総説
亜臨界流体中での還元糖の異性化による希少糖の生産
2025 年 26 巻 3 号 p. 85-101
詳細
2025 年 26 巻 3 号 p. 85-101
In recent years, interest in rare sugars has been increasing. The conversion of common sugars into rare ones can be broadly classified into two approaches: biochemical methods which utilize biocatalysts such as enzymes and microorganisms, and chemical methods which rely on the Lobry de Bruyn-Alberda van Ekenstein (LBAE) transformation under alkaline conditions. Although the biochemical approach offers high selectivity, it is labor-intensive requiring extensive effort to identify suitable catalysts. This paper focuses on the chemical approach, which, while less selective, offers greater versatility. Specifically, this paper provides an overview of several methods for producing rare sugars from common reducing sugars under subcritical water conditions. These methods include homogeneous reactions in subcritical water, aqueous organic solvents, buffer solutions, and basic amino acid solutions, as well as heterogeneous reactions using eggshells or seashells which are primarily composed of calcium carbonate.
近年希少糖に対する関心が高まっている．稀少糖は，狭義には自然界に少量しか存在しない単糖およびその誘導体と定義される[1]が，広義には単糖に限らず自然界における存在量が少ない糖を意味する[2]．本稿では，後者の観点から希少糖という用語を使用する．
自然界における存在量の多い糖（汎用糖と表記）から希少糖を生産する方法は，酵素や微生物などの生体触媒を用いる方法[1,7]とLobry de Bruyn-Alberda van Ekenstein（LBAE）転位として知られるアルカリ異性化法[12]に大別される．前者は温和な条件で反応が進行し選択性が高いという利点があるが，それぞれの希少糖の生産に適した触媒を見出すことは容易ではない．一方，アルカリ異性化法は副反応が併発し，選択性が高くないという欠点はあるが，多くの糖の異性化に適用できる汎用性がある．本稿では，アルカリ異性化法[2]のうち，亜臨界流体中での汎用糖の異性化（エピメリ化を含む）による希少糖の生産について概説する．なお亜臨界流体とは，常圧における沸点から臨界温度までの範囲で加圧することにより液体状態を保った流体をいう[15]．
亜臨界流体中では，糖の異性化とともに分解反応などによりアルデヒドや有機酸が副生し（Fig. 1）[26]，pHが低下する（Fig. 2）[27]．NaClなどの塩の存在は糖の分解を促進し[29,30]，反応初期には異性化が優先するが，後半には分解が起こる[31]．亜臨界状態に保ったエタノールなどの水可溶性有機溶媒と水との混合物中では，純水中に比べ希少糖の収率が大きく向上する[42-44,49,50,52,53]．LBAE転位による異性化はpH 6.3以下ではほとんど進行しない（Fig. 8）ので，各種緩衝液中での糖の異性化を検討したところ，リン酸緩衝液がもっとも適していた[56-62]．また，リン酸緩衝液と有機溶媒との混合液中で希少糖の収率が向上した[63]．塩基性アミノ酸は水溶液がアルカリ性を示し，食品素材に含まれる天然物であり，安全であるので，green catalyst[64]とよばれる．各種の塩基性アミノ酸による糖の異性化を比較したところアルギニンがもっとも高い希少糖の収率を与えた[68-75]．なお，マイクロ波で加熱したときには，ワット数によらず反応液に吸収された単位体積あたりのエネルギー（エネルギー密度）で整理できることを示した（Fig. 10）[72]．
炭酸カルシウムを主成分とする卵殻[79]や貝殻[82,83]は糖の異性化過程で副生した有機酸を中和し，pHの低下を抑えることにより希少糖の収率を向上させた．
Rare sugars are narrowly defined as monosaccharides and their derivatives that exist only in small quantities in nature [1]. However, in a broader sense, rare sugars refer to sugars that are scarce in nature, not limited to monosaccharides [2]. In this paper, the term “rare sugars” follows the broader definition. Sugars that are abundant in nature are described as conventional sugars, natural sugars, or common sugars. The term “common sugars” or “common saccharides” is used here.
Many rare sugars exhibit physiological functions or serve as food additives. For example, d-tagatose has health-promoting properties such as antioxidant and prebiotic effects, low digestibility, reduced glycemic and insulinemic responses, and the potential to improve lipid profile [3]. d-Allulose (also known as d-psicose) has been reported to have physiological functions such as anti-obesity, anti-diabetes, anti-inflammatory, antioxidant, and neuroprotective effects [4]. Smith et al. [5] reported health benefits, mechanisms of action and potential uses of d-allulose and d-tagatose. Since most of the monosaccharides mentioned in this paper are d-enantiomers, the letter d is omitted to indicate optical isomerism, except in the case of l-enantiomers. Ahmed et al. [6] reviewed physiological and cardiometabolic effects of five rare sugars, allulose, arabinose, tagatose, trehalose, and palatinose (isomaltulose), in humans, and concluded that rare sugars could offer an opportunity for commercialization as an alternative sweetener, particularly for individuals who are at high cardiometabolic risk. The potential use of rare sugars in agriculture has also been reported [7].
Isomerization or epimerization of common sugars to rare ones can be broadly classified into biochemical methods using enzymes or microorganisms and chemical methods using base catalysts. In the 1950s, isomerization of aldose to ketose by enzymatic methods was reported [8,9]. Isomerization of glucose to fructose by glucose isomerase is carried out on an industrial scale in the production of high fructose corn syrup, with annual global production reaching 14 million metric tons by dry weight [10]. A systematic strategy for the biochemical production of various rare sugars, named “Izumoring”, was proposed [1], and Fukuda and Izumori [11] reported a comprehensive overview of the methods, advancements and development in the enzymatic production of various rare sugars by the strategy. Enzymatic production of rare sugars has the advantage of proceeding under mild conditions with high selectivity. On the other hand, the biochemical production of rare sugars requires much effort to discover an enzyme that specifically catalyze the reaction. Also, the enzymes found are not always stable and may require divalent cations for exhibiting their activities.
In contrast, isomerization of sugars in caustic alkaline solutions has been known since the 19th century and is referred to as Lobry de Bruyn-Alberda van Ekenstein (LBAE) transformation [12]. Aqueous solutions of alkaline metal hydroxide and alkaline-earth metal hydroxide such as NaOH and Ca(OH)2 have been used as base catalysts. Anion exchange resins have also been used [13], and some examples have been industrialized [2,14].
