Extraction of antioxidant compounds from Jabuticaba (Myrciaria cauliflora) skins: Yield, composition and economical evaluation

Journal of Food Engineering 101 (2010) 23–31

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Extraction of antioxidant compounds from Jabuticaba (Myrciaria cauliflora) skins: Yield, composition and economical evaluation Diego T. Santos, Priscilla C. Veggi, M. Angela A. Meireles * LASEFI/DEA/FEA (School of Food Engineering)/UNICAMP (University of Campinas), R. Monteiro Lobato, 80, 13083-862 Campinas, SP, Brazil

a r t i c l e

i n f o

Article history: Received 28 December 2009 Received in revised form 20 May 2010 Accepted 5 June 2010 Available online 9 June 2010 Keywords: Anthocyanins Antioxidant compounds Cost of manufacturing Extraction methods Jabuticaba Myrciaria cauliflora Phenolic compounds

a b s t r a c t Obtaining an extract with high antioxidant activity using environmentally friendly technologies and lowcost raw materials is of great interest. In the present work, a combined extraction process developed by our research group involving ultrasound treatment and agitated solvent extraction was evaluated. This method was compared in terms of yield, composition, and economical feasibility to traditional extraction methods, including ultrasound assisted, agitated bed and soxhlet extraction with ethanol (acidified or not). The proposed method maximizes the extraction of phenolic compounds with acceptable degradation of anthocyanin pigments from an unusual source: Brazilian jabuticaba (Myrciaria cauliflora) skins. The use of ultrasonic irradiation continuously supporting a main extraction process has demonstrated increased performance but implies in high consumption of energy and consequently, money. However, the procedure described in this paper appears to be a viable option because it uses shorter ultrasonic irradiation and results in high antioxidant activity extracts, and the anthocyanin profile corroborates literature data (cyanidin-3-glucoside and delphinidin-3-glucoside). Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The desire for a healthier diet allied with the increasing concern of consumers over the use of synthetic additives in food has pushed the food industry to search for new sources of natural pigments (Montes et al., 2005). Anthocyanins are a type of functional pigment responsible for a wide range of colors present in vegetables, flowers, fruits, and derived products. It is known that anthocyanin pigments act as strong antioxidants and are antiinflammatory, with antimutagenic and cancer chemopreventive activities (Kong et al., 2003). These bioactive properties have already been demonstrated in in vitro and in vivo studies (Galvano et al., 2004), and an increase of publications in this field has been observed in recent years. In a recent review paper, Santos and Meireles (2009) compiled the recent studies on the health-promoting properties of anthocyanins. This review demonstrated that consumption of dietary phytochemicals, of which anthocyanins represent a considerable part, may promote several health benefits: reduction in the risk of cardiovascular diseases, diabetes and cancer; a protective effect against hepatic and gastric damage and collagen degradation; an increase of cognitive performance, etc. Grape peels, grape by-products (constituted mainly by peels) and berries are well known for their antioxidant properties due * Corresponding author. Tel.: +55 1935214033; fax: +55 1937884027. E-mail address: [email protected] (M. Angela A. Meireles). 0260-8774/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2010.06.005

to the presence of anthocyanins and other phenolic compounds. Many studies have been done to extract and evaluate these compounds on the industrial scale (Santos and Meireles, 2009). In Brazil another source seems promising; jabuticaba (Myrciaria cauliflora) is grape-like in appearance and texture, although its skin is thicker and tougher. This fruit has a dark purple to almost black skin color due to a high content of anthocyanins that cover a white gelatinous flesh inside (Terci, 2004). As the extraction procedure is of great importance for obtaining natural colorants, different research groups have made an effort to develop an efficient extraction procedure. An efficient extraction should maximize anthocyanin recovery with minimal degradation and result in an extract with high antioxidant activity using environmentally friendly technologies and low-cost raw materials. For this purpose this paper aims to demonstrate a potential technique to extract antioxidant compounds (anthocyanins and other phenolic compounds) from jabuticaba skins: utilizing a short ultrasonic irradiation as pre-treatment before conventional solvent extraction. This method is proposed based on the fact that ultrasonic irradiation facilitates the release of extractable compounds and enhances mass transport of the solvent from the continuous phase into plant cells of target compounds (Lee and Row, 2006), mainly during the initial minutes (Zhang et al., 2009). Ethanol was used in all experiments because it is a GRAS (generally-recognized-as-safe) solvent. It is well known that many flavonoids and related phenolic compounds contribute significantly to the total antioxidant

