Phase behavior and process parameters effect on grape seed extract encapsulation by SEDS technique

Industrial Crops and Products 50 (2013) 352–360

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Phase behavior and process parameters effect on grape seed extract encapsulation by SEDS technique Daiane L. Boschetto a , Irede Dalmolin b , Alana M. de Cesaro c , Aline A. Rigo c , Sandra R.S. Ferreira a, M. Angela A. Meireles b, Eduardo A.C. Batista b, J. Vladimir Oliveira a,∗ a

Department of Chemical and Food Engineering, UFSC, Florianópolis, SC 88040-900, Brazil Department of Food Engineering, School of Food Engineering, UNICAMP, Campinas, SP 13083-862, Brazil c Department of Food Engineering, URI-Campus de Erechim, Erechim, RS 99700-000, Brazil b

a r t i c l e

i n f o

Article history: Received 9 May 2013 Received in revised form 6 July 2013 Accepted 25 July 2013 Keywords: SEDS technique Grape seed extract PHBV Supercritical carbon dioxide Phase equilibrium data

a b s t r a c t Grape seed extract (GSE) was encapsulated in biocompatible poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) using the solution enhanced dispersion by supercritical fluids (SEDS) technique. In order to help selecting the appropriate operating conditions for the encapsulation mechanism, the fluid phase behavior of pseudo-ternary carbon dioxide (CO2 ) + dichloromethane (DCM) + GSE systems was studied employing a static synthetic method. The investigated SEDS operating parameters were in the following range: pressure (8–12 MPa), temperature (308–318 K), GSE to PHBV mass ratio (1:1–1:3), with concentrations of organic solution formed by GSE (6.67–20.00 kg m−3 ) in DCM, and fixed PHBV 20.00 kg m−3 in DCM. The organic solution flow rate was fixed at 1.67 × 10−8 m3 s−1 and the antisolvent flow rate at 3.33 × 10−7 m3 s−1 . The effect of operating parameters on particle size, particle morphology and encapsulation efficiency was checked. The best result was obtained at 8 MPa, 308 K, GSE to PHBV mass ratio of 1:1, with concentration of GSE and PHBV, both of 20.00 kg m−3 , leading to spherical particles with the smallest size of ∼0.70 ␮m and encapsulation efficiency of 66.01%. SEM-EDS micrographs showed that, besides spherical particles, agglomerated particles were observed at temperatures higher than 308 K, which might be due to a more favorable extract re-dissolution at higher temperatures. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Grape seed is a residue of the wine fermentation and, in many countries such as Brazil, it is generally used for animal feed or discarded by burning. The use of residues and environmentally safe and benign technologies comprise a general research trend of scientific community, stimulated by government requirements and appeal to the consumers market which increasingly seek highquality natural products. An alternative destination to this residue may be the extraction of seed oil, diversifying and adding value to products of wine industry. Conventional extraction processes consist of obtaining vegetable oil from a solid matrix by mechanical pressing and/or organic solvent extraction. Traditionally, n-hexane has been employed, but in spite of the process being very efficient, possible thermal degradation of the oil and incomplete solvent elimination residue, from (500 to 1000) × 10–6 mg kg−1 , are the main drawbacks of such techniques (Reverchon and De Marco, 2006).

∗ Corresponding author. Tel.: +55 48 37212508; fax: +55 48 37219687. E-mail address: [email protected] (J. Vladimir Oliveira). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.07.044

Extraction of seed oils by supercritical carbon dioxide (SC-CO2 ) represents an attractive alternative process to the conventional organic solvent-based techniques commonly employed, in terms of extraction yield, improved selectivity and leaving essentially no toxic traces in the extracts and environment. The solvent CO2 is safe, readily available at low cost and, in addition, allows operation at relatively low pressures and near-room temperatures, which helps minimize the product degradation due to thermal treatment (Bravi et al., 2007). Grape seed extract (GSE) presents several benefits for human health due to the high content of unsaturated fatty acids and antioxidant compounds (Prado et al., 2012; Dalmolin et al., 2010; Passos et al., 2009, 2010; Prasain et al., 2009). Therefore, this product is commercialized as food and also for cosmetic and pharmaceutical applications. Vegetable extracts in its crude form may undergo chemical and sensorial changes due to conditions of storage and processing. Thus, microencapsulation offers protection to the extract, to prevent undesirable changes regarding physico-chemical and functional properties (Martín et al., 2010). The selection of solute, as well as the technique used for the precipitation and encapsulation may be relevant to the final product characteristics. The particle formation and encapsulation using traditional techniques (spray-drying,

