Structural analysis of novel kestose isomers isolated from sugar beet molasses

Carbohydrate Research 424 (2016) 1–7

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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 Research Center, Nippon Beet Sugar Mfg. Co., Ltd., Obihiro 080-0831, Japan c Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan b

A R T I C L E

I N F O

Article history: Received 12 October 2015 Received in revised form 31 January 2016 Accepted 1 February 2016 Available online 11 February 2016 Keywords: Beet molasses Kestose isomer Oligosaccharide

A B S T R A C T

Eight kestose isomers were isolated from sugar beet molasses by carbon–Celite column chromatography and HPLC. GC–FID and GC–MS analyses of methyl derivatives, MALD-TOF-MS measurements and NMR spectra were used to confirm the structural characteristics of the isomers. The 1H and 13C NMR signals of each isomer saccharide were assigned using COSY, E-HSQC, HSQC–TOCSY, HMBC and H2BC techniques. 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, α-D-fructofuranosyl-(2- > 4)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, β-D-fructofuranosyl(2- > 4)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, β-D-fructofuranosyl-(2- > 3)-α-Dglucopyranosyl-(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 compounds are novel saccharides. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction We previously reported the structural analysis of several oligoand poly-saccharides composed of fructose and other monosaccharide 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, matrixassisted laser desorption ionisation/time-of-flight mass spectrometry; NMR, nuclear magnetic resonance; SPT, selective population transfer; TOCSY, total correlation spectroscopy. 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: [email protected] (T. Abe). http://dx.doi.org/10.1016/j.carres.2016.02.002 0008-6215/© 2016 Elsevier Ltd. All rights reserved.

D-fructopyranosyl-(2- > 6)-D-glucopyranose,8 α-D-fructofuranosyl(2- > 6)-D-glucopyranose, 9 β-D-fructopyranosyl-(2- > 6)-β-Dglucopyranosyl-(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)-βD-fructofuranoside11 were isolated from a fermented vegetable juice as new saccharides. Further studies are in progress to form the oligosaccharides containing 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)-Dglucopyranose15 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 absorption and a suppressive effect on the elevation of blood glucose and insulin levels in rats given sucrose or soluble starch.17 The characteristics of β-D-fructopyranosyl-(2- > 6)-D-glucopyranose,8 α-Dfructofuranosyl-(2- > 6)-D-glucopyranose, 15 and 1 F (1-β-Dfructofuranosyl)m-6G(α-D-galactopyranosyl)n sucrose include low

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2. Results and discussion

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.

Sugar beet molasses diluted with water was passed through a 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 obtained, 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 retention 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 corresponding 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 corresponding 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-tetraO-methyl-D-glucoside (tR, 1.03 and 1.46), and methyl 1,3,4-tri-Omethyl-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-Dglucoside (tR, 1.03 and 1.46) and methyl 3,4,6-tri-O-methyl-Dfructoside (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 2,4,6tri-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 reported. The methanolysates of permethyl saccharides 4, 5, and 7 gave four peaks corresponding to methyl 1,3,4,6-tetra-O-methylD-fructoside (t R , 1.04 and 1.10) and methyl 2,3,4,6-tetra-Omethylglucoside (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

Saccharide 1 Saccharide 2 Saccharide 3 Saccharide 6 Saccharide 8 6-α-Fructosyl D-glucoseb Kojibioseb Nigeloseb 1-Kestoseb Neokestoseb Timosy Levanb Methyl α-D-glucosideb Methyl β-D-glucosideb

1.08 1.08 1.08 1.09 1.06 1.08

1.09 1.08 1.08

1.29 1.29 1.29 1.30 1.28 1.30

1.30 1.30 1.28

2,3,4,6-Glc

3,4,6-Glc 3.02

1.03 1.03

2,4,6-Glc

3,4,6-Fru

1.43 1.42 1.46 1.43 1.45

2.98

1,3,4-Fru

3.63

1.46 1.46 3.18

1.00 1.00 1.08

2,3,4-Glc

2.56

3.67

2.56

3.66

2.55

3.65

2.72

3.67

2.74

4.11

1.90

2.52

3.96

4.47

1.90

2.49

3.98

4.49

4.70

3.59 3.19

4.71

1.00

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

b

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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

