Hydrolysis of disaccharides containing glucose residue in subcritical water

Biochemical Engineering Journal 18 (2004) 143–147

Hydrolysis of disaccharides containing glucose residue in subcritical water Toshinobu Oomori, Shabnam Haghighat Khajavi, Yukitaka Kimura, Shuji Adachi∗ , Ryuichi Matsuno Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan Received 6 June 2003; accepted 7 August 2003

Abstract The hydrolysis of disaccharides consisting of two glucose residues or of glucose and fructose or galactose residues in subcritical water was measured using a tubular reactor at 10 MPa and 180–260 ◦ C. The relationship between the fraction of remaining disaccharide and the residence time could be expressed by the Weibull equation for all the disaccharides tested at any temperature. The susceptibility to hydrolysis largely depended on the type of disaccharide, and it was found that the electrostatic potential charge of a glucosidic oxygen atom was an indication for the susceptibility of disaccharide to the hydrolysis. It was also shown that the enthalpy–entropy compensation held for the hydrolysis of disaccharides in subcritical water. © 2003 Elsevier B.V. All rights reserved. Keywords: Degradation; Disaccharides; Subcritical water; Weibull equation

1. Introduction Water that is in a liquid state under a pressurized condition at temperatures above its boiling point under ambient pressure and below the critical point is called subcritical water [1]. Subcritical water has properties different from water at room temperature and ambient pressure [2]: one is a low relative dielectric constant and another is a large ion product. The relative dielectric constant of subcritical water is comparable with that of methanol or acetone at ambient conditions. Due to this property, there are some reports on the use of subcritical water for the extraction of hydrophobic substances such as aromas from rosemary [3], environmental pollutants from soil [4], hydrocarbons from oil shales [5] and marjoram essential oil [6]. The latter property, a large ion product, means that the concentrations of hydrogen and hydroxide ions in subcritical water are high and that the subcritical water has the potential as a catalyst for the degradation of organic compounds. The hydrolysis of various compounds such as fish meat [7], plant oils [8] and ethyl acetate [9] has been reported. Carbohydrates are naturally occurring and renewable resources. Food and woody wastes abundantly contain car∗ Corresponding author. Tel.: +81-75-753-6286; fax: +81-75-753-6285. E-mail address: [email protected] (S. Adachi).

1369-703X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2003.08.002

bohydrates. The enzymatic [10] or acid [11] hydrolysis of waste carbohydrates to yield mono- or oligosaccharides that are resources potentially used as raw materials in many fields has been reported. Recently, attention has been paid to the hydrolysis or decomposition of carbohydrates in sub- or supercritical water [12–14]. Polysaccharides are composed of certain kinds of hexoses and pentoses that are bound to each other via many types of glycosidic bonds. Their susceptibility to hydrolysis in subcritical water would depend on the constituent monosaccharides and the type of glycosidic bond. In this context, the hydrolysis of disaccharides consisting of two glucose residues or of glucose and fructose or galactose residues via various glucosidic bonds in subcritical water was measured using a tubular reactor in the temperature range of 180–260 ◦ C at 10 MPa. The hydrolysis of a disaccharide in subcritical water would proceed in consecutive reactions: the disaccharide is first hydrolyzed to the constituent monosaccharides, and the produced monosaccharides are then degraded to 5-hydroxymethylfurfural and other compounds. Because only the disappearance of a disaccharide in subcritical water was observed throughout this study, the term hydrolysis is used to describe this disappearance. The kinetics for the hydrolysis of the disaccharides and the difference in the susceptibility to hydrolysis among the disaccharides are discussed.

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T. Oomori et al. / Biochemical Engineering Journal 18 (2004) 143–147

