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Supercritical extraction of petals and pellets of marigold flowers using ethanol-modified CO2

K. Araus,a F. Temelli,b J.M. del Valle,a J.C. de la Fuente,c,* & P. Robertd


a

Departamento de Ingeniera Qumica y Bioprocesos, Pontificia Universidad Catlica de Chile, Santiago


(kaaraus@uc.cl, delvalle@ing.puc.cl).
b
Departament of Agricultural, Food and Nutritional Science, University of Alberta, Canada
(Feral.Temelli@ales.ualberta.ca).
c
Departamento de Ingenera Qumica y Ambiental, Universidad Tcnica Federico Santa Mara, Valparaso, Chile
*
Centro Regional de Estudios en Alimentos Saludables, Blanco 1623, Valparaso, Chile
(juan.delafuente@usm.cl).
d
Departamento de Ciencia de Alimentos y Tecnologa Qumica, Universidad de Chile, Santiago, Chile
(proberts@uchile.cl).

ABSTRACT
The food industry requieres green technologies such as supercritical extraction using innocuous and environmentallyfriendly solvent (e.g., CO2) to isolate high-value compounds from biological substrates (e.g., lutein esters from marigold)
to fulfill consumer-driven demands of bioactive compounds for nutraceuticals and function foods. In this work, oleoresin
was extracted from marigold (Tagetes erecta L.) flower petals using pure and ethanol-modifed CO2. Extraction yields
following 6-h extraction increased when pelletizing dry petals, increasing extraction temperature (40 or 60 C),
increasing extraction pressure (25 or 55 MPa), or adding 5% (w/w) ethanol to CO2. Reconstitution of petals and pellets
with saponified oleoresin increased apparent solubility, especially of petals, that had a larger surface per unit volume
(smaller particle size) than pellets, and when using ethanol-modified CO2, because of the added polarity of lutein
following de-esterification. As compared to its untreated counterpart, yield of saponified oleoresin from reconstituted
petals increased because there was some residual oleoresin in the petroleum-ether-extracted substrate. On the other hand,
as compared to its untreated counterpart, yield of saponified oleoresin from reconstituted pellets decreased, because of
difficulties in infiltrating the petroleum-ether-extracted substrate. There was no effect of sample pretreatment and
extraction conditions on the selectivity of supercritical CO2 for the extraction of carotenoids. The carotenoid content
increased from 8-12% to 40-50% of the extract as a result of the saponification of the oleoresin. On the other hand, the
lutein content in extracts decreased from 12-17% to 2-6% of all carotenoids as a result of oleoresin saponification. This
work findings demonstrate the potential for supercritical fluid extraction of lutein from marigold flowers and provide
insight into the mechanism of extraction of this high-value compound.
Keywords: Apparent solubility; carbon dioxide; co-solvent; marigold flower petals; pelletization.

INTRODUCTION
The main xanthophyll in petals of marigold (Tagetes erecta L.) flowers is lutein, which represents ca. 80-90% of all
carotenoids [1]. Lutein is used as a natural colorant in foods and feeds because of its yellow colour, and as an antioxidant
in nutraceuticals because it protects retina against photic damage. Consequently, the food and pharmaceutical industries
are currently interested in isolating lutein from natural matrices using green technologies, so as to comply with
tightening legal regulations. One of those technologies is SuperCritical (SC) carbon dioxide (CO 2) extraction, which
produces organic-solvent-free extracts with minimal thermal damage and improved quality. Skerget et al. [2] extracted
marigold flower petals using SC CO2 at 30 MPa and found small differences in yield of lutein diesters (and none in total
oleoresin yield) between 40 and 80 C.
There are reports on the use of supercritical CO2 extraction of low-solubility (high-molar-mass and/or polar) solutes such
as carotenoids using co-solvents, whose effectiveness depends on the solid matrix and target solute. Because such cosolvents should be highly soluble in CO2 and have GRAS status, ethanol has been applied extensively as co-adjuvant for
the extraction of heavy, polar solutes. Ethanol has been successfully added to SC CO2 as a co-solvent to extract several
carotenoids from paprika (Capsicum annuum L.) [3], and -carotene and lutein from stinging neetle (Urtica dioica L.)
[4]. Additional successful examples include several carotenoids from microalgae (-carotene and xanthophylls from
Spirulina pacifica [5], and astaxanthin from Haematococcus pluvilis [6]), and astaxanthin from crustacean shells [7]. In
marigold, lutein is free or esterified with lauric, myristic, palmitic, and stearic acid, having the degree of esterification a
large effect on the molecular weight and polarity, and thus in the solubility of marigolds lutein. Free (unesterified) lutein
is possibly more soluble in SC CO2 than the less polar mono- or diesterified counterparts. The solubility of lutein diesters
in SC CO2 at 40 C and 28 MPa increases 1.4-fold when adding 7% (v/v) ethanol to the CO2 [8], and a larger effect in
solubility would be expected in the case of the more polar free lutein. Despite the positive effect of ethanol in the
solubility of lutein and derivatives, to the best of the authors knowledge there are no reports on the extraction of lutein
from marigold flowers using ethanol-modified SC CO2.

