Journal of Food Engineering 128 (2014) 167–173

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Journal of Food Engineering

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j f o o d e n g

Características De Adsorción / Desorción Y La Separación De Los Antocianos Y Polifenoles De Los Arándanos Utilizando Resinas Adsorbentes Macroporosas

Timothy J. Buran, Amandeep K. Sandhu, Zheng Li, Cheryl R. Rock, Weihua W. Yang, Liwei Gu ⇑Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, United States

a r t i c l e i n f o

Article history:Received 21 August 2013Received in revised form 4 December 2013 Accepted 24 December 2013Available online 4 January 2014

Palabras clave: Arándanos Las antocianinas Resinas Adsorción Desorción

a b s t r a c t

Los arándanos contienen fitoquímicos antioxidantes, como las antocianinas , flavonoles y procianidinas , con muchos beneficios para la salud . En el presente estudio , los fitoquímicos de arándanos se extrajeron mediante ecografía asistida extracción de agua caliente y se concentraron con resinas de adsorción Amberlite . Ensayos de adsorción estática demostraron que la resina FPX66 tenía una mayor capacidad de adsorción y desorción de relación que las resinas XAD7HP y XAD4 . XAD761 y XAD1180 mostraron la capacidad de desorción más baja y la relación . Ensayos de adsorción y de isotermas Kinetic revelado que FPX66 tenía la más alta eficiencia de adsorción y requiere un tiempo más corto para alcanzar el equilibrio de adsorción . Adsorción dinámica en FPX 66 resina en una columna de vidrio demostrado que las antocianinas en el extracto de arándanos agua comenzaron a romper después de 16 volúmenes de lecho de extracto se cargó . Un desorción completa se consiguió usando 3 volúmenes de lecho de 95 % ETH - Anol . Cien gramos de arándanos frescos dio 0,80 g extracto concentrado de arándanos . Azúcares no se detectaron en el extracto .

2013 Elsevier Ltd. All rights reserved.

11 . Introducción

Arándanos frescos se sabe que tienen mayor capaci-dad antioxidante que otras frutas (Wu et al. , 2004 ) . Los fitoquímicos de los arándanos son reportados a reducir el colesterol en la sangre ( Prior et al, 2009 . ) Y prevenir el cáncer y la aterosclerosis (Adams et al, 2010 ; . . Wu et al, 2010 ) . La investigación sugiere que los fitoquímicos extraídos de los arándanos eran más eficaces que las frutas enteras en - ing prevenir el aumento de peso corporal ( Prior et al. , 2008 ) . Los arándanos contienen 9,96 g de azúcar por 100 g de fruta fresca ( USDA , 2012 ) . Cerca de 8.3 % y el 35 % de la población de los EE.UU. son diabéticos o pre -diabéticos (CDC, 2011 ) . Este grupo de consumidores y público en general puede beneficiarse de un producto con poco o ningún contenido de azúcar. Por lo tanto , hay una demanda de su- - gar libre de arándanos fitoquímicos y concentrada en forma de un suplemento dietético para presumir la ingesta de antioxidantes y la prevención de enfermedades .

Tecnología de adsorción de la resina se está explorando para concentrar polifenoles y para eliminar los azúcares ( Kammerer et al , 2005 ; . . Soto et al , 2011 ) . Resinas sintéticas permiten la adsorción de los polifenoles de solución acuosa a través de apilamiento de unión y aromático hidrófobo . Ellos desorben fitoquímicos en disolventes orgánicos, tales como metanol o etanol . Debido a que los azúcares no interactúan con resinas , que se pueden eliminar fácilmente por elución en agua . El ultrasonido fue investigado para aumentar la eficiencia de extracción. Los polifenoles atrapados en la matriz de la planta fueron puestos en libertad de manera efectiva por el ultrasonido en anteriores

⇑ Autor para correspondencia. Tel : +1 (352) 392 1991 x210 ; . fax: . +1 (352) 392 9467 E -mail: LGu@ufl.edu (L. Gu ) .

