Industrial Crops & Products 111 (2018) 615–624

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Industrial Crops & Products

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Two-step isolation of hemicelluloses from Chinese quince fruit: Effect of T

hydrothermal treatment on structural features
⁎
Hua-Min Liua, Yao-Ran Lia, Min Wub, Hui-Shuang Yina, Xue-De Wanga,
a
College of Food Science and Technology, Henan University of Technology, Zhengzhou 450001, China
b
Institute of Physical Science and Engineering, Zhengzhou University, Zhengzhou 450001, China

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

Keywords: Two-step isolation of hemicelluloses from Chinese quince fruit was carried out in this investigation. At the first
Chinese quince treatment stage, hydrothermal treatment was developed to enhance the xylose amount in the solid residue
Hemicelluloses remaining after pectin extraction. Second, the alkali-extractable hemicelluloses were isolated from the solid
Hydrothermal treatment residue obtained from hydrothermal treatment and were successively sub-fractionated by precipitation at graded
Characterization
ethanol concentrations of 30%, 50%, 70% and 90%. The structural properties of the isolated hemicelluloses from
raw fruits and fruits treated with the hydrothermal process were characterized by sugar analysis, FT-IR, mole-
cular weights, thermo-gravimetric analysis, morphological analysis, and NMR. Results showed that the xylose
amount in the solid residue increased from 58.2% to 62.2% when the hydrothermal treatment temperature was
increased from 120 °C to 150 °C. Arabinose and mannose were found to be hydrolyzed by hydrothermal treat-
ment, while the crystalline cellulose and lignin were not affected. Compared with the raw Chinese quince, the
treatment had an important effect on the sugar composition distributions and average molecular weights of
hemicelluloses obtained at various ethanol concentrations. Structural characterization revealed that the isolated
hemicellulosic sub-fractions precipitated at a final ethanol concentration of 30% had a backbone of D-xylose
residues with a β-(1 → 4)-linkage. This work reveals a novel hemicelluloses isolation route from Chinese quince
fruit, which was important in the development of biomass refining industry. This work reveals a novel hemi-
celluloses isolation route from Chinese quince fruit, which was important in the development of biomass refining
industry.

1. Introduction polysaccharides that contain 1,4-linked α-D-galacturonic acid (GalpA)

Chinese quince (Chaenomeles sinensis) is deciduous spiny shrub,
belonging to the Rosaceae family and native to eastern Asia, including
China, Japan, and Korea (Kim et al., 2013). The fruits of Chinese quince
are not palatable raw due to their strong astringency and woody flesh;
however, the fruits are widely consumed after cooking or processing as
products such as glutinous starch syrup, jam, and fruit liquor (Hamauzu
et al., 2007). Dried Chinese quince fruit has been commonly used in
traditional medicine to treat throat diseases in China (Hui et al., 2010).
Additionally, the extracts from Chinese quince have been used in-
dustrially in beverages or candies sold with health claims (Hamauzu
et al., 2007).
Although many components of Chinese quince fruit (e.g., procya-
nidins, polyphenols, lectin) have been investigated for use as beneficial
food additives (Jang et al., 2011; Hamauzu et al., 2008, 2007; Ishiwari
et al., 2010; Sawaikuroda et al., 2013), pectin is still considered the
most useful and widely applicable. Pectin is a family of complex
residues. It is widely used in pharmaceutical, food and cosmetic in-
dustries as thickener, gelling agent and stabilizer (Wang and Lü, 2014;
Voragen et al., 2009). The traditional method of extracting pectin from
plant tissues is heat acid extraction (Liu et al., 2012). Recently, many
novel extraction methods, such as ultrasound-assisted, microwave-as-
sisted, alkali and enzymatic extraction, have been investigated seeking
for effective method to increase production (Zuzana et al., 2010;
Ptichkina et al., 2008; Wang et al., 2007; Zykwinska et al., 2006).
Hydrothermal treatment is an effective technology to obtain poly-
saccharides from plant materials under mild operational conditions. It
is also known as an economical and environmentally friendly procedure
(Yan et al., 2017; Xiao et al., 2013). It is friendly to the environment
because no chemical reagents are used. It is economical because the raw
material does not need to be dried prior to hydrothermal treatment. The
water present in the raw material serves as a reactant as well as a
solvent in the treatment process. This eliminates the typically high
energy cost of drying wet materials (Liu et al., 2012). Hydrothermal

⁎
Corresponding author.
E-mail addresses: wangxuede1962@126.com, liuhuamin5108@163.com (X.-D. Wang).