Besides the use of modifiers, substrate pretreatment is instrumental in increasing extraction rate and yield which, in the
case of vegetable substrates, improves when the accessibility of SC CO2 to the extract increases as a result of cell wall
fracturing during sample conditioning and extraction [9]. The yield of lutein esters from marigold using SC CO2 at 55 C
and 32.5 MPa increases from ca. 4.98 g/kg (dry basis) to 6.87 g/kg when simultaneously applying ultrasound to help
release oleoresin from the cells [10]. Size reduction during milling increases the specific surface (surface-to-volume
ratio) of vegetable substrates, as well as the fraction of easily available superficial extract [11], but milling does not break
the walls of inner cells in the tissue. Pelletization, extrusion, and related processes, on the other hand, break cell walls
relying in shear efforts, in such a way that their effectiveness does no depend on a reduction in particle size. Pelletization
is ideally suited for SC CO2 extraction of vegetables because the inner microstructure of the pelletized substrate becomes
a network of interconnected, open pores containing all the solute originally contained in the cells [12]. Another advantage
of pelletization is that increases the bulk density of the solid substrate in the packed bed, so as to eventually increase the
volumetric productivity (weight of extract per unit vessel volume and per unit time) of the extraction vessel [13,14]. To
the best of the authors knowledge there are no reports on the use of pelletization or alternative densifying treatments
prior to the extraction of marigold flower petals.
The objective of this work was to study the effects of substrate pelletization, temperature, pressure, and ethanol addition
on the extraction rate and yield of oleoresin, total carotenoids, and lutein from marigold flower petals.
MATERIALS & METHODS
Fresh petals of marigold flowers were dried in the shadow (11.9% moisture) or pelletized and dried (4.36% moisture).
Both the petals and pellets were stored in a freezer at -18 C until analysis. In selected experiments, petals and pellets
were reconstituted with saponified oleoresin having high-solubility (because of replacement of lutein esters by more
soluble free lutein). For that, intact petals or pellets were extracted with petroleum ether, and the resulting oleoresin was
saponified as informed at the end of this section. Ten-gram samples of petroleum-ether-extracted petals and pellets were
submerged under continuous agitation in a beaker with a diethyl ether solution containing 0.114 g saponified oleoresin,
that was placed in a closed (light-protection purposes) fume hood at room temperature. A 20-g sample of diethyl-ethercleaned glass beads was similarly impregnated in a diethyl ether solution containing 0.3 g of saponified extract. Diethyl
ether was removed from impregnated petals, pellets, and beads by a nitrogen stream under vacuum.
SC CO2 extraction experiments were carried out in duplicate in a one-pass, laboratory unit using 0.060 m3 (NPT)/h of
CO2. Dried petals and pellets were extracted with pure CO2 at 40 or 60 C and 25 or 55 MPa, or with CO2 complimented
with 5% (w/w) ethanol (at 60 C and 25 MPa only). Reconstituted petals and pellets and impregnated glass beads were
also extracted at 60 C and 25 MPa using pure and ethanol-modified SC CO2. Extracted oleoresin was collected in vials
at regular time intervals during extraction for 6 h, and quantified gravimetrically. Oleoresin samples of single
experiments were pooled in a single vial, sealed under a nitrogen stream, and kept in a freezer at -18 C up to analysis.
Untreated samples of dried marigold petals and pellets were analyzed to determine water, oleoresin, total carotenoids,
lutein, and -carotene contents; extracted petal and pellet samples were analyzed to determine water and oleoresin
contents; and pooled extract samples were analyzed to determine total carotenoids, lutein, and -carotene contents.
Moisture (water content) was measured by drying samples (finely milled in a coffee grinder) in an oven to constant
weight. Oleoresins were measured by extracting the finally milled samples with petroleum ether to exhaustion in a
Soxhlet apparatus. Total carotenoids were measured by UV/VIS spectrophotometry at 445 nm [15]. Lutein and carotene in saponified oleoresin samples were measured by HPLC using the method of Giuffrida et al. [16]. For
saponification purposes, done as described by Rodrguez-Amaya [15], oleoresin samples were dissolved in diethyl ether
and the solution was added to a 20% KOH methanolic solution containing BHT (to prevent oxidation). Saponification
was done at room temperature overnight, with the reaction mixture under continuous agitation and protected from light.
At the end, the organic (diethyl ether) phase containing saponified oleoresin was washed up with water up to reaching
neutral pH, and filtered over a layer of anhydrous Na2SO4.
RESULTS & DISCUSSION
Figure 1 shows cumulative extraction plots of oleoresin yield (g/kg substrate) versus specific CO2 consumption (kg/kg
substrate) for marigold flower petals as a function of sample pretreatment, extraction temperature, extraction pressure,
and use of ethanol as co-solvent. The initial slope of these cumulative plots (in g oleoresin/kg CO2) corresponds to a socalled apparent solubility of the oleoresin in CO2 under process conditions (temperature and pressure), that should
coincide with the corresponding thermodynamic solubility when the content of easily available (free) solute in the
substrate and the contact time between the substrate and CO2 are large enough to allow true equilibrium conditions to be
reached between the oleoresin and SC CO2 [17]. The apparent solubility is a relevant parameter (defines extraction rate)
in the initial stages of solubility-controlled processes. On the other hand, the horizontal asymptote of cumulative plots
(maximal yield, in g oleoresin/kg substrate) defines the fraction of the oleoresin that is soluble in SC CO2 under process
conditions. Figure 1A and Figure 1B show that the initial extraction rate (apparent solubility) and yield (soluble

