Structural Analysis of a Novel Trisaccharide Containing Fructopyranoside Residue Isolated from a Fermented Beverage of Plant Extracts

Abstract

Super Ohtaka^® (fermented beverage of plant extracts) is prepared from approximately 50 vegetables and fruits. Natural fermentation is primarily performed using lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp.). An unidentified oligosaccharide was isolated from this beverage using carbon-Celite^® column chromatography and high performance liquid chromatography. The oligosaccharide structure was confirmed by MALDI-TOF MS and NMR measurements. This oligosaccharide was identified as β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, it was newly found from natural source. We speculated that this oligosaccharide was generated or enhanced during the fermentation, and was barely degraded by artificial gastric juice and rat intestinal enzymes, although it was slightly hydrolyzed by pig pancreatic enzymes.

Abbreviations

COSY, correlation spectroscopy; HPAEC, high performance anion-exchange chromatography; HPLC, high performance liquid chromatography; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum coherence/correlation; TOCSY, total correlation spectroscopy; MALDI-TOF MS, matrix assisted laser desorption ionization time of flight mass spectrometry; NMR, nuclear magnetic resonance.

Super Ohtaka^® (Ohtakakohso Co., Ltd., Otaru, Japan; fermented beverage of plant extracts) is produced by fermentation of an extract from 50 fruits and vegetables [1]. The extract was obtained using sucrose osmotic pressure in a cedar barrel for seven days followed by fermentation by lactic acid bacteria and yeast for 180 days. High performance anion-exchange chromatography (HPAEC) showed that Super Ohtaka^® contained high levels of monosaccharides (550-590 g/L), mainly glucose and fructose, and a small amount of undetermined oligosaccharides. We have previously examined the preparation of fructopyranoside series oligosaccharides in Super Ohtaka^®, such as β-D-fructopyranosyl-(2→6)-D-glucopyranose [2], β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→3)-D-glucopyranose [3], β-D-fructopyranosyl-(2→6)-[β-D-glucopyranosyl-(1→3)]-D-glucopyranose [3], β-D-fru­ctopyranosyl-(2→6)-D-fructofuranose [4], β-D-fru­ctopyranosyl-(2→1)-D-fructopyranose [4], β-D-fructopyranosyl-(2→1)-β-D-fructofuranosyl-(2↔1)-α-D-glucopyranoside [4], and β-D-fructopyranosyl-(2→6)-α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside [4]. Distinctive characteristics of β-D-fructopyranosyl-(2→6)-D-glucopyranose include non-cariogenicity and low digestibility. Further, unfavorable bacteria that produce mutagenic substances do not use the saccharide [5]. In addition, we isolated and identified novel non-reducing trisaccharides from Super Ohtaka^®, such as 1^F-β-glucosylsucrose and 1^F-β-galactosylsucrose [6]. Two oligosaccharides containing an α-fructofuranoside linkage were also detected in this beverage [7]. Furthermore, we observed that novel saccharides were produced during fermentation [7], two of which, β-D-fructopyranosyl-(2→6)-D-glucopyranose and α-D-fructofuranosyl-(2→6)-D-glucopyranose, were synthesized from D-glucose and D-fructose using a thermal treatment [8, 9].

We have isolated and identified many kinds of novel oligosaccharides from Super Ohtaka^®, including oligosaccharides with pyranose-type fructose residues. In this study, the structure of a novel oligosaccharide isolated from Super Ohtaka^® was determined using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR). In addition, we investigated the properties of this oligosaccharide, including their digestibility.

