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In comparison with traditionally enzymatic treatment, sonication treatment increased extraction yield 3.4% and shortened treatment time over three times; combined ultrasound and enzyme t

Trang 1

Application of ultrasound in grape mash treatment in juice processing

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

Keywords:

Enzymatic treatment

Grape mash

Optimization

Ultrasound

a b s t r a c t

Recently, application of ultrasound has attracted considerable interest as an alternative approach to tra-ditional 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 indi-cated that optimal conditions were the temperature of 74 °C and the time of 13 min for sonication treat-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 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 sug-ars, total acids and phenolics as well as color density of grape juice

Ó 2009 Elsevier B.V All rights reserved

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

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]

However, there are no studies on application of ultrasound in enzy-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.

* 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).

Contents lists available atScienceDirect

Ultrasonics Sonochemistry

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

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2.1.2 Grape mash

Grape (Red Cardinal) used in this study was purchased from a

lo-cal 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

refriger-ated 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

tempera-ture 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  151  150 mm) with the maximal volume of 5.75 L, to

which 35 kHz transducers are annealed at the bottom so that

ultra-sonic waves are transmitted from the bottom to above The

equip-ment operated at an ultrasound intensity of 2 W/cm2 and an

ultrasound power of 360 W The sonotrode of the bath had a

sur-face area of about 180 cm2which 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

pre-sented inTable 2 At the end of the process, the mash was also

fil-tered and centrifuged in the same way of Section2.2.1

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,

qua-dratic central composite circumscribed (CCC) response surface

de-sign was also used to study the effect of enzyme concentration and

treatment time on the extraction yield The software Modde ver-sion 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 be-fore 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 Section2.2.1

2.2.4 Enzymatic treatment after sonication The samples obtained from the experiments of ultrasonic treat-ment (Section2.2.2) 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 fol-lowing steps were similar to those in Section2.2.1

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 meth-ods, all experiments were carried out again at the appropriate con-ditions obtained from Section2.2.1 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 1

Independent variables and their levels in the response surface design.

 ffiffiffi 2 p

2 p

Combined ultrasound and enzyme treatment Enzyme concentration (%v/v) 0.012 0.02 0.04 0.06 0.068

Table 2 Experimental planning and results of extraction yield for sonication treatment of grape mash.

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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:

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 m2and 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:

E ¼Y2 Y1

where Y1and Y2were 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:

grel¼ t

to

qo

 

ð3Þ where t andqwere the flow time and the specific mass of juice,

respectively; toandqowere 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 1shows independent variables selected for these two

treat-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

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-tion yield as results ofFig 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

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 cencen-tral composite rotary design (CCRD).Table

2shows 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:

where Y1, X1, X2were the extraction yield of grape mash treatment

by ultrasound (%), the sonication temperature (°C) and the sonica-tion time (min), respectively

Table 3presents 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 inFig 2

Table 4presents 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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on yield of the treatment process, while their obvious quadratic

ef-fects 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

in-cludes 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 tem-perature, the intensity of bubble collapse is weak by the higher va-por 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 tem-perature at which the viscosity is low enough to form enough vio-lent 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 previ-ous 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 in-crease 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 fromFig 2

was 13 min Under optimal conditions, the model predicted a

max-73

74

75

76

77

78

79

80

81

0 0.02 0.04 0.06 0.08 0.1 0.12

Enzyme concentration (%v/v)

72

73

74

75

76

77

78

79

80

Treatment time (min)

A

B

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.

Listed F-value a

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.

81.5 82.3 80.5 81.5 79.5 80.5

78.5 79.5 77.5 78.5 74.0 77.5

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

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

X 1 : sonication temperature, X 2 : sonication time (min).

P indicates significance of linear regressions.

a

Significant factors at 95% of confidence level.

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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

predic-tive equation obtained is as given below:

where Y2, X3and X4were the extraction yield of grape mash

treat-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 F-value according to

anal-ysis of variance which is presented inTable 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 inTable 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

As a result, combination of ultrasound and enzyme in grape mash treatment increased extraction yield 2.0% more than tradi-tionally enzymatic treatment and the process time was shortened over four times; however, its yield was slightly lower than that in the sonication treatment (Section3.2) The results of Section3.2

showed that the optimal temperature of the sonication treatment was 74 °C while the temperature of 50 °C was kept in this experi-ment to maintain enzyme activity Consequently, effect of ultra-sound 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 en-zymes is very little because contradictory results of inactivation and activation of enzymes upon ultrasound treatment have been reported Unlike traditional heat denaturation, the sonication pro-cess does not destroy all of enzymes[42] According to Yachmenev

et al.[25], when ultrasound was specifically used to inactivate en-zymes, its actual efficiency was quite low and contrary to common belief, low intensity and uniform sonication does not damage or

Table 6

Analysis of variance of the regression model in experiments of combined ultrasound

and enzyme treatment.

Listed F-value a

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.

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.

X 3 : enzyme concentration (%v/v), X 4 : treatment time (min).

P indicates significance of linear regressions.

a

Table 5

Experimental planning and results of extraction yield for combined ultrasound and

enzyme treatment.

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

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inactivate sensitive structures of enzyme protein macromolecules

[25] In this study, ultrasound with intensity of 2 W/cm2improved

the transport of enzyme macromolecules but does not generate an

excessive amount of high reactive intermediates which cause

deac-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

the products of hydrolytic reaction from the reaction zone[25]

Therefore, ultrasound increased the efficiency of enzymatic

treat-ment with higher extraction yield and lower treattreat-ment time

3.4 Enzymatic treatment after sonication

As results of Section3.2, sonication increased extraction yield of

grape mash treatment, but it also increased content of

polysaccha-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-ined 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 inFig 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

3.5 Comparison in physico-chemical characteristics of grape juice

obtained from different grape mash treatments

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 inTable 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 8also 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 differdiffer-ence

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 8shows 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

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 8also shows that application of all treatment methods improved color of the obtained grape juice due to higher values

70 73 76 79 82 85 88

0 0.02 0.04 0.06 0.08 0.1 0.12

Enzyme concentration (%v/v)

Treated sample Control sample

70 73 76 79 82 85 88

Treatment time (min)

Treated sample Control sample

A

B

Fig 4 Effects of enzyme concentration and treatment time on enzymatic treatment after sonication.

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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 8also 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 extracapplica-tion

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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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*

122.8 ± 0.5 a

4.13 ± 0.01 a

2.56 ± 0.01 a

50.6 ± 0.5 a

24.7 ± 0.7 a

57.7 ± 1.0 a

130.4 ± 0.3 b

4.54 ± 0.01 b

4.93 ± 0.03 b

49.4 ± 0.9 ab

30.7 ± 0.3 b

49.0 ± 1.6 b

ST 1.67 ± 0.01 c 137.5 ± 0.6 c 4.69 ± 0.01 c 5.48 ± 0.01 c 48.4 ± 0.6 bc 29.9 ± 0.8 bc 43.5 ± 0.7 c

141.8 ± 0.3 d

4.72 ± 0.01 d

5.64 ± 0.04 d

49.0 ± 1.0 bc

29.7 ± 0.3 c

43.1 ± 0.4 c

136.1 ± 0.8 e

4.58 ± 0.01 e

4.84 ± 0.03 e

48.1 ± 0.1 c

30.0 ± 0.0 bc

43.7 ± 0.6 c

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.

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