Figure 1. Supercritical CO2 extraction of marigold flower (A) petals and (B) pellets at ( ) 40 C and 25 MPa, ( ) 40 C and 55 MPa,
( ) 60 C and 25 MPa, and ( ) 60 C and 55 MPa. (C) Supercritical extraction of marigold flower ( , ) petals or ( , ) pellets
using ( , ) pure of ( , ) ethanol-modified (5% w/w) CO2 at 60 C and 25 MPa.

fraction of oleoresin) increase with extraction temperature and pressure. The effect of an increase in temperature from 40
to 60 C is more pronounced than the effect of an increase in pressure from 25 to 55 MPa. Solubility increases as a result
of an increase in the solvent power of SC CO2 (that increases as its density increases) or the volatility of the solute (that
increases as its vapor pressure increases) [17]. Results are as expected in that the density of CO2 increases with system
pressure, and the volatility of carotenoids and other components in marigold oleoresin increases with system temperature.
At near-critical pressures (ca. 7-10 MPa in the case of CO2) density decreases pronouncedly when increasing temperature
and this negative effect overshadows the positive one of temperature on vapor pressures, with the end result of a
reduction in solubility [17]. Farther away from the critical pressure, above a solute-dependent crossover pressure
(typically 15-20 MPa), the increase in volatility predominates over the decrease in density and solubility increases as
temperature increases [17]. Thus, results in Figure 1 are consistent with an increase in solubility of oleoresin components
with an increase in extraction temperature and pressure, and a crossover pressure below 25 MPa.
Extraction rate and yield of oleoresins from marigold flowers depended on sample pretreatment in that the apparent
solubility was smaller and the final yield larger using pellets (Fig. 1B) than petals (Fig. 1A), with the end result of
extraction curves that crossed each other. (This can be more clearly appreciated in Fig. 1C for extractions at 60 C and 25
MPa using pure SC CO2.) Extractions at 40 C and 25 MPa were exceptional in that cumulative yield was higher for
petals than pellets throughout. If the chemical make-up of oleoresins from petals and pellets coincides, apparent
solubilities should coincide for same extraction conditions between the two, unless it is not determined by the
thermodynamic solubility but rather availability in the substrate. Overall, the oleoresin contents in marigold flower petals
determined by Soxhlet extraction using petroleum ether did not depend on sample pretreatment (88.5 g/kg in petals
versus 94.6 g/kg in pellets). However, a more relevant quantity is free oleoresin in broken surface cells, which increases
with the specific surface of the particles (or the decrease in equivalent diameter). Thus, experimental results are
consistent with milled petals containing more freely available oleoresin (positive feature) and more tied-up oleoresin
(negative feature) than pellets, respectively because of comparatively large specific surface, on one hand, and entrapment
of oleoresin in intact inner cells, on the other. In SC CO2 extractions of marigold petals at 60 C and 25 MPa,
pelletization increased oleoresin yield 17% (46.8 g/kg in pellets versus 40.1 g/kg in petals) as a result of the large shear
stresses that destroyed the original cellular matrix with the end result of releasing virtually all oleoresins from the cells.
More than 6 h are required to fully extract all marigold oleoresins using SC CO 2, because at that time cumulative
extraction curves are still moving upwards, and the petroleum-ether-soluble oleoresin content in marigold petals (ca. 9192 g/kg) was larger than experimentally observed using as the solvent SC CO2 at the most favorable condition tested (cf.
curve for 60 C and 55 MPa in Fig. 1B).
Extraction rate and yield improved as a result of pelletization of marigold flower petals and addition of ethanol as cosolvent (Fig. 1C). In the case of pure SC CO2, the initial extraction rate at 60 C and 25 MPa is faster, but yield after 6-h
extraction is lower using petals than pellets. Ethanol addition increased oleoresin yield, especially of pellets (a 46%
increase, from 46.8 g/kg for pure CO2 to 68.6 g/kg for ethanol-modified CO2). Overall, the combination of pelletization