Super Ohtaka^® (3,000 g: contains approximately 1,400 g of carbohydrates) was loaded onto a carbon-Celite^® [1:1; activated charcoal powder (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and Celite^® No. 535 (Wako Pure Chemical Industries, Ltd.)] column (8.2 × 53 cm) and successively eluted with distilled water (15 L), 5 % ethanol (15 L), 30 % ethanol (10 L), and 50 % ethanol (5 L). Almost all the glucose and fructose were eluted with water (3-6 L). The 30 % ethanol fraction, which was suspected to contain unidentified oligosaccharides, was concentrated in vacuo and freeze-dried to give 2.65 g. This fraction was eluted with 80 % acetonitrile applied at a flow rate of 1.0 mL/min at 80 °C using a high-performance liquid chromatography (HPLC) system (Tosoh Corporation, Tokyo, Japan) equipped with an Amide-80 column (4.6 mm × 25 cm, Tosoh Corporation) and refractive index detection. The fraction eluting between 50 and 60 min included the unidentified peak indicated by the arrow in Fig. 1a and yielded 29.8 mg. Subsequently, the fraction was eluted with distilled water at a flow rate of 0.5 mL/min at room temperature (approximately 25 °C) using an HPLC system equipped with an ODS-100V column (4.6 mm × 25 cm × 2 [connect two columns], Tosoh Corporation) and refractive index detection. The peak indicated by the arrow in Fig. 1b was collected and designated as Saccharide 1, and yielded 3.5 mg of a white powder. The amounts of oligosaccharides containing fructopyranoside residues recovered from one kg of Super Ohtaka^® are summarized in Table 1. The amount of Saccharide 1 isolated in this study was not as high as that of the other oligosaccharides recovered. Retention duration of Saccharide 1 did not correspond with that of any of the following known saccharides: glucose, fructose, sucrose, maltose, trehalose, laminaribiose, raffinose, 1-kestose, maltotriose, panose, nystose, β-D-fructopyranosyl-(2→6)-D-glucopyranose, β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→3)-D-glucopyranose, or β-D-fructopyranosyl-(2→6)-[β-D-glucopyranosyl-(1→3)]-D-glucopyranose. The degree of polymerization of Saccharide 1 was 3 according to the measurements of [M+Na]⁺ (m/z 527) by MALDI-TOF MS. Acid hydrolysates of Saccharide 1 resulted in the detection of glucose and fructose. The structural conformation of Saccharide 1 was determined by ¹H and ¹³C NMR analyses, followed by the complete assignment of ¹H and ¹³C NMR signals for the saccharide using 2D NMR techniques, including heteronuclear single quantum coherence/correlation (HSQC), band-selective HSQC, HSQC-total correlation spectroscopy (TOCSY), correlation spectroscopy (COSY), and heteronuclear multiple bond correlation (HMBC) (Fig. S1-S14; see J. Appl. Glycosci. Web site). The NMR spectrum of Saccharide 1 indicated that it was an anomeric mixture in which the β anomer was predominant. HMBC analysis revealed Fru C-2 in the sample correlated with H-6 but not with H-5; therefore, it was determined to be fructopyranose (Frup). In addition, the chemical shifts of the carbon atoms supported a pyranose rather than a furanose type. HMBC correlations were obtained between the C-6 of Glc and H-1 of Glc’, and between C-1 of Glc’ and H-6 of Glc6. Furthermore, the coupling constant of H-1 of Glc’ (J = 7.9 Hz) indicated that it was a β bond. Therefore, the gentiobiose portion was assigned (Fig. S11; see J. Appl. Glycosci. Web site). Although a correlation between C-2 of Frup and H-6 of Glc’ was predicted, the chemical shifts of one of the methylene proton H-6 of Glc’ (3.91 ppm) and H-3 of Frup, and/or other H-6 proton (3.73 ppm) and H-6 of Frup overlapped, making it unclear which (or both) contributed to the correlation between C-2 of Frup and HMBC. Therefore, the chemical shift of carbon was measured and compared when the sample was dissolved in D₂O or a H₂O:D₂O = 9:1 solution (Fig. S12; see J. Appl. Glycosci. Web site). The comparison between the resulting sample signals in H₂O and D₂O allowed us to assign Fru C-1 and C-3 to the free hydroxyl groups. The signal for C-6 of Glc’ did not move, indicating that it was bound to other saccharide residues. Therefore, the correlation observed in the HMBC was between C-2 of Frup and H-6 of Glc’. The δ values of H-3 and H-4 of Frup were very close in the proton NMR spectrum, and the J values between the protons could not be read from ¹H NMR spectrum. Therefore, the J values for H-3 and H-4 of Frup were measured using two-dimensional non-decoupling HSQC (Fig. S14; see J. Appl. Glycosci. Web site). Comparing the chemical shifts of Saccharide 1 with those of ethyl α-fructopyranoside and ethyl β-fructopyranoside revealed that the J value at positions 3/4 differed from that of ethyl α-fructopyranoside, while the carbon signal at positions 5/6 was closer to that of ethyl β-fructopyranoside. Therefore, we inferred that this was a β bond (unpublished data, Table S1; see J. Appl. Glycosci. Web site).

