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J. of Supercritical Fluids 45 (2008) 189–194

Selective fractionation of carbohydrate complex mixtures by

supercritical extraction with CO2 and different co-solvents
F. Montañés a , N. Corzo a , A. Olano a , G. Reglero b , E. Ibáñez a,∗ , T. Fornari b
a Instituto de Fermentaciones Industriales (CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain
b Sección Departamental de Ciencias de la Alimentación, Universidad Autónoma de Madrid,

Campus de Cantoblanco, 28049 Madrid, Spain

Received 29 May 2007; received in revised form 27 August 2007; accepted 28 August 2007

Abstract
Nowadays consumers hold high standards for the foods they consume and they also demand for products that can provide added healthful
benefits; two of these products with an observed prebiotic activity, tagatose and lactulose, are the core of our study. These two carbohydrates are
ketoses and have important biological activities, such as the stimulation of the growth of beneficial microorganisms in the colon. They are currently
produced by alkaline isomerization of the corresponding aldose, followed by several purification steps to remove carbohydrate by-products and
the unreacted aldose (around 30%).
In this work, the possibility of using supercritical fluid extraction (SFE) technology to fractionate complex carbohydrate mixtures is ana-
lyzed. The study is based on previous results obtained for the fractionation of solid binary carbohydrate mixtures such as lactulose–lactose and
tagatose–galactose. The appropriate selection of the co-solvent employed, together with the most suitable extraction conditions (including temper-
ature, pressure and co-solvent flow rate) allows the selective extraction of the prebiotic ketose with high selectivity and recovery. The fractionation
by SFE of a complex commercial carbohydrate mixture, containing around 74 wt.% of lactulose and 24 wt.% of different aldose carbohydrates,
was carried out at the optimal extraction conditions attained in the study of binary mixtures. The selective extraction of the ketosugar lactulose was
also observed in this case, obtaining an extract with 81 wt.% lactulose and around 67% yield.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Lactulose; Tagatose; Carbohydrate complex mixture; Supercritical fluid extraction; Co-solvents

1. Introduction expensive separation and purification steps to remove catalyst,

Prebiotics are generally defined as non-digestible food ingre-
dients, which stimulate the growth and activity of certain bacteria
in the intestine or colon. These friendly bacteria seem to perform
many important functions such as protection from food-borne
illnesses and allergies, regulating hormone balance, and enhanc-
ing immunity. Many carbohydrates are reported to be prebiotic.
This work is focused in two of these prebiotic carbohydrates,
lactulose and tagatose.
Lactulose (4-o-␤-d-galactopyranosyl-d-fructose) is a syn-
thetic ketose disaccharide obtained from lactose by alkaline
isomerization [1]. The ability of lactulose to stimulate bifidobac-
teria present in the gastrointestinal tract is known since 1957
[2]. Current methods for preparing high-purity lactulose involve
∗ Corresponding author. Tel.: +34 91 5618806x385; fax: +34 91 5644853.
E-mail address: elena@ifi.csic.es (E. Ibáñez).
more than 20% of unreacted lactose as well as small amounts of
by-products such as galactose and tagatose [3] rising the price
of the final product [4].
Tagatose is a low-calorie carbohydrate sweetener, stereoiso-
mer of d-fructose, naturally occurring in some lichens [5] and
in some dairy products [6,7]. Its physical properties ensure ease
of use in a wide range of functional foods, drinks and other
applications. In US is considered a GRAS product (generally
recognized as safe) and its use in foods and beverages has been
approved in several countries [8]. Amongst its known benefits,
tagatose has a minimal impact on blood glucose, has prebi-
otic effects and does not promote tooth decay. Tagatose may
be obtained via a patented two-step process from galactose
using chemical methods involving isomerization of galactose
in the presence of basic catalysts such as calcium hydroxide [9]
and aluminates [10]. The chemical methods have disadvantages,
including complex purification steps, by-products formation due
to the alkaline reaction conditions, and chemical waste forma-

