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FORMULATION OPTIMIZATION OF ANTIOXIDANT-RICH JUICEPOWDERS BASED ON EXPERIMENTAL MIXTURE DESIGNMI-BO KIM,1 JEONG-YEON KO2 and SANG-BIN LIM1,2,3

1Jeju Wellbeing Vegetables RIS System, Jeju National University, Jeju 690-756, Republic of Korea

2Department of Food Bioengineering, Jeju National University, Jeju 690-756, Republic of Korea

3Corresponding author.

TEL:1 82 64 754 3617;

FAX:1 82 64 755 3601;

EMAIL: [email protected]

Received for Publication August 11, 2015

Accepted for Publication January 26, 2016

doi:10.1111/jfpp.12897

ABSTRACT

This study optimized the mixing ratio of broccoli (BroMP), cabbage (CabMP),

and carrot-mixed powders (CarMP) for the development of juice powders con-

taining high total phenolic content (TPC), high antioxidant activities, and prefer-

able sensory properties using a mixture design. TPC and antioxidant activities of

juice powders were increased with a higher proportion of BroMP and lower pro-

portions of CabMP and CarMP. However, the overall acceptance was increased

with a higher CarMP proportion and lower BroMP and CabMP proportions. The

optimal mixing ratio was 67.4% BroMP, 16.7% CabMP, and 15.9% CarMP. Atthis ratio, the predicted response values of TPC, ABTS radical scavenging activity

(IC50), FRAP activity, and overall acceptance were 9.51 mg GAE/g, 5.35lg/mL,

81.3 mM FSE/g, and 4.62, respectively. The optimized mixed juice powder

showed high sums of individual phenolic compounds, with chlorogenic acid

(121.16 mg/100 g) and quercetin (71.74 mg/100 g) as the major phenolics.

PRACTICAL APPLICATIONS

Vegetable juice powder containing BroMP, CabMP, and CarMP can be utilized as

a functional drink having high antioxidant activities and preferable sensory prop-

erties. The optimized juice powder contained higher contents of individual phe-

nolic compounds than those of the broccoli or cabbage powder. Thus, mixed

vegetable powders can provide large amounts of diverse phenolic compoundsfrom different vegetable sources. The mixture design approach was found to be a

suitable method for optimizing a healthy and delicious juice formulation, and can

be effectively applied in other food mixture systems.

INTRODUCTION

Detoxification is defined as the physiological or medicinal

elimination of toxic substances from the human body, and

it promotes health and well-being as well as weight loss

(Klein and Kiat 2014). Detox diets involve the use of diu-

retics, laxatives, and cleansing foods and are recommendedfor individuals with toxin exposure, inflammation, gastro-

intestinal disorders, autoimmune disease, and chronic

fatigue syndrome (Klein and Kiat 2014). Vegetables and

fruits are representative detox foods, contain biologically

active antioxidants such as polyphenols, glucosinolates,

carotenoids, vitamins (C, E), and minerals, produce detoxi-

fying enzymes, and scavenge potentially mutagenic free rad-

icals (Shimazuet al. 2014).

Broccoli is an excellent detox food that aids toxin removal

from cells and contains polyphenolic compounds and glu-

cosinolates. These bioactive compounds support a detox

process in the human body, including the neutralization

and elimination of unwanted contaminants (Harris and

Johnson 2012; Col 2013). Cabbage is also a major detoxfood that plays an important role in the elimination of

potential carcinogens from the human body (Zulpa 2014).

Carrot contains high levels of beta-carotene and fiber, which

support the digestive tract to slough off toxins (Harris and

Johnson 2012).

Mixed vegetable juice powders are good sources of many

biologically active antioxidative compounds and can provide

large amounts of diverse phenolic compounds from different

1Journal of Food Processing and Preservation00(2016) 0000VC 2016 Wiley Periodicals, Inc.

Journal of Food Processing and Preservation ISSN 1745-4549


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vegetable sources (Wootton-Beard et al. 2011; Wootton-

Beard and Ryan 2011). Antioxidant powder and microbial

safety of fruit and vegetable juices can be improved by

the processing. Total phenolics, DPPH, total antioxidant

capacity, and total carotenoids of grapefruit juice were

increased with the increase in pulsed electric strength (Aadil

et al. 2015a). The quality of grapefruit juice was improvedwith the treatment of thermosonication by inactivating pec-

tin methylesterase, peroxidase and polyphenolase, and micro-

organisms (Aadil et al. 2015b). Growth of microbial flora

during storage of apple, orange and strawberry juices treated

with pulsed light was prohibited (Ferrarioet al. 2015).

