DSpace at VNU: Application of ultrasound in grape mash treatment in juice processing

Ultrasonics Sonochemistry 17 (2010) 273–279

Contents lists available at ScienceDirect

Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultsonch

Application of ultrasound in grape mash treatment in juice processing
Le Ngoc Lieu, Van Viet Man Le *
Dep. of Food Tech., Ho Chi Minh City University of Technology, Ho Chi Minh City, Viet Nam

a r t i c l e

i n f o

Article history:
Received 15 January 2009
Received in revised form 25 April 2009
Accepted 7 May 2009
Available online 13 May 2009
PACS:
43.35.+d
47.35.Rs
62.60.+v
81.40.Gh
83.80.Mc
83.85.Jn

a b s t r a c t
Recently, application of ultrasound has attracted considerable interest as an alternative approach to traditional methods. In this study, response surface methodology (RSM) was used to optimize the conditions
for grape mash treatment by ultrasound and by combination of ultrasound and enzyme. The results indicated that optimal conditions were the temperature of 74 °C and the time of 13 min for sonication treatment; and were the enzyme concentration of 0.05% and the time of 10 min for combined ultrasound and

enzyme treatment. In comparison with traditionally enzymatic treatment, sonication treatment
increased extraction yield 3.4% and shortened treatment time over three times; combined ultrasound
and enzyme treatment increased extraction yield slightly, only 2%, but shortened treatment time over
four times. After sonication treatment, enzymatic treatment increased extraction yield 7.3% and total
treatment time of this method was still shorter than that of traditionally enzymatic treatment method.
Besides, application of ultrasound improved the grape juice quality because it increased contents of sugars, total acids and phenolics as well as color density of grape juice.
Ó 2009 Elsevier B.V. All rights reserved.

Keywords:
Enzymatic treatment
Grape mash
Optimization
Ultrasound

1. Introduction
Grape juice is not consumed in large amounts because it is too
sweet or too acidic [1]. However, grape is the single most abundant
fruit harvested in the world [2] because grape wines are produced
in greatest volume [1]. Traditionally, grape mash is treated with
enzymes to increase volume of free-run juice and to reduce pressing time. However, enzymatic maceration takes much time [3] and
therefore the cost of energy is increased.
Recently, application of ultrasonic technology in food processing has widely attracted attentions. Ultrasound was applied in
extraction of plant materials because of enhancement of yield
and shortening of extraction time [4–6]. There are several studies
on application of ultrasound in extraction, but the authors were
interested in one or two valuable components in the plant extract
such as phenolics [7–9], tartaric and malic acids [10], flavors [11–
13], lycopene [14], oil [15,16], polysaccharides [17–20]. None of
these studies mentioned simultaneous extraction of many compounds by ultrasound in juice processing. In addition, ultrasound
was applied in enzymatic treatment because of its ability of violent

agitation and its positive effects on enzyme activity [21–26].
* Corresponding author. Tel.: +84 8 38 64 62 51; fax: +84 8 38 63 75 04.
E-mail address: (V.V.M. Le).
1350-4177/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultsonch.2009.05.002

However, there are no studies on application of ultrasound in enzymatic treatment of fruit mash in juice processing.
The objective of this study was to determine optimal conditions
of ultrasound assisted process and combined ultrasound and enzyme process for grape mash treatment by using response surface
methodology as well as to compare efficiency of these treatment
methods with that of traditionally enzymatic method.
2. Materials and methods
2.1. Materials
2.1.1. Enzyme source
Pectinex Ultra SP-L from Aspergillus aculeatus obtained from
Novozymes Switzerland AG, Dittengen, Switzerland – was used
in this study. This enzyme preparation contains different pectinolytic enzymes [endo-polygalacturonase (EC 3.2.1.15; C.A.S. No.
9032-75-1), pectin-lyase (EC 4.2.2.10; C.A.S. No. 9033-35-6),
pectin esterase (EC 3.1.1.11; C.A.S. No. 9025-98-3)], and other
activities, such as b-galactosidase, cellulase, chitinase and transgalactosidase [27]. The activity of Pectinex Ultra SP-L is 26,000
PG per mL (polygalacturonase activity per mL). The catalytic temperature and pH of this enzyme preparation are 50 °C and 4.5,
respectively [28–30].