As mentioned above, base-catalyzed isomerization and epimerization of sugars are not substrate specific, i.e., they are versatile and potentially applicable to the production of various rare sugars. Delidovich [2] reviewed in detail the base-catalyzed isomerization of sugars, including the reaction mechanism. Although the isomerization of sugars in subcritical fluids is mentioned in the review, its description is limited. In this context, this paper reviews the isomerization of sugars in subcritical fluids and its application to production of rare sugars under various conditions, primarily based on our findings.
A subcritical fluid is defined as a fluid at temperatures and pressures below its thermodynamic critical point. While the upper temperature limit of a subcritical fluid is clearly defined by the critical temperature, no rigorous definition exists for the lower temperature boundary. Therefore, for the sake of clarity and convenience, we define it as follows in this paper. A subcritical fluid is a fluid that is kept in a liquid state by pressurization in the range from the boiling point at ambient pressure to the critical temperature [15]. For example, if the fluid is water, subcritical water is water in a liquid state in the temperature range from 100°C to 374°C. Compared to water at room temperature, subcritical water has two characteristics: a low relative permittivity and a large ion product [16]. The latter feature increases the concentration of hydronium and hydroxide ions in subcritical water to be several to 30 times higher than those in water at room temperature. Therefore, subcritical water acts as both an acid or a base catalyst, facilitating hydrolysis [17-19], pyrolysis [20,21], condensation [22,23], isomerization. In this paper, we review the formation of rare sugars by isomerization and epimerization of sugars in water or water-soluble organic solvents under subcritical water conditions. The formation of rare sugars using eggshell and seashell powders, primarily composed of CaCO3 and capable of producing slightly alkaline solution, is also discussed. Since epimerization is a subset of isomerization, it is also referred to as isomerization in this paper.
As mentioned above, subcritical water has a high concentration of hydroxide ion and acts as a base catalyst to isomerize sugars through LBAE transformation. For example, glucose, an aldo-hexose, is isomerized to fructose and mannose via the 1,2-enediol intermediate, and fructose is further isomerized to allulose via the 2,3-enediol intermediate (Fig. 1(a)). Since the isomerization of glucose into fructose is an endothermic reaction, higher temperatures shift the equilibrium toward the ketose form, fructose [24,25]. This is also true for the isomerization of other aldoses to ketoses. The higher the temperature, the faster the reaction rate. These are the advantages of rare sugar production in subcritical water. Figure 1(b) shows the structures of sugars presented in this paper.
Conversion of sugars under subcritical water conditions. (a) Possible reactions occurring during the treatment of glucose under subcritical water conditions. (b) Structures of the sugars presented in this paper. Glc and Gal represent glucose and galactose residues, respectively. α-1,4 and β-1,4 indicate that two hexose molecules are bonded via α-1,4 and β-1,4 glycosidic bonds.
On the other hand, at high temperatures, the substrate, product, and intermediate enediol are decomposed into various aldehydes and further converted into organic acids by the retro-aldol reaction and double-bond rule (Fig. 1(a)) [26]. The double-bond rule refers to a phenomenon in which the bond between a carbon next to a double bond and the carbon next to the carbon next to the double bond is cleaved. Thus, compounds with two and four carbons are formed from aldo-hexose, and two compounds with three carbons are formed from keto-hexose. These compounds further react to produce byproducts such as organic acids, and the pH of the reaction solution decreases. As described below, LBAE transformation does not proceed below pH 6.3, and isomerization stops. When reducing sugars and amino acids are present at high temperatures, the Maillard reaction occurs, and caramelization takes place even in the absence of amino acids, resulting in coloration of the reaction mixture. These are disadvantages in the production of rare sugars by subcritical water.
When aqueous solutions of mannose, fructose, or glucose were treated in water at 220°C in a continuous flow-type reactor at various residence times (reaction times), reversible isomerization proceeded as well as degradation reactions of each sugar [27]. The isomerization of mannose to fructose proceeded most readily, followed by that of mannose to glucose. Assuming that each process at 180-220°C followed first-order reaction kinetics, the rate constants were determined, and the activation energy and frequency factor were evaluated using the Arrhenius equation. The enthalpy-entropy compensation of each process was established. It should be noted that the rate constants obtained here are apparent values because the pH changes during these reaction processes, as described below. This applies to all rate constants reported hereafter.
The above findings were obtained at relatively low sugar concentrations, ranging 0.5 to 5.0% (w/v). The isomerization of sugars in various media, as described below, has also been performed at similarly low concentrations, and little is known about the reaction behavior at high substrate concentrations. Therefore, we measured the reaction behavior at a wide range of substrate concentrations from 0.1 to 40% (w/w) at 200°C using a continuous flow-type reactor [28]. The isomerization and decomposition behavior of glucose greatly depended on the initial glucose concentration. The rate of glucose consumption decreased as the reaction time increased. Although glucose continued to decrease over time, the yield of fructose leveled off, and a small amount of mannose produced exhibited the same trend. Figures 2(a) and (b) show the relationship between the selectivity of fructose and the pH of the reaction solution (measured at room temperature) and the conversion of glucose. In pure subcritical water, the pH of the reaction mixture decreased rapidly to a value at which LBAE transformation did not proceed. This tendency was more pronounced at higher initial glucose concentrations. At higher initial glucose concentrations, more fructose was degraded into byproducts, leading to lower selectivity of fructose. Although increasing the substrate glucose concentration is desirable for obtaining a higher concentration of target product (fructose in this case), excessively high substrate concentrations may not always be preferable due to selectivity concerns, suggesting the existence of an optimal initial substrate concentration. These findings will be useful for the investigation of the conditions for isomerization of other sugars.
Changes in (a) the selectivity of fructose and (b) pH during the conversion of glucose at 200°C in subcritical water. The initial glucose concentrations were (
) 0.1 % (w/w), (
) 1.0 %, (
) 10 %, and (
) 40%. The curves were drawn empirically.