2. Materials and methods 2.1. Materials Maltose (␣-Glc(1 → 4)Glc) and lactose (␤-Gal(1 → 4)-Glc), where Glc and Gal represent glucose and galactose residues, respectively, were purchased from Nacalai Tesque, Kyoto, Japan. Cellobiose (␤-Glc(1 → 4)Glc), isomaltose (␣-Glc(1 → 6)Glc) and palatinose (␣-Glc(1 → 6)Fru), where Fru represents fructose, were purchased from Tokyo Chemical Industry, Tokyo, Japan. Gentiobiose (␤-Glc(1 → 6)Glc) and leucrose (␣-Glc(1 → 5)Fru) were obtained from Fulka, Buchs, Switzerland. Turanose (␣-Glc(1 → 3)Fru) and melibiose (␣-Gal(1 → 6)Glc) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sucrose (␣-Glc(1 → 2)␤-Fru) was purchased from Wako Pure Chemical Industries, Osaka, Japan. Trehalose (␣-Glc(1 → 1)Glc) was supplied by Hayashibara, Okayama, Japan. 2.2. Apparatus Fig. 1 illustrates the apparatus used in this study. A coiled stainless steel (SUS316) tube (0.8 mm i.d.) was immersed in an oil bath filled with SRX310 silicone oil (ca. 5.4 l; Toray Dow Corning Silicone, Tokyo) and in an iced water bath (ca. 4.8 l). The length of the tube immersed in the oil bath was 1 m or 6 m, which corresponded to 0.5 ml or 3.0 ml of the tubular reactor. The length of the tube immersed in the iced water bath was ca. 1 m. The tube was connected to an LC6A pump (Shimadzu, Kyoto) that fed the saccharide solution. A 26-1700 back-pressure valve (70 MPa maximum pressure, Tescom Corp., Elk River, MN, USA) was connected to the line after the cooling coil to control the pressure in the system. The pressure was regulated at 10 MPa. 2.3. Hydrolysis of disaccharide

to prevent re-dissolution of oxygen in the solution. The solution was fed by the pump at a constant flow rate to give a specific residence time in the reaction coil of 12–360 s. The residence time was calculated from the flow rate, the inner volume of the tubular reactor, and the water densities [15] at room and reaction temperatures. The temperature in the oil bath was regulated between 180 and 260 ◦ C. After about 10 residence times had elapsed, the effluent (ca. 1 ml) was sampled in a test tube. The concentration of the remaining disaccharide in the effluent was determined using an HPLC with a Supelcogel K column (7.5 mm i.d. × 300 mm; Supelco, Bellofonte, PA, USA) and a YRU-880 refractometer (Shimamuratech, Tokyo). The eluent used was 15 mmol/l K2 HPO4 , and its flow rate was 0.55 ml/min. The determination was repeated at least three times and averaged. 2.4. Evaluation of electrostatic potential charge of glucosidic oxygen atom The electrostatical potential charge of the glucosidic oxygen atom of each disaccharide was estimated using the molecular orbital calculation software, MOPAC 2000 (Fujitsu, Chiba, Japan). The options used in the calculation were PM3 (calculation type) and ESP (geometry optimization).

3. Results and discussion 3.1. Hydrolysis of disaccharides Fig. 2a shows the relationships between the fraction of the remaining disaccharide, C/C0 , and the residence time, τ, at 220 ◦ C and 10 MPa for glucobioses consisting of two glucose residues: C and C0 represent the disaccharide concentrations in the effluent and feed reservoir, respectively. 1.0

Each disaccharide was dissolved in distilled water at a concentration of 0.5% (w/v). The solution in a bottle (ca. 500 ml) was sonically degassed under reduced pressure, and the bottle was connected to a balloon filled with helium gas C / C0

(a)

(b)

0.5

0 0

Fig. 1. Schematic of the apparatus used: (1) reservoir of feed solution, (2) pump, (3) tubular reactor (coiled stainless steel tube), (4) oil bath, (5) cooling coil, (6) iced water bath, (7) back-pressure valve, and (8) test tube for collecting the effluent.

100

200

300 0 100 Residence time [s]

200

300

Fig. 2. Relationships between the fraction of remaining disaccharide, C/C0 , and the residence time, τ, in the tubular reactor at 220 ◦ C and 10 MPa for: (a) the disaccharides consisting of two glucose residues, and (b) those consisting of glucose and fructose or galactose residues. Symbols: (a) (䊐) cellobiose, () gentiobiose, (䉫) isomaltose, (䊊) maltose, and () trehalose; and (b) () lactose, (䉫) leucrose, ( ) melibiose, (䊐) palatinose, (䊊) sucrose, and () turanose. The curves were drawn according to the Weibull equation.

