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Influence of the Bed Height on the Kinetics of Watermelon Seed
  Influence of the bed height on the kinetics of watermelon seedoil extraction with pressurized ethanol  J. Colivet a,b , A.L. Oliveira a , R.A. Carvalho a, ⇑ a Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, FZEA/USP, Caixa Postal 23, CEP: 13635-900, Pirassununga, São Paulo, Brazil b Escuela de Zootecnia y Tecnologia de Alimentos, Universidad de Oriente, Campus Los Guaritos, código postal 6201, Maturín, Monagas, Venezuela a r t i c l e i n f o  Article history: Received 15 April 2016Received in revised form 3 June 2016Accepted 6 June 2016Available online 7 June 2016 Keywords: OilExtraction Citrullus lanatus Green processPolyphenols a b s t r a c t The influence of the bed volume on the kinetics of watermelon seed oil extraction was evaluated in thisstudy. Watermelon seed oil was extracted using different extraction cells (S 1  =34mL, S 2  =66mL andS 3  =100mL) at different temperatures (40, 60 and 80  C) using the sample mass/solvent volume ratioas the fixed sizing criteria (w/s=0.30). The extraction kinetics were mathematically described usingthe Peleg, Fick and second-order models. Samples were extracted in batches using pressurized liquidextraction (PLE) with ethanol as the solvent for different extraction times. Oil extraction yields rangedfrom24.69 to 37.21g oil/100g of seeds, and the concentration of total phenolic compounds ranged from2.44 to 3.84mg of gallic acid equivalents (GAE)/g of seeds. All kinetic models showed a good fit to theexperimental data, but the second-order model better predicted the behavior of data, with high coeffi-cientofdetermination(R  2ajd )andlowroot-mean-squaredeviation(RMSD)values.Thedifferentextractioncells did not affect the total extraction yield, but affected the extraction parameters obtained in themodels. The effective diffusivities were dependent on temperature and ranged from 9.10  10  6 to2.07  10  5 m 2 /s. The activation energy ranged from 11.43 to 18.54J/mol.   2016 Elsevier B.V. All rights reserved. 1. Introduction Watermelonis a freshfruit consumedworldwide andis usedinthe productionof juices, jellies, marmalades, sauces and salads [1].Inaddition, watermelonhas beenwidelyusedas a medicinal plantin African and Asian cultures due to the presence of severalcompounds with phytochemical activities [2]. Watermelon seedshave strong antioxidant activity [3,4], and due to their diuretic and purgative activity, have also been used in the treatment of gastrointestinal diseases [5], urinary infections [6], gonorrhea, leukorrhea [7] and prostatic hyperplasia [8]. Watermelon seed oils are classified as high quality oils due to the presence of   x -3 and x -6 fatty acids [9] and phenolic compounds such as gallic,protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic,p-coumaric and ferulic acids [10]. The processing of the water-melon fruit yields a large amount of seeds, which are usually trea-ted as waste. The use of these seeds to produce oil and otherfunctional ingredients will add value to this waste produce thatcurrently has no specific use, so that it can become a raw materialused for the generation of a product with active compounds.The extractionandpurificationof activecompoundsfromnatu-ral sources are important steps in the production of phytochemi-cals for use in food supplements or nutraceuticals, functionalfoods and pharmaceuticals [11]. In industry, oil extraction pro-cesses are based on conventional methods that involve the use of organic solvents which are heated at atmospheric pressure condi-tions, and production can also occur via mechanical processingsuch as pressing. High-pressure extraction processes, such as pres-surized liquid extraction (PLE) and extraction with supercriticalCO 2 , are extraction techniques that are becoming more prominentdue to their ability to obtain specific target molecules and reducedloss of solvents during the production