0896-8446/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2007.08.012
190 F. Montañés et al. / J. of Supercritical Fluids 45 (2008) 189–194
tion [11]. Biological tagatose production from galactose using
l-arabinose isomerase has received much research attention dur-
ing the last few years and commercially viable processes have
been reported [11–14]. Although considerably bioconversion
yield can be achieved, removal of remaining galactose is still
required.
In this work, the recovery of ketosugars from solid carbohy-
drate mixtures using SFE technology has been studied. Carbon
dioxide, which is by far the most widely used supercritical fluid,
is especially useful, as it is non-toxic, non-flammable, and non-
ozone depleting, it has a supercritical temperature near ambient
(good for temperature sensitive substances) and it is relatively
inexpensive [15]. On the other side, the solubility of carbohy-
drates in the supercritical phase may considerably be increased
combining supercritical carbon dioxide (SC-CO2 ) with polar co-
solvents [16] that allow changing the polarity of the supercritical
fluid while increasing its solvating power towards the analyte.
The hypothesis of selectively extract a ketosugar from
ketose–aldose mixtures using SFE is supported by the fact that
the solubility of ketoses in alcohols is considerably greater than
those of aldoses [17]. Additionally, previous studies [18,19]
have demonstrated that the selective recovery of ketosugars from
binary solid ketose + aldose carbohydrate mixtures is possible,
providing the adequate co-solvent and extraction conditions.
Fig. 1. Molecular structures of carbohydrates discussed in this study.
Thus, the aim of the present work was to develop an extrac-
tion process, based on the use of SC-CO2 with alcohol-type
co-solvents, for the selective recovery of lactulose from a com-
plex solid carbohydrate mixture, commercially available as
Duphalac® , which contains around 74 wt.% of lactulose and
24 wt.% of aldose carbohydrates. Duphalac® action has greatly
varying effects depending on the dosage: prevention and treat-
ment of portosystemic encephalopathy and constipation, and the
promotion of defecation and fecal excretion of salmonellae in
enteritis. The manufacture of a product with high lactulose purity
from a commercial product, and using a clean technology such
as supercritical fluids, could be of great commercial interest con-
sidering that the dosage and effects is mainly dependant on the
final concentration of lactulose.
2. Materials and methods
2.1. Materials
Sugar standards (d-(+)-galactose, d-(−)-tagatose, d-lactose
monohydrate and lactulose; molecular structures shown in
Fig. 1), internal standard (phenyl-␤-glucoside), methanol and
two derivatizing reagents (N-trimethyl-sylyl-imidazole, and
Chlorotrimethylsilane redistilled) were obtained from Sigma
 F. Montañés et al. / J. of Supercritical Fluids 45 (2008) 189–194
(St. Louis, MO, USA). The other derivatizing agent, pyridine
dried, was supplied by Merck (Darmstadt, Germany). n-
Propanol extra pure and isopropanol extra pure were purchased
from Scharlau Chemie S.A. (Barcelona, Spain). Duphalac®
(Duphar Nezel Solvay, Weesp, The Netherlands) is a medic-
inal product in aqueous solution containing 74 wt.% lactulose,
24 wt.% of aldose carbohydrates (12.2 wt.% galactose, 6.6 wt.%
lactose and 5.2 wt.% epilactose) and 2 wt.% of other ketoses
(just tagatose). Composition data were taken from the label
of Duphalac® syrup. Sea sand, glass wool washed chemically
pure and n-butanol were acquired from Panreac Quı́mica S.A.
(Barcelona, Spain). Ethanol absolute was from Prolabo (Fonte-
nay sous Bois, France). 18.2 M cm Ultrapure water quality
with 1–5 ppb TOC and <0.001 EU/mL pyrogen levels (Milli-Q)
was produced in-house using a Laboratory water purification
Milli-Q Synthesis A10 system (Millipore, Bellerica, MA, USA)
and was used throughout. The carbon dioxide liquefied at high
pressure used in supercritical extraction was supplied by Praxair
Inc. (Danbury, CT, USA).
2.2. Supercritical fluid extraction system
The scheme of the supercritical fluid extraction device
employed to carry out all the experiments can be found else-
where [19]. Extraction processes were performed on samples