0260-8774 / $ - see front matter 2013 Elsevier Ltd. Todos los derechos reservados . http://dx.doi.org/10.1016/j.jfoodeng.2013.12.029 

estudios ( Chemat et al. , 2011 ) . En el presente estudio , la alta capacidad de extrac-ción de ultrasonidos de potencia se acopló con la capacidad de concentración de la adsorción de la resina para producir extractos concentrados fitoquímicos de arándanos.

2 . Materiales y métodos

2.1 . Productos Químicos

AAPH ( 2,20 - azotis ( 2 - amidinopropano ) ) era un producto de Wako Chemicals Inc. ( Bellwood , RI ) . Trolox ( ácido 5,7,8 - tetrametilbenceno thylchroman - 2 - carboxílico 6 - hidroxi - 2 ) y cianidina 3 - rutinósido se adquirieron de Sigma - Aldrich ( St. Louis, MO ) . Normas de los 3 -OB -glucósidos de pelargonidina , cianidina , peonidina , delfinidina , petunidina y malvidin (seis norma antocianina mixta , grado HPLC) , se compraron de polifenoles Laboratories ( Sandnes, Noruega). Reactivo de Folin -Ciocalteu , ácido clorogénico y otros productos químicos eran productos de Fisher Scientific ( Pittsburg , PA ) . Resinas Amberlite ( XAD761 , XAD4 , XAD1180 ,

XAD7HP y FPX66 ) fueron producto de Rohm Hass ( Philadelphia , PA). Propiedades químicas y físicas de estas resinas se resumen en la Tabla 1 .

2.2 . Pre- tratamiento de las resinas

Resinas se suspendieron en agua para expandir las perlas antes de su envasado en una columna de vidrio ( L ID : 22 350 mm ) . El etanol ( 140 ml , 95 % ) se usó para lavar la columna con un caudal de 400 ml / h . Columna se lavó con agua hasta que el eluyente era

168 T.J. Buran et al. / Journal of Food Engineering 128 (2014) 167–173

Table 1Propiedades químicas y físicas de las resinas

Amberlite Chemical matrix Surface Particle Poreresins area (m2/g) size (mm) envelope

(Å)

XAD4 Crosslinked aromatic 750 0.3–1.2 100polymer

XAD7HP Crosslinked aromatic 500 0.6 450polymer

XAD761 Crosslinked 200 0.7 600formophenolicpolymer

XAD1180 Crosslinked aromatic 500 0.2–1.9 400polymer

FPX66 Crosslinked aromatic 700 0.60–0.75 200–250polymer

clear. The resin was then eluted with 140 mL of 4% HCl followed by distilled water until pH of the eluent became neutral. The column was then washed with 140 mL of 5% NaOH, followed by distilled water until the eluent reached a pH of 7.0. Samples of the pre-trea-ted resins were weighed into aluminum dishes and kept in an oven at 60 LC for 24 h. Weight loss was calculated as moisture content.

2.3. Extraction of phytochemicals from blueberries

Frozen southern high bush blueberries (160 g) were mixed with 400 mL of hot water (90 LC, acidified with 0.5% v/v acetic acid) and blended for 1 min in a blender. A power ultrasound probe was placed into the blueberry mash and the mixture was sonicated (20 kHz) at 100% amplitude for 5 min. The extraction mixture was filtered through cheese cloth to separate water extract and blueberry residue. The residue was re-extracted with an additional 400 mL of acidified water by using ultrasound. The water extracts were combined and filtered through Whatman No. 4 filter papers.

Desorption ratio

D% ¼ Cd

Vd

100 ð3ÞðC0 CeÞViDesorption capacity

qd ¼ Cd

Vd

ð4Þð1 MÞW% Recovery

CdVd

R ¼ 100% ð5ÞC0V0

where D is the desorption ratio (%), qd is the desorption capacity (mg/g dry resin), and R is the recovery (%) after desorption is com-plete. Cd is the concentration of anthocyanins or total phenolics in the desorption solution (mg/L). Vd is the volume of the desorption solution (mL). C0, Ce, M, W, and Vi are the same as above.

