Precipitation of β-carotene and PHBV and co-precipitation from SEDS technique

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J. of Supercritical Fluids 47 (2008) 259–269

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The Journal of Supercritical Fluids

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recipitation of �-carotene and PHBV and co-precipitationrom SEDS technique using supercritical CO2

lton Franceschia, Alana M. De Cesaroa, Mirian Feitena, Sandra R.S. Ferreirab, Cláudio Darivac,arcos H. Kunitad, Adley F. Rubirad, Edvani C. Munizd, Marcos L. Corazzaa, J. Vladimir Oliveiraa,∗

Department of Food Engineering, URI-Campus de Erechim, Av. Sete de Setembro, 1621 Erechim, RS 99700-000, BrazilEQA-CTC/UFSC, Chemical Engineering and Food Engineering Department, Federal University of Santa Catarina, C.P. 476, CEP 88040-900, Florianópolis, SC, BrazilInstituto de Pesquisa e Tecnologia-ITP, Programa de Mestrado em Engenharia de Processos PEP/UNIT, Campus Farolândia, Av. Murilo Dantas, 300 Aracaju, SE 49032-490, BrazilDepartment of Chemistry, Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900 Maringá, PR, Brazil

r t i c l e i n f o

rticle history:eceived 15 April 2008eceived in revised form 31 July 2008ccepted 1 August 2008

eywords:ncapsulationEDS techniqueluid phase behaviorarbon dioxide-CaroteneHBV

a b s t r a c t

The objective of this work was to investigate the application of the solution enhanced dispersion bysupercritical fluids (SEDS) technique for the precipitation of pure �-carotene and copolymer poly(3-hydroxybutirate-co-hydroxyvalerate) (PHBV), as well as some encapsulation tests of the solute in thebiopolymer. The following parameters were investigated in the precipitation of pure �-carotene: pressure(8.0 and 12.0 MPa), anti-solvent flow rate (20–40 mL/min) and concentration of �-carotene in an organicdichloromethane solution (4 and 8 mg/mL). For pure �-carotene, the results showed that the mean particlesize varied from 3.8 to 246.8 �m, depending on the processing conditions. The morphology of �-carotenewas modified from plate-like to leaf-like particles, as verified by micrographs of scanning electronicmicroscopy (SEM). For the PHBV precipitation, the SEM micrographs showed that for all experimentalconditions the morphology of polymer was different from the unprocessed material. The precipitatedpolymer presented a quasi-spherical shape with interconnected particles in the sub-micrometric range(particle size in the range of 278–570 nm), while the unprocessed material was composed of films andlarge blocks. The co-precipitation tests showed that the best ratio of �-carotene to PHBV in solution was
1:3 (w/w), which resulted in approximately 80% of encapsulation. Fluid phase behavior of the ternary sys-tems CO2 + dichloromethane + �-carotene and CO2 + dichloromethane + PHBV was also investigated withthe aim of elucidating the region of the phase diagram in which the precipitation occurs. Phase equilib-rium data were measured in the temperature range of 303–343 K, with CO2 compositions ranging from 40to 90 wt% for �-carotene, and from 30 to 90 wt% for PHBV. Vapor–liquid and also solid–vapor–liquid phasetransitions were observed in the phase equilibrium study. It was observed that the presence of �-carotene
xture

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. Introduction

Incorporation of bioactive compounds such as vitamins, probi-tics, bioactive peptides and antioxidants into polymeric matricesrovide a simple way to develop novel functional foods thatay have physiological benefits or reduce risks of diseases [1].

arotenoids, considered nutraceutical compounds, are the most
ommon group of pigments in nature. The main role of carotenoidsn human diet is to function as vitamin A precursors and its directse as antioxidants [2–4,5]. Distinct from synthetic carotenoids, theatural ones are more easily oxidized [6] and their oxidation prod-
∗ Corresponding author. Fax: +55 54 35209090.E-mail address: [email protected] (J.V. Oliveira).

sodtcoti

896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.supflu.2008.08.002

had a little influence on the fluid phase behavior of the systems.© 2008 Elsevier B.V. All rights reserved.

cts have little or negligible pigmentation, provitamin-A activitynd singlet oxygen quenching activity [7].

The application properties and the color strength of pigmentsre strongly dependent on their physical properties such as particleize, particle size distribution and morphology, as well as the routehat pigments are obtained [8]. It should be emphasized, however,hat carotenoid particles with much reduced size are still more sen-itive to degradation due to the high superficial area that is peculiarf particles with micro or nanometric size. Thus, it is desirable theevelopment of preservation techniques to avoid degradation and
o increase the dissolution rate of these compounds in water, asarotenoids are lipossoluble. The stability of carotenoids againstxidation can be considerably increased through their encapsula-ion in biopolymers and even an increase in their dissolution raten water can be achieved [4].
http://www.sciencedirect.com/science/journal/08968446

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dx.doi.org/10.1016/j.supflu.2008.08.002

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60 E. Franceschi et al. / J. of Supe

There is a range of polymers that can be employed to encap-ulate bioactive compounds, due to their biocompatibility andiodegradability. Several biopolymers were already used for encap-ulate pharmaceutical compounds [9,10], proteins [11–13], andarotenoids [4,14]. The polymer used for encapsulation plays a cru-ial role, as the characteristics of polymer used determine howhe encapsulated solute will be released. The delivery can occurn two ways: diffusion release or degradation release. The first

echanism takes place when the incorporated compound passeshrough the polymer pores or through the polymer chain. Degra-ation release occurs when a polymer degrades within the foodatrix, as a result of natural biological process such as hydroly-

is. In this case, the polymer degradation is strongly dependentn the chemical structure and molecular mass of the polymer10].