Foods and their ingredients are generally multi-component systems in which multiple reactions occur simultaneously during their processing under subcritical water conditions, making it difficult to elucidate individual reaction pathways. Sodium chloride (NaCl), a common component of foods, has been reported to promote the degradation and hydrolysis of mono- and disaccharides [29,30]. The reaction kinetics of glucose and fructose under subcritical water conditions containing 1-20% (w/w) NaCl were analyzed [31]. When glucose was treated at 220°C, the decrease of glucose in 1% (w/w) NaCl solution was larger than that in pure water, and fructose and a small amount of mannose were also produced. Fructose increased in the early stage of the reaction, reached its peak, and then decreased. A similar trend was observed at 180°C and 200°C, although the effect of NaCl was minimal at these temperatures. On the other hand, when fructose was treated in 1% (w/w) NaCl solution and in pure water at 180-220°C, fructose decreased faster at higher temperatures, and NaCl accelerated the decrease. However, neither glucose nor mannose formation was observed under any of the condition. Therefore, the decrease in fructose under subcritical water conditions can be attributed solely to degradation.
When the effect of NaCl concentration was examined during the treatment of 1.0% (w/v) glucose at 200°C, the yield of fructose increased with increasing the conversion of glucose, but after reaching the maximum value, the yield of fructose decreased in the latter half of the reaction (Fig. 3). At low NaCl concentrations (1-5% (w/w)), the effect of concentration was small and the maximum yield of fructose was about 4%, but in the presence of 10% (w/w) and 20% (w/w) NaCl, the maximum yield of fructose decreased to about 2%. This was enhanced fructose degradation at higher NaCl concentrations.
Effect of the sodium chloride concentration on the relationships between yield of fructose and conversion of 1.0% (w/v) glucose at 200°C. The initial sodium chloride concentrations were () 1% (w/w), () 2%, () 5%, () 10%, and (
) 20%. The symbol, 
, represents the relationship in pure water. The curves were drawn empirically.
The effects of various salts, including chlorides of alkali metal ions (Li+, Na+, K+, and Cs+) and alkaline earth metal ions (Mg2+, Ca2+, and Sr2+) on the reaction behavior of glucose and fructose in subcritical water (190°C) have been investigated using a flow-type reactor (Fig. 4) [32]. The conversion of glucose was ca. 6% in pure water, 7-20% in alkali metal salt solutions, and 27-31% in alkaline earth metal salt solutions at a residence time (reaction time) of 120 s. In other words, alkali metal salts promoted glucose conversion, with alkaline earth metal salts exhibiting an even greater effect. Under all conditions, the decrease in glucose did not follow first-order kinetics and tended to approach a constant value as the reaction time was prolonged.
Changes in the yield of fructose over the residence (reaction) time during the treatment of 5.0% (w/w) glucose dissolved in (
) water, and () lithium, () sodium, () potassium, () cesium, (
) magnesium, (
) calcium, and (
) strontium chlorides at 190°C. The concentration of each salt was 0.50 mol/L. The curves were drawn empirically.
For all salts, fructose was rapidly formed in the early stage of the reaction, reaching a peak, followed by a decline in the yield of fructose in the latter half of the reaction. This trend was more pronounced in the alkaline earth metal salt solutions, except in pure subcritical water, and a slightly similar trend was observed in the alkali metal salt solutions, with a lower yield of fructose in the latter half of the reaction than in pure subcritical water. In the latter half of the reaction, fructose yields in the salt solution were lower than those in pure subcritical water. These results indicate that cation valence affected isomerization and accelerated degradation. It was reported that calcium ions coordinate to C1-3 oxygen atoms to promote retro-aldol condensation (α-dicarbonyl cleavage), resulting in the degradation of sugars and the formation of organic acids [33]. The presence of divalent ions in the subcritical fluid would have caused similar phenomena, and treatment of fructose in subcritical salt solutions accelerated the degradation of fructose compared to that in pure subcritical water.
3.3 Aqueous organic solventIt was reported that isomerization of glucose to fructose was promoted in the aqueous ethanol [34-36]. The promotion observed upon adding ethanol is attributed to changes in both the conformation and configuration of saccharides in aqueous ethanol [37], as well as to change in the apparent chemical equilibrium [38].
The influence of water-miscible primary and secondary alcohols on the isomerization of glucose to fructose and mannose, which is a C2 epimer of glucose, was investigated under subcritical aqueous conditions [39]. Isomerization of mannose and fructose was also examined in similar aqueous alcohols under subcritical conditions [40]. Figure 5 shows the relationship between the conversion of glucose and the yield of fructose in pure subcritical water and in aqueous methanol, ethanol, 1-propanol, 2-propanol, and tert-butyl alcohol at 180°C. All of the plots in Fig. 5 lie approximately on a straight line, and the slope of the line, representing the selectivity of glucose to fructose, is about 0.8. However, as shown in the inset of Fig. 5, the yield of fructose in aqueous methanol, ethanol, 1-propanol, and 2-propanol was much higher than that in pure water. This may be because the ratio of cyclic to acyclic structures of glucose is affected by the presence of alcohol [41]. However, the reason for the extremely low yield of fructose in tert-butyl alcohol is unclear. The yield of mannose was only about 1/6 that of fructose under all conditions, although it is not shown in Fig. 5.
Relationship between the conversion of glucose and the yield of fructose during the treatment at 180°C of 0.5% (w/v) glucose dissolved in () water, and aqueous solutions of () methanol, () ethanol, () 1-propanol, () 2-propanol, and () tert-butyl alcohol. The concentration of each alcohol was 60% (v/v). Inset: Changes in the yield of fructose with residence time. The curves were drawn empirically.
The influence of ethanol concentration (0-80% (v/v)) on the mutual isomerization of glucose, fructose, and mannose (each initially at 0.5% (w/v)) at 180, 190, and 200°C was investigated using a tubular reactor [42]. At 180°C, the isomerization of glucose to fructose was enhanced until the ethanol concentration exceeded 40% (v/v). Mannose was undetectable at ethanol concentrations below 40% (v/v); however, its formation was observed in 60% (v/v) aqueous ethanol, yielding approximately 1/7 the amount of fructose. At 200°C in 80% (v/v) aqueous ethanol, the consumption of fructose was the slowest among the three hexoses, with mannose and glucose produced in nearly equal, albeit low, yields. The isomerization of mannose occurred more rapidly than that of fructose or glucose. The fructose yield initially surpassed that of glucose, peaked, and then gradually declined. The yields of glucose and mannose derived from fructose remained low, at approximately 5%.
