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Ultrasonics Sonochemistry xxx (2009) xxx–xxx

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 a b s t r a c t

Article history: Recently, application of ultrasound has attracted considerable interest as an alternative approach to tra-
Received 15 January 2009 ditional methods. In this study, response surface methodology (RSM) was used to optimize the conditions
Received in revised form 25 April 2009 for grape mash treatment by ultrasound and by combination of ultrasound and enzyme. The results indi-
Accepted 7 May 2009
cated that optimal conditions were the temperature of 74 °C and the time of 13 min for sonication treat-
Available online xxxx
ment; 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
PACS:
increased extraction yield 3.4% and shortened treatment time over three times; combined ultrasound
43.35.+d
47.35.Rs
and enzyme treatment increased extraction yield slightly, only 2%, but shortened treatment time over
62.60.+v four times. After sonication treatment, enzymatic treatment increased extraction yield 7.3% and total
81.40.Gh treatment time of this method was still shorter than that of traditionally enzymatic treatment method.
83.80.Mc Besides, application of ultrasound improved the grape juice quality because it increased contents of sug-
83.85.Jn ars, 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 However, there are no studies on application of ultrasound in enzy-

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 press-
ing time. However, enzymatic maceration takes much time [3] and
therefore the cost of energy is increased.
Recently, application of ultrasonic technology in food process-
ing 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 com-
pounds 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: lvvman@hcmut.edu.vn (V.V.M. Le).
matic 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 en-
zyme 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 pectino-
lytic 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 trans-
galactosidase [27]. The activity of Pectinex Ultra SP-L is 26,000
PG per mL (polygalacturonase activity per mL). The catalytic tem-
perature and pH of this enzyme preparation are 50 °C and 4.5,
respectively [28–30].

1350-4177/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultsonch.2009.05.002