https://doi.org/10.1016/j.indcrop.2017.11.035
Received 13 September 2017; Received in revised form 7 November 2017; Accepted 18 November 2017
Available online 24 November 2017
0926-6690/ © 2017 Elsevier B.V. All rights reserved.
H.-M. Liu et al.
treatment has been proved to be effective for extracting pectin from
apple pomace and citrus peel (Wang and Lü, 2014; Tanaka et al., 2012).
However, hydrothermal treatment of these materials yielded a high
solid residue amount, which indicated the majority of material was not
converted over the whole range of temperatures tested. For an eco-
nomic perspective, high amount yield of solid residue would not be
favorable for the application of these lignocellulosic materials. Our
investigation of hydrothermal treatment of soy hulls showed that the
xylose composition in the residues increased from 26.7% to 35.4%
when the temperature reached 180 °C (Liu et al., 2016). Thus, these
residues from hydrothermal treatment of quince fruit may be excellent
raw material for xylan-type hemicelluloses production.
Hemicellulosic polymers are a large group of complex poly-
saccharides found in plant cell walls. Extracted, they are used widely in
the food, medicine and chemical industries (Peng et al., 2012; Peng
et al., 2011a). Many methods have been applied to obtain hemi-
celluloses from plant materials, such as isolation with organic solvent,
alkali, or alkaline peroxide. Among these methods, alkaline extractions
have been widely used because the ester linkages present between
hemicelluloses and other components can be effectively hydrolyzed by
simple and effective alkaline agents (Bian et al., 2012). Zhang et al.
(2016) reported using a GTP (glycerol thermal processing) pretreat-
ment for isolation of xylan-type hemicelluloses from hard-wood at
temperatures ranging from 200 to 240 °C in the presence of anhydrous
glycerol. The investigation showed that the xylan content in the raw
material was enhanced to 80% after GTP pretreatment and subsequent
sequential water and organic solvent isolation. To the best of our
known, there have been no studies on xylan-type hemicellulose isola-
tion from the solid residue of Chinese quince after pectin extraction by
hydrothermal treatment. In particular, there have been no studies of the
chemical structure and properties of these hemicelluloses.
In this investigation, the objective was to extract hemicellulosic
polymers from the solid residue remaining after pectin extraction from
Chinese quince by hydrothermal treatment. Thus, the isolation of
hemicelluloses from Chinese quince proceeded in two steps. First,
Chinese quince was pretreated by hydrothermal method to obtain
pectin. Second, xylan-type hemicelluloses were isolated from the solid
residue left after hydrothermal treatment. The structural properties of
the hemicelluloses isolated from both raw and hydrothermally treated
quince fruit were characterized by sugar analysis, FT-IR, molecular
weights, thermo-gravimetric analysis, morphological analysis, and
NMR, and then compared.
2. Experimental
2.1. Materials
The ripe fruits of Chinese quince were purchased from a local
orchard in Nanyang Prefecture, China. The fruits were sliced, seeds
were removed, and the pulp was freeze-dried. The samples were milled
and then screened into fractions of 40-mesh particle diameter. The
extracts were removed from the powder using a Soxhlet extractor and
subsequently air-dried. All solvents and chemicals used were of either
chromatographic grade or analytical grade.
2.2. Hydrothermal treatment
Hydrothermal treatment of Chinese quince was carried out in a
pressurized glass reactor (250 mL). The glass reaction system was he-
ated by a magnetic heating stirrer (IKA, Germany) and the reaction
temperature was measured by a type-J thermocouple. For a typical run,
Chinese quince powder and water were loaded in the reactor with a
solid to liquid ratio of 1:10 (g/mL). It was heated to the reaction tem-
perature at a rate of 3 °C/min, and then held constant for the specified
period. When the treatment was completed, the glass reaction system
was cooled down to room temperature by air and the solid residue was
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Industrial Crops & Products 111 (2018) 615–624
extracted with water until the solvent in the thimble become colorless.
The solid residue (SR) was dried at 60 °C and designated as pretreat-
ment material.
2.3. Isolation and purification of xylan
The untreated and pretreated materials were first isolated with 10%
KOH for 2 h at 80 °C (solid to liquor ratio of 1:20, g/mL). The two so-
lutions were filtered, and the filtrates were neutralized using 6 M acetic
acid until the pH reached 7.0. Each mixture was centrifuged to obtain
supernatant. Then appropriate volumes of 95% ethanol was added into
the filtrate with continuous stirring until a final ethanol concentration
of 30% (v/v) was achieved. At that point, the mixture was left at room
temperature for 24 h. The precipitates were obtained by freeze-drying
after centrifugation and named X30. Next the filtrate was subjected to a
series of centrifugations with increasing concentrations of ethanol.
First, the mixture itself was centrifuged; the precipitate was freeze-dried
and named X50. Then, the supernatant was brought to an ethanol
concentration of 70%, centrifuged, and the precipitate labeled as X70.
This procedure was repeated with concentrations of 90% alcohol,
yielding xylan subfraction X90. Thus, the eight hemicellulosic fractions
obtained from raw materials were defined as UPX30, UPX50, UPX70,
UPX90, while the hemicellulosic fractions obtained from pretreated
materials were defined as PX30, PX50, PX70, PX90.
2.4. Characterization
Crystallinities of various Chinese quince before and after hydro-
thermal treatment were evaluated using an X-ray diffractometer
(Shimadzu XRD-6000, Tokyo, Japan) with Cu Ká radiation source at
40 kV and 30 mA. The crystallinity index (CrI) of various samples was
determined by the formula as description previously (Segal et al.,
1959).
I 002 − Iam
CrI = × 100
I 002 (1)
in which I002 is the intensity for the crystalline portion of biomass (i.e.,
cellulose) at about 2θ = 22.5, and Iam is the peak for the amorphous
portion (i.e., cellulose, hemicelluloses, and lignin) at about 2θ = 18.7
in most literatures. It should be noted in this study that the second
highest peak after 2θ = 22.5 was at 2θ = 16.8, and this was assumed
to correspond to the amorphous region.
The sugar analyses of hemicelluloses were performed using a
HPAEC (high performance anion exchange chromatography) system
(Dionex, ICS-3000, USA) coupled with an AS50 autosampler and a
CarbopacTM PA-20 column (4 mm × 250 mm, Dionex). After the hy-
drolysis treatment, the mixtures were diluted 50-fold, filtered and then
injected into the HPAEC system. Calibration was carried out with
standard solutions of D-galactose, D-mannose, D-glucose, D-xylose, D-
glucose, L-arabinose, glucuronic acid, and galacturonic acids.
Fourier transform infrared spectrometer (FT-IR) spectra of the
samples were recorded on a Nicolet 510 type FT-IR spectrophotometer
in the range 4000–500 cm−1 at a resolution of 2 in the transmission
mode using a KBr disk containing 1% finely ground samples.
GPC with an Agilent 1200 series HPLC system was used to de-
termine the molecular weights of various hemicelluloses fractions,
which equipped with a Tskgel G3000PWXL column (300 × 7.8 mm,
Tosoh, Japan) with a differential refractive index detector (RID). The
analysis process was described previously (Wang et al., 2017).
Thermal degradation of all samples was evaluated by thermo-
gravimetric analyzer (Shimadzu, Japan) from 30 to 670 °C at a heating
rate of 10 °C/min with the protection of nitrogen. The mass of the
samples used varied from 3 to 5 mg. Thermal denaturation of all sam-
ples was measured via DSC (Q200, TA instrument, Schaumburg, IL).
The scanning electron micrographs reveal the microstructure of the
hemicellulosic fractions, and the shape and surface features of the
H.-M. Liu et al. Industrial Crops & Products 111 (2018) 615–624

