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Unfrozen water in amylosic molecules is dependent on the molecular structures—A differential scanning calorimetric study
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HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 862–867 www.elsevier.com/locate/foodhyd
Unfrozen water in amylosic molecules is dependent on the molecular structures—A differential scanning calorimetric study Shiho Suzuki, Shinichi Kitamura Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Gakuencho Nakaku, Sakai 599-8531, Japan Received 28 August 2006; accepted 6 April 2007
Abstract The amount of unfrozen water (UFW) associated with linear and cyclic (1-4)-a-D-glucans and oligomers thereof, including maltopentaose, two enzymatically synthesized amyloses; A-55 (Mw ¼ 54,500) and A-820 (Mw ¼ 820,000), three cyclodextrins; a-CD, b-CD and g-CD and two cycloamyloses; CA-5 (Mw ¼ 4700) and CA-13 (Mw ¼ 13,100) were determined by differential scanning calorimetry (DSC). UFW, estimated from a plot of the melting enthalpy of ice against the weight fraction of water, showed that the glucans examined contain surprisingly different amounts of UFW. The molar ratio of UFW per glucose residue was increased in the order of a-CD (1.24), b-CD (1.78), g-CD (1.96), A-55 (2.77), A-820 (2.98), CA-5 (2.93) and CA-13 (3.52). These values can be interpreted in terms of their hydrated conformational structures. r 2007 Elsevier Ltd. All rights reserved. Keywords: Unfrozen water; Amylose; Cycloamylose; Cyclodextrin; Differential scanning calorimetry; Bound water; Hydration.
1. Introduction In a solute–water system, some water molecules interact more closely with solutes. This hydrated water is generally referred to as ‘‘bound water’’ (Nakamura, Hatakeyama, & Hatakeyama, 1981; Wootton & Bamunuarachchi, 1978). When a solute–water system is subjected to subzero temperatures, a portion of the water does not freeze out as ice. Such unfrozen water (UFW) associated with bound water, is considered to be water molecules that strongly interact with the solute through hydrogen bonding, although they may not be totally immobilized or bound (Franks, 1986; Li, Dickinson & Chinachoti, 1998). Differential scanning calorimetry (DSC) has been used to study the state or the amount of bound water for some glucans, including malto-oligosaccharides (Schenz, Courtney, & Israel, 1993), starch (Chung, Lee, & Lim, 2002; Liu & Lelievre, 1992; Tananuwong & Reid, 2004; Wootton & Bamunuarachchi, 1978), dextran (Murase, Shiraishi, Koga, & Gonda, 1982), cellulose (Nakamura et al., 1981) and other polymers and oligomers (Higuchi & Iijima, 1985; Corresponding author. Tel.: +81 72 254 9457; fax: +81 72 254 9458.
E-mail address: [email protected] (S. Kitamura). 0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2007.04.011
Ichikawa, Mitsumura, & Nakajima, 1994; Liesebach, Rades, & Lim, 2003; Murase, Fujita, & Gonda, 1983; Murase, Inoue, & Ruike, 1997; Murase, 1993; Nakamura, Hatakeyama, & Hatakeyama, 1991). For a-(1-4)-linked glucans, previous studies have shown that the amount of bound water in starch–water systems varies, depending on the source of the starch (Ishida, Kobayashi, & Keiji, 1988; Wootton & Bamunuarachchi, 1978). The gelatinization of natural starch (Tananuwong & Reid, 2004) or the retrogradation of a starch gel (Ishida et al., 1988) increases the UFW. This shows that the gel structure, which undergoes disruption or the restructuring of the crystal part reflects, not only conformational change in the (1-4)-a-D-glucans, but also the state of the water molecules. The objective of this study was to estimate the amount of UFW and analyze the state of water molecules for linear and cyclic a-(1-4)-linked glucans and its oligomers thereof using DSC. 2. Materials and methods 2.1. Materials Two amylose samples; A-55 and A-820 were enzymatically synthesized by a previously reported method using
ARTICLE IN PRESS S. Suzuki, S. Kitamura / Food Hydrocolloids 22 (2008) 862–867
potato phosphorylase (EC 2.4.1.1.) (Kitamura, Yunokawa, Mitsuie, & Kuge, 1982; Nakanishi, Norisuye, Teramoto, & Kitamura, 1993). The number- and weight-average molecular weights (Mn, Mw) and molecular weight distributions were evaluated by high-performance gel permeation chromatography (GPC) using a low-angle laser-light-scattering photometer (LALLS; LS8000; Tosoh Co., Ltd., Japan) and a differential refractometer (RI; RI-8011; Tosoh Co., Ltd.) as detectors. Amylose was dissolved in 0.25 M aqueous CH3COOK at a concentration of 1% and injected onto GPC columns that were thermostatically controlled at 55 1C. Three sequentially connected columns of G2000PW, G3000PW and G5000PW (Tosoh Co., Ltd., Japan) were used for A-55. For high molecular weight amylose; A-820, a column combination of G3000PW, G5000PW and G6000PW was used. The flow rate was 0.7 mL/min for both column systems. The LALLS photometer used in this system measures scattering intensity at y ¼ 51, where the correction for angular dependence to y ¼ 0 can be ignored. The RI and LALLS signals at each point of the chromatogram were analyzed by a software package provided by the Tosoh Co., Ltd., to determine Mw, Mn and molecular weight distribution curves. a- and g-CDs were provided by the Nihon Shokuhin Kako Co., Ltd. (Japan). Maltopentaose and b-CD were provided by Nacalai Tesque, Inc. (Japan). Mixtures of large-ring cyclodextrin (denoted as cycloamylose in this paper), CA-5 and CA-13 were produced by the action of recombinant Thermus aquaticus amylomaltase (4-a-glucanotrasferase; EC 2.4.1.25) on a synthetic linear amylose sample A-480 (Mw ¼ 482,000) according to the method reported previously (Terada, Fujii, Takaha, & Okada, 1999). The enzyme was kindly provided by the Ezaki Glico Co., Ltd. (Japan). The reaction time for CA-5 and CA-13 were 24 and 1 h, respectively. The enzyme was deactivated and removed by centrifugation. CA-5 was prepared which was phase separated as the dilute phase at 67 vol% ethanol and the concentrated phase at 80 vol% ethanol. CA-13 was precipitated with 50 vol% ethanol. The average Mw of cycloamyloses was determined by highperformance liquid chromatography with a charged aerosol detection (CAD) system (ESA Inc., MA) (Dixon & Peterson, 2002; Gamache et al., 2005). Cycloamyloses were dissolved in solvent at a concentration of 0.36% or 0.625% and injected into Cadenza CD-C18 column (Imtakt Corporation, Japan) that were thermostatically controlled at 35 1C. The flow rate was 0.5 mL/min.