Dietary antioxidants are defined as substances that scav-

enge reactive oxygen/nitrogen species and stop radical chain

reactions. Cumulative biological exposure to antioxidants

provides health benefits by preventing cardiovascular dis-

ease, cancer, and age-related degeneration (Wootton-Beard

and Ryan 2011; Yuanet al. 2013).

A mixture design is an effective method for food product

development and ingredient optimization to obtain the

desired properties of food products. When several food

ingredients are involved, it is difficult to determine their

mixing ratio. A mixture design can be used to determine the

optimal mixing ratio of ingredients that maximizes or mini-

mizes the response level of the dependent variables (Jang

et al. 2011; Tokeret al. 2013). Numerical and graphical anal-

yses of a mixture design can easily be performed to select

ingredient combinations that optimize the product proper-

ties, and have been applied for various food products (Toker

et al. 2013).

Several studies are available regarding the development of

food products using a mixture design. Kim et al. (2012)developed a citrus peel drink and Jang et al. (2011) devel-

oped a salad dressing with a Chinese quince juice using a

mixture design. Lawlesset al. (2013) also applied a mixture

design for consumer optimization of black cherry, concord

grape, and pomegranate juice blends. Mensah-Brown et al.

(2014) produced a chocolate-flavored soy-peanut beverage

with acceptable chemical and physicochemical properties,

and Shibyet al. (2013) developed a whey-fruit-based energy

drink using a mixture design.

There are several commercial juices that consist of vegeta-

bles and fruits, such as broccoli, cabbage, cauliflower, kale,

beet, parsley, celery, onion, spinach, tomatoes, basil, aspara-

gus, olive oil, carrot, cucumber, lime, strawberry, blueberry,apple, lemon, kale, beet, orange, ginger, and berries. Never-

theless, they did not provide detailed information on the

functional compounds and properties of the products. In

addition, there are also no systematic studies on the devel-

opment of antioxidant-rich juice powders using cruciferous

vegetables (broccoli and cabbage) as major ingredients with

high health-promoting properties and delicious formula-

tions using a mixture design.

The objective of this study was to optimize the mixing

ratio of broccoli, cabbage, and carrot powders for the devel-

opment of juice powders with large amounts of diverse phe-

nolic compounds, high antioxidant activities, and favorable

sensory preference using a mixture design.

MATERIALS AND METHODS

Materials and Chemical Reagents

Broccoli, cabbage, carrot, tomato, and radish harvested in

Jeju, Korea and apple harvested in Gyeongsangbuk-do,

Korea were purchased in a local store in Jeju, Korea.

Folin-Ciocalteus phenol reagent (2 M), sodium carbonate,

gallic acid standard, 2,20-azino-bis-3-ethylbenzthiazoline-

6-sulphonic acid (ABTS), trolox, and sodium acetate were

purchased from SigmaAldrich (St. Louis, MO). Potassium

persulfate was purchased from Daejung Chemicals, Ltd.

(Gyeonggi-do, Korea). Compound 2,4,6-tripyridyl-s-triazine

(TPTZ) was purchased from Santa Cruz Biotechnology, Inc.

(Santa Cruz, CA). Ferric chloride hexahydrate was purchased

from Junsei Chemical Co., Ltd. (Tokyo, Japan). Ferrous sul-

fate heptahydrate was purchased from Wako Pure Chemicals

Industries, Ltd. (Osaka, Japan). Acetic acid and hydrochloric

acid were purchased from Oriental Chemical Industries

(Incheon, Korea). All phenolic standards were purchased

from Fluka (Steinheim, Switzerland) except for quercetin

(Sigma, St. Louis, MO).

Preparation of Vegetable and Fruit PowdersVegetables and fruit were sorted, washed, and cut into pieces

approximately 23cm thick. They were blanched for 3 min in

boiling water, cooled with cold tap water, spread on sieves to

drain excess water, and freeze-dried using a deep freezer

(SFDSM24L; Samwon Freezing Engineering Co., Gyeonggi-do,

Korea). The freeze-dried samples were ground into a fine pow-

der using a grinder (MF10 basic; IKA-Werke GmbH & Co. KG,

Staufen, Germany) and passed through a 60-mesh sieve. The

powdered samples were then stored in a freezer at -20C until

needed.