process [12]. PLE is a tech-nique that has emerged as an alternative to conventional extrac-tion methods, such as maceration, percolation or reflux, as itoffersadvantagesforparametersincludingextractiontime,solventconsumption, extraction yield and reproducibility [13]. PLE usesorganic solvents at elevated pressure and temperature in order toincrease the extraction process efficiency, reduce viscosity andimprove solvent penetration and diffusivity, thereby reducingextraction times and avoiding possible thermal degradation[13,14]. PLE can be used for the extraction of polar compoundssuch as polyphenols, however, this is a little studied techniquewhich still requires more research before its implementation ona larger scale [12]. An advantage of PLE is the possibility of using   2016 Elsevier B.V. All rights reserved. ⇑ Corresponding author. E-mail address: (R.A. Carvalho).Separation and Purification Technology 169 (2016) 187–195 Contents lists available at ScienceDirect Separation and Purification Technology journal homepage:  any solvent for the fractionationof phytochemicals. An example of this is the ability to perform successive extraction steps in thesame sample using the same or different solvent for each extrac-tionstep, whichyieldsavarietyofcompoundswithdifferentprop-erties. Another advantage of using PLE is that it significantlymodifiestheproperties oftheextractionsolvents, offeringthepos-sibility of using polar solvents for the extraction of compoundswith hydrophobic characteristics (e.g. lipids, pigments and vita-mins). Pronyk and Mazza [15] reported that pressurized liquidshave high versatility due to the physicochemical properties of the solvent, as the density, diffusivity and dielectric constant canbe controlled by varying the pressure and extraction temperature,therefore,changingthesepropertiescaneffectivelycontrolthesol-vation power and selectivity of solvents.Considering the advantages of using PLE as an extraction tech-nique, it is important to carry out studies to establish equipmentdesigncriteria, inadditiontotheindustrialandpilotscale, inorderto obtain the information required for its implementation. Thedesign criteria takes into account the extraction processes andtheeffectoftheextractionbedgeometryonthekineticparametersthat define the process profile used, and this information can beused in relation to the physicochemical properties of the productsobtained. Thus, the aim of this study was to investigate the influ-ence of the bed height on the kinetics of watermelon seed oilextraction with pressurized ethanol at different temperatures,through the use of mathematical models that describe the extrac-tion process. In addition, we also studied the effect of bed heightandtemperatureonthekineticparameters,totalcontentofpheno-lic compounds and antiradical capacity of the oil obtained. 2. Materials and methods  2.1. Sample preparation Watermelons of the Ruby variety were purchased from a localmarket in the city of Pirassununga (Sao Paulo, Brazil). The seedswere manually extracted by slicing the watermelons with a stain-less steel knife, after which the juice was extracted from the pulp.The seeds were separated from the residue using a sedimentation-flotationsysteminplasticcontainerswithpotablewater.Theseedswere then dried at 60  C for 24h, after which they were groundusing a Wiley mill (Thomas Scientific, Philadelphia, PA, USA) witha 1mm sieve. Finally, the powder (1% moisture) was packagedand stored under refrigeration at 5  C [16].  