Separation and purification of fructo-oligosaccharide by high-speed counter-current chromatography coupled with precolumn derivatization

Article abstract of DOI:10.3390/molecules23020381

High-speed counter-current chromatography (HSCCC) coupled with precolumn derivatization was developed for isolating and purifying fructo-oligosaccharides (FOSs). Firstly, the total FOSs were precolumn derivatized and then separated by high-speed counter-current chromatography (HSCCC) with two-phase solvent system petroleum ether-n-butanol-methanol-water (3:2:1:4, v/v). Secondly, the obtained compounds were deacetylated and the fructo-oligosaccharides (FOSs) with high purity were obtained. Their structures were identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). This research successfully established a novel strategy for separation and purification of FOS. There is no doubt that the application of the research will be beneficial for the quantitative and qualitative analysis of products containing FOSs.

Full text of DOI:10.3390/molecules23020381

molecules
Article
Separation and Purification of Fructo-Oligosaccharide
by High-Speed Counter-Current Chromatography
Coupled with Precolumn Derivatization
1
1
2
1
2
1
Wenjuan Duan , Wenhua Ji , Yuanan Wei , Ruixuan Zhao , Zijian Chen , Yanling Geng ,
Feng Jing 1 and Xiao Wang *
1,
ID
1
Qilu University of Technology (Shandong Academy of Sciences), Shandong Analysis and Test Center,
Shandong Key Laboratory of TCM Quality Control Technology, 19 Keyuan Street,
Jinan 250014, Shandong, China; duanwj4048@126.com (W.D.); jwh519@163.com (W.J.);
zhaoruixuan2016@126.com (R.Z.); gengyanling@126.com (Y.G.); jingfeng20052009@163.com (F.J.)
Quantum Hi-Tech (China) Biological Co., Ltd., 133 Gaoxin Road West, Hi-tech Zone,
Jiangmen 529081, Guangdong, China; yawei@qht.cc (Y.W.); chenzj@qht.cc (Z.C.)
Correspondence: wxjn1998@126.com; Tel.: +86-531-8260-5304
2
*
Received: 15 December 2017; Accepted: 1 February 2018; Published: 10 February 2018
Abstract: High-speed counter-current chromatography (HSCCC) coupled with precolumn
derivatization was developed for isolating and purifying fructo-oligosaccharides (FOSs).
Firstly, the total FOSs were precolumn derivatized and then separated by high-speed
counter-current chromatography (HSCCC) with two-phase solvent system petroleum
ether–n-butanol–methanol–water (3:2:1:4, v/v).
Secondly, the obtained compounds were
deacetylated and the fructo-oligosaccharides (FOSs) with high purity were obtained. Their structures
were identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). This research
successfully established a novel strategy for separation and purification of FOS. There is no doubt
that the application of the research will be beneficial for the quantitative and qualitative analysis of
products containing FOSs.
Keywords: fructo-oligosaccharide; high-speed counter-current chromatography; precolumn
derivatization; separation and purification
1
. Introduction
Prebiotics are ‘non-digestible compounds which through metabolisation by gut microbiota,
modulate the compositions and/or activities of the gut microorganisms, thereby conferring beneficial
physiological effects on host’ [ ]. In recent decades, the bioactivities of prebiotics have attracted
1
more and more attention, worldwide, because of physiological functions such as improving the
gut ecosystem, restoring endothelial dysfunction, increasing heme bioavailability, barrier protective
function in human intestinal organoids, modulating lipogenesis, reducing triglyceridemia,
immunomodulatory and cerebral protective effects [2–8]. In addition, they could meet the requirements
for both sweet tooths and diabetics, due to their non-digestion and non-absorption in body [9].
Fructo-oligosaccharides (FOSs), one of the most significantly used commercial prebiotics in
practice, is a mixture (homopolymer) composed of 2–6 fructose monomers connected via
β (2–1)
glycosidic bonds linked to a terminal glucose residue. In recent years, FOSs have become one of the
most important healthy food ingredients, have great potential to improve the quality of many foods,
and have been applied in a wide range of fields such as infant milk powder, health care products,
pharmaceuticals and cosmetics. Due to the fact that the newer types of functional food ingredients
are increasingly popular, high-quality fructo-oligosaccharides (FOSs) are urgently needed to meet the
Molecules 2018, 23, 381; doi:10.3390/molecules23020381
www.mdpi.com/journal/molecules

Molecules 2018, 23, 381
2 of 9
demand for quality control and activity research. To date, a series of methods have been established to
purify FOSs, such as fast protein liquid chromatography, size-exclusion chromatography, hydrophilic
interaction chromatography, and anion exchange chromatography [10–12]. However, due to their
low yield, time-consuming processes, and small sample amounts, commercialized reference materials
of FOS are expensive. Therefore, it is necessary to develop an efficient method for isolating and
purifying FOSs.