Polyhydoxyalkanoates (PHAs) are polyesters produced byicroorganisms under unbalanced growth conditions. PHA is

enerally biodegradable, with good biocompatibility, being anttractive polymer for use as encapsulating agent [15]. The mostommon type of PHA is poly(3-hydroxibutirate) (PHB). However,HB is stiff and brittle thus restricting its range of application. Onhe other hand, PHB copolymers with 3-hydroxyvalerate (PHBV)re less stiff, tougher and crystalline [16]. The use of poly(3-ydroxybutirate-co-hydroxyvalerate) (PHBV) in biomedical fieldas increased mainly due to the fact that it is possible to pre-are an appropriate controlled drug delivery system that graduallyegrades in the body [15]. Thus, the copolymer PHBV can also besed as encapsulation medium for the protection and controlledelivery of carotenoids in foods.

The particle formation and encapsulation using traditionalechniques (spray-drying, coacervation, freeze-drying, interfacialolymerization, etc.) can suffer from some drawbacks like the poorontrol of particle size and morphology, degradation of thermo sen-itive compounds, low encapsulation efficiency, and/or low yield.

In this sense, several supercritical fluid-based techniques,mploying mainly carbon dioxide, have been proposed to explorehe solvent strength, high diffusivity and near-zero interfacial ten-ion that are peculiar in the vicinity of the critical point [17]. The usef supercritical or near critical fluids as solvents or anti-solvents inhe particles precipitation/encapsulation was demonstrate by sev-ral researchers as a useful means to the modification of materialroperties such as particle size, size distribution and morphology.nother advantage of such techniques is the efficient separation of

he solvent and anti-solvent of the particles after precipitation, pre-enting organic solvent residues in the final product and permittingeutilization of solvent and anti-solvent [18].

Independently of the technique employed for solid materialsecrystallization (SAS, SEDS, GAS, ASES, PCA, etc.), the phase behav-or of the involved system plays a crucial role in understandinghe precipitation mechanism, such as jet breakup, nucleation androwth kinetics, and mass transfer, as well as to determine the mostatisfactory operating conditions during the precipitation [19–22].

In this context, the objective of this work was to investigatehe effect of processing parameters (pressure, flow rate of solu-ion and anti-solvent, initial concentration of the solid in solutionnd mass ratio between �-carotene and PHBV) on the precipita-ion of pure �-carotene and PHBV and co-precipitation of themsing the solution enhanced dispersion by supercritical fluid (SEDS)echnique, focusing on particle size, particle size distribution, mor-hology and encapsulation percentage. The phase behavior of the
ystems solvent/solutes/anti-solvent was also investigated to sit-ate the operating point in the phase diagrams, and helping the
nterpretation of the results. The morphology of precipitated pow-ers was characterized by scanning electronic microscopy (SEM)nd the particle size and size distribution was calculated employing

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al Fluids 47 (2008) 259–269

he software Size Meter (version 1.1). The encapsulation percentageas verified by UV–vis spectrophotometry.

. Experimental

.1. Materials

trans-�-Carotene, with a purity of 95% and mean particle size of�m, was purchased from Sigma–Aldrich (USA). Dichloromethane

DCM—99.5%) was purchased from Merck (Germany), carbon diox-de (99.9% in liquid phase) was supplied by White Martins S.A.,nd the co-polymer PHBV, with a fibrous aspect, was kindly sup-lied by the PHB Industrial S.A. All materials were used as received.he polymer average molecular weight (MW) and polydispersityere found to be 1.96 × 105 g gmol−1 and 1.85, respectively, as mea-

ured by GPC using a calibration curve obtained from polystyrenetandards.

.2. High-pressure phase equilibrium apparatus andxperimental procedure

Phase behavior experiments of ternary systems were con-ucted employing the static synthetic method in a high-pressureariable-volume view cell. A detailed description of the experimen-al apparatus and procedure can be found elsewhere [23–25].

.3. Experimental design

A full factorial 23 experimental design was adopted to orga-ize the experiments and to evaluate the main effects of processarameters on the precipitation of �-carotene and PHBV fromichloromethane by the SEDS technique. In this work, it was inves-igated the influence of precipitation pressure (P), concentrationf �-carotene or PHBV in the solution (C) and anti-solvent flowate (qCO2 ). The temperature, nozzle diameter and solution flowate were maintained constant at 313 K, 100 �m and 1.0 mL/min,espectively. The variables ranges were selected based on pre-iminary precipitation tests, on previous results of the group,n the apparatus limitations, and on the fluid phase behaviorf the ternary systems (CO2 + dichloromethane + �-carotene andO2 + dichloromethane + PHBV). The pressure range adopted was–12 MPa, which means pressure values near and above the mix-ure critical point. The values of solute concentration were from

to 8 mg/mL for �-carotene and from 10 to 40 mg/mL for PHBVt room temperature, allowing the operation with concentratednd diluted solutions. Regarding the anti-solvent flow rate, theelected range was from 20 to 40 mL/min with the aim of increas-ng the turbulence inside the precipitation chamber (PC), ando promote a more intense mixing between the solution andnti-solvent.

The main effects of above-mentioned variables on particle sizend particle size distribution were evaluated using the commer-ial software Statistica® 6.0, adopting a confidence level of 95%p < 0.05). In this work, it was defined the particle length as thearameter to compute the characteristic size of the precipitated �-arotene, whereas the diameter was the obvious choice for PHBVarticles.