2.5. Adsorption kinetics

Pre-treated resin (1 g) was mixed with 25 mL of blueberry water extract in a flask and placed in a water bath shaker (45 rpm) at room temperature. An aliquot of supernatant (1 ml) was obtained every 30 min for the first 6 h and then every 60 min from 6 to 12 h. Adsorption kinetics was evaluated using the pseudo first and second order models.

Pseudo first order model

lnðqe qt Þ ¼ ln qe kf t ð6ÞPseudo second order model

t 1 1

t ð7Þ¼ þq

t ksqe2 q

e

where kf is the rate constant of the pseudo-first-order-model and ks is the rate constant of the pseudo-second-order-model, qt (mg/g) is the amount of anthocyanins or total phenolics adsorbed at time t and qe is the adsorption capacity at equilibrium.

2.4. Static adsorption/desorption testing

Pre-treated hydrated resin (1 g) and 25 mL of blueberry water extract were added to 250 mL Erlenmeyer flasks with stoppers. Flasks were kept for shaking in a water bath shaker (Stovall Life Sciences Inc.; Greensboro, NC) at a rate of 45 rpm. Adsorption was conducted at room temperature (25 LC) for 24 h. For static desorption testing, the phytochemical-laden resins were washed with 25 mL distilled water. After water wash, 50 mL of 95% ethanol was added in the flasks. The flasks were kept in a water bath shaker at 45 rpm for 24 h at room temperature. Adsorption and desorp-tion ratios and capacities were calculated using the following equations:

Adsorption ratio

A %Þ ¼

ðC0 CeÞ

ð1

Þð C0Adsorption capacity

q

e ¼C

0

Ce

ðViÞ

ð2

Þð1 MÞWwhere A is the adsorption ratio (%) and qe is the adsorption capacity (mg/g dry resin) at equilibrium. C0 and Ce is the initial and equilib-rium concentrations of anthocyanins or total phenolics in the blue-berry extract solutions (mg/L), respectively. M is the moisture content of the resin (w/w, %) whereas W is the initial weight of the resin being used (g). Vi is the volume (mL) of blueberry extract used.

2.6. Adsorption isotherms and thermodynamics

FPX66 resin was selected for adsorption isotherms and thermo-dynamics testing. Hydrated resin (1 g) was added to 25 mL blue-berry water extracts of different concentrations. Adsorption was conducted at three different temperatures (25, 35, or 45 LC) in a shaking water bath at 45 rpm. The equilibrium adsorption iso-therms for total phenolics and total anthocyanins were determined using Langmuir and Freundlich equations.

Langmuir equation

qmCe

ð8Þqe

¼ KL þ Ce

where qm (mg/g) is the maximum amount of adsorption, KL is the affinity constant in Langmuir model which can be calculated from the slope and intercept of the linear plot of Ce/qe versus Ce, respectively.

Freundlich equation

qe ¼ KF C1e=n

ð9Þ

where by potting log qe vs log Ce, constant KF and an exponent of 1/n can be calculated.

2.7. Dynamic adsorption/desorption testing

A glass column with a fritted disk (I.D. L, 19 400 mm) was loaded with FPX66 resin with a resin bed volume of 30 mL. Blue-berry water extract was loaded into this column using a flow rate

T.J. Buran et al. / Journal of Food Engineering 128 (2014) 167–173 169

of 2, 5 or 10 BV/h, respectively. The adsorbate-laden column was then washed with water (150 mL) to remove sugars and other compounds that did not absorb on the resin. Phytochemicals were desorbed using 95% ethanol at a flow rate 2, 4, or 6 BV/h, respec-tively. Eluent was collected and analyzed for total anthocyanin and phenolic content.

