microwave assisted extraction (UMAE) and ultrasonic assisted extraction (UAE) of lycopene from tomatoes

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Ultrasonics Sonochemistry 15 (2008) 731–737 www.elsevier.com/locate/ultsonch

Optimization and comparison of ultrasound/microwave assisted extraction (UMAE) and ultrasonic assisted extraction (UAE) of lycopene from tomatoes Zhang Lianfu a,b,*, Liu Zelong a a

School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

b

Received 6 September 2007; received in revised form 19 November 2007; accepted 11 December 2007 Available online 23 December 2007

Abstract The extracting technology including ultrasonic and microwave assisted extraction (UMAE) and ultrasonic assisted extraction (UAE) of lycopene from tomato paste were optimized and compared. The results showed that the optimal conditions for UMAE were 98 W microwave power together with 40 KHz ultrasonic processing, the ratio of solvents to tomato paste was 10.6:1 (V/W) and the extracting time should be 367 s; as for UAE, the extracting temperature was 86.4 °C, the ratio of the solvents to tomato paste was 8.0:1 (V/W) and the extracting time should be 29.1 min, while the percentage of lycopene yield was 97.4% and 89.4% for UMAE and UAE, respectively. These results implied that UMAE was far more efficient extracting method than UAE. Ó 2008 Published by Elsevier B.V. Keywords: Lycopene; Ultrasound/microwave assisted extraction (UMAE); Ultrasound assisted extraction (UAE)

1. Introduction Lycopene is one of most important commercial and medicinal plant pigments found in nature. It is accumulated in photo-synthesis pigment–pigment complex of plants and can be most easily seen in ripe tomato, watermelon, pink grapefruit, guava and papaya, giving them a characteristic red color [1,2]. Tomato and tomato products are the major sources for lycopene production [3]. Lycopene has been used as natural food colorant for many years and it is only recently that lycopene attracts considerable attentions as pharmaceutical components [3–5]. Lycopene is an acyclic carotene with 11 conjugated double bonds occuring in various geometrical isomers: the alltrans-isomers are of the dominant percentage in most raw * Corresponding author. Address: School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China. Tel./ fax: +86 510 85917081. E-mail address: [email protected] (Z. Lianfu).

1350-4177/$ - see front matter Ó 2008 Published by Elsevier B.V. doi:10.1016/j.ultsonch.2007.12.001

materials but the cis-isomers are more familiar in our body and with even stronger bioactivities [1,2,6–8]. The biological activities of lycopene include antioxidant activity (singlet oxygen quenching and peroxyl radical scavenging), induction of intercellular communication and growth control, but no provitamin A activity [2,9,10]. In vitro and in vivo studies showed that lycopene was a promising bioactive component on lowering down the risk of some chronic diseases including certain cancer (e.g., prostate cancer) and coronary heart diseases [11,12]. The supplying of this product relies on the extraction of lycopene from plants and zymotic liquid via conventional solvent extraction, super critical fluid (CO2) extraction (SCFE) and supersonic assisted extraction. Conventional method requires long processing time with low efficiency while SCFE represents non-organic solvents residing as advantage but the equipment is expensive and the energy consumption is very high. By now we know that ultrasound and microwave radiation could accelerate the extracting process and this may

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improve bioactive compound extraction [13–19]. The interest on applying sonochemistry to natural product extraction has increased because of its advantage (e.g., reduction in extraction time, saving in energy, increased yield, etc.) [15]. Surely ultrasonic extraction is preferable to saponification, which break the cell wall through alkaline condition due to no chemical involvement in the process [20]. Microwave assisted extraction heat the extracts quickly and accelerate the extraction process for adsorption and desorption of the targeted compounds from matrix, while its disadvantage is inhomogeneous heating [17]. Hence, coupling microwave with ultrasound extraction is a complementary technique and may present some more advantages. Here the authors optimize and compare the extraction of lycopene by ultrasound and microwave assisted extraction (UMAE) and ultrasound assisted extraction (UAE) in order to illuminate their advantages and disadvantages. 2. Materials and methods 2.1. Materials Fig. 1. Schematic diagram of UMAE apparatus.

