1.0 1.0 1.0 1.0 11.1 16.7 22.2 27.8 11.1 11.1 11.1 11.1 11.1 11.1 1.0 11.1

10 10 10 10 10 10 10 10 10 10 15 20 25 30 10 10 10 10

1.1 12.2 16.7 35.7 52.6 69.0 16.7 16.7 16.7 16.7 24.1 31.1 37.5 43.6 16.7 16.7 16.7 16.7

73.8 ± 5.5 76.6 ± 4.9 78.± 4.2 82.8 ± 10.7 78.8 ± 5.2 79.7 ± 13.5 81.4 ± 3.8 78.6 ± 1.0 80.9 ± 6.7 75.8 ± 14.0 75.3 ± 5.0 74.0 ± 2.7 70.6 ± 2.2 67.6 ± 2.2 79.2 ± 1.8 80.6 ± 0.3 77.8 ± 0.3 75.6 ± 0.6

0.6 ± 0.0 6.1 ± 0.7 1.5 ± 0.4 10.5 ± 3.9 13.3 ± 0.1 15.7 ± 4.9 10.6 ± 0.9 7.8 ± 3.0 6.9 ± 0.0 4.4 ± 1.4 12.6 ± 0.3 11.7 ± 0.2 10.3 ± 0.2 9.4 ± 0.8 6.2 ± 0.0 8.5 ± 0.1 2.5 ± 0.1 5.5 ± 0.0

54.7 ± 3.1 50.3 ± 5.5 9.2 ± 2.3 29.5 ± 10.9 25.3 ± 0.3 22.8 ± 7.1 63.4 ± 5.4 47.1 ± 18.1 41.6 ± 0.3 26.5 ± 8.4 52.2 ± 1.1 37.7 ± 0.7 27.6 ± 0.5 21.6 ± 1.8 37.3 ± 0.3 50.9 ± 0.9 15.0 ± 0.8 33.0 ± 0.2

40.6 ± 5.3 38.8 ± 6.7 7.1 ± 1.5 25.6 ± 12.1 20.0 ± 1.1 17.2 ± 2.7 51.4 ± 2.0 36.9 ± 13.7 33.7 ± 3.0 18.9 ± 2.6 39.4 ± 3.4 27.9 ± 0.5 19.5 ± 0.9 14.7 ± 1.7 29.5 ± 0.9 41.0 ± 0.5 11.7 ± 0.5 25.0 ± 0.1

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) or mixture (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.

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 powders Spray-dried powders were dissolved at a concentration of 8 mg/ mL in 40% v/v aqueous methanol for quantification of the thymol content using high performance liquid chromatography (HPLC) in accordance with a literature method (Ghosheh et al., 1999). The 1200 series HPLC system from Agilent Technologies, Inc., (Santa Clara, CA) was equipped with a 1200 series quaternary pump, a diode array detector, and a 1200 series vacuum degasser. A ZORBAX Eclipse Plus-C18 column (Agilent Technologies, Inc., Santa Clara, CA) was used. HPLC grade water mixed with methanol at a volume ratio of 40:60 was used as the mobile phase in an isocratic mode at a flow rate of 1 mL/min. A 20 lL volume of sample was injected. UV spectra were acquired in the wavelength range between 190 and 370 nm and the chromatogram at 254 nm was extracted and analyzed using Chemstation Plus software (Agilent Technologies, Inc., Santa Clara, CA). A calibration curve previously established using standard solutions with various thymol concentrations was used to 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 efficiency Thymol retention was defined as the ratio of the actual loading of thymol (Eq. (3)) to the theoretical loading (Eq. (1)).

Thymol retention ð%Þ ¼ ðActual loading=Theoretical loadingÞ  100 ð4Þ The encapsulation efficiency (EE) was defined as in Eq. (5) to compare total thymol mass in a spray-dried product and the corresponding thymol mass in the feed prior to spray-drying.

EE ð%Þ ¼ ðMass of thymol in collected product=Mass of thymol in 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 morphology 2.4.4.1. Scanning electron microscopy (SEM). Spray-dried capsules were coated onto a two-way adhesive tape mounted on a stainless steel stub. After sputter-coating with a gold layer of 5 nm, structures of powders were imaged using a LEO 1525 surface scanning electron microscope (LEO Electron Microscopy, Oberkochen, Germany).

2.4.4.2. Atomic force microscopy (AFM). Spray-dried capsules prepared from formulation 7 were hydrated at a powder concentration 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. The aqueous dispersion was heated at 80 °C for 15 min, diluted to a dry matter concentration of ca. 10 ppm, drop-deposited on freshly cleaved mica sheets, air-dried, and imaged using an atomic force microscope (XE-100, Park Systems Inc., Santa Clara, CA) at room temperature. A sample assembly was mounted on the closed-loop XY scanner, brought into contact with a beam-shaped nanoprobe cantilever tip (Bruker, CA) oscillated at a preset amplitude and frequency around a resonance frequency, and scanned using an independently operated Z scanner. Topographical images were generated based on vertical movements of the Z scanner.