2.8. Folin–Ciocalteu assay

Total phenolic content was determined by the Folin–Ciocalteu assay (Lee et al., 2005). Blueberry extracts were mixed with diluted Folin–Ciocalteu reagent and 15% sodium carbonate. Absorbance at 765 nm was measured on a SPECTRAmax 190 microplate reader (Molecular Devices, Sunnyvale, CA) after 30 min of incubation at room temperature. Gallic acid was used to generate a standard curve. Results of total phenolic content for blueberries were ex-pressed as milligram gallic acid equivalent per gram of frozen fresh blueberry samples (mg GAE/g).

2.9. Total anthocyanin content

Total anthocyanin content was measured by using a pH differ-ential assay. Absorbance at 520 and 700 nm was measured on a Life Science UV/Vis spectrophotometer (DU 730, Beckman Coulter, Fullerton, CA) after 15 min of incubation at room temperature. Absorbance (A) was calculated using (A520–A700)pH 1.0 – (A520– A700)pH 4.5. Total anthocyanin content (mg Cy–G/g) was calculated using (A 449.0 80 1000)/(29,740 1) for blueberry and was expressed as milligram cyanidin 3-glucoside equivalent per gram of frozen fresh blueberry (mg Cy–G/g).

2.10. Oxygen radical absorbance capacity (ORAC)

The ORAC assay for blueberry samples was conducted on a Spectra XMS Gemini plate reader (Molecular Devices, Sunnyvale, CA). Briefly, 50 lL of standard and samples were added to the des-ignated wells of a 96-well black plate. This was followed by the addition of 100 lL of fluorescein (20 nM). The mixture was incu-bated at 37 LC for 7 min before the addition of 50 lL of AAPH. Fluo-rescence was monitored using 485 nm excitation and 530 nm emissions at 1 min intervals for 40 min. Trolox was used to gener-ate a standard curve. The antioxidant capacities of extracts were expressed as micromoles of Trolox equivalents (TE) per gram (lmol of TE/g).

2.11. HPLC analyses of phytochemicals and sugars

An Agilent 1200 HPLC system consisting of an autosampler, a binary pump, a column compartment, a diode array detector and a refractive index detector (Agilent Technologies, Palo Alto, CA) was interfaced to a HCT ion trap mass spectrometer (Bruker Dal-tonics, Billerica, MA). Samples were centrifuged at 13,300 rpm for 10 min and 10 lL of supernatant was injected for phytochemical analysis. A Zorbax SB-C18 column (250 mm 4.6 mm, 5 lm parti-cle size, Agilent Technologies, Palo Alto, CA) was used for the sep-aration. The binary mobile phase consisted of (A) formic acid: water (5:95 v/v) and (B) methanol. For the analysis of flavonols, chlorogenic acid derivatives and anthocyanins, a 70-min gradient was adapted from a published paper (Cho et al., 2004). The gradi-ent is described as follows: 0 min-5% B, 2 min-5% B, 10 min-20% B, 15 min-20% B, 30 min-25% B, 35 min-25% B, 50 min-33% B, 55 min-40% B, 60 min-60% B, 65 min-70% B, 70 min-5% B isocratic; fol-lowed by 5 min of re-equilibration of the column before the next run. The detection wavelength was 520 nm for anthocyanins, and 360 nm for chlorogenic acid and flavonols. Electrospray ionization in alternating mode was performed using nebulizer 30 psi, drying

gas 11 L/min, drying temperature 300 LC, and capillary of 4000 V, allowing compounds to be detected in both positive and negative mode in the same run. The full scan mass spectra of the anthocya-nins were recorded from m/z 350 to 650. Auto MS2 was conducted with 100% compound stability and 100% trap drive level. Pure com-pounds of chlorogenic acid, rutin, myricetin, quercetin, and kaempferol were used as external standards to quantify chlorogen-ic acid and flavonols. Anthocyanin glucoside mixture was used as an external standard for the quantification of anthocyanins. Data was analyzed using Chemstation software (Version B. 01.03, Agi-lent Technologies, Palo Alto, CA). Blueberry phytochemicals were identified on the basis of full scan and product ion mass spectra, UV/VIS spectra on diode array detector, and comparison with pub-lished papers (Cho et al., 2004; Wu and Prior, 2005).

