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Journal of Food Engineering xxx (2011) xxxxxx

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Journal of Food Engineering


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Effect of boldo (Peumus boldus M.) pretreatment on kinetics of supercritical CO2 extraction of essential oil
Edgar Uquiche a,b, Elizabeth Huerta a, Alicia Sandoval a, Jos Manuel del Valle c,d,
a

Department of Chemical Engineering, Universidad de La Frontera (UFRO), Temuco, Chile Center of Food Biotechnology and Bioseparations, Scientic and Technological Bioresource Nucleus (BIOREN), UFRO, Temuco, Chile c Department of Chemical and Bioprocess Engineering, Ponticia Universidad Catlica (UC) de Chile, Santiago, Chile d ASIS-UC Interdisciplinary Research Program on Tasty and Healthy Foods, UC, Santiago, Chile
b

a r t i c l e

i n f o

a b s t r a c t
This work examined the effect of the solid matrix on supercritical carbon dioxide (SC CO2) extraction of essentials oils from boldo leaves (Peumus boldus M.) subjected to rapid decompression of a CO2-impregnated sample, conventional milling, and low-temperature milling. Low-temperature conditioning prior to milling decreased heat-driven losses of volatile compounds during milling, as attested by a higher extract yield for low-temperature (10.8 g extract/kg extract-free substrate) than conventionally (9.63 g/kg) milled sample. Extract yield was even larger for the rapidly decompressed sample (11.4 g/kg). Results of SC CO2 extraction experiments carried out at 40 C and 10 MPa were adjusted to a diffusional model using the effective diffusivity of the extract in the solid matrix (De) and a single partition of essential oils between solid substrate and SC CO2 as best-tting parameters. A microstructural factor (FM), which was estimated as the ratio between the binary diffusion coefcient of the essential oil in SC-CO2 under extraction conditions and De, was used to characterize the effect of sample pretreatment on extraction rates. Values of FM for rapidly decompressed (202) and low-temperature milled (1740) samples were smaller than value for conventionally milled samples (2200), which revealed that the two rst treatments disrupted secretory cavities in boldo leaves more effectively than the third. This was conrmed by light microscopy observations. The work included also measurements using oregano bracts (Origanum vulgare L.) as the substrate to conrm literature reports on the SC CO2 extraction of pretreated bracts and to serve as a reference to our main results with boldo leaves. Trends with oregano coincided with those of boldo. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 2 May 2011 Received in revised form 3 October 2011 Accepted 8 October 2011 Available online xxxx Keywords: Boldo Essential oils Mass-transfer Microstructure Milling Modeling Rapid decompression Supercritical CO2 extraction

1. Introduction Boldo (Peumus boldus M.) is a native plant from Chile whose leaves contain bioactive essential oils, alkaloids, and avonoids having interesting medicinal properties (Bisset, 1994; Speisky and Cassels, 1994). Essential oils are complex mixtures of terpenes, a widespread and chemically diverse group of natural compounds that derive from isoprene, a ve-carbon, unsaturated hydrocarbon molecule (Brielmann et al., 2006). They generally include the 10carbon (or 2-isoprene) monoterpenes and the 15-carbon (or 3-isoprene) sesquiterpenes, as well as oxygen-containing derivatives (e.g., oxygenated monoterpenes) that are responsible for characteristic plant aromas (del Valle et al., 2011). Supercritical (SC) uid extraction has emerged as a viable alternative of conventional extraction techniques for plant essential oils, such as hydrodistillation, steam distillation, and solvent
Corresponding author at: Department of Chemical and Bioprocess Engineering, Ponticia Universidad Catlica (UC) de Chile, Avenida Vicua Mackenna 4960, Macul, Chile. Tel.: +56 2 3544418; fax: +56 2 354580. E-mail address: delvalle@ing.puc.cl (J.M. del Valle).
0260-8774/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.10.013

extraction, because it circumvents the use of organic solvents, and/or high temperatures (Quirin and Gerard, 2007; Reverchon and De Marco, 2007). Carbon dioxide (CO2) is the uid most widely used in SC uid extraction applications because of it is non-corrosive, non-ammable, non-reactive, and innocuous; it is available highly puried at low cost; it is effective at room temperature; and it can be removed completely from treated substrates and extracts (del Valle and Aguilera, 1999). del Valle et al. (2004, 2005) attempted the SC CO2 extraction of natural antioxidants in boldo barks and leaves. Main boldo antioxidants are boldine in the alkaloid fraction, and catechin in the avonoid fraction (Schmeda-Hirschmann et al., 2003; Quezada et al., 2004). On the other hand, boldo essential oils and other fatty compounds have pro-oxidant activity (Quezada et al., 2004). Obtaining antioxidant extracts from boldo leaves using SC CO2 as the solvent requires >70 MPa and/or polar ethanol as co-solvent (del Valle et al., 2004, 2005) because of the low solubility in SC CO2 of boldine and especially catechin (Berna et al., 2001; de la Fuente et al., 2005). An alternative to isolate boldo antioxidants is to remove aroma compounds using moderate-pressure CO2 (del Valle et al., 2005; Mazutti et al., 2008), and then extract the antioxidants using