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activity of many fruits and vegetables (Luo et al., 2002). Given that some recent papers have demonstrated that the biological (antioxidant, antiradical, etc.) activities of jabuticaba skin extracts are due to their compositions (Reynertson et al., 2008) the study of the effect of the extraction method on the extract antioxidant activity was evaluated. To compare the effect of the extraction method on the process in terms of yield, composition and economic feasibility, different extraction methods were carried out for comparison purposes: ultrasound assisted, agitated bed, and soxhlet extraction using ethanol and acidified ethanol as solvent. 2. Materials and methods 2.1. Plant material Jabuticaba fruits (Myrciaria cauliflora) harvested from a plantation in the State of São Paulo, Brazil, were acquired from a fruit and vegetable market center (CEASA-Campinas, Brazil). Immediately after acquisition, the fruits were stored in the dark in a domestic freezer (10 °C) (Double Action, Metalfrio, São Paulo, Brazil) until sample preparation. Before extraction, the fruits were manually peeled. 2.2. Extraction procedures 2.2.1. Ultrasound assisted extraction (UAE) Jabuticaba skins were added to 50-cm3 Erlenmeyer flasks and then mixed with different volumes of ethanol 99.5% (Ecibra, Santo Amaro, Brazil) to give a feed to solvent ratio of 1:10. Immediately after the addition of the solvent, the flasks were sonicated in an ultrasonicator bath with a 40-kHz frequency (81 W) (model T 1440, Thornton, São Paulo, Brazil) at room temperature for 2 h. After ultrasound extraction, the solvent was separated from the plant residue by simple filtration and evaporated using a rotary evaporator (Laborota, model 4001, Vertrieb, Germany), with vacuum control (Heidolph Instruments Gmbh, Vertrieb, Germany) and a thermostatic bath at 40 °C. The extracts were stored (10 °C) in the dark until analysis. 2.2.2. Agitated bed extraction (ABE) The extraction was carried out at 30 °C by placing jabuticaba skins into 125-cm3 Erlenmeyer flasks containing ethanol (99.5%, Ecibra, Santo Amaro, Brazil) using a feed to solvent ratio of 1:10. Extractions were carried out in a shaker (model MA 420, Piracicaba, Brazil) with agitation (150 rpm) for 2 h. After extraction, the solvent was separated from the plant residue and evaporated, and the extract was stored as described before. 2.2.3. Combined UAE + ABE Erlenmeyer flasks (125-cm3) containing jabuticaba skins and ethanol 99.5% (Ecibra, Santo Amaro, Brazil) (feed to solvent ratio of 1:10) were sonicated for 10 min at a frequency of 40 kHz at room temperature; afterwards, the flasks were incubated in a rotary shaker (150 rpm) at 30 °C for 2 h. The solvent–plant residue separation, solvent evaporation and extract storage were done as described before. 2.2.4. Soxhlet extraction Approximately 25 g of jabuticaba skins and 250 cm3 of ethanol or acidified ethanol (acidified to pH 3 with HCl) (99.5% Ecibra, Santo Amaro, Brazil) were used (feed to solvent ratio of 1:10). The extraction was done in a soxhlet apparatus for 8 h, and after that the solvent was evaporated, and the extract was stored as described before.

2.3. Extract characterization 2.3.1. Antioxidant activity (AA) The evaluation of antioxidant activity of the extracts was based on the coupled oxidation of b-carotene and linoleic acid. The technique developed by Marco (1968) consisted of measuring the bleaching of b-carotene resulting from oxidation by the degradation products of linoleic acid. One milligram of b-carotene (97%, Sigma–Aldrich, St. Louis, USA) was dissolved in 10 cm3 of chloroform (99 %, Ecibra, Santo Amaro, Brazil). The absorbance was tested after adding 0.2 cm3 of the solution to 5 cm3 of chloroform, then reading the absorbance of this solution at 470 nm using a UV–Vis. spectrophotometer (Hitachi, model U-3010, Tokyo, Japan). A reading between 0.6 and 0.9 indicated a workable concentration of b-carotene. One milliliter of b-carotene chloroform solution was added to a flask that contained 20 mg of linoleic acid (99%, Sigma–Aldrich, St. Louis, USA) and 200 mg Tween 40 (99%, Sigma–Aldrich, St. Louis, USA). Chloroform was removed using a rotary evaporator (Laborota, model 4001, Vertrieb, Germany), with vacuum control (Heidolph Instruments Gmbh, Vertrieb, Germany) and a thermostatic bath at 40 °C; then 50 cm3 of oxygenated distilled water (oxygenation for 30 min) was added to the flask with vigorous agitation to form an emulsion. Five milliliters of the emulsion was added to 0.2 cm3 of the antioxidant solution (7.5 mg of extract/1 cm3 of distilled water) in assay tubes. To the control solution, 0.2 cm3 of pure distilled water was added. A blank consisting of 20 mg linoleic acid, 200 mg Tween 40 and 50 cm3 oxygenated distilled water was used to bring the spectrophotometer to zero. Tubes were manually shaken, and absorbance measurements made at 470 nm immediately after the addition of the emulsion to the antioxidant solution. The tubes were placed in a water bath (model TE 159, Tecnal, Piracicaba, Brazil) at 50 °C. Absorbance measurements were made at 30 min intervals during 3 h. The average deviation of duplicated experiments never exceeded 8%, therefore, no additional statistical analysis was considered necessary. The antioxidant activities (AAs) were calculated by Eq. (1):