Dark red tomatoes were purchased from local market. Damaged and over mature fruits were abandoned; whole washed tomatoes were sorted and trimmed to remove any visible defects and the part of pedicle to be removed. Then chopped into smaller pieces and milled into puree by colloid grind at maximum clearance. Tomato puree was centrifuged at 3000 rpm by horizontal scroll-bowl centrifuge for 5 min. The supernatant was removed and the sedimentary was collected to next step. After centrifugation, the moisture content of the dehydration treatments were adjusted by vacuum-drying at 60 °C and 0.01 MPa pressure for 8–10 h. The final moisture content of the samples in all experiments was controlled at 78.86 ± 0.2% (W/W). The sample was stored in the refrigerator at 2–8 °C until use. 3. Methods 3.1. Ultrasonic and microwave assisted extraction of lycopene UMAE experiment was carried out with ultrasonic and microwave extracting apparatus (CW-2000, Shanghai Xintuo Microwave Instrument Co. Ltd.). The schematic diagram of UMAE apparatus is shown (Fig. 1). An open microwave with maximal power of 800 W at a frequency of 2450 MHz, and an ultrasonic transducer with a fixed power of 50 W at a frequency of 40 KHz were used simultaneously to extract lycopene from tomato paste: transfer the sample (about 2 g) into the flask, add proper volume (assigned according to the experiment planning) of ethyl acetate and then transfer the flask into the chamber of the apparatus and connect it with condensing tubes. Finally, close the door of chamber and set the program

of the parameters (microwave power and extraction time) according to the experimental design. When the extraction completed, the flask was removed from apparatus and the mixture was filtrated, then the volume of the filtrate and lycopene content in it were measured. Triplicate experiments were done for each design. 3.2. Ultrasound assisted extraction of lycopene Ultrasonic assisted extraction was carried out in an open rectangular ultrasonic processor (KJ-300, Wuxi Kejie Ultrasonic Electronic Equipment Co. Ltd., 325  210  140 mm internal dimension) with maximal power of 300 W at frequency of 40 KHz while operating at 50 W in practical process: take 2 g of tomato paste into 250 mL flask, add proper volume (assigned according to the experiment planning) of ethyl acetate, agitate the mixture, connect the flask with fitted condensing works and then immerse the flask in the ultrasonic water bath, the temperature of the water bath was controlled automatically. When the extraction finished, the flask was taken out and cooled to room temperature, the mixture was filtered, and finally the volume of the filtrate and lycopene content in it were measured. To avoid lycopene decomposing during the extraction, these processes should be done in darkroom and each design was triplicate. The UAE experimental design is presented in Table 2. 3.3. Determination of the total lycopene content in the extract Lycopene content in the extract was determined according to the absorbance measured at 502 nm via UV–VIS

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Table 1 Independent variable values and their corresponding levels Coded

UMAE experiment

1.682 1 0 1 1.682 Xi

UAE experiment

Time (Z1)

Power (Z2)

Solvent:tomato paste (v/w, Z3)

Time (Z1)

Temperature (Z2)

Solvent:tomato paste (v/w, Z3)

511 s 450 s 360 s 270 s 209 s X1 = (Z1  360)/90

110 W 100 W 85 W 70 W 60 W X2 = (Z2  85)/15

11.5:1 10.5:1 9:1 7.5:1 6.5:1 X3 = (Z3  9)/1.5

46.8 min 40 min 30 min 20 min 13.2 min X1 = (Z1  30)/10

86.8 °C 80 °C 70 °C 60 °C 53.2 °C X2 = (Z2  70)/10

9.5:1 8.5:1 7:1 5.5:1 4.5:1 X3 = (Z3  7)/1.5

Spectrophotometer (UNICO 2000, Shanghai) [21]. Total lycopene content in the raw material (known as 100%) was determined by conventional extraction method and the extraction procedure was repeated until the filtrate was colorless, then combine the filtrate and determine its lycopene content in the same way. The determining parameters was as follows: temperature, 60 °C; extracting period, 1 h; the ratio of solvent to tomato paste, 5:1 (V/W). Yield of lycopene from raw material was indicated according to Y ð%Þ ¼

AV  100 At  V t

ð1Þ

where Y, relative lycopene yield; A, absorbance of extractant at 502 nm; V, volume of the extractant (mL); At, absorbance of total extractant at 502 nm and Vt, volume of total extractant (mL). 3.4. Response surface methodology The experimental results were analyzed by response surface methodology (RSM) using the software Statistical

Analysis System (SAS version 8.0, SAS Institute Inc., USA). Calculations were done at 95% of confidence level. In these two different extracting methods, the combination effect of extraction temperature as X1 (power of microwave in UMAE), extraction time as X2 and the ratio of solvent to tomato paste as X3 was studied using RSM. In order to optimize the extracting conditions and investigate effects of above independent variables on the yield of lycopene, a central-composite rotary design with the variables at five levels (Table 1) was used in the experiments. Table 2 presents the design matrix for the experiment and the regression model proposed for response was given below: Y ¼ b0 þ