placed into the extraction cell (8 cm3 ), consisting of one part of
tagatose–galactose (70:30 mg) or lactulose–lactose (70:30 mg)
or 200 mg of lyophilized Duphalac® mixed with nine parts of
sea sand in a laboratory mill (Janke and Kunkel IKA A-10,
Labortechnik, Staufen, Germany). Samples were packed with
glass wool into the extraction cell. The 70:30 weight ratio in
the binary solid mixtures was selected in order to represent the
ketose:aldose ratio obtained after the isomerization reactions.
A continuous flow rate (1.2 g/min) of CO2 mixed with different
amounts of the polar co-solvent employed in the particular assay
was maintained through the extraction cell for 2 h.
The effects of different factors, such as extraction pressure,
extraction temperature and co-solvent flow rate, on the amount
of carbohydrates extracted was first studied in binary mixtures
in order to select the optimal conditions to approach the purifi-
cation of lactulose from a complex mixture of carbohydrates
(Duphalac® ). In brief, tagatose–galactose and lactulose–lactose
mixtures were independently studied considering extraction
pressures between 100 and 300 bar, extraction temperatures
between 60 and 100 ◦ C, and co-solvent flow rates ranging
from 0.2 to 0.4 mL/min. The optimal conditions were tested
with Duphalac® considering different type of alcohols and
alcohol–water mixtures as co-solvents.
2.3. GC analysis of supercritical extracts
Prior to analyze the collected extracts, we analyzed
Duphalac® without any extraction by gas chromatography. Our
tests confirmed composition data given in the label. Samples as
well as collected extracts were prepared for gas chromatography.
Half a milliliter of the sample was added to 0.5 mL of
a solution of 0.01% phenyl-␤-d-glucoside in methanol/water
191
(70/30 v/v) as internal standard (w/v). Previous to derivatiza-
tion, samples were dried in a rotavapor R-200 (from Büchi
Labortechnik AG, Flawil, Switzerland).
The dried mixtures were added to 100 ␮L of pyridine, 100 ␮L
of N-trimethyl-sylyl-imidazole, and 100 ␮L of chlorotrimethyl-
silane for sylilation; the reaction was carried out instantly at
room temperature. Sylilated carbohydrates were extracted with
0.1 mL of hexane and 0.2 mL of water. Samples derivatized
were injected into a gas chromatograph using a 30 m × 0.32 mm
inside diameter and 0.5 ␮m film fused silica capillary column
SPBTM -17, bonded, crosslinked phase poly (50% diphenyl/50%
dimethylsiloxane) (Supelco, 595 North Harrison Road, Belle-
fonte, PA, USA). Data was acquired by means of HP
ChemStations (Agilent Technologies Inc., Wilmington, DE,
USA).
3. Results and discussion
3.1. SFE of tagatose–galactose solid mixtures
The extraction of tagatose from solid tagatose–galactose mix-
tures has been previously studied by our research group using
SC-CO2 and isopropanol as polar co-solvent [19]. Fig. 2 shows
the total yield of the extraction processes-expressed as the per-
centage of both carbohydrates extracted with respect to the total
amount of carbohydrates placed into the extraction cell together
with the tagatose purity obtained in the extracts, as a function of
the amount of isopropanol dissolved into the SC-CO2 extraction
solvent. Experimental results depicted in Fig. 2 correspond, as
mentioned previously, to different processing conditions, cover-
ing pressures from 100 to 300 bar and temperatures in the range
of 60–100 ◦ C. As can be observed in the figure, the extraction
Fig. 2. Yield (symbols with dot) and composition (empty symbols) obtained
in the SC-CO2 extraction of 70:30 tagatose:galactose solid mixture using iso-
propanol as co-solvent and different temperatures (60, 80 and 100 ◦ C): total yield
of carbohydrates (tagatose and galactose) extracted at ( ) 100 bar, ( ) 200 bar
and ( ) 300 bar; tagatose purity (wt.% tagatose in the extract) at () 100 bar,
() 200 bar and () 300 bar.
192 F. Montañés et al. / J. of Supercritical Fluids 45 (2008) 189–194
Fig. 3. Total yield (dark gray columns) and tagatose purity (light gray columns)
in the supercritical extraction of 70:30 tagatose–galactose solid mixtures
(300 bar, 80 ◦ C and 0.6 mL/min co-solvent) using different alcohols as co-
solvents.
yield linearly increases with the amount of co-solvent and is
almost independent of the extraction pressure and temperature.