274

L.N. Lieu, V.V.M. Le / Ultrasonics Sonochemistry 17 (2010) 273–279

2.1.2. Grape mash
Grape (Red Cardinal) used in this study was purchased from a local market in Ninh Thuan, Vietnam. Grape was destemmed,

washed and crushing in a blender (National, Vietnam) for 2–
3 min. Then the pH of grape mash was adjusted to value of 4.5.
2.2. Experimental methods
2.2.1. Enzymatic treatment
Samples of 250 mL grape mash were taken for each assay. The
samples were placed into 500 mL flasks.
First series: Different amounts of Pectinex Ultra SP-L were
added into flasks of samples. Enzyme concentration was varied
from 0%v/v to 0.1%v/v. The samples were then kept in the period
of 40 min.
Second series: Pectinex Ultra SP-L (0.04%v/v) was added into
flasks of samples. The treatment time was varied from 10 to
60 min.
In both series, treatment temperature was adjusted to 50 °C by
using a thermostatic water bath (Memmert, WNB 45, Yogyakarta,
Indonesia). At the end of the process, enzymes in the sample were
inactivated by heating the mash at 90 °C for 5 min in a water bath.
The mash was then filtered through a cheese cloth. The obtained
suspension was centrifuged at 6500 rpm for 10 min by a refrigerated centrifuge (Sartorius, Sigma 3K30, Geneva, Switzerland) and
the supernatant was collected for further analysis.
2.2.2. Sonication treatment
A randomised, quadratic central composite circumscribed (CCC)
response surface design was used to study the effect of temperature and treatment time on the extraction yield of grape mash
treatment by ultrasound. The software Modde version 5.0 was
used to generate the experimental planning and to process data.
For each assay, 2 L grape mash with total solid content of
approximately 20% was directly poured into an ultrasonic bath.
The height of the mash in the bath was about 4.5 cm. The bath
(ElmaÒ, T 660/H, Singen, Germany) is a rectangular container
(300 Â 151 Â 150 mm) with the maximal volume of 5.75 L, to

which 35 kHz transducers are annealed at the bottom so that ultrasonic waves are transmitted from the bottom to above. The equipment operated at an ultrasound intensity of 2 W/cm2 and an
ultrasound power of 360 W. The sonotrode of the bath had a surface area of about 180 cm2 which was large enough for ultrasonic
wave to distribute homogeneously in the height of the treated
sample. The bath was equipped with a thermostatic system.
The treatment temperature was ranged from 60 to 80 °C and the
time was ranged from 5 to 15 min. The experimental design is presented in Table 2. At the end of the process, the mash was also filtered and centrifuged in the same way of Section 2.2.1.
2.2.3. Combined ultrasound and enzyme treatment
In this treatment, grape mash was simultaneously treated by
ultrasound and enzyme in the ultrasonic bath. A randomised, quadratic central composite circumscribed (CCC) response surface design was also used to study the effect of enzyme concentration and

treatment time on the extraction yield. The software Modde version 5.0 was also used to generate the experimental planning
and to process data.
For each assay, 2 L grape mash was added directly into the
ultrasonic bath. A determined amount of Pectinex SP-L (from
0.02%v/v to 0.06%v/v) was added and the mixture was stirred before treatment. The treatment time was ranged from 4 to 12 min.
The experimental design is presented in Table 5. Temperature
was maintained at 50 °C. At the end of the treatment, enzymes in
the sample were inactivated by heating the mash at 90 °C for
5 min in a water bath. The following steps were similar to those
in Section 2.2.1.
2.2.4. Enzymatic treatment after sonication
The samples obtained from the experiments of ultrasonic treatment (Section 2.2.2) were then treated with Pectinex Ultra SP-L.
This part consisted of two series of experiments. For each assay,
samples of 250 mL grape mash were taken and placed into
500 mL flasks.
First series: different amounts of Pectinex Ultra SP-L were
added into flasks of samples. Enzyme concentration was varied
from 0%v/v to 0.1%v/v. The samples were then kept in the period
of 20 min.
Second series: Pectinex Ultra SP-L (0.06%v/v) was added into

flasks of samples. The treatment time was ranged from 10 to
40 min.
In both series, temperature was maintained at 50 °C. The following steps were similar to those in Section 2.2.1.
2.2.5. Comparison in physico-chemical characteristics of grape juice
obtained from different grape mash treatment methods
In order to compare some physico-chemical characteristics of
grape juice obtained from different grape mash treatment methods, all experiments were carried out again at the appropriate conditions obtained from Section 2.2.1 to 2.2.4. The obtained samples
were further analyzed in reducing sugar content, total acid content,
total phenolic content and color density. Control samples without
any treatments were also carried out.
Table 2
Experimental planning and results of extraction yield for sonication treatment of
grape mash.
Run

Temperature (°C)

Time (min)

Yield (%)

