Saccharides 1, 2 and 3 were isolated from the fermented beverage of plant extracts using carbon-Celite column chromatography, and were shown to be homogeneous using anion exchange HPLC [tR, sucrose (relative retention time; retention time of sucrose = 1.0): 1.89, 2.23 and 2.40 respectively]. The retention time of saccharides 1, 2 and 3 did not correspond to that of any authentic saccharides [glucose (0.62), fructose (0.68), sucrose (1.00), maltose (1.43), trehalose (0.58), laminaribiose (1.33), raffinose (1.23), 1-kestose (1.47), 6-kestose (1.75), neokestose (1.90), maltotriose (2.59), panose (1.87), nystose (2.06), fructosylnystose (3.81), O-β-D-fructopyranosyl-(2->6)-D-glucopyranose (0.83) [2], O-β-D-fructopyranosyl-(2->6)-O-β-D-glucopyranosyl-(1->3)-D-glucopyranose (1.74) [6], O-β-D-fructopyranosyl-(2->6)-O-[β-D-glucopyranosyl-(1->3)]-D-glucopyranose (1.72) [6], O-β-D-glucopyranosyl-(1->1)-O-β-D-fructofuranosyl-(2<->1)- α-D-glucopyranoside (1.24) [9], O-β-D-galactopyranosyl-(1->1)-O-β-D-fructofuranosyl-(2<->1)-α-D-glucopyranoside (0.84) [9], 2(2-α-D-glucopyranosyl)isokestose (1.57) [10], 2(2-α-D-glucopyranosyl)2isokestose (1.79) [10], 2(2-α-D-glucopyranosyl)3isokestose (2.09) [10], 2(2-α-D-glucopyranosyl)nystose (2.17) [10], 2(2-α-D-glucopyranosyl)2nystose (2.63) [10], O-α-D-glucopyranosyl-(1->2)-O-α-D-xylopyranosyl-(1->2)-β-D-fructofuranoside (1.51) [11], O-α-D-glucopyranosyl-(1->2)-O-α-D-glucopyranosyl-(1->2)-O-α-D-xylopyranosyl-(1->2)-β-D-fructofuranoside (1.80) [11].
The degree of polymerization of saccharides 1, 2 and 3 was established as 3 by measurements of [M+Na] ions (m/z: 527) using TOF-MS (see Fig. 2), and analysis of the molar ratios of D-glucose to D-fructose in the acid hydrolysates. Acid hydrolysates of saccharides 1 and 3 were liberated to glucose and fructose, and saccharide 2 was liberated to glucose. From the GC analysis, relative retention times of the methanolysate of the permethylated saccharides were investigated [tR (relative retention time; retention time of methyl 2, 3, 4, 6-tetra-O-methyl-β-D-glucoside = 1.0; retention time, 9.60 min)]. The methanolysate of permethylated saccharide 1 exhibited six peaks (see Additional file 2) corresponding to methyl 2,3,4,6-tetra-O-methyl-D-glucoside (tR, 0.94 and 1.48), methyl 2,4,6-tri-O-methyl-D-glucoside (tR, 3.27 and 4.81) and methyl 1,3,4,6- tetra-O-methyl-D-fructoside (tR, 1.06 and 1.32). The methanolysate of permethylated saccharide 3 also exhibited six peaks (see Additional file 2) corresponding to methyl 2,3,4-tri-O-methyl-D-glucoside (tR, 2.58 and 3.59), methyl 2,4,6-tri-O-methyl-D-glucoside (tR, 3.22 and 4.73), and methyl 1,3,4,6-tetra-O-methyl-D-fructoside (tR, 1.07 and 1.29). On the other hand, the methanolysate of permethylated saccharide 2 exhibited two peaks (see Additional file 2) corresponding to methyl 2,3,4,6-tetra-O-methyl-D-glucoside (tR, 0.97 and 1.47). GC-MS analysis on the retention times and fragmentation patterns of the methyl glucosides [12] showed the two peaks (10.08 min and 10.21 min) from the methanolysate of permethylated saccharide 2 to be methyl 3,6-di-O-methyl-D-glucoside. From these findings above, saccharides 1, 2 and 3 were proved to be, O-D-fructofuranosyl-(2->1)-O-[D-glucopyranosyl-(1->3)]-D-glucopyranoside, O-D-glucopyranosyl-(1->2)-O-[D-glucopyranosyl-(1->4)]-D-glucose and O-D-fructofuranosyl-(2->6)-O-D-glucopyranosyl-(1->3)-D-glucose, respectively.