High-speed counter-current chromatography (HSCCC) is a support-free liquid-liquid partition
chromatographic technique, which has been successfully applied in the isolation and purification
of many compounds [13–17]. Compared with many traditional liquid-solid separation methods,
HSCCC benefits from a number of advantages: (1) no irreversible adsorption; (2) complete recovery
of the injected sample; (3) tailing minimized; (4) low risk of sample denaturation; (5) low solvent
consumption; and (6) favorable economics, as there are no expensive columns needed, and only
common solvents are required once the initial investment in the instrument has been made [18].
Although there are many significant advantages to HSCCC, it is difficult to apply on FOSs because
of their high polar. In addition, to the best of our knowledge, no report on the separation of FOSs
by HSCCC has been published. In our previous research, the target compounds were eluted close
to each other near the solvent front, even the two-phase solvent system containing n-butanol or salt.
In this article, the polarity of FOSs was lowered by precolumn derivatization; then, three FOSs were
purified by HSCCC. Their structures were identified by mass spectrometry (MS) and nuclear magnetic
resonance (NMR).
The aim of the present work was to establish an efficient method for the separation and purification
of FOSs, and to provide a novel strategy for the separation and purification of oligosaccharides.
2
. Results and Discussion
2
.1. Strategy for Separation and Purification of FOSs
Because of structural complexity, high polarity, and difficulty of detection, it is difficult to isolate
and separate FOSs by traditional methods. In addition, separation is difficult to realize by HSCCC due
to their strong hydrophilicity, which leads to the target compounds staying in lower phase, even in
n-butanol–water solvent systems. Therefore, a modified strategy for preparative separation of FOSs is
needed. Firstly, the polarities of FOSs were lowered by means of acetylation, which was able to satisfy
the requirements of separation by HSCCC. Then, the obtained compounds were deacetylated, and the
separation of FOSs was realized (the roadmap is shown in Figure 1).
2
.2. Optimization of HPLC for FOSs
HPLC was used to analyze the total FOSs and purified FOS. According to the structure of the
target compounds, three kinds of detectors (DAD, ELSD and RID) were tested. The ELSD was selected
due to its high sensitivity and the short time required to achieve system balance. Due to the high
polarity of FOSs, the resolution of samples eluted with solvent in C18 chromatographic column is poor.
The Xamide 100A and NH inersil columns were compared, and the Xamide 100A was selected for its
2
good resolution and short processing time. Several elution systems were evaluated, such as isocratic
elution of acetonitrile–water and methanol–water. The results showed that the target compounds were
able to achieve suitable separation when the mobile phase consisted of acetonitrile–water (70:30, v/v).
◦
The column temperature was 30 C, the flow rate of the mobile phase was 1.0 mL/min, and the
◦
◦
evaporate temperature was 80 C, the Neb temperature was 90 C, the gas flow rate was 1.2 SLM.
Figure 2 shows that the purities of compounds I, II and III are 98.5%, 98.2%, 98.7%, respectively,
as determined by HPLC.

Molecules 2018, 23, 381
3 of 9
2
.3. Selection of Precolumn Derivatization Method
In our research, the HSCCC was operated after the precolumn derivatization of FOSs. On suitable
method of precolumn derivatization is ether-forming groups, which can be removed easily after
completion of the separation process. According to references [19 21], the common derivatization
–
methods for hydroxyl groups are methyl, acetyl, and phenyl. Comparing the degree of difficulty of the
experiment, the length of operation time, and the degree of hydroxyl group substitutions, acetylation of
hydroxyl groups in FOSs was selected.
Figure 1. Roadmap of separation of FOSs (
a) total FOSs; (
b) the structure of kestose; (c) the structure of
1
,1-kestotetraose; (d) the structure of 1,1,1-kestopentaose.
a
b
c
d
Figure 2. HPLC chromatograms of total acetylated FOSs and separated compounds: (
chromatograms of total acetylated FOSs; ( ) HPLC chromatograms of compound I; (
chromatograms of compound II; (d) HPLC chromatograms of HSCCC peak of compound III.