samples were recorded using an S-570 SEM (Hitachi). All hemicellulosic
fractions were placed on a silicon wafer and coated with gold powder.
The Nanofirst 3600A AFM (atomic force microscope) was used to
observe the molecular topographies of the hemicellulosic fractions.
Each hemicellulosic fraction was dissolved in ultrapure water (1 μL/
mL) with magnetic stirring. Next 5 μL of the preparation solution was
dropped onto freshly cleaved mica substrate and allowed to dry at room
temperature for at least 12 h. The atomic force microscope was oper-
ated in the tapping mode at room temperature. The scanning size was
5 × 5 μL.
The 1H and HSQC (heteronuclear single quantum) spectra NMR
(nuclear magnetic resonance) was obtained on a Bruker Avance 500 M
NMR spectrometer (III HD, Switzerland). Each hemicellulosic sample
(30 mg) was dissolved in 0.7 mL D2O and placed in a 5 mm NMR tube.
HSQC spectra were acquired by HSQCETGP experiment mode, over a t1
spectral width of 10, 000 Hz and a t2 width of 1800 Hz. Data processing
was performed using standard Bruker Topspin-NMR software.
composition in the solid residues remaining after hydrothermal treat-
ment of soy hulls (Xiao et al., 2013). For soy hulls, the glucose in the
solid residue initially increased with the pretreatment temperature and
then decreased. It was noteworthy that the xylose amount in the SR of
quince after hydrothermal treatment increased from 58.2% to 62.2% as
the temperature increased from 120 °C to 150 °C. After hydrothermal
treatment, the contents of arabinose, galactose, mannose, glucose, and
uronic acids were lower as compared with the untreated sample.
Therefore, the increase of the amount of xylose in SR from 120 °C to
150 °C was caused by the degradation of other sugars. From an eco-
nomic perspective, higher pretreatment temperature would favor
higher xylan content in SR. However, higher pretreatment temperature
requires more energy and reduces SR yield. Coupling these results of
pretreatment yield and xylan content in SR, pretreatment at 135 °C was
therefore chosen for the further extraction of high purity xylan from SR
and characterization of its structure.
3.2. Crystallinity of Chinese quince before and after pretreatment

