Original papers
Production of Tagatose from Galactose in a Batch-type Reactor Using a Phosphate Buffer under Subcritical Water Conditions
2020 年 26 巻 6 号 p. 695-699
詳細
2020 年 26 巻 6 号 p. 695-699
Isomerization of galactose to produce rare sugars, tagatose and talose, was performed in a Teflon screw-lid container, used as a batch reactor, at approximately 120 °C. Galactose was dissolved at 5% (w/w) in NaH2PO4-Na2HPO4 buffer at pH 6.0, 7.0 or 8.0. A high yield (approximately 13%) for the conversion of galactose to tagatose was achieved at pH 7.0, with slight changes in the solution color. The tagatose yield increased with increasing buffer concentration of 10 mmol/L; however, it was nearly constant at concentrations of >10 mmol/L. Conversely, little talose was produced at buffer concentrations of <10 mmol/L; however, its yield significantly increased at buffer concentrations of 10 mmol/L. When the galactose concentration was ≥5%, the tagatose yield decreased; however, the tagatose concentration increased with increasing galactose concentration; for example, the tagatose concentration was 1.71% (w/w) when the initial galactose concentration was 25% (w/w).
Sugars and their derivatives that exist in extremely small quantities in nature are defined as rare sugars. Some of them are used as low-calorie sweeteners (Soy et al., 2018; Bhuiyan et al., 1998; Beerens et al., 2012) and they have important physiological functions (Lim et al., 2011; Zhang et al., 2017). For example, daily intake of tagatose, which is 92% as sweet as sucrose (Levin et al., 1995), can facilitate weight loss (Donner et al., 1999). Furthermore, its effectiveness in preventing type II diabetes, hyperglycemia, anemia, and hemophilia has also been reported (Levin, 2002; Kim, 2004). Rare sugars have recently attracted considerable attention because of their potential use as functional food ingredients. However, only a few rare sugars have been produced (Chattopadhyay et al., 2014); for most rare sugars, no industrial production method has yet been established. Rare sugars can be obtained from common sugars via an enzymatic reaction (Beerens et al., 2012; Zhang et al., 2017; Izumori, 2006) or an alkaline isomerization reaction (Takamine et al., 2015; Nagasawa et al., 2019). Alkaline isomerization is based on the Lobry de Bruyn-Alberda-van Ekenstein rearrangement (LBAE rearrangement), and it occurs via mutual isomerization between the epimeric aldose and its ketose in the C-2 position through the formation of an enediol from the reducing sugars under alkaline conditions (Kabyemela et al., 1999).
Previously, we proposed a method to produce rare sugars via isomerization using subcritical fluids, with subcritical aqueous alcohol being the most effective solvent (Usuki et al., 2007; Gao et al., 2015a; Gao et al., 2016; Soisangwan et al., 2017; Khuwijitjaru et al., 2018). Although this method is straightforward, the concentration of rare sugars obtained is low due to the low solubility of the sugar substrate (Gao et al., 2015b). Additionally, during the isomerization reaction, the pH decreases substantially, hindering the LBAE rearrangement (Hirayama and Kobayashi, 2019). To overcome these limitations, we explored the isomerization of galactose for producing rare sugars (tagatose and talose) using a buffer solution under subcritical water conditions (Onishi et al., 2020). Tagatose is a ketose and talose is the epimer in the galactose C-2 position. In our previous work, we used a flow-type reactor operated at 100 °C to 160 °C (most often set at 140 °C). In this study, we investigated isomerization at ≤120 °C using a simple and easy-to-operate batch-type reactor. The isomerization was buffer-dependent, and the most efficient results were obtained in a phosphate buffer, although the MOPS and HEPES buffers suppressed the pH more effectively than the phosphate buffer (NaH2PO4-Na2HPO4). Therefore, the phosphate buffer was used in this study.
Materials d-Galactose, sucrose, anhydrous sodium dihydrogen phosphate, disodium hydrogen phosphate, and acetonitrile were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan).
Isomerization in a batch-type reactor Isomerization of galactose to tagatose was performed using a Teflon screw-lid container (inner volume 50 mL; As One, Osaka, Japan). Galactose (1.25 g) and NaH2PO4-Na2HPO4 buffer (10 mmol/L, pH 7.0, 23.75 g) were poured into the container, and six containers were prepared. After closing the lid tightly, the containers were placed in an oven (SVO-300, Shimadzu, Kyoto, Japan) set at 120 °C. After the predetermined time, the Teflon container was taken out from the oven and quenched with tap water. Next, the reaction mixture was taken out of the container, and the concentrations of the substrate and product, the pH, and the absorbance at 440 nm were measured by the methods described below. Additionally, three containers were heated in a hot dry bath (THB-1, As One) set at 120 °C to examine the effect of the temperature-increase rate on the isomerization. A container was removed from the bath at 60, 90 and 120 min, and cooled to room temperature to stop the reaction. The temperature in the container increased faster when heated in the bath than in the oven.