a
) HPLC
b
c) HPLC

Molecules 2018, 23, 381
4 of 9
2
.4. Selection of Two-Phase Solvent System and Other Conditions of HSCCC
An ideal range of K value for the applied material is a key factor in successfully selecting a
D
suitable two-phase solvent system, which is also the basis for obtaining a good separation effect of the
target compounds using HSCCC. As reference [22] describes, a K value in the range of 0.5 to 2 is a
D
requirement for selecting a suitable two-phase solvent system. In this experiment, the structures of
FOSs were modified by acetylation. After acetylation, the hydroxyls became acetyls, and their polarities
were remarkably reduced. According to the experience of separation of low-polar compounds, a
series of solvent systems composed of petroleum ether–ethyl acetate–methanol–water with different
volume ratios (1:1:1:1, 4:5:4:5, v/v), and petroleum ether–n-butanol–methanol–water with different
volume ratios (3:2:2:3, 3:2:1:4, v/v) were designed to achieve an ideal K for the target compounds.
D
The evaluated K values for the three compounds are shown in Table 1.
D
Table 1. The KD values of compound I, compound II, and compound III in different two-phase solvent
systems used in HSCCC.
KD Values
Solvent Systems
I
II
III
petroleum ether–ethyl
acetate–methanol–water
1:1:1:1
4:5:4:5
1.29
2.04
1.47
2.41
1.61
2.93
petroleum
ether–n-butanol–methanol–water
3:2:2:3
3:2:1:4
0.28
1.34
0.35
1.97
0.52
2.68
Figure 3. HSCCC chromatogram of the acetylated fructo-oligosaccharides. (
a
) Solvent system:
petroleum ether–ethyl acetate–methanol–water (1:1:1:1, v/v); stationary phase: upper phase;
mobile phase: lower phase; flow rate: 2.0 mL/min; revolution speed: 800 rpm; retention of
stationary phase: 57.0%; sample load: 200 mg; detection: ELSD; (b) Solvent system: petroleum
ether–n-butanol–methanol–water (3:2:1:4, v/v); stationary phase: upper phase; mobile phase:
lower phase; flow rate: 2.0 mL/min; revolution speed: 800 rpm; retention of stationary phase: 53.0%;
sample load: 200 mg; detection: ELSD.

Molecules 2018, 23, 381
5 of 9
As reference [22] describes, when the KD value is smaller than 0.5, the solutes will be eluted
close to each other and near the solvent front. As shown in Table 1, the K values of the two-phase
D
solvent systems consisting of ethyl petroleum ether–n-butanol–methanol–water (3:2:2:3, v/v) was
smaller than 0.5, which led to bad peak resolution. When the two-phase solvent systems were used
for separation, the solutes eluted close to each other and near the solvent front. When the two-phase
solvent systems composed of petroleum ether–ethyl acetate–methanol–water (4:5:4:5, v/v) were used,
the K value was greater than 2.0. When the two-phase solvent systems were used for separation,
D
the target compounds were able to be isolated; however, the separation time was too long, which led
to excessively broad peaks. The K values of the two-phase solvent systems composed of petroleum
D
ether–ethyl acetate–methanol–water (1:1:1:1, v/v) and petroleum ether–n-butanol–methanol–water
(3:2:1:4, v/v) were between 0.5 and 2, which were suitable for separating the target compounds.
Therefore, the HSCCC separation was carried out with the above two solvent systems. When the
two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (1:1:1:1, v/v)
was used for isolation, compound I, II and III were able to be eluted in a suitable time, but the
separation effect of the target compounds was not perfect. As shown in Figure 3, when the two-phase
solvent systems composed of petroleum ether–n-butanol–methanol–water (3:2:1:4, v/v) were used,
acceptable separation time and good resolution were able to be obtained. The collected fractions of
HSCCC were analyzed by HPLC-ELSD. 25.6 mg of compound I, 29.4 mg of compound II, 12.8 mg of
compound III with the purities of 98.5, 98.2%, and 98.7%, respectively, were obtained from 200 mg of
the crude sample.
3
. Materials and Methods
3
.1. Reagents and Materials
The FOSs were supplied by Quantum Hi-Tech Biological Co., Ltd. (Jiangmen, China).
◦
Ethanol, ethyl acetate, methanol, isopropanol, and petroleum ether (60–90 C) were all of analytical
grade, and were purchased from Jinan Xinhuicheng Chemical Factory, Jinan, China. Acetic anhydride
of analytical grade was purchased from China National Pharmaceutical Group Corporation (No. 20
Zhichun Road, Haidian District, Beijing, China). Methanol and acetonitrile of chromatographic grade
were purchased from Tedia Company, Inc. (Fairfield, CT, USA), and were used for HPLC analysis.
American ultrapure water (18.2 M
Ω) was used for all solutions and dilutions, and was obtained with
an osmosis Milli-Q water system purchased from Millipore (Bedford, MA, USA).