T. Oomori et al. / Biochemical Engineering Journal 18 (2004) 143–147

The resistance to hydrolysis of the disaccharides largely depended on the type of glucosidic bond. Threhalose with an ␣-1,1-glucosic bond was very resistant to hydrolysis in subcritical water. Gentiobiose with a ␤-1,6-glucosidic bond was the most fragile among the glucobioses tested. Isomaltose with an ␣-1,6-glucosidic bond was more resistant to hydrolysis than gentiobiose with a ␤-1,6-glucosidic bond. On the other hand, cellobiose with a ␤-1,4-glucosidic bond was more resistant than maltose with an ␣-1,4-glucosidic bond. Fig. 2b shows the relationships at 220 ◦ C for disaccharides consisting of glucose and galactose or fructose residues. There was a tendency for the disaccharides to be less resistant to the hydrolysis than glucobioses consisting of two glucose residues. Especially, sucrose was easily hydrolyzed at the short residence times. A kinetic expression for describing the fraction of remaining disaccharide, C/C0 , as a function of the residence time, τ, was considered. Although the first-order kinetics was roughly applicable to the relationship at short residence times for each disaccharide, the fractions at longer residence times did not obey the kinetics. The Weibull model given by Eq. (1) is flexible and has a potential for describing many degradation kinetics [16]. C = exp[−(kτ)n ] C0

(1)

where k is the rate constant, the reverse of which is called the scale constant, and n is the shape constant. The applicability of the equation to the relationship was examined. Eq. (1) can be rearranged as follows:
  C ln −ln = n(ln τ + ln k) (2) C0 Fig. 3 shows the plots of ln[−ln(C/C0 )] versus ln τ for each disaccharide. A linear regression was obtained for every disaccharide, indicating that the Weibull equation was applicable for describing the relationship. Although the

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parameters, k and n, can be obtained from the line, they were estimated based on a non-linear regression using the Origin Ver. 6.1 (Microcal Software Inc., Northampton, MA, USA). The curves in Fig. 2 were drawn using the k and n values estimated by the method. 3.2. A parameter correlating the rate constant As mentioned above, the susceptibility to hydrolysis was quite different among the disaccharides. There seemed to be no tendency for the ␣- or ␤-glucosidic bond or for the bond at a specific position to be more easily hydrolyzed. The first step in the cleavage of the glucosidic bond in subcritical water would be the attack of hydrogen or hydroxide ion on the glucosidic oxygen atom [17]. Therefore, we postulated that the electron density of the oxygen atom would correlate to the rate constant k at 220 ◦ C. The electrostatic potential charge of the oxygen atom, which was estimated using the molecular orbital calculation software, was used as an indication for the negativity of the atom. Fig. 4 shows the plots of the rate constant, k, and the shape constant, n, versus the electrostatic potential charge of the glucosidic oxygen atom. A tendency was found such that the k value was larger for the disaccharide with the more negatively charged oxygen atom. This would suggest that the positively charged hydrogen ion attacks the glucosidic oxygen atom to cleave the bond. The correlation of the k value to the charge enables us to predict the susceptibility of a disaccharide to hydrolysis by calculating the charge of the glucosidic oxygen atom of the disaccharide. For the disaccharides consisting of glucose and fructose residues, the n value was larger for the disaccharide, the glucosidic oxygen atom of which had a larger negativity. There was no tendency between the n value and the charge

10 –1

4

3

0

Pal

10

–2

Suc

Tur Mel Gen Mal Iso

Leu

2

n

k [s –1 ]

ln[–ln( C /C0 )]

2

Tre

Lac

1 Cel

–2

10 –3 –0.3

–4

(a) 2

3

4

5

(b) 2

3

4

5

0 –0.2

–0.1

0

Electrostatic potential charge 6

ln

Fig. 3. Applicability of the Weibull equation to the degradation of disaccharides at 220 ◦ C and 10 MPa. Symbols: (a) (䊐) cellobiose, () gentiobiose, (䉫) isomaltose, (䊊) maltose, and () trehalose; and (b) () lactose, (䉫) leucrose, ( ) melibiose, (䊐) palatinose, (䊊) sucrose, and () turanose. The residence time, τ, is in units of s.

Fig. 4. Relationships between (closed symbols) the rate constant, k, and (open symbols) the shape constant, n, in the Weibull equation, and the electrostatic potential charge of the glucosidic oxygen atom of the disaccharide. The k and n values were estimated at 220 ◦ C and 10 MPa. The symbols (䊉, 䊊), (䉱, ), and (䊏, 䊐) represent disaccharides consisting of: two glucose residues, glucose and fructose residues, and glucose and galactose residues, respectively. Disaccharides are represented by the first three letters of their names.

