9
J. Korean Soc. Appl. Biol. Chem. 53(3), 342-350 (2010) Article
Optimization of Carbohydrate-hydrolyzing Enzyme AidedPolyphenol Extraction from Unripe Apples
Hu-Zhe Zheng1,2, In-Wook Hwang1, Suk-Kyung Kim3, Sang-Han Lee1, and Shin-Kyo Chung1*
1Department of Food Science and Technology, Kyungpook National University, Daegu 702-701, Republic of Korea2Department of Engineering, Liaoning Agricultural College, Yingkou 115-009, China
3Food and Bio-industry Research Institute, Kyungpook National University, Daegu 702-701, Republic of Korea
Received January 12, 2010; Accepted march 9, 2010
Unripe apples lie scattered about the orchards because of manual thinning out or falling, however
they contain a high amount of polyphenol. In order to enhance the extraction of polyphenol from
the unripe apples, carbohydrate-hydrolyzing enzyme (Viscozyme L) aided polyphenol extraction
techniques have been studied with response surface methodology. The optimum conditions were as
follows: the ratio of Viscozyme L to substrate was 0.0195 (1.95 fungal beta-glucanase units), the
reaction temperature was 47.12oC, and the reaction time was 12.52 h. The experimental values of
total phenolic content and caffeic acid content of the unripe apples were well matched with the
response surface methodology predicted values. The levels of total phenolic content and caffeic acid
content obtained from Viscozyme L treatment were about 2 and 13 folds greater, respectively than
those of the control treatment.
Key words: caffeic acid content, carbohydrate-hydrolyzing enzyme, optimum hydrolysis conditions,response surface methodology, total phenolic content, unripe apples
Apples, which are one of the most frequently consumed
fruits, contain abundant levels of polyphenol compound,
that have been reported to exert a variety of biological
actions, such as free radical scavenging activity [Adil et
al., 2007], metal chelation [Chien et al., 2007], anti-
allergic activity [Kojima et al., 2000; Akiyama et al.,
2005], anti-cancer activity [Marian et al., 2000], and anti-
arteriosclerosis activity [Stefania et al., 2007]. Unripe
apples in the orchards, which have resulted from thinning
out or falling, were typically discarded even though they
accounted for 20~30% of Korea’s total apple production
[Park et al., 2004]. A small part of them are utilized as
animal feed and fertilizer, in spite of environmental
contamination [Sudha et al., 2007]. However, unripe
apples contain phytochemicals, such as total phenolic
content (TPC) [Park et al., 2004; Renard et al., 2007],
proanthocyanidin [Akiyama et al., 2005] and flavonoid
[Mohamed et al., 2001], at levels 10 times higher than
those of ripe apples [Akiyama et al., 2005; Adil et al.,
2007; Sudha et al., 2007; Wu et al., 2007].
Over the past few years, not only by-products, but also
a number of other agricultural wastes of plant origin have
attracted considerable attention as potential sources of
bioactive phytochemicals, which can be used for various
purposes in the pharmaceutical, cosmetic and food
industry. However, in many cases, there have been a
rather significant lack of appropriate feasibility studies on
the extraction and exploitation of such bioactive compounds
[Makris et al., 2007], because photochemicals extraction
techniques have been often regarded as bottlenecks in the
food processing industry [Wang and Weller 2006].
Response surface methodology (RSM) was used for
several extraction techniques, such as ultrasound-assisted
extraction [Kashif and Choi, 2009], supercritical fluid
extraction [Adil et al., 2007], microwave-assisted
extraction [Hayat et al., 2009], and solvent extraction
[Pompeu et al., 2009]. Recently, carbohydrate-hydrolyzing
enzymes have been introduced to release cell-wall
complex polyphenol [Landbo and Meyer 2001; Zheng et
al., 2008; Barberousse et al., 2009]. Viscozyme L is a
multi-enzyme complex containing a wide range of
carbohydrate-hydrolyzing enzyme, that has been used to
extract protein from oat bran [Xiao and Yao 2008], and
arabinose and xylose from wheat [Sørensen et al., 2005].
