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Extraction optimization for antioxidant phenolic compounds in red grape jam using ultrasound with a response surface methodology
Ultrasonics Sonochemistry 19 (2012) 1144–1149
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Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Extraction optimization for antioxidant phenolic compounds in red grape jam using ultrasound with a response surface methodology Lucíula Lemos Lima Morelli ⇑, Marcelo Alexandre Prado State University of Campinas, Faculty of Food Engineering, Laboratory of Instrumental Food Analysis, Campinas, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 27 November 2009 Received in revised form 27 September 2011 Accepted 21 March 2012 Available online 29 March 2012 Keywords: Ultrasound Flavonoids Antioxidants Jam Extraction Grape
a b s t r a c t Optimization of the extraction methodology for antioxidant phenolic compounds in red grape jam was performed with an ultrasound-assisted system. The antioxidant phenolic compounds were extracted and analyzed by determining the total phenolic content (Folin Ciocalteu), as well as by employing free radical DPPH and the beta-carotene/linoleic acid system. To optimize the parameters of solvent concentration, time and extraction temperature, the experiments were carried out using the central composite rotatable design (CCRD) method. Using response surface methodology (RSM), the best combinations achieved were with 60% ethanol and water for 20 min at 50 °C. The optimized parameters for this method were compared to an extraction method that has been commonly noted in the literature, which used to be the standard method, and the results were expressed in the milligram equivalent of quercetin per gram of jam (mg E.Q/g Jam). With the new method, the antioxidant potential measured by DPPH was 70% higher than that obtained with the standard extraction method, and the antioxidant potential measured using the beta-carotene/linoleic acid system was 65% higher. In addition, a significant decrease in the total analysis time was achieved (from 10 h to 30 min), when compared to the standard method. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Among fruits, grapes are a major source of antioxidant phenolic compounds, specifically flavonoids. These compounds demonstrate a great capacity for capturing free radicals, which can cause ‘‘oxidative stress’’, and aid in the prevention of cardiovascular diseases and some types of cancer [1–7]. In recent years, interest in natural antioxidants and their affects on health and human nutrition has greatly increased. The most commonly found flavonoids in grapes and its derivative products are flavanols (catechin, epicatechin, epigallocatechin), flavonols (kaempferol, quercetin, myricetin) and anthocyanins, in addition to other antioxidant phenolic compounds that are non-flavonoids, such as stilbenes (resveratrol) and cinnamic acid derivatives [8,9]. In a study concerning the effect of flavonoids on human health, red wine, catechin and quercetin solutions were administrated in patients to verify their action as antioxidants. It was concluded that the red wine and quercetin solutions reduced in more than 33% the levels of induced oxidation by LDL cholesterol, under oxidative stress [10,11]. Martinez et al. [12] have carried out a study ⇑ Corresponding author. Address: Cidade Universitária ‘‘Zeferino Vaz’’ s/n, Departamento de Ciência de Alimentos, Faculdade de Engenharia de Alimentos – UNICAMP, P.O. Box 6121, Campinas 13083-862, SP, Brazil. Tel.: +55 19 3521 2152; fax: +55 19 3521 2153. E-mail address: [email protected] (L.L.L. Morelli). 1350-4177/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2012.03.009
using rats with acute pulmonary injuries and concluded that quercetin decreased the pulmonary oxidative stress and anti-inflammatory response associated with these injuries. Grape jam, among other grape derivatives, is a viable alternative to the economic exploitation of fruits, adding value to the fruit and promoting access to its beneficial constituents for the entire year [13,14]. In addition, when compared with wine, grape jams and grape juices can present an advantage due to the absence of alcohol, enabling the consumption of this grape derivative by children and people with certain types of diseases, such as hepatitis [15,16]. Fruit jams are commonly used with breads, cookies, and cake fillings, among others. The main ingredients employed to prepare jam are fruit (in natura, pieces, juice or pulp), sugar, pectin and citric acid [13]. Although the processing of fruit can decrease the quantity of total anthocyanins and other antioxidant phenolic compounds, some studies have shown that significant amounts of these compounds can be found after three months of storage in the presence of light and at room temperature [17]. It has also been demonstrated [18] that, during jam processing, temperatures of up to 70 °C result in the inactivation of enzymes that degrade antioxidant phenolic compounds. The most common varieties of grapes used to fabricate jams in the state of São Paulo, Brazil, are the Niagara and Isabel varieties. However, in the present work, jams were prepared with the Máximo variety (IAC 138-22), which is a result of genetically crossing the
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Siebel and Syrah varieties [19]. As this is a relatively new cultivar in the Brazilian market, the antioxidant capacity of this variety was not found in the literature; thus, it was tested in the laboratory. An increased amount of phenolic compounds and an increased antioxidant potential was found for this variety, compared to the other grape varieties used to make grape jams for the Brazilian market. For grapes, the synthesis of resveratrol occurs mainly in the skin, and this compound is absent or is found at very low concentrations in the pulp. When the winemaking process for red grapes begins, maceration with the skin and seeds during fermentation is the principal factor that contributes to the elevated levels of resveratrol found in red wines compared to white wines. Extraction of resveratrol from the skin can be facilitated by the ethanol produced during the fermentation process [20–25]. This information indicates that it is important to create non-alcoholic grape derivatives that include grape skin in their formulations. Antioxidant phenolic compounds are commonly extracted from plants [26], and has been developed using new environmentally friendly methods. These techniques promote a decrease in solvent consumption with an increase in the extraction ratio [27,28]. Among these techniques is ultrasound-assisted extraction, which is a simple, efficient and inexpensive alternative [27–31]. The greater efficiency achieved with ultrasound-assisted extraction is due to the acoustic cavitations effects produced in the solvent when the ultrasonic wave passes through it. This effect permits better penetration of the solvent into the sample, increasing the release of the analyzed compound from the matrix to the solvent [30]. This study was carried out to optimize the parameters of solvent concentration, temperature and extraction time using a surface response methodology (RSM) and employing central composite rotatable design (CCDR), in conjunction with ultrasound bath equipment, to obtain the best extraction conditions for antioxidant phenolic compounds in grape jam produced with skin in the formulation. 