.4. Precipitation apparatus and procedure

Fig. 1 presents a schematic diagram of the experimental appara-us employed for �-carotene, PHBV and combined carotene/PHBVrecipitation [26]. Briefly it consists of a cylindrical vessel, withn internal volume of 60 mL and internal diameter of 4 cm,

E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269 261

Fig. 1. Schematic diagram of the precipitation apparatus. CV—check-valve; V1, V2, V3 and V4—ball valve; V5, V6 and V7—needle valve; BPR—back pressure regulator;P mpert

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hich was used as PC; two syringe pumps for CO2 displace-ent (ISCO, Model 500D), operated independently by a set of

all valves—V1 to V4 (Swagelok, Model SS-83KS4), and a digitalPLC liquid pump (Acuflow, Series III) used for organic solutionelivery.

The organic solution was sprayed into the precipitation chamberhrough a silica capillary fusing tube, with an internal diame-er of 100 �m, connected to a polyetheretherketone tubing (Peekubing, Upchurch Scientific). This arrangement was linked to aPR (back pressure regulator, GO-Regulator, Series BP-66, ModelA11QEQ151) and to a tee (T) connector (Swagelok), to link thenti-solvent and the solution flows.

During the experiments, the temperature into the precipitationhamber was kept constant at 313 K by an ultrathermostatic bathNova Ética, Model 521/2D), while the pressure was varied and con-rolled by two needle valves (HIP, Model 15-11AF1). The first valveV5) controlled the anti-solvent flow rate, while the other one con-rolled the depressurization. A second vessel (SC) connected afteralve V6 was used to keep the stream that leaves the precipitationhamber at a relatively low pressure (about 4 MPa) to prevent thelockage of valve V6. A system for powder collection was located

n the bottom of the precipitation chamber, and was composed byintherized metal filter (superficial porosity of 1.0 �m) as a supporto the polytetrafluorethylene membrane filter linked to a high den-ity polyethylene support (Millipore, model FGLP with a porosityf 0.22 �m).

The experimental procedure started with CO2 filling the precip-tation chamber up to the desired pressure. The anti-solvent flowate was controlled by setting V5 and V6 valves, and monitored
y the syringe pump. When the temperature, pressure and anti-olvent flow rate were stabilized, the organic solution was addedhrough the capillary tubing. The pressure for solution spray intohe precipitator was controlled by BPR manipulation and monitoredy the liquid pump. The solution volume added to the chamber
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ature control; PC—precipitation chamber; SC—separation chamber; CT—capillary

as 20 mL, which enable the collection of sufficient amount of pre-ipitated powder for analysis. After the solution addition, around00 mL of CO2 was continuous flowed in order to dry the precipi-ated particles inside the precipitation chamber. The precipitationhamber was slowly depressurized to atmospheric pressure andarticles were collected and stored at appropriate conditions forubsequent analysis and characterization.

.5. Analysis and characterization

Precipitated particles were analyzed by a Shimadzu model SS-50 Superscan scanning electron microscope (SEM) to determinearticle morphology and shape. Particle size was measured by theize Meter software (version 1.1), using at least 500 particles forach experiment, while the Statistica® 6.0 software was used toerform the statistics of particle size distribution.

The encapsulation percentage was verified by an UV–vis spec-rophotometer. Initially, a sample of co-precipitated �-carotenend PHBV was weighed. It was assumed that the ratio between-carotene and PHBV remained constant after the precipitation.he co-precipitated sample was suspended in ethanol and main-ained under sonication for 5 min to extract the �-carotene notncapsulated. Then, the resulting suspension was filtered and driednder controlled temperature (323 K) and vacuum for 24 h. After-ards, the dried powder was dissolved in dichloromethane and

he absorbance of �-carotene was measured at 450 nm. Comparinghe results with a pattern curve of absorbance vs. concentration of-carotene in the solvent, the amount of �-carotene encapsulatedas evaluated by a straightforward calculation of the encapsulation
ercentage by the expression:
-carotene encapsulated (%)

= mass of �-carotene encapsulatedtotal mass of �-carotene in the initial sample

× 100 (1)

262 E. Franceschi et al. / J. of Supercritical Fluids 47 (2008) 259–269

Table 1Experimental phase equilibrium data for the system CO2(1) + dichloromethane(2) + �-carotene(3)

T (K) P (MPa) �a (MPa) Transition type

(CO2-free) �-carotene concentration in DCM solution at room temperature: 4 mg/mLw1 = 0.401

303 4.01 0.01 BP313 4.97 0.01 BP323 5.79 0.02 BP333 6.79 0.04 BP343 7.67 0.03 BP

w1 = 0.601303 4.90 0.03 BP313 6.02 0.04 BP323 6.96 0.06 BP333 7.96 0.01 BP343 9.32 0.05 BP

w1 = 0.800303 5.42b 0.01 BP313 6.96b 0.02 BP323 8.16b 0.02 BP333 9.26b 0.01 BP343 10.01b 0.01 DP

w1 = 0.900303 6.54b 0.06 BP313 7.63b 0.03 BP323 8.40b 0.04 DP

(CO2-free) �-carotene concentration in DCM solution at room temperature: 8 mg/mLw1 = 0.301

303 3.54 0.06 BP313 4.02 0.03 BP323 4.66 0.01 BP333 5.41 0.03 BP343 6.16 0.01 BP

w1 = 0.501303 4.49 0.03 BP313 5.38 0.04 BP323 6.40 0.01 BP333 7.26 0.03 BP343 8.43 0.05 BP

w1 = 0.701303 5.67b 0.04 BP313 6.62b 0.03 BP323 7.57b 0.02 BP333 8.61b 0.03 BP343 9.89b 0.02 BP

w1 = 0.900303 6.29b 0.01 BP313 7.74b 0.01 BP323 8.09b 0.03 DP

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1, CO2 mass fraction; DP, dew point; BP, bubble point; P, pressure; T, temperaturea Standard deviation.b Phase transition with the presence of solid �-carotene.

