Characteristics of α-D-Fructofuranosyl-(2→6)-D-glucose Synthesized from D-Glucose and D-Fructose by Thermal Treatment

Abstract

We have previously observed that the Super Ohtaka^®, produced by fermenting extracts from 50 types of fruits and vegetables, contained the disaccharide, α-D-fructofuranosyl-(2→6)-D-glucose (α-Ff2→6G), which was produced during the fermentation process. α-Ff2→6G was also formed from equal amounts of D-glucose and D-fructose under melting conditions at 130°C for 45 min or at 140°C for 30 min. This disaccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative- high performance liquid chromatography. It was confirmed to be α-Ff2→6G by matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis and nuclear magnetic resonance measurements. The characteristics of α-Ff2→6G were investigated. The saccharide showed low digestibility and was 0.25 times as sweet as sucrose. Furthermore, unfavorable bacteria such as Enterobacter cloacae 1180, Escherichia coli 1099 and Clostridium perfringens 1211 that produce mutagenic substances did not break down the synthetic oligosaccharide.

Abbreviations

β-Fp2→6G, β-D-fructopyranosyl-(2→6)-Dglucopyranose; HPLC, high performance liquid chromatography; HPAEC, high performance anion-exchange chromatography; ABEE, p-aminobenzoic acid ethyl ester.

INTRODUCTION

Super Ohtaka^® is produced by fermenting extracts from 50 types of fruits and vegetables. The extract is obtained after sucrose-osmotic pressure treatment in a cedar barrel for 7 days and fermentation by lactic acid bacteria and yeast at 37°C for 180 days. The fermented beverage shows scavenging activity against the 1,1′-diphenyl-2-picrylhydrazyl radical and significantly reduces ethanol-induced damage of gastric mucosa in rat.¹⁾ This beverage primarily includes glucose and fructose, but it also contains various oligosaccharides. We previously reported structural analyses of the oligosaccharides containing fructosyl bound to position 6 of glucose, namely β-D-fructopyranosyl-(2→6)-D-glucopyranose (β-Fp2→6G),²⁾ β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→3)-D-glucopyranose,³⁾ β-D-fructopyranosyl-(2→6)-D-fructofuranose⁴⁾ and β-D-fructo­pyranosyl-(2→6)-α-D-glucopyranosyl-(1↔2)-β-D-fructo­furanoside⁴⁾ from the fermented beverage of this plant extract. We also previously reported the characteristics of β-Fp2→6G,⁵⁾ which includes non-cariogenicity and low digestibility.

Furthermore, a previous study demonstrated that β-Fp2→6G can be synthesized from D-glucose and D-fructose by caramelization with thermal treatment.⁶⁾ This saccharide was predominately formed from D-glucose and D-fructose under melting conditions in a test tube at 140°C for 60 to 90 min. No reports are currently available on a disaccharide synthesized from D-fructose and D-glucose, except on di-D-fructose dianhydrides synthesized from fructose, sucrose, levan, or inulin by thermal treatment^{7) 8)} and galactooligosaccharides produced by thermal degradation of lactose.⁹⁾

We previously found that the fermented beverage contained the disaccharide α-D-fructofuranosyl-(2→6)-D-glucose (α-Ff2→6G) produced by fermentation.¹⁰⁾ However, there are no reports currently available on the characteristics of the disaccharide, except for information on the chemical synthesis of the saccharide.¹¹⁾

In this study, we investigated some of the characteristics of α-Ff2→6G formed from D-glucose and D-fructose by caramelization by thermal treatment.

MATERIALS AND METHODS

Reagents. p-Aminobenzoic acid ethyl ester (ABEE) labeling kit was purchased from Seikagaku Kogyo Co. (Tokyo, Japan). D-(+)-Glucose, D-(－)-fructose, sucrose and turanose [α-D-glucopyranosyl-(1→3)-D-fructose] were purchased from Sigma Chemical Co. (St. Louis, USA). Palatinose [α-D-glucopyranosyl-(1→6)-D-fructose] was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 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⁾ All other chemicals used in this study were of analytical grade. All human intestinal bacteria that we used were donated by Rakuno Gakuen University.

High Performance Liquid Chromatography (HPLC).