2. Experimental 2.1. Materials Grapes of the Máximo variety (IAC 138-22), employed in the preparation of jams, were obtained from a producer in the Campinas region, in the state of São Paulo, Brazil. The formulation ingredients were 60 parts grape, 40 parts refined sugar and inverted sugar syrup, 1% pectin (m/m) and 6% dry skin (m/m). The jams were produced in triplicate. The dry skin was obtained by an air-forced drier with 3 m/s of air for 3 h. The skins were then milled and added to the jam formulation. Ethanol and methanol solvents were of chromatography grade and were obtained from J.T. Baker. The chloroform was of analytical grade and was obtained from Alkimia. Water was purified using a Milli-Q system. Folin Ciocalteu was from Fluka. The reagents DPPH (2,2-diphenyl-1-picrylhydrazyl), beta-carotene, linoleic acid and Tween 20 were purchased from Sigma–Aldrich (Brazil). The quercetin standard was also purchased from Sigma–Aldrich (Brazil). 2.2. Extraction of antioxidant phenolic compounds Extractions were carried out in an ultrasonic bath (Ultra Cleaning 1400, 40 hz, 80 W, Unique, Ind. e Com. Ltd., Brazil). Samples (2 g) were placed into Erlenmeyer flasks (125 mL) with 100 mL of the extraction solvent and sonicated at various times and temperatures, as the experiment required. The temperature was
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controlled using a thermostat that was added to the ultrasonic bath (Fulgauge). After extraction, samples were cooled with tap water and filtered under vacuum through Whatman No. 1 paper. Subsequently, the samples were placed in a 100 mL volumetric flask, which was then filled to the mark, and used in antioxidant determination tests. 2.3. Determination of total phenolic compounds The method used to determine the total phenolic compound (TPC) levels employed the Folin–Ciocalteu (FC) reagent, according to a procedure described in the literature [32]. An aliquot of 500 lL of sample was added to 6 mL of FC reagent and diluted to 10% (v/v) in distilled water. After 5 min, 2.5 mL of 7.5% (m/v) sodium carbonate solution in distilled water was added to the tube and the solution was shaken. The tubes were kept still for 2 h at room temperature. Subsequently, the solutions were transferred to cuvettes to measure their absorbance at 740 nm. All of the analyses were carried out in triplicate. Using quercetin as a standard, the calibration curve was prepared with 67, 100, 150, 300, 450, 600 and 750 lmols of quercetin, and the results were expressed in quercetin equivalents (mg E.Q/g Jam). 2.4. Determination of the antioxidant potential through free radical DPPH The antioxidant potential of the extracts was assessed using the DPPH method. This parameter was determined to elucidate the ability of the sample to reduce free radicals [32]. The organic radical DPPH, which is very stable, has an intense purple color that fades when it is transformed into the reduced form. A solution of 0.039 mg/mL DPPH was prepared [26] to present an absorbance value of 1.000 when measured at 517 nm. Volumes of 3.9 mL of DPPH solution and 100 lL of the extract were added to each tube for the analyses. For the control solution, 100 lL of the extraction solvent was added to the tube. The absorbance readings were taken after 80 min of reaction time at room temperature and in the absence of light. All of the analyses were carried out in triplicate. The decrease in absorbance of the sample (S) tubes was correlated to the decrease in absorbance of the control (C), resulting in an inhibition percentage of the free radical DPPH (I DPPH), which can be expressed through the following Eq. (1):
I DPPHð%Þ ¼
ðC SÞ 100 C
ð1Þ
The calibration curve for quercetin was prepared with 67, 100, 150, 300, 450, 600 and 750 lmols of quercetin, and the results were expressed in quercetin equivalents (mg E.Q/g Jam). 2.5. Determination of the antioxidant potential through the betacarotene/linoleic acid system The method used for the determination of the total antioxidant potential through the beta-carotene/linoleic acid system followed the procedure described in the literature [34]. For the reactive mixture, 20 lL of linoleic acid, 200 mg of Tween 20, and 1 mg of beta-carotene were added to 5 mL of chloroform and placed in a round roto-evaporator flask. This solution was kept at 40 °C until all the chloroform had evaporated. Then, 50 mL of distilled water was added to the mixture and it was vigorously shaken. In each tube, 6 mL of the mixture was added to 50 lL of the jam extract. In the control tube, the mixture was added to 50 lL of the extraction solvent. After these additions, the absorbance was measured (Abs 0) at 470 nm, and the tubes were placed in a water bath at 50 °C for 2 h to catalyze the oxidation reaction and
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discoloring of beta-carotene. Subsequently, the tube mixtures were measured again (Abs 120) to calculate the decreased absorbance in each sample. This measurement was carried out using Eq. (2) of the antioxidant activity index (AAI):
ðAbs0 Abs120 Þsample 100 AAIð%Þ ¼ 1 ðAbs0 Abs120 Þcontrol
ð2Þ
All of the analyses were performed in triplicate. The calibration curve for quercetin was prepared with 67, 100, 150, 300, 450, 600 and 750 lmols of quercetin, following the same procedure employed for the samples, and the results were expressed in quercetin equivalents (mg E.Q/g Jam). 2.6. Experimental design A CCDR was applied with three independent variables across two levels, including six axial points and four replicates of the central point, totaling 18 extractions [35]. This experimental design has also been used in other studies [27,29–31]. Codified values, levels and real values are listed in Table 1. The experimental parameters and Y responses related to each test, as well as the respective averages, are given in Table 2. Using the obtained results, it is possible to determine the regression coefficients and build the equation that best fits each test realized in this study according to the following Eq. (3):
Y ¼ B0 þ B1 x1 þ B2 x2 þ B12 x1 x2 þ B11 x21 þ B22 x22 þ e
ð3Þ B22 x22
where the values of the variables B0, B1, B2, B12, B11 and are the values generated by the calculation of the regression coefficients. To find these values, the software STATISTICA 7.0 was used, and the explained variation percentage was expressed by the determination coefficient R2 at 5% and 10% statistical significance levels. 3. Results and discussion The results for TPC, IDPPH and AAI were based on the investigation into the effects of ethanol concentration, temperature and extraction time, being the variables of the CCDR, for the antioxidant phenolic compounds in grape jam. To optimize the extraction, the central conditions chosen were 60% ethanol (v/v) and an extraction temperature of 50 °C for 20 min. Table 2 shows the results of the extractions according to factorial design. The highest level of TPC (53.95 mg E.Q/g jam) was obtained with 50% ethanol, at 60 °C for 25 min. For the antioxidant potential measured with DPPH, the best values were obtained under the parameters of the center points (4.08 mg E.Q/g jam). The antioxidant potential through the beta-carotene/linoleic acid system also showed the best results under the center point parameters (5.92 mg E.Q/g jam). It is known from other studies [33,36,37] that TPC is not a specific method, due to the fact that it determines other reductive substances beyond the antioxidant phenolic compounds present in foods that can interfere in the results. Only this test resulted in a different profile, compared to the other two tests, and thus it is not considered in the discussion of the results.