A temperature logger placed in a pressure-resistant waterproof capsule (HyperThermochron #1922E, KN Laboratories, Osaka, Japan) was installed in the container to record the temperature of the reaction mixture. The recorded temperature was retrieved from the logger to a personal computer with the analysis software RhManager (KN Laboratories).
Isomerization at different pH levels The isomerization was carried out in 10 mmol/L NaH2PO4-Na2HPO4 buffer at pH 6.0, 7.0 or 8.0. The other reaction conditions were as described in the previous section. Isomerization in distilled water was also performed as a reference experiment.
Isomerization at different galactose concentrations Different amounts of galactose were dissolved in 10 mmol/L NaH2PO4-Na2HPO4 buffer (pH 7.0) for a total weight of 50 g. The galactose concentration ranged from 1.0% to 30% (w/w). The Teflon containers were placed in the oven at 120 °C for 180 min.
Effect of buffer concentration on isomerization NaH2PO4-Na2HPO4 buffer solutions with different concentrations (1 to 100 mmol/L, pH 7.0) were prepared. Galactose (1.25 g) and the buffer solution (23.75 g) were added to the Teflon containers, and the containers were placed in the oven at 120 °C for 180 min.
Analyses The concentrations of the remaining galactose and the produced tagatose and talose were measured using high-performance liquid chromatography (HPLC). The HPLC system consisted of an eluent delivery pump (LC-10AT), a differential refractive index detector (RID-6A), a Chromatopac integrator (C-R8A) (all from Shimadzu), and a packed column for separation (Cosmosil Sugar-D, 4.6 mmφ × 250 mm, Nacalai Tesque, Kyoto, Japan). Sucrose was used as an internal standard. The eluent was 80% (v/v) acetonitrile at 1.0 mL/min. The volume of injected sample was 10 µL. The pH of the reaction mixture was measured using a pocket-type pH meter (S2K922, Isfetcom, Saitama, Japan). The absorption spectrum of the reaction mixture at a wavelength of 350 to 700 nm was measured using an ultraviolet-visible spectrophotometer (UV-1200, Shimadzu). In preliminary experiments, the absorbance of the reaction mixture was higher at shorter wavelengths, and a small peak was observed near 440 nm in the absorption spectrum. Since the absorbance at 440 nm became large in a reaction time-dependent manner, the absorbance at 440 nm was used as an index for measuring the color intensity of the reaction mixture.
Effect of pH on the isomerization Figure 1 shows the time variation of the temperature, fraction of remaining galactose and produced tagatose, pH and absorbance at 440 nm during the isomerization of galactose dissolved at 5% (w/w) in 10 mmol/L phosphate buffer solution at pH 6.0, 7.0 or 8.0. Because talose was undetectable by HPLC, and its concentration was extremely low under any of the conditions used, its results are not shown in Fig. 1. Formation of tagatose was not observed for the isomerization of galactose dissolved in distilled water. For these experiments, the oven temperature was set at 120 °C, but the maximum temperature of the reaction mixture ranged from 113 to 115 °C. Although tagatose was generated in all buffer solutions prepared, the best results were obtained at pH 7.0 and 8.0 (yield: approximately 13%).
Isomerization of galactose dissolved at 5% (w/w) in 10 mmol/L NaH2PO4-Na2HPO4 buffer (a) pH 6.0, (b) pH 7.0 or (c) pH 8.0. The Teflon containers used as reaction vessel were placed in an oven set at 120 °C. The temperature of the reaction mixture is shown by the solid curve. The symbols: (□) the fraction of remaining galactose, CGal/CGal0; (○) tagatose yield, CTag/CGal0; (△) absorbance at 440 nm, A440, and (◊) pH. CGal0 indicates the initial galactose concentration.
A batch-type Teflon container with low thermal conductivity and a thick wall was used for the isomerization reaction; therefore, it took approximately 150 min for the container to reach the set temperature after being placed in the oven. Isomerization occurred during the temperature-rising process, and when the temperature reached the set value, the tagatose yield was nearly at the maximum, and the pH of the reaction mixture dropped to 6 or lower.
As described in a previous study using a flow-type reactor (Onishi et al., 2020), the pH of the reaction mixture decreased with the progress of the reaction, and almost no tagatose was formed below pH 6.0. Although the tagatose concentration did not increase after 150 min, the galactose concentration gradually decreased, and an increase in the absorbance at 440 nm was observed. This suggests that thermal decomposition of galactose occurred concomitantly with the formation of the colored compounds. Using a buffer solution at pH 7.0 is preferable because the color intensity increases considerably at pH 8.0, and the tagatose yield is similar under both pH conditions.