Please cite this article in press as: Uquiche, E., et al. Effect of boldo (Peumus boldus M.) pretreatment on kinetics of supercritical CO2 extraction of essential oil. Journal of Food Engineering (2011), doi:10.1016/j.jfoodeng.2011.10.013

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Nomenclature Cfo Cso dpi (dp)ref dpS De DL D12 FM i (j) kf K l solute concentration in the SC CO2 phase at the beginning of the experiment (g/kg CO2) solute concentration in the solid phase at the beginning of the experiment (g/kg substrate) size of fraction i of particles (mm) particle size parameter of the RosinRommlerBennett size distribution (mm) mean Sauter diameter of solid substrate particles (mm) effective diffusivity of solute in the solid matrix (m2/s) axial dispersion coefcient (m2/s) binary diffusion coefcient of the solute in SC CO2 (m2/ s) microstructural factor () index characterizing particles of a given size () lm mass transfer coefcient (m/s) solute partition coefcient between the solid matrix and SC CO2 phase () half-thickness of the slab-shaped solid particle (mm) mi MW2 n T Tc Tr Vc2 mass of fraction of particles of size i molecular weight of the extract (g/mol) uniformity coefcient of the RosinRommlerBennett particle size distribution () process temperature (K) critical temperature of SC CO2 (304.3 K) reduced temperature of SC CO2 () critical volume of the extract (cm3/mol)

Greek letters e void fraction in the bed () l viscosity of pure CO2 at high pressure (kg m1 s1) q density of pure CO2 at high pressure CO2 (kg/m3) qb apparent (bulk) density of the substrate (kg/m3) qc critical density of CO2 (468 kg/m3) qr reduced density of CO2 () qs true density of the substrate (kg/m3)

ethanol, water, or their mixtures (Pasquel et al., 2000; Yoda et al., 2003). Boldo essential oil, however, may have commercial value on its own because it has the characteristic aroma of boldo leaves and contains some unusual compounds such as ascaridole (Bruns and Khler, 1974; Bisset, 1994; Miraldi et al., 1996; Vila et al., 1999; Mazutti et al., 2008), a bicyclic monoterpene with a peroxide bridge having sedative, pain-relieving, antifungal, and antitumor effects (Pare et al., 1993; Efferth et al., 2002; Dembisky, 2008). A factor that affects the SC CO2 extraction rate and yield of essential oils is the pretreatment of the substrate. In most studies, the substrate is milled, which is eventually complimented by separation according to particle size by sieving. These pretreatments are ineffective when they do not selectively disrupt essential-oil-holding structures (Gaspar et al., 2000, 2001), or cause essential oil losses by evaporation and/or thermal/oxidative degradation (Pruthi, 1980; Pesek et al., 1985; Murthy and Bhattacharya, 2008). Gaspar et al. (2000) proposed cryogenic milling to reduce thermal degradation and evaporation of essential oils during sample pretreatment. Effective treatments prior to essential oil extraction should be aimed at a selective destruction of cellular barriers holding essential oils in the herb but not at the expense of causing losses of volatile compounds. Many essential oils are produced in glandular cells on the surface of leaves, bracts, petals, and other organs of aromatic herbs, and are stored in the subcuticular space formed at the gland anex (Bosabalidis and Tsekos, 1982, 1984; Gaspar et al., 2001; Zizovic et al. 2005). The secretory structures that encapsulate plant essential oils in vegetable tissue depend on the plant family and species, with other specialized encapsulating structures including ducts and cavities (Zizovic et al., 2007; Stame nic et al., 2008). Gaspar et al. (2000, 2001) proposed disrupting glandular trichomes of oregano (Origanum virens L.) bracts by contact with high-pressure CO2 followed by a rapid decompression. This work evaluated the effect of the pretreatment (conventional milling, low-temperature milling, and fast decompression of CO2impregnated sample) on the SC CO2 extraction of essential oils from boldo (P. boldus M.) leaves. Because the effect of sample pretreatment on extraction rate and yield can be quantied by mathematical modeling using the effective diffusivity of a relevant pseudo-solute in the solid matrix (De) as a tting parameter (Araus et al., 2009), authors evaluated the effectiveness of the pretreatments by light microscopy and estimation of De values from cumulative extraction plots of essential oil yield versus time. To validate the methods proposed by Gaspar et al. (2000) additional experiments were carried out using oregano (Origanum vulgare L.) bracts as the substrate.