Table 1 Independent variables and codified values employed for optimization of the extraction procedure. Independent variables
Ethanol concentration (%) Extraction temperature (°C) Time (min)
Levels
1 2 3
a (1, 68)
1
0
+1
+a (+1, 68)
43 33 11
50 40 15
60 50 20
70 60 25
77 67 29
The statistical analysis revealed that the most important variable (p < 0.05) for the DPPH test was the temperature; the same result was obtained using the beta-carotene/linoleic acid system (p < 0.10) (Table 3). For the DPPH method, higher concentrations of ethanol (70%) led to lower values of free radical stabilization (1.83 mg E.Q/g jam). For the beta-carotene/linoleic acid system, higher concentrations of ethanol (above 65%) combined with lower extraction times (up to 15 min) resulted in the lowest values for this parameter (2.18–3.35 mg E.Q/g jam). The assays performed at 25 min of extraction time, compared to those realized at 15 min of extraction time for the beta-carotene/linoleic acid system, demonstrated that an increase in extraction time can also imply an increase in the antioxidant potential for this method (see Table 4). The experimental data was subjected to a multiple regression analysis, and the model coefficients were evaluated in relation to their significance. For the case of the DPPH method, the significant coefficients were from the values related to quadratic ethanol quantities and linear and quadratic temperature values. The coefficient of determination (R2) of this method was 0.845, meaning that the model adequately fit the chosen parameters. Although there were some non-significant parameters for this model when the codified model was transferred to the real one, it was necessary to maintain all of the coefficients to build Eq. (4) for the stabilization of the free radical DPPH (IDPPH), as shown here:
I DPPH ¼ 26:8760 þ 0:3829E 0:0039E2 þ 0:5926T 0:0067T2 þ 0:5561t 0:0095t2 þ 0:0017ET 0:0017Et 0:0015Tt
ð4Þ
The ANOVA results (Table 3) shows that it is possible to plot the response surfaces for this experimental design. Although the calculated value of F shows that the lack of fit did not tend toward zero, when compared to the F value in the table, one should note that the pure error between the measurements at the center points was very low. This scenario results in a higher value at the moment of the division of its residue. In this case, it is possible to consider that the lack of fit does tend to zero, and it is possible to plot the response surfaces. The same analysis was performed for the beta-carotene/linoleic acid system. The significant coefficients for the codified model were within the ethanol quadratic value, linear and quadratic temperature values, linear time and the interaction of time and temperature (p < 0.1). The coefficient of determination for this method (R2) was 0.8122, which also implies an adequate correlation between the model and the experimental results for the chosen parameters. The following Eq. (5) represents the real model for the inhibition of the oxidation of beta-carotene:
AAI ¼ 29:3425 þ 0:4247E 0:0053E2 þ 0:5099T 0:0037T2 þ 0:8909t 0:0087t2 þ 0:0025ET þ 0:0025Et 0:00125Tt
ð5Þ
The ANOVA table (4) for the analysis performed using the betacarotene/linoleic acid system indicates that it is possible to plot the response surfaces. According to Eqs. (4) and (5), optimized levels of the independent variables for the antioxidant phenolic compounds extracted from grape jam were determined, and tridimensional response surfaces were built. Fig. 1 shows the effects of ethanol concentration and extraction temperature for both the DPPH and beta-carotene/ linoleic acid methods. For the DPPH method, the extraction was better when applying temperatures between 40 and 60 °C, as the ethanol concentration was under 65%. For the beta-carotene/
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Table 2 Experimental parameters of the model and Y response for tests: total phenolic compounds (Y1), antioxidant potential by DPPH (Y2) and beta-carotene/linoleic acid system (Y3): Test
Ethanol (%)
Temperature (°C)
Time (min)
TPC (mg E.Q/g geléia)
I DPPH (mg E.Q/g geléia)
AAI (mg E.Q/g geléia)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 (50) 1 (70) 1 (50) 1 (70) 1 (50) 1 (70) 1 (50) 1 (70) 1.68 (43) 1.68 (77) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)
1 (40) 1 (40) 1 (60) 1 (60) 1 (40) 1 (40) 1 (60) 1 (60) 0 (50) 0 (50) 1.68 (33) 1.68 (67) 0 (50) 0 (50) 0 (50) 0 (50) 0 (50) 0 (50)
1 (15) 1 (15) 1 (15) 1 (15) 1 (25) 1 (25) 1 (25) 1 (25) 0 (20) 0 (20) 0 (20) 0 (20) 1.68 (11) 1.68 (29) 0 (20) 0 (20) 0 (20) 0 (20)
46.45 31.22 52.54 40.98 49.67 35.76 53.95 39.95 50.31 38.51 42.41 49.93 45.76 50.01 47.87 48.26 47.48 48.28
3.12 1.83 2.67 2.57 3.14 2.03 2.92 1.97 3.52 3.04 2.53 2.46 3.63 3.77 4.08 4.05 4.00 3.96
3.64 2.18 5.15 5.40 5.66 5.40 5.36 5.42 5.27 3.35 4.22 5.33 5.46 4.93 5.92 5.88 5.81 5.84
Table 3 ANOVA for design by the DPPH method. Factor
SQ
GL
MQ
F calc
F tab
Regression Resídue Lack of fit Pure error Total
8.125054 1.490342 1.482 0.009 9.615
9 8 5 3 17
0.902784 0.186293 0.2963 0.0029
4.846048
3,39
102.0538
9,01
R2 = 0.85.
Table 4 ANOVA for design by the beta- carotene/linoleic acid system. Factor
SQ
GL
MQ
F calc
F tab
Regression Resídue Lack of fit Pure error Total
14.260324 3.298199 3.292 0.006 17.559
9 8 5 3 17
1.584480427 0.412274857 0.6584 0.0020
3.843262
3,39
327.7271
9,01
R2 = 0.81.
linoleic acid method, the poorest extractions were found with concentrations above 65% ethanol and at temperatures under 50 °C. The effect of ethanol concentration with extraction time is shown in Fig. 2. For both methods, this analysis shows that the ethanol concentration is a more significant variable than the extraction time (between 45% and 60%).