Using the same tubular reactor described above, xylose and ribose, dissolved in 80% (v/v) aqueous ethanol at a concentration of 0.5% (w/v), were treated at 180°C. Galactose, at concentrations ranging from 0.5% to 8.5% (w/v), was treated at temperatures between 160°C and 200°C [43]. Galactose, xylose, and ribose (0.5% w/v) underwent isomerization to their corresponding ketoses, tagatose, xylulose, and ribulose, respectively, in high yields in 80% (v/v) aqueous ethanol at 180°C. The yields of tagatose and talose from galactose increased with higher ethanol concentrations; however, selectivity remained unaffected by ethanol concentration. The influence of initial galactose concentration on isomerization in 60% (v/v) aqueous ethanol was also investigated. It was observed that higher concentrations of galactose led to decreases in galactose conversion, as well as in the yield and selectivity of the primary product, tagatose. Nevertheless, the productivity of tagatose was highest at a concentration of 8.5% (w/v) galactose, the highest concentration tested.
Maltose, isomaltose, cellobiose, lactose, melibiose, palatinose, sucrose, and trehalose were treated in 60% (w/w) aqueous ethanol at 220°C [44]. The aldo-disaccharides except for the latter three (palatinose, sucrose and trehalose) isomerized to the corresponding keto-disaccharides (maltulose, palatinose, cellobiulose, lactulose, and melibiulose), along with hydrolysis of the substrates and products. The maximum yield of keto-disaccharides produced from aldo-disaccharides linked by β-glycosidic bond was higher than that from those linked by α-glycosidic bond. The keto-disaccharide palatinose was hardly isomerized and mainly degraded, while the non-reducing disaccharides, sucrose and trehalose, were not isomerized at all, and sucrose was hydrolyzed to glucose and fructose. On the other hand, trehalose, a disaccharide consisting of two molecules of glucose bonded by α-1,1-glucosidic bond, was hardly hydrolyzed. The effect of maltose concentration in the feed was examined in 60 % (w/w) aqueous ethanol at 220°C. As in the isomerization of galactose, the conversion of maltose and the yield of maltulose were lower, but the productivity of maltulose was higher at the higher maltose concentration.
Lactulose possesses some physiological functions and therapeutic benefits [45-47]. The common production of lactulose is performed by alkaline isomerization of lactose via the LBAE transformation [48]. Isomerization of lactose to lactulose was investigated in subcritical aqueous ethanol (0-60% (w/w)) at 160-220°C [49]. The isomerization and hydrolysis of lactose were promoted and suppressed, respectively, by increasing ethanol concentration. The maximum yield of lactulose was obtained in 60% (w/w) aqueous ethanol at 200°C. The effect of lactose concentration on the productivity was examined in the range of 0.5 to 2.5% (w/w), and the maximum lactulose productivity was obtained at the lactose concentration of 2.5% (w/w). Production of lactulose from lactose was kinetically analyzed under the assumption of first-order kinetics for both the reversible isomerization between lactose and lactulose and the hydrolysis of these disaccharides [50]. As an example, the changes in the fraction of remaining lactose and the yield of lactulose in 40% (w/w) ethanol at 200°C are shown in Fig. 6. Lactose is reversibly isomerized to lactulose, and the disaccharides are hydrolyzed to produce monosaccharides. Although the monosaccharides produced may be isomerized or degraded, a simplified mechanism was assumed as shown in the inset of Fig. 6 to analyze the changes over the residence time of lactose and lactulose concentrations. First-order kinetics was applied to all the steps shown. The reaction rates for the formation of lactose and lactulose are given by Eqs. (1) and (2), respectively, where CA and CK indicate the respective concentrations of lactose and lactulose, ki (i ＝ 1, 2, 3, or 4) is the rate constant, and τ is the residence time in a tubular reactor.
 | (1) |
 | (2) |
The concentration-time integrals method [51] was employed among several methods for estimating the rate constant ki so that the measured values fit the calculated ones. Integrating Eq. (1) with residence time and lactose concentration under the conditions CA ＝ CA0 at τ ＝ 0 and CA ＝ CA at τ ＝ τyields Eq. (3).
 | (3) |
Equation (3) can be rewritten as:
 | (4) |
where
 | (5) |
Similarly, Eq. (2) can be rewritten to give Eq. (6):
 | (6) |
The concentration-time integrals θj,τ at different times were approximated using the trapezoidal rule based on available data points. Then, the rate constants were estimated using the Solver of Microsoft® Excel to adjust the relevant equation parameters to minimize the residual sum of squares for the difference between the experimental and the predicted concentration-time data. The curves in Fig. 6 are calculated lines using the rate constants ki (i ＝ 1 to 4) estimated by this method, and they represent the measured values well.
Changes in () the fraction of remaining lactose, CA/CA0, an () the yield of lactulose, CK/CA0, over the residence (reaction) time during the treatment of 0.5% (w/w) lactose dissolved in 40% (w/w) ethanol at 220°C. A and K represent lactose and lactulose. CA0 is the concentration of lactose in the feed solution. The curves were calculated using the estimated rate constants ki (i ＝1 to 4). Inset: A simplified reaction mechanism for the formation of lactulose in subcritical aqueous ethanol.
The isomerization of cellobiose to cellobiulose, their hydrolysis, and degradation of glucose and fructose, which were produced through the hydrolysis were investigated in subcritical aqueous ethanol (0 to 60% (w/w)) at 170 to 200°C [52]. The maximum yield of cellobiulose was obtained from the treatment of cellobiose in 60% (w/w) aqueous ethanol at 190°C. The rate constants for the mutual isomerization, hydrolysis, and degradation were evaluated using the concentration-time integrals method.
Xylose, ribose, glucose, mannose, fructose, galactose, maltose, cellobiose, and melibiose were treated in 60% (w/w) aqueous acetonitrile at 180°C and 200°C for monosaccharide and disaccharide, respectively, using a tubular reactor [53]. All aldoses except fructose, which is ketose, were isomerized to the corresponding ketoses, most of which are rare sugars. The yield of ketoses tended to be higher in the order of disaccharides > hexoses > pentoses. Isomerization of fructose to glucose or mannose was unfavorable, and fructose was mainly decomposed. Increasing acetonitrile concentration and elevating reaction temperature improved the conversion of glucose and yield of fructose. This trend was also observed for isomerization in subcritical aqueous ethanol.
