Carbohydrate Research 424 (2016) 1–7

Contents lists available at ScienceDirect

Carbohydrate Research
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a r r e s

Structural analysis of novel kestose isomers isolated from sugar beet

molasses
Norio Shiomi a, Tatsuya Abe b,*, Hiroto Kikuchi b, Tsutomu Aritsuka b, Yusuke Takata c,
Eri Fukushi c, Yukiharu Fukushi c, Jun Kawabata c, Keiji Ueno a, Shuichi Onodera a
a Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, Ebetsu 069-8501, Japan
b Research Center, Nippon Beet Sugar Mfg. Co., Ltd., Obihiro 080-0831, Japan
c
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

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

Article history: Eight kestose isomers were isolated from sugar beet molasses by carbon–Celite column chromatogra-
Received 12 October 2015 phy and HPLC. GC–FID and GC–MS analyses of methyl derivatives, MALD-TOF-MS measurements and NMR
Received in revised form 31 January 2016 spectra were used to confirm the structural characteristics of the isomers. The 1H and 13C NMR signals
Accepted 1 February 2016
of each isomer saccharide were assigned using COSY, E-HSQC, HSQC–TOCSY, HMBC and H2BC tech-
Available online 11 February 2016
niques. These kestose isomers were identified as α-D-fructofuranosyl-(2- > 2)-α-D-glucopyranosyl-(1 < -
>2)-β-D-fructofuranoside, α-D-fructofuranosyl-(2- > 3)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside,
Keywords:
α-D-fructofuranosyl-(2- > 4)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, β-D-fructofuranosyl-
Beet molasses
Kestose isomer (2- > 4)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, β-D-fructofuranosyl-(2- > 3)-α-D-
Oligosaccharide glucopyranosyl-(1 < ->2)-β-D-fructofuranoside, α-D-fructofuranosyl-(2- > 1)-β-D-fructofuranosyl-(2 < -
>1)-α-D-glucopyranoside, α-D-fructofuranosyl-(2- > 6)-α-D-glucopyranosyl-(1 < ->2)-β-D-fructofuranoside,
and α-D-fructofuranosyl-(2- > 6)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside. The former five com-
pounds are novel saccharides.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction D-fructopyranosyl-(2- > 6)-D-glucopyranose,8 α-D-fructofuranosyl-

We previously reported the structural analysis of several oligo-
and poly-saccharides composed of fructose and other monosac-
charide residue(s). These included 1-kestose, nystose, neokestose,
and lF(l-β-D-fructofuranosyl)m-6G(l-β-D-fructofuranosyl)n sucrose (4b:
m = 0, n = 2; 4c: m = 1, n = 1; 5a: m = 3, n = 0; 5b: m = 0, n = 3; 5c:
m = 2, n = 1; 5d: m = 1, n = 2; 6a: m = 4, n = 0; 6b: m = 0, n = 4; 6c:
m = 3, n = 1; 6d 1 : m = 1, n = 3; 6d 2 : m = 2, n = 2; asparagus
fructopolysaccharides: m + n = 12–22, and onion fructan: m + n = 7–
13) from asparagus roots 1–4 and onion bulbs. 5–7 Moreover, β-
Abbreviations: COSY, correlation spectroscopy; E-HSQC, CH2-selected edited
heteronuclear single quantum coherence; GC–FID, gas chromatography flame
ionisation detector; GC–MS, gas chromatography mass spectrometry; HMBC,
heteronuclear multiple bond correlation; HPAEC, high-performance anion-exchange
chromatography; HPLC, HR-HMBC, high resolution-HMBC, high-performance liquid
chromatography; H2BC, heteronuclear two-bond correlation; MALDI-TOF-MS, matrix-
assisted laser desorption ionisation/time-of-flight mass spectrometry; NMR, nuclear
magnetic resonance; SPT, selective population transfer; TOCSY, total correlation spec-
troscopy.
The English in this document has been checked by at least two professional editors,
both native speakers of English. For a certificate, please see: http://www
.textcheck.com/certificate/8gp1ir.
* Corresponding author. Tel.: +81 155 48 4106; fax: +81 155 47 0711.
E-mail address: abet@nitten.co.jp (T. Abe).
(2- > 6)-D-glucopyranose, 9 β-D-fructopyranosyl-(2- > 6)-β-D-
glucopyranosyl-(1- > 3)-D-glucopyranose,10 β-D-fructopyranosyl-
(2- > 1)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, 11
and β-D-fructopyranosyl-(2- > 6)-α-D-glucopyranosyl-(1 < ->2)-β-
D-fructofuranoside11 were isolated from a fermented vegetable juice
as new saccharides.
Further studies are in progress to form the oligosaccharides con-
taining fructose residue(s) by enzymatic and non-enzymatic
reactions. Several oligosaccharides such as fructosylxyloside or 1F
(1-β-D-fructofuranosyl) m -6 G (α-D-galactopyranosyl) n sucrose
were synthesised using fructosyltransferases from microorganisms12
or plant sources. 13 Conversely, β-D-fructopyranosyl-(2- >
6)-D-glucopyranose 14 and α-D-fructofuranosyl-(2- > 6)-D-
glucopyranose15 were produced from D-fructose and D-glucose by
thermal treatments.
Fructooligosacharides are known to exhibit health benefits
such as prebiotic effects, enhancement of mineral absorption, and
improvements in lipid metabolism.16 Previous studies also showed
that fructosylxyloside had an enhancing effect on mineral absorp-
tion and a suppressive effect on the elevation of blood glucose and
insulin levels in rats given sucrose or soluble starch.17 The charac-
teristics of β-D-fructopyranosyl-(2- > 6)-D-glucopyranose,8 α-D-
fructofuranosyl-(2- > 6)-D-glucopyranose, 15 and 1 F (1-β-D-
fructofuranosyl)m-6G(α-D-galactopyranosyl)n sucrose include low