3. Results and discussion
3.1. Structural constituents of Chinese quince before and after pretreatment
Hydrothermal treatment has been widely applied because of the
advantages of water, which is a non-toxic, environmentally benign, and
inexpensive medium. Generally, hydrothermal treatment is carried out
at the temperature range of 150–220 °C. Over the range, the xylose
composition in solid residue remaining after the process can be as high
as 88–98% because of the depolymerization of other type sugar com-
positions (Peng et al., 2011a). In the present investigation, 120, 135,
and 150 °C were selected as the pretreatment temperatures in this in-
vestigation. The pretreatment yield and structural constituents of un-
treated and pretreated Chinese quince samples are presented in Table 1.
As shown in Table 1, increasing the pretreatment temperature led to a
decrease in the SR yield, particularly in the range of 120–150 °C. The
yields of SR reached 67.1%, 65.9%, and 62.9% at 120, 135, and 150 °C,
respectively. Higher temperature of the hydrothermal treatment re-
duced the yield of SR because large-chain compounds such as cellulose,
lignin, xylan, and other sugar compositions were broken down into
simpler and smaller molecules by thermal degradation. Hydrothermal
treatment also led to increases in the remaining lignin content in SR
compared with the untreated Chinese quince due to the extraction of
pectin polysaccharides.
Table 1 shows that the main sugars in Chinese quince are xylose
(from xylan; 48.5%), followed by glucose (from cellulose; 17.7%) and
arabinose (mainly from hemicelluloses; 17.5%). Small amounts of ga-
lactose (7.3%), mannose (6.0%), and uronic acid (3.6%) were also
found. The amount of glucose (cellulose) in SR initially decreased after
pretreatment at 120 °C and 135 °C and then increased to 18.0% after
the hydrothermal treatment temperature reached 150 °C. The result
was not consistent with our previous investigation on sugar
The structures of cellulose crystals in cellulosic materials can be
changed by various treatment methods due to disrupting intra- and
inter-chain hydrogen bonding of cellulose fibrils (Peng et al., 2012).
The features of Chinese quince samples after hydrothermal treatment
were also investigated using X-ray diffraction (XRD) and compared with
the corresponding untreated samples. Fig. 1 shows the XRD spectra of
the pretreated and untreated Chinese quince at various temperatures
(120, 135, and 150 °C). Clearly, the XRD pattern of the samples showed
two peak at 2θ = 16.8° and 22.8°, typical of cellulose I (Guo et al.,
2016). The crystallinity index (CrI) for all samples was calculated from
the XRD data, and the results are shown in Fig. 1. As shown, the un-
treated Chinese quince is lowly crystalline (15.9%). Furthermore, the
CrI of Chinese quince after pretreatment at 120 °C was even lower.
Probably, the heat dissolved a certain amount of the crystalline glucose
(as shown Sugar analysis results). The CrI of the sample increased,
however, from 13.6% to 24.5% to 22.7% as the hydrothermal treatment
temperature increased from 120 to 135 to 150 °C, respectively. In other
words, temperatures above 120 °C increased the crystallinity of the
sample. The increases in CrI at 135 and 150 °C were probably due to the
partial removal of amorphous lignin and polysaccharides. However,
hydrothermal pretreatment processes under the given conditions were
unable to break down the intra-and inter-chain hydrogen bonding in
the cellulose fibrils of Chinese quince.
3.3. Yield and sugar composition of hemicelluloses fractions
Chinese quince was subjected to hydrothermal treatment to remove
other polysaccharides and degrade some of the lignin-hemicelluloses

Table 1
Yield (% dry initial material) and sugar composition of the Chinese quince before and
after pretreatment.