High Performance Anion-Exchange Chromatography (HPAEC). The saccharides were analyzed using a Dionex Bio LC Series apparatus equipped with an HPLC carbohydrate column (Carbo Pak PA1, inert styrene divinyl benzene polymer) and pulsed amperometric detection (PAD).^{13) 14) 15)} The mobile phase consisted of eluent A (150 mM NaOH) and eluent B (500 mM sodium acetate in 150 mM NaOH) with a sodium acetate gradient as follows: 0 to 1 min, 25 mM; 1 to 2 min, 25 to 50 mM; 2 to 20 min, 50 to 200 mM; 20 to 22 min, 500 mM; and 22 to 30 min, 25 mM at a flow rate of 1.0 mL/min. The applied PAD potentials for E1 (500 ms), E2 (100 ms) and E3 (50 ms) were 0.1, 0.6 and －0.6 V, respectively, and the output range was 1 μC.

Analytical-HPLC. The synthesized saccharide solution was analyzed with an HPLC system (Tosoh Co., Tokyo, Japan) equipped with an ODS-80Ts column (4.6 mm × 25 cm × 2) at room temperature and eluted with distilled water at a flow rate of 0.4 mL/min using refractive index detection (RID).

Preparative-HPLC. The saccharide fraction was repeatedly purified at 35°C (100 times; 200 μL injection volume) using a preparative HPLC system equipped with an ODS-80Ts column (20 mm × 25 cm), and eluted with distilled water at a flow rate of 3.0 mL/min using RID.

p-Aminobenzoic acid ethyl ester (ABEE)-HPLC. The converted saccharide was analyzed with an HPLC system equipped with an Honenpak C-18 column (75 mm × 4.6 mm i.d.; Honen Corp., Tokyo, Japan), at room temperature, and eluted with 0.1 M ammonium acetate buffer (pH 4.0) containing 10.5% acetonitrile at a flow rate of 0.5 mL/min using UV at 305 nm detection.

ABEE conversion method. Saccharides were converted at the reducing end and ABEE was performed as previously reported.^{16) 17)} A 10-μL aliquot of standard saccharide solution was added to ABEE reagent solution (40 μL). The mixture was incubated at 80°C for 1 h. Distilled water (0.2 mL) and chloroform (0.2 mL) were added and the mixture was centrifuged at 2,000 × G for 1 min. The aqueous layer was diluted (100-fold) with water and subjected to ABEE-HPLC analysis.

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS spectra were measured using a Shimadzu-Kratos mass spectrometer (KOMPACT Prove) in positive ion mode with 10% 2,5-dihydroxylbenzoic acid as the matrix. Ions were formed by a pulsed UV laser beam (nitrogen laser, 337 nm). Calibration was using 1-kestose as the external standard.

Nuclear magnetic resonance (NMR) measurements. The oligosaccharide was dissolved in 0.5 mL D₂O. NMR spectra were recorded at 27°C with a Bruker AMX 500 spectrometer (¹H 500 MHz, ¹³C 126 MHz) equipped with a 5-mm diameter C/H dual probe. Chemical shifts of ¹H (δ_H) and ¹³C (δ_C) in ppm were determined in ppm relative to the external standard of sodium [2,2,3,3-²H₄]-3-(trimethylsilyl) propanoate in D₂O (δ_H 0.00 ppm) and 1, 4-dioxane (δ_C 67.40 ppm) in D₂O, respectively. ¹H-¹H COSY,^{18) 19)} HSQC²⁰⁾ and HMBC^{21) 22)} spectra were obtained using gradient selected pulse sequence. The phase-sensitive, heteronuclear single quantum coherence-total correlation spectroscopy (HSQC-TOCSY)^{20) 23)} spectra were determined with the sequence including inversion of direct resonance. The TOCSY mixing time (0.1 ms) composed of DIPSI-2 composite pulses. The coupling patterns of overlapped ¹H were analyzed by SPT method.^{24) 25)}

Investigation of synthesis conditions. Powdered fructose (100 mg) and an equal amount of powdered glucose were mixed by a vortex and heated under various conditions in test tubes within a dry thermo bath heater (MG-2000, Tokyo Rikakikai, Tokyo, Japan). After cooling to room temperature, the saccharide melt was dissolved in 1.0 mL distilled water. The resultant saccharide solution was analyzed with an analytical-HPLC.

Investigation of cariogenicity. Cariogenicity was examined as previously described by Miyamura.²⁶⁾ Saliva was separately collected from 3 persons, 2 h after a meal. The oral cavity of each subject was rinsed with tap water and then the subject was requested to gargle with distilled water. The naturally secreted saliva (15 mL) was collected and shaken well at room temperature. A 0.5-mL aliquot of 1.0% saccharide 1, palatinose, glucose or distilled water was then added to a mixture of 1.5 mL fresh saliva and 0.5 mL brain heart infusion broth. These mixtures were incubated at 37°C to determine the time-dependent variation in pH as indicative of cariogenicity. The examination was repeated 3 times.