3 X i¼1

bi X i þ

3 X

bii X 2i þ

i¼1

3 X

bij X i X j

i
where b0 is the value of the fixed response at the central point of the experiment which is the point (0, 0, 0); bi, bii and bij are the linear, quadratic and cross-products coefficients, respectively. While demonstrating the significant effects 3-dimensional fitted surfaces were drawn. 4. Results and discussion

Table 2 Experimental planning (central-composite rotary design) Run

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

X1 1 1 1 1 1 1 1 1 1.682 1.682 0 0 0 0 0 0 0 0 0 0

X2 1 1 1 1 1 1 1 1 0 0 1.682 1.682 0 0 0 0 0 0 0 0

X3 1 1 1 1 1 1 1 1 0 0 0 0 1.682 1.682 0 0 0 0 0 0

4.1. Optimization of UMAE conditions of lycopene

Response (% yield of lycopene) UMAE

UAE

78.5 91.9 88.0 93.5 85.1 92.5 89.6 94.1 78.8 88.9 83.3 96.0 77.4 97.0 93.9 93.4 93.3 93.1 93.1 93.6

69.7 78.3 79.9 86.7 75.0 76.9 81.9 87.6 76.8 76.4 71.3 85.9 69.9 80.3 79.8 80.5 79.3 80.3 79.4 80.3

Twenty experimental points run randomly according to the UMAE experiment planning (Table 2). The percentage of relative extraction yield of lycopene in extracts ranged from 77.4% to 97.0%. The predicted model can be described by Y ð%Þ ¼ 93:31 þ 1:93X 1 þ 2:82X 2 þ 4:67X 3  0:63X 1 X 2  0:88X 1 X 3  0:77X 22  1:35X 2 X 3  1:63X 23

ð2Þ

R2 ¼ 0:9142 The estimated effects of each variables and analysis of variance for the model are given in Table 3. According to the ANOVA table, the fitted model was significant at consider confidence level since the F-value was more than three times that of the listed F-value [22]. According to the model, all the terms including linear terms of microwave power (X2), solvent to tomato paste ratio (X3), quadratic terms of extraction time ðX 23 Þ, solvent to tomato paste ðX 23 Þ were

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Table 3 ANOVA of response for UMAE experiments Source

DF

1 X1 X2 1 X3 1 1 X1 * X1 X1 * X2 1 X1 * X3 1 1 X2 * X2 X2 * X3 1 X3 * X3 1 Model 9 Error 10 Total 19 F listed value (95%)

SS

MS

F

Pr > F

50.98017 108.8669 297.7069 114.4762 3.125 6.125 8.495979 14.58 38.47843 624.5117 58.63373 683.1454

50.98017 108.8669 297.7069 114.4762 3.125 6.125 8.495979 14.58 38.47843 69.39019 5.863373

8.694683 18.56728 50.77401 19.52395 0.53297 1.044621 1.448992 2.486623 6.562509 11.83452

0.014568 0.00154 0.0001 0.001297 0.482113 0.330838 0.25641 0.145897 0.028289 0.000307

F9,10 = 3.02

significant. It is obvious that the changing of microwave power and solvent to tomato paste ratio had more effects on lycopene extraction than the others. The optimal conditions of UMAE were given by RSM as following: microwave power, 98 W; extracting time, 367 s; solvent to tomato paste ratio, 10.6:1. Under these conditions, the practical yield of lycopene was 97.4%, which was close to the predicted yield (97.1%) calculated according to the regression model presented in Eq. (2). The correlative surface response graphs are shown in Figs. 2–4. According to the fitted surface graphs, the maximal lycopene yield was achieved at higher microwave power, higher solvent to tomato paste ratio in relatively longer extracting time. It is well known that the chemical effect of decomposition of some active natural products is likely happened owing to the negative effect ultrasonic irradiation for a long time [20]. Further work should be progressed to reveal whether alternative of microwave and ultrasound or

Fig. 3. Surface graph for the effects of microwave power and the ratio of solvent to tomato paste on lycopene yield.

Fig. 4. Surface graph for the effects of extracting time and the ratio of solvent to tomato paste on lycopene yield.