D.L. Boschetto et al. / Industrial Crops and Products 50 (2013) 352–360

coacervation, freeze-drying, interfacial polymerization, etc.) can suffer some drawbacks like the poor control of particle size and morphology, degradation of thermo sensitive compounds, low encapsulation efficiency, and/or low yield (Franceschi et al., 2008a). The application of supercritical fluids as an alternative to these conventional processes has been an active field of research and innovation mainly motivated by the possibility of exploiting the peculiar properties of these fluids, in particular the SC-CO2 , the most used supercritical fluid for precipitation processes. Several precipitation processes based on supercritical fluids have been developed. These processes can be classified according to the role of the supercritical fluid in the process (Martín and Cocero, 2008; Jung and Perrut, 2001): solvent, antisolvent, co-solvent or solute, or even propellant gas. Our research group recently studied the precipitation of theophylline, ␤-carotene and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and the encapsulation of ␤-carotene in PHBV using the SEDS technique (Priamo et al., 2010, 2011; Franceschi et al., 2008b, 2009). Therefore, we selected the PHBV as coating material and the SEDS technique to carry out the GSE encapsulation. Application of pressurized fluids as an alternative to traditional precipitation and encapsulation together with the growing demand of the industrial sector for new technologies motivated this research. In this context, the objective of this work was to investigate the effect of processing parameters (pressure, temperature, GSE to PHBV mass ratio, with different concentrations of GSE in DCM) on the encapsulation of GSE using the SEDS technique, focusing on particle size, particle morphology and encapsulation efficiency. 2. Materials and methods 2.1. Materials Carbon dioxide (mass fraction purity 0.999 in the liquid phase) was supplied by White Martins S.A. (São Paulo, SP, Brazil). Dichloromethane (minimum mass fraction purity 0.995) and Ethanol (minimum mass fraction purity 0.999) were purchased from Merck (Darmstadt, HE, Germany) and used without any further treatment. The co-polymer poly(hydroxybutyrateco-hydroxyvalerate) (PHBV), with average molar mass (Mw) of 196,000 and polydispersity index of 1.85 (measured by GPC using a calibration curve obtained from polystyrene standards), was kindly supplied by the PHB Industrial S.A. (Serrana, SP, Brazil) and was submitted to a purification by dissolving in chloroform (minimum mass fraction purity 0.995) and re-precipitation in n-heptane (minimum mass fraction purity 0.995), both purchased from Quimex (São Paulo, SP, Brazil). 2.2. Raw material and grape seed extraction Grape seeds of Malbec and Cabernet Franc varieties (1:1) were kindly supplied by Villa Francioni winery (São Joaquim, SC, Brazil) – such mix of grape seeds is used in the commercial production of wine. Grape seeds collected in April 2009, after wine fermentation, were dried under the sun for 7 days to achieve a humidity of 12.0 wt % (dry basis – d.b.), determined by xylene distillation (Jacobs, 1973) in duplicate. The seeds were triturated in a knife mill (Marconi, model MA 340, Brazil), and the mean particle diameter (0.779 mm) was determined according to ASAE Standards (ASAE, 1998). The material was stored in a domestic freezer at temperature below 255 K. Crude grape seed oil was extracted using a laboratory-scale supercritical fluid extraction (SFE) equipment (Applied Separations, model 7071, USA) with a 0.29 × 10−3 m3 extraction vessel. The fixed bed was formed inside the extraction vessel with 0.280 kg

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of particulate raw material, resulting in an apparent bed density of 966 kg m−3 , calculated by dividing the feed mass by the vessel volume. The extraction vessel containing the raw material was adapted into the SFE unit. The extraction conditions selected were 313 K and 35 MPa according to data published in literature concerning SFE of grape seed oil (Molero Gómez et al., 1996; Reverchon and Marrone, 2001; Fiori, 2007). The solvent used was CO2 (mass fraction purity 0.999 purity, Gama Gases, São Bernardo do Campo, SP, Brazil). After pressurization, the bed was submitted to a static period of 20 min. Once the system reached the operating pressure and stabilized, the valve from the extractors outlet was opened and the extraction process began. CO2 was charged into the system at 1.255 × 10−4 kg s−1 flow rate during 450 min, resulting in a process yield of 13.42 wt% (d.b., weight percentage of extract obtained with respect to the initial charge of raw material in the extractor vessel). The extract was collected in a separator that consists of a 0.5 × 10−4 m3 glass vial immersed in an ice bath at ambient pressure. A more detailed description of the apparatus and experimental procedure can be found in the work of Prado et al. (2012). The GSE using in this work was characterized in terms of fatty acid and sterol composition, tocopherol and tocotrienol content, peroxide value, free fatty acid and water content, and density. Detailed results of GSE chemical characterization were reported in a recent work of our research group (Dalmolin et al., 2010). Such results demonstrate that GSE was obtained from grape seeds of the variety Vitis vinifera L., according to the Codex Alimentarius (1999) identity specifications. In the same work, the authors observed considerable amounts of trans-resveratrol, indicating that this compound may remain in grape seeds after the fermentation process in wine production and can be recovered by SC-CO2 . In fact, it may be important to remember that these characteristics can vary due to the agronomic and climatic conditions, seed quality, extraction technique applied and storage conditions. 2.3. Phase equilibrium apparatus and experimental procedure Phase equilibrium experiments were conducted employing the static synthetic method in a high-pressure variable-volume view cell. The experimental apparatus and procedure have been described in detail in a variety of studies (Bender et al., 2010; Canziani et al., 2009; Michielin et al., 2009; Rodriguez-Reartes et al., 2009; Benazzi et al., 2006; Franceschi et al., 2006; Ndiaye et al., 2006), and were extensively validated (Esmelindro et al., 2008; Borges et al., 2007; Franceschi et al., 2004; Stuart et al., 2000). Basically, the apparatus consists of a view cell, with a maximum internal volume of 27 × 10−6 m3 , with two sapphire windows for visual observations, a magnetic stirrer together with a Teflon-coated stirring bar to promote the agitation in the mixture, an absolute pressure transducer (accuracy 30 kPa, Smar, LD 301, Brazil), a portable programmer (Smar, HT 201, Brazil) for the cell pressure data acquisition, and a syringe pump cylinder (ISCO, Model 260D, USA) jacketed vessel that was coupled to an ultra thermostatic bath. The equilibrium cell includes a movable piston, which permits pressure control inside the cell. Phase transitions (cloud points) were observed visually through the pressure manipulation using the syringe pump with the solvent (carbon dioxide) as pneumatic fluid. A metallic jacket surrounds the cell. Water from a thermostatic bath is used as heating/cooling fluid, which flows through the jacket, so that the cell is kept at the desired temperature. The temperature of the mixture inside the cell is measured within 0.01 K using a “PT 100” type thermocouple. In this work the fluid phase behavior of the pseudo-ternary CO2 + DCM + GSE system was investigated at temperature ranging from (313 to 343) K. The system was named pseudo-ternary because, in fact, GSE is a complex mixture of many components,