2. Materials and methods 2.1. Substrates and pretreatments Dry boldo (P. boldus M.) leaves and oregano (O. vulgare L.) bracts were locally acquired in a market in Temuco (Chile). They were hand-picked to eliminate damaged parts, and subjected to conventional milling, low-temperature milling, and rapid decompression. Samples were placed in a desiccator with silica gel prior to treatments to reduce and homogenize their moisture content. For conventional milling, substrates were treated in 100-g batches in a hammer mill (M2 Assa, Assa Tecnologa S.A., Santiago, Chile) with 1-mm openings for 15 min (boldo leaves) or 10 min (oregano bracts). Low-temperature milling was carried out in a similar fashion, but in this case samples were pre-cooled prior to milling. For that, 200-g batches of each substrate were placed in a metallic basket that was in turn placed inside a 10-dm3 vessel containing 1 dm3 of liquid nitrogen for 3 min (the time that took evaporating all the liquid N2). Conventionally milled and low-temperature milled samples were packed in polyethylene bags, and stored at 5 C up to analysis. For the rapid decompression of CO2-impregnated matrices, ca. 12 g of boldo leaves (weighed accurately) manually cracked to pass through a mesh Tyler number 7 screen (openings of 2.36 mm), or ca. 5 g of oregano bracts (weighed accurately) of approximately the same size, were loaded in a 50-cm3 extraction vessel (inner diameter = 1.4 cm; height = 32.5 cm). The extraction vessel was placed in the air-convection oven of a Spe-ed SFE unit (Applied Separations, Allentown, PA), pressurized with high-purity (99.95% pure) CO2 (AGA S.A., Santiago, Chile), and held at 40 C and 7 MPa for 60 min (Gaspar et al., 2003). After substrates were fully swollen with SC CO2, glands or cavities were disrupted by a fast decompression step; CO2 was released by opening a valve which let the pressure diminish to the normal atmospheric level in 120 s (boldo leaves) or 80 s (oregano bracts). 2.2. Substrate characterization Density, particle size distribution, and microstructural characteristics of pretreated substrates were evaluated. True density (qs) was estimated by weighing the material loaded into a 1-dm3 graduated cylinder using a standard tapping procedure (del Valle et al., 2003). Apparent density (qb) was estimated by weighing a nely milled sample of the material that could be tightly packed into

Please cite this article in press as: Uquiche, E., et al. Effect of boldo (Peumus boldus M.) pretreatment on kinetics of supercritical CO2 extraction of essential oil. Journal of Food Engineering (2011), doi:10.1016/j.jfoodeng.2011.10.013

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a 10-cm3 graduated cylinder (del Valle et al., 2003). The particle size distributions of the milled substrates were determined in a Ro-Tap test sieve shaker (W.S. Tyler, Mentor, OH) using 80, 48, 35, 28, 24, and 20 mesh Tyler screens. These measurements were performed in triplicate. Light microscopy of pretreated boldo leaves and oregano bracts was carried out using the procedures of Uquiche et al. (2004). Following sample xing, samples were embedded with parafn prior to cutting thin slices (30-lm thick) using a manual microtome Jung (Heidelberg, Germany). Following removal of parafn with xylol, samples were stained with safranine and fast green. Photomicrographs of the stained samples were captured using a light microscope Olympus BX50 (Tokyo, Japan) equipped with a camera JVC TK-1280E (Yokohama, Japan). 2.3. Supercritical carbon dioxide extraction experiments Pretreated samples were extracted in the Spe-ed SFE unit using 7.2 g/min SC-CO2 at 40 C and 10 MPa (Reverchon et al., 1993). The temperature of the expansion valve was kept constant at 100 C, and the total extraction time was 120 min in all experiments. In the case of conventionally or low-temperature milled boldo leaves and oregano bracts, ca. 15 g samples (weighed accurately) were loaded in the extraction vessel, whereas those samples subjected to rapid decompression were extracted under the aforementioned conditions following 10min standing in the extraction vessel at normal atmospheric pressure after pretreatment. Ten extract aliquots were periodically collected during extraction in pre-weighed glass vials (15-cm3 capacity) and dried in a desiccator with silica gel to remove co-extracted water, and the recovered extract was assessed gravimetrically by difference with cleaned and dried vials. The weight loss (initial minus nal) of the substrate was measured and compared to the total recovery in the ten glass vials to detect random experimental errors, and then the extraction yield (or mass of recovered solute per unit mass of water-free substrate) was computed as a function of extraction time. Extraction experiments were carried out in duplicate. Pretreated and SC-CO2-extracted samples of boldo leaves and oregano bracts were nely milled with a mortar and pestle before quantication of moisture content. Sample moisture was determined gravimetrically by drying in an air-convection oven (Memmert model UM-400, WTB Binder, Tutlingen, Germany) set at 105 C to a constant nal weight (15 h).

101

ln (1 - F )-1 (dimensionless)

Conventionally milled boldo Low-temperature milled boldo Conventionally milled oregano Low-temperature milled oregano

100

10-1 0.2 0.4 0.5 0.6 0.7 0.8

Particle diameter (dp, mm)


Fig. 1. RosinRommlerBennett plot of cumulative undersize population versus particle size for conventionally and low-temperature milled boldo leaf and oregano bract particles, where F represents the weight fraction of particles of size dp or less.

