2018 年 24 巻 5 号 p. 795-801
The structure of fine starch prepared via a compressed hot water process at different temperatures (160–180 °C) was analyzed using dynamic light scattering and size-exclusion chromatography with multi-angle light scattering and differential refractive index detection. Changes in the molecular weight, polydispersity, hydrodynamic radius, and radius of gyration were assessed. The intrinsic viscosity of the fine starch solution was derived from the Flory-Fox and Ptitsyn-Eizner equations. The weight-averaged molecular weight decreased to 7.29×105 g/mol, and the average hydrodynamic radius and weight-averaged radius of gyration decreased by 34.9 nm and 14.6 nm, respectively, at 180 °C. In fine starch prepared at 160 °C, 165 °C, and 170 °C, tails in the multi-angle light scattering peaks, upswings in the conformation plots, and upturns in the plots of gyration radii and elution volumes were all the result of branching structures. At 175 °C and 180 °C, amylopectin branching was diminished and symmetric scattering peaks were observed. We propose a pathway for waxy rice starch hydrolysis by a compressed hot water process.
Starch is a renewable and biodegradable polymer that is stored in many plants as an energy source from photosynthesis, and is the second most abundant biomass in nature. The starch industry commonly extracts from corn, wheat, and tapioca in native and modified forms, for applications in food, paper, and pharmaceuticals (Whistler and Paschall, 1965; Buléon et al., 1998; Shahidi and Han, 1993; Gharsallaoui et al., 2007). New applications and characteristics of starch are continuously being investigated.
Starch consists of amylose α(1–4)-linked glucose units, amylopectin α(1–4)-linked glucose units, and branched α(1–6)-linkages. There are many reported molecular weights of amylose and amylopectin from various plants. Amylose and amylopectin from normal corn have molecular weights (Mw)=1.4×106 and 39×106 g/mol, respectively (Juna and Huber, 2012); amylose from rice has Mw=5.1–6.9×105 g/mol (Chen and Bergman, 2007); waxy barley starch has Mw=1.06×108 g/mol (Rojas et al., 2008); and Amioca (waxy corn starch) has an apparent molar mass of 107–109 g/mol (Bruijnsvoort et al., 2001).
Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) and field-flow fractionation with multi-angle light scattering (FFF-MALS) are powerful methods for fractionation and characterization of macromolecules. Physicochemical properties of polymers such as the molecular weight, polydispersity, radius of gyration, hydrodynamic radius, and the molecular structure in a solution are then derived (Nilsson, 2013; Podzimek, 2011). Evaluation of not only raw starch, but also modified starch is important for industrial use (Ono et al., 2005; Cave et al., 2009).
The intrinsic viscosity [η] is the quantity of a polymer's contribution to enhance the viscosity of a solution, which depends on its molecular weight, particle size, and particle conformation in solution. Also, the critical concentration c*, where polymers start to overlap in a solution, can be estimated by c* ≈ 2.5/[η] (Nilsson, 2013; Podzimek, 2011). [η] can be calculated from the Flory-Fox and Ptitsyn-Eizner equations by substituting the values of the radius of gyration, molecular weight, and the slope of the conformation plot. These can be derived from light scattering data without a viscometer (Flory and Fox, 1951; Ptitsyn and Eizner, 1960; Podzimek, 2011).
To use starch effectively, techniques for preparing nanoscale starch have been studied. Nanoscale waxy rice starch particles can be prepared using a compressed hot water process via hydrolysis. Starch nanoparticles are prepared by acid hydrolysis, enzymatic treatments, and physical treatments such as high-pressure homogenization, ultrasonication, reactive extrusion, and gamma irradiation (Kim et al., 2015). Biomass conversion is performed by only heating water under high pressure, without the need for catalysts, acids, and enzymes. The compressed hot water process is one of the most useful reaction fields. The smallest average hydrodynamic radius of 75.2 nm was obtained using compressed hot water at 180 °C, with a starch concentration of 0.1% (w/w), and an initial pressure of 3.0 MPa. It was determined with a zeta potential and a submicron particle size analyzer (Yoshioka and Shimizu, 2014). A fine waxy rice starch solution prepared with 160 °C compressed hot water was spray-dried as a wall material for microencapsulation (Ushiyama and Shimizu, under review). Knowledge of starch macromolecular structures is necessary for practical uses in industry. This information for waxy rice starch has been determined with asymmetrical-flow-field flow fractionation, and the weight-averaged molecular weight of 2.30×108 g/mol, with a Z-average radius of gyration of 212.2 nm (Rolland-Sabaté et al., 2007). However, changes in the hydrodynamic particle size induced by the compressed hot water process are unknown.
Here, the particle conformation of fine starch (waxy rice starch), prepared by a compressed hot water process at 160–180 °C, was measured using dynamic light scattering (DLS) and SEC-MALS. The intrinsic viscosity was calculated from the Flory-Fox and Ptitsyn-Eizner equations, and correlations with the particle conformation were derived. In SEC-MALS measurements, the branching amylopectin polymer affects the column separation and the MALS-signal. Therefore, changes in branching structure in waxy rice starch by the compressed hot water process were deduced from SEC-MALS data.
2.1 Chemicals Waxy rice starch was obtained from Joetsu Starch Co., Ltd. (Niigata, Japan). Pullulan, with a weight-average molecular weight of 48800 from Polymer Standards Service GmbH (Mainz, Germany), was used as a standard sample for SEC-MALS. Sodium nitrate from Wako Pure Chemical Industries Ltd. (Osaka, Japan) was used for a buffer solution in SEC-MALS. Milli-Q water (Merck, Germany) was used throughout.
