Food Chemistry 129 (2011) 747–752
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Process optimisation for recovery of carotenoids from tomato waste Irini F. Strati ⇑, Vassiliki Oreopoulou Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou 5, 15780 Zografou, Athens, Greece
a r t i c l e
i n f o
Article history: Received 17 December 2010 Received in revised form 8 March 2011 Accepted 3 May 2011 Available online 8 May 2011 Keywords: Carotenoids Tomato waste Extraction yield Solvent mixtures Optimisation Response surface methodology
a b s t r a c t Carotenoids constitute an important component of waste originating from tomato processing plants. Studies were carried out to assess the extraction yield of tomato waste carotenoids in different solvents and solvent mixtures and to optimise the extraction conditions for maximum recovery. A mixture of ethyl acetate and hexane gave the highest carotenoid extraction yield among the others examined. Extraction conditions, such as percentage of hexane in the solvent mixture of ethyl acetate and hexane, ratio of solvent to waste and particle size were optimised using a statistically designed experiment. A regression equation for predicting the carotenoid yield as a function of three extraction variables was derived by statistical analysis and a model with predictive ability of 0.97 was obtained. The optimised conditions for maximum carotenoid yield (37.5 mg kg1 dry waste) were 45% hexane in solvent mixture, solvent mixture to waste ratio of 9.1:1 (v/w) and particle size 0.56 mm. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The industrial processing of tomato leads to by-products, namely tomato seeds and peels, representing 10–40% of total processed tomatoes (Al-Wandawi, Abdul-Rahman, & Al-Shaikhly, 1985; Topal, Sasaki, Goto, & Hayakawa, 2006). These by-products represent a major disposal problem for the industry, intended mainly for animal feed or fertiliser (Knoblich, Anderson, & Latshaw, 2005), whereas they usually constitute a promising source of compounds that can be used for their nutritional properties and biological potential (Schieber, Stintzing, & Carle, 2001). Baysal, Ersus, and Starmans (2000) reported that a large quantity of carotenoids is lost as waste in tomato processing. According to Knoblich et al. (2005), the carotenoid content of dry tomato byproducts collected from a commercial tomato processing plant amounted to 793.2 and 157.9 lg g1, for peel and seed by-product, respectively. Carotenoids are natural pigments that provide the natural yellow, orange, and red colours of fruits, vegetables, plants, birds, and marine animals. These colours are a result of the presence of conjugated double bonds, also providing carotenoids with antioxidant properties. Additionally, carotenoids are well credited with important health-promoting functions or actions, such as provitamin A activity, enhancement of the immune system and reduction of the risk of degenerative diseases, e.g. cancer and cardiovascular disease (Fraser & Bramley, 2004; Rodriguez-Amaya, 2001; Van den Breg et al., 2000). ⇑ Corresponding author. Tel.: +30 210 7723166. E-mail addresses: [email protected], [email protected] (I.F. Strati). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.015
Most extraction methods of carotenoids from plant sources use organic solvents such as hexane, ethanol, acetone, methanol, tetrahydrofuran, benzene, and petroleum ether (Lin & Chen, 2003; Sadler, Davis, & Dezman, 1990; Sharma & Maguer, 1996). Additionally, mixtures of hexane with acetone, ethanol or methanol are often used (Shi & Le Maguer, 2000; Van den Breg et al., 2000) because other components, such as diethyl ether and tetrahydrofuran, may contain peroxides that react with carotenoids (Van den Breg et al., 2000). Taungbodhitham, Jones, Wahlqvist, and Briggs (1998) observed that the stability of carotenoid extracts obtained with hexane/acetone or hexane/ethanol was higher than that of extracts obtained with other organic solvents, such as chloroform, methanol or dichloromethane. Also, supercritical fluids are suitable for the extraction of compounds that can easily become degraded by light, oxygen and high temperatures like carotenoids, but the solubility of these substances is still relatively low compared to their solubility in organic solvents, and high pressures must be used to obtain reasonable extraction yields (Mattea, Martin, & Cocero, 2009). Therefore, from an industrial point of view, solvent extraction has been always the first option because of its simplicity and low costs. Comparison of efficiency among different solvents for carotenoid extraction from various plant materials is presented in literature, but few of them concern tomato waste (Lin & Chen, 2003; Riggi & Avola, 2008; Taungbodhitham et al., 1998). Moreover, limited reports deal with the optimisation of extraction conditions (Kaur, Wani, Oberoi, & Sogi, 2008; Nunes & Mercadante, 2004; Periago, Rincon, Aguera, & Ros, 2004). In previous work (Strati & Oreopoulou, 2011) the efficiency of several organic solvents to extract carotenoids from dry tomato waste was examined, and the effect of temperature, time and