Fig. 2. Surface graph for the effects of extracting time and microwave power on lycopene yield.

both of them lead to accelerating the oxidative decomposition of lycopene, as extracting time prolongs. When extracting time was fixed, effects of the other two factors on the extraction are shown in Fig. 4. From which we can see that the amount of lycopene yield increases gradually with the increasing of solvent to tomato paste ratio and microwave power. However, the increasing of these two

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parameters could hardly promote any more lycopene yield after the response has reached its peak value, perhaps due to decreased driving force of mass transfer. 4.2. Optimization of UAE conditions of lycopene The aim of UAE experiments was to find the relationship between independent variables and response and the regression model was given in Y ð%Þ ¼ 79:84 þ 0:45X 1 þ 4:45X 2 þ 2:96X 3  0:56X 21  0:13X 1 X 2  0:98X 1 X 3 þ 0:15X 22 þ 0:25X 2 X 3  1:09X 23

ð3Þ

R2 ¼ 0:9217 The estimated effects of each variables and the ANVOA of the fitted model are described in Table 4, where we could see that the square of correlation coefficient (R2 = 0.9217) demonstrating a closed agreement between experimental results and predicted values. The results of ANOVA suggested that the change of microwave power (X2), ratio of solvent to tomato paste (X3) had significant effects on the yield of lycopene. The RSM indicated that the estimated stationary point was a saddle point and the Ridge Analysis need be done in order to get the maximal response value [22]. The results of this analysis are shown in Table 5 and from which we could get the optimal parameters in the extraction of lycopene by UAE: extracting temperature (the temperature of water in bath), 86.4 °C; extracting period, 29.1 min; the ratio of solvent to tomato paste, 8.0:1. Under these conditions, the yield of lycopene by UAE was 89.4%. The surface graphs of the independent variables on response variable are shown in Figs. 5–7. Fig. 5 depicts the effects of extracting temperature and the time of this processing on lycopene yield, which indicating a quadratic effect for irradiation time on the response variable, the similar effect could be observed in Fig. 7 as well. The graphs suggested that extracting temperature behaving a linear effect on the yield of lycopene and the increasing of the ratio of solvent to tomato paste could enhance lycopene yield more effectively.

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Table 5 Estimated ridge of maximum response for variable % Y Coded radius

Estimated response

Standard error

Coded factor values X1

X2

X3

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10

79.839279 80.289288 80.73734 81.183921 81.629461 82.07434 82.518896 82.963425 83.408188 83.853416 84.299312 84.746055 85.193805 85.642701 86.092869 86.54442 86.997452 87.452055 87.908308 88.366281 88.826039 89.28764 89.751135

0.77297 0.772315 0.770442 0.767636 0.764375 0.761342 0.759425 0.759718 0.763493 0.772151 0.78715 0.809891 0.841615 0.883302 0.935618 0.998915 1.073271 1.158558 1.254514 1.360805 1.477073 1.602968 1.738161

0 0.006124 0.010523 0.013336 0.014698 0.014737 0.013573 0.011315 0.008065 0.003915 0.001054 0.006766 0.013155 0.02016 0.027728 0.03581 0.044364 0.053349 0.062732 0.072481 0.082566 0.092964 0.10365

0 0.070571 0.142669 0.216091 0.290659 0.366213 0.442616 0.519746 0.597501 0.675792 0.754542 0.833688 0.913173 0.99295 1.072979 1.153226 1.23366 1.314256 1.394994 1.475853 1.556817 1.637874 1.71901

0 0.045332 0.088465 0.129546 0.16872 0.206133 0.241926 0.276231 0.309172 0.340863 0.37141 0.40091 0.429449 0.457108 0.48396 0.510069 0.535494 0.560291 0.584507 0.608186 0.631369 0.654092 0.676388

Table 4 ANOVA of response for UAE experiments Source

DF

X1 1 X2 1 1 X3 X1 * X1 1 X1 * X2 1 X1 * X3 1 1 X2 * X2 X2 * X3 1 X3 * X3 1 Model 9 Error 10 Total 19 F listed value (95%)

SS

MS

F

Pr > F

2.749073 270.2723 120.0491 4.538366 0.125 7.605 0.306898 0.5 17.1694 422.7327 35.9253 458.658

2.749073 270.2723 120.0491 4.538366 0.125 7.605 0.306898 0.5 17.1694 46.9703 3.59253

0.765219 75.23174 33.41632 1.263279 0.034794 2.116893 0.085427 0.139178 4.779194 13.07444

0.402221 0.0001 0.000178 0.287288 0.855757 0.176342 0.776048 0.716886 0.053686 0.000199

F9,10 = 3.02

Fig. 5. Surface graph for the effects of extracting time and extracting temperature on lycopene yield.