Fig. 3 illustrates the effect of temperature within the extraction time for both methods. For the DPPH method, the temperature should be between 40 and 60 °C for all extraction times. However, for the beta-carotene/linoleic acid method, there was a time band (under 18 min and combined with temperatures under 45 °C) when the extraction was less efficient, compared with other extractions. Using these results and the model equation, one can conclude that the best combination of parameters is at the center point conditions of 60% ethanol concentration at 50 °C for 20 min. Placement of these optimized values into the equations for each method yields, for the DPPH method, a theoretical result of 4.053 mg E.Q/ g jam and, for the beta-carotene/linoleic acid method, a result of 5.860 mg E.Q/g jam. An evaluation was carried out to compare the theoretical results with experimental results. Toward this end, the extraction was repeated with the optimized parameters, and the results were expressed as the average values obtained by triplicates of each antioxidant potential method. For the DPPH method, the average was 4.219 ± 0.09 mg E.Q/g jam. For the beta-carotene/ linoleic acid method, the average was 5.667 ± 0.59 mg E.Q/g jam. The good correlation achieved between the data obtained confirms that the response surface model is adequate to illustrate the optimization of the parameters (Table 5). There are many consolidated methods in the literature for the extraction of antioxidant phenolic compounds from food matrices. Among them, the method used by Scherer and Godoy [26] was used in this study as the standard method for comparison with the results from the experimental design. This method was used because of its large use for extractions from vegetables, compared
Fig. 1. Effect of the ethanol concentration and temperature of extraction for stabilization of DPPH (a) and for the prevention of beta-carotene discoloration (b).
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Fig. 2. Effect of the ethanol concentration within the extraction time for the stabilization of DPPH (a) and for the prevention of beta-carotene discoloration (b).
Fig. 3. Effect of temperature within time of extraction for the stabilization of DPPH (a) and for the prevention of beta-carotene discoloration (b).
Table 5 Comparison between experimental results and results obtained by the equation model, as well as the results obtained when applying the standard method. Optimized conditions
I DPPH (mg E.Q/g jam)
AAI (mg E.Q/g jam)
Ethanol (%)
T ( °C)
Time (min)
Standard method
Equation
Experimental
Standard method
Equation
Experimental
60
50
20
2.39 ± 0.12
4.053
4.219 ± 0.09
3.416 ± 0.22
5.860
5.667 ± 0.587
with other references from the literature. According to the results observed with the DPPH method, there was an increase of 70% in the antioxidant potential for grape jam. For the beta-carotene/ linoleic acid method, the increase was up to 65%. Beyond this increase in extraction efficiency, there was a significant decrease in the extraction time. For the standard method, this effect was a decrease of approximately 10 h because of the large amount of sugar in the jam formulation. These values indicate that the application of ultrasound and temperature for the extraction of antioxidant phenolic compounds in grape jam can strongly decrease extraction times, leading to efficient results, as shown in the 30 min time achieved here for the entire optimized process. The efficiency of ultrasound has already been explained in the literature [27,29,31] as having worked with other food matrices. These authors have also concluded that ultrasound-assisted methods are fast and yield positive results. Other results that are worth mentioning include the standard method and the ultrasound-assisted method that includes the substitution of methanol with ethanol. Ethanol is more environmentally friendly and a smaller volume is necessary for extraction,
and the total amount required for the actual ultrasound-assisted method was only 60 mL. In contrast, with the standard method, 250 mL is required for each sample [26]. This decrease in solvent use implies a significant decrease in residue generation.
4. Conclusions Within the evaluated parameters, the utilization of ultrasound equipment shows higher efficiency than the standard method that has been used previously. In addition, the response surface methodology for the extraction of antioxidant phenolic compounds was excellent at determining the best combination of parameters, which were found to be 60% ethanol at 50 °C for 20 min. Temperature was the most important variable, followed by ethanol concentration. Although time was not found to be significant, it was found that extraction times above 18 min should be used for one of the beta-carotene/linoleic acid method surfaces. A significant increase in the antioxidant potential for the ultrasound-assisted extracts was found when evaluated by both methods, including
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DPPH (up to 70%) and beta-carotene/linoleic acid (up to 65%), when compared to the extracts obtained by the standard method. The present study also indicates that grape jams formulated with the Máximo variety (IAC 138-22) can be considered as good sources of antioxidant phenolic compounds.
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[18] A.P. Falcão, E.S. Chaves, E.M. Kuskoski, R. Fett, L.D. Falcão, M.T. Bordignon-Luiz, Índice de polifenóis, antocianinas totais e atividade antioxidante de um sistema modelo de geléia de uvas, Ciênc. Tecnol. Aliment. 27 (2007) 637–642. Available from:. [19] J.R.de A. Santos Neto, F.M. Pereira, F.P. Martins, H.de F. Leitão Filho, Características e possibilidades do cultivar de videira IAC 138-22, O Agronômico Campinas 20 (1968) 1–8. [20] S.M.M. Bertagnolli, S.B. Rossato, V.L. Silva, et al., Influence of the carbonic maceration on the levels of trans-resveratrol in cabernet sauvignon wine, Rev. Bras. Cienc. Farm. 43 (2007) 71–77. Available from: . [21] S. Kallithraka, I. Arvanitoyannis, A. El-Zajouli, P. Kefalas, The application of an improved method for trans-resveratrol to determine the origin of Greek red wines, Food Chem. 75 (2001) 355–363. [22] R.T. Threlfall, J.R. Morris, A. Mauromoustakos, Effect of variety, ultraviolet light exposure, and enological methods on the trans-resveratrol level of wine, Am. J. Enol. Vit. 50 (1999) 57–64. [23] R.M. Lamuela-Raventos, A.I. Romero-Pérez, et al., Resveratrol and piceid levels in wine production and in finished wine: wine nutritional and therapeutic benefits, American Chemical Society, Washington 1997 (ACS Simposium Series 661), pp. 56–68. Available from: . [24] G.J. Soleas, D.M. Goldberg, E.O. Diamandis, A. Karumanchiri, J. Yan, E. NG, A derivatized gas chromatographic-mass spectrometric method for the analysis of both isomers of resveratrol in juice and wine, Am. J. Enol. Vit. 46 (3) (1995) 346–352. [25] P. Jeandet, R. Bessis, B.F. Maume, P. Meunier, D. Peyron, P. Trollat, Effect of enological practices on the resveratrol isomer content of wine, J. Agri. Food Chem. 43 (1995) 316–319. [26] R. Scherer, Godoy, T. Helena, Antioxidant activity index (AAI) by the 2,2diphenyl-1-picrylhydrazyl method, Food Chem. 112 (2009) 654–658. [27] J. Wang, B. Sun, Y. Cao, Y. Tian, X. Li, Optimization of ultrasounded-assisted extraction of phenolic compounds from wheat bran, Food Chem. 106 (2008) 804–810. [28] L. Wang, C.L. Weller, Recent advances in extraction of nutraceuticals from plants, Trends Food Sci. Technol. 