Fig. 1. High performance liquid chromatography (HPLC) of 30 % ethanol fraction separated from Super Ohtaka^® by carbon-Celite^® column chromatography.

　Super Ohtaka^® was loaded on to a carbon-Celite^® column and successively eluted with 30 % ethanol. The following operating conditions were used for HPLC: (a) column, Amide-80 (4.6 × 250 mm; Tosoh Corporation); column temperature, 80 °C; elution with 80 % acetonitrile at a flow rate of 1 mL/min; detection, by refractive index; (b) column, ODS-100V (4.6 × 250 mm × 2; Tosoh Corporation); column temperature, room temperature (approx. 25 °C); elution with distilled water at a flow rate of 0.5 mL/min; detection by refractive index.

Table 1. Content of oligosaccharides containing fructopyranoside residues in Super Ohtaka^®.

Oligosaccharides	mg/kg	Reference
β-D-Fructopyranosyl-(2→6)-D-glucopyranose	980	[2]
β-D-Fructopyranosyl-(2→6)-D-fructofuranose	2.2	[4]
β-D-Fructopyranosyl-(2→1)-D-fructopyranose	1.3	[4]
β-D-Fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→3)-D-glucopyranose	10	[3]
β-D-Fructopyranosyl-(2→6)-[β-D-glucopyranosyl-(1→3)]-D-glucopyranose	8.0	[3]
β-D-Fructopyranosyl-(2→6)-α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside	5.0	[4]
β-D-Fructopyranosyl-(2→1)-β-D-fructofuranosyl-(2↔1)-α-D-glucopyranoside	3.5	[4]
β-D-Fructopyranosyl-(2→6)-α-D-fructofuranosyl-(2↔1)-α-D-glucopyranoside	3.5	[7]
β-D-Fructopyranosyl-(2→6)-β-D-fructofuranosyl-(2↔1)-α-D-glucopyranoside	1.8	[10]
Saccharide 1	1.2	This study

Using this method, the unassigned Saccharide 1 was assigned and all ¹H and ¹³C NMR signals of Saccharide 1 were assigned (Table 2). Given the above findings, the Fru residue of non-reducing terminal of this saccharide was in the pyranose form, and Saccharide 1 isolated from Super Ohtaka^® was confirmed to be a new oligosaccharide, namely, β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose (Fig. 2).

Table 2. ¹H and ¹³C NMR spectral data (δ^a in ppm, J in Hz) of Saccharide 1.

		δC [ppm]	δH [ppm]		J H, H
β Glc	1	96.77	4.63	d	8.3
	2	74.83	3.24	dd	9.0, 8.3
	3	76.45	3.48	dd	9.2, 9.0
	4	70.30	3.43	dd	9.3, 9.2
	5	75.72	3.59	ddd	9.3, 6.2, 2.2
	6	69.52	4.18	dd	11.5, 2.2
			3.83	dd	11.5, 6.2
β Glc'	1	103.42	4.50	d	7.9
	2	73.91	3.31	dd	9.1, 7.9
	3	76.31	3.50	mb
	4	70.04	3.50	m
	5	75.60	3.53	m
	6	60.79	3.91	dd	11.3, 1.7
			3.73	dd	11.3, 4.5
Fru	1	62.15	3.79	d	12.1
			3.78	d	12.1
	2	101.51
	3	69.22	3.93	d	10.7
	4	70.36	3.93	dd	10.7, 3.2
	5	69.97	4.00	m
	6	64.82	3.99	m
			3.72	m
α Glcc	1	92.93	5.21	d	3.8
	2	72.24	3.53	dd	9.8, 3.8
	3	73.47	3.70	dd	9.8, 9.4
	4	70.30	3.46	dd	10.1, 9.4
	5	71.33	3.95	ddd	10.1, 5.5, 1.8
	6	69.33	4.12	dd	11.1, 1.8
			3.86	dd	11.1, 5.5
α Glc'c	1	103.39	4.48	d	7.9
	2	73.91	3.31	dd	9.1, 7.9
	3	76.35	3.50	m
	4	70.00	3.50	m
	5	75.53	3.52	m
	6	60.73	3.90	dd	11.1, 1.7
			3.73	dd	11.1, 4.5

