Industrial Crops & Products 108 (2017) 355–362

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Industrial Crops & Products

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Extraction, isolation and characterization of inulin from Agave sisalana boles MARK
a b
Alexsandra Conceição Apolinário , Erika Martins de Carvalho ,
⁎
Bolivar Ponciano Goulart de Lima Damascenoc,d, Paulo César Dantas da Silvac, Attilio Convertie, ,
a a,f
Adalberto Pessoa Jr. , José Alexsandro da Silva
a
Department of Biochemical and Pharmaceutical Technology, University of São Paulo, Av. Prof. Lineu Prestes, 580 – Bloco 16, Cidade Universitária, 05508-000, São
Paulo, Brazil
b
Institute of Pharmaceutical Technology, Farmanguinhos, Fundação Oswaldo Cruz – Fiocruz, Rua Sizenando Nabuco, 100, 21041-250, Rio de Janeiro, RJ, Brazil
c
Department of Pharmacy, State University of Paraíba, Brazil
d
Graduation Program in Pharmaceutical Sciences, State University of Paraíba, Rua Juvêncio Arruda, S/N – Bairro Universitário, 58429-600, Campina Grande, Paraíba,
Brazil
e
Department of Civil, Chemical and Environmental Engineering, Chemical Engineering Pole, Genoa University, Via Opera Pia 15, 16145, Genova, Italy
f
Department of Agrarian and Exact Sciences, Campus IV – UEPB, Postgraduate Program in Agroindustry Systems, Federal University of Campina Grande, Pombal,
Paraíba, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Agave sisalana Perrine is a widespread species of the Brazilian Northeast region, where it is exploited only as a
Agave sisalana source of hard fiber (sisal). Although some other Agave species are sources of fructans, there is no study on this
Sisal issue on A. sisalana. This paper aimed at extracting and isolating inulin from aqueous extract of A. sisalana boles.
Aqueous extract After preparation of extracts, crude inulin was precipitated with acetone at low temperature (4 °C). After pur-
Polysaccharide
ification by ion-exchange chromatography, a white powder was obtained by freeze-drying and characterized by
Inulin
X-Ray Diffraction (XRD), thermal analysis, Circular Dichroism (CD) and Matrix-Assisted Laser Desorption/
Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). Moreover, its polysaccharide structure was
confirmed by Fourier Transformed Infrared (FT-IR) spectroscopy and Nuclear Magnetic Resonance (NMR). FT-IR
analysis pointed out absorption at 1420 cm−1, corresponding to deformation of CH2–OH lying on fructose ring,
while absorption at 1075 cm−1 was assigned to CeO and CeC stretching vibrations of inulin pyranose ring. NMR
showed the presence of one signal in the anomeric region at δ 5.4 ppm and others between 3.1 and 4.2 ppm in
the 1H spectrum, besides a chemical shift at 104.4 ppm corresponding to the anomeric region of the 13C spec-
trum of an internal β-fructofuranose unit. XRD highlighted the amorphous state of inulin-rich powder, thermal
analysis a glass transition temperature in the range between 50.0 and 55.8 °C, CD a good thermal stability, and
MALDI-TOF-MS a prevalence of oligosaccharides with degree of polymerization in the range 5–13. These
techniques revealed that A. sisalana boles contain inulin with features similar to those extracted from other
commercial sources such as Agave tequilana or Agave atrovirens, which extends the economic importance of this
species beyond its simple use as a fiber source.

1. Introduction pulque), drugs, construction materials, weaving, paper.

The Agave genus, belonging to the order Asparagales and to the
family Agavaceae, gathers more than 200 monocotyledonous and
monocarpic species, which grow mostly in China, Brazil, Mexico,
Tanzania, South Africa and Mozambique, although native to North
America, with their center of origin in present-day Mexico (Escamilla-
Treviño, 2012; Silva and Beltrão, 1999). Many species of Agave have
been used to produce spirits (e.g., tequila), forage, food, drinks (e.g.,
Species of the genus Agave have been investigated with different
aims in mind, mainly for biotechnological and pharmacological appli-
cations. These plants are known to accumulate large quantities of su-
gars and other commercially important substances, hence proving to be
a potential raw material for the production of biofuels (Arrizon et al.,
2010; Ávila-Fernández et al., 2011) and the recovery of compounds of
concern for pharmaceutical and food industries, including excipients
(emulsifiers, stabilizers, diluents) among others (Branco et al., 2010;

