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 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 for sonication treatment; and were the enzyme concentration of 0.05% and the time of 10 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 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 38 64 62 51; fax: +84 38 63 75 04 E-mail address: lvvman@hcmut.edu.vn (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 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– 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 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 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 in a water bath The mash was then filtered through a cheese cloth The obtained suspension was centrifuged at 6500 rpm for 10 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, 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 ultrasonic waves are transmitted from the bottom to above The equipment operated at an ultrasound intensity of 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 to 15 The experimental design is presented in Table 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, 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 to 12 The experimental design is presented in Table 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 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 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 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 Experimental planning and results of extraction yield for sonication treatment of grape mash Run Temperature (°C) Time (min) Yield (%) 10 11 12 13 60 80 60 80 55.9 84.1 70 70 70 70 70 70 70 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 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 p +1 p ỵ 55.9 2.9 0.012 2.3 60 0.02 70 10 0.04 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  C  100 m1  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 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 factors and levels Table 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 factorial points, axial points and 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 Results and discussion 3.1 Enzymatic treatment The enzymatic treatment of grape mash increased the extraction yield as results of Fig The graphs show that the enzyme concentration of 0.04%v/v and the treatment time of 40 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 were chosen as the central conditions of the central composite rotary design (CCRD) Table 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 ẳ 81:44 ỵ 1:70X ỵ 1:75X 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 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 times more than the F listed value Surface response graph, obtained by using the fitted model presented in Eq (4), is presented in Fig Table 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 0.02 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 Fitted surface for yield of ultrasound assisted treatment of grape mash as a function of temperature and time 77 76 Table Estimated effect of independent variables on extraction yield of sonication treatment 75 74 73 72 10 20 30 40 50 60 70 Factora Effect Standard error P X1 X2 X1  X1 X2  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 Effects of enzyme concentration (A) and treatment time (B) on extraction yield of enzymatic treatment of grape mash Table 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 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 was 13 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 were chosen as the central conditions of the CCRD according to our preliminary results (unpublished data) Table 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 ẳ 80:42 ỵ 1:86X ỵ 0:61X 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 times more than the F listed value according to analysis of variance which is presented in Table 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 Experimental planning and results of extraction yield for combined ultrasound and enzyme treatment Run Enzyme concentration (%v/v) Time (min) Yield (%) 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 12 12 8 2.3 13.7 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 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 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 Fitted surface for yield of combined ultrasound and enzyme treatment of grape mash as a function of enzyme concentration and treatment time Table Estimated effect of independent variables on yield of ultrasound assisted enzymatic treatment Factora Effect Standard error P X3 X4 X3  X3 X4  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 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 The results are presented in Fig The graphs show that the enzyme concentration of 0.06%v/v and the time of 20 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 0.02 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 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 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 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 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 10 20 30 40 50 Treatment time (min) Fig 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 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 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 also reports that lightness of all treated samples decreased because of the increase in color 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(A) and treatment time (B) on extraction yield of enzymatic treatment of grape mash Table Analysis of variance of the regression model in experiments of sonication treatment Source of variation... 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... 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