3
.2. Precolumn Derivatization
The precolumn derivatization of FOS was conducted by the method described in reference [19].
Acetic anhydride (20.4 g, 0.2 mol) was added to a solution of FOSs (5 g) in pyridine (50 mL) at room
temperature. After the reaction mixture was stirred overnight, the solution was concentrated under
reduced pressure to obtain the raw product as a yellow syrup, which was used for further purification.
3
.3. HSCCC Separation
3
.3.1. Apparatus
Model TBE-300A commercial instrument (Shanghai Tauto Biotech Co., Ltd., Shanghai, China),
which comprises a multilayer coil of 150 m in length and 1.6 mm id with a 300 mL total capacity,
was employed as the preparative HSCCC instrument in the present study. The values of this
preparative column ranged from 0.5 at the internal to 0.8 at the external ( = r/R, where r is the rotation
β
β
radius or the distance from the coil to the holder shaft, and R (R = 8 cm) is the revolution radius or the
distance between the holder axis and central axis of the centrifuge). A Model HX-1050 constant-flow
pump (Beijing Bokang Experimental Equipment Co., Ltd., Beijing, China) was used to pump the
solvent into the column. A 30 mL loop manual sample injection (Shanghai Tauto Biotech Co., Ltd.,

Molecules 2018, 23, 381
6 of 9
Shanghai, China) was used to inject the sample to the column. Spectra were achieved with a Model
8
2
823A-UV Monitor (Beijing Institute of New Technology Application, Beijing, China) at a wavelength
54 nm. The pH value was monitored by a Model 320 pH meter (Mettler Toledo Instruments,
Shanghai, China). The chromatogram was obtained by a portable recorder (Yokogawa Model 3057,
Sichuan Instrument Factory, Chongqing, China).
3
.3.2. Measurement of Partition Coefficient
The selection of the composition of the two-phase solvent system was based on the distribution
constant (K ) value of the target compounds. HPLC analysis was applied to determine the K values
D
D
by the following steps [22]: crude extract (5 mg) was dissolved in a test tube (20 mL) to which each
phase (5 mL) of the equilibrated two-phase solvent system was added. The capped tube was shaken
vigorously for several minutes to thoroughly equilibrate the sample with two phases. Then, 1 mL
upper phase and 1 mL lower phase were evaporated separately to dry by the gentle nitrogen stream.
The residues were diluted with methanol (1 mL) and analyzed by HPLC to determine the K of the
D
target compound. The KD value was calculated by the following equation: KD = A /A (A : the peak
U
L
U
area of the upper phase A : the lower phase).
L
3
.3.3. Preparation of Sample Solution and Two-Phase Solvent
The crude extract was dissolved in the mixed two-phase solvent system (lower phase and upper
phase, 1:1, v/v) to obtain the sample solution. Each solvent was added to a separatory funnel and
then repeatedly and vigorously shaken at room temperature to obtain the two-phase solvent system.
Shortly before use, the two phases were separated, following which the upper phase was used as
stationary phase and the lower phase was used as mobile phase.
3
.3.4. Separation Procedure
The separation procedure was carried out in accordance with [23]: firstly, the upper phase
(stationary phase) was pumped into the multilayer coil column until it was entirely filled at a flow
rate of 20 mL/min, while the lower phase (mobile phase) was pumped into the headend of the inlet
column at a flow rate of 2 mL/min, and the rotate speed of the apparatus was 800 rpm. After reaching
hydrodynamic equilibrium, the sample dissolving a clear mobile phase (200 mg of total FOSs in 8 mL
of both phases) was injected into the column via the injection valve. The effluent of the column was
continuously monitored with a UV detector at 254 nm, which was collected at 3 min intervals using
a fraction collector. After running, the residual solvent in the column was pushed out, and then the
retention of the upper phase was measured. The collected fractions were analyzed by HPLC-ELSD.
After analysis, the fractions with high purity were collected and dried separately.
3
.4. Reduction Reaction
The purified acetylated FOSs were reduced by the following method [24]. The target compounds
were added to a round-bottom flask while dissolved in MeOH. NaOMe (2 g, 0.037 mol) was added in a
single portion and stirred at room temperature for 1 hour. After stirring for 20 min, the reaction
+
was neutralized with Dowex 50WX8 (H form) and then filtered. The resulting solution was
concentrated under reduced pressure till thick oil was obtained. Purification by Sephadex LH-20
column chromatography (eluting with methanol) afforded the target compounds as white powder.