1
2
3
4
5
6
7
8
9

10
11
12
13

60
80
60
80
55.9
84.1
70
70
70
70
70
70
70

5
5
15
15
10
10
2.9
17.1
10
10
10

10
10

74.9
80.3
79.3
81.0
75.8
80.4
75.9
82.2
81.5
81.4
81.2
81.8
81.3

Table 1
Independent variables and their levels in the response surface design.
Process

Ultrasound assisted treatment
Combined ultrasound and enzyme treatment

Independent variables

Temperature (°C)
Time (min)
Enzyme concentration (%v/v)
Time (min)

Factor level
pffiffiffi
À 2

À1

0

+1

pffiffiffi
þ 2

55.9
2.9
0.012
2.3

60
5
0.02
4

70
10
0.04
8

80

15
0.06
12

84.1
17.1
0.068
13.7


L.N. Lieu, V.V.M. Le / Ultrasonics Sonochemistry 17 (2010) 273–279

2.3. Analytical methods
2.3.1. Extraction yield
The extraction efficiency of the treatment methods was evaluated by using the extraction yield as an index, which was calculated according to the following equation:

Y¼

m2 Â C
 100
m1 Â ð100 À wÞ

ð1Þ

where Y was the extraction yield (%) of the treatment method, m1
and w were the mass (g) and the moisture (%) of the initial grape
mash, respectively; and m2 and C were the mass (g) and the total
soluble solid content (%) of the obtained grape juice after centrifugation, respectively.
To compare the extraction yields obtained from treatment
methods, extraction enhancement E (%) was calculated according

to the following equation:

E¼

Y2 À Y1
 100
Y1

ð2Þ

where Y1 and Y2 were the extraction yields (%) of two compared
treatment methods.
2.3.2. Relative viscosity
Relative viscosity of juice (grel) was determined by using 15 mL
Ostwald viscometer under temperature of 30 °C [31] and was calculated as follow:

grel ¼

  
t
q
qo
to

ð3Þ

where t and q were the flow time and the specific mass of juice,
respectively; to and qo were the flow time and the specific mass
of distilled water, respectively.
2.3.3. Reducing sugars

Reducing sugar content of grape juice was determined by spectrophotometric method using 3,5-dinitrosalicylic acid reagent. This
method was proposed by Miller [32].
2.3.4. Total acids
Titratable acidity determination, expressed in equivalent of tartaric acid content (g/L), was carried out by diluting a 10 mL aliquot
of each sample with 90 mL of distilled water and subsequently
titrating the sample with 0.1 N NaOH to a pH endpoint of 8.1 [33].
2.3.5. Total phenolics
Total phenolic content of grape juice was determined as by
spectrophotometric method using Folin–Ciocalteu reagent. This
method was proposed by Slinkard and Singleton [34].
2.3.6. Color
The color of grape juice was measured with a Konica Minolta
Colorimeter (CR-410, Osaka Japan). Grape juice was placed on
the light port using a 5 cm diameter plastic dish with cover. Color
parameters were recorded as L* (lightness), a* (redness) and b*
(yellowness). The hue angle (h) (h* = arctan b*/a*) and chroma
(C) (C = [(a*)2 + (b*)2]0.5) were also calculated [35].
2.4. Statistical analysis
Response surface methodology was used to find out optimal
conditions of ultrasound assisted treatment and of combined ultrasound and enzyme treatment. The experiments were carried out
according to a central composite design with 2 factors and 5 levels.
Table 1 shows independent variables selected for these two treat-

275

ments. For each factor, an experimental range was based on our results of a preliminary study (unpublished data). Extraction yield
was the dependent variable. The complete design consisted of 13
experimental points including 4 factorial points, 4 axial points
and 5 center points and the experiment was carried out in a random order. The software Modde version 5.0 was used to generate
the experimental planning and to process data.

