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Physical and storage properties of spray-dried blueberry pomace extractwith whey protein isolate as wall material

Floirendo P. Flores a,b, Rakesh K. Singh a, Fanbin Kong a,⇑aDepartment of Food Science and Technology, The University of Georgia, Athens, GA 30602-2610, USAb Institute of Food Science and Technology, University of the Philippines, College, Laguna 4031, Philippines

a r t i c l e i n f o

Article history:Received 2 January 2014Received in revised form 26 March 2014Accepted 31 March 2014Available online 8 April 2014

Keywords:BlueberriesPomaceWhey protein isolateMicroencapsulationStorageAntioxidant capacity

a b s t r a c t

In the food industry, attempts have been made to extract and encapsulate bioactives from pomace usingorganic solvents, e.g., ethanol. Ethanolic extracts contain high concentrations of bioactives, but encapsu-lation of them with whey proteins presents challenges arising from ternary phase equilibrium. This studyaimed to prepare and characterize spray-dried powders made from blueberry pomace extract and wheyproteins. The resulting microcapsules measured 48.5 lm in diameter, had 5% moisture content, and con-tained 1.32 mg cyanidin-3-O-glucoside (C3G), 2.83 mg gallic acid equivalents (GAE) and 48.52 nmol Fe(II) equivalents per gram powder. Sorption data obeyed Guggenheim–Anderson–De Boer isotherm. Stor-age tests revealed first-order degradation kinetics for monomeric anthocyanins, a two-fold increase intotal phenolics and slight increase in antioxidant capacity. Exposure to light was comparable to storageat 37 �C, but slightly more severe in decomposing monomeric anthocyanins. The spray-dried encapsu-lated powder could be used as a suitable health-promoting food ingredient.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The health-promoting properties of blueberries (Vaccinium sp.)have generated considerable interest after a report was publishedabout its high antioxidant activity among 42 fruits and vegetables(Lee and Wrolstad, 2004). Blueberries were found to be a richsource of bioactive compounds such as anthocyanins and otherflavonoids. Anthocyanins can be considered ‘‘signature’’ healthcompounds because of their greater abundance than the otherflavonoids found in blueberries, and their capability to cross theblood–brain barrier (Kalt et al., 2008). Higher concentrations ofanthocyanins and phenolics are found in the pomace, which is acommon by-product of juice processing (Khanal et al., 2012; Leeand Wrolstad, 2004). Pomace was used as an ingredient inextruded products with health benefits in vivo, such as reductionof plasma cholesterol and abdominal fat (Khanal et al., 2009,2012). Purified blueberry anthocyanin extracts were found to bemore effective than whole berries in altering the development ofobesity (Prior et al., 2010) and this could also be true with pomace.We previously compared several solvent systems in the extractionof anthocyanins from whole blueberries and pomace. Resultsshowed that the ethanolic pomace extract possessed the highest

amount of total monomeric anthocyanins and total phenolics(Flores et al., 2013).

Purified anthocyanins are labile compounds and susceptible todegradation in the presence of high pH, oxygen, heat, light andmetallic ions, among others (Castañeda-Ovando et al., 2009).Hence, microencapsulation, such as by spray drying, can be carriedout to impart protection and facilitate targeted release (Betz andKulozik, 2011). Both ethanolic and aqueous extracts from blue-berry pomace were successfully spray-dried, but the anthocyanincontent from the aqueous extract was 10-fold lower than that ofthe alcoholic extract (Jiménez-Aguilar et al., 2011; Ma and Dolan,2011). Elsewhere, ethanolic extracts were also used in spray dryingof anthocyanin-rich extracts from other botanical sources (Burinet al., 2011; Ersus and Yurdagel, 2007). Food-grade ethanolicextracts may be further processed for human consumption.

Whey proteins are by-products of cheese manufacturing withsignificant commercial potential. They possess superior gellingand emulsification properties, and an amino acid profile suitablefor protein fortification in beverages. Other health benefits associ-ated with whey proteins include antimicrobial activity, inhibitionof angiotensin-converting enzyme, and anticarcinogenic activity,among others (Chatterton et al., 2006). They can also be processedinto pH-sensitive hydrogels or nanoparticles for the controlledrelease of bioactive compounds such as anthocyanins. Whey pro-teins can thus be used as alternatives to polysaccharide-based wall

⇑ Corresponding author. Tel.: +1 (706) 542 7773; fax: +1 (706) 542 1050.E-mail address: fkong@uga.edu (F. Kong).

Reproduced from Journal of Food Engineering 137: 1-6 (2014). Floirendo Flores: Participant of the 3rd UB, 2006-2007.