Table 1 Amyloses and cycloamyloses used in this study
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Weight–average molecular weights (Mws) for amyloses and cycloamyloses are shown in Table 1. Prior to the DSC measurements, each sample was kept in the desiccator at room temperature for 1 week. The water content in each sample was estimated from weight loss of sample after drying at 110 1C until a constant weight was reached. 2.2. DSC measurements DSC measurements were performed with a Micro DSC VII (Setaram, France) in most cases at a scan rate of 0.5 K/min. For pure water, the onset temperature and melting enthalpy of ice were determined, respectively to be 0.0170.01 1C and 335.0170.95 J/g (n ¼ 3), as expected for the melting of ice (see also Fig. 1A). Nine milligrams of purified water was placed in the sample cell, the sample added with requirements for each concentration. It was then heated to 120 1C and kept at 120 1C for 20 min, to produce a fully swollen sample. It was then cooled to 45 1C and kept for 1 h to form crystal ice. It was then heated to 20 1C, to measure the change in heat flow for the melting of ice. An empty cell was used as reference. The total water content, expressed as the weight fraction of water (WH), was calculated from the sum of the added water (g) and the water content in glucan sample (g). In this study, we used the temperature at the heat capacity minima: Tp as the melting temperature of ice for frozen water. The amount of UFW was estimated from a plot of melting enthalpy against WH, as described for starches (Wootton & Bamunuarachchi, 1978) or collagen (Haly & Snaith, 1971). The value of WH indicating the amount of UFW was determined at the point of zero enthalpy where the extrapolated fitted line intercepted the x-axis. 2.3. X-ray diffraction X-ray diffraction patterns were obtained for two amylose samples before and after the DSC measurements. For the swollen sample with WH 0.5 after DSC, it was dried in a desiccator at room temperature for 1 week. Each sample was hermetically sealed up in a silica capillary tube. X-ray powder diffraction patterns were recorded by means of a flat film camera under vacuum with a Geigerflex X-ray diffractometer (Rigaku, Japan) using Ni-filtered CuKa radiation generated at 40 kV and exposured at 15 mA for 1 h. The measured 2y was corrected using an internal reference of NaF (2y ¼ 38.831). 3. Results and discussion
Sample
Mw
Mw/Mn
Amylose
A-55 A-820
54,500 815,000
1.06 1.05
Cycloamylose
CA-5 CA-13
4700 13,100
1.01 1.03
3.1. The amount of UFW The DSC curves for each system with various WH are shown in Fig. 1. Although a typical sharp peak at 0 1C can be seen in the DSC curve at higher water content, the peak is reduced in size and increased in breadth with decreasing WH.
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A
-30
B
-20
-10
0
10 -30
C
-20
-10
0
10 -30
E
-10
0
10
-20
-10
0
10
F
Endothermic heat flow
D
-20
-30
-20
-10
0
G
-30
10 -30
-20
-10
0
10 -30
-20
-10
0
10
H
-20
-10
0
10 -30
Temperature(°C) Fig. 1. DSC curves for the melting of ice in linear and cyclic (1-4)-a-D-glucans and their oligomer—water systems with various weight fractions of water (WHo1.0) indicated in the figure. Maltopentaose, A; A-55, B; A-820, C; a-CD, D; b-CD, E; g-CD, F; CA-5, G and CA-13, H. Bars indicating difference of heat capacity; 100 JK1g1.
The endothermic peaks disappeared at lower WH for all systems, indicating that a certain amount of water is present as UFW. The melting enthalpy, calculated from the peak area, is plotted against WH in Fig. 2. Values for the molar ratio of UFW per glucose residue, determined from
the x-intercept of the plot, are listed in Table 2. These show different values ranging from 1.24 for a-CD to 3.52 for CA-13. For maltopentaose, no data are listed because the DH could not be obtained below WH 0.250, due to the absence of ice formation under these experimental conditions.
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300
A
ΔH / Jg-1
200
100
0 300
B
ΔH / Jg-1
200
100
0
0.0
0.2
0.4
0.6
0.8
1.0
WH Fig. 2. Correlation between the heat of fusion (DH) and the weight fraction of water (WH) in glucan–water systems. (A) Amyloses and cycloamyloses: CA-5, –&–; CA-13, –n–; A-55, –’–; and A-820, –K–. (B) Cyclodextrins: a-CD, –n–; b-CD, –J–; and g-CD, –’–.
Table 2 Amount of UFW in linear and cyclic glucans Sample
UFWa g H2O/g dry glucan
Molar ratio of H2O/ glucose residue
Linear A-55 A-820
0.307 0.332
2.77 2.98
Cyclic a-CD b-CD g-CD CA-5 CA-13
0.137 0.198 0.218 0.326 0.391
1.24 1.78 1.96 2.93 3.52
a
Determined by extrapolated value of the DH against WH.