3.4 Buffer solutionAs mentioned previously, the isomerization of common sugars in subcritical aqueous alcohol (ethanol) reduces the reaction time required to produce rare sugars. However, a limitation exists in increasing the concentration of rare sugars using subcritical aqueous alcohol treatment due to the limited solubility of common sugars. Additionally, sugars in subcritical fluid decompose to form some organic acids, which lower the pH [54,55]. The use of a buffer solution is expected to counteract the pH drop and improve the yield of rare sugars. In this context, the effect of buffer type on the performance for galactose isomerization to tagatose and talose, which are the major and minor products in the isomerization, respectively, was investigated [56]. Figure 7 illustrates the changes in the fraction of remaining galactose, the yield of tagatose (the major product in the isomerization of galactose), and pH over time during the treatment of 5.0% (w/w) galactose dissolved in 0.01 mol/L sodium phosphate, PIPES (piperazine-N,N’-bis(2-ethanesulfonic acid)), and MOPS (3-(N-morpholino)propanesulfonic acid) buffers (pH 7.0) at 140°C. Since the yield of talose was low in all buffers, it is not shown in Fig. 7. Sodium phosphate buffer is hereafter simply referred to as phosphate buffer. In phosphate buffer, the pH decreased, but the conversion of galactose and the yield of tagatose were the highest. In contrast, in PIPES buffer, the pH remained relatively stable, and the conversion of galactose and the yield of tagatose were moderate. Furthermore, in MOPS buffer, the pH showed minimal change, the conversion of galactose changed only slightly, and the yield of tagatose was very low. The reason for the differences observed among the buffer types remains unclear.
Changes in the fraction of remaining galactose, CGal/CGal,0, the yield of tagatose, CTag/CGal,0, and pH over the residence time during the treatment of 5.0% (w/w) galactose dissolved in 0.01 mol/L () phosphate buffer, () PIPES buffer, and () MOPS buffer (each at pH 7) at 140°C. The curves were drawn empirically.
Galactose dissolved in 0.01 mol/L phosphate buffer was treated at various temperatures, and the relationships between pH and the fraction of remaining galactose, the yield of tagatose, or the yield of talose are shown in Fig. 8. At all temperatures, as the residence (reaction) time was extended, the pH decreased, the fraction of remaining galactose decreased, and the yields of tagatose and talose increased. However, once the pH of the reaction solution reached ca. 6.3, the yields of tagatose and talose leveled off, and the fraction of remaining galactose continued to decrease only slightly. As previously mentioned, the isomerization of sugars via the LBAE transformation is facilitated by base catalysis. Consequently, at pH values below approximately 6.3, the hydroxide ion concentration becomes insufficient to drive the reaction forward. This accounts for the observed plateau in the yields of tagatose and talose at pH values lower than 6.3. The yield of tagatose slightly decreased at pH 4.5 or lower. These results suggest that the isomerization of galactose to tagatose and talose does not occur below pH 6.3, and that galactose is gradually converted into minor byproducts. The fact that the plots for all temperatures follow a single curve for each quantity indicates that the reaction mechanism is consistent across the temperature range tested.
Relationships between pH and the fraction of remaining galactose, CGal/CGal,0, the yield of tagatose, CTag/CGal,0, or the yield of talose, CTal/CGal,0 in 0.01 mol/L phosphate buffer at () 120, () 130, () 140, () 150, and () 160°C. CGal,0 represents the galactose concentration in the feed solution. The curves were drawn empirically.
Galactose dissolved in phosphate buffers of various concentrations (0.0001 to 0.5 mol/L, pH 7) was treated at 140°C using a tubular reactor [57]. The isomerization of galactose to tagatose proceeded more efficiently at higher buffer concentrations. The highest yield of the main product, tagatose, was achieved at 0.05 mol/L buffer concentration, while no further increase in the yield was observed at higher buffer concentrations. On the other hand, the yields of talose and sorbose were higher at higher buffer concentration. The formation of byproducts, including organic acids and colored substances, was also accelerated at high buffer concentrations.
Isomerization of galactose dissolved in phosphate buffer at pH 6, 7, or 8 (5% (w/w)) to tagatose and talose was performed at 120°C using a batch reactor [58]. The highest yield of tagatose was obtained at pH 7, with slight changes in the solution color. The yield of tagatose increased with increasing buffer concentration up to 0.01 mol/L but remained nearly constant at concentrations higher than 0.01 mol/L. Little talose was produced at buffer concentrations lower than 0.01 mol/L, but its yield significantly increased at 0.01 mol/L.
Maltulose was produced with a yield of approximately 20% through a batch reaction using 5% (w/v) maltose dissolved in 0.01 mol/L phosphate buffer (pH 7.0) and a heat block set to 110°C (actual temperature: 108°C) [59]. The effect of buffer concentration on the reaction was investigated while maintaining a fixed reaction time of 90 min. At buffer concentrations of 0.01 mol/L or lower, increases in buffer concentration resulted in higher maltulose yield and greater coloration, as assessed by absorbance at 440 nm. However, at buffer concentrations exceeding 0.01 mol/L, side reactions became prominent, leading to a decline in the yield of maltulose and a significant increase in coloration.
A continuous flow-type reactor is preferred for large-scale production of rare sugars. An empty stainless steel HPLC column with an inner diameter of 7.6 mm and a length of 500 mm was used as the reactor to investigate the effects of reaction temperature, average residence (reaction) time, and maltose concentration on the isomerization of maltose to maltulose in 0.01 mol/L phosphate buffer [60]. The optimal conditions were determined to be a temperature of 115°C (the highest tested temperature) and an average residence time of 10 min. Considering both substrate utilization efficiency and maltulose concentration, the optimal maltose concentration was found to be 10% (w/v).