 Fdp 1 exp

dp dp ref

n

where (dp)ref is a particle size parameter (the 36.8th percentile of the cumulative size distribution), and n, a uniformity coefcient related to the steepness of the size distribution (as the value of n increases the scattering of the distribution decreases). Linearization of Eq. (1) suggest presenting particle size distribution data in a loglog plot of ln [1 F(dp)]1 versus dp, as done in Fig. 1. The RRB function tted the size distribution of the milled samples more closely than alternative models such as the lognormal or loglog (or Gaudin Schumann) plots, as also observed by Manohar and Sridhar (2001) for conventionally- and cryogenically-milled turmeric samples. Fig. 1 shows differences in particle size distribution between the substrates, and also between milling treatments for oregano bracts, but not for boldo leaves. The particle size distribution can be also reduced to a single mean value such as the Sauter diameter (dpS) that represents the diameter of a sphere having the same surface-to-volume ratio as the entire population of particles in a milled sample, and can be estimated using Eq. (2):

3. Results and discussion This section describes the effect of sample pretreatment on the physical properties of boldo leaves and oregano bracts, including densities, particle sizes and size distributions, and microstructural features (Section 3.1), and then focus attention to the effect of those pretreatments on SC CO2 extraction rate and yield (Section 3.2), and the parameters of a mathematical model of the extraction process (Section 3.3). 3.1. Characterization of pretreated herbs Fig. 1 presents RosinRommlerBennett (RRB) function plots of cumulative undersize distribution, F(dp), for conventional and lowtemperature milled samples of boldo and oregano. The RRB function plot assumes the following relationship between F and dp (Manohar and Sridhar, 2001; Nagy et al., 2008):

dpS P
i

mi 3

P
i

mi
1 t

P
j

mj P
j mj dpj

d2

pi

where i represents a fraction of particles (mass mi) having a thickness (t) smaller than the average sieve-opening (dpi), which were treated as disks of diameter dpi and height (2l), and j, a fraction of particles (mass mj) whose thickness was above the average sieveopening and which were treated as spheres of diameter dpj. The mean Sauter diameter of rapidly-decompressed samples was estimated by assuming they were shaped as 2.36-mm-diameter disks of height (2l), where thickness (2l) was estimated as the average cross section height of seven light-microscopy images of untreated boldo leaf and oregano bract samples. Table 1 summarizes physical properties of the milled samples, including the RRB model parameters derived from Fig. 1, mean Sauter diameters, and total porosities (e) estimated using Eq. (3) (Nagy et al., 2008):

e1

qb : qs

Please cite this article in press as: Uquiche, E., et al. Effect of boldo (Peumus boldus M.) pretreatment on kinetics of supercritical CO2 extraction of essential oil. Journal of Food Engineering (2011), doi:10.1016/j.jfoodeng.2011.10.013

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Table 1 Physical properties of pretreated boldo leaves and oregano bracts. Sample pretreatment Conventionally-milled boldo leaves Low-temperature-milled boldo leaves Rapidly-decompressed boldo leaves Conventionally-milled oregano bracts Low-temperature-milled oregano bracts Rapidly-decompressed oregano bracts Moisture (g water/100 g) 5.07 0.08 5.82 0.18 5.51 0.12 5.84 0.12 5.63 0.38 5.25 0.26 2l (mm) 0.432 0.037 0.432 0.037 0.432 0.037 0.369 0.061 0.369 0.061 0.369 0.061 n () 3.71 3.52 3.58 3.88 (dp)ref (mm) 0.480 0.476 0.445 0.408 dpS (mm) 0.385 0.380 0.949 0.354 0.339 0.844