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materials with relatively greater functionality (Betz and Kulozik,2011; Betz et al., 2012; Gunasekaran et al., 2007; Oidtmannet al., 2012). Consequently, we compared the in vitro release prop-erties of anthocyanin extracts spray-dried with either gum arabicor whey protein isolates (Flores et al., 2014). Results showed arapid increase in phenolics content and antioxidant activity withgum arabic particles during simulated gastric digestion, followedby a drastic decrease in antioxidant activity after simulated intes-tinal digestion. In contrast, whey protein microcapsules promoteda gradual increase in phenolics content and maintained a high levelof antioxidant activity throughout the entire digestion process.Thus, whey protein microcapsules could be utilized as an encapsu-lant for sustained-release of phenolics with high antioxidant activ-ity. However, to the best of our knowledge, whey protein isolateshave yet to be used as wall material in the spray drying of aqueous,alcoholic anthocyanin extracts. This could be due to the complextemperature and pH dependence of a ternary mixture of alcohol,water, and b-lactoglobulin, the major protein in whey (Abascaland Lencki, 2004).

The economic potential of encapsulating aqueous, alcoholicanthocyanin extracts with whey protein isolates includes greaterconcentration of bioactives and reduced energy costs due to theabsence of a lyophilization step to remove the solvent. In thisstudy, our main goal was to prepare spray-dried powder madewith whey protein isolate and an aqueous, ethanolic extract fromblueberry pomace. We also characterized the powder propertiesincluding particle size, moisture sorption isotherm, and encapsula-tion efficiency, and investigated physicochemical propertiesincluding total monomeric anthocyanin content, total phenolicscontent and antioxidant capacity as affected by storage conditions.Results of this study can be used in the development of health-promoting food ingredients.

2. Materials and methods

2.1. Materials

Ripe rabbiteye (‘‘Powderblue’’ cultivar) blueberries were har-vested from the Horticulture Research Farm of the University ofGeorgia in July 2013 and immediately frozen at �20 �C prior toprocessing. The berries were processed within two months. Wheyprotein isolate, containing at least 95% protein (BiPro�) was a kindgift from Davisco Foods International (Eden Prairie, MN).Chemicals used were reagent-grade and obtained from theSigma–Aldrich Chemical Company (St. Louis, MO).

2.2. Methods

2.2.1. Processing of anthocyanin-rich extractThe berries were thawed between 4 and 6 �C and blanched in

boiling water for 3 min. The juice was expressed using a commer-cial 2.5�L centrifugal Kuvings NJ-9310U juicer (Elk Grove Village,IL). The pomace was collected and extracted with 80% (v/v) aque-ous ethanol at a mass:volume ratio of 1:10 (pomace:solvent).Extraction was performed at room temperature (22 �C) for 24 hand at an agitation rate of 300 rpm. The extraction flasks were cov-ered with aluminum foil to protect against photodegradation. Themixture was filtered and the supernate was collected. The alcoholcontent of the supernate was measured using a Fisherbrand Model11-590 alcohol hydrometer (Fisher Scientific, Pittsburgh, PA) andwas found to be 67% (v/v). Next, the residual solvent was evapo-rated in vacuum for 30 min (Rotavapor R-124, Büchi Corp., New-castle, DE) under the following conditions: 10 kPa total pressure,40 �C bath temperature, 5 �C cooling water temperature, and

180 rpm agitation rate. The alcohol content of the resulting con-centrate was found to be 12% (v/v) and the pH was 3.4.

2.2.2. Spray drying of the blueberry extracts (BBE)Examination of the ternary diagrams developed for mixtures of

ethanol–water-b-lactoglobulin showed that at a pH of 3.0 and tem-perature of 20 �C, a transparent liquid could be obtained at low eth-anol and low-to-moderate b-lactoglobulin concentrations (Abascaland Lencki, 2004). Changes in pH during addition of whey proteinwere also considered. Results of our preliminary trials revealed anoptimummass ratio of 8:67:25 of ethanol:water:whey protein iso-late tomaximize the amount of both BBE andwhey protein at 22 �C.The final pH of the ternary mixture was 6.8. Consequently, the ter-narymixture was spray dried using a Model B-290mini spray dryer(Büchi Corporation, Flawil, Switzerland) under the following pro-cess conditions: 6 mL/min peristaltic pump speed (correspondingto 20% pump rate), 160 �C inlet air temperature; 86–90 �C outletair temperature; 100% aspirator rate (corresponding to a maximumair flow of 35 m3/h), actual air flow rate of 0.667 m3/h (40 mm Qflow), and a nozzle setting of 1 cleaning cycle/min. The powderswere collected and stored in polypropylene bottles at �20 �C.

2.2.3. Particle size distributionParticle size distribution was measured using a laser diffraction

analyzer (Model LS 13 320, Beckman Coulter Inc., Fullerton, CA)under the following conditions: 30% pump speed, 10% obscurationrate, 10 s wait before the first run, 10 s sonication at power settingof 2 before the first run, and 50 s run time. Polarization intensitydifferential scanning (PIDS) was turned on. An optical model wasdeveloped with refractive indexes of 1.333 for the fluid (water)and 1.473 for the solid particles. Volume mean diameters (D4,3),and cumulative mean diameter values corresponding to 10th and90th percentile of the distribution (d10 and d90) were reported.