Sugar analysis was conducted using a Restek ultra amino col-umn (5 lm, 250 4.6 mm). Acetonitrile: water (65:35 v/v) was used as the mobile phase at a constant flow rate of 1.0 mL/min. The column temperature was maintained at 30 LC and 5 lL of sam-ple was injected. Calibration curves were constructed using pure standards of glucose and fructose.

2.12. Statistical analyses

Data were expressed as mean ± standard deviation. One-way analyses of variance with Tukey–Kramer HSD comparison of means were performed using JMP software (Version 8.0, SAS Insti-tute Inc., Cary, NC). A difference of p 6 0.05 was considered as significant.

3. Results and discussion

3.1. Static adsorption and desorption

Adsorption and desorption behaviors of anthocyanins on resins are depicted in Fig. 1(A1 and A2). Amberlite resin FPX66 and XAD1180 had the highest adsorption ratio of 99.8% and 96.7%, respectively (Fig. 1A1). XAD761 showed the lowest at 79.8%. XAD7HP showed the highest adsorption capacity (25.8 mg/g) with FPX66 and XAD1180 following at 24.1 and 23.9 mg/g, respectively. XAD4 had the lowest adsorption capacity at 17.9 mg/g when com-pared to the other resins

despite that XAD4 had the highest surface area of 700 m2/g (Table 1). However, the low adsorption capacity of XAD4 may be explained by its very small pore envelope size of 100 Å. Previous research suggested that the surface area in combi-nation with pore size were the determining factors in predicting the adsorption capacity (Xu et al., 1999). In desorption tests, FPX66 and XAD761 had a higher desorption ratio (121.1% and 119.9%) and greater recovery than the other resins (Fig. 1A2). FPX66 and XAD761 have relatively large particle sizes at 0.75 mm and 0.70 mm, respectively. Resins with larger particle sizes tend to have higher mass exchange rates and allow more material to be transferred to and from the resin, thereby increasing the desorption ratio and recovery.

The adsorption and desorption behaviors of total phenolics on resins are described in Fig. 1(B1 and B2). XAD761 had the highest adsorption capacity and ratio of 53.4 mg/g and 88.6%, respectively. XAD7HP showed the lowest adsorption ratio at 70.6%, whereas FPX66 and XAD4 were in between (Fig. 1B1). In contrast to a high adsorption ratio, XAD761 had the lowest desorption ratio of 21.0% and a recovery of only 16.5%. XAD761 is a polar phenol formalde-hyde polymer (dipole moment = 1.80), whereas the other adsor-bents are made of styrene–divinylbenzene, a hydrophobic polyaromatic polymer (dipole moment = 0.30). Therefore XAD 761 resin has higher adsorption affinity towards phenolic com-pounds with high polarity than hydrophobic resins (Silva et al.,

170 T.J. Buran et al. / Journal of Food Engineering 128 (2014) 167–173

Fig. 1. (A1) Static adsorption capacity and ratio, and (A2) recovery and desorption ratio of total anthocyanins. (B1) Static adsorption capacity and ratio, and (B2) recovery and desorption ratio of total phenolics. Results are mean of three determinations. Different upper-case letters indicate significant differences of solid bars. Different lower-case letters indicate significant differences of lined bars.

40 A FPX 66XAD 4

(mg/

g) XAD 7HP

30

capa

city

20

Ads

orpt

ion

10

00 200 400 600 800

Time (min)

70

B

(mg/

g) 60

50

capa

city

40

Ads

orpt

ion

30

20

10

0 200 400 600 800Time (min)

Fig. 2. Adsorption Kinetics of total anthocyanins (A) and total phenolics (B) from blueberry water extract. Results are mean of three determinations.

Table 2Pseudo first and second order rate constants of resins calculated on the basis of total phenolics and total anthocyanins.