^a The chemical shifts of ¹H (δ_H) and ¹³C (δ_C) in ppm were respectively determined relative to the external standard of sodium [2,2,3,3-²H₄]-3-(trimethysilyl) propionate in D₂O (δ_H 0.00 ppm) and 1,4-dioxane (δ_C 67.40 ppm) in D₂O.

^b m: multiplet.

^c Some of the signals of the minor anomer (α-anomer of glucose) were separated and could be assigned.

Fig. 2. Structure of β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose.

The production of the oligosaccharides present Super Ohtaka^® was investigated using HPAEC (Fig. 3). To simply remove monosaccharides from the sample before and after fermentation, a batch method using activated carbon was performed as previously reported [4], and most monosaccharides were removed from the sample. Only trace amounts of Saccharide 1 were detected in the plant extracts before fermentation. After fermentation, the peak intensity increased with the same retention time. Therefore, we speculate that Saccharide 1 was produced or increased during fermentation. However, whether this saccharide is enzymatically or non-enzymatically synthesized remains unclear. Ongoing research aims to elucidate the production mechanism and examine the conditions for mass production of this oligosaccharide.

Fig. 3. High performance liquid chromatogram of fermentation products.

　Analysis of Saccharide 1 produced during fermentation was performed using high-performance anion exchange chromatography (HPAEC). Plant extract was fermented for: (a) 0 or (b) 180 days. Super Ohtaka^® (100 mL) fermented for 0 or 180 days was added to activated carbon (10 g), stirred for 3 h, and filtered. The activated carbon was extracted three times with 500 mL of 30 % ethanol. Ethanolic extracts were combined, concentrated to dryness, and solubilized in 1 mL of distilled water. Saccharide solutions were analyzed by HPAEC.

The results of our analysis of the duration of the degradation of Saccharide 1 by small-intestinal enzyme in rats is shown in Fig. 4. When the reaction was performed under specified conditions for 180 min, maltose almost completely disappeared, and after 300 min, approximately 90 % of the sucrose present was decomposed. In contrast, Saccharide 1 was hardly decomposed. Table 3 summarizes the results of the digestibility tests. Indeed, Saccharide 1 was hardly decomposed in the artificial gastric juice and was only lightly hydrolyzed by porcine pancreatin. These results indicate that Saccharide 1 has low digestibility. For the first time, we investigated the degradability of a trisaccharide containing a fructopyranoside residue isolated from Super Ohtaka^®. However, disaccharides (βFrup2-6Glc [5] and βFrup2-1βGlc [11]) have been previously investigated, and both are seemingly indigestible. Oligosaccharides with βFrup2-6G bonds tended to be slightly degraded by pancreatin; however, the details of such degradation are unknown.

Fig. 4. Digestion of Saccharide 1 by rat intestinal acetone powder.

　Briefly, 4 μL of rat small intestine enzyme solution (prepared to 2.5 U/mL as maltase) was added to 20 μL of 10 mM sodium phosphate buffer (pH 6.8) containing 1.0 % (w/v) of each saccharide and reacted at 37 °C.

Table 3. Digestion of Saccharide 1 in vitro.

	Remaining ratio (%)a	Reaction time (min)
Artificial gastric juice	100 ± 4.1	100
Pancreatin	91.8 ± 10.0	360
Rat intestinal acetone powder	100 ± 12.1	300

^a Means ± SD (n = 3).