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D.L. Boschetto et al. / Industrial Crops and Products 50 (2013) 352–360 Table 1 Experimental design of encapsulation of grape seed extract by the SEDS technique.

Fig. 1. Schematic diagram of the encapsulation apparatus based on Solution enhanced dispersion by supercritical fluids (SEDS) technique. Adapted from Franceschi et al. (2009).

however, the triacylglycerols (major compounds) present in the vegetable extract do not differ much in nature (similar chain length and chemical structure), and in this case, as an approach, such compounds can be adequately replaced by a pseudo-component having the corresponding average physico-chemical properties (Ndiaye et al., 2006; Franceschi et al., 2004). 2.4. Precipitation apparatus and procedure The encapsulation of GSE in the biopolymer was carried out using the solution enhanced dispersion by supercritical fluids (SEDS) technique, based on and adapted from previous works (Priamo et al., 2010, 2011; Franceschi et al., 2008a, b, 2009). A schematic diagram of the experimental apparatus is presented in Fig. 1, which consists of a precipitation chamber with an internal volume of 0.6 × 10−3 m3 (inner diameter of 0.08 m) (15), two syringe pumps (5) for CO2 (antisolvent) displacement (ISCO, Model 500D, USA), operated independently by a set of ball valves (Swagelok, Model SS-83KS4, USA) (3; 4; 7 and 8), and a digital HPLC liquid pump (Acuflow, Series III, Canada) (11) used for organic solution (10) delivery. The organic solution (GSE + DCM + PHBV) was continuously mixed by means of a magnetic stirrer (9) and, when the temperature, pressure and antisolvent flow rate were stabilized, the organic solution was added by the capillary tubing, then sprayed into the precipitation chamber through a silica capillary fusing tube, with an internal diameter of 100 ␮m, connected to a polyetheretherketone tubing (Peek Tubing, Upchurch Scientific, USA) (internally to 14). This arrangement was linked to a back pressure regulator (12) and to a tee connector (13), to link the CO2 , flowing in the annular region, and the organic solution. This type of contact between the solution (solute + solvent) and the antisolvent characterizes the SEDS technique, where intense mixing of supercritical fluids with solution/solvent(s) is done via coaxial nozzle, increasing mass transfer rate at the interface (Tabernero et al., 2012). During the experiments, the temperature inside the precipitation chamber was controlled by an ultrathermostatic bath (18), while the pressure was controlled by a needle valve (HIP, Model 1511AF1, USA) (19), hence controlling the depressurization. A system for powder collection was disposed in the output of the precipitation chamber, positioned in the precipitation chamber lid, and it was composed by two polytetrafluorethylene membrane filter. The first filter (superficial porosity of 1.0 ␮m, 8.0 mm of diameter, and 1 mm of thickness) was used as a support to the other filter (superficial porosity of 0.22 ␮m, 8.0 mm of diameter, and 150 ␮m

Run

p (MPa)

T (K)

1 2 3 4 5 6 7 8 9 10 11a 12 13 14 15

8 8 8 8 8 8 8 10 10 10 10 12 12 12 12

308 308 313 313 313 318 318 313 313 313 313 308 308 318 318

Grape seed extract to PHBV mass ratio 1:1 1:3 1:3 1:2 1:1 1:3 1:1 1:2 1:2 1:2 1:2 1:3 1:1 1:3 1:1

Concentration of grape seed extract (kg m−3 )

Concentration of PHBV (kg m−3 )

20.00 6.69 6.67 10.06 20.00 6.69 20.02 10.03 10.01 10.02 10.05 6.70 20.00 6.67 20.00

20.00 20.01 20.03 20.00 20.00 20.01 20.02 20.03 20.03 20.01 20.02 20.02 20.00 20.00 20.00

p – pressure (MPa), T – temperature (K). a With higher drying volume of the precipitated particles: 3 × 10−3 m3 of CO2 .