17 (2006) 300–312. [29] M. Virot, V. Tomao, C. Le Bourvellec, C.M.C.G. Renard, F. Chemat, Towards the industrial production of antioxidants from food processing by-products with ultrasound assisted extraction, Ultrason. Sonochem. 17 (2010) 1066–1074. [30] M.A. Rostagno, M. Palma, C.G. Barroso, Ultrasounded-assisted extraction of soy isoflavones, J. Chromatogr. A 1012 (2003) 119–128. [31] S. Rodrigues, G.A.S. Pinto, F.A.N. Fernandes, Optimization of ultrasound extraction of phenolic compounds from coconut (Cocos nucifera) shell powder by response surface methodology, Ultrason. Sonochem. 15 (1) (2008) 95–100. [32] V.L. Singleton, R. Orthofer, R.M. Lamuela-Raventos, Analysis of total phenols and other oxidation subtrates and antioxidants by means of Folin-Ciocalteau reagent, Methods Enzymol. 299 (1999) 152–178. [33] D. Huang, B. Ou, R. Prior, The chemistry behind antioxidant capacity assays, J. Agric. Food Chem. 53 (2005) 1.841–1.856. [34] K. Dastmalchi, H.J.D. Dorman, P.P. Oinonen, Y. Darwis, I. Laakso, R. Hiltunen, Chemical composition and in vitro antioxidative activity of a lemon balm (Melissa officinalis L.) extract, LWT–Food Sci. Technol. 41 (2008) 391–400. Available from: . [35] M.I. Rodrigues, A.F. Iemma, Planejamento de Experimentos e Otimização de Processos: uma Estratégia Seqüencial de Planejamentos, first ed., Casa do Pão Editora, Campinas, SP, 2005. [36] P.M. Angelo, Jorge, Neuza, Compostos fenólicos em alimentos – uma breve revisão, Rev. Inst. Adolfo Lutz 66 (2007) 232–240. [37] X. He, D. Liu, R.H. Liu, Sodium borohydride/chloranil-based assay for quantifying total flavonoids, J. Agric. Food Chem. 56 (2008) 9337–9344.
Contents lists available at SciVerse ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultson
Extraction optimization for antioxidant phenolic compounds in red grape jam using ultrasound with a response surface methodology Lucíula Lemos Lima Morelli ⇑, Marcelo Alexandre Prado State University of Campinas, Faculty of Food Engineering, Laboratory of Instrumental Food Analysis, Campinas, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 27 November 2009 Received in revised form 27 September 2011 Accepted 21 March 2012 Available online 29 March 2012 Keywords: Ultrasound Flavonoids Antioxidants Jam Extraction Grape
a b s t r a c t Optimization of the extraction methodology for antioxidant phenolic compounds in red grape jam was performed with an ultrasound-assisted system. The antioxidant phenolic compounds were extracted and analyzed by determining the total phenolic content (Folin Ciocalteu), as well as by employing free radical DPPH and the beta-carotene/linoleic acid system. To optimize the parameters of solvent concentration, time and extraction temperature, the experiments were carried out using the central composite rotatable design (CCRD) method. Using response surface methodology (RSM), the best combinations achieved were with 60% ethanol and water for 20 min at 50 °C. The optimized parameters for this method were compared to an extraction method that has been commonly noted in the literature, which used to be the standard method, and the results were expressed in the milligram equivalent of quercetin per gram of jam (mg E.Q/g Jam). With the new method, the antioxidant potential measured by DPPH was 70% higher than that obtained with the standard extraction method, and the antioxidant potential measured using the beta-carotene/linoleic acid system was 65% higher. In addition, a significant decrease in the total analysis time was achieved (from 10 h to 30 min), when compared to the standard method. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Among fruits, grapes are a major source of antioxidant phenolic compounds, specifically flavonoids. These compounds demonstrate a great capacity for capturing free radicals, which can cause ‘‘oxidative stress’’, and aid in the prevention of cardiovascular diseases and some types of cancer [1–7]. In recent years, interest in natural antioxidants and their affects on health and human nutrition has greatly increased. The most commonly found flavonoids in grapes and its derivative products are flavanols (catechin, epicatechin, epigallocatechin), flavonols (kaempferol, quercetin, myricetin) and anthocyanins, in addition to other antioxidant phenolic compounds that are non-flavonoids, such as stilbenes (resveratrol) and cinnamic acid derivatives [8,9]. In a study concerning the effect of flavonoids on human health, red wine, catechin and quercetin solutions were administrated in patients to verify their action as antioxidants. It was concluded that the red wine and quercetin solutions reduced in more than 33% the levels of induced oxidation by LDL cholesterol, under oxidative stress [10,11]. Martinez et al. [12] have carried out a study ⇑ Corresponding author. Address: Cidade Universitária ‘‘Zeferino Vaz’’ s/n, Departamento de Ciência de Alimentos, Faculdade de Engenharia de Alimentos – UNICAMP, P.O. Box 6121, Campinas 13083-862, SP, Brazil. Tel.: +55 19 3521 2152; fax: +55 19 3521 2153. E-mail address: [email protected] (L.L.L. Morelli). 1350-4177/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ultsonch.2012.03.009
using rats with acute pulmonary injuries and concluded that quercetin decreased the pulmonary oxidative stress and anti-inflammatory response associated with these injuries. Grape jam, among other grape derivatives, is a viable alternative to the economic exploitation of fruits, adding value to the fruit and promoting access to its beneficial constituents for the entire year [13,14]. In addition, when compared with wine, grape jams and grape juices can present an advantage due to the absence of alcohol, enabling the consumption of this grape derivative by children and people with certain types of diseases, such as hepatitis [15,16]. Fruit jams are commonly used with breads, cookies, and cake fillings, among others. The main ingredients employed to prepare jam are fruit (in natura, pieces, juice or pulp), sugar, pectin and citric acid [13]. Although the processing of fruit can decrease the quantity of total anthocyanins and other antioxidant phenolic compounds, some studies have shown that significant amounts of these compounds can be found after three months of storage in the presence of light and at room temperature [17]. It has also been demonstrated [18] that, during jam processing, temperatures of up to 70 °C result in the inactivation of enzymes that degrade antioxidant phenolic compounds. The most common varieties of grapes used to fabricate jams in the state of São Paulo, Brazil, are the Niagara and Isabel varieties. However, in the present work, jams were prepared with the Máximo variety (IAC 138-22), which is a result of genetically crossing the
L.L.L. Morelli, M.A. Prado / Ultrasonics Sonochemistry 19 (2012) 1144–1149