Juice powders were produced using different ratios of

broccoli, cabbage, and carrot powders, as the major ingre-dients, and using a fixed ratio of apple, tomato, and radish

powders, as the minor ingredients. Three representative

powders were prepared based on preliminary preference

tests as follows; broccoli mixed powder (BroMP; broccoli

65%, apple 22.75%, tomato 8.75%, radish 3.50%), cabbage

mixed powder (CabMP; cabbage 60%, apple 26%, tomato

10%, radish 4%), and carrot-mixed powder (CarMP; carrot

60%, apple 26%, tomato 10%, radish 4%).

OPTIMIZATION OF JUICE POWDERS BY MIXTURE DESIGN M.-B. KIM, J.-Y. KO and S.-B. LIM

2 Journal of Food Processing and Preservation00(2016) 0000VC 2016 Wiley Periodicals, Inc.


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Experimental Design of Juice Powders

Experimental design and data analysis were performed using

Design-Expert software (Trial version 9.0.3.1, Stat-Ease, Inc.,

Minneapolis, MN). As independent variables, juice powders

with 13 different ratios of BroMP, CabMP, and CarMP were

formulated as shown in Table 1. Antioxidant components

(total phenolic content, TPC), antioxidant activities [ABTS

radical scavenging activity and ferric-ion reducing antioxi-

dant power (FRAP)], and sensory attributes (overall accep-

tance) were selected as dependent variables.

Preparation of 80% Methanol Extracts

for Antioxidant Activity and Individual

Phenolics Assays

Each mixed powder (1 g) was extracted using 80% aqueous

methanol with stirring for 1 h at room temperature. Each

extract was filtered through No. 5A filter paper (Advantec;

Toyo Roshi Kaisha, Ltd., Tokyo, Japan) and evaporated in a

rotary vacuum evaporator (Rotavapor R-124; BUCHI

Labortechnik AG, Flawil, Switzerland) at 40C. The dried res-

idue was dissolved in 20mL extraction solvent, and the solu-

tion was stored at 4C for further analysis.

Assay of Total Phenolic Content

The TPC of each extract was determined using Folin-

Ciocalteus method (Hwang and Lim 2014). A total of

200 lL of the extract, 800 lL distilled water, and 100 lL 2 M

Folin-Ciocalteu phenolic reagent were combined in a glass

vial and mixed by vortexing. After 5 min in the dark, 300 lL

20% NaCO3were added, and the solution was brought to a

total volume of 2 mL with distilled water. This solution was

incubated at room temperature in the dark for 2 h, and the

absorbance was measured at 760nm using a Spectronic

Genesys 2 spectrophotometer (Spectronic Instruments,

Rochester, NY). TPC was expressed as mg gallic acid equiva-

lents (GAE)/g dry weight of the extract.

Assay of ABTS Radical Scavenging Activity

ABTS radical scavenging activity of each extract was meas-

ured as described previously with minor modifications

(Thaipongaet al. 2006; Wootton-Beardet al. 2011). Briefly,

ABTS solution was prepared by mixing 14 mM ABTS1

solution and 4.9 mM potassium persulfate solution in equal

quantities and incubated for 16 h at room temperature in

the dark. This solution was diluted with ethanol to an

absorbance of 0.76 0.03 at 750 nm using the Multiskan EX

microplate reader (Thermo Electron Corp., Vantaa, Fin-

land). Fresh ABTS solution was prepared for each assay. A

total of 100 lL each extract (2, 4, 6, 8, and 10 lg/mL) was

mixed with 900 lL ABTS solution and incubated at 30C for

6 min, and the absorbance was measured at 750 nm using

the Multiskan EX microplate reader (Thermo Electron

Corp.). Ethanol and trolox (0.02, 0.04, 0.06, 0.08, and

0.10 lg/mL) were used as the blank and standard, respec-

tively. ABTS radical scavenging activity (IC50) was expressed

as the concentration (lg/mL) of extract required to decrease

50% of the initial ABTS radical concentration.