Resins KF qe Correlationcoefficient (R2)

Pseudo first orderTotal anthocyanins FPX66 0.004 1.19 0.988

XAD4 0.003 1.50 0.959XAD7HP 0.004 1.21 0.954

Total phenolics FPX66 0.005 1.49 0.934XAD4 0.003 1.77 0.975XAD7HP 0.004 1.51 0.979

Pseudo second orderTotal anthocyanins FPX66 0.032 1.37 0.996

XAD4 0.003 2.07 0.987XAD7HP 0.018 2.01 0.997

Total phenolics FPX66 1.02 104 68.0 0.993XAD4 4.50 104 56.5 0.999

XAD7HP 6.61 10458.8 1.000

2007). A significant amount of phenolic compounds in blueberry extract may have been adsorbed irreversibly on XAD761 resin and attributed to the extremely low recovery and desorption ratio of phenolic compounds. FPX66 was the most efficient resin in desorption with recovery of 82.5% and a desorption ratio of 114.9% (Fig. 1B2).

Static tests indicated that FPX66, XAD7HP and XAD4 were con-sistently more efficient at adsorption and desorption than other two resins, hence they were chosen for further kinetic tests.

3.2. Adsorption kinetics

The adsorption kinetics of anthocyanins and total phenolics on

FPX66, XAD7HP and XAD4 resins is shown in Fig. 2. FPX 66 and XAD7HP reached equilibrium at approximately 4 h and 6 h, respec-tively, which was faster than XAD4 (about 8 h). At

equilibrium, FPX 66 had a higher adsorption capacity than XAD7HP or XAD4

T.J. Buran et al. / Journal of Food Engineering 128 (2014) 167–173 171

Table 3Langmuir and Freundlich equation constants of total anthocyanins and total phenolics on Amberlite FPX66 resin.

Temperature (LC) Langmuir R2 Freundlich R2

equation equation

Total anthocyanins25

qe ¼14:8Ce 0.997 0:025 0.142

351:18þCe

0.998qe ¼ 0:091Ce

0.767qe ¼

14:4Ce 0:244

450:731þCe

0.995qe ¼ 0:176Ce

0.888qe ¼

13:5Ce 0:086

0:386þCe qe ¼ 0:115CeTotal phenolics25

qe ¼10:7Ce 0.922 0:617 0.942

350:011þCe

0.922qe ¼ 0:459Ce

0.882qe ¼

9:76Ce 0:784

450:010þCe

0.964qe ¼ 0:506Ce

0.883

qe ¼16:8Ce 0:859

0:014þCe qe ¼ 0:529Ce

1.8A

1.6

1.4

1.2

Ce/Q

e

1.0

0.8

0.6

0.425oC

0.2 35oC45oC

0.00 5 10 15 20 25 30

Ce (mg/L)

35

B30

25

Ce/

Qe 20

15

10

5

0100 200 300 400 500

Ce (mg/L)

Fig. 3. Adsorption Isotherms based on Langmuir equation of total anthocyanins (A) and total phenolics (B) on Amberlite FPX66 resin from blueberry water extract. Results are mean of three determinations.

(Fig. 2A). Regression of kinetic data using pseudo first order model rendered straight lines between log (qeqt) and time. The correla-tion coefficients lie between 0.954 and 0.988 (Table 2). The pseudo first order model describes the initial stages of the adsorption pro-cess. The pseudo second order model is more useful to predict and describe the entire adsorption process. The pseudo second order model appeared to be better than pseudo first order in describing the adsorption kinetics of anthocyanins from blueberry water ex-tract on resins. Similar observations were made in previous studies using Amberlite resins on different compounds (Abdullah et al., 2009).