2.2.4. Chemical analysesAll powders were dissolved at 0.01 g/mL for about 1 h in deion-

ized water prior to the tests. Whey protein isolate served ascontrol.

2.2.4.1. Total monomeric anthocyanin content (TMAC), total phenolicscontent (TPC) and ferric reducing antioxidant power (FRAP). The testswere conducted according to the procedures in our previous study(Flores et al., 2013). The pH differential method was used to mea-sure the total monomeric anthocyanins. Results were calculated asmg of total cyanidin-3-O-glucoside (C3G) per gram of powder. Thetotal phenolics content was measured using the Folin–Ciocalteumethod and calculated as mg gallic acid equivalent (GAE) per gramof powder. Antioxidant activity was measured by FRAP and com-puted as nmol Fe2+ equivalents per gram powder.

2.2.4.2. Encapsulation efficiency. The method of Idham et al. (2012)was employed with modifications. Fifty milligrams of the spray-dried powder was dissolved in 3 mL of 95% (v/v) ethanol in testtubes, agitated for 1 min with a vortex mixer and centrifuged for10 min at 3823 g. The supernate was assayed for surface TMACas described earlier and reported as mg surface C3G/g powder.The encapsulation efficiency is defined as follows:

% Encapsulation efficiency¼ TotalC3G=g� SurfaceC3G=gTotalC3G=g

� 100%

ð1Þ

2.2.5. Moisture sorption isothermThe integral method was employed to develop sorption iso-

therms. Powder samples were placed in tared 7-mL borosilicate

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vials and placed in 5 chambers equilibrated at different % relativehumidities (%RH). The chambers were subsequently stored awayfrom light. Saturated salt solutions were used to maintain different%RH: lithium chloride (11%), potassium acetate (22.5%), magne-sium chloride (32%), magnesium nitrate (57%) and sodium chloride(75%). The samples were equilibrated for two weeks at room tem-perature and weighed, and then for another week and re-weighed.Equilibrium was assumed when the difference between consecu-tive weights was less than 1 mg. The initial moisture content ofthe samples was obtained by vacuum drying at 65 �C for 24 h.Moisture content was computed as the ratio between the mass lostduring dehydration over the initial mass.

Equilibrium moisture content was plotted against equilibriumrelative humidity and data were modeled using Brunauer–Emmett–Teller (BET) and Guggenheim–Anderson–de Boer (GAB)isotherm equations.

BET equation :aw

ð1� awÞXe¼ 1

XmCþ awðC � 1Þ

XmCð2Þ

GAB equation :awXe

¼ aa2w þ baw þ c ð3Þ

a ¼ kXm

1C� 1

� �b ¼ 1

Xm1� 2

C

� �c ¼ 1

XmCk

where aw is water activity or equilibrium relative humidity/100, Xe

is equilibrium moisture content (dry basis), Xm is the monolayermoisture constant (dry basis), C is an empirical constant and k isrelated to the heat of sorption (Saravacos, 2005).

2.2.6. Storage testsThree different temperatures (45, 37, and 22 �C) and a light

source (40W, 260 lumens, and color temperature of 2650 K) wereused for this study. Control samples were kept at �20 �C. A sam-pling schedule that spanned a total of 6 weeks was made and phys-icochemical tests (TMAC, TPC and FRAP) were conducted at eachstorage condition. Temperature-dependent kinetics was investi-gated by comparing test results at each storage time–temperaturecombination. Samples (1 g) were placed in 7-mL screw-cappedborosilicate vials, sealed with parafilm, and stored at each temper-ature. Light-dependent kinetics was evaluated by conducting thesame tests for samples exposed to light and samples kept in thedark. Samples (0.5 g) were placed in clear polystyrene covereddishes measuring 35 mm diameter and 10 mm deep and sealedwith parafilm. Results are presented as fraction of original C3Gand ratios of mg GAE or nmol Fe2+ equivalents of sample to thatof the control.

2.3. Statistical analysis

Extractions, spray-drying, and sampling were performed induplicate, while assays were conducted in triplicate. The ProcGLM and Proc REG functions of SAS 9.3 (SAS Inst., Cary, NC) wereused to analyze one-way design data and lack-of-fit tests, respec-tively. Tukey’s honestly significant difference was employed asposthoc test and means were considered significantly different atp < 0.05.