For amylose, A-55 contains less UFW than A-820. This might be the result of differences in the gel structure. X-ray diffraction patterns of A-55 before DSC showed a V-type with d-spacings (d) of the major peaks, 1.180, 0.659, 0.431 nm. In contrast, after DSC at WH 0.5, it showed a
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B-type with d ¼ 1.625, 0.512, 0.377 nm, indicating that it contains a B-type crystalline component. For A-55, a junction zone constructed from the double helix after gelatinization may reduce the number of amylose hydroxyl groups which are available for solute–solute interactions. A-820 was found to be almost amorphous before as well as after the DSC, since no remarkable diffraction peak was observed by X-ray analysis. More amylose hydroxyl groups are exposed to solvent in this case, resulting in increased amylose–water interactions. This structural difference might be related to the rate of aggregation of amylose chains, which is strictly dependent on the molecular weight (Gidley, 1989; Gidley & Bulpin, 1989; Kitamura, 1996). Amylose with a higher molecular weight is soluble in water and fairly stable for long periods of time (Kitamura, Hakozaki, & Kuge, 1994). A-55 became entangled during the cooling process in DSC, and formed a partially crystallized matrix, while A-820 was aggregated very slightly. The UFW for all cyclodextrins was lower than that for the other samples. Fig. 2B shows that the DH around WH ¼ 0.5 shows irregular behavior. This can be attributed to the crystallization of the cyclodextrins in systems with WHo0.5. This resulted in a small amount of unfrozen matrix. UFW is similar but slightly higher than the reported value of crystallized water for cyclodextrin hydrates, a-CD 6H2O, b-CD 11H2O and g-CD 16H2O (Saenger, Niemann, Herbst, Hinrichs, & Steiner, 1993). In this cyclodextrin crystallized system, UFW may be correlated with crystallized water for cyclodextrin hydrates. Cycloamylose, CA-5 and CA-13 have a relatively large amount of UFW compared to cyclodextrins, suggesting that it contains a large number of hydrated water molecules in the three dimensional network of the polymer chain. Cycloamylose is much more soluble than cyclodextrin (Ueda, Wakisaka, Nagase, Takaha, & Okada, 2002b; Ueda, 2002a) and is stable in aqueous solution. Cycloamylose has been proposed to have an extended conformation with more flexibility in aqueous solution (Kitamura, et al., 1997; Shimada, Kaneko, Takada, Kitamura, & Kajiwara, 2000). Their model for cycloamylose, calculated by molecular dynamics, indicates that approximately 4.6 water–glucose hydrogen bonds are formed per glucose unit. UFW may be correlated with its high water hydration properties. 3.2. Freezing point depression of water The freezing point depression of water was observed with decreasing water content for all systems (Fig. 3). This was analyzed using the Flory–Huggins theory (Flory, 1953) for polymer solutes and the interaction parameters, m were determined from the correlation between Tp and the volume fraction of the polymer, v2 by curve fitting using the following equation (Kuntz & Kauzmann, 1974): RT 0m lnð1 v2 Þ þ v2 þ mv22 DT m ffi , DH m T 0m
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3.3. Recrystallization of water
Tp / °C
0
-10
0.0
0.2
0.4
0.0
0.2
0.4
0.0
0.2
0.4
ν2
0.6
0.8
1.0
Tp / °C
0
-10
ν2
0.6
0.8
1.0
0.6
0.8
1.0
Tp / °C
0
-10
ν2
Fig. 3. The freezing point depression of water in linear and cyclic glucan–water systems. (A) Amyloses and maltopentaose, maltopentaose, –’–; A-55, –n–; and A-820, –*–. (B) Cycloamyloses: CA-5, –’–; and CA-13, –n–. (C) Cyclodextrins: a-CD, –}–; b-CD, –’–; and g-CD, –n–.
where DTm is the depression in the freezing point, T0m the normal freezing point of the solvent and DHm the heat of fusion of the solvent. It was found that (1-3)-b-D-glucans such as curdlan and schizophyllan show a good fit to this relationship (unpublished data). In contrast to these (1-3)b-D-glucans, the line does not fit well for amylose. This may result from the non-equilibrium system of such molecules under these experimental conditions. A larger depression with a low molecular weight was observed for maltopentaose, compared to A-55 and A-820 using the equation. For all cyclodextrins, Tp shows one high temperature at around WH 0.5. Since cyclodextrin with a WH below 0.5 is crystalline, an increase in freezable water may result in a high Tp. The depression width of b-CD is small.
For all systems, except for b-CD, a small exothermic peak was observed just below Tp, which can be attributed to the recrystallization (devitrification) of water (Fig. 1). Although it was reported in a rapid cooled system (Levine & Slade, 1991), it can also be observed in these systems, in which a slow cooling rate was used, 0.5 K/min. This depends on water content and is apparently observed in systems with about half the weight fraction of water. It is noteworthy that the exothermic peak goes over the baseline on the DSC curve for A-55 at WH 0.531 and A-820 at WH 0.534. This peak for A-55 is larger than that for A-820. Recrystallization occurs immediately after the glass transition during the heating process. This appears to be due to the increased mobility of polymers, which induce ice formation. It may relate to water which remains unfrozen in a nonequilibrium system, and, thus, would be affected by the time scale and thermal history. Annealing at near the Tg results in a smaller exothermic peak and UFW decreases due to new ice formation (Tananuwong & Reid, 2004). The effect of network size was proposed in a study of cross-linked dextran–water systems (Murase et al., 1982). The conformation of amylose chains changed at the time scale considered and the state of water molecules also changed. b-CD showed a single monotonic peak, which shows no apparent evidence of a glass transition or recrystallization. The solubility of b-CD in aqueous solution at 25 1C is less than that of a- and g-CD (Ueda, 2002b). Crystallization may reduce the extent of hydration between solute—water interactions. This study shows that the values of UFW appear to be related to the conformational structure of the amylosic molecules. UFW is affected, not only by the degree of polymerization, but also by the linear or ring structure. Especially for cyclic molecules, CA-13 has a remarkably higher UFW compared with the linear form, indicating a high hydration property. UFW for a series of cyclodextrins is low, indicating that it undergoes crystallization. A DSC study for UFW is useful to know the total state of amylosic chain, including its conformational structure, solute–solute interaction and hydration to water molecules, especially in concentrated solutions or gels. Lastly, it should be noted that the current results are useful for mutual comparison of UFW for several types of amylosic molecules, but not for the determination of the absolute amount of bound water for the individual amylosic molecules measured here. It is difficult to establish to what extent the UFW determined by DSC reflects kinetic effects or actual binding with local water properties, distinct from the bulk water.