Monitored positive ion [m/z]

Saccharide 4 Saccharide 5 Saccharide 7 6-α-Fructosyl-D-glucoseb Lactuloseb 1-Kestoseb Melezitoseb Methyl α-D-glucosideb Methyl β-D-glucosideb

2,3,4,6-Glc

1,4,6-Fru

1,3,6-Fru

205

250

191

191

[M－CH2OCH3]+

[M]+

[M－CH2OCH3]+

[M－CH2OCH3]+

1.04 1.04 1.04 1.04

1.10 1.10 1.10 1.10

1.04

1.10

1.00 1.00 1.00

1.11 1.11 1.11

1.00 1.00 1.00

1.10 1.11 1.11 1.11

1.27

1.27

1.31 1.36 1.35

1.62 1.62

1.36

1.62

1.67

1.31

1.00

Retention time of methyl 2,3,4,6-tetra-O-methyl-β-D-glucoside = 1.0; retention time, 4.15 min. 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; 1,3,6Fru, methyl 1,3,6-tri-O-methyl-D-fructoside. a

b

from saccharides 4 gave two peaks monitored by the [M－CH2OCH3]+ ion (m/z 191) and corresponding to methyl 1,4,6-tri-O-methyl-Dfructoside (tR, 1.27 and 1.31). Those from saccharides 5 and 7 gave two peaks monitored at m/z 191 and corresponding to methyl 1,3,6tri-O-methyl-D-fructoside (tR, 1.35 and 1.62). The above data show that saccharides 1, 2, 3, 6, 8, and 4 were D-fructosyl-(2- > 2)-Dglucosyl-(1 < ->2)-D-fructoside, D-fructosyl-(2- > 6)-D-fructosyl(2 < ->1)-D-glucoside, D-fructosyl-(2- > 1)-D-fructosyl-(2 < ->1)-Dglucoside, D-fructosyl-(2- > 6)-D-glucosyl-(1 < ->2)-D-fructoside, D-fructosyl-(2- > 3)-D-glucosyl-(1 < ->2)-β-D-fructoside, and D-fructosyl-(2- > 3)-D-fructosyl-(2 < ->1)-D-glucoside, respectively. Saccharides 5 and 7 were D-fructosyl-(2- > 4)-D-fructosyl(2 < ->1)-D-glucoside. The structures of all isolated oligosaccharides were determined by NMR techniques as follows. Assignments for all 1H and 13 C signals are shown in Tables S2 and S3. NMR spectral analyses were initiated at the anomeric proton and carbon signals, which exhibited separate characteristic signals in the 1H and 13C NMR spectra, respectively. Saccharide 1 has an anomeric proton (δH 5.46 ppm, d, 3.3 Hz) and three anomeric carbon 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 quaternary 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 saccharide 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 presence 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 assigned to H-2′. Subsequently, the methine proton that exhibited

a correlation peak with C-2′ in the HMBC spectrum was assigned to H-3′. Similarly, the methine carbon atom that yielded a correlation with H-1′ in the HMBC spectrum was assigned to C-5′. The remaining methine proton included in the same spin–spin network with H-1′ was assigned to H-4′. In the case of Fru′, the characteristic doublet proton signal, which exhibited a correlation peak with the quaternary carbon arom (C2″) in the HMBC spectrum, was assigned to H-3″. The methine proton that produced a correlation peak with H-3″ in the COSY spectrum was assigned to H-4″. Similarly, the methine proton that gave a correlation with H-4″ in the COSY spectrum was assigned to H-5″. The methylene proton in the same spin–spin network as H-3″, H-4″, and H-5″ was assigned to H-6″. The methylene proton, which was correlated with C-2″ in the HMBC spectrum, was assigned to H-1″. The intra-residual assignment of Fru was accomplished in a way similar to that described above for Fru′. The correlation peak from C-5 to H-5 in the HMBC spectrum indicates that Fru existed in its furanosyl form. The arrangement of sugar residues was determined by inter-residual HMBC correlation peaks. The quaternary carbon atom

Fig. 2. Selected parts of the COSY (a), HSQC–TOCSY (b), E-HSQC (c), and HMBC (d) spectra of saccharide 1.