of thickness) deposited on a high density polyethylene support (Millipore, model FGLP, USA). The experimental procedure consisted of CO2 filling the precipitation chamber up to the desired pressure. The antisolvent flow rate was controlled and monitored by the syringe pump, and it was pumped at constant pressure of 20 MPa. The antisolvent flow rate and organic solution flow rate were 3.33 × 10−7 m3 s−1 and 1.67 × 10−8 m3 s−1 , respectively. The pressure for solution spray into the precipitation chamber was controlled by a back pressure regulator (12) manipulated and monitored by the HPLC liquid pump. The organic solution volume added to the chamber was 40 × 10−6 m3 , which enabled the production of sufficient amount of precipitated powder for analysis. After the organic solution addition, CO2 was continuous flowed at least 2 × 10−3 m3 in order to dry the precipitated particles inside the precipitation chamber, with a CO2 flow rate of 3.33 × 10−7 m3 s−1 . The precipitation chamber was slowly depressurized to atmospheric pressure and cooled to room temperature (∼298 K). The particles were then collected and stored in a domestic freezer at 255 K for subsequent characterization. Encapsulated particles were analyzed by a scanning electron microscopy combined with energy-dispersive spectrometry (SEMEDS) in a scanning electron microscope (Jeol JSM, model T300, USA) to determine particle morphology, and with the support of Size Meter software (version 1.1, Florianópolis, SC, Brazil), using at least 500 particles for each experiment, was measured the particle size. Briefly, a sample of encapsulated GSE in PHBV was weighed in an analytical balance with precision of 0.00001 g (Mettler Toledo, model XS205 Dual Range, Brazil) and added to different volumes of ethanol to remove the non-encapsulated (excess, free) GSE. Ethanol was selected because GSE presents relatively low solubility in this solvent, hence allowing slow removal of the non-encapsulated material, avoiding possible damages in the polymer wall. The suspensions of particles into ethanol were manually agitated for about 15 s at room temperature (∼298 K) and then all samples were filtered using a membrane filter with porosity of 0.22 ␮m (Millipore, model FGLP, USA). After filtration, the material retained was dried under desiccator for 24 h (Priamo et al., 2010, 2011; Franceschi et al., 2008a, b, 2009). Initially, a sample of the dried powder (co-precipitated GSE and PHBV) was weighed. It was assumed that the ratio between GSE and PHBV remained constant after the precipitation. Afterwards, the co-precipitated was dissolved in dichloromethane and the solution was analyzed in a UV-vis spectrophotometer (FEMTO, model 800 XI, São Paulo, SP, Brazil), in order to determinate the percentage

D.L. Boschetto et al. / Industrial Crops and Products 50 (2013) 352–360 Table 2 Phase equilibrium experimental data for the system {carbon dioxide (1) + dichloromethane (2) + grape seed extract (3)}. Solutions of GSE in dichloromethane with concentration of 5 kg m−3 at room temperature. T/K

p (MPa)

w1 = 0.9600 313.15 w1 = 0.9500 313.15 w1 = 0.9397 313.15 w1 = 0.9200 313.15 w1 = 0.9100 313.15 323.15 w1 = 0.8970 313.15 323.15 w1 = 0.8504 313.15 323.15 333.15 w1 = 0.8201 313.15 323.15 333.15 343.15 w1 = 0.7998 313.15 323.15 333.15 343.15 w1 = 0.7002 313.15 323.15 333.15 343.15 w1 = 0.4999 313.15 323.15 333.15 343.15 w1 = 0.3007 313.15 323.15 333.15 343.15

 (MPa)

Transition type

7.56

0.01

VLE-DP

7.64

0.02

VLE-BP

7.46

0.01

VLE-BP

7.25

0.02

VLE-BP

7.18 8.20

0.01 0.01

VLE-BP VLE-DP

7.11 8.38

0.02 0.00

VLE-BP VLE-DP

6.88 8.17 9.36

0.01 0.01 0.03

VLE-BP VLE-BP VLE-DP

6.60 7.84 9.03 10.18

0.01 0.02 0.02 0.01

VLE-BP VLE-BP VLE-BP VLE-DP

6.41 7.79 8.95 10.22

0.01 0.01 0.01 0.02

VLE-BP VLE-BP VLE-BP VLE-DP

5.87 6.86 8.20 9.63

0.01 0.02 0.00 0.01

VLE-BP VLE-BP VLE-BP VLE-BP

4.91 5.89 6.98 8.01

0.02 0.02 0.00 0.01

VLE-BP VLE-BP VLE-BP VLE-BP

3.56 4.18 5.04 5.76

0.01 0.01 0.01 0.02

VLE-BP VLE-BP VLE-BP VLE-BP

T – temperature (K), p – pressure (MPa),  – standard deviation (MPa), w – stands for the mass fraction of components 1, 2 and 3, VLE – vapor–liquid equilibrium, BP – bubble point, DP – dew point.