Acknowledgments We are very grateful to Dr. K. Fujii of the Ezaki Glico Co., Ltd. for providing amylomaltases. This work was
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supported by grant for the Bio-oriented Technology Research Advancement Institution. References Chung, H. J., Lee, E. J., & Lim, S. T. (2002). Comparison in glass transition and enthalpy relaxation between native and gelatinized rice starches. Carbohydrate Polymers, 48, 287–298. Dixon, R. W., & Peterson, D. S. (2002). Development and testing of a detection method for liquid chromatography based on aerosol charging. Analytical Chemistry, 74, 2930–2937. Flory, P. J. (1953). Principles of polymer chemistry. New York: Cornell University Press. Franks, F. (1986). Unfrozen water—yes—unfreezable water—hardly— bound water—certainly not. Cryo-Letters, 7, 207. Gamache, P. H., McCarthy, R. S., Freeto, S. M., Asa, D. J., Woodcock, M. J., Laws, K., et al. (2005). HPLC analysis of nonvolatile analytes using charged aerosol detection. LCGC Europe, 18, 345–354. Gidley, M. J. (1989). Molecular mechanisms underlying amylose aggregation and gelation. Macromolecules, 22, 351–358. Gidley, M. J., & Bulpin, P. V. (1989). Aggregation of amylose in aqueous systems—the effect of chain-length on phase-behavior and aggregation kinetics. Macromolecules, 22, 341–346. Haly, A. R., & Snaith, J. W. (1971). Calorimetry of rat tail tendon collagen before and after denaturation: The heat of fusion of its absorbed water. Biopolymers, 10, 1681–1699. Higuchi, A., & Iijima, T. (1985). DSC investigation of the states of water in poly (vinyl-alcohol) membranes. Polymer, 26, 1207–1211. Ichikawa, T., Mitsumura, Y., & Nakajima, T. (1994). Water-sorption properties of poly(epsilon-lysine)—carboxymethyl cellulose (cmc) dietary complex films. Journal of Applied Polymer Science, 54, 105–112. Ishida, N., Kobayashi, T., & Keiji, K. (1988). Studies on the physical state of water in starch gel. Nippon shokuhin kogyo gakkaishi, 35, 98–104. Kitamura, S. (1996). Starch polymers, natural and synthetic. In J. C. Salamone (Ed.), The polymeric materials encyclopedia, synthesis, properties and applications, Vol. 10 (pp. 7915–7922). Boca Raton: CRC Press. Kitamura, S., Hakozaki, K., & Kuge, T. (1994). Effects of molecular weight on the retrogradation of amylose. In K. Nishinari, & E. Doi (Eds.), Food Hydrocolloids: Structures, Properties, and Functions (pp. 179–182). New York: Plenum Press. Kitamura, S., Isuda, H., Shimada, J., Takada, T., Takaha, T., Okada, S., et al. (1997). Conformation of cyclomaltooligosaccharide (‘‘cycloamylose’’) of dp21 in aqueous solution. Carbohydrate Research, 304, 303–314. Kitamura, S., Yunokawa, H., Mitsuie, S., & Kuge, T. (1982). Study on polysaccharide by the fluorescence method. II. Micro-Brownian motion and conformational change of amylose in aqueous solution. Polymer Journal (Tokyo, Japan), 14, 93–99. Kuntz, I. D., Jr., & Kauzmann, W. (1974). Hydration of proteins and polypeptides. Advances in protein chemistry, 28, 239–345. Levine, H., & Slade, L. (1991). Polymer physicochemical characterization of oligosaccharides. In R. B. Friedman (Ed.), Biotechnology of Amylodextrin Oligosaccharides (pp. 219–260). Washington, DC: American Chemical Society.
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Li, S., Dickinson, L. C., & Chinachoti, P. (1998). Mobility of ‘‘unfreezable’’ and ‘‘freezable’’ water in waxy corn starch by 2H and 1 H NMR. Journal of Agricultural and Food Chemistry, 46, 62–71. Liesebach, J., Rades, T., & Lim, M. (2003). A new method for the determination of the unfrozen matrix concentration and the maximal freeze-concentration. Thermochimica Acta, 401, 159–168. Liu, H., & Lelievre, J. (1992). Transitions in frozen gelatinized-starch systems studied by differential scanning calorimetry. Carbohydrate Polymers, 19, 179–183. Murase, N. (1993). Origin of an endothermic trend observed prior to ice crystallization exotherm in the dsc rewarming trace for polymer gels. Cryo-Letters, 14, 367–376. Murase, N., Fujita, T., & Gonda, K. (1983). Low-temperature calorimetric studies of compartmentalized water in hydrogel systems. II. Cryo-Letters, 4, 19–22. Murase, N., Inoue, T., & Ruike, M. (1997). Equilibrium and nonequilibrium freezing of water in cross linked dextran gels. Cryo-Letters, 18, 157–164. Murase, N., Shiraishi, M., Koga, S., & Gonda, K. (1982). Lowtemperature calorimetric studies of compartmentalized water in hydrogel systems. I. Cryo-Letters, 3, 251–254. Nakamura, K., Hatakeyama, T., & Hatakeyama, H. (1981). Studies on bound water of cellulose by differential scanning calorimetry. Textile Research Journal, 51, 607–613. Nakamura, K., Hatakeyama, T., & Hatakeyama, H. (1991). Formation of the glassy state and mesophase in the water–sodium alginate system. Polymer Journal, 23, 253–258. Nakanishi, Y., Norisuye, T., Teramoto, A., & Kitamura, S. (1993). Conformation of amylose in dimethyl-sulfoxide. Macromolecules, 26, 4220–4225. Saenger, W., Niemann, C., Herbst, R., Hinrichs, W., & Steiner, T. (1993). Crystal-structures of linear and cyclic oligosaccharides—an overview. Pure and Applied Chemistry, 65, 809–817. Schenz, T. W., Courtney, K., & Israel, B. (1993). Studies on the magnitude of the glass transitions of glucose polymers. Cryo-Letters, 14, 91–96. Shimada, J., Kaneko, H., Takada, T., Kitamura, S., & Kajiwara, K. (2000). Conformation of amylose in aqueous solution: Small-angle X-ray scattering measurements and simulations. Journal of Physical Chemistry B, 104, 2136–2147. Tananuwong, K., & Reid, D. S. (2004). Differential scanning calorimetry study of glass transition in frozen starch gels. Journal of Agricultural and Food Chemistry, 52, 4308–4317. Terada, Y., Fujii, K., Takaha, T., & Okada, S. (1999). Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: Production of cycloamylose. Applied and Environmental Microbiology, 65, 910–915. Ueda, H. (2002a). Physicochemical properties and complex formation abilities of large-ring cyclodextrins. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 44, 53–56. Ueda, H., Wakisaka, M., Nagase, H., Takaha, T., & Okada, S. (2002b). Physicochemical properties of large-ring cyclodextrins (CD18-CD21). Journal of Inclusion Phenomena and Macrocyclic Chemistry, 44, 403–405. Wootton, M., & Bamunuarachchi, A. (1978). Water binding capacity of commercially produced native and modified starches. Staerke, 30, 306–309.