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2.1.2. Grape mash treatment time on the extraction yield. The software Modde ver-
Grape (Red Cardinal) used in this study was purchased from a lo- sion 5.0 was also used to generate the experimental planning
cal market in Ninh Thuan, Vietnam. Grape was destemmed, and to process data.
washed and crushing in a blender (National, Vietnam) for 2– For each assay, 2 L grape mash was added directly into the
3 min. Then the pH of grape mash was adjusted to value of 4.5. 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 be-
2.2. Experimental methods fore treatment. The treatment time was ranged from 4 to 12 min.
The experimental design is presented in Table 5. Temperature
2.2.1. Enzymatic treatment was maintained at 50 °C. At the end of the treatment, enzymes in
Samples of 250 mL grape mash were taken for each assay. The the sample were inactivated by heating the mash at 90 °C for
samples were placed into 500 mL flasks. 5 min in a water bath. The following steps were similar to those
First series: Different amounts of Pectinex Ultra SP-L were in Section 2.2.1.
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 2.2.4. Enzymatic treatment after sonication
of 40 min. The samples obtained from the experiments of ultrasonic treat-
Second series: Pectinex Ultra SP-L (0.04%v/v) was added into ment (Section 2.2.2) were then treated with Pectinex Ultra SP-L.
flasks of samples. The treatment time was varied from 10 to This part consisted of two series of experiments. For each assay,
60 min. samples of 250 mL grape mash were taken and placed into
In both series, treatment temperature was adjusted to 50 °C by 500 mL flasks.
using a thermostatic water bath (Memmert, WNB 45, Yogyakarta, First series: different amounts of Pectinex Ultra SP-L were
Indonesia). At the end of the process, enzymes in the sample were added into flasks of samples. Enzyme concentration was varied
inactivated by heating the mash at 90 °C for 5 min in a water bath. from 0%v/v to 0.1%v/v. The samples were then kept in the period
The mash was then filtered through a cheese cloth. The obtained of 20 min.
suspension was centrifuged at 6500 rpm for 10 min by a refriger- Second series: Pectinex Ultra SP-L (0.06%v/v) was added into
ated centrifuge (Sartorius, Sigma 3K30, Geneva, Switzerland) and flasks of samples. The treatment time was ranged from 10 to
the supernatant was collected for further analysis. 40 min.
In both series, temperature was maintained at 50 °C. The fol-
2.2.2. Sonication treatment lowing steps were similar to those in Section 2.2.1.
A randomised, quadratic central composite circumscribed (CCC)
response surface design was used to study the effect of tempera- 2.2.5. Comparison in physico-chemical characteristics of grape juice
ture and treatment time on the extraction yield of grape mash obtained from different grape mash treatment methods
treatment by ultrasound. The software Modde version 5.0 was In order to compare some physico-chemical characteristics of
used to generate the experimental planning and to process data. grape juice obtained from different grape mash treatment meth-
For each assay, 2 L grape mash with total solid content of ods, all experiments were carried out again at the appropriate con-
approximately 20% was directly poured into an ultrasonic bath. ditions obtained from Section 2.2.1 to 2.2.4. The obtained samples
The height of the mash in the bath was about 4.5 cm. The bath were further analyzed in reducing sugar content, total acid content,
(ElmaÒ, T 660/H, Singen, Germany) is a rectangular container total phenolic content and color density. Control samples without
(300  151  150 mm) with the maximal volume of 5.75 L, to any treatments were also carried out.
which 35 kHz transducers are annealed at the bottom so that ultra-
sonic waves are transmitted from the bottom to above. The equip-
Table 2
ment operated at an ultrasound intensity of 2 W/cm2 and an Experimental planning and results of extraction yield for sonication treatment of
ultrasound power of 360 W. The sonotrode of the bath had a sur- grape mash.
face area of about 180 cm2 which was large enough for ultrasonic
Run Temperature (°C) Time (min) Yield (%)
wave to distribute homogeneously in the height of the treated
1 60 5 74.9
sample. The bath was equipped with a thermostatic system.
2 80 5 80.3
The treatment temperature was ranged from 60 to 80 °C and the 3 60 15 79.3
time was ranged from 5 to 15 min. The experimental design is pre- 4 80 15 81.0
sented in Table 2. At the end of the process, the mash was also fil- 5 55.9 10 75.8
tered and centrifuged in the same way of Section 2.2.1. 6 84.1 10 80.4
7 70 2.9 75.9
8 70 17.1 82.2
2.2.3. Combined ultrasound and enzyme treatment 9 70 10 81.5
In this treatment, grape mash was simultaneously treated by 10 70 10 81.4
ultrasound and enzyme in the ultrasonic bath. A randomised, qua- 11 70 10 81.2
12 70 10 81.8
dratic central composite circumscribed (CCC) response surface de-
13 70 10 81.3
sign was also used to study the effect of enzyme concentration and

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

Process Independent variables Factor level

pffiffiffi pffiffiffi
 2 1 0 +1 þ 2
Ultrasound assisted treatment Temperature (°C) 55.9 60 70 80 84.1
Time (min) 2.9 5 10 15 17.1
Combined ultrasound and enzyme treatment Enzyme concentration (%v/v) 0.012 0.02 0.04 0.06 0.068
Time (min) 2.3 4 8 12 13.7

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2.3. Analytical methods
2.3.1. Extraction yield
The extraction efficiency of the treatment methods was evalu-
ated by using the extraction yield as an index, which was calcu-
lated according to the following equation:
m2  C
Y¼  100
m1  ð100  wÞ
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 centrifu-
gation, respectively.
To compare the extraction yields obtained from treatment
methods, extraction enhancement E (%) was calculated according
to the following equation:
Y2  Y1
E¼  100
Y1
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 cal-
culated as follow:
  