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T. Oomori et al. / Biochemical Engineering Journal 18 (2004) 143–147 10 –1

180 Tre

160 k [s –1 ]

E [kJ/mol]

10 –2

10 –3

(a) 1.9

Mal

2.1

2.2 1.9 2.0 10 3 / [K –1 ]

Mel Pal

Lac

2.1

2.2

2.3

Suc

80 15

Fig. 5. Arrhenius plots for the degradation of disaccharides consisting of: (a) two glucose residues, and (b) glucose and fructose or galactose residues at 10 MPa. Symbols: (a) (䊐) cellobiose, (䉫) isomaltose, (䊊) maltose, and () trehalose; and (b) () lactose, (䉫) leucrose, ( ) melibiose, (䊐) palatinose, (䊊) sucrose, and () turanose.

for the disaccharides consisting of two glucose residues or of glucose and galactose residues. 3.3. Enthalpy–entropy compensation in the hydrolysis The hydrolysis of disaccharides except of gentiobiose was observed at various residence times and at different temperatures. It could be expressed by the Weibull equation for every disaccharide at any temperature, and the k and n values were evaluated. Fig. 5 shows the plots of the rate constant, k, versus the reciprocal of the absolute temperature, 1/T. The temperature dependence of the rate constant could be expressed by the Arrhenius equation:   −E k = k0 exp (3) RT where E is the activation energy, k0 the frequency factor, and R the gas constant. The E and k0 values for each disaccharide were evaluated from the plots shown in Fig. 5. The activation energy of trehalose that was shown in Fig. 2 to be the most resistant to hydrolysis among the tested disaccharides was the highest and that of the most fragile sucrose was the lowest. As shown in Fig. 6, there was a linear relationship between the E value and the logarithm of the k0 value: E = RT␤ ln k0 + γ

Leu

100

(b) 2.0

Cel

120

10 –4 1.8

Tur

Iso

140

(4)

where T␤ is a parameter called the isokinetic temperature and γ is a constant. Eq. (4) is one of the expressions describing the enthalpy–entropy compensation [18,19]. The linear relationship shown in Fig. 6 revealed that the enthalpy–entropy compensation held for the hydrolysis of disaccharides in subcritical water and indicated that the hydrolysis essentially proceeded by the same reaction mechanism. The T␤ was evaluated to be 536 K (263 ◦ C) from the slope of the line. The rate constant should be common for all the disaccharides at this temperature.

20

25 ln 0

30

35

Fig. 6. Enthalpy–entropy compensation for the degradation of disaccharides by subcritical water at 10 MPa. E and k0 are the activation energy in units of kJ/mol and the frequency factor in units of s−1 , respectively, for the rate constant k of the Weibull equation. The symbols and labels are the same as in Fig. 4.

4. Conclusion The susceptibility of a disaccharide to hydrolysis in subcritical water largely depended on the constituent monosaccharides and the type of glucosidic bond. Threhalose, ␣-1,1-glucosylglucoside, was the most resistant to the hydrolysis while sucrose, ␤-2,1-glucosylfructoside, was the most easily hydrolyzed among the 11 tested disaccharides. The hydrolysis process was expressed by the Weibull equation with two parameters for every disaccharide at any temperature. It was demonstrated that the rate constant at 220 ◦ C could be correlated to the electrostatic potential charge of the glucosidic oxygen atom. The temperature dependence of the rate constant was expressed by the Arrhenius equation for every disaccharide, and it was shown that the enthalpy–entropy compensation held for the hydrolysis of the disaccharides in subcritical water.

Acknowledgements Part of this study was financially supported through Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. S.H.K. gratefully acknowledges a Monbukagakusho Scholarship from the Japanese government. References [1] P. Krammer, H. Vogel, Hydrolysis of esters in subcritical and supercritical water, J. Supercrit. Fluids 16 (2000) 189–206. [2] T. Clifford, Fundamentals of Supercritical Fluids, Oxford University Press, New York, 1998, pp. 22–23. [3] A. Basile, M.M. Jiménez-Carmona, A.A. Clifford, Extraction of rosemary by superheated water, J. Agric. Food Chem. 46 (1998) 5205–5209.

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