. Results and discussions

.1. Fluid phase behavior

In this work the fluid phase behavior of ternary sys-ems CO2 + dichloromethane + �-carotene and CO2 + dichloro-

ethane + PHBV was investigated. Solutions of �-carotene andHBV in dichloromethane were prepared at the same concentra-ions used in the precipitation experiments. The CO2 mass fractionas varied from 0.4 to 0.9 for �-carotene solution and from 0.3 to 0.9

or PHBV solution. The range of temperature investigated was from03 to 343 K. Tables 1 and 2 show the experimental results for theuid phase behavior of ternary systems CO2 + dichloromethane + �-arotene and CO2 + dichloromethane + PHBV, respectively, for the

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ve isotherms investigated. It can be observed from these tableshat vapor–liquid equilibria occurred, with bubble and dew points,or both systems studied.

As can be seen in Tables 1 and 2, starting from a certain CO2oncentration, phase transitions for all isotherms occur with thexistence of a solid phase. For the �-carotene ternary systemTable 1), the presence of a solid phase during vapor–liquid phaseransition started from CO2 mass fraction of 0.800 for the moreiluted solution (4 mg/mL), and from 0.701 for the concentratedne (8 mg/mL). For the PHBV ternary system (Table 2), the PHBV
ich-solid phase started from a CO2 mass fraction lower than thaterified for the ternary system with �-carotene. For the dilutedHBV solution, the vapor–liquid phase transition with the pres-nce of solid PHBV started from CO2 mass fraction of 0.439, while


Table 2Experimental phase equilibrium data for the system CO2(1) + dichloromethane(2) + PHBV(3)

T (K) P (MPa) � (MPa) Transition type

(CO2-free) PHBV concentration in DCM solution at room temperature: 10 mg/mLw1 = 0.303303 3.63 0.01 BP313 4.36 0.02 BP323 4.98 0.05 BP333 5.85 0.04 BP343 6.47 0.01 BP

w1 = 0.439303 4.08a 0.05 BP313 5.00a 0.04 BP323 6.00a 0.03 BP333 6.91a 0.04 BP343 7.86a 0.01 BP



w1 = 0.800303 5.63a 0.04 BP313 7.02a 0.04 BP323 8.06a 0.02 BP333 9.26a 0.03 BP343 9.94a 0.02 DP

w1 = 0.900303 6.40a 0.02 BP313 77.9a 0.03 BP323 8.49a 0.01 DP

(CO2-free) PHBV concentration in DCM solution at room temperature: 40 mg/mLw1 = 0.303303 3.43 0.03 BP313 4.10 0.05 BP323 4.87 0.02 BP333 5.65 0.03 BP343 6.29 0.01 BP

w1 = 0.402303 4.11a 0.01 BP313 4.98a 0.03 BP

w1 = 0.402323 5.72a 0.02 BP333 6.45a 0.02 BP343 7.66a 0.01 BP





Table 2 (Continued )

T (K) P (MPa) � (MPa) Transition type

w1 = 0.799303 5.97a 0.01 BP313 7.04a 0.02 BP

w1 = 0.799323 7.98a 0.02 BP333 9.13a 0.02 BP343 9.81a 0.01 DP

w1 = 0.900303 6.62a 0.01 BP313 7.57a 0.01 BP323 8.37a 0.03 DP

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1, CO2 mass fraction; DP, dew point; BP, bubble point; P, pressure; T, temperaturea Phase transition with the presence of solid PHBV.

or the more concentrated solution at CO2 mass fraction of 0.402.s discussed in the literature, this fact is due to the co-solvent

acts enhancing the solute solubility) and anti-solvent (reduceshe solute solubility) effect of CO2 in the mixture [19,21]. At lowO2 concentrations, it acts as a co-solvent and, at high concen-rations as an anti-solvent. The limit concentration in which CO2tarts to behave as anti-solvent depends on the solution concen-ration. For diluted solutions high amounts of CO2 are necessaryo induce the formation of a solid phase. On the other hand, foroncentrated solutions, the amount of CO2 needed for a solidhase formation may be much lower due to the solution saturationroximity.

To check possible modifications in the phase diagram dueo the addition of a solid compound to the binary systemO2 + dichloromethane, two solute concentrations were tested

n the organic solution. Results presented in Figs. 2 and 3rovide a comparison between the ternary system CO2 + dichloro-ethane + �-carotene and CO2 + dichloromethane + PHBV, respec-

ively, for two solute concentrations in the organic solution at 313 Kith the binary system CO2 + dichloromethane [27].

As can be observed from these figures, the presence of the solid�-carotene or PHBV) has negligible influence on the fluid phaseehavior. This fact can be explained as phase transitions occurred

n the presence of solid solutes, for the concentrated solutions,

ig. 2. Experimental data at 313 K for the ternary system CO2 + dichloromethane + �-arotene at (CO2-free) �-carotene concentration of 4 mg/mL and 8 mg/mL at roomemperature. Experimental binary system data from the literature [27].

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here the solid phase can be considered as pure solute. When phaseransitions occurred with solutes completely dissolved in the lowoncentrated solutions, pressure values were not influenced due tohe very low solutes concentration in the mixture.

.2. ˇ-Carotene precipitation

The variables studied in the �-carotene precipitation includedhe precipitation pressure, �-carotene concentration in the organicolution, and anti-solvent flow rate. The experimental runs andesults of particle size and particle size distribution are summarizedn Table 3.