Assay of Ferric Ion-ReducingAntioxidant Power

The FRAP of each extract was measured as described previ-

ously with minor modifications (Thaiponga et al. 2006;

Wootton-Beard et al. 2011). The FRAP solution was pre-

pared by mixing 300 mM acetate buffer (pH 3.6), 10 mM

TPTZ solution (in 40 mM HCl), and 20 mM ferric chloride

hexahydrate (FeCl36H2O) solution at a ratio of 10:1:1 (v/v),

respectively. A total of 50 lL of each extract was mixed with

150 lL distilled water and 1.5 mL FRAP solution. After incu-

bating at 37C in a water bath for 10 min, the absorbance was

measured at 620 nm using a Multiskan EX microplate reader

(Thermo Electron Corp.). FRAP was expressed as mM

ferrous sulfate equivalents (FSE)/g dry weight of the extract.

Sensory Evaluation

For the overall acceptance test, 18 g of each juice powder were

added to 270 mL water, shaken vigorously for 30 sec, and then

used for the tests. Sensory evaluation was performed by 30

panels at the Department of Food Bioengineering, Jeju National

University. A 9-point category scale was used for the overall

acceptance test (15extremely dislike, 95extremely like).

TABLE 1. MIXTURE DESIGN FOR ANTIOXIDANT-RICH JUICE POWDER

AT DIFFERENT RATIOS OF BROCCOLI, CABBAGE, AND CARROT-MIXED

POWDERS

Standard Run

Pseudo component Actual component

A B C A (%) B (%) C (%)

9 1 0.000 0.333 0.667 0.0 33.3 66.78 2 0.000 0.667 0.333 0.0 66.7 33.3

12 3 0.167 0.167 0.667 16.7 16.7 66.7

11 4 0.167 0.667 0.167 16.7 66.7 16.7

7 5 0.333 0.000 0.667 33.3 0.0 66.7

5 6 0.333 0.667 0.000 33.3 66.7 0.0

10 7 0.667 0.167 0.167 66.7 16.7 16.7

6 8 0.667 0.000 0.333 66.7 0.0 33.3

4 9 0.667 0.333 0.000 66.7 33.3 0.0

3 10 0.000 0.000 1.000 0.0 0.0 100

2 11 0.000 1.000 0.000 0.0 100 0.0

1 12 1.000 0.000 0.000 100 0.0 0.0

13 13 0.333 0.333 0.333 33.3 33.3 33.3

A, broccoli mixed powder; B, cabbage mixed powder; C, carrot-mixed

powder.

M.-B. KIM, J.-Y. KO and S.-B. LIM OPTIMIZATION OF JUICE POWDERS BY MIXTURE DESIGN



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Optimization of the Mixing Ratio

of Juice Powders

The optimal mixing ratio of juice powders was determined

based on numerical and graphical optimizations (Derringer

and Suich 1980). Numerical optimization was performed by

determining a goal area of each response of the model coef-ficients based on the canonical model. The overall desirabil-

ity is a geometric mean of all transformed responses and is

calculated based on the desirability of each response using

the following equation:

D5d13d23 3dn1=n5

nYni51

di

o1 n=(1)

whereDis the overall desirability, diis the individual desir-

ability, andnis each response number.

For numerical optimization, each response should have

certain values assigned to each goal, such as maximum,

minimum, target, or in range, and the overall desirabilitycalculated ranges from 0 to 1 (least to most desirable). For

graphical optimization, the minimum and maximum of

each response are indicated, and contour plots are superim-

posed within the possible ranges, after which the best area is

selected.

Gas Chromatography/Mass Spectrometry

(GC/MS) Analysis

Individual phenolics were qualified and quantified using

GC/MS (Kimet al. 2010). Acid hydrolysis of the extract was

performed as follows: an aqueous solution of hydrochloricacid (3 M, 0.25 mL) was added to 0.5 mL extract. The mix-

ture was maintained at 80C for 1 h. After cooling, 0.5 mL of

potassium hydrogen phosphate (1 M) was added. Each phe-

nolic compound was isolated by solid phase extraction on a

C8 cartridge (WAT036780, Waters, Milford, MA). The car-

tridge was preconditioned with ethyl acetate (3 mL), MeOH

(3 mL), and distilled water (6 mL). The solvent in the car-

tridge was dried under reduced pressure, and loaded slowly

with acid-hydrolysed sample (0.5 mL). Thereafter, the phe-

nolics retained in the cartridge were eluted with ethyl acetate

(3 mL). A 2 mL extract was added internal standard, 3-(4-

hydroxy-phenyl)-1-propanol (Sigma, St. Lousi, MO), and

evaporated to dryness in a rotary vacuum evaporator at40C. Dried sample was derivatized by adding 0.25 mL of

BSTFA (Supelco, Bellefonte, PA), followed by incubation at

75C for 20 min. Each sample (1 lL) was injected into the

GC/MS.