Prior to this study, the utilization of carbohydrate-
*Corresponding authorPhone: +82-53-950-5778; Fax: +82-53-950-6772E-mail: [email protected]
doi:10.3839/jksabc.2010.053
Optimization of enzyme-aided polyphenol extraction from unripe apples 343
hydrolyzing enzymes for the enhancement of polyphenol
extraction efficiency from apple pomace was investigated
[Zheng et al., 2008].
In the present study, Viscozyme L aided hydrolysis
variables such as the ratio of Viscozyme L to substrate,
the reaction temperature, and reaction time were
optimized using RSM, by employing a five level, three
variable central composite rotatable design (CCRD), in
order to obtain the optimum conditions for the extraction
of polyphenol from unripe apples.
Materials and Methods
Materials and chemicals. Unripe apples (Malus
pumila cv. Fuji) were collected at the 85 days after full
bloom from the orchard of Kyungpook National University
in Daegu, Korea, in 2009, and stored in a freezer (−70oC)
until the experiment. Viscozyme L (from Aspergillus
aculeatus, 100 fungal beta-glucanase units (FBG)/mL,
Novozymes, Bagsvaerd, Denmark) was used in this
study. FBG unit is determined based on the Christian et
al. [2005] with some modification, and defined as the
Viscozyme L liberating one μmol of glucose from β-
glucan per minute. Folin-Ciocalteu phenol reagent, caffeic
acid, chlorogenic acid, p-coumaric acid, ferulic acid,
phloretin, and phloridzin were obtained from Sigma Co.
(St. Louis, MO). All organic solvents and other chemicals
were at the analytical grade from Duksan Co. (Seoul,
Korea), except for high performance liquid chromatography
(HPLC, J. T. Baker, Phillpsburg, NJ).
Instruments. UV-Visible spectrophotometer (UV 1601
PC, Shimadzu, Co., Kyoto, Japan) and HPLC (LC-10A,
Shimadzu, Co., Kyoto, Japan) associated with UV-
Visible detector (SPD-10A, Shimadzu, Co., Kyoto,
Japan) were used for the determination of TPC and
caffeic acid content (CAC) in the unripe apples.
Viscozyme L aided polyphenol extraction from
unripe apples. One hundred grams of unripe apples were
blanched at 80oC for 10 min for the polyphenol oxidase
(PPO) inhibition [Buckow et al., 2009], and cut into
cylindrical shapes without peeling, added to a half
volume of distilled water (w/v), and then homogenized.
For the enzyme aided hydrolysis reaction, 10 g of unripe
apples homogenate was put into a 30 mL vial, Viscozyme
L solution was added, and then incubated after nitrogen
flushing for the PPO inhibition. Enzyme hydrolyzation
was performed at a selected temperature and at a different
time.
Experimental design. A five level, three variable
CCRD consisting of 16 experimental runs was used to
study the response pattern and to determine the optimum
combination of Viscozyme L aided hydrolysis reaction
variables for the extraction of TPC and CAC from unripe
apples. Based on the preliminary experiments, three
independent variables, the ratio of Viscozyme L to
substrate (from 0 to 0.03, namely from 0 to 3 FBG),
reaction temperature (from 30 to 70oC), and reaction time
(from 4 to 20 h) were selected (Table 1). Regression
analysis was performed after triplicate observations of the
data obtained from the dependent variables, which were
affected by the extraction conditions, and the results of
the analysis were substituted into an empiric second order
polynomial model as shown in the following equation
(Eq.) 1:
(1)
where X1, X2, ..., Xk are the independent variables affecting
the responses Yi; β0, β (i=1, 2, ..., k), βii (i=1, 2, ..., k), and
βij (i=1,2, ..., k; j=1,2, ..., k) are the regression coefficients
for intercept, linear, quadratic, and interaction terms,
respectively; k is the number of variables.
The responses obtained from the experimental design
set (Table 1) were subjected to multiple nonlinear
regression analysis using the Statistical Analysis System
(Version 9.1, Institute Inc., Cary, NC, USA), to obtain the
coefficients of the second-order polynomial model. The
quality of the fit of the polynomial model was expressed
by the coefficient of determination R2, and its statistical
significance was checked using a F-test.
Determination of TPC. TPC was determined using
Folin-Ciocalteu reagent with some modifications [Singleton
et al., 1999], and expressed as gallic acid equivalent in
milligrams per 100 g fresh weight (mg GAE/100 g).
Determination of CAC and polyphenol composition.