It can be noted from Table 3 that, in general, a reduction or anncrease in the particle size were observed: starting from original-carotene with mean particle size of 4.6 �m, the mean particleize of precipitated �-carotene varied from few microns (3.8 �m)o larger sizes (246.8 �m). The size distribution was characterizedy the variation coefficient (VC) as very distinct particle size wasbserved among runs. Variation coefficients in the range of 23–57%round the mean particle size were observed in the experiments.rom replicate measurements shown in Table 3, runs 9–11, one can
ee that a good reproducibility regarding mean particle size waschieved.
The statistical analysis adopting 95% of confidence levelp < 0.05) revealed that precipitation pressure and anti-solvent flow

ig. 3. Experimental data at 313 K for the ternary system CO2 + dichloro-ethane + PHBV at (CO2-free) polymer concentration of 10 mg/mL and 40 mg/mL

t room temperature. Experimental binary system data from the literature [27].

E. Franceschi et al. / J. of Supercritic

Table 3Factorial experimental design (23) of �-carotene precipitation from a DCM/�-carotene solution using CO2 by the SEDS technique and experimental results of meanparticle size (PS) and variation coefficient (VC)

Run C (mg/mL) P (MPa) qCO2(mL/min) PS (�m) VCa (%)

1 4 8.0 20 6.2 532 8 8.0 20 29.1 333 4 12.0 20 246.8 574 8 12.0 20 186.0 425 4 8.0 40 4.6 246 8 8.0 40 3.8 427 4 12.0 40 42.8 248 8 12.0 40 124.7 449 6 10.0 30 88.3 24

10 6 10.0 30 71.5 2311 6 10.0 30 66.5 23

Precipitation temperature 313 K, solution rate 1 mL/min, nozzle diameter 100 �m.Cs

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ate present a significant effect on particle size of �-carotenerecipitated, whereas the solute concentration did not present aignificant effect. Pressure was the variable that more stronglynfluenced the particle size. An increase in pressure leads to anncrease in the mean particle size, while the anti-solvent flow ratead an opposite effect, with a decrease in the mean particle sizeue to a raise in the anti-solvent flow rate.

.3. Pressure effect

Experiments for �-carotene precipitation were carried out byhanging the precipitation pressure from 8 to 12 MPa. This pressureange combined with a fixed temperature (313 K) condition, resultn a single supercritical phase where the organic solvent and thenti-solvent are completely miscible, as can be seen in Fig. 2. Asommented above, as the pressure are increased, the mean particleize is also enhanced, as shown in Table 3 for runs 1 and 3 and, foruns 6 and 8.

From runs 1 to 3, the pressure was increased and the solutiononcentration and anti-solvent flow rate was maintained at theowest values. Comparing these runs, the mean particle size wasncreased from 6.2 to 246.8 �m and the variation coefficient waslso increased from 53 to 57%. When the pressure was increased,
ith the solution concentration and anti-solvent flow rate in thepper limits (runs 6 and 8) the mean particle size was also increasedrom 3.8 to 124.7 �m and the VC enhanced from 42 to 43%. The
orphology of �-carotene precipitated also changed with pressure,here a modification of particles from plate-like to a leaf-like form

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ig. 4. SEM micrographs of the precipitated �-carotene at 8.0 MPa (A—run 6) and 12.00 mL/min.

al Fluids 47 (2008) 259–269 265

as observed as the pressure is increased. The effect of precipita-ion pressure on the particle size and morphology of precipitated-carotene is illustrated in Fig. 4 through SEM micrographs ofxperimental conditions 6 (Fig. 4A) and 8 (Fig. 4B).

The pressure effect on particle size of precipitated �-caroteneound in this work is in agreement with those obtained by Miguelt al. [2] that investigated the precipitation of lycopene fromichloromethane solutions using carbon dioxide as anti-solvent.

n that work, the authors estimated the maximum supersatura-ion that could be achieved in lycopene precipitation, and that theupersaturation decreased as pressure increased above the mixtureritical point due to a decrease of solute concentration in the mix-ure. Considering that the solubility of lycopene and �-carotene inO2 are similar and also due to the similar chemical structure, oneould also expect that the results obtained for lycopene should bepplied to �-carotene [2].

In this sense, pressure effects can be explained in terms of solu-ility, since the supersaturation is defined by the ratio between theolute concentration in the solvent + anti-solvent mixture and theaturation concentration of the solute in the mixture. As the sol-ent + anti-solvent mixture stands above the mixture critical point,n increase in pressure leads to a decrease in solute concentrationn the mixture, hence reducing the supersaturation. This decreasen supersaturation causes a decrease in the nucleation rate, leadingo a larger growth of �-carotene precipitated particles, increasinghe particle size and modifying their morphology from plate-likeFig. 4A) to leaf-like (Fig. 4B) particles.

Though the solubility of CO2 in the organic solvent is diminisheds pressure is decreased from 12 to 8 MPa the mass transfer ratesf the CO2 in the solution droplets generated by the spray and inhe organic solvent in the CO2 bulk phase are enhanced, leading toigher nucleation rates and smaller particles.