and ethanol addition increased oleoresin yield by 71% (40.1 g/kg for the extraction of petals using pure CO 2 versus 68.6
g/kg for the extraction of pellets using ethanol-modified CO2). The positive effects of ethanol may be due to an
improvement in the solubility of marigold carotenoids in CO2 (co-solvent effect) [8], and/or competition with carotenoids
for polar binding sites in the solid matrix (modifier effect) [18], which depend only on the chemistry of extract and the
substrate. An additional positive effect is physical modification of the solid matrix favoring inner mass transfer (e.g.,
ethanol-induced swelling) [19] that may be better in pellets than petals.
Table 1 summarizes apparent solubilities (initial slopes of cumulative extraction curves of oleoresin yield versus
specific CO2 consumption, cf. Fig. 1) for different substrates using pure and ethanol-modified CO2 at 60 C and 25 MPa.
For each solvent, no differences were expected between petals and pellets, on one hand, and between reconstituted petals
and pellets, and impregnated glass beads, on the other. Impregnated glass beads had considerably less apparent
solubilities than the other samples, so that we can conclude that there was too little oleoresin available in the system to
saturate the CO2 phase in this case; this was possibly due to the limited specific surface (because of the comparatively
large diameters of the beads) and absence of pores in glass beads. Because of improved solubility of the saponified
oleoresin as compared with the oleoresin, petals and pellets should have had lower apparent solubilities than reconstituted
petals and pellets. This was observed only in the case of extractions with ethanol-modified SC CO2. A conclusion is that
for extractions with pure SC CO2 apparent solubilities are about 1.0-1.2 g oleoresin/kg CO2, independent of the sample,
in cases where there are no limitations in (free) available oleoresin (in this case the result with pellets cannot be
explained). Another conclusion is that for extractions with ethanol-modified SC CO2 apparent solubilities are higher for
petals than pellets, and higher for reconstituted than original substrates. This may be due to an increase in availability of
(free) oleoresin (because of the higher specific surface of petals than pellets) and/or an increase in thermodynamic
solubility (because of replacement of xanthophyll esters by free xanthophylls and other changes brought about by
saponification that improved dissolution in ethanol-modified SC CO2 of saponified oleoresin as compared to the original
oleoresin).
Table 2 summarizes yields and compositions of marigold flower petal extracts from different substrates as a function of

Table 1. Apparent solubilities of oleoresin from petals, pellets, and glass beads at 60 C and 25 MPa.
Substrate

Apparent solubilities (g oleoresin/kg CO2)


Without EtOH

With 5% (w/w) of EtOH

0.99
1.38
1.19
1.10
0.12

1.67
0.99
1.99
1.68
0.19

Petals
Pellets
Reconstituted petals
Reconstituted pellets
Glass beads

sample pretreatment, extraction temperature, extraction pressure, and use of ethanol as co-solvent. The effects of
pelletization, extraction temperature and pressure, and addition of ethanol as co-solvent on oleoresin yield were
commented and explained before. Yield increased as a result of improved breakage in inner cells walls during
pelletization, increased solute volatility when increasing temperature, and increased solvent power of CO2 when
increasing pressure or adding ethanol to it. Oleoresin yields were higher in petals reconstituted with saponified extract
than the original substrate, which may be due to a large amount of high-solubility (free) extract on the surface of the
reconstituted petals (that had a large specific surface). On the other hand, oleoresin yields were lower in reconstituted
pellets than the original substrate, which may be due to a limited impregnation of the saponified extract within pellets
having small specific surface and thin pores (less easily infiltrated by a liquid solution than SC CO2).
Table 2. Yields of oleoresin and carotenoids of marigold flowers extracts using petals, pellets, and glass beads with pure SC CO2 and
modified with ethanol as co-solvent.
Substrate