Twenty μL of unripe apples polyphenol solution was
injected into a HPLC after filtration (0.45 μm) with an
ODS-HG-5 (Develosil, 150×4.6 mm, i.d.) column, in a
mobile phase of 2% acetic acid in water (solvent A),
0.5% acetic acid, and 45.5% acetonitrile in water (solvent
B) with a flow rate of 1.0 mL/min, and monitored at 290
nm. The CAC was determined by the linear regression
equation used for standard caffeic acid solutions ranges
from 5 mg/kg to 50 mg/kg.
Prediction and validation of optimum condition.
The optimum Viscozyme L aided hydrolysis condition
for both TPC and CAC was predicted within the overlapping
ranges by superimposing the four-dimensional response
surfaces for both components using a Mathematica
program (Version 7.0, Wolfram Research, Inc., USA). To
verify the significance of a regression equation, the
optimum value predicted by setting up the enzyme
Yi β0 βiXi
i 1=
k
∑ βiiXi2
i 1=
k
∑ βij
j 2=
k
∑ XiXj
i 1=
i j<
k 1–
∑+ + +=
344 Hu-Zhe Zheng et al.
hydrolysis conditions at any point within the predicted
ranges and then applying those to the regression equation,
was compared with the actual values from a real
extraction experiment [Kwon et al., 2006].
Results and Discussion
Modeling of the Viscozyme L aided hydrolysis
reaction conditions from unripe apples. In general, the
efficiency of the enzyme aided phytochemical compounds
extraction was influenced by multiple variables including
but not limited to enzyme type and concentration, the
reaction solution pH, the reaction temperature and time,
and their effects were either independent or interactive
[Meyer et al., 1998; Landbo and Meyer 2001; Sørensen
et al., 2005; Pinelo et al., 2006]. From the preliminary
experiments, the ratio of Viscozyme L to substrate of
0.015 (1.5 FBG), the reaction temperature of 50oC, and
the reaction time of 12 h were chosen as the central
condition of the CCRD. Table 1 shows the experimental
conditions and the results of the extraction by the factorial
design. The results of the analysis of variance, goodness
of fit, and the adequacy of the models are summarized in
Table 2. According to Table 2, the regression models
were highly significant (p<0.001 or p<0.01) for all
unripe apples extracts and had a satisfactory coefficient of
determination (R2) that varied from 0.9470 to 0.9555,
analysis of variance was used for the lack of a fit test and
did not reveal an inadequacy of the model with regard to
TPC and CAC having an unacceptable value (p>0.05),
indicating that the model could adequately fit the
experimental data (Table 2). Hence, the data showed a
good fit with Eq. 1.
Effect of Viscozyme L aided hydrolysis variables on
TPC. The TPC of the unripe apples extracts obtained by
Viscozyme L aided extraction and based on the central
composite design are shown in Table 2. Multiple
regression analysis was performed on the experimental
data, and the coefficients of the model were evaluated for
significance. Xiao and Yao [2008] suggested that, for a
good fit of a model, R2 should be at least 0.80. In the
results, the multiple coefficients of correlation R2=0.9470
(p=0.0035) indicated a close agreement between the
experimental and predicted values of the TPC yield. The
corresponding variables were more significant when the
absolute t value became larger and the p value became
smaller [Fu et al., 2006]. The factor t value (7.62) and p
value (p=0.0003) corresponded to the ratio of Viscozyme L
to substrate (β11), while the t values for reaction time (β3)
was smaller at 1.90, but the p value was still significant at
0.096. It was noted that the variable with the largest effect
was the quadratic term of β11, followed by linear term of
β1, quadratic term of reaction temperature (β22), linear
term of β2, quadratic term of β33, and linear term of β3, but