Samplea Lignin (%) Yield Molar composition (%)

Arab Galb Glub Xylb Manb Gluab

CQ 14.6 100 17.5 7.3 17.7 48.0 6.0 3.6

SR-120 20.4 67.1 16.4 6.6 14.3 58.2 2.7 1.9
SR-135 24.6 65.9 9.1 5.0 14. 6 61.7 2.7 6.9
SR-150 28.7 62.9 5.2 5.8 18.0 62.2 3.3 5.6

a
CQ, SR-120, SR-135, and SR-150 represent the untreated and pretreated Chinese
quince by hydrothermal at 120 °C, 135 °C, and 150 °C, respectively. Fig. 1. XRD patterns and crystallinity index of untreated and pretreated Chinese quince
b
Abbreviation: Xyl, xylose; Ara, arabinose; Gal, galactose; Glc, glucose; UA, uronic (A: untreated material; B: pretreated at 120 °C; C: pretreated at 135 °C; D: pretreated at
150 °C).
acid.

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Table 2 treatment. Therefore, it is likely that hydrothermal treatment slightly

Yield (% dry initial material) and sugar composition of the polysaccharide fractions. degrades the macromoleculare structure of hemicelluloses in raw ma-
terials. The xylan structure in Chinese quince was not degraded during
Fractiona Molar composition
hydrothermal treatment process as reveal by sugar analysis of Chinese
Yieldb Xylc Arac Galc Gluc Manc UAc Ara/Xyl quinces before and after pretreatment. As shown in Fig. 2A, the curves
of molar mass distribution for the hemicellulosic fractions precipitated
UPX30 16.3 73.7 7 7.4 3.7 1.3 6.9 0.09
by different final ethanol concentrations were quite dissimilar. The
UPX50 0.63
UPX70 2.2 34.1 40.5 15.4 2.4 2.3 5.3 1.19 hemicellulosic fractions precipitated in the lower ethanol concentra-
UPX90 6.6 21.2 51.8 18.5 1.4 2.3 4.8 2.44 tions clearly showed a higher molar mass compared with the fractions
PX30 4.4 84.3 2.8 6.5 2.9 3 0.7 0.03 obtained from the higher ethanol concentrations. This is in accordance
PX50 6.1 52.9 22 14.6 3.7 1.8 5.1 0.42 with the Mw values.
PX70 8 35.9 42.6 13.1 1.7 1.6 5.2 1.19
PX90 4.7 11.5 58.8 17.1 1.1 2.1 8.4 5.11
3.5. FT-IR analysis
a
UPX30, UPX50, UPX70, UPX90, PX30, PX50, PX70, and PX90 represent the poly-
saccharide fractions from before (UPX) and after (PX) pretreatment Chinese quince by Fig. 2B and C show the FT-IR spectra of various hemicellulosic
precipitation in a final ethanol concentration of 30%, 50%, 70%, and 90%, respectively. fractions from Chinese quince before and after pretreatment by pre-
b
Based on raw Chinese quince (wt%).
c
cipitation in various ethanol comcentrations. As shown, the spectral
Abbreviation: Xyl, xylose; Ara, arabinose; Gal, galactose; Glc, glucose; UA, uronic
profiles and relative intensities were mostly rather similar for most
acid.
bands, which indicated a similar structure of the xylan in the various
samples. The spectra showed a strong hydrogen bond OeH stretching
linkages; it was then extracted with 10% NaOH for 16 h to obtained
absorption at 3420 cm−1 and a prominent CeH stretching vibration
high xylan polysaccharides. The sugar composition and yield of all
absorption at 2923 cm−1. The intensive band at 1046 cm−1 is assigned
hemicellulosic fractions are showed in Table 2. For unpretreatment
to CeO, CeC stretching and the glycosidic (CeOeC) contributions,
Chinese quince, the precipitates collected at 30%, 50%, 70%, and 90%
indicating a dominant xylan structure in the polysaccharide fractions,
ethanol, yielded 16.3%, 0.63%, 2.2%, and 6.6% of dry matter, re-
which corresponded to the results of sugar analysis. The absorption at
spectively. Clearly, the major hemicellulosic fraction was obtained at
1116 cm−1 is attributed to CeC stretching. The appearance of sym-
the ethanol concentration of 30% and the highest yield of the fraction
metric stretching from COO groups at 1417 cm−1 indicated the pre-
was 16.3%, which corresponds to precipitating 65.9% of the total
sence of glucuronic acid. In addition, the wavenumber at 1254 cm−1 is
polysaccharides. Comparatively, the xylan polysaccharides of extrac-
referred to carboxylic acid vibrations, which was attributed to 4-O-
tion from pretreated Chinese quince showed a lower total yield com-
methyl-α-D-glucuronic acid side groups (except for samples PX90 and
pared with that of unpretreatment raw material. The precipitates col-
UPX30) (Tanaka et al., 2012). The sharp absorption at 896 cm−1 is
lected at 30%, 50%, 70%, and 90% ethanol yielded 4.4%, 6.1%, 8.0%,
attributed to the β-configuration of the 1 → 4 glycosidic bond between
and 4.7% of dry matter of the initial amount of pretreatment material.