FOOD
HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 862–867 www.elsevier.com/locate/foodhyd
Unfrozen water in amylosic molecules is dependent on the molecular structures—A differential scanning calorimetric study Shiho Suzuki, Shinichi Kitamura Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Gakuencho Nakaku, Sakai 599-8531, Japan Received 28 August 2006; accepted 6 April 2007
Abstract The amount of unfrozen water (UFW) associated with linear and cyclic (1-4)-a-D-glucans and oligomers thereof, including maltopentaose, two enzymatically synthesized amyloses; A-55 (Mw ¼ 54,500) and A-820 (Mw ¼ 820,000), three cyclodextrins; a-CD, b-CD and g-CD and two cycloamyloses; CA-5 (Mw ¼ 4700) and CA-13 (Mw ¼ 13,100) were determined by differential scanning calorimetry (DSC). UFW, estimated from a plot of the melting enthalpy of ice against the weight fraction of water, showed that the glucans examined contain surprisingly different amounts of UFW. The molar ratio of UFW per glucose residue was increased in the order of a-CD (1.24), b-CD (1.78), g-CD (1.96), A-55 (2.77), A-820 (2.98), CA-5 (2.93) and CA-13 (3.52). These values can be interpreted in terms of their hydrated conformational structures. r 2007 Elsevier Ltd. All rights reserved. Keywords: Unfrozen water; Amylose; Cycloamylose; Cyclodextrin; Differential scanning calorimetry; Bound water; Hydration.
1. Introduction In a solute–water system, some water molecules interact more closely with solutes. This hydrated water is generally referred to as ‘‘bound water’’ (Nakamura, Hatakeyama, & Hatakeyama, 1981; Wootton & Bamunuarachchi, 1978). When a solute–water system is subjected to subzero temperatures, a portion of the water does not freeze out as ice. Such unfrozen water (UFW) associated with bound water, is considered to be water molecules that strongly interact with the solute through hydrogen bonding, although they may not be totally immobilized or bound (Franks, 1986; Li, Dickinson & Chinachoti, 1998). Differential scanning calorimetry (DSC) has been used to study the state or the amount of bound water for some glucans, including malto-oligosaccharides (Schenz, Courtney, & Israel, 1993), starch (Chung, Lee, & Lim, 2002; Liu & Lelievre, 1992; Tananuwong & Reid, 2004; Wootton & Bamunuarachchi, 1978), dextran (Murase, Shiraishi, Koga, & Gonda, 1982), cellulose (Nakamura et al., 1981) and other polymers and oligomers (Higuchi & Iijima, 1985; Corresponding author. Tel.: +81 72 254 9457; fax: +81 72 254 9458.
E-mail address: [email protected] (S. Kitamura). 0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2007.04.011
Ichikawa, Mitsumura, & Nakajima, 1994; Liesebach, Rades, & Lim, 2003; Murase, Fujita, & Gonda, 1983; Murase, Inoue, & Ruike, 1997; Murase, 1993; Nakamura, Hatakeyama, & Hatakeyama, 1991). For a-(1-4)-linked glucans, previous studies have shown that the amount of bound water in starch–water systems varies, depending on the source of the starch (Ishida, Kobayashi, & Keiji, 1988; Wootton & Bamunuarachchi, 1978). The gelatinization of natural starch (Tananuwong & Reid, 2004) or the retrogradation of a starch gel (Ishida et al., 1988) increases the UFW. This shows that the gel structure, which undergoes disruption or the restructuring of the crystal part reflects, not only conformational change in the (1-4)-a-D-glucans, but also the state of the water molecules. The objective of this study was to estimate the amount of UFW and analyze the state of water molecules for linear and cyclic a-(1-4)-linked glucans and its oligomers thereof using DSC. 2. Materials and methods 2.1. Materials Two amylose samples; A-55 and A-820 were enzymatically synthesized by a previously reported method using
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potato phosphorylase (EC 2.4.1.1.) (Kitamura, Yunokawa, Mitsuie, & Kuge, 1982; Nakanishi, Norisuye, Teramoto, & Kitamura, 1993). The number- and weight-average molecular weights (Mn, Mw) and molecular weight distributions were evaluated by high-performance gel permeation chromatography (GPC) using a low-angle laser-light-scattering photometer (LALLS; LS8000; Tosoh Co., Ltd., Japan) and a differential refractometer (RI; RI-8011; Tosoh Co., Ltd.) as detectors. Amylose was dissolved in 0.25 M aqueous CH3COOK at a concentration of 1% and injected onto GPC columns that were thermostatically controlled at 55 1C. Three sequentially connected columns of G2000PW, G3000PW and G5000PW (Tosoh Co., Ltd., Japan) were used for A-55. For high molecular weight amylose; A-820, a column combination of G3000PW, G5000PW and G6000PW was used. The flow rate was 0.7 mL/min for both column systems. The LALLS photometer used in this system measures scattering intensity at y ¼ 51, where the correction for angular dependence to y ¼ 0 can be ignored. The RI and LALLS signals at each point of the chromatogram were analyzed by a software package provided by the Tosoh Co., Ltd., to determine Mw, Mn and molecular weight distribution curves. a- and g-CDs were provided by the Nihon Shokuhin Kako Co., Ltd. (Japan). Maltopentaose and b-CD were provided by Nacalai Tesque, Inc. (Japan). Mixtures of large-ring cyclodextrin (denoted as cycloamylose in this paper), CA-5 and CA-13 were produced by the action of recombinant Thermus aquaticus amylomaltase (4-a-glucanotrasferase; EC 2.4.1.25) on a synthetic linear amylose sample A-480 (Mw ¼ 482,000) according to the method reported previously (Terada, Fujii, Takaha, & Okada, 1999). The enzyme was kindly provided by the Ezaki Glico Co., Ltd. (Japan). The reaction time for CA-5 and CA-13 were 24 and 1 h, respectively. The enzyme was deactivated and removed by centrifugation. CA-5 was prepared which was phase separated as the dilute phase at 67 vol% ethanol and the concentrated phase at 80 vol% ethanol. CA-13 was precipitated with 50 vol% ethanol. The average Mw of cycloamyloses was determined by highperformance liquid chromatography with a charged aerosol detection (CAD) system (ESA Inc., MA) (Dixon & Peterson, 2002; Gamache et al., 2005). Cycloamyloses were dissolved in solvent at a concentration of 0.36% or 0.625% and injected into Cadenza CD-C18 column (Imtakt Corporation, Japan) that were thermostatically controlled at 35 1C. The flow rate was 0.5 mL/min.