.4. Anti-solvent flow rate effect

The anti-solvent flow rate was investigated in the range of0–40 mL/min. According to statistical analysis, an increase in thisariable induces a decrease in mean particle size and variation coef-cient. However, this effect is less pronounced when compared tohe pressure influence. The effect of anti-solvent flow rate was eval-ated by comparing runs 1 and 5 and runs 4 and 8. In runs 1 and 5he anti-solvent flow rate was varied maintaining the pressure andolution concentration at the lowest values (8 MPa and 4 mg/mL,
espectively). In run 1 the mean particle size obtained was 6.2 �mith a variation coefficient of 53%, while in run 5 the mean particle
ize decreased to 4.6 �m with a variation coefficient of 24%.When the anti-solvent flow rate increased with pressure and

olution concentration at the highest values (runs 4 and 8) the

MPa (B—run 8). Solution concentration of 8 mg/mL and anti-solvent flow rate of


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ig. 5. SEM micrographs of the precipitated �-carotene at anti-solvent flow rateoncentration of 4 mg/mL.

ean particle size was reduced from 186 to 124.7 �m with a slightncrease in the variation coefficient of the mean particle size from2 to 43%. However, keeping constant the anti-solvent flow rate athe lowest or at the highest value and increasing simultaneouslyhe pressure and the solution concentration, a notable increase inarticle size was observed. The particle morphology of precipitated-carotene was not modified by the anti-solvent flow rate. SEMicrographs of runs 1 (Fig. 5A) and 5 (Fig. 5B) presented in Fig. 5

llustrate the influence of anti-solvent flow rate on particle size ofrecipitated �-carotene.

From this figure it can be noted that the precipitated particleshow agglomeration, mainly when the anti-solvent flow rate was
ncreased to 40 mL/min. This fact can be attributed to the high inletelocity of the anti-solvent that causes intense collisions of the par-icles and thus the formation of agglomerates. Reduction of particleize with the increase of the anti-solvent flow rate is probably dueo hydrodynamics aspects. The high velocity of CO2 causes a pro-
mopaf

ig. 6. SEM micrographs of the precipitated �-carotene from runs 9 (A), 10 (B) and 11 (C) af 30 mL/min.

mL/min (A—run 1), and 40 mL/min (B—run 5). Pressure of 8.0 MPa and solution

ounced turbulence inside the precipitation chamber leading to anncrease in the kinetic energy of the atomizing CO2. Thus, the jetreak up is intensified increasing the interaction of anti-solvent andolution, and generating fine droplets. In addition, the mass transferates between CO2 and the organic solvent are increased by the highurface area generated; thus the CO2 diffuses more rapidly into theroplet and the solvent evaporates from droplets instantaneouslyausing accelerated supersaturation and nucleation.

The reproducibility of the precipitated �-carotene morphologyan be verified in Fig. 6, from SEM micrographs of runs 9 (Fig. 6A),0 (Fig. 6B) and 11 (Fig. 6C) (Table 3) indicating that the methodol-gy and the experimental unit presented a good reproducibility on
orphology of precipitated �-carotene particles. For the purpose
f illustration, Fig. 7 depicts a histogram of PSD for run 6, whichrovided the smallest particle size, and from where one can seen approximate Gaussian distribution pattern, a common featureound in many works in the literature.

t pressure of 10 MPa, solution concentration of 6 mg/mL and anti-solvent flow rate


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Table 4Factorial experimental design (23) of PHBV (MW = 1.96 × 105 g gmol−1 and polydis-persity = 1.85) precipitation DCM/PHBV solution using CO2 by the SEDS techniqueand experimental results of mean particle size (PS) and variation coefficient (VC)

Run C (mg/mL) P (bar) qCO2(mL/min) PS (nm) VCa (%)

1 10 8.0 20 487 212 40 8.0 20 505 243 10 12.0 20 329 214 40 12.0 20 – –5 10 8.0 40 370 216 40 8.0 40 570 287 10 12.0 40 278 228 40 12.0 40 563 319 25 10.0 30 330 24

10 25 10.0 30 324 2211 25 10.0 30 314 21

Precipitation temperature 313 K, solution rate 1 mL/min, nozzle diameter 100 �m.C, solution concentration; P, precipitation pressure; qCO2

, CO2 flow rate; PS, particlesize.

s

1wAacpooicwfF

Bafateti

Fr

ig. 7. Particle size distribution of �-carotene precipitated at solution concentrationf 8 mg/mL, pressure of 8 MPa and anti-solvent flow rate of 40 mL/min.

.5. PHBV precipitation

The same variables of �-carotene precipitation were inves-igated in the PHBV studies: precipitation pressure, solutiononcentration and anti-solvent flow rate. The range of precipita-ion pressure and anti-solvent flow rate was the same as the onesor �-carotene precipitation; however the solution concentrationas investigated in distinct values due to the higher solubil-

ty of the polymer than that of �-carotene in dichloromethane.able 4 presents the experimental runs and results regarding par-icle size and size distribution of PHBV precipitation, where onean observe from the replicate measurements a good experimentaleproducibility.

From Table 4 it can be noted that the particle size of precipi-ated PHBV was in the sub-micrometric range, varying from 278o 570 nm while the VC varied from 21 to 31%. One can note thatbers, not particles, were produced in run 4. In all other precipi-ation experiments quasi-spherical interconnected particles werebtained, differently from the unprocessed PHBV that is formed bybers. According to statistical analysis at 95% of confidence levelp < 0.05), only the solution concentration had a significant andositive effect on particle size.

.6. Solution concentration effect

The effect of the solution concentration on particle size andorphology of PHBV precipitation was evaluated in the range of

Comac

ig. 8. SEM micrographs of the precipitated PHBV at solution concentration of 10 mg/mLate of 40 mL/min.

a VC is defined as the ratio between the standard deviation and the mean particleize.

0–40 mg/mL. Other variables (pressure and anti-solvent flow rate)ere firstly maintained at the lowest values (runs 1 and 2 of Table 4).n increase in the solution concentration in this case resulted inn increase in mean particle size from 487 to 505 nm and a smallhange in the variation coefficient (21–24%). Runs 7 and 8 of Table 4resent the effect of the solution concentration at the higher valuesf pressure and anti-solvent flow rate. In these cases, the increasef the solution concentration resulted in a more pronounced raisen particle size, from 278 to 563 nm, and in the variation coeffi-ient, from 22 to 31%. The PHBV particles produced in these casesere almost spherical shape and interconnected, as can be seen

rom SEM micrographs of experiments 7 (Fig. 8A) and 8 (Fig. 8B) inig. 8.