AA ð%Þ ¼ 100  1 

t¼0

t

t¼0

t

Absextract  Absextract Abscontrol  Abscontrol

! ð1Þ

2.3.2. Total phenolic compounds Total phenolic content was estimated using the Folin–Ciocalteau method for total phenolics, based on a colorimetric oxidation/reduction reaction of phenols (Singleton et al., 1965). Briefly, 1 cm3 of the sample was mixed with 1 cm3 of Folin and Ciocalteu’s phenol reagent. After 3 min, 1 cm3 of saturated sodium carbonate solution (50% w/w) was added to the mixture, and the volume was adjusted to 10 cm3 with distilled water. The reaction was kept in the dark for 90 min at room temperature, after which the absorbance was read at 725 nm with a UV–Vis. Spectrophotometer Hitachi, model U-3010 (Tokyo, Japan). For the control sample, 1 cm3 of distilled water was taken. The results were calculated on the basis of the calibration curve of gallic acid (GA) and expressed as milligrams of gallic acid equivalents (GAEs)/g dry material. The average deviation of duplicated experiments never exceeded 8%; therefore, no additional statistical analysis was considered necessary. 2.3.3. Total monomeric anthocyanins (TMA) The total monomeric anthocyanin (TMA) content was determined using the pH differential method described by Giusti and Wrolstad (2001), which relies on the structural transformation of the anthocyanin chromophore as a function of pH. A UV– Vis. spectrophotometer (Hitachi, model U-3010, Tokyo, Japan)

D.T. Santos et al. / Journal of Food Engineering 101 (2010) 23–31

was used for spectral measurements at the maximum absorbance wavelength (approximately 512 nm) and 700 nm, using distilled water as a blank. For this purpose, 20 mg of extract was dissolved in 10 cm3 of distilled water. Two dilutions of the sample were prepared: one with hydrochloric acid/potassium chloride buffer pH = 1.0 and the other with sodium acetate/acetic acid buffer pH = 4.5. The pH values of the buffers were measured using a pH-meter (Digimed, model DM-22, São Paulo, Brazil) calibrated with buffers at pH 4.01 and 6.86, and they were adjusted with HCl (99.5% Ecibra, Santo Amaro, Brazil). Aliquots of extract were brought to pH 1.0 and 4.5; 15 min later, the absorbance of each equilibrated solution was measured at the maximum absorption wavelength and 700 nm for haze correction using 1-cm path length glass cells (l). The dilution factor (DF) was determined (final volume over original sample volume). The difference in absorbance values at pH 1.0 and 4.5 is directly proportional to the TMA concentration. The anthocyanin content was calculated as cyanidin-3-glycoside (MW = 449.2 g/mol and e = 26.900 L/mol.cm), and the results were expressed as mg cy3-glycoside/g dry material. The average deviation of duplicated experiments never exceeded 8%; therefore, no additional statistical analysis was considered necessary. The absorbance of the diluted sample (A) and the TMA were calculated with Eqs. (2) and (3):

A ¼ ðAmax  A700 ÞpH1;0  ðAmax  A700 Þp4;5

ð2Þ

TMA ðmg=LÞ ¼ ðA  MW  DF  1000Þ=ðe  lÞ

ð3Þ

2.3.4. Thin-layer chromatography (TLC) The extracts were fractionated by thin-layer chromatography (TLC). The TLC was performed using silica plates (20  20 cm, 1-mm height, Merck, Darmstadt, Germany). To identify which anthocyanins were extracted in each extraction procedure, according to Wagner and Bladt (2001), the mobile phase used was composed by ethyl acetate (99.5%, Merck, São Paulo, Brazil), glacial acetic acid (99.7%, ECIBRA, Santo Amaro, Brazil), formic acid (36.5%, VETEC, Rio de Janeiro, Brazil) and distilled water (100: 11:11:26), and no spray reagent was used. Extracts and anthocyanin standards were diluted in methanol (98%, Synth, Diadema, Brazil) and applied on the plates (TLC). The anthocyanin standards, cyanidin-3-glucoside chloride, delphinidin-3-glucoside chloride, peonidin-3-glucoside chloride, malvidin-3-glucoside chloride,

25

pelargonidin-3-glucoside chloride and petunidin-3-glucoside chloride were purchased from Extrasynthese (Genay, France).