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number of extraction steps. Three extraction steps of 30 min each were sufficient for all solvents, whilst the increase of temperature (from 25 to 70 °C) generally resulted in an increase of carotenoid yield. The present work was undertaken to examine mixtures of solvents and to optimise the extraction conditions as regards the synthesis of the solvent mixture, the ratio of solvent mixture to waste, and the particle size of the dried ground waste. The response surface methodology (RSM) was used for the process optimisation as it allows the evaluation of the effect of multiple variables and their interactions on the output variables with reduced number of trials (Basß & Boyaci, 2007). 2. Materials and methods
degradation reactions of the carotenoids. The mixture was vacuum filtered through filter paper and the solid residue was collected and re-extracted with fresh extraction solvent under the same conditions. The whole procedure was repeated three times. The extracts were combined and centrifuged at 3000 rpm (HERMLE centrifuge Z380, Gosheim, Germany) for 10 min to separate the supernatant. The carotenoid content of the supernatant was measured spectrophotometrically (Varian DMS 80 UV Spectrophotometer, Mulgrave, Victoria, Australia) at kmax for lycopene in each solvent or solvent mixture against the particular solvent or solvent mixture as blank.The following equation was used to calculate the carotenoid concentration, C (mg l1), expressed as lycopene, in each extract:
Akmax 104
2.1. Preparation of tomato waste
C¼
Tomato processing waste, composed of skin and seeds, was collected from NOMIKOS, a Greek tomato-processing factory. Moisture content was determined at fresh tomato processing waste upon arrival at the laboratory and found to be 80.48 ± 0.35%. The material was subsequently air dried at 25 °C, homogenised in a domestic blender and finally ground in a laboratory mill (Type ZM1, Retsch GmbH, Haan, Germany) equipped with different particle size sieves. Moisture content of ground dry tomato waste was 7.65 ± 0.21%. The dry ground material was kept in glass jars wrapped with aluminium foil at 20 °C until needed.
where Akmax is the absorbance of the extract at kmax specified for each solvent and A1% 1 cm is the absorption coefficient (absorbance at a given wavelength of a 1% solution in a spectrophotometer cuvette with a 1-cm light path) of lycopene in the respective solvent. The absorption coefficients were determined experimentally from calibration curves prepared with the lycopene standard in the relevant solvents (Strati & Oreopoulou, 2011) and found: 3450 in hexane at 471 nm, 2967 in acetone at 474 nm, 3950 in ethanol at 472 nm and 2963 in ethyl acetate at 473 nm, whereas the absorption coefficient of lycopene in hexane mixtures was 3450 at 472 nm. Although the accuracy of absorption coefficients is difficult to be obtained and some discrepancies can be noted, the values obtained experimentally were close to the ones already published in the literature (Britton, 1995; Taungbodhitham et al., 1998). The yield of extracted carotenoids (CY), expressed as lycopene, was calculated by Eq. (2):
2.2. Chemicals Hexane and ethyl acetate, analytical grade, were purchased from Fisher Scientific (Fair Lawn, NJ). Acetone and ethanol, p.a., were purchased from Sigma Chemical Co. (Sigma–Aldrich Company, St. Louis, MO). All solvents used for HPLC analysis (acetonitrile, 1-butanol and methylene chloride) were of HPLC grade and were obtained from Merck (Darmstadt, Germany). REDIVIVO Lycopene 10% FS (10% microcrystalline lycopene in corn oil containing a-tocopherol as antioxidant) from DSM Nutritional Products (Kaiseraugst, Switzerland) was used for the preparation of standard solutions for spectrophotometric measurements. All-trans lycopene, all-trans b-carotene and all-trans lutein were all purchased from Sigma Chemical Co. (Sigma–Aldrich Company, St. Louis, MO). 2.3. Carotenoid extraction Carotenoids were extracted using different organic solvents and solvent mixtures by the method described by Strati and Oreopoulou (2011). The polar solvents used were acetone, ethyl acetate and ethanol, whilst the non polar solvent used was hexane. The first series of experiments was conducted with single solvents or mixtures of equal volumes (50:50) at a solvent to waste ratio of 10:1 (v/w), whilst the particle size of the dry tomato waste was 1.0 mm. The second series of experiments, i.e. the factorial designed experiments (as described in Section 2.5) were conducted with mixtures of hexane and ethyl acetate varying among 10:90 and 80:20 (v/v), solvent to waste ratio varying among 3:1 and 10:1 (v/w), and particle size among 0.5 and 1.0 mm. Homogenised dry tomato waste (10.00g) was stirred with the solvent in an extraction vessel equipped with a vertical water cooler and placed in a water bath. The extraction temperature and time were kept constant at 25 ± 1 °C and 30 min, respectively. These experimental conditions were based on the results of previous work (Strati & Oreopoulou, 2011), which indicated that a quasisteady state was obtained in 30 min of extraction, whilst a sufficient yield was obtained at 25 °C. Therefore this temperature was used in this work to limit the operation cost and avoid undesirable