qs (kg/m3)
1057.3 4.1 1057.3 4.1 1057.3 4.1 984.6 1.2 984.6 1.2 984.6 1.2

qb (kg/m3)
284.7 4.2 283.3 7.2 209.7 2.5 283.3 4.2 283.3 5.3 98.0 3.5

e ()
0.730 0.732 0.801 0.713 0.713 0.900

There were only minor differences in the uniformity coefcient of the particle size distribution, which ranged from 3.5 to 3.9, indicating a similar data scattering for all samples, whereas the particle size parameter was approximately the same for the boldo samples (480 lm), but varied between the conventionally (450 lm) and low-temperature (410 lm) milled samples of oregano. These differences translated into similar difference between mean Sauter diameters that were slightly smaller than particle size parameters in all cases. In any case, unlike Manohar and Sridhar (2001), who found that cryogenic milling produced turmeric particles that were about 10 times smaller than those produced by conventional milling (8.56 lm versus 88.2 lm Ferret diameter), authors did not observe large differences between conventional- and lowtemperature-milled samples. This was probably due to differences in mechanical properties of the treated samples, and in process temperatures between the two studies. Indeed, it is reasonable to assume that turmeric stalk samples were more brittle in the cryogenic milling experiments of Manohar and Sridhar (2001) than oregano bracts and boldo leaves used in our low-temperature milling experiments, which resulted in smaller particles in their case than ours (8.56 lm versus 339380 lm). On the other hand, the average particle diameters were much larger for the rapidly decompressed than either of the milled samples for both oregano and boldo (Table 1) because those samples were manually cracked prior to treatment, and the rapid decompression treatment did not result in a further reduction in particle size but a selective destruction of essential-oil-containing structures. The larger particles of rapidly decompressed samples resulted in smaller apparent densities than those of milled samples, because of the increased difculty in accommodating large as compared to small particles in the void spaces between particles in packed beds. This, in turn, resulted in larger total porosities for rapidly decompressed than milled samples especially in the case of oregano bracts (cf. Table 1). It is important stressing that values of e in Table 1 incorporate both the inner porosity of the particles (that should change little with herb pretreatment) and the interparticle porosity of the packed bed (that should decrease considerably as a result of particle size reduction during milling). There is limited information on the specialized structures that store essential oils in boldo, a plant of the Monimiaceae family. Martnez-Laborde (1988) described the glands or hairs on the surface of boldo leaves as being stellate (star-shaped), Bisset (1994) presented microphotographs of stellate trichomes on the surface and oil cells in the spongy parenchyma of boldo leaves, and Uauy (1998) presented microscopic evidence of secretory cavities within boldo leaves. Secretory cavities are spherical structures where plants store the essential oils (Zizovic et al. 2007; Stamenic et al. 2008), resins, and/or other secondary metabolites used to ght against, e.g., microorganism and insects. These substances are synthesized in the epithelial cells that line the cavities, and are subsequently secreted into them (Salisbury and Ross, 1992). Upon rupture of the cavities, resins and essential oils are secreted to offer protection and/or provide the characteristic plant scent. On the other hand, there is extensive information in literature about glandular trichomes or glands; the specialized storage struc-

tures for essential oils on the surface of oregano bracts (Bosabalidis and Tsekos, 1982, 1984; Gaspar et al., 2001) and other Lamiaceae herbs (Zizovic et al., 2005, 2007; Krstic et al., 2006). These glands have the same function as secretory cavities in boldo leaves, and their amount depends on the species and exhibits plant-to-plant variability (Krstic et al., 2006). Fig. 2 presents light microscopy images of conventionally milled (Fig. 2A and B), low-temperature milled (Fig. 2C and D) and rapidly decompressed (Fig. 2E and F) boldo leaves (Fig. 2A, C, and E) and oregano bracts (Fig. 2B, D, and F). There is a stellate trichome clearly visible on the upper surface of the conventionally milled boldo leaf in Fig. 2A. The presence of unruptured cavities/glands could explain a limited extraction rate due to microstructural limitations to intraparticle essential oil transport in the conventionally milled samples. This is very important considering that the extraction rate of plant essential oils has been claimed to be controlled by internal resistance to mass transfer in the vegetable structure (Gaspar et al., 2001; Reverchon and De Marco, 2007). Fig. 2C shows detachment of trichome, supercial shrinkage, and a pronounced change in inner microstructure in the low-temperature milled boldo sample which may have freed essential oils and favor their intraparticle transport during SC CO2 extraction. Advantages of cryogenic-milling for sample preparation prior to extractions have been reported, but there is limited microscopy evidence in literature about the advantages of this size-reduction method. Fig. 2E shows the disruption of a stellar trichome on the upper surface of a rapidly-decompressed boldo leaf treated with CO2, and disruption of the intraparticle cellular structure (cf. lower left side and compare with Fig. 2A) which suggest specicity of this treatment to free entrapped essential oils so as to favor their extraction with SC CO2. Microphotographs showed similar improvements in sample disruption when applying low-temperature milling (Fig. 2D) or rapid-decompression (Fig. 2F) instead of conventional milling (Fig. 2B) to oregano bracts. Shrinking, detachment, and/or disappearance, of the glands seen in the upper and lower face of the bracts in Fig. 2B suggest freeing of essential oil during low-temperature milling or rapid-decompression treatments. The effect of rapid decompression on supercial glands of aromatic herbs was explained by the slow penetration of CO2 into the glands when exposed to high-pressure CO2 until the oil becomes saturated in CO2 which may result in bursting of a fraction of the glands, e.g., 39.2% of the trichomes in mentha leaves (assessed by SEM) following 1 h exposure to SC CO2 at 40 C and 10 MPa, as compared to 34.9% trichome disruption by conventional milling (Zizovic et al., 2007). Stamenic et al. (2010) provided visual evidence of the swelling on a mint leaf upon exposure to SC CO2 at 40 C and 10 MPa. The effectiveness of the treatment increases if the exposure to highpressure CO2 is followed by a rapid decompression because of the pressure gradient that builds up across the walls and epidermis of glands and opposes free ow of CO2 thus resulting in the disruption of the glands when the pressure drop across those barriers surpasses their outbreak pressure (Gaspar et al., 2001). As an example, Gaspar et al. (2003) reported a 85% efciency in the disruption of oregano bract glands following rapid (113 s) decompression of samples held 1 h is SC CO2 at 40 C and 7 MPa.