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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 (H1′) 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 determined by δC values and 3JHH coupling patterns.20,21 The δC values and 3 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.

In the case of saccharide 7, H-4″ and H-5″ of Fru′ were overlapped 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 structure 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. HRHMBC spectra revealed that C-2″ of Fru′ exhibited a correlation not 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 2DNMR correlations of saccharides 7 and 8 are shown in Figs. 5 and 6, respectively.

Fig. 4. Selected parts of the COSY (a), HSQC–TOCSY (b), E-HSQC (c), and HMBC spectra of saccharide 5.

Fig. 6. Selected parts of the COSY (a), HSQC–TOCSY (b), E-HSQC (c), and HMBC spectra of saccharide 8.

Fig. 5. Selected parts of the COSY (a), HSQC–TOCSY (b), E-HSQC (c), and HMBC spectra of saccharide 7.

N. Shiomi et al./Carbohydrate Research 424 (2016) 1–7

5

ring flip

Glc

α-Fru (αF)

Sucrose moiety

β-Fru (βF)

Fru

Saccharides RF1 RF3 RF4 RF6 RG2 RG3 RG6 1* H H H H αF H H 2 H H H αF H H H 3 αF H H H H H H 4* H αF H H H H H 5* H H βF H H H H 6 H H H H H H αF 7* H H αF H H H H 8* H H H H H βF H * novel saccharides Fig. 7. Structures of kestose isomers 1–8 isolated from sugar beet molasses.

The structures of the three remaining oligosaccharides were determined similarly. As shown in Fig. 7, eight oligosaccharides 1–8 isolated from sugar beet molasses were confirmed to be the following 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-Dfructofuranosyl-(2 < ->1)α-D-glucopyranoside, α-D-fructofuranosyl(2- > 1)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, α-Dfructofuranosyl-(2- > 3)-β-D-fructofuranosyl-(2 < ->1)-α-Dglucopyranoside, β-D-fructofuranosyl-(2- > 4)-β-D-fructofuranosyl(2 < ->1)-α-D-glucopyranoside, α-D-fructofuranosyl-(2- > 6)-α-Dglucopyranosyl-(1 < ->2)-β-D-fructofuranoside, α-D-fructofuranosyl(2- > 4)-β-D-fructofuranosyl-(2 < ->1)-α-D-glucopyranoside, and β-Dfructofuranosyl-(2- > 3)-α-D-glucopyranosyl-(1 < ->2)-β-Dfructofuranoside. 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 saccharides 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 substitution of the 3F-α-fructofuranosyl residue, the 4F-β-fructofuranosyl residue, and the 4 F -α-fructofuranosyl residue for the 1 F -βfructofuranosyl 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 preparative 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-Dfructofuranosyl-(2- > 6)-β-D-fructofuranosyl-(2 < ->1)α-Dglucopyranoside, 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-Dfructofuranosyl-(2- > 6)-α-D-glucopyranosyl-(1 < ->2)-β-Dfructofuranosideα-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 corresponding enzyme from Scopulariopsis brevicaulis.23 Neokestose and 6-α-D-fructofuranosyl-D-glucopyranose were isolated from asparagus 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 Reducing 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 carbohydrate 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),

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E2 (120 ms), and E3 (300 ms) were 0.04, 0.60 and −0.80 V, respectively, and the output range was 1 μC as described previously. Quantitative determinations of D-glucose and D-fructose were performed 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 freezedried 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 concentrated 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, raffinose, 1-kestose, 6-kestose, neokestose, nystose and fructosylnystose, as shown in Fig. 1. Each fraction was dissolved in water to a 2% concentration, 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 conditions 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.

4.7. 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 temperature 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 temperature 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 2.5or 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.

4.5. Methylation and methanolysis Authors’ contributions Methylation of saccharides was carried out using the method described 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 atmosphere. 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 chloroform 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)

NS and TA performed data analyses and contributed to the drafting 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. 2. 3. 4. 5. 6. 7. 8.

GC–FID analyses were carried out on a Shimadzu GC 8A gas chromatograph 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.

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