All experiments were performed in triplicate. The experimental
results obtained were expressed as means ± SD. Mean values were
considered significantly different when P < 0.05. Analysis of variance (ANOVA) was performed using the software Statgraphics plus,
version 3.2.
3. Results and discussion
3.1. Enzymatic treatment
The enzymatic treatment of grape mash increased the extraction yield as results of Fig. 1. The graphs show that the enzyme
concentration of 0.04%v/v and the treatment time of 40 min were
the appropriate conditions for the enzymatic treatment, which increased extraction yield of treated samples approximately 9.2% in
comparison with that of the untreated samples. Treatments with
higher enzyme concentration and longer time did not make significant differences in extraction yield.
Pectinase enzymes are known to work on pectic substances
which occur as structural polysaccharides in the middle lamella
and primary cell wall. The presence of macerating side-activities
in the Pectinex Ultra SP-L preparation, such as cellulases and hemicellulases would result in a more complete breakdown of the polysaccharide structure, causing solubilization of the middle lamella
and improving juice extraction.
Our results agreed with conclusions of many previous studies
which suggested that pectolytic and cellulolytic enzymes could improve juice yield of fruit processing such as studies on apple [36],
pineapple [37], carrot [29], elderberry [38], and orange [39].
3.2. Sonication treatment
Based on our preliminary investigations (unpublished data), a
temperature of 70 °C and a time of 10 min were chosen as the central conditions of the central composite rotary design (CCRD). Table
2 shows extraction yield of each run according to the experimental
planning.
Multiple regression analysis was performed on the experimental data and the coefficients of the model were evaluated for significance with a Student t-test. All the linear coefficients were
significant (P < 0.05). One crossproduct coefficient was eliminated
in the refined equation as its effect was not significant. Neglecting
the insignificant term, the final predictive equation obtained is as
given below:

Y 1 ¼ 81:44 þ 1:70X 1 þ 1:75X 2 À 1:60X 21 À 1:12X 22

ð4Þ

where Y1, X1, X2 were the extraction yield of grape mash treatment
by ultrasound (%), the sonication temperature (°C) and the sonication time (min), respectively.
Table 3 presents ANOVA of the fitted model. According to the
ANOVA table, the regression model is significant at the considered
confidence level since a satisfactory correlation coefficient was obtained and the F-value was 7 times more than the F listed value.
Surface response graph, obtained by using the fitted model presented in Eq. (4), is presented in Fig. 2.
Table 4 presents the estimated effect of each variable, as well as
their interactions on the yield of treatment process. The results
show that temperature and time had significantly positive effects


276

L.N. Lieu, V.V.M. Le / Ultrasonics Sonochemistry 17 (2010) 273–279

A

81
80

Yield (%)

79
78
77
76

75
74
73
0

0.02

0.04

0.06

0.08

0.1

0.12

Enzyme concentration (%v/v)

B

80
81.5 .. 82.3
80.5 .. 81.5
79.5 .. 80.5

79

78.5 .. 79.5
77.5 .. 78.5

74.0 .. 77.5

Yield (%)

78
Fig. 2. Fitted surface for yield of ultrasound assisted treatment of grape mash as a
function of temperature and time.

77
76

Table 4
Estimated effect of independent variables on extraction yield of sonication treatment.

75
74
73
72
0

10

20

30

40

50

60

70

Factora

Effect

Standard error

P

X1
X2
X1 Â X1
X2 Â X2

3.40162
3.50258
À3.19049
À2.24022

0.200553
0.200553
0.2151
0.2151

6.26877E-005
5.18922E-005
0.000147368

0.00124288

X1: sonication temperature, X2: sonication time (min).
P indicates significance of linear regressions.
a
Significant factors at 95% of confidence level.

Treatment time (min)
Fig. 1. Effects of enzyme concentration (A) and treatment time (B) on extraction
yield of enzymatic treatment of grape mash.

Table 3
Analysis of variance of the regression model in experiments of sonication treatment.
Source of variation

SS

DF

MS

F

Regression
Residual
Total
Listed F-valuea

76.93
2.25

79.09

5
7
12

14.936
0.322
6.411

46.423

F(4, 4) = 6.4

SS: sum of squares; DF: degrees of freedom; MS: mean square; F: F-value.
a
F-value at 95% of confidence level.

on yield of the treatment process, while their obvious quadratic effects were also observed, but were negative; and temperature had
stronger effect on extraction yield than time.
The enhancement of extraction yield by ultrasound is attributed
to a physical phenomenon called acoustic cavitation which includes the formation, growth, and violent collapse of small bubbles
or voids in liquids as a result of pressure fluctuation [40]. Collapse

of the bubbles causes shock wave that passes through the solvent,
enhancing the mass transfer within the system [5,6]. At high temperature, the intensity of bubble collapse is weak by the higher vapor pressure. However, increased temperature augments the
number of cavitation bubbles as well as decreases the viscosity
resulting to a more violent collapse. Thus, there is an optimal temperature at which the viscosity is low enough to form enough violent cavitation bubbles, yet the temperature is low enough to avoid
the dampening effect on collapse by a high vapor pressure [41]. In
our study, the optimal temperature of the sample during ultrasonic

treatment was about 74 °C (Fig. 2). Our results agreed with previous researches of other authors who reported that sonication at
70 °C had positive effect on extraction of some compounds of other
plant materials such as phenolic compounds [9], anthocyanins [7],
tartaric and malic acids [10]. The higher temperatures resulted in
the lower extraction yield. Ultrasound has been reported to increase the extractability of polysaccharides from plant materials
[13,19]. These substances block drainage channels in the pulp
through which the juice must pass [1]. As a result, the extraction
yield was lower.
The optimal time of sonication treatment obtained from Fig. 2
was 13 min. Under optimal conditions, the model predicted a max-