⁎
Corresponding author.
E-mail addresses: acapolinario@gmail.com (A.C. Apolinário), erikamc@far.fiocruz.br (E.M. de Carvalho), bolivarpgld@pq.cnpq.br (B.P.G. de Lima Damasceno),
paullocds@gmail.com (P.C.D. da Silva), converti@unige.it (A. Converti), pessoajr@usp.br (A. Pessoa), alexuepb@yahoo.com.br (J.A. da Silva).

http://dx.doi.org/10.1016/j.indcrop.2017.06.045
Received 24 August 2016; Received in revised form 6 June 2017; Accepted 17 June 2017
Available online 10 July 2017
0926-6690/ © 2017 Elsevier B.V. All rights reserved.
A.C. Apolinário et al. Industrial Crops & Products 108 (2017) 355–362

Santos et al., 2013). Total carbohydrate content was determined at 480 nm according to
Sterols, steroidal sapogenins, steroidal alkaloids, alkaloidal amines the phenolsulphuric acid method described by Dubois et al. (1956),
and flavonoids have been isolated from some Agave species (Chen et al., using a UV–vis spectrophotometer, model UV-mini-1240 (Shimadzu,
2011; Debnath et al., 2010), most of which demonstrated to possess Kyoto, Japan), and a reference curve (y = 0.0092x + 0.011;
anti-inflammatory, antioxidant, antimycotic, antibacterial, antiviral, R2 = 0.9929) obtained in triplicate with inulin as a standard. Free re-
antituberculosis and gastroprotective activities (Ben Hamissa et al., ducing sugars were quantified at 540 nm by the dinitrosalicylic acid
2012; Cerqueira et al., 2012; Dunder et al., 2010). method described by Miller (1959), using a reference curve
Among the various species of the genus Agave, Agave sisalana (y = 0.6018x + 0.0481; R2 = 0.9954) obtained in triplicate with
Perrine is a species widely present in the Brazilian Northeast region, fructose as a standard. The contents of total polyphenols, flavonoids
where it is exploited only as a source of hard fiber, known worldwide as and other components were already reported in a previous study
sisal, of which Brazil is the world’s largest exporter. Nonetheless, some (Apolinário et al., 2014a).
studies have demonstrated the potential of A. sisalana in different new
applications, for example as a source of secondary metabolites and new
2.3. Isolation and purification of inulin
products of interest for pharmaceutical and food industries (Apolinário
et al., 2014a; Ramasamy et al., 2015; Santos et al., 2013).
Polysaccharides of the aqueous extract were precipitated with
Crassulacean acid metabolism (CAM) of A. sisalana is an evidence
acetone (1:2 v/v) overnight at 4 °C, centrifuged (Excelsa I 206 BL,
that this species can be a source of inulin type-fructans (Apolinário
Fanem, Guarulhos, SP, Brazil) at 1118g for 20 min and submitted to
et al., 2014b). CAM plants do in fact produce photosynthetically these
four cycles of resuspension in distilled water at 80 °C and centrifugation
fructans that act as osmoprotectants during drought; for this reason,
under the same conditions as above. The polysaccharide solution was
inulin and other fructans are present as storage carbohydrate in more
loaded on a 1.5 × 12 cm polypropylene column (Econo-Pac® 732-1010,
than 30,000 plants species (Espinosa-Andrews and Urias-Silvas, 2012).
Bio-Rad, Hercules, CA, USA) packed with 1.0 g of DEAE-cellulose pre-
Inulin is a water-soluble fructose-based polymer resulting from ex-
equilibrated with 10 vol of 0.05 mM Tris HCl buffer (pH 7.0).
tended sucrose metabolism, which consists essentially of fructose units
An aliquot (1.0 mL) of each eluted fraction was collected separately
linked by β-(2→1) fructosyl-fructose bonds and a terminal glucose one.
in a 10 mL-test tube. After polysaccharide precipitation with acetone
Its variable degree of polymerization determines its applications and
(1:2 v/v) and centrifugation under the same conditions as above, the
the crop value (Guggisberg et al., 2009). Inulin is also largely used as a
precipitate was resuspended in distilled water (up to about
prebiotic compound (Oliveira et al., 2011b) and has known beneficial