A1% 1 cm
CY ¼ C V=W
ð1Þ
ð2Þ
where C the concentration in each solvent/solvent mixture (mg l1), calculated by Eq.(1) V the volume of the extract (l). W the dry weight of tomato waste used in the first extraction (kg). 2.4. Carotenoid analysis For the identification of individual carotenoids, the extracts obtained by single solvents or solvent mixtures were further analysed by high performance liquid chromatography (HPLC), according to the method proposed by Sinanoglou, Strati, and Miniadis-Meimaroglou (2011). All extracts were filtered through a 0.45 lm membrane filter to remove particulate residues before injection. 20 ll of extract was injected for HPLC analysis. The HPLC (Hewlett Packard Series 1100, Waldbronn, Germany) system was composed of a HP 1100 Series Diode Array Detector, a HP 1100 Quaternary Pump, an Agilent 1100 Series Micro Vacuum Degasser and a Rheodyne model 7010 Sample Injector. The HPLC system was equipped with a YMC (Tokyo, Japan) C30 column (250 4.6 mm I.D., 5 lm particle). A mobile phase of acetonitrile (A), 1-butanol (B), and methylene chloride (C) with the following gradient elution was used: 69.3% A, 29.7% B and 1.0% C initially, increased to 67.2% A, 28.8% B and 4% C in 10 min, 61.6% A, 26.4% B and 12% C in 20 min, 49% A, 21% B and 30% C in 40 min and returned to 69.3% A, 29.7% B and 1% C in 50 min. The flow rate was maintained at 2 ml min1, the column temperature at 25 °C, and detection was carried out at 472 nm. The analysis of the chromatographic data was carried out on a ChemStation for LC 3D software (Agilent Technologies 1999– 2000, Waldbronn, Germany). The identification of major carotenoids in tomato waste extracts was carried out by comparing the
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retention times and absorption spectra with reference standards and absorption spectra characteristics as described in the literature (Lin & Chen, 2003). The percentage of HPLC identified carotenoids in tomato waste extracts was calculated as the ratio of the concentration of each carotenoid (based on the respective standard curve) to the sum of concentrations of all identified carotenoids in the chromatogram, multiplied by 100. The concentration range for the carotenoid standard curves were: 1–20 lg/ml for all-trans-lutein, 2–40 lg/ml for all-trans-b-carotene and 10–100 lg/ml for all-trans-lycopene. All standard curves constructed had high correlation coefficients (R2 > 0.98).
2.5. Experimental design and statistical analysis Experiments with single solvents or their mixtures (50:50, v/v) were conducted in triplicate and the data were analysed by the software STATISTICA (Statsoft.Inc, 2004). Analysis of variance (ANOVA) and Duncan’s multiple range tests were used to determine the significant difference in extraction yield between different solvent or solvent mixtures, at 95% confidence level (P < 0.05). The conditions for extraction were optimised with respect to hexane percentage in the hexane–ethyl acetate solvent mixture (X1), solvent to waste ratio (X2) and particle size (X3) using the Box-Behnken experimental design with the aid of the software STATISTICA (Statsoft.Inc, 2004). The experimental design involved three factors (process variables) each at three equidistant levels (1, 0, +1) and the response variable was the total carotenoid yield (Y). The levels of three factors were selected based on preliminary experiments. In total, 15 combinations of factors were used. The combination of factors at the centre of the level was run in triplicate. The experimental design determined the effect of the three main factors (X1, X2, X3) and their interactions on the response variable. The effect of each factor and their interactions on the carotenoid yield was assessed by ANOVA technique. A regression model containing 10 coefficients, including linear and quadratic effect of factors and linear effect of interactions, was assumed to describe relationships between response (Y) and the experimental factors (X1, X2, X3) as follows:
Y ¼ bo þ
3 X i¼1
bi X i þ
3 X
bii X 2i þ
i¼1
2 3 X X
bij X i X j ;
ð3Þ
i¼1 j¼iþ1
where bo is the constant coefficient (model intercept), bi is the linear coefficient of main factors, bii is the quadratic coefficient for main factors, and bij is the second order interaction coefficient. The 3D response graphs and profile for predicted values and desirability level for factors were plotted using STATISTICA software.