t q
grel ¼
to qo
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 spec-
trophotometric 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 tar-
taric 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 ultra-
sound 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-
Please cite this article in press as: L.N. Lieu, V.V.M. Le, Application of ultrasound in grape mash treatment in juice processing, Ultrason. Sonochem. (2009),
doi:10.1016/j.ultsonch.2009.05.002
3
ments. For each factor, an experimental range was based on our re-
sults 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 ran-
dom order. The software Modde version 5.0 was used to generate
the experimental planning and to process data.
ð1Þ 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 vari-
ance (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 extrac-
ð2Þ tion 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 in-
creased 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 signif-
icant 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
ð3Þ in the Pectinex Ultra SP-L preparation, such as cellulases and hemi-
cellulases would result in a more complete breakdown of the poly-
saccharide 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 im-
prove 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 cen-
tral 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 experimen-
tal data and the coefficients of the model were evaluated for signif-
icance 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  1:60X 21  1:12X 22 ð4Þ
where Y1, X1, X2 were the extraction yield of grape mash treatment
by ultrasound (%), the sonication temperature (°C) and the sonica-
tion 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 ob-
tained and the F-value was 7 times more than the F listed value.
Surface response graph, obtained by using the fitted model pre-
sented 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
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A 81

80

79

78
Yield (%)

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 78.5 .. 79.5
79 80.5 .. 81.5 77.5 .. 78.5
79.5 .. 80.5 74.0 .. 77.5
78
Fig. 2. Fitted surface for yield of ultrasound assisted treatment of grape mash as a
function of temperature and time.
77
Yield (%)

76
Table 4
75 Estimated effect of independent variables on extraction yield of sonication treatment.

Factora Effect Standard error P

74
X1 3.40162 0.200553 6.26877E-005
X2 3.50258 0.200553 5.18922E-005
73 X1  X1 3.19049 0.2151 0.000147368
X2  X2 2.24022 0.2151 0.00124288
72 X1: sonication temperature, X2: sonication time (min).
0 10 20 30 40 50 60 70 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. of the bubbles causes shock wave that passes through the solvent,
enhancing the mass transfer within the system [5,6]. At high tem-
perature, the intensity of bubble collapse is weak by the higher va-
por pressure. However, increased temperature augments the
Table 3 number of cavitation bubbles as well as decreases the viscosity
Analysis of variance of the regression model in experiments of sonication treatment. resulting to a more violent collapse. Thus, there is an optimal tem-
Source of variation SS DF MS F perature at which the viscosity is low enough to form enough vio-
Regression 76.93 5 14.936 46.423
lent cavitation bubbles, yet the temperature is low enough to avoid
Residual 2.25 7 0.322 the dampening effect on collapse by a high vapor pressure [41]. In
Total 79.09 12 6.411 our study, the optimal temperature of the sample during ultrasonic
Listed F-valuea F(4, 4) = 6.4 treatment was about 74 °C (Fig. 2). Our results agreed with previ-
SS: sum of squares; DF: degrees of freedom; MS: mean square; F: F-value. ous researches of other authors who reported that sonication at
a
F-value at 95% of confidence level. 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
on yield of the treatment process, while their obvious quadratic ef- the lower extraction yield. Ultrasound has been reported to in-
fects were also observed, but were negative; and temperature had crease the extractability of polysaccharides from plant materials
stronger effect on extraction yield than time. [13,19]. These substances block drainage channels in the pulp
The enhancement of extraction yield by ultrasound is attributed through which the juice must pass [1]. As a result, the extraction
to a physical phenomenon called acoustic cavitation which in- yield was lower.
cludes the formation, growth, and violent collapse of small bubbles The optimal time of sonication treatment obtained from Fig. 2
or voids in liquids as a result of pressure fluctuation [40]. Collapse was 13 min. Under optimal conditions, the model predicted a max-