An Agilent (Wallborn, Germany) series GC 6890N,

coupled with an HP 5973 MS detector (EI, 70 eV) and an

HP 7683 autosampler, was used for analysis of each phenolic

compound. Analyses used an HP-5 MS capillary column

(30m3 0.25 mm, 0.25 lm film thickness) at a split ratio of

1:5. Helium was used as a carrier gas at a flow rate of

0.6 mL/min. The injector and transfer line temperatures

were set at 280C and 300C, respectively. The oven tempera-

ture was held at 120C for 1 min, then increased to 220C at

5C/min, then to 300C at 10C/min and held for 10 min.

Chromatographic peaks were identified by comparing the

retention times and three fragment ions of each phenolic

compound with those of reference compounds.

Statistical Analysis

Statistical analyses were performed using SPSS version 18.0

software (SPSS Inc., Chicago, IL). Significant differences

(P< 0.05) among treatment means were determined based

on Duncans multiple range test.

RESULTS AND DISCUSSION

Total Phenolic Contents of the

Juice Powders

The TPC of the juice powders were measured at 13 experi-

mental points (Table 2). The TPCs range from 3.90 to

12.76 mg GAE/g dry extract. The maximum TPC was

observed at 100% BroMP, while the minimum TPC was

observed at 100% CarMP. The TPC of the juice powders

increased when a higher proportion of BroMP and a lower

proportion of CarMP were used.

Statistical analysis and the polynomial regression results

for the TPC of the juice powders are presented in Table 3. A

quadratic model was selected for the TPC, and the validity

was indicated at P< 0.0001. Based on the analysis of the

coefficients in the regression formula, the TPC was most

affected by the proportion of BroMP in the juice powders.

The effects of different proportions of each powder on

the TPC were also expressed as the trace plot and response

surface (Fig. 1). The TPC of the juice powders increased sig-

nificantly as the proportion of BroMP increased (A-A line),

and it increased gradually as the proportion of CarMP

decreased (C-C line). The proportion of CabMP did not

affect the TPC of the juice powders (B-B line).

Antioxidant Activities of the Juice Powders

The antioxidant activities of the juice powders were meas-

ured as ABTS radical scavenging activity and FRAP activity

(Table 2). The ABTS radical scavenging activities (IC50)

ranged from 4.13 to 8.25 lg/mL, while the FRAP activities

ranged from 38.65 to 119.49mM FSE/g dry extract. The

maximum ABTS radical scavenging (lowest IC50value) and

FRAP activities were observed with 100% BroMP. The




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minimum ABTS radical scavenging activity (highest IC50value) was observed in the mixed powder consisting of

33.3% CabMP and 66.7% CarMP, and the minimum FRAP

activity was observed with 100% CarMP. ABTS radical scav-

enging activity and FRAP activity of juice powders increased

at a higher ratio of BroMP. When the ratios of CabMP and

CarMP increased, the activities decreased.

Statistical analysis and the polynomial regression

model for the ABTS radical scavenging activities and

FRAP activities of the juice powders are presented in

TABLE 2. TOTAL PHENOL CONTENT (TPC), ABTS RADICAL SCAVENGING ACTIVITY, FERRIC ION- REDUCING ANTIOXIDANT POWER (FRAP)

ACTIVITY, AND OVERALL ACCEPTANCE OF ANTIOXIDANT-RICH JUICE POWDERS AT DIFFERENT RATIOS OF BROCCOLI, CABBAGE, AND

CARROT-MIXED POWDERS

Standard Run TPC (mg GAE/g) ABTS (IC50, lg/mL) FRAP (mM FSE/g) Overall acceptance