3. Results and discussion 3.1. Extraction of carotenoids The results presented in Table 1 concern the total carotenoid yield of the three successive extraction steps and the percentage of individual carotenoids identified by HPLC analysis. The combination of hexane with ethanol or ethyl acetate improved the total yield compared with that obtained by any of the individual solvents. On the contrary, acetone alone presented higher yield than its mixture. The highest carotenoid yield (36.5 mg kg1) was obtained when carotenoids were extracted with a mixture of ethyl acetate and hexane. All extracts obtained with solvents or solvent mixtures were subjected to carotenoid analysis, and the results, presented in Table 1, showed that all the extracts contain lycopene, b-carotene and lutein, their percentages, however, differ as expected. HPLC analysis of all extracts did not reveal any peaks that would indicate isomerisation or degradation. The lowest (66%) and the highest (84%) percentages for lycopene were observed in ethanol and hexane extracts, respectively, whereas the lowest (13%) and the highest (28%) percentages for b-carotene were noticed in acetone and hexane–ethanol extracts, respectively. On the other hand, lutein was not identified in hexane extracts, whilst its percentage was less than 3% in the extracts of hexane–ethanol and hexane–acetone. The maximum obtained percentage of lutein (20%) was found in ethanol extracts, followed by acetone (15%) and ethyl acetate (12%) extracts. The above results are consistent to the findings of Hakala and Heinonen (1994) who observed that, when carotenoids of tomato puree are extracted with relatively unpolar solvents the amounts of polar xanthophylls decrease and the proportion of lycopene relative to total carotenoids increases from 76% to 87%, thus improving lycopene extraction. The combination of polar solvents with the non polar hexane seems to enhance the solubilisation of the non polar carotenoids (lycopene and b-carotene), whereas individual polar solvents (ethanol, acetone and ethyl acetate) enhance the solubilisation of the polar lutein. This is possibly related to the relative solubility of lutein in ethanol, acetone and ethyl acetate which is 15–40 folds higher than the respective one in hexane. Similarly, the relative solubility of b-carotene in ethanol is 20-fold less than in hexane (Craft & Soares, 1992). In addition to the solubilising capacity, penetration or diffusion of the solvents into the solid matrix plays an important role in extraction efficiency. Acetone alone is a good solvent and a wetting material that penetrates easier in the solid matrix than the hexane–acetone mixture. Therefore, the yield obtained by the mixture is lower than that of acetone, and almost in the middle between acetone and hexane, indicating a cumulative action and no synergistic effect of the solvents. Similar results were obtained by Lin
Table 1 Total carotenoid yield and percentage of HPLC separated carotenoids from tomato waste extracted with different solvents and solvent mixtures, at 25 °C, solvent:waste ratio of 10 v/w, and waste particle size of 1 mm. Solvent/solvent mixture
Ethanol Hexane Ethyl acetate Acetone Hexane–ethanol (50:50) Hexane–acetone (50:50) Hexane–ethyl acetate (50:50)
Carotenoid yield (mg kg1 dry waste)
6.1 ± 0.3a 25.2 ± 0.7b 31.5 ± 0.2d 33.4 ± 0.3e 28.1 ± 0.6c 30.5 ± 0.8d 36.5 ± 1.1f
Values are means ± SD (n = 3). Values with different letters (a < b < c < d < e < f) differ significantly (P < 0.05). Nd, not detected.