2.4. Process simulation SuperPro Designer 6.0Ò was used for process simulation. This software allows the mass and energy balance estimation for all streams of the process, estimates purchase costs, and reports stream and equipment data, as well as capital and manufacturing costs. The conventional extraction denoted in this paper by agitated bed extraction (ABE) was developed using the software in a similar manner to the work of Takeuchi et al. (2009), as shown in Fig. 1. The extraction procedure consists placing a known mass of jabuticaba skins immersed in a known volume of solvent inside an agitated tank. The equipment consists of two extractors, extractsolution tank, pump, evaporator, condenser, and recycled solvent tank. The presence of the second extraction vessel among the equipment permits the simulation of a continuous process: while one of the vessels is under operation, the other one goes through the cleaning and recharging processes. For the simulation of the combined UAE + ABE extraction, in accordance with a possible setup of an ultrasound extraction reactor described by Vinatoru (2001), it was assumed that ultrasonic transducers are bonded to tank external walls. Then, during the first 10 min, the raw material stays immersed in the solvent inside an agitated tank receiving the ultrasound treatment; after that, there is 2 h of agitation without ultrasound irradiation (as in a conventional extraction by agitation). For simulating UAE, the same process was assumed; however, the ultrasound treatment was applied during the entire process time with an intermittent agitation, just for the purpose of homogeneity. Moreover, the excessive energy dissipation in the form of heat may lead to the degradation of the substrate and hence requires cooling; for this reason, the final temperature in the cooling operation step was 30 °C. To simulate soxhlet extraction, some modifications were made to the process, as shown in Fig. 2. The process also consisted of placing a known mass of jabuticaba skins immersed in a known volume of solvent inside an agitated tank, and the solution is heated to 78 °C. The presence of a condenser connected to the extractor simulates the condensation step of solvent extraction

Fig. 1. Flowchart of Agitated Bed Extraction (ABE) developed in SuperPro DesignerÒ.

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and its reflux to the extractor. The reflux operation is exhaustedly repeated.

2.5. Economical evaluation The estimation of the cost of manufacturing (COM) was done for the crude extract, the phenolic compounds fraction and the anthocyanin-rich fraction for the extracts obtained by UAE, UAE + ABE, ABE, and soxhlet (acidified solvent or not). The main costs that compose the COM are similar to the ones described by Turton et al. (2003), which are given by total capital investment cost and operating cost. The total capital investment cost represents the fixed capital investment (FCI), working capital and start-up cost. The first one involves expenses with equipment, installation, territorial taxes, engineering, etc., while the second one represents operating liquidity available to a business, and finally, the start-up cost is associated with the beginning of operation and the validation of the process. The operating cost

represents direct costs that are directly dependent on the production rate; it is composed of the cost of raw materials (CRM), the cost of the lost solvent during the process, utilities cost (CUT), which represents the demand for steam and cooling water required for the evaporator and condenser, electricity, and operational labor cost (COL). According to Pereira and Meireles (2007) the estimation of the COM for the phenolic compound fraction and anthocyanin-rich fraction was done by taking into account that the percentage of these fractions in the extracts can affect their specific cost.

2.6. Scale-up The scale-up procedure assumed that the industrial scale unit has the same performance as the laboratorial scale unit when the solvent to feed ratio between the mass of solid and solvent are kept constant (S/F), as well the true density of substrate and operational conditions. The process was designed to run 7920 h per year,

Fig. 2. Flowchart of soxhlet extraction developed in SuperPro DesignerÒ.

Table 1 Operational data input used to estimate COM by software for UAE, ABE, UAE + ABE, soxhlet and soxhlet_pH3. ExtractionProcess

S/F

Temperature (°C)

Time (min)

Global yield (%)

ABE UAE + ABE UAE Soxhlet Soxhlet_pH 3

1/10 1/10 1/10 1/10 1/10

25 25 25 78 78

120 130 120 480 480

9.01 10.08 11.93 9.92 9.5

Raw material cost1 (US$/kg) Ethanol cost (US$/kg) Operational labor cost (US$/h) Electricity cost2 (US$/kWh) 1 2

CEASA, Campinas, Brazil. CPFL, 2008.