Onishi et al. [26] investigated the isomerization of xylose, arabinose, glucose, galactose, maltose, and lactose dissolved in 0.01 mol/L phosphate buffer (pH 7.0) at 140°C using a tubular reactor made of PEEK resin (inner diameter of 0.75 mm). Each sugar was isomerized into its corresponding ketose and C-2 positional epimer. The yields of keto-disaccharides, keto-hexoses, and keto-pentoses, all classified as rare sugars, were 21-27 mol%, 13-16 mol%, and 5.8-8.9 mol%, respectively. Hydrolysis of disaccharides was negligible, with monosaccharide yields below 1 mol%. For all sugars, isomerization was ineffective when the pH of the reaction solution dropped below 6.3. Furthermore, pathways for the formation of byproducts from each sugar were proposed.
In previous studies, aldoses were predominantly used as substrates. Hashimoto et al. [61] investigated the isomerization of fructose, a ketose, under subcritical water conditions with phosphate buffer (0.01-0.5 mol/L, pH 7.0 and 8.0). Among the tested conditions, the highest yields of mannose and allulose+allose (quantified as a single peak via HPLC due to co-elution) were obtained when 5% (w/v) fructose dissolved in phosphate buffer at an initial pH of 8.0 was treated at 120°C. The yields were approximately 15% for mannose and 18% for allulose+allose.
Lactose was dissolved in 0.1 mol/L phosphate buffer, pH 8, and 0.1 mol/L carbonate-bicarbonate buffer (referred as carbonate buffer), pH 10 or 11, at a concentration of 5 or 10% (w/v), and treated using a batch reactor at 110, 120, or 130°C [62]. The highest yield of lactulose was obtained in phosphate buffer, followed by carbonate buffer at pH 10 and carbonate buffer at pH 11 at any temperature. The formation of some monosaccharides, such as glucose, galactose, fructose, tagatose, talose, allulose, and sorbose, occurred due to the hydrolysis and isomerization during the treatment. High pH was not necessarily effective for production and led to rapid decomposition of lactulose and a significant change in color of the reaction solution.
As previously mentioned, sugar isomerization was significantly accelerated in aqueous ethanol and phosphate buffer at elevated temperatures compared to water alone. Therefore, the isomerization of galactose to tagatose and talose at 140°C was investigated using a tubular reactor and a mixture of 0.01 mol/L phosphate buffer (pH 7.0) and ethanol [63]. Ethanol and the buffer synergistically enhanced the isomerization performance. When ethanol was absent, the yield of tagatose after a reaction time of 300 s was 13%, whereas it increased to 16% in 60% (w/w) ethanol, the highest ethanol concentration tested. Conversely, the yield of talose decreased from 1.5% to 1.2%. The addition of ethanol was found to suppress pH reduction, which is considered one of the reasons for the improved tagatose yield. Additionally, the addition of other organic solvents, such as alkanols, polyols, acetonitrile, and pyridine, also improved tagatose yields.
3.5 Basic amino acid solutionAqueous solutions of basic amino acids exhibit alkalinity, and the basic amino acids such as arginine, lysine, and histidine have been studied as catalysts for the isomerization of sugars. These amino acids are considered “green catalysts” due to their relative safety and environmental friendliness. Yang et al. [64] investigated the isomerization of glucose to fructose using the three amino acids and found that arginine was the most effective catalyst. However, when using amino acids as catalysts for sugar isomerization, there is a concern about coloration due to the Maillard reaction. Kim and Lee [65] studied the mutual isomerization between fructose and glucose at various pH, and Lamberts et al. [66] reported the isomerization of glucose to fructose and xylose to xylulose using arginine, glutamic acid, glutamine, leucine, lysine, phenylalanine, and γ-aminobutyric acid at pH 6.0. However, a detailed investigation under subcritical water conditions has yet to be considered.
As mentioned earlier, ribose was one of the pentoses with a low isomerization yield in subcritical phosphate buffer. Ribulose is used as a starting material in the synthesis of pharmaceuticals and chemicals [67]. 0.2 mol/L ribose was treated with 0.01 mol/L arginine, lysine, or histidine at 110°C using a batch reactor to compare their catalytic abilities and extent of coloration [68]. Figure 9 shows the changes in the fraction of remaining ribose, the yield of ribulose, and absorbance at 420 nm, which indicates coloration or browning, over the reaction time. The highest yield of ribulose was observed when arginine was used as a catalyst, followed by lysine, while histidine resulted in a very low yield. This order is consistent with that reported by Yang et al. [64] for the isomerization of glucose to fructose. On the other hand, the coloration was highest when lysine was used as a catalyst, followed by arginine. Histidine not only exhibited low isomerization ability but also caused minimal coloration. Therefore, arginine is the most suitable catalyst for isomerization of ribose to ribulose, and this would likely apply to the isomerization of other reducing sugars.
Changes in (open symbols) the fraction of remaining ribose (CS/CS0), (black symbols) the yield of ribulose (CP/CS0), and (gray symbols) absorbance at 420 nm of the reaction mixture (A420) during the treatment of ribose with (circles) arginine, (triangles) lysine, and (squares) histidine at 140°C. The solid curves represent CS/CS0 and CP/CS0, while the broken curves represent A420. The curves were empirically drawn.
The effects of reaction conditions on the yield of ribulose and degree of browning were studied using arginine as a catalyst [69]. The conditions investigated included the molar ratio of arginine to ribose (at a constant ribose concentration of 0.2 mol/L) at 110°C, reaction temperature for a solution containing 0.2 mol/L ribose and 0.01 mol/L arginine, and initial ribose concentration at a molar ratio of arginine to ribose of 0.05 at 110°C. A high yield of ribulose and a low degree of browning were achieved with a molar ratio of arginine to ribose of 0.05 at 110°C. The optimal ribose concentration was found to be 0.75 mol/L. The relationship between ribulose yield and pH, within a range of 90 to 120°C followed a single curve. Similarly, the relationship for various ribose concentrations also followed another single curve. Furthermore, the selectivity in the isomerization of ribose to ribulose was independent of the operating conditions. These findings suggest that the reaction mechanism for isomerization is consistent and unaffected by the conditions tested.