HPLC identified carotenoids Lycopene
b-carotene
Lutein
66 ± 3% 84 ± 2% 73 ± 3% 72 ± 2% 69 ± 3% 72 ± 3% 83 ± 3%
14 ± 1% 16 ± 2% 15 ± 1% 13 ± 1% 28 ± 2% 25 ± 1% 13 ± 2%
20 ± 1% Nd 12 ± 1% 15 ± 1%
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Process optimisation for recovery of carotenoids from tomato waste Irini F. Strati ⇑, Vassiliki Oreopoulou Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou 5, 15780 Zografou, Athens, Greece
a r t i c l e
i n f o
Article history: Received 17 December 2010 Received in revised form 8 March 2011 Accepted 3 May 2011 Available online 8 May 2011 Keywords: Carotenoids Tomato waste Extraction yield Solvent mixtures Optimisation Response surface methodology
a b s t r a c t Carotenoids constitute an important component of waste originating from tomato processing plants. Studies were carried out to assess the extraction yield of tomato waste carotenoids in different solvents and solvent mixtures and to optimise the extraction conditions for maximum recovery. A mixture of ethyl acetate and hexane gave the highest carotenoid extraction yield among the others examined. Extraction conditions, such as percentage of hexane in the solvent mixture of ethyl acetate and hexane, ratio of solvent to waste and particle size were optimised using a statistically designed experiment. A regression equation for predicting the carotenoid yield as a function of three extraction variables was derived by statistical analysis and a model with predictive ability of 0.97 was obtained. The optimised conditions for maximum carotenoid yield (37.5 mg kg1 dry waste) were 45% hexane in solvent mixture, solvent mixture to waste ratio of 9.1:1 (v/w) and particle size 0.56 mm. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The industrial processing of tomato leads to by-products, namely tomato seeds and peels, representing 10–40% of total processed tomatoes (Al-Wandawi, Abdul-Rahman, & Al-Shaikhly, 1985; Topal, Sasaki, Goto, & Hayakawa, 2006). These by-products represent a major disposal problem for the industry, intended mainly for animal feed or fertiliser (Knoblich, Anderson, & Latshaw, 2005), whereas they usually constitute a promising source of compounds that can be used for their nutritional properties and biological potential (Schieber, Stintzing, & Carle, 2001). Baysal, Ersus, and Starmans (2000) reported that a large quantity of carotenoids is lost as waste in tomato processing. According to Knoblich et al. (2005), the carotenoid content of dry tomato byproducts collected from a commercial tomato processing plant amounted to 793.2 and 157.9 lg g1, for peel and seed by-product, respectively. Carotenoids are natural pigments that provide the natural yellow, orange, and red colours of fruits, vegetables, plants, birds, and marine animals. These colours are a result of the presence of conjugated double bonds, also providing carotenoids with antioxidant properties. Additionally, carotenoids are well credited with important health-promoting functions or actions, such as provitamin A activity, enhancement of the immune system and reduction of the risk of degenerative diseases, e.g. cancer and cardiovascular disease (Fraser & Bramley, 2004; Rodriguez-Amaya, 2001; Van den Breg et al., 2000). ⇑ Corresponding author. Tel.: +30 210 7723166. E-mail addresses: [email protected], [email protected] (I.F. Strati). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.015
Most extraction methods of carotenoids from plant sources use organic solvents such as hexane, ethanol, acetone, methanol, tetrahydrofuran, benzene, and petroleum ether (Lin & Chen, 2003; Sadler, Davis, & Dezman, 1990; Sharma & Maguer, 1996). Additionally, mixtures of hexane with acetone, ethanol or methanol are often used (Shi & Le Maguer, 2000; Van den Breg et al., 2000) because other components, such as diethyl ether and tetrahydrofuran, may contain peroxides that react with carotenoids (Van den Breg et al., 2000). Taungbodhitham, Jones, Wahlqvist, and Briggs (1998) observed that the stability of carotenoid extracts obtained with hexane/acetone or hexane/ethanol was higher than that of extracts obtained with other organic solvents, such as chloroform, methanol or dichloromethane. Also, supercritical fluids are suitable for the extraction of compounds that can easily become degraded by light, oxygen and high temperatures like carotenoids, but the solubility of these substances is still relatively low compared to their solubility in organic solvents, and high pressures must be used to obtain reasonable extraction yields (Mattea, Martin, & Cocero, 2009). Therefore, from an industrial point of view, solvent extraction has been always the first option because of its simplicity and low costs. Comparison of efficiency among different solvents for carotenoid extraction from various plant materials is presented in literature, but few of them concern tomato waste (Lin & Chen, 2003; Riggi & Avola, 2008; Taungbodhitham et al., 1998). Moreover, limited reports deal with the optimisation of extraction conditions (Kaur, Wani, Oberoi, & Sogi, 2008; Nunes & Mercadante, 2004; Periago, Rincon, Aguera, & Ros, 2004). In previous work (Strati & Oreopoulou, 2011) the efficiency of several organic solvents to extract carotenoids from dry tomato waste was examined, and the effect of temperature, time and