1.80 0.65 6 0.092

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D.T. Santos et al. / Journal of Food Engineering 101 (2010) 23–31

which corresponds to 330 days per year with continuous 24-h per day shifts. The technical information used to estimate the COM of the processes is shown in Table 1. This study considered an industrial setup with extractors of 0.05, 0.1 and 0.3 m3. The amount of jabuticaba skins used per industrial batch immersed in ethanol was determined for each capacity. The solvent loss was considered to be 10% of the total ethanol involved in the process. The true density of the vegetable material was 1450.5 kg/m3. The number of ultrasonic transducers needed for each capacity in the UAE and UAE + ABE processes and, consequently, their price was estimated using information provided by Unique Group (Indaiatuba, Brazil).

3. Results and discussion 3.1. Antioxidant activity Fig. 3 shows the antioxidant activities of the extracts. Although the antioxidant activities of all extracts were superior to the antioxidant activity of b-carotene (control), large differences were observed among them. Even though the solvent (ethanol) and the process time duration (2 h) used in ultrasound assisted and agitated bed extraction were the same, the antioxidant activities of the extracts were different, demonstrating that the extraction method employed is an important parameter. Similar behavior was observed by Corrales et al. (2008) when using different extraction methods (ultrasound, high hydrostatic pressured and pulsed electric fields assisted extractions), with the best antioxidant activity attributed to the extract obtained using pulsed electric fields. Fig. 3 shows that the soxhlet extraction using ethanol and acidified ethanol (pH 3),and the combined UAE + ABE process using ethanol resulted in extracts with the highest antioxidant activities; their deviation was less than the experimental error. It can also be noted that recognized synthetic BHT and the natural antioxidant quercetin presented higher antioxidant activities than all extracts. In the case of soxhlet extraction, most of the time the extraction time is very long in comparison with all the other techniques (Wilga et al., 2007), resulting in a high consumption of energy and therefore higher cost. In this work an extraction time of 8 h was used for soxhlet extraction, which resulted in an extract with AA similar to the extract obtained using combined UAE + ABE.

To completely understand the results, studying the composition of each extract is necessary because in general different extraction methods introduce different combinations of phenolic compounds and anthocyanins into the extract, resulting in different bioactivities (Dai et al., 2009).

3.2. Phenolic compounds Fig. 4 shows the extraction of phenolic compounds using different extraction methods expressed as milligrams of gallic acid equivalents (GAE)/g dry material (left y-axis) and their respective extraction yield (j) expressed as g extract/g wet material100 (%) (right y-axis). Reynertson et al. (2008) investigated the total phenolic content of jabuticaba fruit and found the value of 31.6 mg of GAE/g of dry fruit. Taking into account that the major part of the fruit is pulp and its water content is very high, by approximation, the previous value becomes little higher than 31.6 mg of GAE/g of dry skin, corroborating the results presented in Fig. 4. Because no further effective comparison can be done with the results presented and the results obtained by Reynertson et al. (2008) in terms of which phenolic compound extraction yields, the best methods, among the ones studied in this paper, were also the three that resulted in extracts with the highest antioxidant activities. The correlation between the phenolic compound extraction yields and the antioxidant activities of the extracts was explored. A high and significant (r = 0.88) correlation was found between them. This positive correlation indicates that higher values of antioxidant activities are related to higher yields of phenolic compounds. Dragovic´-uzelac et al. (2007) observed the same direct correlation between phenolic content and antioxidant capacity of several fruit extracts. Two different methods were used for measuring the antioxidant capacity, ABTS [2,2-azinobis(3-ethylbenzothiazoline6-sulfonic acid) radical cation assay] and ORAC (oxygen radical absorbance capacity), which also resulted in a high and significant correlation between them, where r = 0.99 and r = 0.84, respectively. It can be observed in Fig. 4 that the combined UAE + ABE process resulted in phenolic compound yields higher than that obtained by the UAE or ABE process and similar to that obtained by soxhlet extraction. The extraction yields did not correlate strongly

100

Antioxidant Activity (%)

90 80 70 60 50 40 30 20 0

30

60

90 Time (min)

120

150

180

UAE

ABE

Combined UAE + ABE

Soxhlet (Ethanol)

Soxhlet (Ethanol pH 3)

BHT

Quercetin Fig. 3. Antioxidant Activity of jabuticaba skin extracts obtained using different methods (Expressed as % of inhibition of oxidation).