Table 1 Amyloses and cycloamyloses used in this study
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Weight–average molecular weights (Mws) for amyloses and cycloamyloses are shown in Table 1. Prior to the DSC measurements, each sample was kept in the desiccator at room temperature for 1 week. The water content in each sample was estimated from weight loss of sample after drying at 110 1C until a constant weight was reached. 2.2. DSC measurements DSC measurements were performed with a Micro DSC VII (Setaram, France) in most cases at a scan rate of 0.5 K/min. For pure water, the onset temperature and melting enthalpy of ice were determined, respectively to be 0.0170.01 1C and 335.0170.95 J/g (n ¼ 3), as expected for the melting of ice (see also Fig. 1A). Nine milligrams of purified water was placed in the sample cell, the sample added with requirements for each concentration. It was then heated to 120 1C and kept at 120 1C for 20 min, to produce a fully swollen sample. It was then cooled to 45 1C and kept for 1 h to form crystal ice. It was then heated to 20 1C, to measure the change in heat flow for the melting of ice. An empty cell was used as reference. The total water content, expressed as the weight fraction of water (WH), was calculated from the sum of the added water (g) and the water content in glucan sample (g). In this study, we used the temperature at the heat capacity minima: Tp as the melting temperature of ice for frozen water. The amount of UFW was estimated from a plot of melting enthalpy against WH, as described for starches (Wootton & Bamunuarachchi, 1978) or collagen (Haly & Snaith, 1971). The value of WH indicating the amount of UFW was determined at the point of zero enthalpy where the extrapolated fitted line intercepted the x-axis. 2.3. X-ray diffraction X-ray diffraction patterns were obtained for two amylose samples before and after the DSC measurements. For the swollen sample with WH 0.5 after DSC, it was dried in a desiccator at room temperature for 1 week. Each sample was hermetically sealed up in a silica capillary tube. X-ray powder diffraction patterns were recorded by means of a flat film camera under vacuum with a Geigerflex X-ray diffractometer (Rigaku, Japan) using Ni-filtered CuKa radiation generated at 40 kV and exposured at 15 mA for 1 h. The measured 2y was corrected using an internal reference of NaF (2y ¼ 38.831). 3. Results and discussion
Sample
Mw
Mw/Mn
Amylose
A-55 A-820
54,500 815,000
1.06 1.05
Cycloamylose
CA-5 CA-13
4700 13,100
1.01 1.03
3.1. The amount of UFW The DSC curves for each system with various WH are shown in Fig. 1. Although a typical sharp peak at 0 1C can be seen in the DSC curve at higher water content, the peak is reduced in size and increased in breadth with decreasing WH.
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A
-30
B
-20
-10
0
10 -30
C
-20
-10
0
10 -30
E
-10
0
10
-20
-10
0
10
F
Endothermic heat flow
D
-20
-30
-20
-10
0
G
-30
10 -30
-20
-10
0
10 -30
-20
-10
0
10
H
-20
-10
0
10 -30
Temperature(°C) Fig. 1. DSC curves for the melting of ice in linear and cyclic (1-4)-a-D-glucans and their oligomer—water systems with various weight fractions of water (WHo1.0) indicated in the figure. Maltopentaose, A; A-55, B; A-820, C; a-CD, D; b-CD, E; g-CD, F; CA-5, G and CA-13, H. Bars indicating difference of heat capacity; 100 JK1g1.
The endothermic peaks disappeared at lower WH for all systems, indicating that a certain amount of water is present as UFW. The melting enthalpy, calculated from the peak area, is plotted against WH in Fig. 2. Values for the molar ratio of UFW per glucose residue, determined from
the x-intercept of the plot, are listed in Table 2. These show different values ranging from 1.24 for a-CD to 3.52 for CA-13. For maltopentaose, no data are listed because the DH could not be obtained below WH 0.250, due to the absence of ice formation under these experimental conditions.
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300
A
ΔH / Jg-1
200
100
0 300
B
ΔH / Jg-1
200
100
0
0.0
0.2
0.4
0.6
0.8
1.0
WH Fig. 2. Correlation between the heat of fusion (DH) and the weight fraction of water (WH) in glucan–water systems. (A) Amyloses and cycloamyloses: CA-5, –&–; CA-13, –n–; A-55, –’–; and A-820, –K–. (B) Cyclodextrins: a-CD, –n–; b-CD, –J–; and g-CD, –’–.
Table 2 Amount of UFW in linear and cyclic glucans Sample
UFWa g H2O/g dry glucan
Molar ratio of H2O/ glucose residue
Linear A-55 A-820
0.307 0.332
2.77 2.98
Cyclic a-CD b-CD g-CD CA-5 CA-13
0.137 0.198 0.218 0.326 0.391
1.24 1.78 1.96 2.93 3.52
a
Determined by extrapolated value of the DH against WH.