0.5 g mL−1), frozen at −80 °C in an ultrafreezer, model MDF-U72 V
health effects (Barclay et al., 2010).
(Sanyo, Osaka, Japan), and lyophilized at −50 °C by using a freeze
Based on a previous study showing that aqueous extract of A. sisa-
drier, model L101 (Liotop, São Carlos, SP, Brazil). Fig. 1 summarizes the
lana boles are rich in sugars (Apolinário et al., 2014a) and taking into
steps of production of inulin from A. sisalana (INAS) in powder form.
account that inulin extraction from this raw material has never been
INAS powder diluted with distilled water up to 1000 μg mL−1 was
reported before, in this work we extracted crude inulin from A. sisalana
submitted to UV–vis scanning in the wavelength range of 200–300 nm
and performed a first purification by ion-exchange chromatography,
to make quality control of the final product. To this purpose, readings
with the aim of increasing added value to this crop, whose potential has
were performed in triplicate using 1.0 cm-pathlength quartz cuvettes in
been explored only a little so far. To this purpose, in order to contain
the above UV–vis spectrophotometer against water as a blank.
the costs of inulin recovery, we preferred to use the traditional hot
(80 °C) extraction technique under agitation, which is the method most
widely used to extract inulin from plants (Apolinário et al., 2014b). 2.4. Fourier Transformed Infrared analysis
Moreover, a set of analytical techniques was used to characterize the
recovered inulin-rich powder. The Fourier Transformed Infrared (FT-IR) spectra of INAS were re-
corded on an IR spectrophotometer, model Vertex 70 interferometer
2. Materials and methods (Bruker Optics, Ettlingen, Germany). After sample (5.0 mg) homo-
genization with KBr, the resulting mixture was pressed to form tablets,
2.1. Materials and the spectra were collected in the range 4000–400 cm−1 with a
resolution of 4 cm−1.
Six year-old plants of Agave sisalana Perrine were collected in
Monteiro, PB, Brazil (7°52′40.50” S and 37°07′34.91” W), on January
2.5. Nuclear Magnetic Resonance
2013. A voucher was deposited at the Herbarium Manoel de Arruda
Câmara (Campina Grande, PB, Brazil) under number 210. 1
H and 13C Nuclear Magnetic Resonance (NMR) spectra of INAS
Pure inulin used as a standard was purchased from Orafti® HP
were recorded on a spectrometer, model Avance 500 (Bruker, Bremen,
(Tienen, Belgium). Fructose, 3,5-dinitrosalicylic acid (DNS) and die-
Germany), at 500 MHz for 1H and 125 MHz for 13C, in D2O at
thylaminoethyl (DEAE)-cellulose resin were obtained from Sigma-
30 ± 0.1 °C using 30 pulse (12.5 μs for 1H and 7.0 μs for 13C) and a
Aldrich® (São Paulo, SP, Brazil). All the other reagents were of analy-
5 mm switchable probe. 1H NMR spectra were acquired by 1024 scans
tical grade.
with a relaxation delay of 2.0 s, 16 K data points, 8278.1 Hz spectral
width using a digital resolution of 0.30 Hz. The 13C ones were acquired
2.2. Preparation of the extract
by 386,440 scans with 23,980.8 Hz spectral width using a digital re-
solution of 1.0 Hz and 32 K data points.
Plant boles were dried in a forced circulation oven at 40 °C until
constant weight and milled. Based on the scientific literature on inulin
extraction from plants (Apolinário et al., 2014b), after dilution with 2.6. X-Ray Diffraction
distilled water up to 0.143 g mL−1, extraction was performed twice at
80 °C in a thermostatic bath, model SL 155/10 (Solab, Piracicaba, SP, X-Ray Diffraction (XRD) patterns of powder samples were recorded
Brazil), for 2 h. Dynamic maceration was allowed by agitation at at room temperature on a diffractometer, model Miniflex Goniometer
1600 rpm using a mechanical stirrer, model 713 (Fisatom, São Paulo, (Rigaku, Tokyo, Japan). Diffraction spectra were collected within 2 h in
SP, Brazil). To obtain final aqueous extracts, samples were filtered the 2θ range from 10° to 80° with a constant step of 0.04° and a
through qualitative paper. counting time of 1 s per step.