Table 1. Experimental design of the five-variable central composite, TPC, and CAC of Viscozyme L aided hydrolysisreaction from unripe apples
Run NO.Factor valuesa Response values
X1 X2 X3 TPC (mg GAE/100g) CAC (mg/kg)
1 0.0075 (-1) 40 (-1) 8 (-1) 085.88±0.60b 31.59±0.36
2 0.0225 (1) 40 (-1) 8 (-1) 99.46±0.43 36.05±0.37
3 0.0075 (-1) 40 (-1) 16 (1) 89.49±0.56 35.84±0.31
4 0.0225 (1) 40 (-1) 16 (1) 100.78±0.440 35.26±0.35
5 0.0075 (-1) 60 (1) 8 (-1) 90.16±0.09 20.51±0.25
6 0.0225 (1) 60 (1) 8 (-1) 102.42±0.700 23.24±0.22
7 0.0075 (-1) 60 (1) 16 (1) 91.83±0.60 18.91±0.17
8 0.0225 (1) 60 (1) 16 (1) 98.52±0.44 21.63±0.26
9 0.015 (0) 50 (0) 12 (0) 114.90±0.730 43.21±0.45
10 0.015 (0) 50 (0) 12 (0) 115.85±1.070 42.80±0.44
11 0.015 (0) 30 (-2) 12 (0) 90.37±0.15 32.99±0.30
12 0.015 (0) 70 (2) 12 (0) 92.36±0.37 13.48±0.19
13 0.015 (0) 50 (0) 4 (-2) 101.02±0.700 24.93±0.35
14 0.015 (0) 50 (0) 20 (2) 106.86±0.730 31.97±0.37
15 0 (-2) 50 (0) 12 (0) 51.85±0.44 03.14±0.22
16 0.03 (2) 50 (0) 12 (0) 98.25±0.73 20.87±0.42
aNumbers in parentheses are coded symbols for levels of independent parameters, X1; ratio of Viscozyme L to substrate, X2;
reaction temperature (oC), X3; reaction time (h)bMean±SD (n=3).
Optimization of enzyme-aided polyphenol extraction from unripe apples 345
the cross product term was not significant (Table 2). The
regression Eq. 2 of the results from the response surface
analysis is:
YTPC= –151.57375+7401.5X1+6.489625X2
YTPC= +6.122813X3–179222X12–9.86667X1X2
YTPC= –0.060025X22–32.75X1X3–0.022375X2X3
YTPC= –0.178672X32 (2)
In this equation, X1, X2, and X3 represent the conditions
of the ratio of Viscozyme L to substrate, reaction
temperature (oC) and reaction time (h), respectively. Fig.
1 illustrates the four-dimensional response surfaces
drawn using the response surface regression equation of
the yield. The TPC was affected chiefly by the ratio of
Viscozyme L to substrate, reaction temperature (Table 3
and 4). The TPC increased as the ratio of Viscozyme L to
substrate and reaction temperature increased up to a
maximum (the ratio of Viscozyme L to substrate 0.018
(1.8 FBG), reaction temperature 50.27, and reaction time
12.33 h), the predicted stationary point was at the
maximum (116.40 mg GAE/100 g). While TPC was
decreased as the ratio of Viscozyme L to substrate,
reaction temperature, and reaction time were increased
above these maximum values. These results suggested
that an increase of reaction temperature might favor
Viscozyme L activity and enhance the decomposition
activity during the enzyme reaction. Nevertheless, an
excessive decrease or increase of the reaction temperature
partly inhibited the Viscozyme L activity, thereby
decreasing the TPC. Similar results were reported by
Landbo and Meyer [2001], Pinelo et al. [2006], and
Zheng et al. [2008], who found that an optimum given
temperature may enhance the extraction efficiency of
antioxidants, while too high temperature had a
significantly negative effect. Furthermore, an excessive
increase of reaction time reduced TPC, which was
possibly due to the decomposition of antioxidants during
Table 2. Regression coefficients and of t value predicted quadratic polynomial models for the response TPC andCAC of Viscozyme L aided hydrolysis reaction from unripe apples
TermTPC CAC
coefficient t value coefficient t value
β0 –151.6025 –2.78c –137.111250 –3.68b
Linear
β1 7401.5 4.55b 4628.250000 4.16b
β2 6.489625 4.35b 4.569188 4.48b
β3 6.127188 1.90d 7.039844 3.19c
Quadratic
β11 –179222 –7.62a –137778 –8.57a
β22 –0.060025 –4.54b –0.049425 –5.46b
β33 –0.178828 –2.16d –0.227422 –4.02b
Crossproduct
β12 –9.866667 –0.40 2.616667 0.15
β13 –32.75 –0.53 –21.041667 –0.49
β23 –0.022375 –0.48 –0.020844 –0.65
R2e 0.947b 0.9555b
Lack of fit 0.0879 0.0555
aSignificant at p<0.001, bp<0.01, cp<0.05, dp<0.1, eCoefficient of multiple determination.