277

L.N. Lieu, V.V.M. Le / Ultrasonics Sonochemistry 17 (2010) 273–279

imum response of 82.3%. This value of extraction yield was 12.9%
higher than that of the untreated sample. As a result, application
of ultrasound in grape mash treatment increased the extraction
yield 3.4% more than traditionally enzymatic treatment and the
process time was shortened over three times.
3.3. Combined ultrasound and enzyme treatment
In this experiment, an enzyme concentration of 0.04%v/v and a
time of 8 min were chosen as the central conditions of the CCRD
according to our preliminary results (unpublished data). Table 5
shows results of extraction yield for each run obtained from the
experiments.
In order to establish fitted model, multiple regression analysis
was also performed on the experimental data and the final predictive equation obtained is as given below:

Y 2 ¼ 80:42 þ 1:86X 3 þ 0:61X 4 À 1:36X 23 À 0:59X 24

ð5Þ

where Y2, X3 and X4 were the extraction yield of grape mash treatment by combined ultrasound and enzyme method (%), the enzyme
concentration (%v/v) and the treatment time (min), respectively.
The regression model was significant (P < 0.05) because the Fvalue was 8 times more than the F listed value according to analysis of variance which is presented in Table 6.
In order to determine optimal levels of the variables for the
extraction yield of the treatment, three-dimensional surface plots
were constructed according to Eq. (5) (Fig. 3).
According to the estimated effect of each variable as well as
their interactions on the extraction yield in Table 7, change in enzyme concentration or time resulted in significant change in
extraction yield of the treatment process.
From the model, the obtained optimal conditions were the enzyme concentration of 0.05%v/v and the time of 10 min, at which
the model predicted a maximum response of 81.2%. This value of
extraction yield was 11.4% higher than that of untreated sample.

As a result, combination of ultrasound and enzyme in grape
mash treatment increased extraction yield 2.0% more than traditionally enzymatic treatment and the process time was shortened
over four times; however, its yield was slightly lower than that in
the sonication treatment (Section 3.2). The results of Section 3.2
showed that the optimal temperature of the sonication treatment
was 74 °C while the temperature of 50 °C was kept in this experiment to maintain enzyme activity. Consequently, effect of ultrasound on extraction yield decreased and the extraction yield in
this case was lower. However, the treatment time of this method
was lower than that of the sonication method.
The understanding of the actual effect of ultrasound on enzymes is very little because contradictory results of inactivation
and activation of enzymes upon ultrasound treatment have been
reported. Unlike traditional heat denaturation, the sonication process does not destroy all of enzymes [42]. According to Yachmenev
et al. [25], when ultrasound was specifically used to inactivate enzymes, its actual efficiency was quite low and contrary to common
belief, low intensity and uniform sonication does not damage or

Table 5
Experimental planning and results of extraction yield for combined ultrasound and
enzyme treatment.
Run

Enzyme concentration (%v/v)

Time (min)

Yield (%)

1
2
3
4
5
6
7
8
9
10
11
12
13

0.02
0.06
0.02
0.06

0.012
0.068
0.04
0.04
0.04
0.04
0.04
0.04
0.04

4
4
12
12
8
8
2.3
13.7
8
8
8
8
8

76.0
80.1
77.4
81.2
76.0
81.1

79.2
81.2
81.1
81.1
80.8
80.9
80.7

Table 6
Analysis of variance of the regression model in experiments of combined ultrasound
and enzyme treatment.
Source of variation

SS

DF

MS

F

Regression
Residual
Total
Listed F-valuea

44.864
1.144
46.008

5
7
12

8.973
0.163
3.834

54.927

80.5 .. 81.2
79.5 .. 80.5
78.5 .. 79.5

77.5 .. 78.5
76.5 .. 77.5
75.0 .. 76.5

Fig. 3. Fitted surface for yield of combined ultrasound and enzyme treatment of
grape mash as a function of enzyme concentration and treatment time.

Table 7
Estimated effect of independent variables on yield of ultrasound assisted enzymatic
treatment.
Factora

Effect

Standard error

P

X3
X4
X3 Â X3
X4 Â X4

3.72842
1.2115
À2.72067
À1.17021

0.142909
0.142909
0.153274
0.153274

3.62442E-006
0.00384638
4.67098E-005
0.00656462

F(4, 4) = 6.4

SS: sum of squares; DF: degrees of freedom; MS: mean square; F: F-value.
a
F-value at 95% of confidence level.