3. Results and discussion

Generally, processes to microencapsulate extracts vary depend-ing on solvents and wall materials. Aqueous extracts were usedbecause of the simplicity of the process, but the resulting spray-dried powders contained less bioactive compounds (Fang andBhandari, 2012; Ma and Dolan, 2011). Ethanol-based extraction

methods have been used in several papers, usually followed byrotary evaporation to achieve a desired solids concentration. Poly-saccharides are preferred over proteins as encapsulating materialsbecause of higher retention of phenolics content and antioxidantactivity upon storage (Burin et al., 2011; Idham et al., 2012;Jiménez-Aguilar et al., 2011; Tonon et al., 2009). However, wheyprotein was a better encapsulant than gum arabic in terms of sus-taining a high level of antioxidant activity in vitro (Flores et al.,2014). Whey protein isolate and maltodextrin were compared aswall materials for spray drying of bayberry juice and resultsshowed that on a mass basis, less proteins were required to encap-sulate a known amount of extracts (Fang and Bhandari, 2012).Whey proteins were previously used in encapsulating anthocyaninextracts prepared as emulsions (Betz et al., 2012; Oidtmann et al.,2012). A process to microencapsulate alcoholic extracts with wheyprotein, however, has not yet been reported. This is possibly due toa complex temperature- and pH-dependent ternary phase equilib-rium that exists among whey proteins, water and ethanol (Abascaland Lencki, 2004).

In this study, we were able to demonstrate a process to recoverbioactive compounds from blueberry pomace, encapsulate theextracts, and monitor the changes in physicochemical propertieswith time. In contrast to published methods that measured solidscontent (Idham et al., 2012; Jiménez-Aguilar et al., 2011), we con-trolled the concentration of alcohol in the mixture prior to additionof whey protein isolate, so that a stable, homogeneous mixturecould be spray-dried. The final alcohol concentration prior to spraydrying employed in one study (Burin et al., 2011) was too high topermit significant addition of whey protein isolate. The anthocya-nin content of our spray-dried extract from blueberry pomace wasof the same magnitude to that reported for spray-dried juiceextract from cull blueberries without the use of ethanol (Ma andDolan, 2011). We also used a relatively more dilute concentrationof ethanol (80% by volume) compared to that used in other studies(Idham et al., 2012; Jiménez-Aguilar et al., 2011).

3.1. Powder characteristics

Fig. 1 shows the mean particle size distributions of the spray-dried BBE and the whey protein isolate. The volumetric meandiameters (D4,3) of the spray-dried powder and the whey proteinisolate control are 48.5 and 86.8 lm, respectively. Both distribu-tions possessed comparable cumulative mean diameters for 10%of the distribution (d10) [BBE = 11.9 lm, WPI = 13.2 lm] but variedin the 90th percentiles [BBE = 82.8 lm, WPI = 175.2 lm]. The sizedistribution within the 90th percentile was unimodal for spray-dried BBE, while multiple peaks were found in the WPI control.

0

1

2

3

4

5

6

1 10 100 1000 10000

Volu

me

%

Particle size, µm

Fig. 1. Particle size distributions of the spray-dried (SD) alcoholic extract (d) fromblueberry pomace and whey protein isolate (WPI, h).

materials with relatively greater functionality (Betz and Kulozik,2011; Betz et al., 2012; Gunasekaran et al., 2007; Oidtmannet al., 2012). Consequently, we compared the in vitro release prop-erties of anthocyanin extracts spray-dried with either gum arabicor whey protein isolates (Flores et al., 2014). Results showed arapid increase in phenolics content and antioxidant activity withgum arabic particles during simulated gastric digestion, followedby a drastic decrease in antioxidant activity after simulated intes-tinal digestion. In contrast, whey protein microcapsules promoteda gradual increase in phenolics content and maintained a high levelof antioxidant activity throughout the entire digestion process.Thus, whey protein microcapsules could be utilized as an encapsu-lant for sustained-release of phenolics with high antioxidant activ-ity. However, to the best of our knowledge, whey protein isolateshave yet to be used as wall material in the spray drying of aqueous,alcoholic anthocyanin extracts. This could be due to the complextemperature and pH dependence of a ternary mixture of alcohol,water, and b-lactoglobulin, the major protein in whey (Abascaland Lencki, 2004).

The economic potential of encapsulating aqueous, alcoholicanthocyanin extracts with whey protein isolates includes greaterconcentration of bioactives and reduced energy costs due to theabsence of a lyophilization step to remove the solvent. In thisstudy, our main goal was to prepare spray-dried powder madewith whey protein isolate and an aqueous, ethanolic extract fromblueberry pomace. We also characterized the powder propertiesincluding particle size, moisture sorption isotherm, and encapsula-tion efficiency, and investigated physicochemical propertiesincluding total monomeric anthocyanin content, total phenolicscontent and antioxidant capacity as affected by storage conditions.Results of this study can be used in the development of health-promoting food ingredients.