of encapsulation (PE %) and encapsulation efficiency (EE %). The absorbance of the solution was measured at 410 nm. Comparing the results with a pattern curve of absorbance versus concentration of the solution, the PE % and EE % of GSE in each assay were evaluated by the following expressions (Kalogiannis et al., 2006): PE [%] =

EE [%] =

mass of GSE encapsulated × 100 (mass of GSE + mass of PHBV )after filtration percentage of GSE encapsulated × 100 theoretical loading percentage of GSE encapsulated

where the theoretical loading percentage of GSE encapsulated is the ratio between the mass of GSE and the total mass of GSE and PHBV used in the co-precipitation experiments. It was studied the SEDS operating parameters in the following ranges, pressure (8–12 MPa), temperature (308–318 K), GSE to PHBV mass ratio (1:1–1:3), with concentrations of GSE (6.67–20 kg m−3 ) and PHBV in DCM fixed at 30 kg m−3 , in order to investigate the effect of these operating parameters on the particle size, particle morphology and encapsulation efficiency. In this

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Table 3 Phase equilibrium experimental data for the system {carbon dioxide (1) + dichloromethane (2) + grape seed extract (3)}. Solutions of GSE in dichloromethane with concentration of 15 kg m−3 at room temperature. T/K w1 = 0.9600 313.15 w1 = 0.9500 313.15 w1 = 0.9300 313.15 w1 = 0.9200 313.15 w1 = 0.9000 313.15 323.15 w1 = 0.8801 313.15 323.15 w1 = 0.8701 313.15 323.15 333.15 w1 = 0.8500 313.15 323.15 333.15 w1 = 0.8200 313.15 323.15 333.15 343.15 w1 = 0.7999 313.15 323.15 333.15 343.15 w1 = 0.7799 313.15 323.15 333.15 343.15 w1 = 0.6997 313.15 323.15 333.15 343.15 w1 = 0.6001 313.15 323.15 333.15 343.15 w1 = 0.4997 313.15 323.15 333.15 343.15 w1 = 0.3001 313.15 323.15 w1 = 0.3005 333.15 343.15

p/MPa

/MPa

Transition type

7.76

0.01

VLE-DP

7.87

0.02

VLE-BP

7.79

0.02

VLE-BP

7.56

0.01

VLE-BP

7.31 8.36

0.01 0.01

VLE-BP VLE-DP

7.30 8.45

0.01 0.01

VLE-BP VLE-BP

7.10 8.42 9.48

0.02 0.01 0.02

VLE-BP VLE-BP VLE-DP

6.86 8.13 9.55

0.01 0.02 0.00

VLE-BP VLE-BP VLE-DP

6.80 7.99 9.28 10.28

0.01 0.01 0.01 0.01

VLE-BP VLE-BP VLE-BP VLE-DP

6.69 7.90 9.21 10.37

0.02 0.01 0.01 0.00

VLE-BP VLE-BP VLE-BP VLE-DP

6.54 7.85 9.14 10.42

0.01 0.01 0.01 0.01

VLE-BP VLE-BP VLE-BP VLE-DP

6.04 6.91 8.12 9.37

0.01 0.01 0.01 0.01

VLE-BP VLE-BP VLE-BP VLE-BP

5.46 6.51 7.58 8.85

0.01 0.01 0.01 0.02

VLE-BP VLE-BP VLE-BP VLE-BP

5.01 5.81 6.74 7.86

0.01 0.01 0.02 0.01

VLE-BP VLE-BP VLE-BP VLE-BP

3.55 4.13

0.01 0.03

VLE-BP VLE-BP

5.06 5.74

0.02 0.01

VLE-BP VLE-BP

T – temperature (K), p – pressure (MPa),  – standard deviation (MPa), w – stands for the mass fraction of components 1, 2 and 3, VLE – vapor–liquid equilibrium, BP – bubble point, DP – dew point.

work, size was evaluated by the largest characteristic dimension of the particle. Experimental conditions of all runs are summarized in Table 1. To check the reproducibility of the experimental procedure, triplicate runs were conducted at the central point of the experimental design (runs 8–10). Only for run 11, CO2 was continuous flowed inside the precipitation chamber with at least 3 × 10−3 m3 , in order to verify the effect of the increase the amount of CO2 in

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Table 4 Experimental results of mean particle size. Run

X¯ (␮m)

X (min/␮m)

Xmax (␮m)

 (␮m)

PE (%)

EE (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0.70 0.72 – – – – – 0.62 0.71 0.53 – – 0.64 – –

0.21 0.19 – – – – – 0.18 0.23 0.11 – – 0.24 – –

1.54 2.40 – – – – – 2.01 2.04 1.65 – – 1.50 – –

0.27 0.31 – – – – – 0.25 0.29 0.22 – – 0.23 – –

33.01 10.55 6.66 9.56 21.33 5.62 17.86 12.70 12.93 12.65 12.44 12.18 26.01 4.45 13.26

66.01 42.07 26.65 28.56 42.67 22.40 35.72 38.07 38.79 37.93 37.26 48.56 52.01 17.79 26.52

 – standard deviation (␮m), X¯ – Mean particle size (␮m), X min – minimum particle size (␮m), X max – maximum particle size (␮m), PE – percentage of Encapsulation (%), EE – encapsulation efficiency (%).

drying of the precipitated particles, while for the other runs it was kept fixed at 2 × 10−3 m3 .