http://dx.doi.org/10.1016/j.carres.2016.02.002
0008-6215/© 2016 Elsevier Ltd. All rights reserved.
2 N. Shiomi et al./Carbohydrate Research 424 (2016) 1–7

2. Results and discussion

Sugar beet molasses diluted with water was passed through a

Fig. 1. High-performance liquid chromatogram of fractions R5–8, R5–11, R5–16, R5–
19, and R20.
digestibility13,15 and non-cariogenic qualities.8,15 These saccharides
were selectively used by the beneficial bacteria, Bifidobacteria, but
were not used by unfavourable bacteria such as Clostridium
perfringens, Escherichia coli, and Enterococcus faecalis that produce
mutagenic substances.8,13,15
During a search of functional oligosaccharides in food sources,
twenty oligosaccharides were observed in beet sugar molasses.
In the present study, we confirmed the structures of five novel
kestose isomers, and three known compounds, from sugar
beet molasses. This study can be used as a first step in the
assessment of these compounds for use in food chemistry
applications.
carbon–Celite column (5.5 × 47 cm) and successively eluted with
water, 5 v/v% ethanol, 10 v/v% ethanol, and 20 v/v% ethanol. Each
eluted fraction from the sugar beet molasses was concentrated in
vacuo and freeze-dried. Thirty-one powdered fractions were ob-
tained, as shown in Table S1. Eight saccharides, saccharide 1 from
R5–8, saccharides 2 and 3 from R5–11, saccharides 4 and 5 from
R5–16, saccharides 6 and 7 from R5–19, and saccharide 8 from R-20
were purified using a preparative HPLC apparatus equipped with
an ODS (octadecylsilane) column (TSKgel ODS-80Ts, 20 mm × 25 cm),
as shown in Fig. 1.
Isolated saccharides 1–8 were homogeneous by HPAEC [ tR.sucrose
(retention time of sucrose = 1.0, 4.84 min): 2.15, 2.16, 1.68, 1.82, 1.73,
2.16, 2.24, and 2.83, respectively] and showed no reducing power
in the presence of Somogyi–Nelson reagent. The degree of
polymerisation (DP) of all of the saccharides was established as three
by measuring [M + Na]+ ions (m/z: 527) using MALDI-TOF-MS and
by analyses of the molar ratio (2) of fructose to glucose in the
hydrolysates.
To verify the bond structures of the saccharides, relative reten-
tion times of the methyl derivatives of permethylated saccharides
were investigated by GC–FID and GC–MS (Tables 1 and 2). The
methanolysates of permethyl saccharides 1–8 yielded two peaks cor-
responding to methyl 1,3,4,6-tetra-O-methyl-D-fructoside (tR, 1.08
and 1.28–1.30). Moreover, saccharide 1 gave two peaks correspond-
ing to methyl 3,4,6-tri-O-methyl-D-glucoside (tR, 3.02 and 3.63).
Saccharide 2 gave six peaks corresponding to methyl 2,3,4,6-tetra-
O-methyl-D-glucoside (tR, 1.03 and 1.46), and methyl 1,3,4-tri-O-
methyl-D-fructoside (tR, 1.90, 2.52, 3.96, and 4.47). Saccharide 3 gave
four peaks corresponding to methyl 2,3,4,6-tetra-O-methyl-D-
glucoside (tR, 1.03 and 1.46) and methyl 3,4,6-tri-O-methyl-D-
fructoside (t R , 2.72 and 4.09). Saccharide 6 gave two peaks
corresponding to methyl 2,3,4-tri-O-methyl-D-glucoside (tR, 2.56 and
3.67). Saccharide 8 gave two peaks corresponding to methyl 2,4,6-
tri-O-methyl-D-glucoside (tR, 3.14 and 4.70). The methyl derivatives
from permethyl saccharides 4, 5, and 7 were studied using GC–
MS, because analyses of methyl l,4,6-tri-O-methyl-D-fructoside and
methyl 1,3,6-tri-O-methyl-D-fructoside by GC–FID have not been re-
ported. The methanolysates of permethyl saccharides 4, 5, and 7
gave four peaks corresponding to methyl 1,3,4,6-tetra-O-methyl-
D-fructoside (t R , 1.04 and 1.10) and methyl 2,3,4,6-tetra-O-
methylglucoside (tR, 1.00 and 1.10). Furthermore, the methanolysate