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number of extraction steps. Three extraction steps of 30 min each were sufficient for all solvents, whilst the increase of temperature (from 25 to 70 °C) generally resulted in an increase of carotenoid yield. The present work was undertaken to examine mixtures of solvents and to optimise the extraction conditions as regards the synthesis of the solvent mixture, the ratio of solvent mixture to waste, and the particle size of the dried ground waste. The response surface methodology (RSM) was used for the process optimisation as it allows the evaluation of the effect of multiple variables and their interactions on the output variables with reduced number of trials (Basß & Boyaci, 2007). 2. Materials and methods
degradation reactions of the carotenoids. The mixture was vacuum filtered through filter paper and the solid residue was collected and re-extracted with fresh extraction solvent under the same conditions. The whole procedure was repeated three times. The extracts were combined and centrifuged at 3000 rpm (HERMLE centrifuge Z380, Gosheim, Germany) for 10 min to separate the supernatant. The carotenoid content of the supernatant was measured spectrophotometrically (Varian DMS 80 UV Spectrophotometer, Mulgrave, Victoria, Australia) at kmax for lycopene in each solvent or solvent mixture against the particular solvent or solvent mixture as blank.The following equation was used to calculate the carotenoid concentration, C (mg l1), expressed as lycopene, in each extract:
Akmax 104
2.1. Preparation of tomato waste
C¼
Tomato processing waste, composed of skin and seeds, was collected from NOMIKOS, a Greek tomato-processing factory. Moisture content was determined at fresh tomato processing waste upon arrival at the laboratory and found to be 80.48 ± 0.35%. The material was subsequently air dried at 25 °C, homogenised in a domestic blender and finally ground in a laboratory mill (Type ZM1, Retsch GmbH, Haan, Germany) equipped with different particle size sieves. Moisture content of ground dry tomato waste was 7.65 ± 0.21%. The dry ground material was kept in glass jars wrapped with aluminium foil at 20 °C until needed.
where Akmax is the absorbance of the extract at kmax specified for each solvent and A1% 1 cm is the absorption coefficient (absorbance at a given wavelength of a 1% solution in a spectrophotometer cuvette with a 1-cm light path) of lycopene in the respective solvent. The absorption coefficients were determined experimentally from calibration curves prepared with the lycopene standard in the relevant solvents (Strati & Oreopoulou, 2011) and found: 3450 in hexane at 471 nm, 2967 in acetone at 474 nm, 3950 in ethanol at 472 nm and 2963 in ethyl acetate at 473 nm, whereas the absorption coefficient of lycopene in hexane mixtures was 3450 at 472 nm. Although the accuracy of absorption coefficients is difficult to be obtained and some discrepancies can be noted, the values obtained experimentally were close to the ones already published in the literature (Britton, 1995; Taungbodhitham et al., 1998). The yield of extracted carotenoids (CY), expressed as lycopene, was calculated by Eq. (2):
2.2. Chemicals Hexane and ethyl acetate, analytical grade, were purchased from Fisher Scientific (Fair Lawn, NJ). Acetone and ethanol, p.a., were purchased from Sigma Chemical Co. (Sigma–Aldrich Company, St. Louis, MO). All solvents used for HPLC analysis (acetonitrile, 1-butanol and methylene chloride) were of HPLC grade and were obtained from Merck (Darmstadt, Germany). REDIVIVO Lycopene 10% FS (10% microcrystalline lycopene in corn oil containing a-tocopherol as antioxidant) from DSM Nutritional Products (Kaiseraugst, Switzerland) was used for the preparation of standard solutions for spectrophotometric measurements. All-trans lycopene, all-trans b-carotene and all-trans lutein were all purchased from Sigma Chemical Co. (Sigma–Aldrich Company, St. Louis, MO). 2.3. Carotenoid extraction Carotenoids were extracted using different organic solvents and solvent mixtures by the method described by Strati and Oreopoulou (2011). The polar solvents used were acetone, ethyl acetate and ethanol, whilst the non polar solvent used was hexane. The first series of experiments was conducted with single solvents or mixtures of equal volumes (50:50) at a solvent to waste ratio of 10:1 (v/w), whilst the particle size of the dry tomato waste was 1.0 mm. The second series of experiments, i.e. the factorial designed experiments (as described in Section 2.5) were conducted with mixtures of hexane and ethyl acetate varying among 10:90 and 80:20 (v/v), solvent to waste ratio varying among 3:1 and 10:1 (v/w), and particle size among 0.5 and 1.0 mm. Homogenised dry tomato waste (10.00g) was stirred with the solvent in an extraction vessel equipped with a vertical water cooler and placed in a water bath. The extraction temperature and time were kept constant at 25 ± 1 °C and 30 min, respectively. These experimental conditions were based on the results of previous work (Strati & Oreopoulou, 2011), which indicated that a quasisteady state was obtained in 30 min of extraction, whilst a sufficient yield was obtained at 25 °C. Therefore this temperature was used in this work to limit the operation cost and avoid undesirable