Two CO2 + DCM + GSE systems were determined, one with fixed solution of GSE in DCM with concentration of 5 kg m−3 and another with 15 kg m−3 , both at room temperature. The concentration range of GSE in DCM was selected after preliminary determinations to improve the solubility of GSE and PHBV in dichloromethane, according to Tres et al. (2007), at 293 and 298 K, resulting in (17.45 and 17.76) kg m−3 , respectively. The study was carried out with a maximum concentration of GSE in DCM of 15 kg m−3 so as to ensure complete solubilization of the extract. Results for the first pseudo-ternary system, CO2 + DCM + GSE with solution of GSE in DCM with concentration of 5 kg m−3 are shown in Table 2, where the pressure values are in fact average values of at least replicate measurements (observation of cloud point transitions), and the experimental error for each condition is represented by the standard deviation () of triplicates. Transition pressures were observed in the range of (3.55–10.23) MPa for temperature ranging from (313–343) K and, in this range of temperature studied, vapor–liquid equilibrium (VLE) occurred with visual observation of bubble (BP) and dew points (DP). Similarly, Table 3 presents the results for the second pseudoternary system, CO2 + DCM + GSE with solution of GSE in DCM with concentration of 15 kg m−3 . For temperature ranging from (313 to 343) K, transition pressures were observed in the range of (3.54–10.42) MPa and, as in the previous system, visual observation of VLE with BP and DP was noted. Fig. 2 represents experimental pressure versus mass fraction projections (P-w) for the two pseudo-ternary systems investigated, containing CO2 + DCM + GSE with solutions of GSE in DCM with concentrations of (5 and 15) kg m−3 , at (313, 323, 333 and 343) K and, overall mass fractions of CO2 from 0.30 to 0.96. It can be seen from this figure that the two systems have similar phase behavior when the solutions with two different concentration of GSE in DCM are used, indicating that an increase in the concentration of the GSE in DCM, in the range investigated, does not promote a significant increase of the biphasic region. Standard deviations for these systems were less than 0.03 MPa, pointing the good quality of the experimental data. Franceschi et al. (2008a) observed that the presence of solid ␤-carotene or the biopolymer PHBV, at low concentrations,

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

CO2 / mass fraction (wt %) Fig. 2. Experimental data for two ternary systems containing CO2 + DCM + GSE. Solutions of GSE in DCM, at room temperature, with concentrations of 5 kg m−3 : 313.15 K ( VLE-BP and  VLE-DP); 323.15 K ( VLE-BP and 䊉 VLE-DP); 333.15 K ( VLE-BP and  VLE-DP) and 343.15 K ( VLE-BP and  VLE-DP) and, 15 kg m−3 : 313.15 K (♦ VLE-BP and  VLE-DP); 323.15 K (夽 VLE-BP and  VLE-DP); 333.15 K ( VLE-BP and

VLE-DP) and 343.15 K ( VLE-BB and VLE-DP).

12 11 10

p / MPa

3.1. Fluid phase behavior

p / MPa

3. Results and discussion

12 11 10 9 8 7 6 5 4 3 2 1 0 0.0

9 8 7 6 5 4 3 2 1 0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

CO2 / mass fraction (wt %) Fig. 3. Comparison between the ternary system CO2 + DCM + GSE (solution of GSE in DCM with concentration of 15 kg m−3 at room temperature): 313.15 K (♦ VLE-BP and  VLE-DP); 323.15 K (夽 VLE-BP and  VLE-DP); 333.15 K ( VLE-BP and VLEDP) and 343.15 K ( VLE-BP and VLE-DP), with binary system CO2 + DCM (Corazza et al., 2003): 313.15 K ( VLE-BP and  VLE-DP); 323.15 K ( VLE-BP and 䊉 VLE-DP); 333.15 K ( VLE-BP and  VLE-DP) and 343.15 K ( VLE-BP and  VLE-DP).

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Fig. 4. SEM-EDS micrographs of the encapsulated GSE in PHBV. Spherical particles: (a) run 1; (b) run 2; (c) run 8, (d) run 9, (e) run 10, and (f) run 13.

completely dissolved in DCM, had negligible influence on the fluid phase behavior of the binary system CO2 + DCM determined previously by Corazza et al. (2003). Based on the work of Franceschi et al. (2008a) and, for the purpose of a comparison and to check possible modifications in the phase diagram due to the addition of GSE in DCM to the binary system CO2 + DCM (Corazza et al., 2003), Fig. 3 is presented. In this comparison, it was used the system solution of GSE in DCM with the higher concentration, 15 kg m−3 , in order to work closer to the limiting solubility condition.