xylopyranose units of the main xylan chains (except PX90) (Liu et al.,
The data of pretreated Chinese quince showed that the yield of the
2016). The intense signal at 1634 cm−1 is attributed to C]O asym-
hemicellulosic fraction increased with an increasing ethanol con-
metric vibration of carboxyl group, which also the characteristic IR
centration from 30% to 70%, then decreased at the final ethanol con-
absorption of hemicelluloses (Sun et al., 2011).
centration of 90%. The highest polysaccharide yield of 8.0% was ob-
tained at the ethanol concentration of 70%. Compared with the
3.6. Morphological properties
untreated Chinese quince, hydrothermal treatment had an important
effect on the yields distribution of xylan polysaccharides obtained at
SEM and AFM were used to characterize the surface morphology of
various ethanol concentrations. This result could be explained by the
polysaccharides and to predict their physical properties; the images
changes of the structure and chemical components caused by the pre-
provide visual evidence of the structural changes in the samples. Fig. 3A
treatment. Generally, most of the xylose composition resulted from the
depicts the morphologies of untreated and pretreated Chinese quince
xylan backbone in Chinese quince, while glucose, galactose, arabinose,
samples with different molecular weights. The microstructures of
and uronic acids originated from the side chains. Thus, the sugar ana-
polysaccharides in a state of aggregation have irregular lamellar
lysis indicated that hemicellulosic fractions UPX30 and PX30 comprised
structures and porous surfaces, possibly due to the effect of lyophili-
a backbone of xylan. Compared with UPX30 and PX30, hemicellulosic
zation during the drying process. The porous fractions were decreased
fraction PX30 has a lower ratio of Ara/Xyl (0.03) than that of UPX30
and, in some cases, nearly disappeared, while the lamellar structure
(0.09), which demonstrated that UPX30 was more highly branched
increased. The samples subjected to hydrothermal treatment at 120 °C
than PX30, since Ara/Xyl ratios are indicative of the degree of linearity
showed marked structural changes compared to the untreated samples.
or branching of hemicelluloses (Chaikumpollert et al., 2004).
Samples subjected to hydrothermal treatment had broken and damaged
polysaccharide chains, with many smaller and simpler molecules. Fi-
3.4. Average molecular weight lamentous structures were broken and open, and huge lamellar struc-
tures on the external surfaces were observed. Additionally, the hydro-
Fig. 2A presents the GPC profiles and average molecular weight of thermal treatment of Chinese quince changed the orientation of mass
the hemicellulosic fractions. Obviously, an increase in the final ethanol transfer, thereby contributing to higher efficiencies of extracting xylan,
concentration from 30% to 90% led to a decrease in Mw from 120386 as seen in PX30 and PX70.
and 207730–72862 and 4144 Da for untreated and pretreated Chinese These results of morphology analysis were consistent with that of
quince, respectively. This result showed that the hemicellulosic frac- AFM, further confirming that entangled and branched structures exist
tions of higher Mw were precipitated at lower ethanol concentrations, in the polysaccharides molecules of quince. 2D and 3D AFM images of
while the Mw of the precipitated polysaccharide fractions decreased untreated and pretreated Chinese quince samples are shown in
with increasing final ethanol concentration. For the polysaccharide Fig. 3(B,C). Round lumps and chains were observed on the irregular and
fractions precipitated by 30% ethanol, hydrothermal treatment in- rough surface of samples. Due to the intra- and inter-molecular hy-
creased the content of polysaccharides of Mw from 120386 (UP30) to drogen bonds, these aggregates exhibited various sizes, which reflected
207730 (PX30) Da. Ma et al. (2012) reported that hemicellulosic frac- the heterogeneity and complexity of the branched polysaccharides (Li
tions with small molecular structures are released during hydrothermal et al., 2017). The heights of lumps ranged from 9.0 to 11.5 nm,

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Fig. 2. GPC profiles and molecular weights of polysaccharide fractions (A);

and FT-IR spectra of xylan fractions from unpretreatment and pretreatment
Chinese quince by hydrothermal (B: UPX30, UPX70, and UPX90 fractions; C:
PX30, PX50, PX70, and PX90 fractions).