Decreasing ratio was investigated according to the method of Okada et al. [26].

We elucidated the structure of the oligosaccharide, β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose. Seemingly, this oligosaccharide was generated or increased during the fermentation, and was indigestible. In contrast, gentiobiose was detected in the 5 % ethanolic fraction of the activated carbon column, and approximately 40 mg was recovered (data not shown). We previously reported that several oligosaccharides containing fructopyranside residues isolated from Super Ohtaka^® increased during fermentation in a similar manner [4, 5, 7]. Furthermore, among these oligosaccharides, β-D-fructopyranosyl-(2→6)-D-glucopyranose was indigestible [5]. These saccharides may be useful novel materials for the manufacture of foods and chemicals.

EXPERIMENTAL

Materials. Glucose, fructose, sucrose, maltose, trehalose, laminaribiose, and raffinose were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Crystalline 1-kestose (β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside) was prepared from sucrose using a Scopulariopsis brevicaulis enzyme [12]. β-D-fructopyranosyl-(2→6)-D-glucopyranose, β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→3)-D-glucopyranose, and β-D-fructopyranosyl-(2→6)-[β-D-glucopyranosyl-(1→3)]-D-glucopyranose were isolated from Super Ohtaka^® as reported previously [2, 3]. Rat small-intestine acetone powder was purchased from Sigma-Aldrich, Inc. Pancreatin was purchased from Wako Pure Chemical Industries, Ltd. All other chemicals used in this study were of analytical grade.

Preparation of Super Ohtaka^®. The “fermented beverage of plant extracts” (Super Ohtaka^®) was prepared as follows: 50 fruits and vegetables were cut, sliced, diced into small pieces, mixed, and placed in cedar barrels, as previously reported [1, 13, 14].

HPAEC. Saccharide 1 was analyzed using an ICS5000 Plus chromatograph (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a CarboPac PA-1 anion-exchange column (Thermo Fisher Scientific Inc.) and a pulsed amperometric detector, as previously described [15].

Hydrolysis. The complete hydrolysis of Saccharide 1 was analyzed using HPAEC. Specifically, 0.5 mg of Saccharide 1 was dissolved in 0.1 M HCl (0.1 mL) and hydrolyzed by heating at 100 °C for 30 min.

MALDI-TOF MS. MALDI-TOF MS spectra were obtained on a Shimadzu Kratos mass spectrometer (KOMPACT Probe; Shimadzu Corporation, Kyoto, Japan) in positive ion mode with 10 % 2,5-dihydroxybenzoic acid as the matrix. Ions were formed using a pulsed ultraviolet laser beam (nitrogen laser, 337 nm). 1-Kestose was used as an external standard for calibration.

NMR measurement. Saccharide 1 (approximately 1.1 mg) was dissolved in 60 μL of D₂O. NMR spectra were recorded at 27 °C with an AVANCE Neo spectrometer (Bruker, Billerica, MA, USA; ¹H 500 MHz, ¹³C 126 MHz) equipped with Dul-z 2.5 mm φ probe (¹³C spectra) and a TXI-z 1.7 mm φ probe (¹H and 2D spectra). The chemical shifts of ¹H (δ_H) and ¹³C (δ_C) were determined in ppm relative to the external standards, sodium [2,2,3,3-²H₄]-3-(trimethylsilyl) propionate in D₂O (δ_H 0.00 ppm) and 1,4-dioxane (δ_C 67.40 ppm) in D₂O, respectively. Spectra for ¹H-¹H COSY [16, 17], HSQC with and without ¹³C decoupling [18], band-selective HSQC [19], HSQC-TOCSY [18, 20], HMBC [21, 22], and 1D-TOCSY [23, 24, 25] were obtained using gradient-selected pulse sequences. The TOCSY mixing time (170 ms) comprised MLEV composite pulses guarded by a trim pulse (2.5 ms) or a DIPSI-2 sequence.