Fig. 1. Four-dimensional response surfaces for TPC (atconstant values, 70-90-110 mg GAE/100 g) of extractsfrom unripe apples as functions of the ratio ofViscozyme L to substrate, reaction temperature, andreaction time in the Viscozyme L aided extraction.
346 Hu-Zhe Zheng et al.
long Viscozyme L reaction times [Pinelo et al., 2008;
Zheng et al., 2008]. According to Table 2~6 and Fig. 1,
the TPC of the unripe apples were significantly (p<0.01)
affected by the linear and quadratic terms of the ratio of
Viscozyme L to substrate (p<0.001), reaction temperature
(p<0.05), and that reaction time was not significant.
Effect of Viscozyme L aided hydrolysis variables on
CAC. Chlorogenic acid, a major phenolic compound of
apples was hydrolyzed to caffeic acid by carbohydrate-
hydrolyzing enzymes [Zheng et al., 2008]. Hence, caffeic
acid has been suggested as a marker to detect whether
cleavage of the quinic esters of chlorogenic acid by
carbohydrate-hydrolyzing enzymes has occurred [Renard
et al., 2001; Benoit et al., 2006]. Although, caffeic acid
and chlorogenic acids can act as antioxidants in vitro,
caffeic acid is efficiently absorbed through the small
Table 3. Analysis of variance of the regression parameters of the predicted response surface quadratic models
Regression DFc Sum of squares R2 F value p value
TPC
Linear 3 1187.540675 0.3746 14.14b 0.0040
Quadratic 3 1795.843300 0.5665 21.39b 0.0013
Cross product 3 18.511450 0.0058 0.22 0.8788
Total model 9 3001.895425 0.9470 11.92b 0.0035
CAC
Linear 3 684.258369 0.3875 17.42b 0.0023
Quadratic 3 993.724038 0.5628 25.30a 0.0008
Cross product 3 9.057038 0.0051 0.23 0.8720
Total model 9 1687.039444 0.9555 14.32b 0.0021
aSignificant at p<0.001, bp<0.01, cDegrees of freedom.
Table 4. Analysis of variance of the factors obtained from RSM for TPC and CAC
RegressionTPC CAC
F value p value F value p value
Ratio of Viscozyme L to substrate (X1) 25.05a 0.0007 20.81b 0.0012
Reaction temperature (X2, oC) 5.32c 0.0356 18.00b 0.0017
Reaction time (X3, h) 1.41 0.3354 4.46d 0.0518
aSignificant at p<0.001, bp<0.01, cp<0.05, dp<0.1.
Table 5. The optimum conditions for maximum extract of TPC and CAC by ridge analysis and predicted conditionsof response variables by superimposing response surfaces of TPC and CAC from unripe apples in the Viscozyme Laided extraction
Responses X1
a X2
b X3
c Maximum Morphology
TPC 0.018139 50.269607 12.325665 116.401862 Maximum
CAC 0.016243 43.973152 12.710955 45.679476 Maximum
Predicted condition0.0195
(0.018~0.02)47.12
(40~50)12.52
(11~13)
aX1; ratio of Viscozyme L to substrate, bX2; reaction temperature (oC), c X3; reaction time (h).
Table 6. Comparison between the predicted value and experimental values for the response variables at the givenoptimum conditions for unripe applesa
Response variablesMaximum value
B/A×100 (%) Controlb
Predicted value (A) Experimental values (B)
TPC (mg GAE/100g) 115.52 110.52±0.30c 95.67 49.83±0.11
CAC (mg/kg) 43.77 43.13±0.45 98.54 3.21±0.05
aReaction; the ratio of Viscozyme L to substrate 0.0195, 47.12oC, 12.52 h, and pH 3.7bWithout Viscozyme L treatment, cMean±SD (n=3).
Optimization of enzyme-aided polyphenol extraction from unripe apples 347
intestine, increasing the total antioxidant status of plasma
in mammals [Nardini et al., 2002; Gonthier et al., 2006].