2. Materials and methods

2.1. Materials

Ripe rabbiteye (‘‘Powderblue’’ cultivar) blueberries were har-vested from the Horticulture Research Farm of the University ofGeorgia in July 2013 and immediately frozen at �20 �C prior toprocessing. The berries were processed within two months. Wheyprotein isolate, containing at least 95% protein (BiPro�) was a kindgift from Davisco Foods International (Eden Prairie, MN).Chemicals used were reagent-grade and obtained from theSigma–Aldrich Chemical Company (St. Louis, MO).

2.2. Methods

2.2.1. Processing of anthocyanin-rich extractThe berries were thawed between 4 and 6 �C and blanched in

boiling water for 3 min. The juice was expressed using a commer-cial 2.5�L centrifugal Kuvings NJ-9310U juicer (Elk Grove Village,IL). The pomace was collected and extracted with 80% (v/v) aque-ous ethanol at a mass:volume ratio of 1:10 (pomace:solvent).Extraction was performed at room temperature (22 �C) for 24 hand at an agitation rate of 300 rpm. The extraction flasks were cov-ered with aluminum foil to protect against photodegradation. Themixture was filtered and the supernate was collected. The alcoholcontent of the supernate was measured using a Fisherbrand Model11-590 alcohol hydrometer (Fisher Scientific, Pittsburgh, PA) andwas found to be 67% (v/v). Next, the residual solvent was evapo-rated in vacuum for 30 min (Rotavapor R-124, Büchi Corp., New-castle, DE) under the following conditions: 10 kPa total pressure,40 �C bath temperature, 5 �C cooling water temperature, and

180 rpm agitation rate. The alcohol content of the resulting con-centrate was found to be 12% (v/v) and the pH was 3.4.

2.2.2. Spray drying of the blueberry extracts (BBE)Examination of the ternary diagrams developed for mixtures of

ethanol–water-b-lactoglobulin showed that at a pH of 3.0 and tem-perature of 20 �C, a transparent liquid could be obtained at low eth-anol and low-to-moderate b-lactoglobulin concentrations (Abascaland Lencki, 2004). Changes in pH during addition of whey proteinwere also considered. Results of our preliminary trials revealed anoptimummass ratio of 8:67:25 of ethanol:water:whey protein iso-late tomaximize the amount of both BBE andwhey protein at 22 �C.The final pH of the ternary mixture was 6.8. Consequently, the ter-narymixture was spray dried using a Model B-290mini spray dryer(Büchi Corporation, Flawil, Switzerland) under the following pro-cess conditions: 6 mL/min peristaltic pump speed (correspondingto 20% pump rate), 160 �C inlet air temperature; 86–90 �C outletair temperature; 100% aspirator rate (corresponding to a maximumair flow of 35 m3/h), actual air flow rate of 0.667 m3/h (40 mm Qflow), and a nozzle setting of 1 cleaning cycle/min. The powderswere collected and stored in polypropylene bottles at �20 �C.

2.2.3. Particle size distributionParticle size distribution was measured using a laser diffraction

analyzer (Model LS 13 320, Beckman Coulter Inc., Fullerton, CA)under the following conditions: 30% pump speed, 10% obscurationrate, 10 s wait before the first run, 10 s sonication at power settingof 2 before the first run, and 50 s run time. Polarization intensitydifferential scanning (PIDS) was turned on. An optical model wasdeveloped with refractive indexes of 1.333 for the fluid (water)and 1.473 for the solid particles. Volume mean diameters (D4,3),and cumulative mean diameter values corresponding to 10th and90th percentile of the distribution (d10 and d90) were reported.

2.2.4. Chemical analysesAll powders were dissolved at 0.01 g/mL for about 1 h in deion-

ized water prior to the tests. Whey protein isolate served ascontrol.

2.2.4.1. Total monomeric anthocyanin content (TMAC), total phenolicscontent (TPC) and ferric reducing antioxidant power (FRAP). The testswere conducted according to the procedures in our previous study(Flores et al., 2013). The pH differential method was used to mea-sure the total monomeric anthocyanins. Results were calculated asmg of total cyanidin-3-O-glucoside (C3G) per gram of powder. Thetotal phenolics content was measured using the Folin–Ciocalteumethod and calculated as mg gallic acid equivalent (GAE) per gramof powder. Antioxidant activity was measured by FRAP and com-puted as nmol Fe2+ equivalents per gram powder.

2.2.4.2. Encapsulation efficiency. The method of Idham et al. (2012)was employed with modifications. Fifty milligrams of the spray-dried powder was dissolved in 3 mL of 95% (v/v) ethanol in testtubes, agitated for 1 min with a vortex mixer and centrifuged for10 min at 3823 g. The supernate was assayed for surface TMACas described earlier and reported as mg surface C3G/g powder.The encapsulation efficiency is defined as follows:

% Encapsulation efficiency¼ TotalC3G=g� SurfaceC3G=gTotalC3G=g

� 100%

ð1Þ

2.2.5. Moisture sorption isothermThe integral method was employed to develop sorption iso-

therms. Powder samples were placed in tared 7-mL borosilicate

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The particle size of the spray-dried BBE (48.5 lm) was comparableto a range (32–63 lm) reported for another spray-dried blueberrypowder (Ma and Dolan, 2011) and smaller than either the microen-capsulated emulsions (200 lm) made with anthocyanin extractsand whey protein isolates or the spray-dried powder made (250–500 lm) with maltodextrin and pectin (Oidtmann et al., 2012).