3
.5. HPLC-ELSD Analysis
The HPLC equipment was an Agilent system, and included an Agilent 1260 TCC, an Agilent 1260
quat pump, an Agilent 385-ELSD (Evaporative Light Scattering Detector), and an Agilent workstation
Agilent, Palo Alto, CA, USA).
(

Molecules 2018, 23, 381
7 of 9
The prebiotics and FOSs purified from the HSCCC separation were analyzed by HPLC-ELSD
◦
with a Xamide 100A column (250
consisted of acetonitrile–water (75:25, v/v) at a flow rate of 1.0 mL/min.
×
4.6 mm, id) and a column temperature of 25 C. The mobile phase
3
.6. Structural Identification
The identification of target compounds was carried out by ESI-MS on an Agilent 1100/MSD
1
13
and by H-NMR, C-NMR spectra on a Varian-600 NMR spectrometer with D O solvent and
2
1
13
tetramethylsilane (TMS) as internal standard. Comparing the ESI-MS, H-NMR, and C-NMR
data with the literature, the compounds I–III were identified as 1-kestose, 1,1-kestotetraose,
and 1,1,1-kestopentaose, respectively.
+
1
Compound kestose: ESI-MS m/z: 527 [M + Na] . H-NMR (D O, 600 MHz) δ: 5.26 (1H, d, J = 3.6 Hz,
2
G-1), 3.38 (1H, dd, J = 4.2, 9.6 Hz, G-2), 3.67 (1H, m, G-3), 3.30 (1H, t, J = 9.0 Hz, G-4), 3.58 (1H, m,
G-5), 3.64 (2H, m, G-6), 3.67 (1H, m, Fa-1), 3.57 (1H, m, Fa-1), 4.11 (1H, d, J = 9.0 Hz, Fa-3), 3.87 (1H, t,
J = 8.4 Hz, Fa-4), 3.71 (1H, m, Fa-5), 3.56 (1H, m, Fb-1), 3.50 (1H, m, Fb-1), 4.02 (1H, d, J = 8.4 Hz, Fb-3),
3
.91 (1H, t, J = 8.4 Hz, Fb-4), 3.70 (1H, m, Fb-5), 3.68 (1H, m, Fb-6), 3.52 (1H, m, Fb-6). 13C-NMR (D O,
2
150 MHz)
δ
: 92.4 (G-1), 71.0 (G-2), 72.3 (G-3), 69.1 (G-4), 72.4 (G-5), 60.0 (G-6), 60.2 (Fa-1), 103.6 (Fa-2),
76.5 (Fa-3), 73.7 (Fa-4), 81.0 (Fa-5), 62.0 (Fa-6), 60.7 (Fb-1), 103.1 (Fb-2), 76.5 (Fb-3), 74.3 (Fb-4), 81.1
(Fb-5), 62.2 (Fb-6). Compared with the data given in reference [25], peak I was identified as kestose.
+
1
Compound 1,1-kestotetraose: ESI-MS m/z: 689 [M + Na] . H-NMR (D O, 600 MHz)
δ: 5.25 (1H, d,
2
J = 3.6 Hz, G-1), 3.35 (1H, dd, J = 4.2, 9.6 Hz, G-2), 3.57 (1H, m, G-3), 3.29 (1H, t, J = 9.6 Hz, G-4),
3
3
.64 (1H, m, G-5), 3.63 (2H, m, G-6), 3.66 (1H, m, Fa-1), 3.57 (1H, m, Fa-1), 4.10 (1H, d, J = 9.0 Hz, Fa-3),
.87 (1H, t, J = 8.4 Hz, Fa-4), 3.69 (1H, m, Fa-5), 3.63 (1H, m, Fa-6), 3.61 (1H, m, Fa-6), 3.65 (1H, m,
Fb-1), 3.55 (1H, m, Fb-1), 4.05 (1H, d, J = 8.4 Hz, Fb-3), 3.90 (1H, t, J = 8.4 Hz, Fb-4), 3.68 (1H, m, Fb-5),
3
3
.64 (1H, m, Fb-6), 3.57 (1H, m, Fb-6), 3.57 (1H, m, Fc-1), 3.50 (1H, m, Fc-2), 4.01 (1H, d, J = 8.4 Hz, Fc-3),
13
.93 (1H, t, J = 8.4 Hz, Fc-4), 3.68 (1H, m, Fc-5), 3.65 (1H, m, Fc-6), 3.57 (1H, m, Fc-6). C-NMR (D O,
2
150 MHz)
δ
: 92.4 (G-1), 71.0 (G-2), 72.4 (G-3), 69.1 (G-4), 72.3 (G-5), 60.0 (G-6), 60.9 (Fa-1), 103.1 (Fa-2),
76.6 (Fa-3), 73.7 (Fa-4), 81.0 (Fa-5), 62.1 (Fa-6), 60.7 (Fb-1), 102.9 (Fb-2), 76.6 (Fb-3), 74.2 (Fb-4), 81.0
(
Fb-5), 62.1 (Fb-6), 60.2 (Fc-1), 103.6 (Fc-2), 77.3 (Fc-3), 74.3 (Fc-4), 81.1 (Fc-5), 62.1 (Fc-6). Compared with
the data given in reference [26], peak II was identified as 1,1-kestotetraose.