Table 1
GC–FID analysis of methanolysates of permethylated saccharides 1, 2, 3, 6, and 8 isolated from sugar beet molasses

Methanolysate origin Relative retention time of methyl glycosidesa

1,3,4,6-Fru 2,3,4,6-Glc 3,4,6-Glc 2,4,6-Glc 2,3,4-Glc 3,4,6-Fru 1,3,4-Fru

Saccharide 1 1.08 1.29 3.02 3.63

Saccharide 2 1.08 1.29 1.03 1.46 1.90 2.52 3.96 4.47
Saccharide 3 1.08 1.29 1.03 1.46 2.72 3.67
Saccharide 6 1.09 1.30 2.56 3.67
Saccharide 8 1.06 1.28 3.18 4.70
6-α-Fructosyl D-glucoseb 1.08 1.30 2.56 3.66
Kojibioseb 1.00 1.43 2.98 3.59
Nigeloseb 1.00 1.42 3.19 4.71
1-Kestoseb 1.09 1.30 1.08 1.46 2.74 4.11
Neokestoseb 1.08 1.30 2.55 3.65
Timosy Levanb 1.08 1.28 1.43 1.90 2.49 3.98 4.49
Methyl α-D-glucosideb 1.45
Methyl β-D-glucosideb 1.00

Retention time of methyl 2,3,4,6-tetra-O-methyl-β-D-glucoside = 1.00; retention time, 4.92 min.

a
b
Reference methylated saccharides.
2,3,4,6-Glc, methyl 2,3,4,6-tetra-O-methyl-D-glucoside; 1,3,4,6-Fru, methyl 1,3,4,6-tetra-O-methyl-D-fructoside; 3,4,6-Glc, methyl 3,4,6-tri-O-methyl-D-glucoside; 2,4,6-
Glc, methyl 2,4,6-tri-O-methyl-D-glucoside; 2,3,4-Glc, methyl 2,3,4-tri-O-methyl-D-glucoside; 3,4,6-Fru, methyl 3,4,6-tri-O-methyl-D-fructoside; 1,3,4-Fru, methyl
1,3,4-tri-O-methyl-D-fructoside.
 N. Shiomi et al./Carbohydrate Research 424 (2016) 1–7 3

Table 2
GC–MS analysis of methanolysates of permethylated saccharides 4, 5, and 7 isolated from sugar beet molasses

Methanolysate origin Relative retention time of methyl glycosidesa

1,3,4,6-Fru 2,3,4,6-Glc 1,4,6-Fru 1,3,6-Fru

Monitored positive ion [m/z] 205 250 191 191

[M－CH2OCH3]+ [M]+ [M－CH2OCH3]+ [M－CH2OCH3]+

Saccharide 4 1.04 1.10 1.00 1.11 1.27 1.31

Saccharide 5 1.04 1.10 1.00 1.11 1.36 1.62
Saccharide 7 1.04 1.10 1.00 1.11 1.35 1.62
6-α-Fructosyl-D-glucoseb 1.04 1.10
Lactuloseb 1.00 1.10 1.36 1.62 1.67
1-Kestoseb 1.04 1.10 1.00 1.11
Melezitoseb 1.00 1.11 1.27 1.31
Methyl α-D-glucosideb 1.11
Methyl β-D-glucosideb 1.00
a
Retention time of methyl 2,3,4,6-tetra-O-methyl-β-D-glucoside = 1.0; retention time, 4.15 min.
b Reference methylated saccharides.
2,3,4,6-Glc, methyl 2,3,4,6-tetra-O-methyl-D-glucoside; 1,3,4,6-Fru, methyl 1,3,4,6-tetra-O-methyl-D-fructoside; 1,4,6-Fru, methyl 1,4,6-tri-O-methyl-D-fructoside; 1,3,6-
Fru, methyl 1,3,6-tri-O-methyl-D-fructoside.