9 1 4.776 0.04b 8.256 0.43i 42.2062.30ab 5.6361.88bcd

8 2 5.866 0.06cd 7.896 0.21hi 50.0263.06bc 4.6361.99abc

12 3 5.596 0.12c 7.696 0.19h 43.7364.39ab 5.6761.67cd

11 4 7.096 0.09f 6.956 0.37g 58.6463.02cd 4.5362.00abc

7 5 6.076 0.14e 6.596 0.10fg 48.8167.48abc 5.4762.27abcd

5 6 8.516 0.11g 5.886 0.08cd 71.1366.34ef 4.4761.96ab

10 7 9.146 0.23h 5.556 0.46c 71.94610.65ef 4.7061.74abc

6 8 8.496 0.25g 5.976 0.56cde 75.2463.80fg 4.7362.24abc

4 9 9.916 0.42i 5.106 0.08b 85.1565.69g 4.3762.16a

3 10 3.906 0.02a 8.226 0.03i 38.6568.19a 6.3761.59d

2 11 7.046 0.14f 6.426 0.07ef 58.4063.44cd 4.3062.00a

1 12 12.7660.48j 4.136 0.07a 119.496 8.13h 4.4362.22a

13 13 7.406 0.05f 6.286 0.28def 63.9664.33de 4.7361.93abc

*Values followed by different letters are significantly different (P


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Table 3. A linear model and a quadratic model were

applied for the ABTS radical scavenging activity and the

FRAP activity, respectively, and the validities were indi-

cated atP< 0.0001. The ABTS radical scavenging activity

(low IC50 value, low coefficient value) and FRAP activity

were mostly dependent on the proportion of BroMP

according to the analysis of coefficients in the regression

formula.

The trace plots and response surfaces (Figs. 2 and 3) showed

that the ABTS radical scavenging activity and the FRAP activ-

ities increased significantly as the proportion of BroMP

increased (A-A line) and that of CarMP decreased (C-C line).

The ABTS radical scavenging activity decreased slowly as the

proportion of CabMP increased (B-B line), but the proportion

of CabMP did not affect FRAP activity (B-B line). These trends

were similar to those of the TPC.

FIG. 2. TRACE PLOT AND RESPONSE SURFACE FOR ABTS RADICAL SCAVENGING ACTIVITY (IC 50VALUE) AT DIFFERENT RATIOS OF BROCCOLI (A),

CABBAGE (B), AND CARROT (C) MIXED POWDERS

FIG. 3. TRACE PLOT AND RESPONSE SURFACE FOR FERRIC ION-REDUCING ANTIOXIDANT POWER (FRAP) ACTIVITY AT DIFFERENT RATIOS OF BROC-

COLI (A), CABBAGE (B), AND CARROT (C) MIXED POWDERS




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Phenolic compounds contribute to the antioxidant capacity

of vegetables and fruits, which have high ABTS radical scaveng-

ing and FRAP activities (Guoaet al. 2011). Polyphenolic com-

pounds contain functional phenolic hydroxyl (OH) groups

that scavenge reactive oxygen species as a hydrogen-donating

radical scavenger (Soengaset al. 2011; Leeet al. 2013). A high

level of phenolic compounds, specially quercetin, in broccoli is

related to its high antioxidant potential, and broccoli is often

considered the most abundant source of antioxidants in the

diet (Soengas et al. 2011; Porter 2012).In addition to phenolic compounds, broccoli contains

anthocyanins and glucosinolate hydrolysis products, which

also contribute to human health (Guoa et al. 2011). Espe-

cially, sulforaphane, a hydrolysis product of glucoraphanin

found in broccoli, produces phase I and phase II detoxifica-

tion enzymes and other antioxidant proteins, and helps con-

trol toxin removal from the human body (Klein and Kiat

2014; Liu et al. 2014). Furthermore, broccoli contains high

levels of glutathione, which helps expel toxins from the liver

by supporting enzymes that promote detoxification (Harris

and Johnson 2012).

Carrot is rich in hydrophobic carotenoids, and thus shows

poor antioxidant activity (Thaiponga et al. 2006). Carote-

noids respond in a different manner to different radicals or

oxidant sources, which are not particularly good peroxylradical quenchers compared with phenolics and other

antioxidants. The ABTS is oxidized by peroxyl radicals to

ABTS1 radical cation, resulting in the lower ABTS radical

scavenging activity of carrot powder (Prior et al. 2005; da

Silvaet al. 2014).