+
1
Compound 1,1,1-kestopentaose: ESI-MS m/z: 851 [M + Na] . H NMR (D O, 600 MHz)
δ: 5.26 (1H, d,
2
J = 4.2 Hz, G-1), 3.37 (1H, dd, J = 4.2, 9.6 Hz, G-2), 3.58 (1H, m, G-3), 3.30 (1H, t, J = 9.0 Hz, G-4), 3.67
(
(
1H, m, G-5), 3.62 (2H, m, G-6), 3.56 (1H, m, Fa-1), 3.65 (1H, m, Fa-1), 4.10 (1H, d, J = 8.4 Hz, Fa-3), 3.86
1H, t, J = 9.0 Hz, Fa-4), 3.57 (1H, m, Fa-5), 3.54 (2H, m, Fa-6), 3.65 (1H, m, Fb-1), 3.71 (1H, m, Fb-1),
4
(
.07 (1H, d, J = 8.4 Hz, Fb-3), 3.91 (1H, t, J = 8.4 Hz, Fb-4), 3.58 (1H, m, Fb-5), 3.55 (2H, m, Fb-6), 3.55
1H, m, Fc-1), 3.70 (1H, m, Fc-1), 4.05 (1H, d, J = 8.4 Hz, Fc-3), 3.94 (1H, t, J = 8.4 Hz, Fc-4), 3.58 (1H, m,
Fc-5), 3.55 (2H, m, Fc-6), 3.49 (1H, m, Fd-1), 3.58 (1H, m, Fd-1), 4.01 (1H, d, J = 8.4 Hz, Fd-3), 3.94 (1H, t,
J = 8.4 Hz, Fd-4), 3.58 (1H, m, Fd-5), 3.56 (2H, m, Fd-6). 13C NMR (D O, 150 MHz)
: 92.3 (G-1), 71.0
G-2), 72.3 (G-3), 69.1 (G-4), 72.4 (G-5), 60.0 (G-6), 60.3 (Fa-1), 103.5 (Fa-2), 76.5 (Fa-3), 73.7 (Fa-4), 81.0
Fa-5), 62.1 (Fa-6), 60.5 (Fb-1), 103.1 (Fb-2), 76.5 (Fb-3), 74.1 (Fb-4), 81.0 (Fb-5), 62.1 (Fb-6), 60.8 (Fc-1),
02.9 (Fc-2), 77.2 (Fc-3), 74.2 (Fc-4), 81.0 (Fc-5), 62.1 (Fc-6), 60.8 (Fd-1), 102.9 (Fd-2), 77.4 (Fd-3), 74.3
Fd-4), 81.1 (Fd-5), 62.1 (Fd-6). Compared with the data given in reference [27], peak III was identified
as 1,1,1-kestopentaose.
δ
2
(
(
1
(
4
. Conclusions
The present research indicated that a novel strategy had been developed to separate and purify
FOS from total FOSs. Firstly, the sample was structurally modified by acetylation. Then, the
acetylated target compounds were isolated by HSCCC with two-phase solvent system composed of
petroleum ether–n-butanol–methanol–water (3:2:1:4, v/v), and three compounds with high purity were
obtained in one step. Finally, the target compounds were obtained by reduction reaction whose

Molecules 2018, 23, 381
8 of 9
structure was identified by ESI-MS and NMR. In the present research, kestose in 98.5% purity,
,1-kestotetraose in 98.2% purity and 1,1,1-kestopentaose in 98.7% purity were obtained in a single run.
1
Compared with classic methods such as pre-HPLC, HSCCC coupled with precolumn derivatization is
characterized by excellent sample loading, simple operation, and as being less time-consuming and
more environmentally friendly. This study indicated that it is an efficient method for the separation
and purification of FOSs, and provides a novel strategy for the isolation of oligosaccharides.