X3: enzyme concentration (%v/v), X4: treatment time (min).
P indicates significance of linear regressions.

a
Significant factors at 95% of confidence level.


L.N. Lieu, V.V.M. Le / Ultrasonics Sonochemistry 17 (2010) 273–279

inactivate sensitive structures of enzyme protein macromolecules
[25]. In this study, ultrasound with intensity of 2 W/cm2 improved
the transport of enzyme macromolecules but does not generate an
excessive amount of high reactive intermediates which cause deactivation of enzymes [25]. Moreover, ultrasound was also applied to
activate the catalytic performance of the enzyme macromolecules
adsorbed onto the surface of substrate and to enhance removal of
the products of hydrolytic reaction from the reaction zone [25].
Therefore, ultrasound increased the efficiency of enzymatic treatment with higher extraction yield and lower treatment time.

A

3.4. Enzymatic treatment after sonication
As results of Section 3.2, sonication increased extraction yield of
grape mash treatment, but it also increased content of polysaccharides in the treated samples and this phenomenon made difficulties for free-run juice recovery. If these substances were broken
down, the extraction yield would be higher. Therefore, we examined enzymatic treatment after sonication using the optimal
parameters, i.e. the temperature of 74 °C and the time of 13 min.
The results are presented in Fig. 4.
The graphs show that the enzyme concentration of 0.06%v/v
and the time of 20 min were the appropriate conditions for the
enzymatic treatment after sonication. This treatment increased
the extraction yield approximately 3.8% more than sonication
treatment and 7.3% more than enzymatic treatment.

82

Treated sample
79

Control sample

76
73
70
0

0.02

0.04

0.06

0.08

0.1

0.12

Enzyme concentration (%v/v)

B

88
85

3.5. Comparison in physico-chemical characteristics of grape juice

obtained from different grape mash treatments

82

Yield (%)

The above results indicated that treatment by ultrasound or
combination of ultrasound and enzyme improved extraction yield
as well as shortened treatment time in comparison with traditionally enzymatic treatment of grape mash, and enzymatic treatment
after sonication made the extraction yield increase more. In this
experiment, we determined some physico-chemical characteristics
of grape juice obtained from these treatments. The results are presented in Table 8.
Pectinases are able to break down pectin molecules, mainly colloidal compounds of grape juice. As a result, enzymatic treatment
(ET) decreased viscosity of grape juice (Table 8). On the contrary,
sonication treatment (ST) with ultrasound wave of 2 W/cm2 intensity was not only unable to break down pectin molecules but also
extracted macromolecules from cell walls which increased viscosity of the obtained grape juice. Enzymatic treatment after sonication (ETAS) lowered viscosity due to its ability of pectin
breakdown. However, some other colloidal macromolecules extracted by ultrasound were not broken down by Pectinex Ultra
SP-L preparation. This was the reason why the viscosity of grape
juice in this method was still higher than that in the enzymatic
treatment. In combined ultrasound and enzyme treatment (CUET),
enzyme decreased viscosity while ultrasound increased it. Consequently, viscosity of grape juice in this method was similar to that
of the control sample.
Table 8 also shows that the content of reducing sugars in ET, ST,
CUET and ETAS increased 6.2%, 12.0%, 10.9% and 15.4%, respectively
in comparison with that in the control sample. Although the difference in extraction yield of ET and ST was low (3.4%), the difference
in sugar contents between them was higher (5.8%). The reason
could be that although ET generated grape juice with lower sugar
content, it increased volume of the obtained grape juice. Consequently, the difference in extraction yield was lower.
With regards to total acid content, Table 8 shows that its values
in ET, ST, CUET and ETAS increased 9.9%, 13.6%, 10.9% and 14.3%,

respectively in comparison with that in the control sample. These

88
85

Yield (%)

278

Treated sample
Control sample

79
76
73
70
0

10

20

30

40

50

Treatment time (min)
Fig. 4. Effects of enzyme concentration and treatment time on enzymatic treatment

after sonication.

results suggested that ST possessed greater ability of acid extraction than ET.
In comparison with the control sample, all treated samples contained significantly higher total phenolic content which increased
93.0%, 114.3%, 89.3% and 120.8% in ET, ST, UAET and ETAS, respectively. Our results agreed with many previous researches which reported that ultrasound possessed high extractability for phenolic
compounds such as anthocyanins [7] and total phenolics [9]. The
results showed that ST extracted phenolics more effectively than
ET. In grape cells, phenolic compounds can link with various compounds of cell walls such as polysaccharides or proteins. As a result, random breakdown of cell wall by ultrasound was more
effective than selective breakdown by enzymes. That was the reason why the content of phenolics liberated in the ultrasound treatment was higher.
Table 8 also shows that application of all treatment methods
improved color of the obtained grape juice due to higher values