8.0–17.9 nm, 5.9–23.2 nm for UPX30, UPX70, UPX90, respectively. lumps were estimated to be in the range of 0.6–7.8 nm, 0.3–4.3 nm,
These results show that the molecular chains were branched and en- 0.3–0.4 nm for PX30, PX70, PX90, respectively. In other words, the
tangled. The AFM images showed a significant difference between un- heights of the lumps of pretreated samples were significantly lower
treated and pretreated Chinese quince samples. The heights of the than those of untreated samples, however, the number of lumps

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Fig. 3. Scanning electron micrographs (A) and atomic force microscopy images (B-2D image; C-3D image) of various hemicelluloses fractions.

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Fig. 4. The 1H (A and B) spectra of UPX30 and PX30 fractions.

Fig. 5. The 2D HSQC NMR spectra of UPX30 and PX30 fractions.

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Fig. 6. TG and DTG curves of the PX30 and UPX30 fractions.

A
PX30
80 UPX30

60 o
310 C for PX30
o
308 C for UPX30
50%

40
25.0%

20 21.0%

100 200 300 400 500 600

o
Temperature ( C)
-0.062 for PX30
-0.059 for UPX30
B
0.0
o
70 C PX30
UPX30

-0.6

-1.2 -1.16
-1.28

100 200 300 400 500 600

o

increased after pretreatment. These results were attributed to hydro-
thermal degradation which broke down the large-chains of poly-
saccharide into simpler molecules and exposed more branched chains.
In summary, this hydrothermal treatment was related to the formation
of morphology of polysaccharides.
3.7. 1H NMR and 2D NMR analysis
1
H NMR spectroscopy is a fast method for certain tasks, such as the
investigation of the interaction of polysaccharides with solvents and the
determination of branching patterns of polysaccharides. HSQC together
with the 1H NMR spectrum clearly indicated the typical signal patterns
expected for hemicelluloses and could be considered as accurate
quantitative analysis. Therefore, to further elucidate the structural
features of the hemicellulosic fractions extracted from untreated and
pretreated Chinese quince, the hemicellulosic polymers UPX30 and
PX30 were investigated by 1H and 2D HSQC NMR spectroscopy. The 1H
NMR spectra of UPX30 and PX30 fractions are shown in Fig. 4A and B.
As shown in Fig. 4A and B, the two hemicellulosic fractions showed
analogous 1H NMR spectra, indicating that the two hemicelluloses have
similar structures. According to the literature (Peng et al., 2011a,b;
Peng and She, 2014) and sugar analysis, the main signals at δ 4.42 (H-
1), 4.04 (H-5eq), 3.50 (H-3), 3.73 (H-4), 3.42 (H-5ax), and 3.29 ppm
(H-2) are assigned to non-substituted (1 → 4)-β-D-Xylp structure. The
weak signals at δ 5.28 and 5.23 ppm are attributed to the Araf units
attached to 3-O- and 2-O- of the same Xylp residues, respectively. From
1
H NMR spectra of UPX30, the minor signals at δ 5.09 (H-1), 3.69 (H-2),
3.93 (H-3), 4.33 (H-4), and 4.90 (H-5) are considered to have originated
from α-GalpA (1 → ) units, which was in agreement with the results
from sugar analyses.
From 2D HSQC spectra of PX30 and UPX30 as shown in Fig. 5, five
dominant cross-peaks could be expressly indentified at 102.3/4.50,
76.4/3.74, 75.6/3.37, 72.8/3.19, and 62.9/3.33 + 4.01 ppm, and are
assigned to C1eH1, C4eH4, C3eH3, C2eH2, and C5eH5 of the (1 → 4)-
linked-β-D-Xylp residues, respectively. By comparing the 2D HSQC
NMR spectra of UPX30 and PX30, two hemicellulosic fractions showed
slightly different 2D HSQC NMR spectra, indicating some differences in
the structures of the hemicelluloses fractions. For example, some weak
signals at 101.5/5.06, 70.6/3.71, 71.0/3.91, 72.9/4.29, and 73.3/4.86
(no shown) ppm are observed in the 2D NMR spectrum of UPX30,
which represented the C1eH1, C4eH4, C3eH3, C2eH2, and C5eH5 of α-
GalpA (1 → ) units, respectively. In addition, the weak signals of 4-O-
Me-α-D-GlcpA-(1 → 2) units (72.81/3.68 and 59.72/3.46), Xylp units
attached with GlcA (78.39/3.56), and (1 → 4)-β-D-Galp units (105.10/