Acknowledgments: The project was sponsored by the Key Research and Development Plan of Shandong Province
(
20146GSF202020), the Natural Science Foundation of Shandong province (ZR2013HM075) the natural Science
Foundation of Shandong (ZR2016YL006), and the Shandong Province Taishan Scholar Program (Lanping Guo).
Author Contributions: Wenjuan Duan performed the experiment and wrote the manuscript; Wenhua Ji,
Yuanan Wei, Ruixuan Zhao, Zijian Chen, Yanling Geng, Feng Jing participated in carrying out the experiment;
Xiao Wang designed and guided the experiment.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Bindels, L.B.; Delzenne, N.M.; Cani, P.D.; Walter, J. Towards a more comprehensive concept for prebiotics.
Nat. Rev. Gastroenterol. Hepatol. 2015, 12, 303–310. [CrossRef] [PubMed]
2
.
Catry, E.; Bindels, B.L.; Tailleux, A.; Lestave, S.; Neyrinck, M.A.; Goossens, J.F.; Lobysheva, I.; Plovier, H.;
Essaghir, A.; Demoulin, J.B.; et al. Targeting the gut microbiota with inulin-type fructans: Preclinical
demonstration of a novel approach in the management of endothelial dysfunction. Gut 2018, 67, 271–283.
[CrossRef] [PubMed]
3
4
5
6
7
.
.
.
.
.
Kato, T.; Fukuda, S.; Fujiwara, A.; Suda, W.; Hattori, M.; Kikuchi, J.; Ohno, H. Multiple omics uncovers
host-gut microbial mutualism during prebiotic fructooligosaccharide supplementation. DNA Res. 2014, 21,
4
69–480. [CrossRef] [PubMed]
Weinborn, V.; Valenzuela, C.; Olivares, M.; Arredondo, M.; Weill, R.; Pizarro, F. Prebiotics increase heme iron
bioavailability and do not affect non heme iron bioavailability in humans. Food Funct. 2017, 8, 1994–1999.
[
CrossRef] [PubMed]
Wu, R.Y.; Abdullah, M.; Määttänen, P.; Pilar, A.V.C.; Scruten, E.; Johnson-Henry, K.C.; Napper, S.; O’Brien, C.;
Jones, N.L.; Sherman, P.M. Protein kinase C signaling is required for dietary prebioticinduced strengthening
δ
of intestinal epithelial barrier function. Sci. Rep. 2017, 1, 1–10.
Zhou, S.; Liu, X.; Guo, Y.; Wang, Q.; Peng, D.; Cao, L. Comparison of the immunological activities of
arabinoxylans from wheat bran with alkali and xylanase-aided extraction. Carbohydr. Polym. 2010, 81,
7
84–789. [CrossRef]
Yen, C.H.; Kuo, Y.W.; Tseng, Y.H.; Lee, H.L.; Chen, H.L. Beneficial effects of fructo-oligosaccharides
upplementation on fecal bifidobacteria and index of peroxidation status in constipated nursing-home
residents—A placebocontrolled, diet-controlled trial. Nutrition 2011, 27, 323–328. [CrossRef] [PubMed]
Roberfroid, M.B. Fructo-oligosaccharide malabsorption: Benefit for gastrointestinal functions. Curr. Opin.
Gastroenterol. 2000, 16, 173–177. [CrossRef] [PubMed]
Agheli, N.; Kabir, M.; Berni-Canani, S.; Petitjean, E.; Boussairi, A.; Luo, J.; Bornet, F.; Slama, G.; Rizkalla, S.W.
Plasma lipids and fatty acid synthase activity are regulated by short-chain fructo-oligosaccharides in
sucrose-fed insulin-resistant rats. J. Nutr. 1998, 128, 1283–1288. [CrossRef]
8
.
.