Therefore, the hydrolysis of chlorogenic acids to caffeic
acid before consumption or during digestion is very
meaningful to the bioavailability. CAC from unripe apples
under various hydrolysis conditions using Viscozyme L is
presented in the Table 1. Experimental data was subjected
to regression analysis, and the coefficients of the estimate
are presented in Table 2. Regression Eq. 3 was used to
calculate the variation of the contents through the
response surface analysis as follows:
YCAC= –137.11125+4628.25X1+4.569188X2
YCAC= +7.03984X3–13778X1
2+2.61667X1X2
YCAC= –0.049425X2
2–21.041667X1X3
YCAC= –0.020844X2X3–0.227422X3
2 (3)
The R2 of the regression equation for CAC was 0.9555,
which was significant at the 5% level (p<0.05). The CAC
increased as the ratio of Viscozyme L to substrate,
reaction temperature (oC) and reaction time (h) increased
up to a maximum (the ratio of Viscozyme L to substrate
0.016 (1.6 FBG), reaction temperature 43.97, and
reaction time 12.71 h), the predicted stationary point was
at the maximum (45.70 mg/kg) (Table 5). Fig. 2
demonstrates the four-dimensional response surfaces for
CAC obtained from the regression equation. The CAC
was considerably affected by the ratio of Viscozyme L to
substrate, reaction temperature, and reaction time (Table
3 and 4). These results were supported by other research
conducted on olive oil by-products [Bouzid et al., 2005],
coffee pulp and apple marc [Benoit et al., 2006] to release
simple phenolic compounds such as p-coumaric and
caffeic acid using fungal enzymes. However, the
influence of reaction variables on the production of
caffeic acid from plant by-products has not yet been
reported. This research might be the first attempt to
optimize enzyme aided caffeic acid production in apple
by RSM.
Prediction and validation of optimum hydrolysis
condition. To optimize Viscozyme L aided hydrolysis
conditions, quality criteria were established for functional
food materials, these were TPC and CAC. Therefore, the
response surfaces for 110 mg GAE/100 g of TPC and 40
mg/kg of CAC were superimposed to reveal the
overlapped portions, which had the highest response
values in the Viscozyme L aided extraction (Fig. 3). The
ranges of Viscozyme L aided extraction conditions used
for determination of the optimum conditions were ratio of
Viscozyme L to substrate of 0.018~0.02 (1.8~2.0 FBG),
reaction temperature of 40~50oC, and reaction time of
11~13 h (Table 5), these were assumed to maximize TPC
and CAC in the enzyme extraction. In order to validate
the predicted optimum extraction condition for both
components, an optimum point for each condition was
selected within the ranges, i.e. the ratio of Viscozyme L to
substrate 0.0195 (1.95 FBG), reaction temperature 47.12oC, and reaction time 12.52 h (Table 5). Under this
condition, RSM models predicted the yield of TPC and
CAC to be 115.52 mg GAE/100 g and 43.77 mg/kg of
Fig. 3. Superimposed response surface for optimizationof TPC and CAC (at constant values of 110 mg GAE/100g TPC and 40 mg/kg CAC) of extracts from unripeapples as functions of the ratio of Viscozyme L tosubstrate, reaction temperature, and reaction time inthe Viscozyme L aided extraction.
Fig. 2. Four-dimensional response surfaces for CAC (atconstant values, 20-30-40 mg/kg) of extracts fromunripe apples as functions of the ratio of Viscozyme Lto substrate, reaction temperature, and reaction time inthe Viscozyme L aided extraction.
348 Hu-Zhe Zheng et al.
unripe apples, respectively (Table 6). These values showed
good agreement with the experimental values that was
executed and determined at the optimum condition,
which reflects the fitness of the optimization. It was
noteworthy that the TPC and CAC with the Viscozyme L
treatment was 2 and 13 fold higher than that of the
control, respectively (Table 6). HPLC chromatograms of
the unripe apples polyphenol extracted without (A) and
with Viscozyme L (B) are shown in Fig. 4. In contrast to
the control treatment which contains 3 kinds of
polyphenols, 7 kinds of phenolic acids and flavonoids,
such as chlorogenic acid, caffeic acid, p-coumaric acid,
ferulic acid, quercetin-3-glucoside, phloridzin, and
phloretin were detected in the Viscozyme L treatment.
Acknowledgments. This work was supported by
Technology Development Program for Agriculture and
Fishery, Gyeongsangbuk-Do, Republic of Korea.
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