It can be observed that the data of pseudo-ternary system overlap those of the binary system, demonstrating that the presence of GSE has negligible influence on the fluid phase behavior. From this figure, it is also possible to see that the appearance of a dew point is shifted to lower carbon dioxide compositions with increasing temperature, at 313.15 K with approximately 0.96 wt%, 323.15 K around 0.90 wt%, at 333.15 K with 0.85 wt% and at 343.15 K, 0.80 wt%, indicating the region with a maximum pressure value where the mixture critical point is located (Corazza et al.,

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Fig. 5. SEM-EDS micrographs of the encapsulated GSE in PHBV: (a) run 3; (b) run 4; (c) run 5; (d) run 6, (e) run 7, (f) run 11, (g) run 12, (h) run 14, and (i) run 15.

2003). This fact is important because phase equilibrium data for this binary system can be used as relevant information to support the development of technologies, such as the precipitation and encapsulation of bioactive compounds using supercritical-fluid micronization techniques, and play an important role for the scaleup of these processes. 3.2. Encapsulation of grape seed extract in PHBV Table 4 shows the results of GSE encapsulated in PHBV. With ¯ ␮m), minimum particle size (Xmin , regard to mean particle size (X, ␮m), maximum particle size (Xmax , ␮m) and standard deviation (, ␮m), results are presented only for six experimental conditions investigated, where spherical form of the particles was found, as can be seen in Fig. 4, revealed by SEM-EDS micrographs. It can be noted from Table 1 that the first eight experimental runs were conducted at 8 MPa. Comparing these runs, neglecting the pressure effect, and considering the particle morphology of

GSE encapsulated in PHBV, respective results from Table 4 show that only the first two experiments, conducted at lower temperature, 308 K, showed spherical particles (Fig. 4a and b), and so it was ¯ Xmin , Xmax and . possible to determine X, At the central point of the experimental design (runs 8–10), conducted 10 MPa, 313 K and GSE to PHBV mass ratio of 1:2, spherical particle were also obtained. For the last four runs, at the highest pressure, 12 MPa, an exception was observed for run 13, where spherical particle were obtained. This run differs experimentally from run 12 only in terms of GSE to PHBV mass ratio. However, the same effect was not observed from runs 1 and 2, at 8 MPa, which also differ experimentally only in terms of GSE to PHBV mass ratio, but these runs were conducted at lower temperature, 308 K. In general, highest temperatures implicated in agglomeration of the particles and formation of fibrous structure, as can be observed from Fig. 5. This might be due to a more favorable re-dissolution of particles at higher temperatures. Similar results were observed by Cocero and Ferrero (2002) and Kalogiannis et al. (2006), which also

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demonstrated that coalescence is produced as well by the interaction of the liquid solvent dissolved in the supercritical antisolvent and the solute particles. This fact is very important because lower temperature does not produce degradation of the natural product as GSE, and reduces the supplied heat, decreasing operating costs. As previously mentioned, although run 12 was conducted at 308 K, agglomerated particles with fibrous structure were found (Fig. 5g). Comparing run 12 with run 2, which differ experimentally only in terms of operating pressure, particles agglomeration in run 12 can be attributed to the fact that this system been operated at 12 MPa, a region of complete miscibility of the polymer in the mixed organic solvent and CO2 , as can be seen in (P-w) projections shown in Figs. 2 and 3. This increase in pressure leads to the decrease of the achieved supersaturation, and may lead to particles with larger mean sizes and may also cause a great drop in the glass transition temperature, leading to coalescence of the formed particles (Kalogiannis et al., 2006). At the experimental conditions reported here which presented spherical particles, the mean particle size varied from (0.53 to 0.72) ␮m, for runs 10 and 2, respectively. Unfortunately, it was not possible to visualize the effect of the variables on the particle size in this work. A more comprehensive study on the encapsulation of GSE in PHBV using the SEDS technique, assessing the effects of process parameters on the particle size is underway within our research group and will be the subject of a next report. The effect of the temperature parameter on encapsulation efficiency can be observed comparing runs where the other parameters were fixed, as comparison between run 1 with runs 5 and 7, also run 12 with run 14 and finally run 13 with 15. In all cases, an increase in temperature led to a decrease in the encapsulation efficiency. This fact can be explained in terms of solubility of GSE in DCM. With the increase of temperature, GSE increases its solubility in DCM, and are together dragged out with the antisolvent CO2 . In the same way, in general, an increase in pressure led to a decrease in the encapsulation efficiency, noted by comparing run 1 with run 13, run 6 with run 14, and run 7 with run 15. This is in agreement with studies of encapsulation of lavandin essential oil with biopolymers by PGSS technique (Varona et al., 2010). These authors argued that this can be explained considering also that the solubility of the extract in CO2 increases when pressure is increased, becoming completely miscible with CO2 at pressures above the mixture critical point. While for concentration of organic solution, a positive effect on encapsulation efficiency was observed, as can be seen by comparison between run 1 and run 2, as well as run 3, run 4 and run 5, also between run 6 and run 7, run 12 and run 13, and run 14 and run 15. The same was observed by Priamo et al. (2010). Process parameters of SEDS technique which promoted the greatest achieved value of the encapsulation efficiency, 66.01% for run 1, with standard deviation of 0.27 ␮m, were the smallest pressure (8 MPa), as well as the smallest temperature (308 K) and the highest concentration of organic solution (GSE to PHBV mass ratio (1:1)). Results for run 11, wherein CO2 was continuous flowed inside the precipitation chamber with at least 3 × 10−3 m3 , in order to verify the effect of the increase the amount of CO2 in drying of the precipitated particles, compared with results from runs of central point of the experimental design (runs 8–10), with the same operating parameters, demonstrated that, probably, the particle agglomeration occurred (Fig. 5f) was due to more collisions between particles formed by increasing the amount of CO2 . However, the value for encapsulation efficiency was similar to runs of central point of the experimental design. Note that the similar results for encapsulation efficiency of the central point attest the very good reproducibility of the experiments with small differences attributed to random experimental uncertainties. Results presented here, demonstrated that the SEDS technique employed for encapsulation of non-solid materials, such as GSE