Siebel and Syrah varieties [19]. As this is a relatively new cultivar in the Brazilian market, the antioxidant capacity of this variety was not found in the literature; thus, it was tested in the laboratory. An increased amount of phenolic compounds and an increased antioxidant potential was found for this variety, compared to the other grape varieties used to make grape jams for the Brazilian market. For grapes, the synthesis of resveratrol occurs mainly in the skin, and this compound is absent or is found at very low concentrations in the pulp. When the winemaking process for red grapes begins, maceration with the skin and seeds during fermentation is the principal factor that contributes to the elevated levels of resveratrol found in red wines compared to white wines. Extraction of resveratrol from the skin can be facilitated by the ethanol produced during the fermentation process [20–25]. This information indicates that it is important to create non-alcoholic grape derivatives that include grape skin in their formulations. Antioxidant phenolic compounds are commonly extracted from plants [26], and has been developed using new environmentally friendly methods. These techniques promote a decrease in solvent consumption with an increase in the extraction ratio [27,28]. Among these techniques is ultrasound-assisted extraction, which is a simple, efficient and inexpensive alternative [27–31]. The greater efficiency achieved with ultrasound-assisted extraction is due to the acoustic cavitations effects produced in the solvent when the ultrasonic wave passes through it. This effect permits better penetration of the solvent into the sample, increasing the release of the analyzed compound from the matrix to the solvent [30]. This study was carried out to optimize the parameters of solvent concentration, temperature and extraction time using a surface response methodology (RSM) and employing central composite rotatable design (CCDR), in conjunction with ultrasound bath equipment, to obtain the best extraction conditions for antioxidant phenolic compounds in grape jam produced with skin in the formulation. 2. Experimental 2.1. Materials Grapes of the Máximo variety (IAC 138-22), employed in the preparation of jams, were obtained from a producer in the Campinas region, in the state of São Paulo, Brazil. The formulation ingredients were 60 parts grape, 40 parts refined sugar and inverted sugar syrup, 1% pectin (m/m) and 6% dry skin (m/m). The jams were produced in triplicate. The dry skin was obtained by an air-forced drier with 3 m/s of air for 3 h. The skins were then milled and added to the jam formulation. Ethanol and methanol solvents were of chromatography grade and were obtained from J.T. Baker. The chloroform was of analytical grade and was obtained from Alkimia. Water was purified using a Milli-Q system. Folin Ciocalteu was from Fluka. The reagents DPPH (2,2-diphenyl-1-picrylhydrazyl), beta-carotene, linoleic acid and Tween 20 were purchased from Sigma–Aldrich (Brazil). The quercetin standard was also purchased from Sigma–Aldrich (Brazil). 2.2. Extraction of antioxidant phenolic compounds Extractions were carried out in an ultrasonic bath (Ultra Cleaning 1400, 40 hz, 80 W, Unique, Ind. e Com. Ltd., Brazil). Samples (2 g) were placed into Erlenmeyer flasks (125 mL) with 100 mL of the extraction solvent and sonicated at various times and temperatures, as the experiment required. The temperature was
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controlled using a thermostat that was added to the ultrasonic bath (Fulgauge). After extraction, samples were cooled with tap water and filtered under vacuum through Whatman No. 1 paper. Subsequently, the samples were placed in a 100 mL volumetric flask, which was then filled to the mark, and used in antioxidant determination tests. 2.3. Determination of total phenolic compounds The method used to determine the total phenolic compound (TPC) levels employed the Folin–Ciocalteu (FC) reagent, according to a procedure described in the literature [32]. An aliquot of 500 lL of sample was added to 6 mL of FC reagent and diluted to 10% (v/v) in distilled water. After 5 min, 2.5 mL of 7.5% (m/v) sodium carbonate solution in distilled water was added to the tube and the solution was shaken. The tubes were kept still for 2 h at room temperature. Subsequently, the solutions were transferred to cuvettes to measure their absorbance at 740 nm. All of the analyses were carried out in triplicate. Using quercetin as a standard, the calibration curve was prepared with 67, 100, 150, 300, 450, 600 and 750 lmols of quercetin, and the results were expressed in quercetin equivalents (mg E.Q/g Jam). 2.4. Determination of the antioxidant potential through free radical DPPH The antioxidant potential of the extracts was assessed using the DPPH method. This parameter was determined to elucidate the ability of the sample to reduce free radicals [32]. The organic radical DPPH, which is very stable, has an intense purple color that fades when it is transformed into the reduced form. A solution of 0.039 mg/mL DPPH was prepared [26] to present an absorbance value of 1.000 when measured at 517 nm. Volumes of 3.9 mL of DPPH solution and 100 lL of the extract were added to each tube for the analyses. For the control solution, 100 lL of the extraction solvent was added to the tube. The absorbance readings were taken after 80 min of reaction time at room temperature and in the absence of light. All of the analyses were carried out in triplicate. The decrease in absorbance of the sample (S) tubes was correlated to the decrease in absorbance of the control (C), resulting in an inhibition percentage of the free radical DPPH (I DPPH), which can be expressed through the following Eq. (1):
I DPPHð%Þ ¼
ðC SÞ 100 C
ð1Þ
The calibration curve for quercetin was prepared with 67, 100, 150, 300, 450, 600 and 750 lmols of quercetin, and the results were expressed in quercetin equivalents (mg E.Q/g Jam). 2.5. Determination of the antioxidant potential through the betacarotene/linoleic acid system The method used for the determination of the total antioxidant potential through the beta-carotene/linoleic acid system followed the procedure described in the literature [34]. For the reactive mixture, 20 lL of linoleic acid, 200 mg of Tween 20, and 1 mg of beta-carotene were added to 5 mL of chloroform and placed in a round roto-evaporator flask. This solution was kept at 40 °C until all the chloroform had evaporated. Then, 50 mL of distilled water was added to the mixture and it was vigorously shaken. In each tube, 6 mL of the mixture was added to 50 lL of the jam extract. In the control tube, the mixture was added to 50 lL of the extraction solvent. After these additions, the absorbance was measured (Abs 0) at 470 nm, and the tubes were placed in a water bath at 50 °C for 2 h to catalyze the oxidation reaction and
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discoloring of beta-carotene. Subsequently, the tube mixtures were measured again (Abs 120) to calculate the decreased absorbance in each sample. This measurement was carried out using Eq. (2) of the antioxidant activity index (AAI):
ðAbs0 Abs120 Þsample 100 AAIð%Þ ¼ 1 ðAbs0 Abs120 Þcontrol
ð2Þ