from saccharides 4 gave two peaks monitored by the [M－CH2OCH3]+ a correlation peak with C-2′ in the HMBC spectrum was assigned
ion (m/z 191) and corresponding to methyl 1,4,6-tri-O-methyl-D- to H-3′. Similarly, the methine carbon atom that yielded a corre-
fructoside (tR, 1.27 and 1.31). Those from saccharides 5 and 7 gave lation with H-1′ in the HMBC spectrum was assigned to C-5′. The
two peaks monitored at m/z 191 and corresponding to methyl 1,3,6- remaining methine proton included in the same spin–spin network
tri-O-methyl-D-fructoside (tR, 1.35 and 1.62). The above data show with H-1′ was assigned to H-4′.
that saccharides 1, 2, 3, 6, 8, and 4 were D-fructosyl-(2- > 2)-D- In the case of Fru′, the characteristic doublet proton signal, which
glucosyl-(1 < ->2)-D-fructoside, D-fructosyl-(2- > 6)-D-fructosyl- exhibited a correlation peak with the quaternary carbon arom (C-
(2 < ->1)-D-glucoside, D-fructosyl-(2- > 1)-D-fructosyl-(2 < ->1)-D- 2″) in the HMBC spectrum, was assigned to H-3″. The methine proton
glucoside, D-fructosyl-(2- > 6)-D-glucosyl-(1 < ->2)-D-fructoside, that produced a correlation peak with H-3″ in the COSY spectrum
D-fructosyl-(2- > 3)-D-glucosyl-(1 < ->2)-β-D-fructoside, and was assigned to H-4″. Similarly, the methine proton that gave a cor-
D-fructosyl-(2- > 3)-D-fructosyl-(2 < ->1)-D-glucoside, respectively. relation with H-4″ in the COSY spectrum was assigned to H-5″. The
Saccharides 5 and 7 were D-fructosyl-(2- > 4)-D-fructosyl- methylene proton in the same spin–spin network as H-3″, H-4″, and
(2 < ->1)-D-glucoside. H-5″ was assigned to H-6″. The methylene proton, which was cor-
The structures of all isolated oligosaccharides were deter- related with C-2″ in the HMBC spectrum, was assigned to H-1″.
mined by NMR techniques as follows. Assignments for all 1H and The intra-residual assignment of Fru was accomplished in a way
13
C signals are shown in Tables S2 and S3. similar to that described above for Fru′. The correlation peak from
NMR spectral analyses were initiated at the anomeric proton and C-5 to H-5 in the HMBC spectrum indicates that Fru existed in its
carbon signals, which exhibited separate characteristic signals in furanosyl form.
the 1H and 13C NMR spectra, respectively. Saccharide 1 has an The arrangement of sugar residues was determined by
anomeric proton (δH 5.46 ppm, d, 3.3 Hz) and three anomeric carbon inter-residual HMBC correlation peaks. The quaternary carbon atom
atoms (δC 109.50 ppm, 104.70 ppm, and 93.06 ppm). The carbon
signals at δC 109.50 ppm and 104.70 ppm were attributed to qua-
ternary carbon atoms. Subsequently, the carbon signals
corresponding to each proton signal were assigned according to
E-HSQC spectra, such that the assignment of a particular proton
signal was equivalent to the assignment of the corresponding carbon
signal. In addition, methylene carbon signals were distinguished from
other carbon signals in E-HSQC spectra.
The HSQC–TOCSY spectrum of saccharide 1 (Fig. 2 and Fig. S1)
revealed proton and carbon signals in the same aldose unit and from
C-3 and H-3 to C-6 and H-6 in the ketose unit. The anomeric proton
exhibited correlation peaks to six carbon atoms, indicating that sac-
charide 1 includes an aldose unit. As described below, the J coupling
values and chemical shifts indicated that the aldose unit was a
glucosyl residue. There remained two sets of four carbon atoms in
the same spin–spin network: two separated methylene carbon atoms
and two quaternary carbon atoms. These findings suggested the pres-
ence of two fructosyl residues. Among the two fructosyl residues
with anomeric carbons (δH 104.70 ppm and δH 109.50 ppm), the
former was named Fru and the latter was named Fru′. The glucosyl
residue is represented as Glc.
With regard to Glc, the anomeric proton was assigned to H-1′
and the methylene proton was assigned to H-6′. The methine proton
that exhibited a correlation with H-1′ in the COSY spectrum was Fig. 2. Selected parts of the COSY (a), HSQC–TOCSY (b), E-HSQC (c), and HMBC (d)
assigned to H-2′. Subsequently, the methine proton that exhibited spectra of saccharide 1.
4 N. Shiomi et al./Carbohydrate Research 424 (2016) 1–7
Fig. 3. Selected parts of the HSQC–TOCSY (a), E-HSQC (b), and HMBC (c) spectra of
saccharide 4.
(C-2) of Fru exhibited a correlation with the anomeric proton (H-
1′) of Glc (Fig. 2d and Fig. S1). The other quaternary proton (C-2″)
of Fru′ exhibited a correlation with the methine protone (H-2′) of
Glc (Fig. 2d and Fig. S1). These inter-residual HMBC correlation peaks