Galactose (5% (w/v)) were treated using arginine as a catalyst at a molar ratio of arginine to galactose of 0.10 at temperatures ranging from 90 to 120°C [70]. Galactose underwent isomerization to tagatose and talose via 1,2-enediol, with some tagatose further isomerized to sorbose via 2,3-enediol. Maximum yields of tagatose, talose, and sorbose were 16.8%, 2.7%, and 3.3%, respectively, under conditions of 120°C for 20 min. Higher temperatures and reduced reaction time minimized the extent of the Maillard reaction. Variations in arginine concentrations of 0.05, 0.10, and 0.15 mol/mol-galactose led to a modest increase in tagatose yield. In constract, increasing the initial galactose concentration from 5% (w/v) to 20% (w/v) resulted in a decrease in tagatose yield despite a rise in tagatose concentration. The optimal tagatose productivity, 278 g/(L·h), was achieved with 20% (w/v) galactose and 0.10 mol/mol-galactose arginine at 120°C for 4 min.
The isomerization of 5% (w/v) lactose to lactulose in an aqueous solution containing arginine (0.10 mol/mol-lactose) at an initial pH of 9.80 was systematically examined. Elevated reaction temperatures (100°C, 110°C, and 120°C) significantly enhanced lactulose production, with a peak yield of approximately 26% achieved after 12 min at 120°C [71]. This process involved the consumption of lactose and the formation of lactulose and monosaccharides (glucose and galactose). The Maillard reaction, characterized by increasing absorbance at 280, 325, and 420 nm and a gradual decline in pH, underscored the role of arginine as an effective catalyst for the conversion of lactose into its rare isomer, lactulose.
Microwave heating was applied to isomerize maltose to maltulose using arginine as a catalyst. The isomerization of maltose to maltulose under microwave heating was investigated, with the initial molar ratio of arginine to maltose fixed at 0.05 [72]. Maltulose yields of 28-30% were achieved within 90-400 s, depending on the microwave power and substrate solution volume, though the yield was independent of the initial maltose concentration. This demonstrates the efficiency of microwave heating for this reaction. Additionally, the fraction of remaining maltose, the yield of maltulose, pH of the reaction mixture, and absorbance at 280 and 420 nm were all shown to be functions of energy density, defined as the energy absorbed per unit volume of the reaction mixture. Figure 10(a) illustrates the changes in the fraction of remaining maltose and the yield of maltulose during the microwave heating of the substrate solution (50 mL) containing maltose and arginine at 0.2 and 0.01 mol/L, respectively, at powers of 200 W, 500 W, and 700 W. Microwave heating elevates the temperature of the reaction mixture within the vessel, while heat loss occurs due to the temperature difference between the reaction mixture and its surrounding environment. The energy density, EV, is expressed by the following equation [72].
 | (7) |
where A is the heat transfer area, cp is the heat capacity of reaction mixture (approximately equal to that of water), fE is the energy efficiency during microwave heating, P is the power of microwave heater, t is the heating time, U is the overall heat transfer coefficient, w0 is the weight of the reaction mixture, and ρ0 is the initial density of the substrate solution. Under the specific microwave heating conditions employed, the fE values, dependent on both the heater’s power and type, were approximately 0.38, 0.53, 0.55, and 0.57 for reaction solution volumes of 25, 50, 75, and 100 mL, respectively. These results indicate a general trend of improved energy efficiency with increasing solution volume. Figure 10(b) shows the fraction of remaining maltose and the yield of maltulose as functions of the energy density. The changes in the fraction of remaining maltose and the yield of maltulose each follow a distinct curve, suggesting that the isomerization conditions can be effectively optimized based on energy density, offering a reliable framework for controlling the isomerization process.
Synthesis of maltulose from maltose by microwave heating. (a) Changes in (open symboles) the fraction of remaining maltose, CS/CS0, and (closed symbols) the yield of maltulose, CP/CS0, over the heating time during the microwave heating at a power of (, ) 200 W, (, ) 500 W, and (, ) 700 W. (b) Illustration of the observed results as a function of the energy density, EV, of the reaction mixture. The keys are the same as those in (a). The curves were empirically drawn.
In the preceding section, the isomerization of sugars in aqueous arginine solutions and phosphate buffer solutions under subcritical water conditions was discussed. In the following section, the isomerization of sugars in arginine solutions and phosphate buffer solutions is compared. Ribose, xylose, and arabinose (0.2 mol/L) were dissolved in 0.01 mol/L arginine or 0.01 mol/L phosphate buffer (pH 7.0) and subjected to treatment at 110°C. The yields of ribulose, xylulose, and ribulose, along with the absorbance at 280 nm and 420 nm of the reaction mixture and the pH, were measured [73]. The reactions proceeded rapidly in highly alkaline arginine solution, achieving apparent equilibrium yields of ribulose, xylulose, and ribulose at 8.8%, 8.9%, and 3.5%, respectively. In phosphate buffer, the yields were slightly lower at 8.4%, 6.5%, and 2.2%, respectively. Notably, even after the product yields reached equilibrium, the pH continued to decrease, and absorbance at 280 nm and 420 nm increased, particularly in the latter half of the reaction.
The reactions of glucose and galactose (0.2 mol/L) were conducted in 0.01 mol/L arginine solution or 0.01 mol/L phosphate buffer (pH 7.0) using a batch reactor at 110°C. Product yields, pH, and absorbance at 280 nm and 420 nm were monitored throughout the reaction [74]. Fructose, mannose, and allulose were produced from glucose, while tagatose, talose, and sorbose were formed from galactose. The reactions proceeded more rapidly in arginine solution than in phosphate buffer. After 30-min reaction in arginine solution, the yields of fructose and tagatose reached 20% and 16%, respectively, compared to 14% and 10% in phosphate buffer. However, in both media, pH continued to decrease and absorbances increased even after product yields stabilized. The increase in absorbance, attributed to the formation of browning products, was particularly pronounced during the latter half of the reaction.
Maltose, cellobiose, and lactose (0.2 mol/L) were dissolved in 0.01 mol/L arginine solution or 0.01 mol/L phosphate buffer (pH 7.0) and subjected to batch reactions at 110°C [75]. The corresponding keto-disaccharides, maltulose, cellobiulose, and lactulose, were formed with yields of 23.4% and 17.0% for maltose, 19.3% and 13.0% for cellobiose, and 20.0% and 13.7% for lactose in arginine solution after 30 min, and in phosphate buffer after 150 min, respectively. Hydrolysis of all disaccharides occurred at levels ranging from 0.5% to 3.5%, with a higher degree of hydrolysis observed in arginine solution compared to phosphate buffer. The monosaccharides produced through hydrolysis underwent minor isomerization into other monosaccharides. Despite the yields of the main products stabilizing, pH continued to decline, and absorbance at 280 nm and 420 nm increased. This rise in absorbance, especially during the latter stages of the reaction, indicated the formation of browning products. These findings suggest that promptly terminating the reaction upon achieving maximum product yield is crucial to minimizing browning caused by byproducts with high absorption coefficients.