For amylose, A-55 contains less UFW than A-820. This might be the result of differences in the gel structure. X-ray diffraction patterns of A-55 before DSC showed a V-type with d-spacings (d) of the major peaks, 1.180, 0.659, 0.431 nm. In contrast, after DSC at WH 0.5, it showed a
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B-type with d ¼ 1.625, 0.512, 0.377 nm, indicating that it contains a B-type crystalline component. For A-55, a junction zone constructed from the double helix after gelatinization may reduce the number of amylose hydroxyl groups which are available for solute–solute interactions. A-820 was found to be almost amorphous before as well as after the DSC, since no remarkable diffraction peak was observed by X-ray analysis. More amylose hydroxyl groups are exposed to solvent in this case, resulting in increased amylose–water interactions. This structural difference might be related to the rate of aggregation of amylose chains, which is strictly dependent on the molecular weight (Gidley, 1989; Gidley & Bulpin, 1989; Kitamura, 1996). Amylose with a higher molecular weight is soluble in water and fairly stable for long periods of time (Kitamura, Hakozaki, & Kuge, 1994). A-55 became entangled during the cooling process in DSC, and formed a partially crystallized matrix, while A-820 was aggregated very slightly. The UFW for all cyclodextrins was lower than that for the other samples. Fig. 2B shows that the DH around WH ¼ 0.5 shows irregular behavior. This can be attributed to the crystallization of the cyclodextrins in systems with WHo0.5. This resulted in a small amount of unfrozen matrix. UFW is similar but slightly higher than the reported value of crystallized water for cyclodextrin hydrates, a-CD 6H2O, b-CD 11H2O and g-CD 16H2O (Saenger, Niemann, Herbst, Hinrichs, & Steiner, 1993). In this cyclodextrin crystallized system, UFW may be correlated with crystallized water for cyclodextrin hydrates. Cycloamylose, CA-5 and CA-13 have a relatively large amount of UFW compared to cyclodextrins, suggesting that it contains a large number of hydrated water molecules in the three dimensional network of the polymer chain. Cycloamylose is much more soluble than cyclodextrin (Ueda, Wakisaka, Nagase, Takaha, & Okada, 2002b; Ueda, 2002a) and is stable in aqueous solution. Cycloamylose has been proposed to have an extended conformation with more flexibility in aqueous solution (Kitamura, et al., 1997; Shimada, Kaneko, Takada, Kitamura, & Kajiwara, 2000). Their model for cycloamylose, calculated by molecular dynamics, indicates that approximately 4.6 water–glucose hydrogen bonds are formed per glucose unit. UFW may be correlated with its high water hydration properties. 3.2. Freezing point depression of water The freezing point depression of water was observed with decreasing water content for all systems (Fig. 3). This was analyzed using the Flory–Huggins theory (Flory, 1953) for polymer solutes and the interaction parameters, m were determined from the correlation between Tp and the volume fraction of the polymer, v2 by curve fitting using the following equation (Kuntz & Kauzmann, 1974): RT 0m lnð1 v2 Þ þ v2 þ mv22 DT m ffi , DH m T 0m
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3.3. Recrystallization of water
Tp / °C
0
-10
0.0
0.2
0.4
0.0
0.2
0.4
0.0
0.2
0.4
ν2
0.6
0.8
1.0
Tp / °C
0
-10
ν2
0.6
0.8
1.0
0.6
0.8
1.0
Tp / °C
0
-10
ν2
Fig. 3. The freezing point depression of water in linear and cyclic glucan–water systems. (A) Amyloses and maltopentaose, maltopentaose, –’–; A-55, –n–; and A-820, –*–. (B) Cycloamyloses: CA-5, –’–; and CA-13, –n–. (C) Cyclodextrins: a-CD, –}–; b-CD, –’–; and g-CD, –n–.
where DTm is the depression in the freezing point, T0m the normal freezing point of the solvent and DHm the heat of fusion of the solvent. It was found that (1-3)-b-D-glucans such as curdlan and schizophyllan show a good fit to this relationship (unpublished data). In contrast to these (1-3)b-D-glucans, the line does not fit well for amylose. This may result from the non-equilibrium system of such molecules under these experimental conditions. A larger depression with a low molecular weight was observed for maltopentaose, compared to A-55 and A-820 using the equation. For all cyclodextrins, Tp shows one high temperature at around WH 0.5. Since cyclodextrin with a WH below 0.5 is crystalline, an increase in freezable water may result in a high Tp. The depression width of b-CD is small.
For all systems, except for b-CD, a small exothermic peak was observed just below Tp, which can be attributed to the recrystallization (devitrification) of water (Fig. 1). Although it was reported in a rapid cooled system (Levine & Slade, 1991), it can also be observed in these systems, in which a slow cooling rate was used, 0.5 K/min. This depends on water content and is apparently observed in systems with about half the weight fraction of water. It is noteworthy that the exothermic peak goes over the baseline on the DSC curve for A-55 at WH 0.531 and A-820 at WH 0.534. This peak for A-55 is larger than that for A-820. Recrystallization occurs immediately after the glass transition during the heating process. This appears to be due to the increased mobility of polymers, which induce ice formation. It may relate to water which remains unfrozen in a nonequilibrium system, and, thus, would be affected by the time scale and thermal history. Annealing at near the Tg results in a smaller exothermic peak and UFW decreases due to new ice formation (Tananuwong & Reid, 2004). The effect of network size was proposed in a study of cross-linked dextran–water systems (Murase et al., 1982). The conformation of amylose chains changed at the time scale considered and the state of water molecules also changed. b-CD showed a single monotonic peak, which shows no apparent evidence of a glass transition or recrystallization. The solubility of b-CD in aqueous solution at 25 1C is less than that of a- and g-CD (Ueda, 2002b). Crystallization may reduce the extent of hydration between solute—water interactions. This study shows that the values of UFW appear to be related to the conformational structure of the amylosic molecules. UFW is affected, not only by the degree of polymerization, but also by the linear or ring structure. Especially for cyclic molecules, CA-13 has a remarkably higher UFW compared with the linear form, indicating a high hydration property. UFW for a series of cyclodextrins is low, indicating that it undergoes crystallization. A DSC study for UFW is useful to know the total state of amylosic chain, including its conformational structure, solute–solute interaction and hydration to water molecules, especially in concentrated solutions or gels. Lastly, it should be noted that the current results are useful for mutual comparison of UFW for several types of amylosic molecules, but not for the determination of the absolute amount of bound water for the individual amylosic molecules measured here. It is difficult to establish to what extent the UFW determined by DSC reflects kinetic effects or actual binding with local water properties, distinct from the bulk water.