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imum response of 82.3%. This value of extraction yield was 12.9% As a result, combination of ultrasound and enzyme in grape
higher than that of the untreated sample. As a result, application mash treatment increased extraction yield 2.0% more than tradi-
of ultrasound in grape mash treatment increased the extraction tionally enzymatic treatment and the process time was shortened
yield 3.4% more than traditionally enzymatic treatment and the over four times; however, its yield was slightly lower than that in
process time was shortened over three times. the sonication treatment (Section 3.2). The results of Section 3.2
showed that the optimal temperature of the sonication treatment
3.3. Combined ultrasound and enzyme treatment was 74 °C while the temperature of 50 °C was kept in this experi-
ment to maintain enzyme activity. Consequently, effect of ultra-
In this experiment, an enzyme concentration of 0.04%v/v and a sound on extraction yield decreased and the extraction yield in
time of 8 min were chosen as the central conditions of the CCRD this case was lower. However, the treatment time of this method
according to our preliminary results (unpublished data). Table 5 was lower than that of the sonication method.
shows results of extraction yield for each run obtained from the The understanding of the actual effect of ultrasound on en-
experiments. zymes is very little because contradictory results of inactivation
In order to establish fitted model, multiple regression analysis and activation of enzymes upon ultrasound treatment have been
was also performed on the experimental data and the final predic- reported. Unlike traditional heat denaturation, the sonication pro-
tive equation obtained is as given below: cess does not destroy all of enzymes [42]. According to Yachmenev
et al. [25], when ultrasound was specifically used to inactivate en-
Y 2 ¼ 80:42 þ 1:86X 3 þ 0:61X 4  1:36X 23  0:59X 24 ð5Þ
zymes, its actual efficiency was quite low and contrary to common
where Y2, X3 and X4 were the extraction yield of grape mash treat- belief, low intensity and uniform sonication does not damage or
ment 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 F-
value was 8 times more than the F listed value according to anal-
ysis 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 en-
zyme concentration or time resulted in significant change in
extraction yield of the treatment process.
From the model, the obtained optimal conditions were the en-
zyme 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.

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

Run Enzyme concentration (%v/v) Time (min) Yield (%)

1 0.02 4 76.0
2 0.06 4 80.1
3 0.02 12 77.4
4 0.06 12 81.2
5 0.012 8 76.0
6 0.068 8 81.1 80.5 .. 81.2 77.5 .. 78.5
7 0.04 2.3 79.2 79.5 .. 80.5 76.5 .. 77.5
8 0.04 13.7 81.2 78.5 .. 79.5 75.0 .. 76.5
9 0.04 8 81.1
10 0.04 8 81.1
11 0.04 8 80.8 Fig. 3. Fitted surface for yield of combined ultrasound and enzyme treatment of
12 0.04 8 80.9 grape mash as a function of enzyme concentration and treatment time.
13 0.04 8 80.7

Table 7
Table 6 Estimated effect of independent variables on yield of ultrasound assisted enzymatic
Analysis of variance of the regression model in experiments of combined ultrasound treatment.
and enzyme treatment.
Factora Effect Standard error P
Source of variation SS DF MS F
X3 3.72842 0.142909 3.62442E-006
Regression 44.864 5 8.973 X4 1.2115 0.142909 0.00384638
Residual 1.144 7 0.163 54.927 X3  X3 2.72067 0.153274 4.67098E-005
Total 46.008 12 3.834 X4  X4 1.17021 0.153274 0.00656462
Listed F-valuea F(4, 4) = 6.4
X3: enzyme concentration (%v/v), X4: treatment time (min).
SS: sum of squares; DF: degrees of freedom; MS: mean square; F: F-value. P indicates significance of linear regressions.
a a
F-value at 95% of confidence level. Significant factors at 95% of confidence level.

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inactivate sensitive structures of enzyme protein macromolecules 88

[25]. In this study, ultrasound with intensity of 2 W/cm2 improved
A
the transport of enzyme macromolecules but does not generate an
excessive amount of high reactive intermediates which cause deac- 85
tivation 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 82

Yield (%)
the products of hydrolytic reaction from the reaction zone [25].
Therefore, ultrasound increased the efficiency of enzymatic treat- Treated sample
ment with higher extraction yield and lower treatment time. 79 Control sample

3.4. Enzymatic treatment after sonication 76

As results of Section 3.2, sonication increased extraction yield of
grape mash treatment, but it also increased content of polysaccha- 73
rides in the treated samples and this phenomenon made difficul-
ties for free-run juice recovery. If these substances were broken
down, the extraction yield would be higher. Therefore, we exam- 70
ined enzymatic treatment after sonication using the optimal
0 0.02 0.04 0.06 0.08 0.1 0.12
parameters, i.e. the temperature of 74 °C and the time of 13 min.
The results are presented in Fig. 4. Enzyme concentration (%v/v)
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 B 88
the extraction yield approximately 3.8% more than sonication
treatment and 7.3% more than enzymatic treatment.
85