9
1
0. Li, J.; Cheong, K.L.; Zhao, J.; Hu, D.J.; Chen, X.Q.; Qiao, C.F.; Zhang, Q.W.; Chen, Y.W.; Li, S.P. Preparation of
inulin-type fructooligosaccharides using fast protein liquid chromatography coupled with refractive index
detection. J. Chromatogr. A 2013, 1308, 52–57. [CrossRef] [PubMed]
1
1. Yang, Z.M.; Hu, J.; Zhao, M.Y. Isolation and quantitative determination of inulin-type oligosaccharides in
roots of Morinda officinalis. Carbohydr. Polym. 2011, 83, 1997–2004. [CrossRef]
1
2. Ronkart, S.N.; Blecker, C.S.; Fourmanoir, H.; Fougnies, C.; Deroanne, C.; Herck, J.C.V.; Paquot, M. Isolation
and identification of inulooligosaccharides resulting from inulin hydrolysis. Anal. Chim. Acta 2007, 604,
8
1–87. [CrossRef] [PubMed]

Molecules 2018, 23, 381
9 of 9
1
1
3. Shu, X.K.; Duan, W.J.; Liu, F.; Shi, X.G.; Geng, Y.L.; Wang, X. Preparative separation of polyphenols from
the flowers of Paeonia lactiflora Pall. by high-speed counter-current chromatography. J. Chromatogr. B 2014
47–948, 62–67. [CrossRef] [PubMed]
4. Chen, S.Q.; Zhao, X.; Li, Y.; Yang, D.; Zhou, T.T.; Fan, G.R.J. Impurities preparation of sodium
tanshinone IIA sulfonate by high-speed counter-current chromatography and identification by liquid
chromatography/multistage tandem mass spectrometry. J. Chromatogr. B 2013, 1288, 28–34. [CrossRef]
,
9
[PubMed]
1
1
5. Adhami, H.R.; Zehl, M.; Dangl, C.; Dorfmeister, D.; Stadler, M.; Urban, E. Preparative isolation of oleocanthal,
tyrosol, and hydroxytyrosol from olive oil by HPCCC. Food Chem. 2015, 170, 154–159. [CrossRef] [PubMed]
6. Wang, X.Y.; Shu, X.K.; Wang, X.; Yu, J.Q.; Jing, F. Preparative isolation of seven diterpenoid alkaloids from
Aconitum coreanum by pH-zone-refining counter-current chromatograph. Molecules 2014, 19, 12619–12629.
[CrossRef] [PubMed]
1
1
1
7. Wang, X.; Zheng, Z.J.; Guo, X.F.; Yuan, J.P.; Zheng, C.C. Preparative separation of gingerols from Zingiber
officinale by high speed counter-current chromatography using stepwise elution. Food Chem. 2011, 125,
1
476–1480. [CrossRef]
8. Sun, Y.; Yu, Z.Y.; Duan, W.J.; Lei, F.; Xu, S.S.; Wang, X. Isolation and purification of seven lignans from
Magnolia sparengeri by high-speed-counter-current chromatography. J. Chromatogr. B 2011, 879, 3775–3779.
[CrossRef] [PubMed]
9. Hartmann, M.; Betz, P.; Sun, Y.C.; Gorb, S.N.; Lindhorst, T.K.; Krueger, A. Saccharide-modified nanodiamond
conjugates for the efficient detection and removal of pathogenic bacteria. Chemistry 2012, 18, 6485–6492.
[
CrossRef] [PubMed]
0. Ciucanu, J.; Kerek, F.A. Simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res.
984, 131, 209–217. [CrossRef]
1. Patankar, M.S.; Oehninger, S.; Barnett, T.R.; Williams, L.; Clark, G.F. A revised structure for fucoidan may
explain some of its biological activities. J. Biol. Chem. 1993, 268, 21770–21776. [PubMed]
2. Ito, Y. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current
chromatography. J. Chromatogr. A 2005, 1065, 145–168. [CrossRef] [PubMed]
3. Ouyang, X.K.; Jin, M.C.; He, C.H. Preparative separation of four majoralkaloids from medicinal plant of
2
2
2
2
1
Tripterygium Wilfordii Hook F using high-speed counter-current chromatography. Sep. Purif. Technol. 2007
6, 319–324. [CrossRef]
,
5
2
4. Lin, Y.A.; Chalker, J.M.; Davis, B.G. Olefin Cross-Metathesis on Proteins: Investigation of Allylic Chalcogen
Effects and Guiding Principles in Metathesis Partner Selection. J. Am. Chem. Soc. 2010, 132, 16805–16811.
[CrossRef] [PubMed]
2
2
2
5. Bruyn, A.D.; Loo, J.V. The identification by 1H- and 13C-n.m.r. spectroscopy of sucrose, 1-kestose,
and neokestose in mixtures present in plant extracts. Carbohydr. Res. 1991, 211, 131–136. [CrossRef]
6. Timmermans, J.W.; Waard, P.D.; Tournois, H.; Leeflang, B.R.; Vliegenthart, J.F.G. NMR spectroscopy of
nystose. Carbohydr. Res. 1993, 243, 379–384. [CrossRef]
7. Cui, C.B.; Yang, M.; Yao, Z.W.; Cai, B.; Luo, Z.P.; Xu, Y.K. Studies on proton and carbon-13 nuclear magnetic
resonance of inulin-type oligosaccharides. Chin. J. Med. Chem. 1995, 5, 32–39.
Sample Availability: Samples of the compounds are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).