All of the analyses were performed in triplicate. The calibration curve for quercetin was prepared with 67, 100, 150, 300, 450, 600 and 750 lmols of quercetin, following the same procedure employed for the samples, and the results were expressed in quercetin equivalents (mg E.Q/g Jam). 2.6. Experimental design A CCDR was applied with three independent variables across two levels, including six axial points and four replicates of the central point, totaling 18 extractions [35]. This experimental design has also been used in other studies [27,29–31]. Codified values, levels and real values are listed in Table 1. The experimental parameters and Y responses related to each test, as well as the respective averages, are given in Table 2. Using the obtained results, it is possible to determine the regression coefficients and build the equation that best fits each test realized in this study according to the following Eq. (3):
Y ¼ B0 þ B1 x1 þ B2 x2 þ B12 x1 x2 þ B11 x21 þ B22 x22 þ e
ð3Þ B22 x22
where the values of the variables B0, B1, B2, B12, B11 and are the values generated by the calculation of the regression coefficients. To find these values, the software STATISTICA 7.0 was used, and the explained variation percentage was expressed by the determination coefficient R2 at 5% and 10% statistical significance levels. 3. Results and discussion The results for TPC, IDPPH and AAI were based on the investigation into the effects of ethanol concentration, temperature and extraction time, being the variables of the CCDR, for the antioxidant phenolic compounds in grape jam. To optimize the extraction, the central conditions chosen were 60% ethanol (v/v) and an extraction temperature of 50 °C for 20 min. Table 2 shows the results of the extractions according to factorial design. The highest level of TPC (53.95 mg E.Q/g jam) was obtained with 50% ethanol, at 60 °C for 25 min. For the antioxidant potential measured with DPPH, the best values were obtained under the parameters of the center points (4.08 mg E.Q/g jam). The antioxidant potential through the beta-carotene/linoleic acid system also showed the best results under the center point parameters (5.92 mg E.Q/g jam). It is known from other studies [33,36,37] that TPC is not a specific method, due to the fact that it determines other reductive substances beyond the antioxidant phenolic compounds present in foods that can interfere in the results. Only this test resulted in a different profile, compared to the other two tests, and thus it is not considered in the discussion of the results.
Table 1 Independent variables and codified values employed for optimization of the extraction procedure. Independent variables
Ethanol concentration (%) Extraction temperature (°C) Time (min)
Levels
1 2 3
a (1, 68)
1
0
+1
+a (+1, 68)
43 33 11
50 40 15
60 50 20
70 60 25
77 67 29
The statistical analysis revealed that the most important variable (p < 0.05) for the DPPH test was the temperature; the same result was obtained using the beta-carotene/linoleic acid system (p < 0.10) (Table 3). For the DPPH method, higher concentrations of ethanol (70%) led to lower values of free radical stabilization (1.83 mg E.Q/g jam). For the beta-carotene/linoleic acid system, higher concentrations of ethanol (above 65%) combined with lower extraction times (up to 15 min) resulted in the lowest values for this parameter (2.18–3.35 mg E.Q/g jam). The assays performed at 25 min of extraction time, compared to those realized at 15 min of extraction time for the beta-carotene/linoleic acid system, demonstrated that an increase in extraction time can also imply an increase in the antioxidant potential for this method (see Table 4). The experimental data was subjected to a multiple regression analysis, and the model coefficients were evaluated in relation to their significance. For the case of the DPPH method, the significant coefficients were from the values related to quadratic ethanol quantities and linear and quadratic temperature values. The coefficient of determination (R2) of this method was 0.845, meaning that the model adequately fit the chosen parameters. Although there were some non-significant parameters for this model when the codified model was transferred to the real one, it was necessary to maintain all of the coefficients to build Eq. (4) for the stabilization of the free radical DPPH (IDPPH), as shown here:
I DPPH ¼ 26:8760 þ 0:3829E 0:0039E2 þ 0:5926T 0:0067T2 þ 0:5561t 0:0095t2 þ 0:0017ET 0:0017Et 0:0015Tt
ð4Þ
The ANOVA results (Table 3) shows that it is possible to plot the response surfaces for this experimental design. Although the calculated value of F shows that the lack of fit did not tend toward zero, when compared to the F value in the table, one should note that the pure error between the measurements at the center points was very low. This scenario results in a higher value at the moment of the division of its residue. In this case, it is possible to consider that the lack of fit does tend to zero, and it is possible to plot the response surfaces. The same analysis was performed for the beta-carotene/linoleic acid system. The significant coefficients for the codified model were within the ethanol quadratic value, linear and quadratic temperature values, linear time and the interaction of time and temperature (p < 0.1). The coefficient of determination for this method (R2) was 0.8122, which also implies an adequate correlation between the model and the experimental results for the chosen parameters. The following Eq. (5) represents the real model for the inhibition of the oxidation of beta-carotene:
AAI ¼ 29:3425 þ 0:4247E 0:0053E2 þ 0:5099T 0:0037T2 þ 0:8909t 0:0087t2 þ 0:0025ET þ 0:0025Et 0:00125Tt
ð5Þ
The ANOVA table (4) for the analysis performed using the betacarotene/linoleic acid system indicates that it is possible to plot the response surfaces. According to Eqs. (4) and (5), optimized levels of the independent variables for the antioxidant phenolic compounds extracted from grape jam were determined, and tridimensional response surfaces were built. Fig. 1 shows the effects of ethanol concentration and extraction temperature for both the DPPH and beta-carotene/ linoleic acid methods. For the DPPH method, the extraction was better when applying temperatures between 40 and 60 °C, as the ethanol concentration was under 65%. For the beta-carotene/
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Table 2 Experimental parameters of the model and Y response for tests: total phenolic compounds (Y1), antioxidant potential by DPPH (Y2) and beta-carotene/linoleic acid system (Y3): Test
Ethanol (%)
Temperature (°C)
Time (min)
TPC (mg E.Q/g geléia)
I DPPH (mg E.Q/g geléia)
AAI (mg E.Q/g geléia)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1 (50) 1 (70) 1 (50) 1 (70) 1 (50) 1 (70) 1 (50) 1 (70) 1.68 (43) 1.68 (77) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)
1 (40) 1 (40) 1 (60) 1 (60) 1 (40) 1 (40) 1 (60) 1 (60) 0 (50) 0 (50) 1.68 (33) 1.68 (67) 0 (50) 0 (50) 0 (50) 0 (50) 0 (50) 0 (50)
1 (15) 1 (15) 1 (15) 1 (15) 1 (25) 1 (25) 1 (25) 1 (25) 0 (20) 0 (20) 0 (20) 0 (20) 1.68 (11) 1.68 (29) 0 (20) 0 (20) 0 (20) 0 (20)
46.45 31.22 52.54 40.98 49.67 35.76 53.95 39.95 50.31 38.51 42.41 49.93 45.76 50.01 47.87 48.26 47.48 48.28
3.12 1.83 2.67 2.57 3.14 2.03 2.92 1.97 3.52 3.04 2.53 2.46 3.63 3.77 4.08 4.05 4.00 3.96
3.64 2.18 5.15 5.40 5.66 5.40 5.36 5.42 5.27 3.35 4.22 5.33 5.46 4.93 5.92 5.88 5.81 5.84
Table 3 ANOVA for design by the DPPH method. Factor
SQ
GL
MQ
F calc
F tab
Regression Resídue Lack of fit Pure error Total
8.125054 1.490342 1.482 0.009 9.615
9 8 5 3 17
0.902784 0.186293 0.2963 0.0029
4.846048
3,39
102.0538
9,01
R2 = 0.85.