Acknowledgments We are very grateful to Dr. K. Fujii of the Ezaki Glico Co., Ltd. for providing amylomaltases. This work was
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supported by grant for the Bio-oriented Technology Research Advancement Institution. References Chung, H. J., Lee, E. J., & Lim, S. T. (2002). Comparison in glass transition and enthalpy relaxation between native and gelatinized rice starches. Carbohydrate Polymers, 48, 287–298. Dixon, R. W., & Peterson, D. S. (2002). Development and testing of a detection method for liquid chromatography based on aerosol charging. Analytical Chemistry, 74, 2930–2937. Flory, P. J. (1953). Principles of polymer chemistry. New York: Cornell University Press. Franks, F. (1986). Unfrozen water—yes—unfreezable water—hardly— bound water—certainly not. Cryo-Letters, 7, 207. Gamache, P. H., McCarthy, R. S., Freeto, S. M., Asa, D. J., Woodcock, M. J., Laws, K., et al. (2005). HPLC analysis of nonvolatile analytes using charged aerosol detection. LCGC Europe, 18, 345–354. Gidley, M. J. (1989). Molecular mechanisms underlying amylose aggregation and gelation. Macromolecules, 22, 351–358. Gidley, M. J., & Bulpin, P. V. (1989). Aggregation of amylose in aqueous systems—the effect of chain-length on phase-behavior and aggregation kinetics. Macromolecules, 22, 341–346. Haly, A. R., & Snaith, J. W. (1971). Calorimetry of rat tail tendon collagen before and after denaturation: The heat of fusion of its absorbed water. Biopolymers, 10, 1681–1699. Higuchi, A., & Iijima, T. (1985). DSC investigation of the states of water in poly (vinyl-alcohol) membranes. Polymer, 26, 1207–1211. Ichikawa, T., Mitsumura, Y., & Nakajima, T. (1994). Water-sorption properties of poly(epsilon-lysine)—carboxymethyl cellulose (cmc) dietary complex films. Journal of Applied Polymer Science, 54, 105–112. Ishida, N., Kobayashi, T., & Keiji, K. (1988). Studies on the physical state of water in starch gel. Nippon shokuhin kogyo gakkaishi, 35, 98–104. Kitamura, S. (1996). Starch polymers, natural and synthetic. In J. C. Salamone (Ed.), The polymeric materials encyclopedia, synthesis, properties and applications, Vol. 10 (pp. 7915–7922). Boca Raton: CRC Press. Kitamura, S., Hakozaki, K., & Kuge, T. (1994). Effects of molecular weight on the retrogradation of amylose. In K. Nishinari, & E. Doi (Eds.), Food Hydrocolloids: Structures, Properties, and Functions (pp. 179–182). New York: Plenum Press. Kitamura, S., Isuda, H., Shimada, J., Takada, T., Takaha, T., Okada, S., et al. (1997). Conformation of cyclomaltooligosaccharide (‘‘cycloamylose’’) of dp21 in aqueous solution. Carbohydrate Research, 304, 303–314. Kitamura, S., Yunokawa, H., Mitsuie, S., & Kuge, T. (1982). Study on polysaccharide by the fluorescence method. II. Micro-Brownian motion and conformational change of amylose in aqueous solution. Polymer Journal (Tokyo, Japan), 14, 93–99. Kuntz, I. D., Jr., & Kauzmann, W. (1974). Hydration of proteins and polypeptides. Advances in protein chemistry, 28, 239–345. Levine, H., & Slade, L. (1991). Polymer physicochemical characterization of oligosaccharides. In R. B. Friedman (Ed.), Biotechnology of Amylodextrin Oligosaccharides (pp. 219–260). Washington, DC: American Chemical Society.
867
Li, S., Dickinson, L. C., & Chinachoti, P. (1998). Mobility of ‘‘unfreezable’’ and ‘‘freezable’’ water in waxy corn starch by 2H and 1 H NMR. Journal of Agricultural and Food Chemistry, 46, 62–71. Liesebach, J., Rades, T., & Lim, M. (2003). A new method for the determination of the unfrozen matrix concentration and the maximal freeze-concentration. Thermochimica Acta, 401, 159–168. Liu, H., & Lelievre, J. (1992). Transitions in frozen gelatinized-starch systems studied by differential scanning calorimetry. Carbohydrate Polymers, 19, 179–183. Murase, N. (1993). Origin of an endothermic trend observed prior to ice crystallization exotherm in the dsc rewarming trace for polymer gels. Cryo-Letters, 14, 367–376. Murase, N., Fujita, T., & Gonda, K. (1983). Low-temperature calorimetric studies of compartmentalized water in hydrogel systems. II. Cryo-Letters, 4, 19–22. Murase, N., Inoue, T., & Ruike, M. (1997). Equilibrium and nonequilibrium freezing of water in cross linked dextran gels. Cryo-Letters, 18, 157–164. Murase, N., Shiraishi, M., Koga, S., & Gonda, K. (1982). Lowtemperature calorimetric studies of compartmentalized water in hydrogel systems. I. Cryo-Letters, 3, 251–254. Nakamura, K., Hatakeyama, T., & Hatakeyama, H. (1981). Studies on bound water of cellulose by differential scanning calorimetry. Textile Research Journal, 51, 607–613. Nakamura, K., Hatakeyama, T., & Hatakeyama, H. (1991). Formation of the glassy state and mesophase in the water–sodium alginate system. Polymer Journal, 23, 253–258. Nakanishi, Y., Norisuye, T., Teramoto, A., & Kitamura, S. (1993). Conformation of amylose in dimethyl-sulfoxide. Macromolecules, 26, 4220–4225. Saenger, W., Niemann, C., Herbst, R., Hinrichs, W., & Steiner, T. (1993). Crystal-structures of linear and cyclic oligosaccharides—an overview. Pure and Applied Chemistry, 65, 809–817. Schenz, T. W., Courtney, K., & Israel, B. (1993). Studies on the magnitude of the glass transitions of glucose polymers. Cryo-Letters, 14, 91–96. Shimada, J., Kaneko, H., Takada, T., Kitamura, S., & Kajiwara, K. (2000). Conformation of amylose in aqueous solution: Small-angle X-ray scattering measurements and simulations. Journal of Physical Chemistry B, 104, 2136–2147. Tananuwong, K., & Reid, D. S. (2004). Differential scanning calorimetry study of glass transition in frozen starch gels. Journal of Agricultural and Food Chemistry, 52, 4308–4317. Terada, Y., Fujii, K., Takaha, T., & Okada, S. (1999). Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: Production of cycloamylose. Applied and Environmental Microbiology, 65, 910–915. Ueda, H. (2002a). Physicochemical properties and complex formation abilities of large-ring cyclodextrins. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 44, 53–56. Ueda, H., Wakisaka, M., Nagase, H., Takaha, T., & Okada, S. (2002b). Physicochemical properties of large-ring cyclodextrins (CD18-CD21). Journal of Inclusion Phenomena and Macrocyclic Chemistry, 44, 403–405. Wootton, M., & Bamunuarachchi, A. (1978). Water binding capacity of commercially produced native and modified starches. Staerke, 30, 306–309.