Streptococcus mutans JCM 5705^T (1.0 × 10⁶ CFU/mL) was grown overnight at 37°C in trypto-soya agar (Nissui). The CFUs were determined after conducting appropriate serial dilutions in 0.9% NaCl solution. The same experiment was performed with 1.5 mL culture suspension of S. mutans instead of the saliva as appositive control.

Investigation of digestibility. Digestibility of saccharide 1 by human saliva, pig pancreatic amylase, rat intestinal enzyme and artificial gastric juice were investigated as previously described by Okada et al.²⁷⁾ with modifications.

Human saliva was obtained by the same method as that for the cariogenicity test. A 100-μL aliquot of human saliva (43 U/mL) was added to 100 μL of 50 mM Bis-Tris buffer (pH 6.0) containing 1 mM calcium chloride and 10% saccharide 1. Digestion was performed at 37°C for 0, 1, 2, 3, 4, 5 and 6 h and the reaction was terminated by heating in a dry thermo bath heater at 100°C for 10 min. Glucose and fructose formed from saccharide 1 were assayed by HPAEC.

Pig pancreatic amylase was obtained from Wako Pure Chemical Industries, Ltd. A 100-μL aliquot of pig pancreatic amylase suspension (4 U/mL) was added to 100 μL of a 50 mM Bis-Tris buffer (pH 6.6) containing 1 mM calcium chloride and 200 mM saccharide 1. Digestion was performed at 37°C for 1, 2, 3, 4, 5 and 6 h and the enzyme reaction was terminated by heating in a dry thermo bath heater at 100°C for 10 min. Glucose and fructose formed from saccharide 1 were assayed by HPAEC.

Rat intestinal enzyme was prepared from intestinal acetone powder. A suspension of 300 mg rat intestinal acetone powder in 2.7 mL of 10 mM phosphate buffer (pH 6.8) was homogenized for 5 min using a glass homogenizer and then centrifuged at 12,070 × G for 15 min to obtain intestinal enzyme solution in the supernatant. A 100-μL aliquot of rat intestinal enzyme solution (4.0 U/mL) was added to 100 μL of 10 mM phosphate buffer (pH 6.8) containing 200 mM of saccharide 1. Digestion was performed at 37°C for 0, 15, 30, 60 and 120 min and the reaction was stopped by heating in a dry thermo bath heater at 100°C for 10 min. Glucose and fructose formed from saccharide 1 were assayed by HPAEC.

Artificial gastric juice solution (pH 2.0) was prepared from 0.9 mM CaCl₂, 50 mM hydrochloric acid and 50 mM potassium chloride. A 50-μL aliquot of this solution was added to 100 μL of 200 mM saccharide 1 and digestion was performed at 37°C for 0, 15, 30, 60 and 120 min. Digestion was terminated by adding 50 μL of 10 mM sodium hydroxide. Digestibility was determined as the amount of saccharide 1 in the digestive solution using analytical-HPLC.

Enzyme activities and units were defined as follows.

The activities of the saliva and pig pancreatic amylase were assayed by the Somogyi-Nelson method and 1 unit of activity was defined as the amount of enzyme required to provide reducing-power equivalent to that of 1.0 μmol glucose from 0.1% soluble-starch per min at 37°C and pH 6.0. The activity of intestinal enzymes was assayed by analytical-HPLC and 1 unit of activity was defined as the amount of enzyme required to liberate 2 μmol glucose from 200 mM maltose per min at 37°C and pH 6.8.

Utilization by human intestinal bacteria. LB broth²⁸⁾ was used as the basal medium for testing the utilization of the saccharide by intestinal bacteria. D-Glucose, sucrose, palatinose, turanose, 1-kestose or saccharide 1 was added to LB medium (2 mL/tube) at a final concentration of 0.5%. After incubation at 37°C for 72 h under anaerobic conditions (replacement by nitrogen gas) using an anaerobic jar, bacterial growth was measured by analyzing the pH of the medium. The symbols “+++”, “++”, “+”, “±” and “－” indicate²⁹⁾ pH values of < 4.5, 4.5 to 5.0, 5.0 to 5.5, 5.5 to 6.0 and > 6.0, respectively.