A1% 1 cm
CY ¼ C V=W
ð1Þ
ð2Þ
where C the concentration in each solvent/solvent mixture (mg l1), calculated by Eq.(1) V the volume of the extract (l). W the dry weight of tomato waste used in the first extraction (kg). 2.4. Carotenoid analysis For the identification of individual carotenoids, the extracts obtained by single solvents or solvent mixtures were further analysed by high performance liquid chromatography (HPLC), according to the method proposed by Sinanoglou, Strati, and Miniadis-Meimaroglou (2011). All extracts were filtered through a 0.45 lm membrane filter to remove particulate residues before injection. 20 ll of extract was injected for HPLC analysis. The HPLC (Hewlett Packard Series 1100, Waldbronn, Germany) system was composed of a HP 1100 Series Diode Array Detector, a HP 1100 Quaternary Pump, an Agilent 1100 Series Micro Vacuum Degasser and a Rheodyne model 7010 Sample Injector. The HPLC system was equipped with a YMC (Tokyo, Japan) C30 column (250 4.6 mm I.D., 5 lm particle). A mobile phase of acetonitrile (A), 1-butanol (B), and methylene chloride (C) with the following gradient elution was used: 69.3% A, 29.7% B and 1.0% C initially, increased to 67.2% A, 28.8% B and 4% C in 10 min, 61.6% A, 26.4% B and 12% C in 20 min, 49% A, 21% B and 30% C in 40 min and returned to 69.3% A, 29.7% B and 1% C in 50 min. The flow rate was maintained at 2 ml min1, the column temperature at 25 °C, and detection was carried out at 472 nm. The analysis of the chromatographic data was carried out on a ChemStation for LC 3D software (Agilent Technologies 1999– 2000, Waldbronn, Germany). The identification of major carotenoids in tomato waste extracts was carried out by comparing the
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retention times and absorption spectra with reference standards and absorption spectra characteristics as described in the literature (Lin & Chen, 2003). The percentage of HPLC identified carotenoids in tomato waste extracts was calculated as the ratio of the concentration of each carotenoid (based on the respective standard curve) to the sum of concentrations of all identified carotenoids in the chromatogram, multiplied by 100. The concentration range for the carotenoid standard curves were: 1–20 lg/ml for all-trans-lutein, 2–40 lg/ml for all-trans-b-carotene and 10–100 lg/ml for all-trans-lycopene. All standard curves constructed had high correlation coefficients (R2 > 0.98).
2.5. Experimental design and statistical analysis Experiments with single solvents or their mixtures (50:50, v/v) were conducted in triplicate and the data were analysed by the software STATISTICA (Statsoft.Inc, 2004). Analysis of variance (ANOVA) and Duncan’s multiple range tests were used to determine the significant difference in extraction yield between different solvent or solvent mixtures, at 95% confidence level (P < 0.05). The conditions for extraction were optimised with respect to hexane percentage in the hexane–ethyl acetate solvent mixture (X1), solvent to waste ratio (X2) and particle size (X3) using the Box-Behnken experimental design with the aid of the software STATISTICA (Statsoft.Inc, 2004). The experimental design involved three factors (process variables) each at three equidistant levels (1, 0, +1) and the response variable was the total carotenoid yield (Y). The levels of three factors were selected based on preliminary experiments. In total, 15 combinations of factors were used. The combination of factors at the centre of the level was run in triplicate. The experimental design determined the effect of the three main factors (X1, X2, X3) and their interactions on the response variable. The effect of each factor and their interactions on the carotenoid yield was assessed by ANOVA technique. A regression model containing 10 coefficients, including linear and quadratic effect of factors and linear effect of interactions, was assumed to describe relationships between response (Y) and the experimental factors (X1, X2, X3) as follows:
Y ¼ bo þ
3 X i¼1
bi X i þ
3 X
bii X 2i þ
i¼1
2 3 X X
bij X i X j ;
ð3Þ
i¼1 j¼iþ1
where bo is the constant coefficient (model intercept), bi is the linear coefficient of main factors, bii is the quadratic coefficient for main factors, and bij is the second order interaction coefficient. The 3D response graphs and profile for predicted values and desirability level for factors were plotted using STATISTICA software.