Please cite this article in press as: Uquiche, E., et al. Effect of boldo (Peumus boldus M.) pretreatment on kinetics of supercritical CO2 extraction of essential oil. Journal of Food Engineering (2011), doi:10.1016/j.jfoodeng.2011.10.013

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Fig. 2. Light micrographs of cross sectional areas of (A, C, E) boldo leaves and (B, D, F) oregano bracts exposed to (A, B) conventional milling, (C, D) low-temperature milling, or (E, F) rapid decompression. The magnication of all images is the same (bar = 200 lm).

3.2. Effect of sample pretreatment on SC CO2 extraction rate and yield Fig. 3 describes the kinetics of extraction of pretreated boldo leaves (Fig. 3A) and oregano bracts (Fig. 3B) using 7.2 g/min of SC CO2 at 40 C and 10 MPa (U = 1.24 mm/s). Curves approach horizontal asymptotes (not shown) that represent the yield of the process (Cso) in grams of extract per kilogram of dry substrate. Based on longer preliminary SC CO2 extraction experiments, the values of Cso reported in Table 2 were adopted as 5% above those observed after 2-h treatment that can be read from Fig. 3. Reported values of essential oil content in boldo leaves range from 4 g/kg (del Valle et al., 2005) to 31 g/kg (Quezada et al., 2004), which illustrates typical variations in biologic samples due to genetic variations or differences in harvest time, drying treatment, and storage condition (Miraldi et al., 1996; Vogel et al., 1999). The essential oil content also depends on sample pretreatment being larger in the low-temperature milled (10.8 g/kg) or rapidly decompressed samples (11.4 g/kg) than in the conventionally milled samples (9.63 g/kg). This was as expected from microscopy results in Section 3.1 that

suggested a more pronounced liberation of essential oils in lowtemperature milled or rapidly decompressed than conventionally milled samples. The heat generated during grinding results in losses of volatile compounds, which can be reduced by lowering the initial temperature of the material prior to grinding, as done in cryogenic grinding operations (Murthy and Bhattacharya, 2008). Indeed, the use of liquid nitrogen for sample cooling allows samples to absorb the heat generated during grinding without experiencing a temperature that will result in volatile losses or thermal degradation reactions (Krejcov et al., 2008). Data in Fig. 3A also suggests a faster initial extraction of essential oils from rapidly decompressed boldo leaves than either milled sample, and only following a short initial extraction period does the yield from the low-temperature milled sample approach that of the rapidly decompressed sample. These differences in initial extraction rate may be due to the selective destruction of mass transfer barriers to essential oil extraction during the rapid decompression of the CO2-impregnated samples (Gaspar et al., 2000, 2001, 2003). Gaspar et al. (2001) reported the effectiveness of

Please cite this article in press as: Uquiche, E., et al. Effect of boldo (Peumus boldus M.) pretreatment on kinetics of supercritical CO2 extraction of essential oil. Journal of Food Engineering (2011), doi:10.1016/j.jfoodeng.2011.10.013

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12

10

B
5 4 3 2 1 0 0 20 40 60 80 100 120 Conventional-milling Cryogenic-milling Rapidly-expanded

Thus, values of Cfo reported in Table 2 do not correspond to true solubilities; they represent the fraction of essential oils freed by sample pretreatment and released during the static extraction period. Thus, the slightly higher value of Cfo of the rapidly decompressed sample as compared to the milled samples in spite of a 20% reduction in the load of the extraction vessel supports the claim of an improved release of essential oil components from secretory cavities in boldo leaves by that treatment. Qualitatively, the trends observed for the pretreated oregano samples (Fig. 3B) are quite similar to those observed for the boldo samples (Fig. 3A), with the only differences being due to the lower essential oil content in oregano bracts than boldo leaves. Thus, our results conrm trends already reported by Gaspar et al. (2000). Conditioning to low-temperature prior to milling reduces losses of volatile compounds as a result of heat generation during grinding, whereas rapid decompression of CO2-impregnated samples selectively disrupts glandular trichomes holding essential oils in selected plants, thus facilitating SC CO2 extraction processes. There is another difference between oregano bracts and boldo leaves regarding apparent solubility that was considerably lower in rapidly decompressed than milled samples (Table 2). In this case, Cfo is probably dened by essential oil availability instead of essential oil solubility, which paralleled a decrease in the loading capacity of the extraction vessel and an increase in total porosity for oregano bracts as compared to boldo leaves. The negative impact on essential oil availability of the rapid decompression pretreatment of the decrease in oregano bract load as compared to boldo leaf load was compounded by the lower essential oil content in the former than the latter.