indicated a connectivity of Fru′ (2″- > 2′) to Glc (1′<->2) to Fru.
Finally, the configuration of saccharide 1 was determined by 3JHH
coupling patterns and carbon chemical shifts. 3JHH coupling values
were extracted from SPT difference spectra18,19 (Fig. S2). Small JHH
values (J = 2–5) between H-1′ and H-2′, and large JHH values (J = 8–
10 Hz) between H-2′ and H-3′, H-3′ and H-4′, and H-4′ and H-5′
indicated that an aldose unit is an α-glucosyl residue, as shown in
Fig. S3. The anomeric configuration and differentiation between the
pyranosyl and furanosyl forms of the fructose unit were deter-
mined by δC values and 3JHH coupling patterns.20,21 The δC values and
3 HMBC spectra revealed that C-2″ of Fru′ exhibited a correlation not
JHH coupling patterns of Fru and Fru′ indicated that they are in the
β anomer form and α anomer form of fructofranoside, respectively.
The structures of saccharides 4 and 5 were determined using the
same techniques used to determine the structure of saccharide 1.
Each 2D-NMR correlation is shown in Figs. 3 and 4.
Fig. 4. Selected parts of the COSY (a), HSQC–TOCSY (b), E-HSQC (c), and HMBC spectra
of saccharide 5.
Fig. 5. Selected parts of the COSY (a), HSQC–TOCSY (b), E-HSQC (c), and HMBC spectra
of saccharide 7.
In the case of saccharide 7, H-4″ and H-5″ of Fru′ were over-
lapped in the 1H NMR spectrum. For this reason, separation of H-4″
and H-5″ was established by acquiring an H2BC spectrum (Fig. S4).
The methine carbon, which exhibited a correlation to H-3″ in the
H2BC spectrum, was assigned to C-4″. Except for this, the struc-
ture of saccharide 7 was determined using the same techniques used
for saccharides 1, 4, and 5.
With saccharide 8, H-3′ of Glc, H-3″ and H-4″ of Fru′, and H-3
of Fru were intricately overlapped in the 1H NMR spectrum. HR-
with H-3″ of Fru′ but with H-3′ of Glc (Fig. S5). 3JHH values of H-3″
and H-4″ were also determined by the HR-HMBC spectrum. The 2D-
NMR correlations of saccharides 7 and 8 are shown in Figs. 5 and
6, respectively.
Fig. 6. Selected parts of the COSY (a), HSQC–TOCSY (b), E-HSQC (c), and HMBC spectra
of saccharide 8.
 N. Shiomi et al./Carbohydrate Research 424 (2016) 1–7
Glc
Sucrose moiety
Fru
Saccharides RF1 RF3 RF4 RF6 RG2 RG3 RG6
1* H H H H αF H
2 H H H αF H
3 αF H H H
4* H αF H H H
5* H H βF H
6 H H H H
7* H H αF H
8* H H H H
* novel saccharides
Fig. 7. Structures of kestose isomers 1–8 isolated from sugar beet molasses.
The structures of the three remaining oligosaccharides were de-
termined similarly. As shown in Fig. 7, eight oligosaccharides 1–8
isolated from sugar beet molasses were confirmed to be the fol-
lowing kestose isomers, which consist of a sucrose moiety and a
fructosyl residue: α-D-fructofuranosyl-(2- > 2)-α-D-glucopyranosyl-
(1 < ->2)-β-D-fructofuranoside, α-D-fructofuranosyl-(2- > 6)-β-D-
fructofuranosyl-(2 < ->1)α-D-glucopyranoside, α-D-fructofuranosyl-
(2- > 1)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, α-D-
fructofuranosyl-(2- > 3)-β-D-fructofuranosyl-(2 < ->1)-α-D-
glucopyranoside, β-D-fructofuranosyl-(2- > 4)-β-D-fructofuranosyl-
(2 < ->1)-α-D-glucopyranoside, α-D-fructofuranosyl-(2- > 6)-α-D-
glucopyranosyl-(1 < ->2)-β-D-fructofuranoside, α-D-fructofuranosyl-
(2- > 4)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, and β-D-
fructofuranosyl-(2- > 3)-α-D-glucopyranosyl-(1 < ->2)-β-D-
fructofuranoside. Saccharides 1, 4, 5, 7, and 8 are novel saccharides.
Although saccharides 2, 3, and 6 are reportedly produced by the
pyrolysis of sucrose,22 the formation mechanisms of all the saccha-
rides isolated from beet sugar molasses is not clear and has become
a subject of considerable interest.
Saccharides 4, 5, and 7, which are derived from the substitu-
tion of the 3F-α-fructofuranosyl residue, the 4F-β-fructofuranosyl
residue, and the 4 F -α-fructofuranosyl residue for the 1 F -β-
fructofuranosyl residue in 1-kestose were named α-3-kestose,
4-kestose, and α-4-kestose, respectively.
3. Conclusions
In this study, eight oligosaccharides were isolated from sugar beet
molasses using carbon–Celite column chromatography and pre-
parative HPLC. Structural confirmation of these saccharides was
provided by methylation analysis, MALDI-TOF-MS, and NMR
measurements.
The eight saccharides 1–8 shown in Fig. 7 were isolated from sugar
beet molasses and identified as the following kestose isomers, which
consist of a sucrose moiety and fructosyl residue: α-D-fructofuranosyl-
(2- > 2)-α-D-glucopyranosyl-(1 < ->2)-β-D-fructofuranoside, α-D-
fructofuranosyl-(2- > 6)-β-D-fructofuranosyl-(2 < ->1)α-D-