Evaluation of sweetness. The degree of sweetness was measured as previously described by Takenaka et al.³⁰⁾ Sweetness was tested individually by 10 volunteers using 1 mL saccharide. The sweetness of sucrose solutions ranging from 1.0% to 3.5% concentrations in steps of 0.5%, was compared at room temperature with that of 10% saccharide 1 by the volunteer, to give the degree of sweetness of saccharide 1 as relative to that of sucrose.

RESULTS AND DISCCUSION

Synthesis and isolation of saccharide 1.

A powdered mixture of 30 g each, of D-glucose and D-fructose was carefully heated at 150°C for 60 min in an electric furnace. Then, the saccharide melt was dissolved in 250 mL distilled water and the resulting saccharide solution was analyzed by analytical-HPLC. As shown in Figs. 1(A) and 1(B), several saccharides were produced during the thermal treatment.

Fig. 1.

High-performance liquid chromatogram of saccharides synthesized under thermal treatment.

The products formed by the thermal treatment were analyzed by analytical-HPLC. (A) no heating. (B) heating for 60 min at 150°C. Glc, glucose; Fru, fructose.

The saccharide solution (300 mL) was loaded onto a 4.8 × 36-cm carbon-Celite column (1:1, charcoal: Celite-535) and successively eluted with water (4.4 L). Almost all of the D-glucose and D-fructose was eluted first, within the first 600 mL and saccharide 1 was subsequently eluted with the remaining 3.2 L. The water fraction containing saccharide 1 was concentrated to 20 mL (Fig. 2). This fraction was repeatedly purified with preparative-HPLC. Purified saccharide 1 (916 mg) was finally obtained as a white powder, confirmed as homogeneous by HPAEC with a retention time of 9.90 min and relative retention time of 1.74 (the retention time of sucrose being 1.0). The saccharide was confirmed to be a reducing sugar by ABEE-HPLC with a retention time of 25.52 min and relative retention time of 0.93 (the retention time of glucose being 1.0). The degree of polymerization of saccharide 1 was 2 by [M + Na]⁺ (m/z 365) MALDI-TOF MS measurements. Complete hydrolysis of saccharide 1 was investigated using analytical-HPLC, where saccharide 1 (3.0 mg) was dissolved in 0.1 N HCl (0.2 mL) and hydrolyzed by heating at 100°C for 30 min. As a result, glucose and fructose were detected in an equal amount. The structure of saccharide 1 was confirmed to be α-Ff2→6G by NMR (Table 1).

Fig. 2.

Preparative-HPLC of the disaccharide fractions eluted by carbon-Celite column chromatography.

Table 1.

¹H- and ¹³C-NMR spectral data (δ^a in ppm, J in Hz) for saccharide 1.

^＊1H- and ¹³C-NMR spectral data for α-D-fructofuranosyl-(2→6)-D-glucose are quoted from the data of Okada et al.^{10) ＊＊}The chemical shift (δ_C) of α Fru f-6 was revised from the previous paper.^{10) 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-(trimethylsilyl) propanoate in D₂O (δ_H 0.00 ppm) and 1,4-dioxane (δ_C 67.40) in D₂O. ^bm, multiplet.

Optimal conditions for saccharide 1 (α-Ff2→6G) synthesis was investigated. The effects of temperature, at 110, 120, 130, 140, 150 and 160°C for 60 min, on saccharide synthesis are shown in Fig. 3 (A). The saccharide could be synthesized by heating for 60 min at 110 to 160°C, with maximum yield 130°C. The time-course for saccharide synthesis at 110, 120, 130, 140 and 150°C in 0, 15, 30, 45, 60, 90, 120 and 180 min, respectively, and for 240 min at 110°C was investigated (Fig. 3 (B)). The saccharide was efficiently synthesized at 130°C for 45 min as well as 140°C for 30 min. Although the quantity of saccharide increased to the same level at 110°C for 240 min as that with the previous 2 conditions, this level then slowly decreased.

Fig. 3.

Temperature and time conditions for the synthesizing of saccharide 1.

(A) Temperature. The saccharide was synthesized by heating at 110, 120, 130, 140, 150 or 160°C for 60 min. The saccharide formed was analyzed by HPLC using an ODS-80Ts column. Each column is presented as mean with SD of three determinations. (B) Time. The saccharide was synthesized by heating at 110, 120, 130, 140 or 150°C for 0, 15, 30, 45, 60, 90, 120 or 180 min, respectively. The formed saccharide was analyzed by analytical-HPLC. Each point represents the mean with SD of three determinations. The maximum amounts of synthesized saccharides were shown by 100% in conditions (A) and (B). Symbols: ○, 110°C; ●, 120°C; △, 130°C; ▲, 140°C; □, 150°C.