356
A.C. Apolinário et al.
Fig. 1. Scheme of isolation of inulin from Agave sisalana boles, showing the steps of precipitation, resuspension, purification and obtaining of inulin-rich solution.
2.7. Thermal analysis
The thermogravimetric (TG) curve was obtained using an Exstar
TG/DTA equipment, model 7200 (SII Nano Technology, Tokyo, Japan),
using a platinum crucible. The instrument was calibrated with calcium
oxalate monohydrate. The sample mass was 5.0 mg. Runs were per-
formed in the temperature range 30–500 °C and at a heating rate of
20 °C min−1 under nitrogen flowrate of 100 mL min−1. Differential
Scanning Calorimetry (DSC) curves were obtained using an Exstar DSC
equipment, model 7020 (SII Nano Technology), calibrated with stan-
dard indium (m.p. 156.6 °C). The sample mass (2.0 mg) was placed in a
hermetic aluminum sample pan and heated from 25 to 300 °C at a
heating rate of 5 °C min−1 under nitrogen flowrate of 50 mL min−1.
2.8. Circular Dichroism
A spectropolarimeter, model J-810 (Jasco, Tokyo, Japan), was used
to collect Circular Dichroism (CD) spectra of INAS at a concentration of
0.6 mg mL−1. Spectra were collected in triplicate at 25 °C in the wa-
velength range from 200 to 800 nm at 0.5 nm intervals. Samples con-
tained in 1.0 cm long quartz cuvettes were heated from 40 to 80 °C at a
heating rate of 10 °C min−1, and a new scanning was carried out.
2.9. MALDI-TOF-MS analysis
Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass
Spectrometry (MALDI-TOF-MS) analysis was done in positive linear
mode using an Autoflex III (Bruker Daltonics, Billerica, MA, USA).
Samples were dissolved in water (4.0 mg mL−1) and solubilized in a
matrix of α-cyano-4-hydroxycinnamic acid and acetonitrile
(10.0 mg mL−1); then, 1.0 mL of mixture was applied into the probe
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Industrial Crops & Products 108 (2017) 355–362
and quickly dried under vacuum.
3. Results and discussion
3.1. Total and free sugar contents of the extract
The general profile of extract sugars, either in terms of free reducing
or total sugars, was determined spectrophotometrically before isolating
inulin. The contents of free reducing and total sugars were 27.7 ± 0.1
and 347.6 ± 0.1 mg of sugar/g of dried bole, respectively (Table 1).
This total sugar content is close to those reported in the literature for
other plants that are considered potential sources of prebiotics like
Undaria pinnatifida (370 mg/g) and Cordyline australis (480 mg/g)
(Vidanarachchi et al., 2009). Higher concentrations of total sugars
(867 mg/g) were reported by Waleckx et al. (2008) for Agave tequilana,
but no similar study was made about these metabolites in Agave sisa-
lana. Nonetheless, these promising results suggest that boles of this
species could be a potential source of water-soluble carbohydrates like
those of other agave species.
3.2. Isolation and purification of inulin
Since this is the first time that A. sisalana inulin is described, our
Table 1
Sugar contents and yields of the aqueous extract of Agave sisalana boles.
Free sugars (mg/g) 27.7 ± 0.1
Total sugars (mg/g) 347.6 ± 0.1
Yield of crude inulin (% w/w) 4.8
Yield of recovered inulin (% w/w) 0.52
A.C. Apolinário et al.
Fig. 2. Fourier Transformed Infrared (FT-IR) spectrum of Agave sisalana inulin powder
obtained by freeze-drying.
main goal here was to isolate and characterize it. Crude inulin was
obtained as a brown pellet by precipitation of the extract with acetone,
with a yield of 4.8% (w/w) of dry bole (Table 1). After first purification
in DEAE-cellulose column, inulin from A. sisalana (INAS) was recovered
as white pellet and resuspended in water to obtain a freeze-dried
powder, with yields of 0.52 and 10.9% (w/w) with respect to raw
material and crude inulin, respectively. No absorbance at 270 nm was
detected by UV–vis scanning, which suggested the absence of proteins
or nucleic acids as contaminants.
3.3. FT-IR spectrum of A. sisalana inulin
The FT-IR spectrum illustrated in Fig. 2 gives clear evidence of the
fructan structure of isolated INAS, which was revealed by typical peaks
at 3492, 2924, 1603, 1420, 1292, 1075 and 887 cm−1 (Grube et al.,
2002).
In particular, the major broad stretching peak around 3492 cm−1
indicated the presence of hydroxyl groups (-OH) of carbohydrates,
while the small band at around 2924 cm−1 was attributed to CeH
stretching. The relatively strong absorption peak at around 1603 cm−1,
which is absent in pure inulin (Supplementary Material, Fig. S1), was
likely due to the absorption of some impurities commonly met in
polysaccharide extracts such as lignin (Cui et al., 2014; Tang et al.,
2017). These results confirm that the obtained powder consisted of not
totally pure inulin. On the other hand, the band at around 1420 cm−1
can be assigned to deformation of CH2–OH lying on fructose ring
(Panchev et al., 2011), those between 1292 and 887 cm−1 are typical of
polysaccharide structure with CeOeC bonds between monomers