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used in the present work, in biopolymers can be considered for formation of micronic or sub-micronic products for food and pharmaceutical industries as an alternative to conventional techniques. 4. Conclusions New phase equilibrium (cloud point) experimental data for the system containing carbon dioxide + dichloromethane + grape seed extract measured up to 343.15 K and pressures up to 10.5 MPa were presented in this work. Phase transitions, bubble and dew points, were observed in the temperature and pressure ranges studied. The addition of grape seed extract to the system consisting of CO2 and dichloromethane did not influence the transition pressure, which allows the consideration of a binary system to select the operating conditions for the encapsulation in the phase equilibrium diagram. This work presented a study of the influence of SEDS operating parameters, pressure, temperature, grape seed extract to PHBV mass ratio and concentrations of grape seed extract and PHBV in dichloromethane, on the morphology and size of particle grape seed extract encapsulated in PHBV by a SEDS process. Temperature presented a greater influence on particle morphology. Lower temperatures led to less agglomeration and smaller particles. The best result generated spherical particles with the smallest size of 0.70 ␮m, obtained at pressure of 8 MPa, temperature of 308 K, mass ratio (1:1) and concentration of grape seed extract and PHBV, both of 20 kg m−3 , keeping fixed all other processing variables. Results obtained in the present work are relevant toward developing new technologies for the encapsulation of extracts in biopolymers, and also as a support for the successful design of controlled delivery and protection systems applied to food products. Acknowledgements The authors thank Villa Francioni winery (São Joaquim, SC, Brazil) for raw material donation, CAPES/PROCAD (840/2008), Pronex/FAPERGS/CNPq and FAPESP/BIOEN (2008/56258-8) for financial support and for the Ph.D. assistantship. We also thank Dr. J. M. Prado, Dr. R. M. Dallago and Dr. R. L. Cansian for their help and advice. References ASAE – American Society of Agricultural Engineers, 1998. Method of Determining and Expressing Fineness of Feed Materials by Sieving. ASAE S319.3, pp. 447–550. Benazzi, T., Franceschi, E., Corazza, M.L., Oliveira, J.V., Dariva, C., 2006. High-pressure multiphase equilibria in the system glycerol + olive oil + propane + AOT. Fluid Phase Equilibria 244, 128–136. Bender, J.P., Feitein, M., Mazutti, M.A., Franceschi, E., Corazza, M.L., Oliveira, J.V., 2010. Phase behaviour of the ternary system {poly(-caprolactone) + carbon dioxide + dichloromethane}. The Journal of Chemical Thermodynamics 42, 229–233. Borges, G.R., Junges, A., Franceschi, E., Corazza, F.C., Corazza, M.L., Oliveira, J.V., Dariva, C., 2007. High-pressure vapor–liquid equilibrium data for systems involving carbon dioxide + organic solvent + ␤-carotene. Journal of Chemical and Engineering Data 52, 1437–1441. Bravi, M., Spinoglio, F., Verdone, N., Adami, M., Aliboni, A., D’Andrea, A., De Santis, A., Ferri, D., 2007. Improving the extraction of ␣-tocopherol-enriched oil from grape seeds by supercritical CO2 . Optimisation of the extraction conditions. Journal of Food Engineering 78, 488–493. Canziani, D., Ndiaye, P.M., Franceschi, E., Corazza, M.L., Oliveira, J.M., 2009. Phase behaviour of heavy petroleum fractions in pure propane and n-butane and with methanol as co-solvent. The Journal of Chemical Thermodynamics 41, 966–972. Cocero, M.J., Ferrero, S., 2002. Crystallization of ␤-carotene by a GAS process in batch. Effect of operating conditions. Journal of Supercritical Fluids 22, 237–245. Codex Alimentarius FAO/WHO Food Standards, 1999. Codex Standard for Named Vegetable Oils Codex-Stan 210., pp. 1–13. Corazza, M.L., Cardozo, Filho, L., Antunes, O.A.C., Dariva, C., 2003. High pressure phase equilibria of the related substances in the limonene oxidation in supercritical CO2 . Journal of Chemical and Engineering Data 48, 354–358. Dalmolin, I., Mazutti, M.A., Batista, E.A.C., Meireles, M.A.A., Oliveira, J.V., 2010. Chemical characterization and phase behavior of grape seed oil in compressed carbon dioxide and ethanol as co-solvent. Journal of Chemical Thermodynamics 42, 797–801.

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