2.4.5. Thermal stability Spray-dried capsules were hydrated at a powder concentration of 5% w/v in deionized water at room temperature (20 °C) for 14 h and adjusted to pH 3.0, 5.0, and 7.0 using 1 N NaOH or 1 N HCl and an NaCl concentration of 0 and 50 mM. Two mL volumes of the aqueous dispersions were placed in 4 mL glass vials and heated for 15 min in a water bath maintained at 80 °C, followed by cooling in 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 photographed before and after heating at 80 °C for 15 min.

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2.4.5.2. Particle size distribution. Particle size distributions in the heated and unheated aqueous dispersions of spray-dried capsules were determined using Delsa™ Nano-Zeta Potential and Submicron Particle Size Analyzer (Beckman Coulter, Inc., Brea, CA). The volume– length mean particle diameter (d4,3) was calculated using Eq. (6) in which ni is the number of particles corresponding to diameter di:

X 4 ni di i¼1 d4;3 ¼ X 3 ni di

ð6Þ

i¼1

3. Results and discussion 3.1. Encapsulation performance The encapsulation performance is summarized in Table 1. The preparations with an oil phase volume fraction of 10% v/v (samples 1–10, 15, 16, C-3, and C-7) resulted in similar mass yields ranging from 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 in spray-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 that an increase in the content of WPI–MD conjugates from 1.0% w/v to 11.1% w/v in the aqueous phase of emulsion significantly improved the retention of thymol in spray-dried capsules. The thymol retention decreased from 63.4% to 26.5% and EE from 51.4% to 18.9% when the thymol content in the oil phase increased from 20% w/v (sample 7) to 50% w/v (sample 10), regardless of the conjugate level (16.7–27.8% w/v). The thymol retention and EE also decreased from 63.4% to 21.6% and from 51.4% to 14.7%, respectively, when the oil phase volume fraction increased from 10% v/

v (sample 7) to 30% v/v (sample 14). The vapor pressure of thymol is 8.0 kPa at 150 °C (Syracuse Research Corporation, 2011), which was the inlet temperature during spray-drying, which causes the loss of thymol during spray-drying. In terms of the retention of thymol, the best formulation was found to consist of 20.0% w/v thymol 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 formulation (81.4%) was the second best among all examined formulations. Effects of DE of MD on thymol retention can be deduced from comparisons between samples 7, 15, and 16. The DE is a measure of the reducing power of MD relative to that of glucose with an identical mass. The DE is inversely correlated with the average molecular weight of MD as MD consists of 1 ? 4 and 1 ? 6 linked a-d-glucose. From the DE values of 4, 10, and 18, theoretical number average molecular weights of the MD used in this study are expected to be 4000, 2500, and 1000, respectively. In Table 1, both the thymol retention and EE are shown to increase with increasing DE or decreasing molecular weight of MD. It was confirmed that sample 7, the DE of which was the largest, had an advantage over samples 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 characteristics such as shape, size, and surface defects (Fig. 2). Most samples demonstrated spherical shell structures varying from 1 to 5 lm in size. Extensively agglomerated capsules were observed for samples 1–6 that contained a relatively small amount (1% w/v) of WPI–MD conjugates in the aqueous phase during emulsion preparation. It is possible that these capsule shells were mechanically weaker than those prepared from higher contents of WPI–MD conjugates and forced to aggregate upon evaporation of hexane and water during

Fig. 2. Scanning electron micrographs of thymol-containing capsules prepared from formulations in Table 1. The scanned dimension is 30  40 lm.

B. Shah et al. / Journal of Food Engineering 113 (2012) 79–86

spray-drying. Defects such as holes and cracks were detected on the surface of several samples (e.g. samples 9 and 14), presumably due to evaporation of hexane. Many capsules also showed partially collapsed spherical structures or dents on shell structures (e.g. samples 10 and 16). Ruptured capsules were also observed (e.g., samples 7 and 8). Ruptures may have occurred upon rapid evaporation of volatile components within emulsion droplets during spray-drying or manual scraping and collection of capsules using a spatula. The observation based on SEM was verified using AFM. For SEM imaging, spray-dried capsules were sputter-coated with gold, while those for AFM imaging were first hydrated in water, airdried, and then imaged without metal coating. Most particles were smaller than 100 nm but there were several bigger structures composed of loosely-aggregated smaller particles (Fig. 3) assembled around a void space similar to the shell-type structure observed in SEM (Fig. 2). This likely resulted from spray-dried capsules that were not fully dissociated into individual capsules upon rehydration and heating at 80 °C. The presence of ruptured capsules was evident (Fig. 3), confirming that rupture occurred during spraydrying, not during sample preparation for SEM. Furthermore, individual particles in the flocculated structure are much bigger than individual whey protein molecules that are 2–3 nm based on AFM (not shown) and have hydrodynamic radii of 2.6–4.9 nm for b-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 molecules in the shell of spray-dried capsules underwent self-assembling process during hydration and possibly heating.