FIG. 4. TRACE PLOT AND RESPONSE SURFACE FOR OVERALL ACCEPTANCE AT DIFFERENT RATIOS OF BROCCOLI (A), CABBAGE (B), AND CARROT

(C) MIXED POWDERS

TABLE 4. CONSTRAINT VALUES OF INDEPENDENT VARIABLES AND PREDICTED VALUES OF DEPENDENT VARIABLES AT THE MAXIMUM

DESIRABILITY

Independent and dependent variables Goal

Predicted values

Numerical

optimization

Graphical

optimization

Independent BroMP (%) In range (0-1) 67.4 67.4

CabMP (%) In range (0.167-1) 16.7 16.7

CarMP (%) In range (0-1) 15.9 15.9

Dependent TPC (mg GAE/g) Maximum 9.51 9.51

ABTS (IC50, lg/mL) Minimum 5.35 5.35

FRAP (mM FSE/g) Maximum 81.34 81.32

Overall acceptance* Target (4.7) 4.62 4.62

BroMP, broccoli mixed powder; CabMP, cabbage mixed powder; CatMP, carrot-mixed powder.

* 1; extremely dislike, 9; extremely like.




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Overall Acceptance of Juice Powders

Sensory evaluation was performed in juice powders in terms

of overall acceptance using a 9-point category scale (Table

2). The overall acceptance of the samples evaluated ranged

from 4.30 to 6.37 (1: extremely dislike, 9: extremely like).

The maximum overall acceptance was observed with 100%

CarMP and the minimum overall acceptance with 100%

CabMP. The overall acceptance of juice powders increasedwith a higher proportion of CarMP due to sugary taste.

When the ratios of BroMP and CabMP increased, the overall

acceptances decreased.

Statistical analysis and the polynomial regression equa-

tion for the overall acceptance of juice powders are pre-

sented in Table 3. A linear model was applied for the overall

acceptance. The high coefficient values of the regression for-

mula indicated that the ratio of CarMP in juice powders

greatly affected the overall acceptance.

The trace plot and response surface for the overall accep-

tance at different ratios of juice powders showed that the

overall acceptance decreased as the proportions of BroMP

(A-A line) and CabMP increased (B-B line) (Fig. 4). This

was probably due to the hydrolyzed products isothiocya-

nates, generated from the precursor glucosinolates enriched

in Brassica vegetables, including broccoli and cabbage(Ghawi et al. 2014). Isothiocyanates are largely responsible

for the bitter taste and characteristic sulfurous aromas and

lead to limited consumer acceptability and low acceptance

(Ghawiet al. 2014; Grnbk 2014).

This bitterness may be compensated by increasing the

proportion of CarMP within the juice powder. The overall

acceptance of juice powders increased as the proportion of

CarMP increased (C-C line) (Fig. 4). The high sugar content

of carrots may mask the bitter taste of broccoli and cabbage

and decrease the perceived bitter taste (Grnbk 2014).

Optimization of the Juice PowderMixing Ratio

The mixing ratio of juice powders was optimized by the

method outlined in Derringer and Suich (1980). The opti-

mal mixing ratio of juice powders was determined based on

the phytochemical content (TPC), antioxidant activities

(ABTS radical scavenging and FRAP activities), and sensory

properties. As independent variables, the ratios of BroMP

and CarMP were set at a lower limit of 0 and upper limit

of 1, and that of CabMP was at a lower limit of 0.167

and upper limit of 1 based on preliminary preference

tests. The dependent variables TPC and FRAP activity were

set to the maximum value, ABTS radical scavenging activity(IC50value) was set to the minimum value, and the overall

acceptance was set to the target value of 4.7, which was the

middle value of 13 runs for the overall acceptance scores

(Table 4).