Adsorption capacity of total phenolics on FPX66 and XAD7HP increased quickly within the first 3 h, whereas a gradual increase was observed on XAD4 (Fig. 2B). At 4 h both the FPX66 and

0.07 2 Bed Volume/h A5 Bed Volume/h

0.06 10 Bed Volume/h

Ant

hocy

anin

Con

tent 0.05

(mg/

L)

0.04

0.03

0.02

0.01

0.005 10 15 20 250

Volume Eluted (mL)

5B 2 Bed Volume/h

4 Bed Volume/h

46 Bed Volume/h

Anth

ocya

nin

Cont

ent

3

(mg/

L)

2

1

0

0 20 40 60 80 100 120 140 160 180 200Volume Eluted (mL)

Fig. 4. Dynamic adsorption (A) and desorption (B) curves of total anthocyanins on Amberlite FPX66 resin at different flow rates. Results are mean of two determinations.

XAD7HP resins reached equilibrium. FPX66 had a higher adsorp-tion capacity than XAD7HP. At the end of 7 h XAD4 matched the adsorption capacity of FPX66. Kinetic data using pseudo first and second order models are depicted in Table 2. Similar to total antho-cyanins, the pseudo second order model appeared to be better than pseudo first order in describing the adsorption kinetics of total phenolics from blueberry water extracts on resins.

3.3. Adsorption isotherms and thermodynamics of anthocyanins on FPX66 resin

The total anthocyanins and phenolics data for adsorption was regressed according to the Langmuir and Freundlich isotherms equations. Equation constants and correlation coefficients are listed in Table 3. The Langmuir and Freundlich isotherms are the most common models for exploring adsorption equilibrium data. The Freundlich model assumes that the surface of the resin is het-erogeneous in nature that is characterized by sorption sites at dif-ferent energies. It describes the adsorption behavior of a monomolecular layer as well as a multi-molecular layer. The Lang-muir model describes a monolayer adsorption with energetically identical sorption sites as well as without mutual interactions be-tween the adsorbed molecules (Duran et al., 2011). The correlation coefficients in Table 3 depict that the Langmuir model was a better fit for the data. Results based on total anthocyanins showed that the maximum adsorption capacity (qm) remains consistent, imply-ing that temperature did not significantly affect the adsorption capacity. However, the KL values decreases as temperature in-creases, suggesting adsorption of anthocyanins was decreased as

172 T.J. Buran et al. / Journal of Food Engineering 128 (2014) 167–173

Table 4Content of anthocyanins, flavonols, chlorogenic acid, and sugars in frozen fresh blueberries and the concentrated blueberry extract.

Compounds Frozen fresh Concentratedblueberries blueberry extract(mg/g berries) (mg/g extract)

AnthocyaninsPeonidin 3-galactoside 0.37± 0.02 38.8 ± 1.6Cyanidin 3-glucoside 0.04± 0.00 3.76 ± 0.18Peonidin 3-arabinoside 0.12± 0.07 19.4 ± 0.1Petunidin 3-galactoside 0.23± 0.01 21.1 ± 0.1Cyanidin 3-arabinoside 0.04± 0.00 3.45 ± 0.19Peonidin 3 galactoside 0.04± 0.00 3.69 ± 0.21Petunidin 3-arabinoside 0.08± 0.00 8.94 ± 0.19Malvidin 3-galactoside 0.74± 0.01 52.2 ± 1.5Malvidin 3-glucoside 0.04± 0.00 3.39 ± 0.16Malvidin 3-arabinoside 0.24± 0.00 21.2 ± 0.2Total anthocyanins 1.94± 0.03 175.9 ± 2.4

Chlorogenic acid and flavonolsChlorogenic acid 0.02± 0.00 1.45 ± 0.10Myricetin 3-rhamnoside 0.01± 0.00 0.26 ± 0.01Quercetin 3-glucoside 0.01± 0.00 0.19 ± 0.01Myricetin 3-glucoside 0.01± 0.00 0.44 ± 0.09

SugarsFructose (mg/g) 69.97± 0.45 Not detectedGlucose (mg/g) 65.84± 1.39 Not detected

OthersTotal phenolic content (mg GAE/g) 3.77± 0.12 215 ± 2.13ORAC (lmoL TE/g) 68.3± 0.7 7660 ± 1.1

Data are mean ± standard deviation for three determinations.

temperature rose. Degradation of the heat sensitive anthocyanins at higher temperatures may explain these results. The Langmuir adsorption isotherms on FPX66 at 25, 35, and 45 LC are shown in Fig. 3.