The structural confirmations of saccharides 1, 2 and 3 according to 1H and 13C NMR analyses and the subsequent complete assignment of 1H and 13C NMR signals of the three saccharides were carried out using 2D-NMR techniques.
First, the NMR spectra of saccharide 1 were analyzed. The HSQC-TOCSY spectrum revealed the1H and 13C signals of each Glc, Glc' and Fru. The isolated methylene was assigned as H-1 and C-1 in Fru. The other three methylene carbons were assigned as C-6 in these residues. The COSY spectrum assigned the spin systems of these residues; from H-1 to H-3 and H-1' to H-3' (Fig. 3(a)), and from H-3" to H-6". The corresponding 13C signals were assigned by HSQC spectrum (Fig. 3(b)). These results clarified the assignment of 1H and 13C NMR signals of each residue. The position of the glucosidic linkage and fructosidic linkage was analyzed as follows. The C-3' showed the HMBC [13, 14] correlations between H-1 (Fig. 3(c)). The J (H-1/H-2) value was 7.9 Hz. These results indicated the Glc 1β ->3' Glc linkage, namely the laminaribiose moiety. The C-2" showed the HMBC correlations to H-1'. The J (H-1'/H-2') value was 7.4 Hz. These results indicated the Glc' 1β ->2 β Fru linkage, and all 1H and 13C NMR signals were assigned as shown in Additional file 3.
The coupling patterns of overlapped 1H were analyzed by the SPT method [15, 16]. Due to strong coupling between H-4' and H-5', these couplings could not be analyzed in first order.
The NMR spectra of saccharide 2 showed that it was an anomeric mixture at the Glc. The α anomer was predominant. The COSY spectrum was assigned from H-1 to H-6. The C-4 showed the HMBC correlations between H-1" (Fig. 4(a) and 4(b)). The J (H-1"/H-2") value was 7.6–7.8 Hz. These results indicated the Glc" 1β ->4 Glc linkage, namely the cellobiose moiety. The C-2 showed the HMBC correlations to H-1'. The J (H-1'/H-2') value was 7.6 Hz. These results indicated the Glc' 1β ->2 Glc linkage, and all 1H and 13C NMR signals were assigned as shown in Additional file 3.
The NMR spectra of saccharide 3 were analyzed in the same manner as those of saccharide 2. Saccharide 3 was also an anomeric mixture at the Glc'. The β anomer was predominant. The HSQC-TOCSY spectrum revealed the1H and 13C signals of each Glc, Glc' and Fru. The isolated methylene was assigned as H-1" and C-1". The other three methylene carbons were assigned as C-6 in these residues (Fig. 5(a)). The position of the glucosidic linkage and fructosidic linkage was analyzed as follows. The C-3' showed the HMBC correlations between H-1 (Fig. 5(b)). The J (H-1/H-2) value was 7.9 Hz. These results indicated the Glc 1β ->3 Glc' linkage, namely the laminaribiose moiety. The C-2 showed the HMBC correlations to H-6. These results indicated the Glc 6 <-2 β Fru linkage, and all 1H and 13C NMR signals were assigned as shown in Additional file 3.
From all of these findings, saccharides 1, 2, and 3 from the fermented beverage of plant extracts were confirmed to be new oligosaccharides (Fig. 1):O-β-D-fructofuranosyl-(2->1)-O-[β-D-glucopyranosyl-(1->3)]-β-D-glucopyranoside (named "3G-β-D-glucopyranosyl β, β-isosucrose"), O-β-D-glucopyranosyl-(1->2)-O-[β-D-glucopyranosyl-(1->4)]-D-glucopyranose (41-β-D-glucopyranosyl sophorose) and O-β-D-fructofuranosyl-(2->6)-O-β-D-glucopyranosyl-(1->3)-D-glucopyranose (62-β-D-fructofuranosyl laminaribiose).
Synthesis of the saccharides by fermentation of plant extracts was investigated using HPAEC. Almost all of the monosaccharides were removed from the fermented and unfermented beverages of plant extracts by the batch method with Charcoal. The saccharides 1, 2, and 3 were observed in the fermented beverage, but were not present in the unfermented one. Therefore, saccharides 1, 2, and 3 were confirmed to have been produced during fermentation of the beverage of plant extracts (Fig. 6).