glucopyranoside, α-D-fructofuranosyl-(2- > 1)-β-D-fructofuranosyl-
(2 < ->1)-α-D-glucopyranoside, α-D-fructofuranosyl-(2- > 3)-β-D-
5
ring
flip
α-Fru
(αF)
β-Fru
(βF)
H
H H
H H H
H H
H H H
H H αF
H H H
H βF H
fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, β-D-fructofuranosyl-
(2- > 4)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, α-D-
fructofuranosyl-(2- > 6)-α-D-glucopyranosyl-(1 < ->2)-β-D-
fructofuranosideα-D-fructofuranosyl-(2- > 4)-β-D-fructofuranosyl-
(2 < ->1)-α-D-glucopyranoside, and β-D-fructofuranosyl-(2- > 3)-
α-D-glucopyranosyl-(1 < ->2)-β-D-fructofuranoside.
Saccharides 1, 4, 5, 7, and 8 are novel saccharides.
4. Experimental
4.1. Materials
The sugar beet molasses was produced by Nippon Beet Sugar Mfg.
Co., Ltd., Hokkaido, Japan. Standard sugars were prepared as follows.
Crystalline 1-kestose was prepared from sucrose using the corre-
sponding enzyme from Scopulariopsis brevicaulis.23 Neokestose and
6-α-D-fructofuranosyl-D-glucopyranose were isolated from aspar-
agus root1 and a fermented plant extract beverage.9,14,15 Levan was
obtained from the roots of the timothy plant (Phleum pratense L).24
Methyl α/β-D-glucoside, kojibiose, lactulose, melezitose, and nigerose
were purchased from Nakalai Tesque (Kyoto, Japan).
4.2. Quantitative determination of sugar
Total sugars were determined by the anthrone method.25 Re-
ducing sugars were quantified using the methods described by
Somogyi26,27 and Nelson.28
4.3. High-performance anion-exchange chromatography (HPAEC)
The saccharides, from monomer to oligomer, were analysed using
a Dionex Bio LC Series apparatus, equipped with an HPLC carbo-
hydrate column (CarboPac PAl, inert styrenedivinylbenzene polymer)
by pulsed amperometric detection (PAD).29,30 The elution gradient
was established by mixing eluent A (150 mM NaOH) with eluent
B (500 mM sodium acetate in 150 mM NaOH) as follows:31 0–1 min,
25 mM; 1–2 min, 25–50 mM; 2–20 min, 50–200 mM; 20–22 min,
500 mM; 22–30 min, 25 mM. Elution was performed at a column
flow rate of 1 mL/min. The applied PAD potentials for E1 (300 ms),
6 N. Shiomi et al./Carbohydrate Research 424 (2016) 1–7
E2 (120 ms), and E3 (300 ms) were 0.04, 0.60 and −0.80 V, respec-
tively, and the output range was 1 μC as described previously.
Quantitative determinations of D-glucose and D-fructose were per-
formed over the range from 5 to 50 μg/mL by HPAEC. The kestose
isomer (0.5–1 mg) was hydrolysed in 0.5 N hydrochloric acid (0.5 mL)
at 100 °C for 30 min.
4.4. Isolation of saccharides
Sugar beet molasses, diluted five times with water, was freeze-
dried to give a light brown powder. Thirteen grams of the powder
was dissolved in 100 mL of water and the solution was loaded onto
a carbon–Celite [charcoal (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan) and Celite-535 (Nakarai Chemical Industries, Ltd.,
Osaka, Japan); 1:1] column (5.5 × 47 cm) and successively eluted with
water (7.0 L), 5 v/v% ethanol (21.4 L), 10 v/v% ethanol (5.1 L), and
20 v/v% ethanol (3.0 L). Each oligosaccharide fraction was concen-
trated in vacuo and freeze-dried. Thirty-one different powdered
fractions were obtained as shown in Table S1. Fractions R5–8, R5–
11, R5–16, R5–19, and R-20 contained several saccharides that
differed from the standard saccharides: maltose, maltotriose, raf-
finose, 1-kestose, 6-kestose, neokestose, nystose and fructosylnystose,
as shown in Fig. 1. Each fraction was dissolved in water to a 2% con-
centration, and 0.5-mL aliquots were repeatedly applied to a
preparative HPLC system (JASCO GULLIVER, Tokyo, Japan) equipped
with an ODS column (TSKgel ODS-80Ts, 20 mm × 25 cm, Tosoh,
Tokyo, Japan) at 35 °C. The samples were eluted with water at a flow
rate of 3.0 mL/min. Saccharide 1 from R5–8 (0.39 g), saccharides 2
and 3 from R5–11 (0.61 g), saccharides 4 and 5 from R5–16 (0.24 g),
saccharides 6 and 7 from R5–19 (0.33 g), and saccharide 8 from R-20
(0.37 g) were separated using preparative HPLC under the same con-
ditions as above. Furthermore, all of the saccharides were purified
using the same preparative HPLC method. Purified saccharides 1
(5.2 mg), 2 (14.5 mg), 3 (6.0 mg), 4 (3.5 mg), 5 (3.9 mg), 6 (12.8 mg),
7 (4.5 mg), and 8 (2.1 mg) were obtained as white powders.
4.5. Methylation and methanolysis
Methylation of saccharides was carried out using the method de-
scribed by Hakomori. 32 Solution of reference saccharides (3–
5 mg) or kestose isomers (1–5 mg) in 1 mL of dimethyl sulfoxide
(DMSO) were prepared with stirring under a nitrogen atmo-