Therefore, differences cannot be shown in Fig. 2 considering dif-
ferent extraction temperatures using isopropanol as co-solvent.
Additionally, tagatose purity in the extracts is almost constant
(ca. 90%) what means that extraction pressure and temperature
have a slight effect on selectivity.
Fig. 3 shows the effect of the type of co-solvent employed
(different alcohols) on the SFE process. In this case, all
extractions were carried out at 300 bar, 80 ◦ C and 0.6 mL/min
(20 wt.%) of co-solvent. As can be seen, the total yield consid-
erably increases from 40 to 65% as the polarity of the co-solvent
increases (from n-butanol, n-propanol, isopropanol to ethanol)
with a consequent tagatose purity decrease. As a result, it can
be concluded that the SFE of tagatose–galactose mixtures, con-
taining 70 wt.% tagatose, at 300 bar, 80 ◦ C and with 20 wt.%
of ethanol or isopropanol as co-solvent, produce extracts with
higher tagatose concentrations (ca. 87%) and yields around 60%.
The introduction of small amounts of water in the alcohol co-
solvent was also explored and resulted in a significant increase
of the extraction yield with the consequent decrease of tagatose
purity. This can be observed in Fig. 3, by comparing the use
of pure ethanol and ethanol:water (95:5) as co-solvents; extrac-
tion yield increase from 65 to 78% while a slightly decrease of
tagatose purity from 87 to 85% was observed.
3.2. SFE of lactulose–lactose solid mixtures
Considering the differences in molecular size between
monosaccharides and disaccharides, it is expected that the
disaccharide solubility in SC-CO2 should be lower than the
monosaccharide solubility. Thus, the increase of yield produced
by the inclusion of water in the alcohol co-solvent (observed
in the SFE of tagatose–galactose mixtures) drove us to con-
sider the inclusion of water in the alcohol co-solvent, to study
the SFE of disaccharides. Thus, different ethanol–water mix-
Fig. 4. Yield (symbols with dot) and composition (empty symbols) obtained
in the SC-CO2 extraction of 70:30 lactulose–lactose solid mixture using 95:5
ethanol:water as co-solvent and different pressures (100, 200 and 300 bar): total
yield of carbohydrates (lactulose and lactose) extracted at ( ) 100 ◦ C, ( ) 80 ◦ C
and ( ) 60 ◦ C; lactulose purity (wt.% lactulose in the extract) at () 100 ◦ C,
() 80 ◦ C and () 60 ◦ C.
tures were employed as co-solvent to study the possibility of a
selective recovery of lactulose from lactulose–lactose mixtures.
Nevertheless, preliminary results [18] showed that increasing
the amount of water from 5 to 10 vol.% in the ethanol–water
co-solvent produced a considerably decrease of lactulose purity
in the extracts from 97 to 77%. Thus, extractions were carried
out using 95:5 ethanol:water as co-solvent and exploring the
same extraction pressure and temperature ranges employed in
the monosaccharides supercritical extraction study.
Results obtained are shown in Fig. 4, in a plot similar to
that previously presented for the tagatose–galactose extrac-
tions (Fig. 2). Also in this case, the selectivity of the process
towards the extraction of the ketosugar was almost independent
of the extraction pressure, temperature or amount of co-solvent
employed. Nevertheless, higher ketose purity was achieved since
the wt.% of lactulose increased from 70% to values higher than
94% in all experiments.
Regarding process yield, as expected, the total amount of
disaccharides extracted is considerably lower than in the case
of monosaccharides. Additionally, high temperatures consider-
ably favor the extraction, being the temperature employed more
important even than the amount of co-solvent used. A total yield
of 33% with a low flow rate of co-solvent (0.2 mL/min) was
achieved at 100 ◦ C and 100 bar.
3.3. SFE of Duphalac®
As mentioned previously, Duphalac® is a commercial prod-
uct containing around 74 wt.% of lactulose and 24 wt.% of
aldoses (galactose, lactose and epilactose). Thus, extraction
conditions which resulted favorable for the lactulose–lactose
mixture (100 ◦ C, 100 bar and 0.2 mL/min co-solvent) were
selected to carry out experiments, and different alcohol and
alcohol–water mixtures were employed. The results obtained are
 F. Montañés et al. / J. of Supercritical Fluids 45 (2008) 189–194 193