Degradability by artificial gastric juice. Oligosaccharide digestibility was investigated as described by Okada et al. [26]. Hydrochloric acid-potassium chloride buffer (50 mM, pH 2.0) was used as artificial gastric juice. Untreated Saccharide 1 was used as a 100 % control and the remaining saccharide was measured using HPAEC to calculate digestibility.

Degradability by pig pancreatic amylase and rat small intestinal enzymes. Briefly, 2 μL of a pancreatin suspension prepared with 4 U/mL amylase was added to 20 μL of 50 mM Bis-Tris buffer (pH 6.6) containing 1 mM calcium chloride and 1.0 % (w/v) Saccharide 1. The reaction was conducted at 37 °C for 6 h and stopped by heating at 100 °C for 5 min. Digestibility was calculated by assuming that the undigested oligosaccharide content was 100 %.

Subsequently, 300 mg of rat small intestinal acetone powder was suspended in 2.7 mL of 10 mM sodium phosphate buffer (pH 7.0), homogenized with a glass homogenizer for 5 min on ice, and centrifuged at 9,000 × G for 15 min at 4 °C. The resulting supernatant was used as a rat small intestinal enzyme solution. Briefly, 4 μL of rat small intestine enzyme solution (prepared with 2.5 U/mL maltase) was added to 20 μL of 10 mM sodium phosphate buffer (pH 6.8) containing 1.0 % (w/v) Saccharide 1. The reaction was conducted at 37 °C, and samples were collected over time and examined for rat small intestine enzyme-mediated extent of degradation. The reaction was stopped by dilution with distilled water and immediately heating at 100 °C for 5 min. Sucrose and maltose were used as controls instead of Saccharide 1. Untreated saccharides were used as a 100 % control.

CONFLICTS OF INTEREST

Hideki Okada, Akira Yamamori, and Naoki Kawazoe are employees of Ohtakakohso Co., Ltd.