40

16

35

14

30

12

25

10

20

8

15

6

10

4

5

2

Extraction Yield (%)

D.T. Santos et al. / Journal of Food Engineering 101 (2010) 23–31

mg GAE / g dry material

28

0

0 UAE

ABE

Combined UAE + ABE

Soxhlet (Ethanol)

Soxhlet (Ethanol pH 3)

Fig. 4. Extraction of phenolic compounds (bars/left y-axis) using different extraction methods (mg GAE/g dry material) and their respective yield extraction (%) (j/right y-axis).

3.3. Anthocyanin yields

mg Cy-3-Glycoside / g dry material

Anthocyanins are one of the most attractive plant phenolic pigments of the group of flavonoids (Gould and Lee, 2002). Some fac-

tors, such as pH, temperature, light, oxygen, metals, etc., may limit the usage of anthocyanins as food colorants. As seen in Fig. 5, the method least aggressive to the anthocyanin recovery was ABE due to the use of low temperature. It has been reported that elevated temperature improves extraction efficiency due to the enhanced solubility and diffusion rate of compounds into the solvent (Dai et al., 2009). However, as may have occurred in anthocyanin soxhlet extraction, the high temperature employed accelerates anthocyanin degradation in the extraction process. This phenomenon was well studied by Ahmed et al. (2004). Although thermal degradation occurred during anthocyanin soxhlet extraction using acidified ethanol, probably due to the solvent acidification, more anthocyanins were extracted or the anthocyanins extracted maintained stable (reducing the degradation), or both of these factors affected the yield. According to Sadilova et al. (2007), to obtain a high yield of anthocyanins in the extract, solvents are usually mildly acidified to facilitate liberation and solubilization of anthocyanins from the fruit tissue and to stabilize anthocyanins as well because the highest stability of these compounds is achieved in an acidic medium. Some articles have demonstrated that low pH (around pH = 3) of the extracting solvent can prevent the oxidation of phenolics and may preserve the anthocyanin stability (Ruenroengklin et al., 2008), and thus the solvent was acidified to pH 3. In agreement with these papers, in Figs. 4 and 5, respectively, it can be observed

7

16

6

14 12

5

10 4 8 3 6 2

4

1

Extraction Yield (%)

with the phenolic compound content (r = 0.18), suggesting that the extraction methods employed extract phenolic and non-phenolic compounds simultaneously, with UAE being less selective to phenolic compounds. This is in contrast to the results of Chen et al. (2007), where UAE, in general, could increase the extraction yield of targeted compounds. Thus, in extracting complex mixtures from biological sources, other operating parameters must be taken into account to narrow or to broaden the range of selectivity of the process. In relation to the benefic effect of the ultrasonic treatment (10 min), a theory can be proposed based on the fact that ultrasonic irradiation facilitates the release of extractable compounds and enhances mass transport of the solvent from the continuous phase into plant cells of target compounds (Lee and Row, 2006), mainly during the initial minutes (Zhang et al., 2009). Hence, it is possible that during the 10 min of ultrasonic treatment, a high release of phenolic compounds occurs, or, at least, as these compounds are enclosed in plant cell structures with complicated access (Corrales et al., 2009), the frequency of ultrasound could break down the sample micelle or matrix, facilitating the second step, that is, the 2 h of ABE.

2 0

0 UAE

ABE

Combined UAE + ABE

Soxhlet (Ethanol)

Soxhlet (Ethanol pH 3)

Fig. 5. Extraction of total anthocyanins (bars/left y-axis) using different extraction methods (mg Cy-3-Glycoside/g dry material) and their respective yield extraction (%) (j/right y-axis).

D.T. Santos et al. / Journal of Food Engineering 101 (2010) 23–31

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that the mentioned acidified solvent prevented phenolic compound oxidation and inhibited the anthocyanin degradation. In the case of UAE and UAE + ABE, the operational conditions may have promoted the degradation of certain anthocyanins due to the ultrasonic frequency used because these compounds are highly sensitive. In spite of the fact that some amount of anthocyanins degraded even during the short ultrasonic treatment, this method continues to be a potential extraction procedure for antioxidant compound recovery from jabuticaba skin. No strong correlation between anthocyanin content and extract antioxidant activity (r = 0.40) was observed, possibly because the anthocyanins make up only a portion of the overall phenolic profile. This result suggests that the antioxidant activity of these extracts is mainly due to the other phenolic compounds not identified in this work and their complicated synergy. 3.4. Anthocyanin identification To identify the anthocyanins extracted by different methods, the jabuticaba skin extracts were fractionated by thin-layer chromatography (TLC). Fig. 6 shows that, independent of the method and condition (acidified solvent or not), the anthocyanin profiles are similar. The phytochemistry of jabuticaba skin has not been extensively reported in the literature; it has been reported to contain tannins (Morton, 1987), cyanidin 3-glucoside (CYA) (Einbond et al., 2004), delphinidin-3-glucoside (DEL) (Terci, 2004), peonidin 3-glucoside (PEO) and its aglycone (Trevisan et al., 1972). At the moment in which TLC (Fig. 6) was visualized, two colored bands situated at the same height of CYA and DEL standard bands were detected, possibly indicating the presence of these anthocyanins in the extracts. 4. Economic evaluation The COM obtained for the extract from jabuticaba skins extraction in UAE, ABE, UAE + ABE, and soxhlet (acidified solvent or not) processes for extractor capacities of 0.05, 0.1 and 0.3 m3 can be observed in Fig. 7. In general, it can be observed that the COM is inversely proportional to the extractor capacity. When the extractor capacity is raised, the COM diminishes, representing an advantage for the investment on a large industrial scale. For all extraction processes,