Table 1
SC-CO2 extraction of Duphalac® (200 mg) at 100 bar, 100 ◦ C and 0.2 mL/min (6 wt.%) co-solvent, using different polar co-solvents
Co-solvent Extract composition

Ga (mg) La (mg) Lu (mg) Ga (%) La (%) Lu (%) Total yield (%)

Ethanol 6.16 ± 0.68 0.97 ± 0.10 30.69 ± 4.76 16.29 2.55 81.16 18.91
Ethanol:water (95:5) 10.17 ± 0.49 7.23 ± 0.25 62.62 ± 1.97 12.71 9.04 78.25 40.01
Isopropanola 0.01 ± 0.00 0.02 ± 0.00 – – – 0.02
Isopropanol:water (95:5) 3.65 ± 0.30 0.06 ± 0.01 2.95 ± 0.63 54.81 0.85 44.34 3.33
a Composition is not reported since the amount of carbohydrates recovered is in the order of the experimental error.

Table 2
SC-CO2 extraction of Duphalac® (200 mg) at 300 bar, 80 ◦ C and 0.6 mL/min (20 wt.%) co-solvent using different polar co-solvents
Co-solvent Extract composition

Ga (mg) La (mg) Lu (mg) Ga (%) La (%) Lu (%) Total yield (%)

Ethanol 21.14 ± 0.69 3.63 ± 0.74 105.62 ± 13.14 16.21 2.78 81.00 66.99
Ethanol:water (95:5) 17.55 ± 0.18 10.13 ± 0.30 93.32 ± 2.15 14.50 8.37 77.12 59.19
Isopropanol 3.81 ± 0.85 1.42 ± 0.42 17.95 ± 5.23 16.44 6.14 77.42 11.39
Isopropanol:water (95:5) 18.24 ± 1.35 1.30 ± 0.01 43.75 ± 2.95 28.83 2.05 69.13 27.75
Isopropanol:water (90:10) 25.86 ± 1.89 3.24 ± 0.13 92.10 ± 3.03 21.34 2.67 75.99 57.42

shown in Table 1. As in the case of the binary lactulose–lactose
SFE, extractions with 95:5 ethanol:water co-solvent produce
yields around 40%, but in this case the composition of lactu-
lose in the extract slightly increased from 74 to 78%. This poor
increase of lactulose purity in the extract, in comparison with
the high increase obtained in the binary lactose–lactulose SFE
(from 70 to 94%), can be attributed to the 12 wt.% of galactose
present in Duphalac® . Although galactose is an aldose carbo-
hydrate it is also a monosaccharide and thus, it can be easier
extracted than the ketodisaccharide lactulose.
The results depicted in Table 1 also show that very low yields
were obtained when isopropanol was used as co-solvent. Fur-
thermore, only a slight enhance of yield was observed when
5 vol.% of water was dissolved in isopropanol, and thus an
increase of water content was not explored. In turn, optimal
extraction conditions obtained in the tagatose–galactose SFE
study (i.e. when isopropanol was employed as co-solvent) was
also employed for Duphalac® , and new experimental assays
were carried out. The results obtained (see Table 2) show that
using these process conditions (300 bar, 80 ◦ C and 0.6 mL/min
co-solvent, meaning a 20 wt.% co-solvent) the extraction yields
considerably increase for all co-solvents employed. Although
the lactulose recovery is quite acceptable, particularly using
pure ethanol as co-solvent, again lactulose purity could only be
increased from 74 to 81% due to the fact that galactose extraction
occurs with almost a 100% recovery.
4. Conclusions
lactulose–lactose) mixtures derived the general conclusion that
supercritical conditions (temperature, pressure and amount of
polar co-solvent dissolved in the SC-CO2 solvent) affects recov-
eries but not selectivity, which is mainly influenced by the
co-solvent composition. Under optimal conditions, satisfactory
recoveries and high purity of the ketosugars, tagatose or lactu-
lose, were achieved.
These optimal extraction conditions were applied to the
supercritical extraction of a complex carbohydrate mixture
(commercial Duphalac® ), and also selectivity towards the
extraction of ketoses over aldoses was observed, although in this
case a smaller increase of the wt.% lactulose in the extract was
achieved. The high amounts of the monosaccharide galactose
extracted, present in Duphalac® in a 12 wt.% concentration, did
not allowed a complete purification of lactulose in the extracts.
Further work is being carried out in our laboratory directed to
a two-step SFE process that could achieve the elimination of
galactose first, followed by the purification of lactulose.
Acknowledgments
This work has been financed under a R+D program of
the Spanish Ministry of Education and Science (AGL2004-
07227-CO2-02) and of the Comunidad Autónoma de Madrid
(S-0505/AGR/000153). F.M. thanks MEC for a FPI grant. T.F.
would like to acknowledge the financial support of the Ramon
y Cajal Program from the Ministry of Education and Science.

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