sphere. To prepare the carbanion solution, a mixture of 500 mg of
sodium hydride and 5 mL of DMSO was stirred in a flask under a
nitrogen atmosphere for 1 h at 50 °C. A 1-mL aliquot of the latter
solution was added to 1 mL of the former and stirred for 3.5–5.0 h
at 20 °C. Subsequently, 0.8 mL of methyl iodide was added and the
solution stirred for an additional 15 h. The reaction mixture was
diluted with water and extracted with chloroform. The chloro-
form extract was washed with water and concentrated in vacuo to
give the methylated products in a syrupy residue. The permethylated
saccharides were methanolysed by heating with 1.5% methanolic
hydrochloric acid at 92 °C for 5–20 min. The reaction mixture was
treated with Amberlite IRA-410 (OH-) to remove hydrochloric acid
and evaporated in vacuo to dryness. The resulting methanolysate
was dissolved in a small quantity of methanol or chloroform prior
to GC–FID or GC–MS analyses.
4.6. Gas chromatography flame ionisation detector (GC–FID)
GC–FID analyses were carried out on a Shimadzu GC 8A gas chro-
matograph using a glass column (2.6 mm × 2 m) packed with 15%
butane-1,4-diol succinate polyester on acid-washed Celite at 175 °C.
The flow rate of the nitrogen gas carrier was 40 mL/ min.
4.7. Gas chromatography mass spectrometry (GC–MS)
GC–MS analyses were performed using a JMS-T100GCV mass
spectrometer (JEOL, Japan) with a VF-23ms (30 m × 0.25 mm I.D.,
0.25 um film) capillary column (Agilent, USA). The injection tem-
perature was 250 °C. Helium was used as the carrier gas with a
ramped flow rate. The flow was initially constant at 1.4 mL/min for
6 min and then ramped to 2 mL/min at 6 mL/min/min. The oven tem-
perature program was as follows: initial temperature, 100 °C (1 min),
then 35 °C/min to 170 °C, 10 °C/min to 210 °C, 40 °C/min to 250 °C,
and 2.5 °C/min to 260 °C. Mass spectra were obtained by field
ionisation. The interface was heated to 250 °C and the ion source
was held at 80 °C.
4.8. Matrix-assisted laser desorption ionisation/time of flight mass
spectrometry (MALDI-TOF-MS)
MALDI-TOF-MS spectra were obtained using a Shimadzu–
Kratos mass spectrometer (KOMPACT Probe) in positive ion mode
with 2.5%-dihydroxybenzoic acid as a matrix. Ions were formed by
a pulsed UV laser beam (nitrogen laser, 337 nm). Calibration was
done using 1-kestose as an external standard.
4.9. Nuclear magnetic resonance (NMR) measurements
Saccharides (1–6 mg) were each dissolved separately in 0.06 or
0.5 mL D2O. NMR spectra were recorded at 27 °C with a Bruker AMX
500 spectrometer (1H 500 MHz, 13C 125 MHz) equipped with a 2.5-
or 5-mm diameter C/H dual probe (1D spectra) and a TXI triple probe
(2D spectra). Chemical shifts in ppm for 1H (δH) and 13C (δC) spectra
were determined relative to an external standard of sodium [2, 2,
3, 3-2H4]-3-(trimethylsilyl)-propionate in D2O (δH 0.00 ppm) and 1,
4-dioxane (δC 67.40 ppm) in D2O, respectively. 1H–1H COSY,33,34
H2BC,35,36 E-HSQC,37–39 HSQC–TOCSY,37,40 HMBC,41,42 and HR-HMBC43
spectra were obtained using gradient-selected pulse sequences. The
TOCSY mixing time (0.17 s) was determined using the decoupling
in the presence of scalar interactions (DIPSI)-2 method.
Authors’ contributions
NS and TA performed data analyses and contributed to the draft-
ing of the manuscript. YT, EF, YF, and JK collected the NMR data.
NS, TA, SO, and KU conceived of the study, participated in its design,
and contributed to the drafting of the manuscript. All authors read
and approved the final manuscript.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.carres.2016.02.002. These data include MOL files and
InChiKeys of the most important compounds described in this article.
References
1. Shiomi N, Yamada J, Izawa M. Agric Biol Chem 1976;40:567–75.
2. Shiomi N, Yamada J, Izawa M. Agric Biol Chem 1979;43:1375–7.
3. Shiomi N. Phytochemistry 1981;20:2581–3.
4. Shiomi N. New Phytol 1993;123:263–70.
5. Shiomi N, Onodera S, Sakai H. New Phytol 1997;136:105–13.
6. Fujishima M, Furuyama K, Ishiguro Y, Onodera S, Fukushi E, Benkeblia N, et al.
Int J Carbohydr Chem 2009;1–9. Article ID 493737.
7. Yamamori A, Okada H, Kawazoe N, Ueno K, Onodera S, Shiomi N. J Appl Glycosci
2015;62:95–9.
8. Okada H, Fukushi E, Yamamori A, Kawazoe N, Onodera S, Kawabata J, et al.
Carbohydr Res 2006;341:925–9.
9. Okada H, Fukushi E, Yamamori A, Kawazoe N, Onodera S, Kawabata J, et al.
Carbohydr Res 2011;346:2633–7.
10. Kawazoe N, Okada H, Fukushi E, Yamamori A, Onodera S, Kawabata J, et al.
Carbohydr Res 2008;343:549–54.
 N. Shiomi et al./Carbohydrate Research 424 (2016) 1–7 7