Isomerization of galactose in various concentrations of phosphate buffer solutions Figure 2 shows the time variation of the temperature, fraction of remaining galactose and produced tagatose, pH, and absorbance at 440 nm during the isomerization of galactose dissolved in 1.0 mmol/L or 100 mmol/L phosphate buffer solution at 5% (w/w) and pH 7.0. For the 1.0 mmol/L buffer solution, the pH of the reaction mixture drastically decreased after a reaction time of 100 min, during which the temperature exceeded 100 °C, and the yield of tagatose was 7%. Conversely, with the 100 mmol/L buffer concentration, the pH decreased slowly, and the tagatose yield was approximately 13%.
Isomerization of galactose dissolved at 5% (w/w) in (a) 1 mmol/L and (b) 100 mmol/L NaH2PO4-Na2HPO4 buffer, pH 7.0. The other experimental conditions and symbol descriptions are given in Fig. 1.
The effects of the buffer solution concentration on the tagatose and talose yields and the color intensity were examined for the isomerization of 5% (w/w) galactose at 120 °C and a reaction time of 240 min (Fig. 3). The initial pH of all buffer solutions was 7.0. At a buffer concentration of ≥5 mmol/L, the amount of remaining galactose decreased, the yield of tagatose increased, and the color intensity (absorbance at 440 nm) increased. At a buffer concentration of ≥10 mmol/L, the tagatose yield was nearly constant; however, the remaining galactose concentration decreased and an increase in the color intensity was observed. Therefore, 10 mmol/L phosphate buffer was considered to be the most suitable once the conversion of galactose to the colored substances was taken into account, but the tagatose yield did not improve. The talose yield was also dependent on the concentration of the phosphate buffer, and it increased significantly at a phosphate buffer concentration of ≥20 mmol/L, when the tagatose yield was nearly constant. The reason why the concentration of talose increased with increasing concentration of the buffer solution remains unclear.
Effect of the buffer concentration on the isomerization of tagatose. The initial pH of the buffer solution was 7.0, and the galactose concentration was 5% (w/w). The batch-type reactor was placed in an oven set at 120 °C for 180 min. The symbol (▽) indicate the talose yield, CTal/CGal0. Other keys are given in Fig. 1.
Effect of galactose concentration on isomerization Galactose solutions dissolved in 10 mmol/L phosphate buffer (pH 7.0) at various concentrations were reacted at 120 °C for 180 min. The concentration of remaining galactose and produced tagatose, the pH, and the color intensity (absorbance at 440 nm) were measured (Fig. 4). The fraction of remaining galactose and the tagatose yield were nearly constant at a galactose concentration of ≤10% (w/w), while at a galactose concentration of ≥20% (w/w), the tagatose yield decreased as the galactose concentration increased. Therefore, Fig. 3 shows that the galactose concentration does not affect the formation of the colored compounds, whereas the buffer concentration does.
Effect of the initial galactose concentration, CGal0, on the isomerization of tagatose dissolved in 10 mmol/L NaH2PO4-Na2HPO4 buffer, pH 7.0. The isomerization was performed in the Teflon container placed in the oven set at 120 °C for 180 min. The symbol (●) indicate the tagatose concentration. Other keys are given in Fig. 1.
In the production of tagatose, the product concentration and yield are important. Although the tagatose concentration was not proportional to the initial galactose concentration, a higher tagatose concentration was achieved with a higher galactose concentration. The tagatose concentration was 1.71% (w/w) for an initial galactose concentration of 25% (w/w).
Effect of temperature-increase rate on isomerization A Teflon container containing a 5% (w/w) galactose solution (25 g) was placed in an oven or heat block set at 120 °C. The temperature and the fraction of remaining galactose and produced tagatose were measured (Fig. 5). For comparison purposes, the results obtained for the containers placed in the oven (Fig. 1b) are also shown in Fig. 5. Compared to the Teflon container placed in the oven, the temperature of the reaction mixture increased more rapidly with the heat block, and it reached the set temperature in approximately half the time. Regardless of the temperature-increase rate, the tagatose yield was 13% when the temperature of the reaction mixture reached 120 °C.
Effect of temperature-rising rate on the isomerization of (□, ■) galactose into (○, ●) tagatose. The Teflon containers were heated in a heat block (solid symbols), and in the oven (open symbols), both were set at 120 °C. The galactose was dissolved at 5% (w/w) in 10 mmol/L NaH2PO4-Na2HPO4 buffer solution, pH 7.0. The solid curves indicate the temperature of the reaction mixture. Data for the container placed in the oven is given in Fig. 1b.
Acknowledgments We thank Mr. T. Ohta and Mr. M. Yamagata for their technical assistance.