Similar to the isotherm data on anthocyanins, the Langmuir equa-tion fitted the data very well (Table 3 ) for total phenolics as well. The maximum adsorption capacity (qm) of total phenolics and the KL values were not affected by an increase in temperature except at 45 LC, where both qm and KL increased. For both anthocyanins and total phenolics, FPX66 resin had the highest adsorption efficiency at lowest tempera-ture (25 LC) and therefore, was chosen for dynamic testing.

3.4. Dynamic adsorption/desorption on FPX66 resin

When blueberry water extract was loaded onto a FPX66 filled column, no anthocyanins were detected in the eluent in the begin-ning (Fig. 4A). After more extract was loaded, the resin in the col-umn slowly reached adsorption saturation. Anthocyanins started to breakthrough and appeared in the eluent. Breakthrough volume is defined as the volume of extract loaded on the column when the concentration of anthocyanins in the eluent is 5% of that in water extract. At a flow rate of 10 BV/h and 5 BV/h, the breakthrough vol-umes were 8 BV and 16 BV, respectively. At a flow rate of 2 BV/h, breakthrough point was not reached even after 23 BV of extract was loaded on the column. The adsorption ratios of anthocyanins were 98.2%, 89.3%, or 82.2%, respectively, using a flow rate of 2, 5, or 10 BV/h. Similar results were reported in an earlier study that investigated the adsorption of lycopene on resins (Liu et al., 2010). The contact time between anthocyanins and resin became shorter when the flow rate was increased. Shorter contact time subse-quently led to an incomplete adsorption (Ma et al., 2009). Results suggested that 5 BV/h was a suitable flow rate as it allowed for maximum adsorption of phenolic compounds in the shortest amount of time.

Ethanol (95%, v/v) was used to desorb polyphenols from column using a flow rate of 2, 4, or 6 BV/h (Fig. 4B). Regardless of flow rate, a complete desorption of anthocyanins was achieved with less

than 3 BV of ethanol. Because of the shortest time used for desorp-tion, highest flow rate (6 BV/h) was chosen.

3.5. Phytochemical and sugar composition of extracts

Contents of anthocyanins, flavonols, chlorogenic acid, and sug-ars in frozen fresh blueberries or concentrated blueberry extract are shown in Table 4. Hot water extract of blueberries contained fructose and glucose. Sugar was not detected in concentrated ex-tracts using HPLC analysis. The anthocyanins content in the extract was 17.6% (w/w) compared with 0.19% (w/w) in fresh blueberries. The concentration of chlorogenic acid and flavonols in the extract was also increased by over 20 folds compared with fresh berries. The yield of concentrated blueberry extract was 0.80 g per 100 g fresh berries. The recovery rate of total phenolics was 45.6%. The low recovery of total phenolics was likely due to unspecific nature of Folin–Ciocalteu assay. For example, ascorbic acid is counted as phenolic when using Folin–Ciocalteu assay, however, ascorbic acid is not absorbed on resins. The recovery rate of total anthocyanins was 72.5%, which was comparable to a previous study (Jia and Lu, 2008).

4. Conclusions

Amberlite FPX66 resin was the most suitable resin for the recovery of anthocyanins and flavonoids from blueberry water ex-tract. This was attributed to its high adsorption and desorption capacity towards these compounds. Resin adsorption resulted in a concentrated blueberry extract that contained 17.6% (w/w) anthocyanins with no detectable sugars. This extract could poten-tially find application as a natural colorant, dietary supplement, antioxidant ingredient for functional foods, and/or as a raw mate-rial in the cosmetic and pharmaceutical industry preparations.

Acknowledgements

This research is supported in part by a Grant from Florida Department of Agriculture and Consumer Services, and Straughn Farms at Waldo Florida.

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