279

L.N. Lieu, V.V.M. Le / Ultrasonics Sonochemistry 17 (2010) 273–279
Table 8
Comparison in physico-chemical characteristics of grape juice obtained from different grape mash treatments.
Treatment method
C
ET
ST
CUET
ETAS

Relative viscosity
a

1.35 ± 0.01
1.31 ± 0.01b

1.67 ± 0.01c
1.38 ± 0.01d
1.35 ± 0.01a

Reducing sugars (g/L)
a

122.8 ± 0.5
130.4 ± 0.3b
137.5 ± 0.6c
141.8 ± 0.3d
136.1 ± 0.8e

Total acidity (g tartaric acid/L)
a

4.13 ± 0.01
4.54 ± 0.01b
4.69 ± 0.01c
4.72 ± 0.01d
4.58 ± 0.01e

Phenolics (g/L)
a

2.56 ± 0.01
4.93 ± 0.03b
5.48 ± 0.01c
5.64 ± 0.04d
4.84 ± 0.03e

C*

L*
a

50.6 ± 0.5
49.4 ± 0.9ab
48.4 ± 0.6bc
49.0 ± 1.0bc
48.1 ± 0.1c

H*
a

24.7 ± 0.7
30.7 ± 0.3b
29.9 ± 0.8bc
29.7 ± 0.3c
30.0 ± 0.0bc

57.7 ± 1.0a
49.0 ± 1.6b
43.5 ± 0.7c
43.1 ± 0.4c
43.7 ± 0.6c

C: control sample, ET: enzymatic treatment, ST: sonication treatment, CUET: combined ultrasound and enzyme treatment, ETAS: enzymatic treatment after sonication, C*:
chroma; L*: lightness; H*: hue angle.
Each value is expressed as mean and standard deviation.

Values are significantly different (P = 0.05) from other values within a column unless they have at least one similar superscript letter.

of C* and lower values of H*. It should be noted that ST, CUET and
ETAS produced grape juices with lower values of H* than ET. These
results illustrated that red pigment content of grape juice obtained
from ST, CUET and ETAS was higher than that from ET. In other
words, application of ultrasound in grape mash treatment improved color of the obtained grape juice more effectively than
application of commercial enzyme. Table 8 also reports that lightness of all treated samples decreased because of the increase in
color density.
4. Conclusions
In comparison with traditionally enzymatic treatment, application of ultrasound in grape mash treatment enhanced extraction
yield and shortened treatment time. Besides, these methods improved quality of the obtained grape juice because they increased
sugar content, total acid content, phenolics content as well as color
density of grape juice.
References
[1] D.R. Kashyaq, P.K. Vohra, S. Chopra, R. Tewari, Bioresource Technol. 77 (3)
(2001) 215–227.
[2] O. Munoz, M. Sepúlveda, M. Schwartz, Food Chem. 87 (2004) 487–490.
[3] F.S.S. Rogerson, E. Vale, H.J. Grande, M.C.M. Silva, Cien. Technol. Alim. 2 (5)
(2000) 222–227.
[4] T.J. Mason, E.D. Cordemans, Trans. Inst. Chem. Eng. 74 (1996) 511–516.
[5] M. Toma, M. Vinatoru, L. Paniwnyk, T.J. Mason, Ultrason. Sonochem. 8 (2001)
137–142.
[6] J. Wu, L. Lin, F. Chau, Ultrason. Sonochem. 8 (4) (2001) 347–352.
[7] Fang Chen, Yangzhao Sun, Guanghua Zhao, Xiaojun Liao, Xiaosong Hu, Jihong
Wu, Zhengfu Wang, Ultrason. Sonochem. 14 (2007) 767–778.
[8] A.H. Goli, M. Barzegar, M.A. Sahari, Food Chem. 92 (2005) 521–525.
[9] Jing Wang, Baoguo Sun, Yanping Cao, Yuan Tian, Xuehong Li, Food Chem. 106
(2008) 804–810.
[10] M. Palma, C.G. Barroso, Anal. Chim. Acta 458 (2002) 119–130.