Extract yield (g / kg dry substrate)

Extraction time (min)


Fig. 3. Effect of substrate conditioning on cumulative extraction plots of essential oil versus time for (A) boldo leaves and (B) oregano bracts using supercritical CO2 at 40 C and 10 MPa (U = 1.24 mm/s). Samples were exposed to (} ) conventional milling, (s ) low-temperature milling, or (4 ) rapid decompression. Symbols and bars represent average and standard deviation of experimental data (duplicates) and lines best-tting curves.

3.3. Mathematical modelling of SC CO2 extractions Cumulative extraction curves in Fig. 3 were best-tted to the diffusion model of Araus et al. (2009), which describes the concentration of extract in the substrate and the SC CO2 phases as a function of extraction time and distances from the particle axis and entrance of the extraction vessel. This model assumes non-porous, homogeneous, slab-shaped particles of thickness (2l) containing a single pseudo-solute; a constant partition coefcient (K) of the pseudo-solute between the solid matrix and SC CO2; and constant physical properties of pretreated substrate and SC CO2 in the packed bed during extraction. In this work, authors solved numerically the differential mass balance equations of the model using a single value of K for each substrate, and a pretreatment-dependent effective diffusivity coefcient (De) as adjustable model parameters. K is sometimes estimated as the ratio between the initial extract content Cso and apparent solubility of the extract Cfo. Although for a single extraction condition K should be a function of the extract and substrate surface chemistries only, which for pretreatments such as those studied here do not change, values of the ratio Cso/Cfo varied depending on herb pretreatment in this work (cf. Table 2). Thus, K was made independent of the Cso/Cfo ratio in this work as

the rapid decompression pretreatment in disrupting glandular trichomes in oregano bracts, and our results suggest that this pretreatment in also effective to disrupt secretory cavities in boldo leaves. An alternative to characterize initial extraction rates is using the so-called apparent solubility concept (Cfo), which is the initial slope of a plot of cumulative yield (gram of extract per kilogram of dry substrate) versus specic CO2 consumption (kilogram of CO2 used per kilogram of dry substrate) (data not shown). Units of these slopes (gram extract per kilogram of CO2) coincide with the ones of true solubilities, but the property is named apparent solubility because it is inuenced by extract availability and binding to the solid matrix. Because the critical pressure of binary (CO2 + essential oil component) systems at 40 C in slightly below 10 MPa (del Valle et al., 2011), they are miscible in SC CO2 under process conditions.

Table 2 Substrate characteristic, extraction yield and model parameters for supercritical CO2 extraction experiments (40 C and 10 MPa) of essential oils from boldo leaves and oregano bracts. Sample pretreatment Conventionally-milled boldo leaves Low-temperature-milled boldo leaves Rapidly-decompressed boldo leaves Conventionally-milled oregano bracts Low-temperature-milled oregano bracts Rapidly-decompressed oregano bracts Cso (g/kg) 9.63 10.8 11.4 5.06 5.33 5.42 Cfo (g/kg CO2) 0.855 0.887 0.934 0.507 0.475 0.127 K () 20.2 20.2 20.2 23.4 23.4 23.4 kf 105 (m/s) 10.8 10.8 9.24 11.1 11.0 9.50 DL 108 (m2/s) 9.27 9.28 9.55 9.24 9.30 9.67 De 1012 (m2/s) 6.02 7.64 65.6 6.22 6.95 38.5 FM () 2204 1737 202 2162 1935 349

Please cite this article in press as: Uquiche, E., et al. Effect of boldo (Peumus boldus M.) pretreatment on kinetics of supercritical CO2 extraction of essential oil. Journal of Food Engineering (2011), doi:10.1016/j.jfoodeng.2011.10.013