REFERENCES

• [1]  Okada H, Kudoh K, Fukushi E, Onodera S, Kawabata J, Shiomi N. Antioxidative activity and protective effect of fermented plant extract on ethanol-induced damage to rat gastric mucosa. J Jpn Soc Nutr Food Sci. 2005; 58: 209-15.
• [2]  Okada H, Fukushi E, Yamamori A, Kawazoe N, Onodera S, Kawabata J, et al. Structural analysis of a novel saccharide isolated from fermented beverage of plant extract. Carbohydr Res. 2006; 341: 925-9.
• [3]  Kawazoe N, Okada H, Fukushi E, Yamamori A, Onodera S, Kawabata J, et al. Structural analysis of two novel oligosaccharides isolated from fermented beverage of plant extract. Carbohydr Res. 2008; 343: 549-54.
• [4]  Okada H, Fukushi E, Yamamori A, Kawazoe N, Onodera S, Kawabata J, et al. Novel fructopyranose oligosaccharides isolated from fermented beverage of plant extract. Carbohydr Res. 2010; 345: 414-8.
• [5]  Okada H, Kawazoe N, Yamamori A, Onodera S, Kikuchi M, Shiomi N. Characteristics of O-β-D-fructopyranosyl-(2→6)-D-glucopyranose isolated from fermented beverage of plant extract. J Appl Glycosci. 2008; 55: 179-82.
• [6]  Kawazoe N, Okada H, Fukushi E, Yamamori A, Onodera S, Kawabata J, et al. Structural analysis of two trisaccharides isolated from fermented beverage of plant extract. Open Glycosci. 2008; 1: 25-30.
• [7]  Okada H, Fukushi E, Yamamori A, Kawazoe N, Onodera S, Kawabata J, et al. Isolation and structural confirmation of the oligosaccharides containing α-D-fructofuranoside linkages isolated from fermented beverage of plant extracts. Carbohydr Res. 2011; 346: 2633-7.
• [8]  Yamamori A, Okada H, Kawazoe N, Onodera S, Shiomi N. Synthesis of β-D-fructopyranosyl-(2→6)-D-glucopyranose from D-glucose and D-fructose by a thermal treatment. Biosci Biotecnol Biochem. 2010; 74: 2130-2.
• [9]  Yamamori A, Okada H, Kawazoe N, Muramatsu K, Onodera S, Shiomi N. Characteristics of α-D-fructofuranosyl-(2→6)-D-glucose synthesized from D-glucose and D-fructose by thermal treatment. J Appl Glycosci. 2014; 61: 99-104.
• [10]  Yamamori A, Takata Y, Fukushi E, Kawabata J, Okada H, Kawazoe N, et al. Structural analysis of a novel oligosaccharide isolated from fermented beverage of plant extracts. J Appl Glycosci. 2017; 64: 123-7.
• [11]  Yamamori A, Takata Y, Fukushi E, Kawabata J, Okada H, Kawazoe K, et al. Structural analysis of novel low-digestible sucrose isomers synthesized from D-glucose and D-fructose by thermal treatment. J Appl Glycosci. 2017; 64: 15-9.
• [12]  Takeda H, Sato K, Kinoshita S, Sasaki H. Production of 1-kestose by Scopulariopsis brevicaulis. J Ferment Bioeng. 1994; 77: 386-9.
• [13]  Okada H, Yamamori A, Kawazoe N, Ueno K, Onodera S, Hirata M. Polysaccharides from a fermented beverage induce nitric oxide and cytokines in murine macrophage cell line. J Appl Glycosci. 2024; 71: 47-54.
• [14]  Okada H, Yamamori A, Kawazoe N, Ueno K, Onodera S. Hyaluronidase inhibitory activity of polysaccharides separated from a fermented beverage of plant extracts. J Appl Glycosci. 2025; 72: 7202202.
• [15]  Shiomi N. Structure of fructopolysaccharide (asparagosin) from roots of asparagus (Asparagus officinalis L.). New Phytol. 1993; 123: 263-70.
• [16]  Aue WP, Bartholdi E, Ernst RR. Two-dimensional spectroscopy. Application to nuclear magnetic resonance. J Chem Phys. 1976; 64: 2229-46.
• [17]  von Kienlin M, Moonen CTW, von der Toorn A, van Zijl PCM. Rapid recording of solvent-suppressed 2D COSY spectra with inherent quadrature detection using pulsed field gradients. J Magn Reson. 1991; 93: 423-9.
• [18]  Willker W, Leibfritz D, Kerssebaum R, Bermel W. Gradient selection in inverse heteronuclear correlation spectroscopy. Magn Reson Chem. 1993; 31: 287-92.
• [19]  Gaillet C, Lequart C, Debeire P, Nuzillard JM. Band-selective HSQC and HMBC experiments using excitation sculpting and PFGSE. J Magn Reson. 1999; 139: 454-9.
• [20]  Domke T. A new method to distinguish between direct and remote signals in proton-relayed X,H correlations. J Magn Reson. 1991; 95: 174-7.
• [21]  Bax A, Summers MF. ¹H and ¹³C Assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J Am Chem Soc. 1986; 108: 2093-4.
• [22]  Hurd RE, John BK. Gradient-enhanced proton-detected heteronuclear multiple-quantum coherence spectroscopy. J Magn Reson. 1991; 91: 648-53.
• [23]  Claridge TDW. Chapter 5-Correlations through the chemical bond I: Homonuclear shift correlation. Tetrahedron Org Chem Ser. 2009; 27: 129-88.
• [24]  Claridge TDW. Chapter 9-Diffusion NMR spectroscopy. Tetrahedron Org Chem Ser. 2009; 27: 303-34.
• [25]  Lucio-Gutierrez JR, Delgado-Montemayor C, Coello-Bonilla J, Waksman-Minsky N, Saucedo AL. Selective 1D-TOCSY and chemometrics to evaluate authenticity of Turnera diffusa and related botanical extracts. Phytochem Lett. 2019; 30: 62-8.
• [26]  Okada K, Yoneyama M, Mandai T, Aga H, Sakai S, Ichikawa T. Digestion and fermentation of pullulan. J Jpn Soc Nutr Food Sci. 1990; 43: 23-9.