[11] T. Furuki, S. Maeda, S. Imajo, T. Hiroi, T. Amaya, T. Hirokawa, J. Appl. Phycol. 15
(2003) 319–324.
[12] R. Ilda Caldeira, M. Pereira, A.P. Cristina Cl´ımaco, R. Belchior, Bruno de Sousa,
Anal. Chim. Acta 513 (2004) 125–134.

[13] L. Paniwynk, E. Beaufoy, P. Lorimer, J. Mason, Ultrason. Sonochem. 8 (2001)
299–301.
[14] Zhang Lianfu, Liu Zelong, Ultrason. Sonochem. 15 (5) (2008) 731–737.
[15] Ai-jun Hu, Shuna Zhao, Hanhua Liang, Tai-qiu Qiu, Guohua Chen, Ultrason.
Sonochem. 14 (2007) 219–224.
[16] Haizhou Li, Lester Pordesimo, Jochen Weiss, Food Res. Int. 37 (2003) 731–738.
[17] A. Ebringerová, Z. Hromádková, J. Alfödia, B. Hrˇíbalová, Carbohyd. Poly. 37
(1998) 231–239.
[18] A. Ebringerová, Z. Hromádková, Ultrason. Sonochem. 9 (2002) 225–229.
[19] Z. Hromádková, A. Ebringerová, Ultrason. Sonochem. 10 (2003) 127–133.
[20] Z. Hroma´dkova´, J. Kova´c’ikova´, A. Ebringerova, Ind. Crops Prod. 9 (1999) 101–
109.
[21] Chengzhou Li, Makoto Yoshimoto, Haruki Ogata, Naoki Tsukuda, Kimitoshi
Fukunaga, Katsumi Nakao, Ultrason. Sonochem. 12 (2005) 373–384.
[22] H. Entezari, H. Nazary, H. Khodaparast, Ultrason. Sonochem. 11 (2004) 379–
384.
[23] Stephen Barton, Clive Bullock, Deborah Weir, En. Micro. Technol. 18 (1996)
190–194.
[24] Val G. Yachmenev, Eugene J. Blanchard, Allan H. Lambert, Ultrasonics 42
(2004) 87–91.
[25] Val G. Yachmenev, B.D. Condon, A. H. Lambert, in: The 19th International
Congress on Acoustics, Madrid, Spain, 2007.
[26] Yaxuan Liu, Qingzhe Jin, Liang Shan, Yuanfa Liu, Wei Shen, Xingguo Wang,
Ultrason. Sonochem. 15 (2008) 402–407.
[27] G. Iraj, G.de S. Aránzazu, F.A. Lucia, A. Miguel, Y. Malcolm, R.C.M. Luisa, P.

Fancisco, B. Antonio, J. Mol. Catal. 35 (1–3) (2005) 19–27.
[28] Y. Aslan, A. Tanrıseven, J. Mol. Catal. 45 (2007) 73–77.
[29] N. Demir, J. Acar, K. Sarõoglu, M. Mutlu, J. Food Eng. 47 (2001) 275–280.
[30] K. Sarioglu, N. Demir, J. Acar, M. Mutlu, J. Food Eng. 47 (2001) 271–274.
[31] S.E. Harding, Prog. Biophys. Mol. Bio. 68 (1997) 207–262.
[32] G.L. Miller, Anal. Chem. 31 (1959) 426–428.
[33] Margaret A. Cliff, Marjoire C. King, Jimmy Schlosser, Food Res. Int. 40 (2007)
92–100.
[34] K. Slinkard, V.L. Singleton, Am. J. Enol. Viticult. 28 (1977) 49–55.
[35] B. Ancos, E. Gonzalez, M.P. Cano, Z. Lebensm, Unters. Forsch. A 208 (1999) 33–38.
[36] I. Alkorta, C. Garbisu, M.J. Llama, J.L. Serra, Pro. Biochem. 33 (1998) 21–28.
[37] K. Chen Chin, A. Yuguwa, H. Yamaoto, J. Food Sci. 49 (1984) 1327–1329.
[38] A.K. Landbo, K. Kaack, A.S. Meyer, Innov. Food Sci. Em. Technol. 8 (2007) 135–
142.
[39] H. Rebeck, Processing of citrus juices, in: D. Hick (Ed.), Production and
Packaging of Non-Carbohydrate Fruit Juices and Fruit Beverages, Van Nosrand
Reinhold, New York, 1990.
[40] K.S. Suslick, Ultrasounds: Its Chemical, Physical and Biological Effects, VHC,
New York, 1988.
[41] A. Patist, D. Bates, Innov. Food Sci. Em. Technol. 9 (2008) 147–154.
[42] D. Güzey, I. Gülseren, B. Bruce, J. Weiss, Food Hydrocolloids 20 (2006) 669–
677.