622
H.-M. Liu et al.
4.54) are also observed in the 2D HSQC NMR spectrum of UPX30
(Lisboa et al., 2005). For 2D HSQC NMR spectrum of PX30, some weak
signals, such as those at 106.7/4.62 (not shown), 74.1/3.51, 75.5/3.67,
71.7/3.90, 78.2/3.71 (not shown), and 64.1/3.82-3.76 (not shown)
ppm which corresponded to C1eH1, C2eH2, C3eH3, C4eH4, C5eH5, and
C6eH6 β-Galp-(1 → ) residues, respectively. Furthermore, the 2D HSQC
spectra of UPX30 and PX30 also showed the cross-peaks characteristic
of the structural element: Xylp units attached with acetyl groups
(72.53/3.76) and (1 → 4)-α-D-Glcp units (61.52/3.69) (Vanhazendonk
et al., 1996; Shukry et al., 2008). These were also confirmed by the
sugar analysis.
3.8. Thermo-gravimetric analysis
Fig. 6 shows the thermal gravimetric analysis (TGA) and the first
derivative (DTG) thermograms of PX30 and UPX30. As can be seen
from Fig. 6, three weight loss phases can be found in the TGA curves. At
the first stage, the initial decreases in the weights of PX30 and UPX30
below 150 °C was undoubtedly due to water desorption existence in the
samples. The second stage of weight loss ranging from 200 to 320 °C
relates to condensation of the degraded hemicellulosic fractions and
combustible gases, such as HCOOH, CH3COOH, CH4, CO2, CO, and
water (Soliman et al., 1997). In the last step (above 320 °C), the slope in
the TGA cures changes slowly, which might be attributed to the further
degradation of the small amount of hemicelluloses and the release of
flammable volatiles. However, it should be noted that there were 25.0%
and 21.0% solid residues at 670 °C for PX30 and UPX30, respectively.
At weight loss of 50%, the degradation temperatures of PX30 and
UPX30 were found to be 310 and 308 °C, respectively. In addition, the
DTG curves (Fig. 6B) show that the maximum peak was observed at
300 °C with the degradation rate of 1.16%/°C for PX30, whereas the
maximum peak occurred at 300 °C with the degradation rate of 1.28%/
°C for UPX30. Therefore, by relating the values of the maximum peak in
the DTG curve, the temperature at 50% weight loss and residue yields,
it can be concluded that hemicellulosic PX30 had higher thermal sta-
bility as compared with the UPX30 fraction. The result could be ex-
plained by the higher molecular weight of PX30 fraction as compared
with UPX30 fraction, since the thermal stability of the hemicellulosic
fraction was parallel to molecular weight (Fig. 2), as shown by Oihana
Gordobil et al. (Gordobil et al., 2016).
4. Conclusion
In the present investigation, hemicelluloses were successfully frac-
tionated from Chinese quince by applying two-step isolation tech-
nology. After the first stage of treatment, when the hydrothermal
treatment temperature increased from 120 °C to 150 °C, the amount of
xylose in the solid residue increased from 58.2% to 62.2%.
Hydrothermal treatment had an important effect on the distributions of
sugars and on the average molecular weight of hemicelluloses obtained
at various ethanol concentrations. Hemicellulosic sub-fractions from
both before and after hydrothermal treatment and precipitated at a
final ethanol percentage of 30% had a backbone of D-xylose residues
with a β-(1 → 4)-linkage. Thermo-gravimetric analysis showed that
hemicellulosic PX30 had higher thermal stability than that of UPX30
fraction. It is believed that the novel two-step hemicelluloses isolation
technology from Chinese quince will contribute to the development of
biomass refining industry.
Acknowledgement
We sincerely acknowledge the financial support by the earmarked
fund for Modern Agro-industry Technology Research System (CARS14-
1-29).
623
Industrial Crops & Products 111 (2018) 615–624
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