3. Results and discussion 3.1. Extraction of carotenoids The results presented in Table 1 concern the total carotenoid yield of the three successive extraction steps and the percentage of individual carotenoids identified by HPLC analysis. The combination of hexane with ethanol or ethyl acetate improved the total yield compared with that obtained by any of the individual solvents. On the contrary, acetone alone presented higher yield than its mixture. The highest carotenoid yield (36.5 mg kg1) was obtained when carotenoids were extracted with a mixture of ethyl acetate and hexane. All extracts obtained with solvents or solvent mixtures were subjected to carotenoid analysis, and the results, presented in Table 1, showed that all the extracts contain lycopene, b-carotene and lutein, their percentages, however, differ as expected. HPLC analysis of all extracts did not reveal any peaks that would indicate isomerisation or degradation. The lowest (66%) and the highest (84%) percentages for lycopene were observed in ethanol and hexane extracts, respectively, whereas the lowest (13%) and the highest (28%) percentages for b-carotene were noticed in acetone and hexane–ethanol extracts, respectively. On the other hand, lutein was not identified in hexane extracts, whilst its percentage was less than 3% in the extracts of hexane–ethanol and hexane–acetone. The maximum obtained percentage of lutein (20%) was found in ethanol extracts, followed by acetone (15%) and ethyl acetate (12%) extracts. The above results are consistent to the findings of Hakala and Heinonen (1994) who observed that, when carotenoids of tomato puree are extracted with relatively unpolar solvents the amounts of polar xanthophylls decrease and the proportion of lycopene relative to total carotenoids increases from 76% to 87%, thus improving lycopene extraction. The combination of polar solvents with the non polar hexane seems to enhance the solubilisation of the non polar carotenoids (lycopene and b-carotene), whereas individual polar solvents (ethanol, acetone and ethyl acetate) enhance the solubilisation of the polar lutein. This is possibly related to the relative solubility of lutein in ethanol, acetone and ethyl acetate which is 15–40 folds higher than the respective one in hexane. Similarly, the relative solubility of b-carotene in ethanol is 20-fold less than in hexane (Craft & Soares, 1992). In addition to the solubilising capacity, penetration or diffusion of the solvents into the solid matrix plays an important role in extraction efficiency. Acetone alone is a good solvent and a wetting material that penetrates easier in the solid matrix than the hexane–acetone mixture. Therefore, the yield obtained by the mixture is lower than that of acetone, and almost in the middle between acetone and hexane, indicating a cumulative action and no synergistic effect of the solvents. Similar results were obtained by Lin
Table 1 Total carotenoid yield and percentage of HPLC separated carotenoids from tomato waste extracted with different solvents and solvent mixtures, at 25 °C, solvent:waste ratio of 10 v/w, and waste particle size of 1 mm. Solvent/solvent mixture
Ethanol Hexane Ethyl acetate Acetone Hexane–ethanol (50:50) Hexane–acetone (50:50) Hexane–ethyl acetate (50:50)
Carotenoid yield (mg kg1 dry waste)
6.1 ± 0.3a 25.2 ± 0.7b 31.5 ± 0.2d 33.4 ± 0.3e 28.1 ± 0.6c 30.5 ± 0.8d 36.5 ± 1.1f
Values are means ± SD (n = 3). Values with different letters (a < b < c < d < e < f) differ significantly (P < 0.05). Nd, not detected.
HPLC identified carotenoids Lycopene
b-carotene
Lutein
66 ± 3% 84 ± 2% 73 ± 3% 72 ± 2% 69 ± 3% 72 ± 3% 83 ± 3%
14 ± 1% 16 ± 2% 15 ± 1% 13 ± 1% 28 ± 2% 25 ± 1% 13 ± 2%
20 ± 1% Nd 12 ± 1% 15 ± 1%