2.2. Pressurized liquid extraction Extractions were performed using an accelerated solventextraction system, ASE 150 (Dionex, Sunnyvale, CA, USA). Extrac-tions were performed at three different temperatures (40, 60 and80  C), using three cylindrical cells (S 1 , S 2  and S 3 ), keeping themass/solvent ratio constant (0.30) over 8 extraction cycles withtotal solvent renovation at different times (3, 6, 12, 18, 24, 30, 36and 42min). The volume of solvent used in each extraction cycle,wasapproximately31,52and78mLforcellsS1,S2andS3,respec-tively.Inthisstudy, ethanolwasselectedasitis a‘generallyrecog-nized as safe’ (GRAS) solvent [17,18].Initially, the fixed bed was packed with ground sample, thensolvent was placed into the cell and the system pressure was con-trolled at 1500psia (102.4atm). Temperature conditions wereadjustedaccordingtoexperimentalconditions.N 2  wasusedtodis-chargethecellsolvent,andthesystemwasfinallydepressurizedinorder to avoid the presence of the remaining extract in the cell.After extraction, the cell was flushed thoroughly before the nextextraction cycle. A rotary evaporator (model IKA RV 05 IKA; IKA-Werke, Staufen, Germany) was used to evaporate the solvent fromthe extract at 40  C, and the yield was determined as the ratiobetween the extract obtained from the seed mass present in thefixed bed extractor.  2.3. Total phenolic content  The phenolic content was determined using the Folin-Ciocalteau reagent [19]. An aliquot of extract sample was mixedwith distilled water (2mL) and 1mL of Folin-Ciocalteu reagent.After3min,2mLofsodiumcarbonate(20%)wasaddedandstirredwithavortex.Solutionswerestoredatroomtemperaturefor1hinthedarkandtheabsorbancewasdeterminedat760nm.Gallicacid(diluted in ethanol) was used as a standard solution for the prepa-ration of the calibration curve at concentrations from 0 to 80mg/L (R  2 =0.997). Results were expressed as mg of gallic acid equiva-lents (GAE) per gram of seed. The quantification of total phenolicswas performed in triplicate.  2.4. Antiradical activity The antioxidant activity was determined using the 2,2-diphenyl-l-picrylhydrazyl (DPPH  ) reagent as the free radical [20].For each extract obtained, different concentrations were tested Nomenclature  A  average yield of oil extracted in the washing step(g oil/100 g db )  A n ,  B n  model-fitting parameters (  A n , dimensionless;  B n , s  1 ) B 0  extraction rate at the beginning of extraction ( t   =  t  o ) C  0  yield of oil extracted in the washing step (g oil/100 g db ) C  s  extraction capacity at equilibrium (g oil/100 g db ) C  sp  extraction capacity of the Peleg model at equilibrium (goil/100 g db ) C  t   extraction yield at any time (g oil/100 g db ) D 0  pre-exponential diffusion constant (m 2 /s) D e  effective diffusivity coefficient (m 2 /s) IC  50  antiradical activity (mg/L) h  initial extraction rate for the second-order model(g/100 g db  minute) k  second-order extraction rate (100 g db /g oil minute) K  1  constant of the Peleg model (min 100 g db /g oil) K  2  constant capacity of the Peleg model (100 g db /g oil) R  p  average radius (m) R  g   gas constant (J/mol K) T   temperature (  C or K) t   extraction time (min) D E  a  activation energy (J/mol)  Abbreviations Abs absorbance to 765 nmDPPH 2,2-diphenyl-1-picrylhydrazyl radicalGAE gallic acid equivalentsGRAS generally recognized as safePLE pressurized liquid extractionRMSD root-mean-square deviationS 1  34 mL extraction cell (5.2  2.8 cm)S 2  66 mL extraction cell (9.8  2.8 cm)S 3  100 mL extraction cell (15.8  2.8 cm) 188  J. Colivet et al./Separation and Purification Technology 169 (2016) 187–195  (in mg extract/L of solution). An aliquot (1mL) of each of theextracts was added to 3.0mL of an ethanolic DPPH  solution(40mg/L, diluted in ethanol). The decrease in absorbance wasdeterminedat517nmafter90min.TheexactDPPH  concentration(C DPPH  ) in the reaction medium was calculated using a calibrationcurve Abs 517nm  ¼ 0 : 0227C DPPH  þ 0 : 13 (R  2 =0.998), obtained by thedilution of DPPH  in ethanol to concentrations ranging from 31 to8ppm. The antiradical activity was defined as the amount of extract required to reduce the DPPH  concentration by 50% (IC 50 ,mg/L).  