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3.3. Thermal stability of nanodispersions Photographs of heated and unheated aqueous dispersions of spray-dried capsules prepared from samples 3 and 7 are presented in 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 °C for 15 min, with noticeable increases in visual clarity after heating. Such observation was consistent with the results from particle size analysis. The d4,3 determined from particle size distribution data (Fig. 5) for selected samples are summarized in Table 2. Dispersions of sample 3 or 7 demonstrated d4,3 less than 250 nm before heating and less than 100 nm after heating (Table 2). The reduced particle sizes after heat treatment indicate improved hydration of spray-dried powders at an elevated temperature, which is characteristic of disruption of inter-particle hydrogen bonding. The d4,3 of sample 7 increased slightly after addition of 50 mM NaCl. The insignificant difference between samples containing 0 and 50 mM NaCl indicates the dominance of steric repulsion, the strength of which is independent on the ionic environment for non-ionizable MD, over electrostatic repulsion that is weakened by 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 those containing samples 15 and 16 became turbid after heating. It is well known that as pH approaches isoelectric point of whey proteins (pH 5.2 for b-lactoglobulin, the most abundant whey protein), electrostatic repulsion between protein molecules is remarkably reduced, due to reduced surface net charges, and protein

Fig. 3. Topographical AFM image of thymol-containing capsules prepared from sample 7 in Table 1. The scanned dimension is 8.4  8.4 lm in (A) and 3.8 3.8 lm in (B).

Fig. 4. Photographs of aqueous dispersions of thymol-containing capsules before and after heating at 80 °C for 15 min. Spray-dried capsules prepared from samples 3 and 7 (labeled on vials) 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.

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aggregation is facilitated (Tziboula and Donald Muir, 1993). The present results suggest that steric repulsion provided by MD conjugated with whey proteins increased with increasing DE. 3.4. Significance of conjugates to nanodispersion properties When whey proteins are conjugated with carbohydrates, their denaturation properties, solubility, emulsification and emulsion stabilization properties can be significantly affected. Akhtar and Dickinson, (2007) demonstrated improved solubility of WPI upon conjugation with MD even at pH 5.0, where the net charge

(B)

40 pH 3-before pH 3-after pH 5-before pH 5-after pH 7-before pH 7-after

20

10

0

0 10

100 Diameter (nm)

1

1000

(D)

(C) 35

pH 3-before pH 3-after pH 5-before pH 5-after: gel pH 7-before pH 7-after

30 25

pH 3-before pH 3-after pH 5-before pH 5-after pH 7-before pH 7-after

20

10

1

Frequency (%)

40

30

Frequency (%)

Frequency (%)

30

10

100 Diameter (nm)

50

20 15 10

1000

pH 3-before pH 3-after pH 5-before pH 5-after: gel pH 7-before pH 7-after

40

Frequency (%)

(A)

of b-lactoglobulin is close to zero and the electrostatic repulsion is not strong enough to prevent protein aggregation (Kulmyrzaev and Schubert, 2004). To illustrate the significance of conjugates on improvement of dispersion properties, conjugated (samples 3 and 7) and non-conjugated (samples C-3 and C-7) WPI and MD were compared for encapsulation performance and heat stability. At the lower level of conjugates (1% w/v) in emulsion preparation (samples 3 and C-3), the retention of thymol and EE were both relatively low (<15%) (Table 1), presumably because the low amount of solids resulted in the formation of thin and/or coarse

30

20

10

5 0

0 10

100

1000 Diameter (nm)

10000

10

100

1000 Diameter (nm)

10000

Fig. 5. Particle size distributions of aqueous dispersions of thymol-containing capsules before and after heating at 80 °C for 15 min. Spray-dried capsules prepared from samples 3 (A), 7 (B), C-3 (C) and C-7 (D) were hydrated at a content of 5% w/v in deionized water.