Analysis of total and surface anthocyanins revealed that thespray drying process was 70% efficient in encapsulating mono-meric anthocyanins. In addition, the spray-dried BBE was foundto contain an average of 1.32 mg C3G, 2.83 mg GAE, and48.52 nmol Fe (II) per g spray-dried powder. Our spray-dried BBEaveraged 5% moisture content dry basis, which is comparable tothat reported in other papers (Fang and Bhandari, 2012; Pitaluaet al., 2010). In contrast, the spray-dried blueberry extract reportedin another paper contained much higher moisture (15–20%) andconsequently higher monolayer values (Jiménez-Aguilar et al.,2011). The final moisture content of the spray-dried product influ-ences the extent of water diffusion, and values less than 7% aregenerally considered to reduce moisture migration (Pitalua et al.,2010). Due to the variety of characterization methods and unitsof measurement available in the literature, it was difficult to makea comprehensive comparison of other powder characteristics, suchas phenolics content and antioxidant capacity.

The sorption isotherms of spray-dried BBE and correspondingmodels are shown in Fig. 2. Lack-of-fit tests were conducted forthe entire water activity range. Results showed that both GABand BET isotherms have comparable values of coefficient of deter-mination, R2 (0.92 and 0.96, respectively) but the GAB is the ade-quate model (p > 0.05). Further, one coefficient (the y-intercept)of the BET equation was found to be statistically insignificant. Datawere also truncated to remove the values at the highest testedwater activity (0.75). On the basis of higher R2 of the linear model(R2 = 0.99) and the lack-of-fit test (R2 = 0.99), the BET equation wasmore adequate than the GAB equation. Consequently, the parame-ters of the GAB equation for the entire data range are as follows:C = 9.35, k = 0.84 and Xm (monolayer value) = 7.42 g water/100 gdry solids. For the BET equation, the values of the parameters areas follows: C = 13.85 and Xm = 5.92 g water/100 g dry solids. In bothcases, the calculated monolayer values were greater than the mois-ture content of the spray-dried BBE.

3.2. Changes in TMAC, TPC and FRAP during storage at differentconditions

The rate of anthocyanin degradation as a function of time isshown in Fig. 3. Anthocyanin loss followed the first-order kinetics

and the following rate constants were calculated:k45�C = 0.0182 d�1, klight = 0.0083 d�1, k37�C = 0.0069 d�1 andk22�C = 0.0031 d�1. The rate of monomeric anthocyanin loss undervarious storage conditions was in the order: 45 �C > light >37 �C > 22 �C. The disadvantage of using whey protein as a wallmaterial in spray drying arises from significantly higher rates ofanthocyanin degradation compared with polysaccharide-basedwall materials (Idham et al., 2012; Tonon et al., 2009). The temper-ature-dependence of the rate constants was modeled by Arrheniusequation (Fig. 4). The calculated energy of activation (Ea) was equalto 62.46 kJ/mol, while the preexponential factor A was equivalentto 2.81 � 108 d�1. The parameters of the modeled Arrhenius equa-tion were statistically significant and the equation was adequatebased on lack-of-fit tests (R2 = 0.82, p = 0.19). Higher values of Eaare usually associated with resistance to thermal degradation(Fischer et al., 2013).

Fig. 5 shows the variation of the total phenolics as a function oftime. The total phenolics increased across all test conditions andeventually the ratio of final to initial GAE ranged between 2 and2.5. The initial increase in total phenolics was greater at elevatedtemperatures compared to lower temperatures. The effect of pho-todegradation was comparable to storage at 37 �C. Similarly, theantioxidant capacity of the samples increased upon storage(Fig. 6). Interestingly, however, the final ferric reducing antioxidantpower varied proportionally with increasing temperatures. Fur-ther, the effect of photodegradation was again comparable to stor-age at 37 �C. The final concentration of Fe (II) equivalents rangedfrom unity (22 �C) to a peak of 1.25 (45 �C).