2.5. Kinetic models In the modeling study of extraction kinetics, three mathemati-cal models were used (Table 1). The second-order model (Table 1) describes the diffusion rate of oil present in the ground water-melon seeds. In this model,  k  is the second-order extraction rateexpressed as 100g of seeds/g oil min,  C  s  is the extraction capacityand  C  t   is the oil concentration at time  t   (minutes). Note that theexperimental data for oil concentrations are expressed in g of oilper 100g of seeds.Considering the limiting conditions from  t   =0 to  t   and  C  t   =0 to C  t  , the second-order model may be transformed into Eq. (1), whichbecomes Eq. (2) when linearized. C  t   ¼  C  2 s kt  1 þ  C  s kt   ð 1 Þ t C  t  ¼  1 kC  2 s þ  t C  s ð 2 Þ In the arrangement of Eq. (2), the extraction rate can be deter-mined according to Eq. (3). C  t  t   ¼  1 1 kC  2 s   þ  t C  s    ð 3 Þ The initial extraction rate ( h ), with  C  t  / t   at a time of approxi-mately 0, is defined in Eq. (4). h  ¼  kC  2 s  ð 4 Þ Similarly, the oil concentration at any time can be calculatedaccording to Eq. (5). C  t   ¼  t  1 h   þ  t C  s    ð 5 Þ All parameters of the second-order extraction model weredetermined by the intersection of the line and the slope of the C  t  / t   vs.  t   graph.Another model tested was the modification of Fick’s law(Table 1) [21]. In this model,  C  t   and  C  s  are the extraction yields attime  t   and at equilibrium, and are both expressed as g oil/100gof solids (ground seeds).  A  and  B 1  are model coefficients where  A is associated with the average concentration of oil extracted fromthe surface of particles that are dragged during extraction (wash-ing), described by Eq. (6). The value of   A 1  when a spherical surfaceis adopted is described by Eq. (7) [22].  A  ¼  1   C  0 C  s    A 1    e B 1 t  0 ð 6 Þ  A 1  ¼  6 p 2  ð 7 Þ The  B 1  coefficient is associated with the effective diffusivity,defined in Eq. (8) B 1  ¼  D e p 2 R 2  p ð 8 Þ R  p  is the average particle radius (m) and  D e  is the effective diffusiv-ity coefficient (m 2 /s) for the experimentally determined  B 1  valuesafter linearization of the Fick model (Table 1).Finally, the Peleg model was also applied to predict the water-melon seed oil extraction behavior. This model was used byBucic´-Kojic´ et al. [23] in polyphenol extraction experiments witha good fit to the experimental data. In this model,  C  t   is the oil con-centrationattime t  , K  1  isthePelegconstantratio(min100gseeds/g of oil) and  K  2  is the Peleg capacity constant (100g seeds/g oil).Constant  K  1  is related with extraction rate  B 0  at the beginning of extraction, ( t   =0), as shown in Eq. (9). B 0  ¼  1 K  1 g of oil = 100 g of seed min ð Þ ð 9 Þ The Peleg capacity constant  K  2  is related to the maximumextraction yield, defined in Eq. (10). C  sp  ¼  1 K  2 g of oil = 100 g of seed ð Þ ð 10 Þ  2.6. Statistical methods To evaluate the effect of the extraction cell and temperature, arandomizedblock design for two factors (extraction cells and tem-perature) was used. Analysis of variance (ANOVA) followed byTukey’s test were performed to determine the effect of these fac-tors on the dependent variable at a 95% significance level usingthe Statistica Software 10.0 (Stat Soft Inc., USA). All experimentswere performed in triplicate.The mathematical models for the study parameters were deter-minedbyfittingthe experimental datausingnon-linear regressionby the Levenberg Marquardt method [24] using the StatgraphicsCenturion XVI software (Statpoint Technology Inc., USA). Theagreement between the experimental data and calculated valueswere determined by the determination coefficient ( R 2 adj ) and theroot-mean-square deviation (RMSD), according to Eq. (11). RMSD  ¼  ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 n X ni ¼ 1  experimental    calculated ð Þ 2 r   ð 11 Þ 3. Results and discussion  3.1. Total yield of oil and phenolic compounds from watermelon seeds The aim of this study was to compare the size of the extractionbed on oil extraction yield from watermelon seeds at differenttemperatures, using the sample mass/solvent volume ratio as thefixed sizing criteria. The different lengths of the fixed bed