(Dalonso et al., 2009), and that at 1075 cm−1 can be assigned to C−O
and CeC stretching vibrations of inulin pyranose ring (Gómez-Ordóñez
and Rupérez, 2011).
3.4. Nuclear magnetic resonance
In addition to the FT-IR analysis, both the 1H and 13C NMR spectra
of INAS shown in Fig. 3 confirm that powder obtained from A. sisalana
boles was mainly constituted by inulin-type fructans. Proton and carbon
signals were assigned by comparison with the spectrum of inulin HP®
taken as a reference and chemical shifts detected in previous study
(Oliveira et al., 2011a).
The INAS 1H NMR spectrum showed the presence of one signal in
the anomeric region at δ 5.4 ppm and other signals between 3.1 and
4.2 ppm, while the main results of 13C NMR spectrum are summarized
in Table 2.
The signal at δ 104.4 ppm likely corresponded to the anomeric
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Industrial Crops & Products 108 (2017) 355–362
region of the spectrum of internal β-fructofuranose (β-D-Fruf) units.
Lopez et al. (2003) did, in fact, report a chemical shift at 104.54 ppm
owing to the C-2 of these units with β(2→1) bond, even though no
resonance was detected around 63 ppm to confirm it, and a resonance
downfield at δ 64.00 ppm (C-6), which is characteristic of β(2→6)
linkages. Therefore, in the present study we can assign it to both C-1 (δ
60.9 ppm) and C-6 (δ 61.6 ppm). Another important region is that from
δ 79 to 84 ppm, where C-5 signals can be found. Finally, also the signal
at 81.6 ppm may be attributed to β(2→6) linkage (Lopez et al. 2003).
These branched mixed-type [(2→1, 2→6)]–linked β-D-Fruf residues
of inulin-type fructan isolated from A. sislana differ from unbranched
inulins comprising linear chains of β(2→1) fructans. These results
suggest that A. sisalana fructan might have a structure similar to that of
A. tequilana described by Lopez et al. (2003) as inulin, levan, and neo-
inulin. The C-1 signal from the agave fructan spectrum (δ 60.9 ppm)
can be attributed to the presence of β-D-Fruf in 6-kestose, while the
resonance at 104.4 ppm can been assigned to an internal C-2 of β-D-Fruf
of 1-kestose, which is preferentially used for inulin elongation. Like-
wise, the resonance at δ 61.6 ppm is typical of a polyfructan with a
β(2→6) linkage, so that it might be due to internal β(2→6)-D-Fruf re-
sidues.
In accordance with our results, Mancilla-Margalli and López (2006)
found in other Agave species (A. tequilana, A. angustifolia, A. potatorum,
A. cantala and A. fourcroydes) fructo-oligosaccharides with degree of
polymerization (DP) of 3, either of the inulin type (1-kestose) or the
neo-inulin one (6-kestotose), whose presence might be indicative of two
existing fructan types: inulin as in chicory, Jerusalem artichoke and
dahlia, and neo-inulin as in Asparagales like onion, garlic and aspar-
agus.
3.5. X-Ray Diffraction
The results of X-Ray Diffraction (XRD) analysis of lyophilized INAS
illustrated in Fig. 4 clearly show a halo pattern, with a broad band
which is a typical feature of predominantly amorphous materials
(Saavedra-Leos et al., 2014). The broader peaks at 17.25° (Yi et al.,
2013), 29° and 40° (2θ) are characteristic of amorphous crystalline
regions of semicrystalline inulin, unlike crystalline inulin that exhibits
sharp, defined peaks (Aravind et al., 2012; Silva and Meireles, 2015).
The amorphous state of INAS may have been due to the fact that
inulin molecules did not have enough time to rearrange during the
precipitation step, and are frozen in their original position during fast
freeze-drying cooling (Ronkart et al., 2009). The molecular weight of
inulin is another factor that plays a crucial role in crystallinity, in that
inulins with degree of polymerization (DP) > 25 are relatively crys-
talline, whereas smaller DPs are usually associated to amorphous state
(Zimeri and Kokini, 2002).
Similar amorphous state with interesting properties was already
reported for fructans of other agave species like A. tequilana (Espinosa-
Andrews and Urias-Silvas, 2012). For instance, whereas crystalline in-
ulin-rich powder forms milky suspensions, the amorphous one dis-
persed in water can cause the formation of strong inulin gel at room
temperature or clear solutions at 70 °C (Glibowski and Pikus, 2011).
Taking into account that amorphous and semi-crystalline forms are
those in which inulin can be purchased (Glibowski et al., 2014), one can
expect similar properties and marketability for amorphous INAS ob-
tained in this work.
3.6. Thermal analysis
The dynamic thermogravimetric curve (TG) illustrated in Fig. 5A
shows two thermal steps of INAS decomposition, which are similar to
those observed for fructans isolated from A. tequilana (Espinosa-
Andrews and Urias-Silvas, 2012).
The first decomposition stage, occurred since the beginning of the
run from 24.0 to 119.0 °C, showed a maximum peak at 66.6 °C and a
A.C. Apolinário et al. Industrial Crops & Products 108 (2017) 355–362