11. Okada H, Fukushi E, Yamamori A, Kawazoe N, Onodera S, Kawabata J, et al. 28. Nelson N. J Biol Chem 1944;153:375–80.
Carbohydr Res 2010;345:414–8. 29. Rocklin RD, Pohl CA. J Liq Chromatogr 1983;6:1577–90.
12. Takeda H, Kinoshita S. J Ferment Bioeng 1995;79:242–6. 30. Johnson DC. Nature 1986;321:451–2.
13. Yamamori A, Onodera S, Kikuchi M, Shiomi N. Biosci Biotechnol Biochem 31. Shiomi N, Onodera S, Chatterton NJ, Harrison PA. Agric Biol Chem 1991;55:1427–
2002;66:1419–22. 8.
14. Yamamori A, Okada H, Kawazoe N, Onodera S, Shiomi N. Biosci Biotechnol Biochem 32. Hakomori S. J Biochem 1964;55:205–8.
2010;74:2130–2. 33. Aue WP, Bartholdi E, Ernst RR. J Chem Phys 1976;64:2229–46.
15. Yamamori A, Okada H, Kawazoe N, Muramatsu K, Onodera S, Shiomi N. J Appl 34. Von Kienlin M, Moonen CTW, Von der Toorn A, Van Zijl PCM. J Magn Reson
Glycosci 2014;61:99–104. 1991;93:423–9.
16. Benkeblia N, Shiomi N. Curr Nutr Food Sci 2006;2:181–91. 35. Nyberg NT, Duus JØ, Sørensen OW. J Am Chem Soc 2005;127:6154–5.
17. Fukumori Y, Onodera S, Shiomi N. J Appl Glycosci 2001;48:205–13. 36. Petersen BO, Vinogradov E, Kay W, Würtz P, Nyberg NT, Duus JØ, et al. Carbohydr
18. Pachler KGR, Wessels PL. J Magn Reson 1973;12:337–9. Res 2006;341:550–6.
19. Uzawa J, Yoshida S. Magn Reson Chem 2004;42:1046–8. 37. Willker W, Leibfritz D, Kerssebaum R, Bermel W. Magn Reson Chem
20. Allerhand A, Doddrell D. J Am Chem Soc 1971;93:2779–81. 1993;31:287–92.
21. Agrawal PK. Phytochemistry 1992;31:3307–30. 38. Fukushi E, Onodera S, Yamamori A, Shiomi N, Kawabata J. Magn Reson Chem
22. Manley-Harris M, Richards GN. Carbohydr Res 1991;219:101–13. 2000;38:1005–11.
23. Takeda H, Sato K, Kinoshita S, Sasaki H. J Ferment Bioeng 1994;77:386–9. 39. Davis DG. J Magn Reson 1991;91:665–72.
24. Yamamoto S, Mino Y. Plant Physiol 1985;78:591–5. 40. Domke T. J Magn Reson 1991;95:174–7.
25. Morris DL. Science 1948;107:254–5. 41. Bax A, Summers MF. J Am Chem Soc 1986;108:2093–4.
26. Somogyi M. J Biol Chem 1945;160:69–73. 42. Hurd RE, John BK. J Magn Reson 1991;91:648–53.
27. Somogyi M. J Biol Chem 1952;195:19–23. 43. Furihata K, Tashiro M, Seto H. Magn Reson Chem 2010;48:179–83.