extrac-tors did not significantly affect the watermelon seed oil yield(p>0.05) (Table 2). The extraction yield was mostly affected bythe temperature at which the extraction was performed. Overall,the average extraction yields for all cells were 25.7, 30 and 37goil per 100g of seeds on a dry base for extraction temperaturesof 40, 60 and 80  C, respectively. The extraction yield values are  Table 1 Models used to describe the kinetics of watermelon seed oil extraction. Model Equation Parameters ReferenceSecond-order  dC  t  dt   ¼ k C  s  C  t  ð Þ 2 k , h  [44]Fick’s law  C  t  C  s ¼ 1   Ae  B 1 t   A ,  B 1  [21]Peleg  C  t   ¼  t K  1 þ K  2 t   K  1 , K  2  [23,45]  J. Colivet et al./Separation and Purification Technology 169 (2016) 187–195  189  comparable to the 32.16% yield previously reported by Conto et al.[25] from seeds of the ‘Top Gun’ watermelon variety, achievedusing Soxhlet extraction with hexane as the solvent. However,theextractionyieldvalueswerelowerthanthoseobtainedbyAcaret al. [3] (52.34%) and Rai et al. [1] (52.37%), who used the Soxhlet extraction and supercritical fluid methods, respectively. This vari-ation could be attributed to differences in the species of water-melon and differences in growth and geographical conditions,including soil properties and nutrients, water availability and sea-sonal temperature, in addition to other relevant factors [26].The concentration of phenolic compounds ranged from 2.4 to3.8mg GAE/g of the seeds, regardless of the different extractioncells (Table 2), and this yield is higher than values whichhave pre-viously been reported ( 6 2mg GAE/g of seed) [3,27,28]. However,when the effect of processing temperature on the concentrationof phenoliccompoundswas considered, wefoundthat the concen-trationofthesecompoundswashigherforextractiontemperaturesbelow 80  C, which could be related to thermal degradation of phenols by the effects of high temperatures and long extractiontimes [29].Kryzˇ evicˇ i  ute˙ et al. [30] suggested that the extraction tempera-ture can affect the content of phenolic compounds extracted frompomace raspberry using PLE. However, this performance maydependonthesolventused.Theuseofpressurizedhexaneresultedin higher yields when temperatures were above 90  C, however,the temperature did not significantly affect the results for pressur-ized methanol, although the highest total yield of phenolics wasobtained at temperatures below 70  C with a longer extractiontime (15min).  3.2. Antiradical activity The effect of extraction time on the antioxidant activity of watermelon seed oils is shown in Fig. 1(A–C). The analysis demon-strated that the different extraction cells did not affect the antiox-idant activity of oils obtained. However, extraction temperatureandtimehadasignificanteffectontheantioxidantactivityofcom-pounds in the oil extracts. Oil obtained at 60  C had a higherantioxidant activity compared to that obtained at temperaturesof 40 or 80  C (Fig. 1A–C). The oil obtained at 40  C showed higheractivity than that obtained at 80  C. Therefore, the antioxidantbehavior was found to be related to the extraction temperature,as lower temperatures produced decreased amounts of bioactivecompounds. According to Mustafa and Turner [31], high tempera- turesdecreasetheviscosityandincreasediffusivityoftheliquidbythe matrix, thus facilitating extraction, which was not observed at40  C.Forhighertemperatures,althoughthediffusivitywashigher,extraction at 80  C may cause degradation of compounds withantioxidant activity [29].After the matrix was submitted to successive extractions withpressurizedethanol, the antioxidant activitywas foundto increaseconsiderably for the 24 and 18min extractions at temperatures of 40 and 60  C, respectively (equivalent to 4 and 5 extraction cycles,respectively), and the activity was found to increase at a slowerrate up to 42min for the extraction temperatures of 40 and60  C. However, when the temperature used was 80  C, the maxi-mum antioxidant