3.5. Comparison in physico-chemical characteristics of grape juice

obtained from different grape mash treatments 82
Treated sample
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 tradition-
ally 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 pre-
sented in Table 8.
Pectinases are able to break down pectin molecules, mainly col-
loidal 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 inten-
sity was not only unable to break down pectin molecules but also
extracted macromolecules from cell walls which increased viscos-
ity of the obtained grape juice. Enzymatic treatment after sonica-
tion (ETAS) lowered viscosity due to its ability of pectin
breakdown. However, some other colloidal macromolecules ex-
tracted 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. Conse-
quently, 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 differ-
ence 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. Conse-
quently, 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
79 Control sample
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 extrac-
tion than ET.
In comparison with the control sample, all treated samples con-
tained significantly higher total phenolic content which increased
93.0%, 114.3%, 89.3% and 120.8% in ET, ST, UAET and ETAS, respec-
tively. Our results agreed with many previous researches which re-
ported 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 com-
pounds of cell walls such as polysaccharides or proteins. As a re-
sult, random breakdown of cell wall by ultrasound was more
effective than selective breakdown by enzymes. That was the rea-
son why the content of phenolics liberated in the ultrasound treat-
ment was higher.
Table 8 also shows that application of all treatment methods
improved color of the obtained grape juice due to higher values

Please cite this article in press as: L.N. Lieu, V.V.M. Le, Application of ultrasound in grape mash treatment in juice processing, Ultrason. Sonochem. (2009),
doi:10.1016/j.ultsonch.2009.05.002
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L.N. Lieu, V.V.M. Le / Ultrasonics Sonochemistry xxx (2009) xxx–xxx 7

Table 8
Comparison in physico-chemical characteristics of grape juice obtained from different grape mash treatments.

Treatment method Relative viscosity Reducing sugars (g/L) Total acidity (g tartaric acid/L) Phenolics (g/L) C* L* H*
a a a a a a
C 1.35 ± 0.01 122.8 ± 0.5 4.13 ± 0.01 2.56 ± 0.01 50.6 ± 0.5 24.7 ± 0.7 57.7 ± 1.0a
ET 1.31 ± 0.01b 130.4 ± 0.3b 4.54 ± 0.01b 4.93 ± 0.03b 49.4 ± 0.9ab 30.7 ± 0.3b 49.0 ± 1.6b
ST 1.67 ± 0.01c 137.5 ± 0.6c 4.69 ± 0.01c 5.48 ± 0.01c 48.4 ± 0.6bc 29.9 ± 0.8bc 43.5 ± 0.7c
CUET 1.38 ± 0.01d 141.8 ± 0.3d 4.72 ± 0.01d 5.64 ± 0.04d 49.0 ± 1.0bc 29.7 ± 0.3c 43.1 ± 0.4c
ETAS 1.35 ± 0.01a 136.1 ± 0.8e 4.58 ± 0.01e 4.84 ± 0.03e 48.1 ± 0.1c 30.0 ± 0.0bc 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 im-
proved color of the obtained grape juice more effectively than
application of commercial enzyme. Table 8 also reports that light-
ness of all treated samples decreased because of the increase in
color density.
4. Conclusions
In comparison with traditionally enzymatic treatment, applica-
tion of ultrasound in grape mash treatment enhanced extraction
yield and shortened treatment time. Besides, these methods im-
proved quality of the obtained grape juice because they increased
sugar content, total acid content, phenolics content as well as color
density of grape juice.
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Please cite this article in press as: L.N. Lieu, V.V.M. Le, Application of ultrasound in grape mash treatment in juice processing, Ultrason. Sonochem. (2009),
doi:10.1016/j.ultsonch.2009.05.002