Fig. 3. Nuclear Magnetic Resonance (NMR) spectra

of Agave sisalana inulin powder. (a) 1H NMR spec-
trum. (b) 13C NMR spectrum.

Table 2
13
C NMR chemical shifts (ppm) of inulin HP® and inulin from Agave sisalana boles.

Carbon Inulin HP® Inulin from A. sisalana boles

β-Fructofuranose
C-1 61.3 60.9
C-2 103.3 104.4
C-3 77.5 77.9
C-4 74.9 74.7
C-5 81.3 81.6
C-6 62.3 61.6

β-Glucopyranose
C-1 92.6 nd
C-2 73.0 72.1
C-3 74.5 74.4
C-4 71.5 70.5
C-5 72.7 71.9
C-6 69.8 69.0

nd = not detected.
Fig. 4. X-Ray diffractogram of Agave sisalana inulin powder.

significant mass loss probably due to the high moisture content of the
sample (13%) (Ronkart et al., 2010). Taking into account that inulin
crystals need water in their structure (Kawai et al., 2011; Zimeri and
Kokini, 2003), such a weight loss may also be due to the water content
of amorphous state inulin. The second decomposition stage, occurred in
the temperature range 247.0–355.5 °C with a peak at 322.3 °C, implied
a 47% mass loss likely related to decomposition of branched chains of
agave fructans and subsequent thermal degradation (Espinosa-Andrews
and Urias-Silvas, 2012). Both endothermic events are consistent with
early-described amorphous state and thermal properties of inulin from
other sources (Saavedra-Leos et al., 2014), which are essential for fu-
ture commercial applications of this product (Dan et al., 2009; Leone
et al., 2014; Zimeri and Kokini, 2003).
Both peaks correspond to the endothermic events evidenced at 55.6
and 165.8 °C by the DSC curve (Fig. 5B), which shows a glass transition