activity was found to occur after 12min of extraction (3rd cycle), which was reduced due to the higher heatexposure time. In PLE, static cycles are used to introduce the sol-vent when the solubility of target compounds is limited, whichfacilitates thepenetrationof thesolvent intothe matrix[32]. Thus,this study showed that using different static cycles improved theextraction capacity of the inhibitor compounds of the DPPH freeradical activity. Furthermore, according to Carabias-Martinezet al. [33], the application of several extraction cycles and theuseofethanolassolventhelpstoreducethepresenceofhydropho-bic compounds (e.g. resins) in the extracts, which interfere withthe subsequent extraction of compounds with phytochemicalactivity.  3.3. Extraction kinetics Fig.2showstheoilextractionyieldsforthedifferentcells(S 1 ,S 2 and S 3 ) at the different extraction temperatures tested (40, 60 and80  C). The experiments suggested that the extraction yields weredependent on time for all three extraction cells. We observed thatthe extraction rate was higher in the first 6min, which wasreduced until equilibrium was reached (  18min). This behavioris associated with the concentration gradient produced in the ini-tial extraction stage, where the solvent extracts components tothe surface of the matrix and begins to penetrate into the solid[34]. Accordingtopreviousstudies,therapidincreaseinextractionperformance in the initial extraction stage is mainly attributed towashing of extracted components located on the external surfaceof particles anddiffusion of the solutedissolved inside brokenpar-ticles [35–37]. However, the decrease in the extraction rate ismainly controlled by diffusion of oil into the particles [35–37].Moreover, whenthethreeextractioncells werecomparedtherewere no differences in the behavior of the process. In this study,the mass/solvent ratio was considered to be constant, indicatingthat this can be used for scaling up in PLE. According to Zabotet al. [38], assessment of the bed geometry in the extraction pro-cess is important for scaling an extraction process. Similar extrac-tion profiles should be obtained regardless of the geometriccharacteristicsofthebed. However,theappropriatescalingcriteriafor similar kinetic parameters must be chosen, in addition to mod-eling the process.  3.4. Mathematical models 3.4.1. Second-order model The second-order model showed a good fit to the experimentaldata (Fig. 2A), demonstrating that it is easily adjustable to extrac-tion processes with pressurized liquids (R  2adj  > 0.964). The parame-ters  C  s  (oil concentration at equilibrium) and  k  (extraction rateconstant) were determined by fitting the second-order model(Table 3) to the experimental data. The parameter  C  s  was used tomeasure the extraction efficiency compared to the total yieldobtained. Overall, these differences decreased with increasing tem-perature, which indicates that a more severe heat treatmentimproves the extraction efficiency, however, the extraction of com-pounds other than oil cannot be excluded. In relation to the constant  k  and extraction parameter  h  (initialextraction rate) we found that the values of these parametersincreasedwithtemperature,buttherewasnotalinearrelationship.  Table 2 Extraction yields of oils and phenolic compounds in watermelon seeds. Temperature (  C) Bed Yield (mean±SD) * Oil (g oil/100g db ) Total phenolic (mg GAE/g db )40 S 1  25.8±0.9 a 3.8±0.06gS 2  24.7±0.5 a 3.1±0.06 c.d.eS 3  26.6±1.1 a.b 3.4±0.07 f 60 S 1  28.7±1.4 b.c 3.0±0.04 c.dS 2  30.3±0.7 c 3.2±0.01 e.f S 3  31.1±0.6 c 3.0±0.07 b.c80 S 1  36.8±1.4 d 2.8±0.04 bS 2  37.0±0.8 d 2.4±0.04 aS 3  37.2±0.4 d 3.2±0.14 d.e.f  * Experimentswereperformedintriplicate. Themeanswithinacolumnfollowedby the same letter are not significantly different at the p=0.05 level. S 1  =34mL,S 2  =66mL, S 3  =100mL.190  J. Colivet et al./Separation and Purification Technology 169 (2016) 187–195
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