Fig. 6. Anthocyanin identification of extracts obtained using different extraction methods by TLC (Sox, soxhlet; pH 3, soxhlet pH 3; ABE, Agitated Bed Extraction; UAE, Ultrasound Assisted Extraction; CYA, Cyanidin-3-Glucoside; DEL, Delphinidin3-Glucoside; PEO, Peonidin-3-Glucoside; MAL, Malvidin-3-Glucoside; PEL, Pelargonidin-3-Glucoside Standard; PET, Petunidin-3-Glucoside).

Fig. 7. Impact of extractor capacity on the extract’s COM obtained by different methods.

the COM decreased between 67% and 50% when the extractor capacity was raised from 0.05 to 0.1 m3 and between 50% and 25% when it was raised from 0.05 to 0.3 m3. Table 2 shows the variation of the COM estimated for the crude extract, the phenolic compound and anthocyanin fractions with extractor capacity. The soxhlet extraction, using acidified ethanol or not, presented higher COM of all extraction methods used from US$ 1001.00/kg to US$ 778.50/kg for 0.3 m3, probably due to its extraction time (8 h). The use of acidified ethanol increased the COM; nonetheless, this promoted an increase of phenolic compound and anthocyanin recovery. For the phenolic compound fraction, the COM ranged from US$ 10.00/g to US$ 2.65/g for the soxhlet process using acidified ethanol and US$ 9.23/g and US$ 2.38/g for soxhlet with ethanol, for 0.05 and 0.3 m3 of capacity; and for anthocyanin fraction, the COM varied between US$ 33.2/ g and US$ 8.8/g for the soxhlet process using acidified ethanol and US$ 60.4/g and US$ 15.57/g for soxhlet with ethanol, also for 0.05 and 0.3 m3 of capacity. These results show that the variation of the percentage of phenolic compound and anthocyanin fraction in the extracts directly affects the specific cost. It was observed that extracts with higher amounts of each fraction have lower specific COM. These results corroborate those found in previous studies done by our research group (Pereira and Meireles, 2007). In spite of the fact that soxhlet extraction using acidified ethanol is more efficient for obtaining phenolic compounds among these processes, it is not attractive in terms of cost when compared with UAE + ABE, even though these processes have similar global yield (9.5% and 10.08% for soxhlet and UAE + ABE, respectively) and low difference in phenolic compound concentration. This can be explained mainly due to the fact that the soxhlet processes is time-consuming, leading to a high expense of energy and making the process economically expensive. On the other hand, when comparing UAE, ABE, UAE + ABE, in terms of the COM estimated for the extract and for the phenolic compounds fraction, these processes showed obvious advantages mainly due to the short duration of these extraction methods (approximately 2 h). Among the three processes, the UAE + ABE presented the smallest COM for the extract and for the phenolic compounds fraction. When 10 min of ultrasonic treatment was applied as pretreatment, the amount of phenolic compounds recovered by ABE was enhanced as well as the global yield of the process. Thus, in terms of economics the combined UAE + ABE process seems to be the best extraction method for phenolic compound recovery. In contrast, UAE + ABE presented the second smallest COM for the anthocyanin fraction, with the ABE process being the best extraction method for anthocyanin recovery in terms of economics. In this context, a comparison of ABE and UAE + ABE costs was done in more detail, considering all components (CUT, CRM, FCI

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Table 2 COM for extract´s global yield, fraction of phenolic compounds and anthocyanins in the extracts estimated for UAE, ABE, UAE + ABE, soxhlet and soxhlet_pH3.