As can be noted in Fig. 8 in concentrated solutions (micrographin Fig. 8) show that besides almost spherical particles there are

lso fibers and more agglomerated particles were observed thanor the diluted solution (micrograph A in Fig. 8). These resultsre in agreement with those obtained by Costa et al. [16] inhe precipitation of PHBV. The formation of fibers and agglom-rated particles is characteristic of concentrated solutions due tohe high viscosity of these solutions. The high viscosity impliesn an increase in shear forces thus hindering the diffusion of
O2 into droplets generated by the jet break up. Thus, evap-ration of the organic solvent from droplets by CO2 action isore affected, causing a decrease in the mass transfer rate, and
ccordingly in the precipitation kinetics, generating larger parti-les.

(A—run 7), and of 40 mg/mL (B—run 8). Pressure of 12.0 MPa and anti-solvent flow


Fig. 9. SEM micrographs of the co-precipitated �-carotene and PHBV with mass ratio of 1:2 (A), 1:3 (B) and 1:4 (C) at 8.0 MPa and anti-solvent flow rate of 40 mL/min.

3

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.7. Encapsulation of ˇ-carotene into PHBV

Preliminary tests of �-carotene encapsulation into PHBV werearried out by co-precipitation using the SEDS technique. The maindea for co-precipitation is that the particles of the material to bencapsulated (core) are smaller than those of the encapsulatingaterial (shell). In the precipitation of pure �-carotene and PHBV,

o experimental conditions presented such a result. Nevertheless,xperimental condition 6 in the �-carotene and in the PHBV precip-tation steps (Tables 3 and 4) that led, respectively, to the smallestnd highest particle sizes of these materials was chosen to accom-lish the co-precipitation experiments. Pressure and anti-solventow rate were maintained at 8.0 MPa and 40 mL/min, respectively,arying the mass ratio between �-carotene and PHBV in the organicolution.

The carotene to polymer mass ratio investigated was 1:2, 1:3 and:4. Fig. 9 presents SEM micrographs of the co-precipitation experi-ents for �-carotene to PHBV mass ratio of 1:2, 1:3 and 1:4. Results

howed that for the mass ratio of 1:2, the encapsulated caroteneas around 5%, indicating that the amount of polymer was not suf-cient to effectively encapsulate the carotene. For the mass ratiof 1:3, the encapsulation percentage was close to 80%, suggestinghat �-carotene was precipitated earlier than the polymer, actings seed particles, for subsequent covering of the latter. For the massatio of 1:4, it seems that �-carotene particles remained adsorbedn the polymer surface, indicating that the polymer could precipi-ate early due to its higher concentration, and then the �-carotene
tick on its surface.
A more comprehensive study on the PHBV precipitation ando-precipitation of �-carotene and PHBV, assessing the effects ofrocess parameters is underway within our research group and wille the subject of a next report.

ppcas

. Conclusions

This work investigated the precipitation of pure �-carotenend PHBV from dichloromethane solutions by the SEDS tech-ique with carbon dioxide as anti-solvent. Phase equilibriumata involving CO2, dichloromethane and the solutes (�-carotenend PHBV) were measured. It was verified that the addi-ion of solutes to the binary system consisting of the organicolvent and CO2 had negligible influence on transition pres-ures.

Precipitation of pure �-carotene presented particles with meanize in the range from 3.8 to 246.8 �m and variation coefficientrom 23 and 57% around the mean particle size. For the variablesnvestigated, (precipitation pressure, solution concentration andnti-solvent flow rate), pressure and anti-solvent flow rate pre-ented influence on particle size and morphology with a strongerffect of precipitation pressure. The precipitation pressure presentpositive effect on the mean particle size of �-carotene, whereas

he anti-solvent flow rate showed a negative effect on this variable.he �-carotene morphology changed from plate-like to leaf-likearticles.

For PHBV precipitation, sub-micrometric almost spherical parti-les, were generated except for the highest polymer concentrationn the solution where fibers were produced. The mean particle sizef PHBV precipitated varied from 278 to 570 nm with variationoefficient between 21 and 31%. A raise in solution concentrationed to and increase in particle size. The anti-solvent flow rate and
recipitation pressure were not significant on PHBV particle sizerecipitated at 95% of confidence. The co-precipitation tests indi-ated that encapsulation of �-carotene in PHBV up to 80% werechieved at mass ratio of 1:3 (�-carotene to polymer) in the organicolution.

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cknowledgments

The authors thank CNPq and CAPES for the financial support andcholarships.

eferences

[1] L. Chen, G.E. Remondetto, M. Subirade, Food protein-based materialsas nutraceutical delivery systems, Trends Food Sci. Technol. 17 (2006)272.

[2] F. Miguel, A. Martín, T. Gamse, M.J. Cocero, Supercritical anti solvent precip-itation of lycopene. Effect of the operating parameters, J. Supercrit. Fluids 36(2006) 225.

[3] M.D.A. Saldana, L. Sun, S.E. Guigard, F. Temelli, Comparison of the solubilityof �-carotene in supercritical CO2 based on a binary and a multicomponentcomplex system, J. Supercrit. Fluids 37 (2006) 342.

[4] A. Martín, F. Mattea, L. Gutiérrez, F. Miguel, M.J. Cocero, Co-precipitation ofcarotenoids and bio-polymers with the supercritical anti-solvent process, J.Supercrit. Fluids 41 (2007) 138.