Generally, it is accepted that the concentration of monomericanthocyanins decreases with an increase in temperature. However,thermal effects on phenolics content and antioxidant activity arenot clear. The phenolics content and antioxidant activities ofspray-dried açai and blueberry both decreased under prolongedstorage (Jiménez-Aguilar et al., 2011; Tonon et al., 2009). The rateof decrease in phenolics content and antioxidant activity appearedto be linear for the non-encapsulated control and non-linear for theencapsulated extracts, but mathematical information regarding theobserved trends was not discussed (Jiménez-Aguilar et al., 2011).With our samples, there was a slight increase in antioxidant activ-ity and a two-fold increase in phenolics content. The plots in Fig. 5suggest that the phenolics content ratio stabilizes at a valuebetween 2 and 2.5. A linear relationship may be proposed for22 �C but may need prolonged storage conditions longer than thatnormally considered in the literature. Similarly, the considerablyhigher standard deviations in antioxidant activity observed forsamples stored at 37 �C and 22 �C (Fig. 6) seem to support the con-clusion that bioactivity may be conserved because an increase in

Fig. 2. Modeling of the experimental sorption isotherm based on the BET and GABequations.

Fig. 3. Rate of monomeric anthocyanin loss modeled after first-order kinetics.Legend: r 45 �C, 37 �C, 4 22 �C, light.

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phenolics content compensates for any loss of monomeric antho-cyanins (Sadilova et al., 2007). Divergent results are also foundfor extracts and free polyphenols from other botanical sources.Several papers showed that the total antioxidant capacityremained unchanged or increased only slightly as the total antho-cyanins decreased (Hager et al., 2008; Nayak et al., 2011). Upon

heating, anthocyanins were found to decompose to phloroglucinal-dehyde and benzoic acid derivatives such as syringic acid or 4-hydroxybenzoic acid (De Villiers et al., 2009; Patras et al., 2010).However, the rate-limiting step, which is usually used for kineticmodeling, has not yet been identified. Further, anthocyanidinshave varying sensitivity to temperature increase, with cyanidinsbeing more sensitive to thermal decomposition than delphinidin.The use of FRAP to measure antioxidant capacity may also leadto a redox reaction between ferric ions and some anthocyaninsand accelerate degradation of delphinidin but not cyanidin(Xiong et al., 2006). This could impact processing of blueberriesfrom different cultivars. Besides decomposition, anthocyaninsmay also polymerize upon heating or prolonged storage (Hageret al., 2008). Thermally produced phenolic compounds from degra-dation or polymerization may partially or fully compensate for lossin antioxidant activity arising from decreased monomeric anthocy-anins (Fischer et al., 2013; Sadilova et al., 2007). In our case, theincrease in both phenolics content and antioxidant capacity ofthe spray-dried BBE upon storage implies that the powdered BBEcan be used as a food ingredient with health-promoting properties.

Fang and Bhandari (2012) evaluated the surface atomic compo-sition of the encapsulated, spray-dried whey protein-blueberryextracts and concluded that the encapsulated extracts promotedthe surface migration of proteins compared to the whey proteincontrol. Using the same technique, our preliminary results (datanot shown) revealed no significant variation between samplesand control, even after accelerated shelf life tests. Hence, the extentof thermal- and photo-degradation on surface phenolic compoundsand the encapsulated fraction remains unclear and may be the sub-ject of future investigations.

4. Conclusions

Microencapsulated blueberry powder was successfully pre-pared from an aqueous, ethanolic pomace extract and whey pro-tein isolate. Anthocyanin degradation followed the first-orderkinetics. Photodegradation was comparable to storage at 37 �C interms of rate of phenolic increase and antioxidant activity, butwas more severe in the rate of loss of monomeric anthocyanins.Upon storage, the total phenolics concentration increased approx-imately two-fold across all storage conditions, while the antioxi-dant capacity increased only slightly. Based on these results, thespray-dried product may be further studied in developing a foodingredient that promotes health.

Acknowledgment

We are deeply grateful to Dr. Anish Malladi of the Departmentof Horticulture, College of Agriculture and Environmental Sciences,University of Georgia for the blueberry fruits used in this study.

References

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Fig. 4. Arrhenius plot showing (–) natural logarithm of rate constants formonomeric anthocyanin loss vs inverse of absolute temperature.

Fig. 5. Changes in phenolics content with time of the spray-dried powder uponstorage. Legend: r 45 �C, 37 �C, 4 22 �C, light.

Fig. 6. Changes in ferric reducing antioxidant power with time of the spray-driedpowder upon storage. Legend: r 45 �C, 37 �C, 422 �C, light.

The particle size of the spray-dried BBE (48.5 lm) was comparableto a range (32–63 lm) reported for another spray-dried blueberrypowder (Ma and Dolan, 2011) and smaller than either the microen-capsulated emulsions (200 lm) made with anthocyanin extractsand whey protein isolates or the spray-dried powder made (250–500 lm) with maltodextrin and pectin (Oidtmann et al., 2012).