Table 4 ANOVA for design by the beta- carotene/linoleic acid system. Factor
SQ
GL
MQ
F calc
F tab
Regression Resídue Lack of fit Pure error Total
14.260324 3.298199 3.292 0.006 17.559
9 8 5 3 17
1.584480427 0.412274857 0.6584 0.0020
3.843262
3,39
327.7271
9,01
R2 = 0.81.
linoleic acid method, the poorest extractions were found with concentrations above 65% ethanol and at temperatures under 50 °C. The effect of ethanol concentration with extraction time is shown in Fig. 2. For both methods, this analysis shows that the ethanol concentration is a more significant variable than the extraction time (between 45% and 60%).
Fig. 3 illustrates the effect of temperature within the extraction time for both methods. For the DPPH method, the temperature should be between 40 and 60 °C for all extraction times. However, for the beta-carotene/linoleic acid method, there was a time band (under 18 min and combined with temperatures under 45 °C) when the extraction was less efficient, compared with other extractions. Using these results and the model equation, one can conclude that the best combination of parameters is at the center point conditions of 60% ethanol concentration at 50 °C for 20 min. Placement of these optimized values into the equations for each method yields, for the DPPH method, a theoretical result of 4.053 mg E.Q/ g jam and, for the beta-carotene/linoleic acid method, a result of 5.860 mg E.Q/g jam. An evaluation was carried out to compare the theoretical results with experimental results. Toward this end, the extraction was repeated with the optimized parameters, and the results were expressed as the average values obtained by triplicates of each antioxidant potential method. For the DPPH method, the average was 4.219 ± 0.09 mg E.Q/g jam. For the beta-carotene/ linoleic acid method, the average was 5.667 ± 0.59 mg E.Q/g jam. The good correlation achieved between the data obtained confirms that the response surface model is adequate to illustrate the optimization of the parameters (Table 5). There are many consolidated methods in the literature for the extraction of antioxidant phenolic compounds from food matrices. Among them, the method used by Scherer and Godoy [26] was used in this study as the standard method for comparison with the results from the experimental design. This method was used because of its large use for extractions from vegetables, compared
Fig. 1. Effect of the ethanol concentration and temperature of extraction for stabilization of DPPH (a) and for the prevention of beta-carotene discoloration (b).
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Fig. 2. Effect of the ethanol concentration within the extraction time for the stabilization of DPPH (a) and for the prevention of beta-carotene discoloration (b).
Fig. 3. Effect of temperature within time of extraction for the stabilization of DPPH (a) and for the prevention of beta-carotene discoloration (b).
Table 5 Comparison between experimental results and results obtained by the equation model, as well as the results obtained when applying the standard method. Optimized conditions
I DPPH (mg E.Q/g jam)
AAI (mg E.Q/g jam)
Ethanol (%)
T ( °C)
Time (min)
Standard method
Equation
Experimental
Standard method
Equation
Experimental
60
50
20
2.39 ± 0.12
4.053
4.219 ± 0.09
3.416 ± 0.22
5.860
5.667 ± 0.587
with other references from the literature. According to the results observed with the DPPH method, there was an increase of 70% in the antioxidant potential for grape jam. For the beta-carotene/ linoleic acid method, the increase was up to 65%. Beyond this increase in extraction efficiency, there was a significant decrease in the extraction time. For the standard method, this effect was a decrease of approximately 10 h because of the large amount of sugar in the jam formulation. These values indicate that the application of ultrasound and temperature for the extraction of antioxidant phenolic compounds in grape jam can strongly decrease extraction times, leading to efficient results, as shown in the 30 min time achieved here for the entire optimized process. The efficiency of ultrasound has already been explained in the literature [27,29,31] as having worked with other food matrices. These authors have also concluded that ultrasound-assisted methods are fast and yield positive results. Other results that are worth mentioning include the standard method and the ultrasound-assisted method that includes the substitution of methanol with ethanol. Ethanol is more environmentally friendly and a smaller volume is necessary for extraction,
and the total amount required for the actual ultrasound-assisted method was only 60 mL. In contrast, with the standard method, 250 mL is required for each sample [26]. This decrease in solvent use implies a significant decrease in residue generation.
4. Conclusions Within the evaluated parameters, the utilization of ultrasound equipment shows higher efficiency than the standard method that has been used previously. In addition, the response surface methodology for the extraction of antioxidant phenolic compounds was excellent at determining the best combination of parameters, which were found to be 60% ethanol at 50 °C for 20 min. Temperature was the most important variable, followed by ethanol concentration. Although time was not found to be significant, it was found that extraction times above 18 min should be used for one of the beta-carotene/linoleic acid method surfaces. A significant increase in the antioxidant potential for the ultrasound-assisted extracts was found when evaluated by both methods, including
L.L.L. Morelli, M.A. Prado / Ultrasonics Sonochemistry 19 (2012) 1144–1149
DPPH (up to 70%) and beta-carotene/linoleic acid (up to 65%), when compared to the extracts obtained by the standard method. The present study also indicates that grape jams formulated with the Máximo variety (IAC 138-22) can be considered as good sources of antioxidant phenolic compounds.
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