The optimal mixing ratio determined from the numerical

optimization (Table 4) was 67.4% BroMP, 16.7% CabMP,

and 15.9% CarMP. This was the same in the graphical opti-

mization models (Fig. 5). In the graphical optimization, the

overlapping region in the graphical plot was selected as the

FIG. 5. THREE-DIMENSIONAL PLOT OF THE COMMON AREA FOR THE

OPTIMIZED JUICE POWDER (A: BROCCOLI MIXED POWDER,

B: CABBAGE MIXED POWDER, C: CARROT-MIXED POWDER)

TABLE 5. QUANTIFICATION OF INDIVIDUAL PHENOLIC COMPOUNDS BY GC/MS (mg/100 g OF DRIED SAMPLE)

Phenolic compound Broccoli Cabbage Carrot Optimized mixed powder

p-Hydroxyl -benzoic acid 3.2460.13* 2.9460.08 8.2360.61 1.8860.16

Vanillic acid 2.7660.29 2.6660.16 8.9860.88 2.2760.08

Ferulic acid 25.316 0.32 13.4460.34 10.0760.08 15.756 0.05

Caffeic acid 9.0460.05 9.0260.08 63.0862.92 12.046 0.05

Sinapic acid 51.606 1.06 51.2660.74 30.286 0.08

Chlorogenic acid 50.1660.45 171.346 10.55 121.16613.47

Quercetin 72.606 0.05 71.746 0.16

Total 164.55 129.48 261.70 255.12

* Data are given as means6 SD.




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optimal range (Fig. 5). The desirability (D) at this optimal

point was calculated to be 0.658. At the optimal mixing

ratio, the predicted response values of TPC, ABTS radical

scavenging activity (IC50), FRAP activity, and overall accep-

tance were 9.51 mg GAE/g dry extract, 5.35lg/mL, 81.3 mM

FSE/g dry extract, and 4.62, respectively (Table 4).

Quantitative Determination of

Individual Phenolics by GC/MS

The levels of individual phenolics present in the extracts are

shown in Table 5. Seven phenolics were identified:p-hydroxy-

benzoic acid, vanillic acid, ferulic acid, caffeic acid, sinapic

acid, chlorogenic acid, and quercetin. The major phenolics

found in broccoli were quercetin (72.60 mg/100g) and

sinapic acid (51.60 mg/100g), those in cabbage were sinapic

acid (51.26 mg/100g) and chlorogenic acid (50.16 mg/100g),

and those in carrot were chlorogenic acid (171.34 mg/100g)

and caffeic acid (63.08 mg/100 g). In the optimized mixedjuice powder produced from BroMP, CabMP, and CarMP,

the major phenolic compounds found were chlorogenic acid

(121.16 mg/100g), quercetin (71.74 mg/100g), and sinapic

acid (30.28mg/100g). Chlorogenic acid is originated mainly

from the carrot powder, while quercetin is mainly originated

from the broccoli powder. Quercetin has 2.33.4-fold more

antioxidant activity than those of ferulic acid, chlorogenic

acid, and caffeic acid (Caiet al. 2006).

The sum of the individual phenolic compounds ranged

from 129.48 to 261.70 mg/100 g. The optimized juice pow-

der contained higher contents of individual phenolic com-

pounds than those of the broccoli or cabbage powder. Thus,

mixed vegetable powders can provide large amounts of

diverse phenolic compounds from different vegetable sour-

ces. However, the individual phenolic compounds quanti-

fied by GC/MS after acid hydrolysis may not be related

directly to their antioxidant capacities, which were assessed

using extracts (which contained more complex molecules).

CONCLUSIONS

Antioxidant-rich juice powders were developed with

BroMP, CabMP, and CarMP. The high proportion of BroMP

positively affected the TPC and antioxidant activities and

resulted in high functionality of the juice powders. The highproportion of CarMP positively affected the overall accep-

tance and resulted in high sensory quality of the juice pow-

ders. The optimized mixed juice powder showed high sums

of individual phenolic compounds, with chlorogenic acid

(121.16 mg/100g) and quercetin (71.74 mg/100g) as the

major phenolics. An optimized mixing ratio will enhance

both functionality and consumer acceptance and can pro-

vide a source of diverse phenolic compounds, present at

high levels, from different vegetable sources. The mixture

design approach was found to be a suitable method for opti-

mizing a healthy and delicious juice formulation.

ACKNOWLEDGMENTS

This research was supported by the Ministry of Trade,

Industry and Energy (MOTIE) and Korean Institute for

Advancement of Technology (KIAT) through the Research

and Development for Regional Industry Program.

NOMENCLATURE

ABTS 2,20-Azino-bis-3-ethylbenzthiazoline-6-sulphonic

acid

GAE Gallic acid equivalent

OH Hydroxyl

TPC Total phenolic content

TPTZ 2,4,6-Tripyridyl-s-triazine

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