[5] W.A. Schroeder, E.A. Johnson, Singlet oxygen and peroxyl radicals regu-late carotenoid biosynthesis in Phaffia rhodoxyma, J. Biol. Chem. 270 (1995)18374.

[6] S. Qing, K.G. Rowley, N.D.H. Balazs, Carotenoids: separation methods applicableto biological samples, J. Chromatogr. B 781 (2002) 393.

[7] C.J. Chang, A.D. Randolph, N.E. Craft, Separation of �-carotene mixtures pre-cipitated from liquid solvents with high-pressure carbon dioxide, Biotechnol.Progr. 7 (1991) 275.

[8] Q.L. Suo, W.Z. He, Y.C. Huang, C.P. Li, H.L. Hong, Y.X. Li, M.D. Zhu, Micronization ofthe natural pigment-bixin by the SEDS process through prefilming atomization,Powder Technol. 154 (2005) 110.

[9] C.G. Kalogiannis, C.M. Michailof, C.G. Panayiotou, Microencapsulation of amox-icillin in poly(l-lactic acid) by supercritical antisolvent precipitation, Ind. Eng.Chem. Res. 45 (2006) 8738.

10] Y. Wang, Y. Wang, J. Yang, R. Pfeffer, R. Dave, B. Michniak, The application of asupercritical antisolvent process for sustained drug delivery, Powder Technol.164 (2006) 94.

11] N. Elvassore, A. Bertucco, P. Caliceti, Production of protein-loaded polymericmicrocapsules by compressed CO2 in a mixed solvent, Ind. Eng. Chem. Res. 40(2001) 795.

12] I. Ribeiro dos Santos, J. Richard, B. Pech, C. Thies, J.P. Benoit, Microencapsulationof protein particles within lipids using a novel supercritical fluid process, Int. J.Pharm. 242 (2002) 69.

[

al Fluids 47 (2008) 259–269 269

13] S. Salmaso, N. Elvassore, A. Bertucco, A. Lante, P. Caliceti, Nisin-loaded poly-l-lactide nano-particles produced by CO2 anti-solvent precipitation for sustainedantimicrobial activity, Int. J. Pharm. 287 (2004) 163.

14] W. He, Q. Suo, H. Hong, A. Shan, C. Li, Y. Huang, Y. Li, M. Zhu, Production of nat-ural carotene-dispersed polymer microparticles by SEDS-PA co-precipitation,J. Mater. Sci. 42 (2007) 3495.

15] G.Q. Chen, Q. Wu, The application of polyhydroxyalkanoates as tissue engineer-ing materials, Biomaterials 26 (2005) 6565.

16] M.S. Costa, A.R.C. Duarte, M.M. Cardoso, C.M.M. Duarte, Supercritical antisol-vent precipitation of PHBV microparticles, Int. J. Pharm. 328 (2007) 72.

17] E. Reverchon, I. De Marco, G. Caputo, G. Della Porta, Pilot scale micronizationof amoxicillin by supercritical antisolvent precipitation, J. Supercrit. Fluids 26(2003) 1.

18] M. Rantakylä, M. Jäntti, O. Aaltonen, M. Hurme, The effect of initial drop sizeon particle size in the supercritical antisolvent precipitation (SAS) technique, J.Supercrit. Fluids 24 (2002) 251.

19] A. Shariati, C.J. Peters, Measurements and modeling of the phase behavior ofternary systems of interest for the GAS process. I. The system carbon dioxide + 1-propanol + salicylic acid, J. Supercrit. Fluids 23 (2002) 195.

20] E. Reverchon, G. Caputo, I. De Marco, Role of phase behavior and atomization inthe supercritical antisolvent precipitation, Ind. Eng. Chem. Res. 42 (2003) 6406.

21] B. De Gioannis, A.V. Gonzalez, P. Subra, Anti-solvent and co-solvent effect of CO2on the solubility of griseofulvin in acetone and ethanol solutions, J. Supercrit.Fluids 29 (2004) 49.

22] Y. Pérez de Diego, F.E. Wubbolts, G.J. Witkamp, Th.W. de Loos, P.J. Jansens, Mea-surements of the phase behavior of the system dextran/DMSO/CO2 at highpressures, J. Supercrit. Fluids 35 (2005) 1.

23] E. Franceschi, M.H. Kunita, A.F. Rubira, E.C. Muniz, M.L. Corazza, J.V. Oliveira, C.Dariva, Phase behavior of binary and ternary systems involving carbon diox-ide, propane, and glycidyl methacrylate at high pressure, J. Chem. Eng. Data 51(2006) 686.

24] T. Benazzi, E. Franceschi, M.L. Corazza, J.V. Oliveira, C. Dariva, High-pressuremultiphase equilibria in the system glycerol + olive oil + propane + AOT, FluidPhase Equilibr. 244 (2006) 128.

25] P.M. Ndiaye, E. Franceschi, D. Oliveira, C. Dariva, F.W. Tavares, J.V. Oliveira, Phasebehavior of soybean oil, castor oil and their fatty acid ethyl esters in carbondioxide at high pressures, J. Supercrit. Fluids 37 (2006) 29.

26] E. Franceschi, M.H. Kunita, M.V. Tres, A.F. Rubira, E.C. Muniz, M.L. Corazza, C.
Dariva, S.R.S. Ferreira, J.V. Oliveira, Phase behavior and process parameterseffects on the characteristics of precipitated theophylline using carbon dioxideas antisolvent, J. Supercrit. Fluids 44 (2008) 8.
27] M.L. Corazza, L. Cardozo Filho, O.A.C. Antunes, C. Dariva, High pressure phaseequilibria of the related substances in the limonene oxidation in supercriticalCO2, J. Chem. Eng. Data 48 (2003) 354.

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