E. Uquiche et al. / Journal of Food Engineering xxx (2011) xxxxxx

proposed by Perrut et al. (1997) in their sorption isotherm/isobar model. Additional parameters of the mathematical model include the lm mass transfer coefcient (kf), that was estimated using the dimensionless relationship of Puiggen et al. (1997), and the axial dispersion coefcient (DL), that was estimated using the equation of del Valle et al. (2011). These calculations required in turn estimates of the physical properties of the SC CO2 phase. The effect of dissolved essential oil on the density (q) and viscosity (l) of the SC CO2 was neglected, being these properties estimated using NIST Standard Reference Database (2007) for pure CO2 (q = 628.6 kg/m3; l = 4.78 105 kg m1 s1 at 40 C and 10 MPa). Values of the binary diffusion coefcient of essential oil in CO2 (D12) were estimated using the correlation of Catchpole and King (1994) as a function of the reduced temperature (Tr = T/Tc = 1.029, where Tc is the critical temperature of CO2, 304.3 K) and reduced density (qr = q/qc = 1.351, where qc is the critical density of CO2, 467.6 kg/m3) of the SC CO2 phase, and the molecular mass (MW2) and critical volume (Vc2) of the essential oil. Using values of MW2 and Vc2 reported by del Valle et al. (2011), that were in turn based on literature reports on essential oil composition of boldo leaves and oregano bracts, the estimated binary diffusion coefcients at process conditions were D12 = 1.33 108 m2/s for boldo essential oil (MW2 = 166.6 g/mol, Vc2 = 521.0 cm3/mol) and D12 = 1.34 108 m2/s for oregano essential oil (MW2 = 151.9 g/mol, Vc2 = 514.5 cm3/mol). Fig. 3 includes best-t curves for the extraction of herb essential oils using CO2 at 40 C and 10 MPa. It is apparent that the diffusional model of Araus et al. (2009) adequately describes the extraction of essential oils from boldo leaves (Fig. 3A) and oregano bracts (Fig. 3B) when using K and De as tting parameters. Best-tted values of K and De were of similar order of magnitude independent of the substrate and substrate pretreatment (Table 2). There were small but consistent differences in effective diffusivity values as a function of sample pretreatment, with the largest value being associated with rapid decompression, and the smallest with conventional milling, which mimics the effect of sample pretreatment in extraction rate. This reveals the effectiveness of rapid decompression as pretreatment to modify herb microstructure (rupturing secretory cavities and glandular trichomes thus releasing essential oils in boldo leaves and oregano bracts, respectively) so as to improve diffusion mechanisms controlling extraction rates, as attested by microscopy evidence (Fig. 2). It is better to discuss the effect of substrate pretreatment on extraction rate as a function of the so-called microstructural factor FM (Araus et al., 2009; del Valle et al., 2011):

at the beginning, whereas less soluble sesqueterpene and oxygenated compounds are extracted later (del Valle et al., 2011). However, changes in D12 differ little between typical essential oil components. For example, D12 of a heavy sesquiterpene ester (farnesyl acetate) is only 75% that of a monoterpenene hydrocarbon (limonene), and only when a large amount of low solubility waxes and triglycerides is incorporated to the extract would D12 decrease to ca. 45% the value of a typical monoterpene hydrocarbon (del Valle et al., 2011). When authors recalculated kf and DL using D12 = 1.00 10-8 m2/s to simulate extraction curves using best t values of De informed in Table 2, differences to curves in Fig. 3 were minimal (data not shown). Authors concluded that small differences in D12 associated to small differences in chemical makeup of extract should not affect best-t values of De to a great extent. Of course the values of FM should change, but not the pretreatment-associated differences between samples. 4. Concluding remarks Low-temperature milling and rapid decompression of CO2impregnated samples were effective pretreatments to increase the rate and/or yield of SC CO2 extraction processes for herb essential oils. Low-temperature conditioning may reduce losses of volatile compounds during milling and other thermally-induced deteriorative reactions, thus increasing extraction yield and improving extract quality. Cryogenic conditions can make a vegetable matrix more brittle thus improving particle size reduction during milling, but this was not observed in our experiments. The reduction in particle size increases the specic surface area thus allowing faster extractions, but there are hydrodynamic limitations to the ow of SC CO2 in a packed bed that may preclude the use of very small particles in extraction experiments. Rapid decompression allows not only a selective disruption of glandular trichomes in the surface of Lamiaceae herbs that hold essential oils, but also of the secretory cavities within the plant tissue having a similar function in other herbs (e.g., boldo leaves). This tissue disruption facilitates mass transfer during SC CO2 extraction as attested by a large decrease of the microstructural factor that characterizes the effect of the solid matrix on the extraction rate. The extraction of the essentials oil from aromatic herbs can be described by a diffusional model which assumes that the partition of essential oils between the solid substrate and CO2 phases is constant, and which uses the effective diffusivity of the extract in the solid matrix as tting parameter. Acknowledgment Funding by Comisin de Investigacin Cientica y Tecnolgica (Fondecyt project 105-0675) from Chile is greatly acknowledged. References
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D12 FM : De

Values of FM in Table 2 suggest that values of D12, which depend only on essential oil composition and extraction conditions, are 2002000 times larger than values of De, which depend also on substrate pretreatment. In the study of Araus et al. (2009) values of FM were from comparable to 10 times as large as those reported in Table 2 (ca. 2.5 104 in the case of oregano). Values covered an ever wider interval (102 < FM < 109) in the review of del Valle et al. (2011), but values of FM depend critically on the assumptions of mass transfer models tted to SC CO2 extraction data, that may differ from actual conditions under which extractions takes place. Being the model applied the same in the two works, values of FM reported in Table 2 approach closely those reported by Araus et al. (2009). Values of D12 in Eq. (4) may be questioned because authors did not measure the chemical make up of boldo and oregano extracts. The composition of extracts typically changes during extraction because more soluble monoterpene hydrocarbons are extracted

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