The influence of the mixing ratio of D-glucose and D-fructose on saccharide synthesis was studied. Efficiency of α-Ff2→6G synthesis was 95, 100 and 80% when the ratio of D-glucose to D-fructose was 0.5, 1 and 2, respectively (The maximum amounts of synthesized α-Ff2→6G were shown by 100%).

The amount of α-Ff2→6G synthesized was about 1.2 times that of α-Fp2→6G.

Characteristics of α-Ff2→6G.

Stability during heating was investigated as follows: A 50-mM aliquot of veronal buffer (pH 3.0, 5.0, 7.0 and 9.0) containing 5% of saccharide [sucrose (Fig. 4 (A)) or α-Ff2→6G (Fig. 4 (B))] was heated in a tube at 100°C for 15, 30, 45 and 60 min in a dry thermal bath heater. α-Ff2→6G was less stable than sucrose at all pH values tested.

Fig. 4.

pH Stability of saccharide 1 and sucrose during heating.

A veronal buffer (pH 3.0, 5.0, 7.0 and 9.0) containing 5% of a saccharide (saccharide 1 or sucrose) was enclosed in a tube and then heated at 100°C for 15, 30, 45 or 60 min. The remained saccharide 1 was measured by analytical-HPLC. (A) sucrose, (B) saccharide 1. Symbols: ○, pH 3.0; △, pH 5.0; ●, pH 7.0; ▲, pH 9.0.

The cariogenicity of α-Ff2→6G is shown in Figs. 5 (A) and 5(B). α-Ff2→6G is a non-cariogenic sugar as both, S. mutans and oral bacteria produce practically no acid.

Fig. 5.

Cariogenicity of saccharide 1.

The cariogenicity was investigated according to the method of Miyamura.²⁵⁾ Details were described in the text. (A) Streptococcus mutans JCM 5705^T. (B) human saliva. Each point represents the mean with SD of three determinations. Symbols: ○, blank; ●, sucrose; ▲, palatinose; ■, saccharide 1.

The saccharide was slightly hydrolyzed by artificial gastric juice, pig pancreatic amylases and rat intestinal enzyme, but not by α-amylase from human saliva. These results indicate that α-Ff2→6G has low digestibility (Table 2).

Table 2.

Digestion of saccharide 1 in vitro.

The conditions for digestion are described in Materials and Methods.

Bifidobacterium and Lactobacillus are bacterial genera that are beneficial for nutrition and health of both, humans and animals, whereas other intestinal bacteria such as Enterobacter cloacae, Escherichia coli and Clostridium perfringens are detrimental. Under normal conditions of bifidobacterial growth, the pH of the medium supplemented with a nonsaccharide (control), α-Ff2→6G, 1-kestose, palatinose, turanose, sucrose and glucose was 6.70 to 6.94, 4.11 to 5.58, 4.01 to 4.28, 3.95 to 4.92, 4.42 to 5.15, 4.02 to 4.43 and 3.99 to 4.21, respectively. α-Ff2→6G was not consumed by E. cloaceae 1180, E. coli 1099, or C. perfringens 1211. In contrast, α-Ff2→6G was selectively consumed by the 6 beneficial bacteria used in this study, including Bifidobacterium and Lactobacillus (Table 3).

Table 3.

Utilization of saccharide 1 and several other saccharides by some human intestinal bacteria.

^＊Pal, palatinose; Tur, turanose; 1-K, 1-kestose, Suc, sucrose; Glc, glucose. ^＊＊+++, < pH 4.5; ++, pH 4.5‒5.0; +, pH 5.0‒5.5; ±, pH 5.5‒6.0; －, > pH 6.0.

When we investigated the sucrose concentration corresponding to sweetness of 10% α-Ff2→6G, 2 volunteers chose 1.5% sucrose, 2 chose 2.0%, 1 chose 2.5%, while 5 chose 3.0%. Therefore, the sweetness of α-Ff2→6G was about 0.25 times that of sucrose.

In this study, we produced α-Ff2→6G from D-glucose and D-fructose by thermal treatment and by demonstrating that α-Ff2→6G. α-Ff2→6G is non-cariogenic with low digestibility, we report that this saccharide could be useful as a novel material for manufacture of foods and chemicals.

ACKNOWLEDGMENTS

We thank Prof. Jun Kawabata and Dr. Eri Fukushi (Hokkaido University, Sapporo) for measuring the NMR spectra.

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