359
A.C. Apolinário et al.
Fig. 5. Thermogravimetric (a) and differential scanning calorimetry (b) profiles of Agave
sisalana inulin powder.
temperature (Tg) in the range between 50.0 and 55.8 °C with a small
peak around 55.6 °C. Such a Tg range, which, as expected, lays within
the temperature range evidenced for water loss by the TGA curve, is
practically coincident with that reported for Jerusalem artichoke inulin
(51–55 °C) (Panchev et al., 2011), but lower than those of inulin from
other sources (Hinrichs et al., 2001; Ronkart et al., 2009, 2010).
Chiavaro et al. (2007) reported for Raftilose® Tg values in the tem-
perature range between 44.7 ± 0.5 °C and 55.5 ± 0.6 °C, and ob-
served a progressive increase in this parameter with molecular weight
(Hinrichs et al., 2001).
It has been reported that an increase in moisture content leads to a
Tg decrease (Kawai et al., 2011; Zimeri and Kokini, 2003); however, if
temperature becomes higher than Tg, the viscoelastic properties of
amorphous polymeric materials change, i.e. the movement of polymer
backbone increases, hence resulting in transition from a glassy to a
rubbery, disordered state (Stelte et al., 2011). As suggested by the DSC
curve (Fig. 5B), this state might have occurred in INAS between 50.0
and 55.8 °C and accelerated physicochemical changes, such as sticking,
collapse, caking, agglomeration, loss of volatiles, browning and oxida-
tion (Bhandari and Howes, 1999).
3.7. Circular Dichroism
Results of Circular Dichroism illustrated in Fig. 6 revealed no sig-
nificant modification in INAS structure with increasing temperature,
i.e. no relevant variation in the ellipticity at temperature lower than Tg,
and suggested thermal stability as for other polysaccharides (Zhang
et al., 2013). To this respect, as suggested by Zhu et al. (2016), car-
bohydrates can exist as disordered, rigid or flexible chains.
Nevertheless, ellipticity in the lowest wavelength range (around
200–215 nm) decreased from 20 to about 5 millidegrees (mdeg) when
temperature was raised from 60 to 80 °C. This effect, which is consistent
with DSC results that pointed out INAS Tg above 50 °C, may be ascribed
to the conformational change related to this event, as suggested for
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Industrial Crops & Products 108 (2017) 355–362
Fig. 6. Circular dichroism spectra of Agave sisalana inulin powder at concentration of
0.6 mg mL−1 obtained in the temperature range between 25 and 80 °C.
other polysaccharides with disordered structures (Eren et al., 2015;
Yang and Zhang, 2009).
3.8. MALDI-TOF-MS analysis
As a final effort, we characterized INAS according to its DP, because
inulin technological properties to be exploited in industrial applications
are influenced by the size of the polysaccharide chain (Lopes et al.,
2015). Short-chain inulin (average DP ≤ 11) is in fact thermally less
stable, more soluble and less viscous than the long-chain one
(DP = 23–25), which, on the other hand, is mainly used as fat sub-
stitute (Guggisberg et al., 2009).
MALDI-TOF-MS analysis of INAS illustrated in Fig. 7 revealed a
molar mass between 671 and 3283 Da (average value of 1641.5 Da),
corresponding to DP in the range 5–20 (average value of 10), which is
close to that detected for fructans isolated from 6 year-old plants of A.
tequilana (4 ≤ DP ≤ 24) (Arrizon et al., 2010), but rather small com-
pared with those reported for inulins from different species (Mensink
et al., 2015). However, the increase in intensity of peaks around
671–1157 Da suggested prevalence of oligosaccharides with DP be-
tween 5 and 13.
Finally, INAS was shown to be an amorphous material with complex
structure: a) linear chain with β(2→1) linkages, b) branched chains
with β(2→6) linkages, c) presence of β-D-Fruf of 1-kestose, and d)
presence of β-D-Fruf of 6- kestose.
4. Conclusions
In this study, which is the first attempt to recover inulin from Agave
sisalana, the structure of inulin contained in boles of this plant was
confirmed by FT-IR and NMR spectra. We found a clear correlation
between the amorphous state of inulin, confirmed by X-Ray Diffraction,
and DSC thermal analysis that indicated a range of glass transition
A.C. Apolinário et al.
temperature of 50.0–55.8 °C. This result appears to be consistent with
the conformational changes evidenced by Circular Dichroism. The
MALDI-TOF-MS analysis revealed that A. sisalana inulin is a short-
length polysaccharide with degree of polymerization (DP) in the range
5–20, with prevalence of oligosaccharides with DP of 5–13, and an
average value of about 10.
Acknowledgements
This work was supported by National Program of Academic
Cooperation (PROCAD), Project N°: 552652/2011-3, Development of
Processes and Pharmaceuticals and Biotechnological Processes Using
the Sustainability Potential of Semiarid. The authors also acknowledge
the Coordination for the Improvement of Higher Education Personnel of
of (CAPES), linked to the Brazilian Ministry of Education, for financial
support of Prof. A. Converti as Special Visiting Researcher (Science
without Borders Program, project n. 2609/2013), Dr. Paul Long of
King’s College London for language polishing, the State of São Paulo
Research Foundation (FAPESP, Process n°: 2013/22212-0), the
National Council for Scientific and Technological Development (CNPq)
and the Brazilian Agricultural Research Corporation (EMBRAPA).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.indcrop.2017.06.045.
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