Analysis of total and surface anthocyanins revealed that thespray drying process was 70% efficient in encapsulating mono-meric anthocyanins. In addition, the spray-dried BBE was foundto contain an average of 1.32 mg C3G, 2.83 mg GAE, and48.52 nmol Fe (II) per g spray-dried powder. Our spray-dried BBEaveraged 5% moisture content dry basis, which is comparable tothat reported in other papers (Fang and Bhandari, 2012; Pitaluaet al., 2010). In contrast, the spray-dried blueberry extract reportedin another paper contained much higher moisture (15–20%) andconsequently higher monolayer values (Jiménez-Aguilar et al.,2011). The final moisture content of the spray-dried product influ-ences the extent of water diffusion, and values less than 7% aregenerally considered to reduce moisture migration (Pitalua et al.,2010). Due to the variety of characterization methods and unitsof measurement available in the literature, it was difficult to makea comprehensive comparison of other powder characteristics, suchas phenolics content and antioxidant capacity.

The sorption isotherms of spray-dried BBE and correspondingmodels are shown in Fig. 2. Lack-of-fit tests were conducted forthe entire water activity range. Results showed that both GABand BET isotherms have comparable values of coefficient of deter-mination, R2 (0.92 and 0.96, respectively) but the GAB is the ade-quate model (p > 0.05). Further, one coefficient (the y-intercept)of the BET equation was found to be statistically insignificant. Datawere also truncated to remove the values at the highest testedwater activity (0.75). On the basis of higher R2 of the linear model(R2 = 0.99) and the lack-of-fit test (R2 = 0.99), the BET equation wasmore adequate than the GAB equation. Consequently, the parame-ters of the GAB equation for the entire data range are as follows:C = 9.35, k = 0.84 and Xm (monolayer value) = 7.42 g water/100 gdry solids. For the BET equation, the values of the parameters areas follows: C = 13.85 and Xm = 5.92 g water/100 g dry solids. In bothcases, the calculated monolayer values were greater than the mois-ture content of the spray-dried BBE.

3.2. Changes in TMAC, TPC and FRAP during storage at differentconditions

The rate of anthocyanin degradation as a function of time isshown in Fig. 3. Anthocyanin loss followed the first-order kinetics

and the following rate constants were calculated:k45�C = 0.0182 d�1, klight = 0.0083 d�1, k37�C = 0.0069 d�1 andk22�C = 0.0031 d�1. The rate of monomeric anthocyanin loss undervarious storage conditions was in the order: 45 �C > light >37 �C > 22 �C. The disadvantage of using whey protein as a wallmaterial in spray drying arises from significantly higher rates ofanthocyanin degradation compared with polysaccharide-basedwall materials (Idham et al., 2012; Tonon et al., 2009). The temper-ature-dependence of the rate constants was modeled by Arrheniusequation (Fig. 4). The calculated energy of activation (Ea) was equalto 62.46 kJ/mol, while the preexponential factor A was equivalentto 2.81 � 108 d�1. The parameters of the modeled Arrhenius equa-tion were statistically significant and the equation was adequatebased on lack-of-fit tests (R2 = 0.82, p = 0.19). Higher values of Eaare usually associated with resistance to thermal degradation(Fischer et al., 2013).

Fig. 5 shows the variation of the total phenolics as a function oftime. The total phenolics increased across all test conditions andeventually the ratio of final to initial GAE ranged between 2 and2.5. The initial increase in total phenolics was greater at elevatedtemperatures compared to lower temperatures. The effect of pho-todegradation was comparable to storage at 37 �C. Similarly, theantioxidant capacity of the samples increased upon storage(Fig. 6). Interestingly, however, the final ferric reducing antioxidantpower varied proportionally with increasing temperatures. Fur-ther, the effect of photodegradation was again comparable to stor-age at 37 �C. The final concentration of Fe (II) equivalents rangedfrom unity (22 �C) to a peak of 1.25 (45 �C).

Generally, it is accepted that the concentration of monomericanthocyanins decreases with an increase in temperature. However,thermal effects on phenolics content and antioxidant activity arenot clear. The phenolics content and antioxidant activities ofspray-dried açai and blueberry both decreased under prolongedstorage (Jiménez-Aguilar et al., 2011; Tonon et al., 2009). The rateof decrease in phenolics content and antioxidant activity appearedto be linear for the non-encapsulated control and non-linear for theencapsulated extracts, but mathematical information regarding theobserved trends was not discussed (Jiménez-Aguilar et al., 2011).With our samples, there was a slight increase in antioxidant activ-ity and a two-fold increase in phenolics content. The plots in Fig. 5suggest that the phenolics content ratio stabilizes at a valuebetween 2 and 2.5. A linear relationship may be proposed for22 �C but may need prolonged storage conditions longer than thatnormally considered in the literature. Similarly, the considerablyhigher standard deviations in antioxidant activity observed forsamples stored at 37 �C and 22 �C (Fig. 6) seem to support the con-clusion that bioactivity may be conserved because an increase in

Fig. 2. Modeling of the experimental sorption isotherm based on the BET and GABequations.

Fig. 3. Rate of monomeric anthocyanin loss modeled after first-order kinetics.Legend: r 45 �C, 37 �C, 4 22 �C, light.

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