UAE

ABE

UAE + ABE

Soxhlet

Soxhlet_pH3

Extractor capacity (m3)

Global yield (%)

0.05 0.10 0.30

11.93

794.46 530.22 401.21

26.335

0.05 0.10 0.30

9.01

1016.88 666.50 422.18

0.05 0.10 0.30

10.08

0.05 0.10 0.30 0.05 0.10 0.30

COM for crude extract (US$/kg)

Content of phenolic compounds (mg/g)

Content of anthocyanins (mg/g)

COM for anthocyanin fraction (USS/g)

3.70 2.46 1.86

3.673

26.50 17.67 13.37

24.103

3.80 2.50 1.58

6.168

14.86 9.74 6.17

932.95 614.28 387.20

33.977

2.77 1.80 1.15

5.339

17.60 11.60 7.30

9.92

3020.00 1800.00 778.42

32.433

9.23 5.50 2.38

4.993

60.40 36.00 15.57

9.50

3776.00 1862.00 1001.00

35.845

10.00 4.94 2.65

5.416

33.20 16.36 8.80

Fig. 8. Contribution of each component in COM of extract obtained by combined UAE+ABE.

and COL) that compose the COM. However, when one takes into account the fractions of the costs, the difference of 1% between these processes did not cause a notable variation; thus, the fraction of COM is presented just for UAE + ABE. Knowing that the contribution of each COM component is different for each extractor capacity, in Fig. 8 it is possible to observe an increase in the fraction of utilities cost (CUT) and raw materials cost (CRM) due to the higher demand as the extractor capacity increases. It is interesting to notice that the CRM did not represent a large fraction of the COM due to its low price. Because that price per kilogram of skins was considered by taking into account the price of the total fruit, including pulp, skin, and seed (US$ 1.8/kg of fruit), this price could be even smaller if the skins were the residue from the production of jabuticaba wine, jabuticaba pulp, etc. On the other hand, the fraction of operational labor (COL) and total capital investment (FCI) diminished with increased capacity; this behavior is due to the increase in extract production leading to the dilution of costs. FCI represents the major fraction of the manufacturing cost for the capacities of the three extractors, ranging between 57.7% and 66.7% of the COM. This behavior is mainly due to the fraction of TDPC (total plant direct cost), which involves the expenses related equipment and contributes to the cost increase. In terms of the FCI of ABE and UAE + ABE, a very small difference (between 0.34% and 1.2%) for all extractor capacities was verified and is due to the ultrasonic transducers added to this process; thus, it is correct to say that the additional cost was insignificant, and a normal industrial agitated extraction tank can be used in a process that permits intermittent ultrasonic irradiation. Otherwise, it was

COM for phenolic compounds fraction (US$/g)

also observed in Table 2 that this treatment decreased the cost of the extract significantly from US$ 422.18/kg to US$ 387.20/kg, despite the increase in processing cost. In addition, this product is not available in the market. On the other hand, one can find glycolic extract of jabuticaba, which costs US$ 9.90/kg (Sarfarm, São Paulo, Brazil). A better comparison would require information about the concentration of anthocyanin in glycolic extract, an information that is not available. In our work, the UAE + ABE process gave US$ 387.20/kg for crude extract and US$ 7.3/g for anthocyanin. Although this price is higher, the processes and the solvent used were different. Therefore, in agreement with this result, Zhang et al. (2009) have proposed that a short time of ultrasound treatment is not cost or time-consuming. Furthermore, the results presented in this paper corroborate the studies that ultrasonic irradiation can assist solvent extraction, improving the efficiency of the extraction (Vinatoru, 2001). Moreover, implementation of ultrasonic treatment in the conventional extraction may improve commercial extract production without considerably increasing the costs, and for scale-up purposes ultrasound seems to be a viable option, particularly when used for shorter processing times before a conventional solvent extraction (Paniwnyk et al., 2009).

5. Conclusion An efficient extraction procedure for antioxidant compound recovery from jabuticaba skin was presented: a combined short ultrasound assisted extraction of 10 min was used as a pre-treatment before conventional solvent extraction under agitation (UAE + ABE). This method maximized the phenolic compound extraction with acceptable anthocyanin degradation, and it is also the best option in terms of economics, with respect to the recovery of AA and phenolic compounds, among the other methods studied in this paper: ultrasound assisted, agitation bed and soxhlet extraction with solvent that was acidified or not. Nonetheless, when contents of anthocyanins are considered, the selected process favors ABE. In agreement with the literature, the antioxidant activity of these extracts was not mainly due to the anthocyanin content, but due to the other compounds not identified in this work and their complicated synergy. Two types of anthocyanins (cyanidin3-glucoside and delphinidin-3-glucoside) were identified in all extracts independently of the extraction methodology, corroborating previous works.

D.T. Santos et al. / Journal of Food Engineering 101 (2010) 23–31

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