4.3. Comparison of UMAE and UAE The results of the UAE experiments indicated that ultrasonic assistance enhanced the efficiency of lycopene extraction by shortening the extract time and lowering the volume

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Fig. 6. Surface graph for the effects of extracting temperature and the ratio of solvent to tomato paste on lycopene yield.

Fig. 7. Surface graph for the effects of extracting time and the ratio of solvent to tomato paste on lycopene yield.

of the solvents needed, but at the same time, UAE did not increase the yield of lycopene comparing with normal solvent extraction [23]. The reason of this phenomenon might be due to the fact that the hydroxyl radicals produced by acoustic cavitation of ultrasound in extracts, with the presentation of a small amount of water, resulted in the decomposition of lycopene. As for UMAE, with the assistances of acoustic cavitation and selected fast heating of microwave, the extraction was obviously improved by even shorter extract time, less volume of the solvents needed and higher

lycopene yield when compared with UAE (see Table 6). The reasonable explanation that the ultrasonic assistance here did not decrease the yield of lycopene was that the extraction was done in a relatively short period of time, the decomposition of lycopene was not too much to influence the yield. This explanation closely met the facts that the yields of lycopene decreased after they reached the maximal value (Figs. 2–4). So we could say that UMAE is a more attractive extracting method when compare with UAE, a relatively new solvent extracting technology developed

Table 6 Comparison of UMAE and UAE Extracting time

UMAE

Heating methods

Solvent/tomato paste

V Opt: a

Trend of yield

VOpt.

Trend of yield

VOpt.

Trend of yield

367 s  6.1 min

To increase (about 200–330 s)

98 Wb

To increase until reach maximal value (in experimental scale: 60–110 W) (>420 s)

10.6

To increase (about 7–9)

Reach peak (330–420 s)

Optimal yield (%) 97.4

Growth slow down and reach peak (>9)

To decrease (>420 s) UAE

29.1 min

To increase (about 13–24 min )

Reach peak (24–36 min)

To decrease (>36 min) a b c

VOpt. – optimal value. Microwave power. Water bath temperature.

86.4 °Cc

To increase until reach the maximized value (in experimental scale: about 56–87°C)

8.0

To increase (7)

89.4

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several decades ago. One main drawback of UMAE is its safety limitation and high initial cost of equipment.

[10]

5. Conclusion [11]

UAE and UMAE extraction of lycopene from tomato paste were optimized using RSM method. The optimal extracting conditions and lycopene yields of UAE and UMAE technology were described as follows: microwave power, 98 W; extracting time, 367 s; the ratio of solvent to tomato paste, 10.6:1 (UMAE) and extracting temperature, 86.4 °C; extracting time, 29.1 min; the ratio of solvent to tomato paste, 8.0:1. The comparison of these two methods showed that UMAE overcomes the shortcomings of UAE and will be a more attractive extract method in the future. References [1] A.V. Rao, S. Agarwal, Role of lycopene as antioxidant carotenoid in the prevention of chronic diseases, Nutrition Research 19 (2) (1999) 305–323. [2] Peter M. Bramley, Is lycopene beneficial to human health, Phytochemistry 54 (2000) 233–236. [3] Enzo Cadoni, M. Rita De Giorgi, Elena Medda, et al., Supercritical CO2 extraction of lycopene and b-carotene from ripe tomatoes, Dyes and Pigments 44 (1) (2000) 27–32. [4] John Shi, Marc Le Maguer, Yukio Kakuda, et al., Lycopene degradation and isomerization in tomato dehydration, Food Research International 32 (1) (1999) 15–21. [5] S.M. Choudhari, L. Ananthanarayan, Enzyme aided extraction of lycopene from tomato tissues, Food Chemistry 102 (1) (2007) 77–81. [6] Kurt B.G. Torssell, Natural Product Chemistry Kemisk Institut, Aarhus Universitet, ISBN 0 471 10378 0 (cloth) AACR2, p. 214. [7] A.C. Boileau, N.R. Merchen, K. Wasson, et al., cis-Lycopene is more bioavailable than trans-lycopene in vitro and in vivo in lymphcannulated ferrets, Journal of Nutrition 129 (1) (1999) 1176–1181. [8] Joseph Schierle, Werner Bretzel, Ilme Bu¨hler, et al., Content and isomeric ratio of lycopene in food and human blood plasma, Food Chemistry 59 (3) (1997) 459–465. [9] L.-X. Zhang, R.V. Conney, J.S. Bertram, Carotenoids enhance gap junctional communication and inhibit lipid peroxidation in C3H/

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