As described above, common sugars can be isomerized into rare ones in hot compressed water; however, side reactions simultaneously produce organic acids and other byproducts. The formation of organic acids lowers the pH of the reaction solution, thereby hindering the progress of isomerization. Consequently, suppressing pH reduction is expected to improve isomerization yields. Reactions conducted in phosphate buffer resulted in significantly higher rare sugar yields compared to those in pure water. One method to suppress pH reduction is the use of neutralizing agents. Montilla et al. [76] reported the production of lactulose from lactose in milk ultrafiltrate using eggshells, whose primary component is calcium carbonate and which also contains magnesium, phosphorus, and various trace elements. Eggshells serve as a source of calcium and have been utilized as food additives [77, 78].
Galactose was dissolved in distilled water at a concentration of 5% (w/w), and 500 mg of eggshell powder or constituent salts of eggshell (calcium carbonate, magnesium carbonate, or calcium phosphate) were added into the galactose solution. The solution was heated to 120°C for 120 min [79]. Figure 11 shows the changes in the fraction of remaining galactose, the yields of isomerization products, pH, and Ca2+ concentration over the reaction time during the treatment of galactose with and without eggshell powder at 120°C. When eggshell powder was added, the fraction of remaining galactose was 70.1 mol% after 120 min of reaction, and tagatose, talose, and sorbose were produced as isomerization products at 15.7 mol%, 1.6 mol%, and 1.9 mol%, respectively. Eggshell powder neutralized the organic acids formed and suppressed the pH decrease. The concentration of water-soluble calcium salts, which could be used as dietary supplements [80], reached 295 mg/L. In contrast, when no eggshell powder was added, 94.4 mol% of galactose remained and 3.4 mol% of tagatose was formed after 120 min of reaction, with no talose or sorbose produced. The efficacy of eggshell components (calcium carbonate, magnesium carbonate, and calcium phosphate) in facilitating the conversion of galactose into rare sugars was also examined. The results demonstrated that rare sugars were successfully produced during treatment with these components. Notably, magnesium carbonate significantly enhanced rare sugar yield; however, its addition also led to increased byproduct formation. Additionally, eggshell powder was reused across multiple treatment cycles. After three cycles, rare sugar yields exhibited minimal variation, yet the selectivity for rare sugars showed substantial improvement.
Changes in (, ) the fraction of remaining galactose (CS/CS0), (CP/CS0) the yields of (, ) tagatose, () talose, and () sorbose, (, ) pH, and (
) Ca2+ concentration over the reaction time during treatment of galactose (open symbols) with and (closed symbols) without eggshell powder at 120°C. The curves were empirically drawn.
The primary component of seashell is calcium carbonate, and an aqueous solution of calcium carbonate is slightly alkaline [81]. To investigate the efficacy of uncalcined seashell powder for the isomerization of aldo-saccharides into keto-saccharides, 50 mL of 0.2 mol/L aqueous solutions containing two aldo-pentoses (ribose and xylose), two aldo-hexoses (glucose and galactose), and two aldo-disaccharides (cellobiose and lactose) were prepared. These solutions, along with 0.5 g of uncalcined scallop shell powder, were placed in a pressure-resistant vessel and heated using a microwave oven at 700 W [82]. The reaction successfully converted the aldo-saccharides into the corresponding keto-saccharides: ribulose and xylulose, fructose and tagatose, and cellobiulose and lactulose. The yields of keto-saccharides were approximately 10% for pentoses, 18% for hexoses, and 25% for disaccharides within 105 s. The selectivity trend followed the order of disaccharides > hexoses > pentoses. Measurements of pH, calcium ion concentration, and absorbance at 280 nm and 420 nm confirmed that the acidic byproducts formed during heating were effectively neutralized by the scallop shell powder. This neutralization maintained the pH of the reaction mixture above 7, thereby facilitating isomerization via the LBAE transformation. Therefore, this approach provided an efficient and selective method for producing keto-saccharides from various aldo-saccharides, with the reaction parameters demonstrating a clear influence on yield and selectivity.
Uncalcined scallop shell powder and an aqueous maltose solution were placed in a pressure-resistant vessel and heated in a microwave oven to facilitate the isomerization of maltose to maltulose [83]. Heating 50 mL of a maltose solution at 700 W resulted in approximately 30% maltulose within 105 s. As the reaction progressed, the scallop shell powder dissolved, effectively neutralizing acidic byproducts and preventing a significant decrease in pH. This maintained the pH of the reaction mixture above 7, a range favorable for isomerization via the LBAE transformation. The average selectivity for the isomerization of maltose to maltulose under various reaction conditions was determined to be 0.822. This high selectivity can be attributed to the ability of the scallop shell powder to sustain the reaction pH within the optimal range of 7 to 9, thereby minimizing the occurrence of side reactions. Notably, the coloration of the reaction mixture intensified rapidly as the yield of maltulose approached its maximum value. As stated previously, this suggests that terminating the reaction at the point of maximum maltulose yield could produce a solution with both a high maltulose concentration and minimal coloration.
Several chemical methods for isomerizing common sugars (reducing sugars) into rare ones under subcritical water conditions were introduced. While the information on chemical equilibrium for each reaction remains insufficient, making it challenging to quantitatively evaluate the yield performance of rare sugars, promising chemical methods for their production are beginning to emerge. Most of the current studies on functional properties of rare sugars has been performed using the sugars with relatively low purity. Moving forward, it is essential to not only identify more efficient production methods and conditions but also to establish reliable separation and purification techniques. Subsequently, it will be necessary to advance to the stage where the physiological and physicochemical functions of each rare sugar with high purity are elucidated.
One of the authors (P.K.) acknowledges the JSPS BRIDGE Fellowship Program 2024 for supporting the visit to Osaka Metropolitan University during the preparation of this manuscript.