Table 2 d4,3 of thymol-containing dispersions adjusted to pH 3.0, 5.0 and 7.0 before and after heating at 80 °C for 15 mina. Sample ID

3 7 15 16 C-3 C-7 a

NaCl (mM)

0 50 0 50 0 50 0 50 0 50 0 50

d4,3 before heating (nm)

d4,3 after heating (nm)

pH 3.0

pH 5.0

pH 7.0

pH 3.0

pH 5.0

pH 7.0

194 ± 4 245 ± 8 67 ± 1 70 ± 2 203 ± 2 227 ± 1 130 ± 2 155 ± 1 353 ± 5 298 ± 6 325 ± 4 343 ± 15

298 ± 9 317 ± 5 100 ± 1 113 ± 6 420 ± 2 367 ± 2 393 ± 1 451 ± 2 1444 ± 21 3631 ± 46 1267 ± 19 1335 ± 50

104 ± 7 138 ± 3 58 ± 1 64 ± 2 124 ± 1 136 ± 2 103 ± 1 91 ± 1 596 ± 13 521 ± 3 328 ± 8 461 ± 17

54 ± 1 52 ± 2 64 ± 1 66 ± 2 99 ± 1 64 ± 1 80 ± 0 76 ± 1 1000 ± 3 988 ± 6 935 ± 14 855 ± 4

148 ± 8 165 ± 3 86 ± 1 89 ± 1 Turbid Turbid Turbid Turbid Gel Gel Gel Gel

35 ± 2 44 ± 1 52 ± 1 60 ± 2 82 ± 2 80 ± 1 96 ± 4 89 ± 1 715 ± 17 751 ± 10 765 ± 5 748 ± 9

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

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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 were hydrated 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, and C-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.

shell structures that were more permeable to volatile thymol during spray-drying. For samples 7 and C-7 that contained 11.1% of WPI–MD conjugates in the aqueous phase, the thymol retention (63.3% vs. 33.0%) and EE (51.3% vs. 25.1%) were higher for sample 7. The positive impact of conjugation on the retention of thymol is consistent with improved emulsifying properties of whey protein after conjugation with MD reported in the literature (Akhtar and Dickinson, 2007, Choi et al., 2010). Spray-dried capsules prepared from samples C-3 and C-7 were hydrated at 5% w/v in deionized water, adjusted for pH and the NaCl concentration, and subjected to heat stability analyses. Visual appearance of dispersions before and after heating is presented in Fig. 7 Dispersions were slightly turbid at pH 3.0 and 7.0 prior to the heat treatment which corresponded to larger d4,3 than those of samples 3 and 7 (Table 2). At pH 5.0, d4,3 of samples C-3 and C-7 were greater than 1 lm even before heating and both samples formed opaque gels after heating. No steric repulsion between capsules prepared from samples C-3 and C-7 is expected because MD molecules are not covalently attached to whey proteins. Due to the absence of steric repulsion and remarkably reduced electrostatic repulsion, the capsules aggregate readily at pH 5.0, eventually forming gels after heating. The enhanced dispersibility and thermal stability of dispersions prepared from WPI–MD conjugates supports the proposed mechanism of stabilization of nanocapsules in which MD molecules covalently attached to whey protein molecules provide steric hindrance against aggregation between nanocapsules even at pH 5.0. 4. Conclusions The present study demonstrated the success of a spray dryingbased emulsion–evaporation method for encapsulating volatile thymol. Upon hydration of the spray-dried powder, transparent

and heat stable nanodispersions were formed at thymol concentrations well above its solubility limit. The advantage of conjugation of WPI with MD over non-conjugated mixtures was demonstrated in improved dispersibility, transparency, and thermal stability. The encapsulation performance of thymol and properties of nanodispersions were impacted by the content of WPI–MD conjugates in the aqueous phase, the content of thymol in the oil phase, and the volume fraction of the oil phase in emulsion preparation as well as the DE of MD. The presented technology is applicable to disperse various lipophilic compounds such as antimicrobials, flavor oils, pigments, nutraceuticals, and fat-soluble vitamins like vitamin D in transparent beverages like clear fruit juices. Acknowledgements This work was supported by the USDA National Institute of Food and Agriculture under the Project Number TEN02010-03476 and The University of Tennessee Institute of Agriculture. We thank Hilmar Cheese Company and Grain Processing Corporation for donating materials. References Akhtar, M., Dickinson, E., 2007. Whey protein–maltodextrin conjugates as emulsifying agents: an alternative to gum arabic. Food Hydrocolloids 21 (4), 607–616. Alamilla-Beltran, L., Chanona-Perez, J.J., Jimenez-Aparicio, A.R., Gutierrez-Lopez, G.F., 2005. Description of morphological changes of particles along spray drying. Journal of Food Engineering 67 (1–2), 179–184. Brownsey, G.J., Noel, T.R., Parker, R., Ring, S.G., 2003. The glass transition behavior of the globular protein bovine serum albumin. Biophysical Journal 85 (6), 3943– 3950. Choi, K.-O., Ryu, J., Kwak, H.-S., Ko, S., 2010. Spray-dried conjugated linoleic acid encapsulated with Maillard reaction products of whey proteins and maltodextrin. Food Science and Biotechnology 19 (4), 957–965.

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