Petal

Pellets

Reconstituted
petals
Reconstituted
pellets
Impregnated
glass beads

Extraction conditions
T / P / Added cosolvent
40 C / 25 MPa
40 C / 55 MPa
60 C / 25 MPa
-- / 5% (w/w) ethanol
60 C / 55 MPa
40 C / 25 MPa
40 C / 55 MPa
60 C / 25 MPa
-- / 5% (w/w) ethanol
60 C / 55 MPa
60 C / 25 MPa
-- / 5% (w/w) ethanol
60 C / 25 MPa
-- / 5% (w/w) ethanol
60 C / 25 MPa
-- / 5% (w/w) ethanol

Oleoresin
g/kg

Total
carotenoids
(% oleoresin)

33.8
39.7
40.1
47.2
56.9
30.1
38.3
46.8
68.6
61.7
53.0
89.0
33.0
51.0
1.55
5.55

7.9
8.2
11.6
12.6
19.1
8.8
11.0
4.4
11.3
12.0
36.7
43.6
32.3
54.7
42.6
92.5

Individual carotenoids (% total)


Lutein

-carotene

17.1
11.0
17.7
17.0
20.3
17.6
16.6
12.9
9.3
17.9
2.1
2.4
3.6
5.0
6.1
6.4

0.5
0.6
0.4
0.5
0.2
0.2
0.1
1.0
0.9
0.4
0.4
0.03
0.1

With few exceptions (petals treated at 60 C and 55 MPa, and pellets treated at 60 C and 25 MPa, both with pure SC
CO2), represented 8-12% of extracted oleoresins, which suggest small effects of extraction parameters on selectivity
(Table 2). There was small differences in selectivity for carotenoids in the extraction of saponified oleoresin from
reconstituted petals and pellets in that their percentage in the extract was relatively constant (40-50%), which is possibly
due to their enrichment during saponification (Table 2). Content of carotenoids was even higher in the extract of
impregnated glass beads. This can be due to the residual oleoresin in petroleum-ether-extracted petals and pellets which
were not finely milled prior to Soxhlet extraction to preserve their original microstructural features. This was confirmed
by HPLC analysis of the extracts, which showed a small amount of xanthophyll esters in the case of reconstituted petals
and pellets, unlike in the case of impregnated glass beads (only un-esterified xanthophylls).
On the other hand, the lutein contents in the carotenoid from of oleoresins extracted from petals and pellets from 12-17%
(Table 2). There were also small differences in the contents of lutein (ca. 2-6%) and -carotene (ca. 0.5-3%) in the
carotenoid fraction of extracts from reconstituted petals and pellets, and impregnated glass beads (Table 2). Values,
however, are too small considering the content of lutein in marigold flower petals.
CONCLUSIONS
Oleoresin yield can increase during SC CO2 extraction as a result of shear-stress induced breakage of inner cell walls
during pelletization of marigold flower petals, at the expense of a lower initial extraction rate that depends on breakage of

surface cells and is more favorably influenced by a reduction in particle diameter. Extraction rate and yield increase as a
result of an increase in solubility due to a pressure-induced increase in the density and solvent power of SC CO2, and a
temperature-induced increase in vapor pressure and volatility of the extract, being this valid above a cross-over pressure
that in the case of marigold oleoresin is below 25 MPa. Addition of 5% (w/w) ethanol to SC CO2 also increases
extraction rate and yield due to an increase in solubility of heavy and/or polar components (e.g., xanthophylls and
xanthophylls esters) in marigold oleoresin. The carotenoid content in CO2-extracted oleoresin varied between 8-12% and
the lutein content between 12 and 17% of all carotenoids, independent of extraction conditions. This indicates that that
the selectivity of the process for high-value lutein is not affected by the experimental variables selected in this study.
An attempt was made in this work to discern the effect of extract composition and substrate microstructure on extraction
rate and yield of marigold oleoresin. For that, petals and pellets were extracted with petroleum ether and reconstituted
with saponified oleoresin. Saponification should increase the solubility of the extract as compared to its unsaponified
counterpart because of the reduction in molecular weight resulting from de-esterification of marigold xanthophylls. This
increase in solubility should be even more pronounced when using ethanol as a co-solvent that can facilitate desorption
from the substrate and dissolution of polar compounds, such as free xanthophylls. Results were not fully as expected
because of differences in impregnation phenomena between petals (surface impregnation favored) and pellets (in depth
infiltration disfavored).
Acknowledgements
This work was funded by Chilean agency Fondecyt (Regular project 1111008).
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