2.2 Preparation of fine starch solution The fine starch solution was prepared with compressed hot water at 160 °C, 165 °C, 170 °C, 175 °C, and 180 °C, using a 48-mm-diameter stainless-steel pressure vessel (Teflon resin inner vessel: ø 36 mm × 120 mm tall) at a concentration of 0.1% (w/w). Starch (0.050 g) was placed in the pressure vessel with Milli-Q water (50 mL) and a stirring chip. The solution was heated using an organic synthesizer (Chemi-Station PPV-3000, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) until reaching a temperature of 160 °C, 165 °C, 170 °C, 175 °C, and 180 °C. Then, the vessel was cooled in a refrigerator at 5 °C. The pressure vessel was filled with nitrogen gas at a primary pressure of 2.5 MPa and the stirring chip speed was 1000 rpm. The solution temperature in the vessel during heating was recorded with a type K thermocouple connected to a data logger (Multichannel Temperature Recorder MCR-4TC, T&D Co., Nagano, Japan).
All of the fine starch solutions were filtered through a 0.45-µm pore-size suction filter made from biologically inert mixtures of cellulose acetate and cellulose nitrate (MF-Millipore membrane filters, Merck KGaA, Darmstadt, Germany) prior to SEC-MALS measurements.
2.3 SEC-MALS analysis The SEC-MALS was a combination of high-performance liquid chromatography (HPLC 1200 Infinity series, Agilent Technologies, Santa Clara, CA, USA), multi-angle light scattering (MALS DAWN8+, Wyatt Technology, Santa Barbara, CA, USA), and a differential refractive index (RI) detector (OptilabrEX, Wyatt Technology). The MALS and RI used a wavelength of 658 nm. The HPLC was equipped with a liquid delivery pump, an automatic sampler, a column compartment, and a UV detector. A LB-G6B guard column and a LB-806M analytical size-exclusion column (Showa Denko K.K., Tokyo, Japan) were connected in tandem. The columns were maintained at 30 °C within the column compartment. The buffer solution for separations was 50 mM NaNO3 in Milli-Q water, which had been passed through a 0.45-µm filter (MF-Millipore membrane filters, Merck KGaA) before use. The 50-µL sample injection was started at a flow velocity of 0.8 mL/min after the laser intensity for detection in MALS and RI was stable. The concentration of pullulan standard solution (PSS-dpul50k, PSS polymer Standard Service GmbH, Mainz, Germany) for detector signal alignment was 3 mL/mg.
Processing of the SEC-MALS data was performed using Astra software 5.3.4 (Wyatt Technology), which used the upper 50% of the light scattering peak intensity. The collection interval in MALS was 0.5 sec, and each data slice was equivalent to 0.67 µL. The refractive index increment (dn/dc) of 0.146 mL/g was used for fine starch prepared by the compressed hot water process (Chen and Bergman 2007 [Please confirm the accuracy of this citation. It appears that the first names of the authors have been used.]; Motawia et al., 2005; Rojas et al., 2008). The molecular weight and the radius of gyration were determined by Zimm plots (Zimm, 1948). The data from the MALS detector were fit to a straight line on the Zimm plots. Inaccurate data, especially that obtained from the low scattering angle detector, were not used. The second coefficient was set to zero because of the low-concentration samples.
The following information was calculated by ASTRA 5.3.4 by integration over one peak: number-average molecular weight Mn; weight-average molecular weight Mw; Z-average molecular weight Mz; number-average radius of gyration 〈Rg〉n; weight-average radius of gyration 〈Rg〉w; and Z-average molecular weight 〈Rg〉z
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where ci, Mi, and 〈Rg〉i were the mass concentration, molecular weight, and radius of gyration of the ith slice, respectively. The two polydispersity values Mw/Mn and Mz/Mn were calculated. The molecular shape information was obtained from the conformation plot, which is a plot of the logarithm of the radius of gyration vs. the logarithm of the molecular weight.
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The value of the exponent v can be obtained from the slope of log Rg vs. log M. The molecular structure can be estimated from this slope. For a spherical structure, ν=0.33, for a rod structure ν=1, and for a random coil structure ν=0.5–0.6 (Nilsson, 2013).
2.4 DLS analysis The hydrodynamic radius Rh was obtained with fiber dynamic light scattering (FDLS-3000, Otsuka Electronics Co., Ltd., Japan). The sample solutions were in a 12-mm-diameter glass cell (Otsuka Electronics Co., Ltd.) that had been cleaned with chloroform. The measurement device was filled with silicone oil and maintained at 25 °C using a thermostat. A 532-nm laser was used and the measurement angle was 90°.
Rh was calculated from the Stokes-Einstein equation:
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where kB, T, η, and Di were Boltzmann's constant, the absolute temperature, the solvent viscosity, and the diffusion coefficient, respectively (Einstein, 1905). A total of 300 particles were counted. Rh distributions and the average particle size were obtained with CONTIN routine. To evaluate the dispersion of the particle size distribution, a relative span factor RSF was derived:
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where Dn was the hydrodynamic radius at n % in the cumulative frequency distribution. Small RSF values indicate uniform particle distributions (Herrero et al., 2006).
The hydrodynamic radius distribution of fine starch was expected to change because of passage through the 0.45-µm filter. Therefore, Rh measurements of both pre-filtration samples and filtered samples were performed.
2.5 Derivation of intrinsic viscosity
The intrinsic viscosity [η] is defined as:
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where η and η0 are the viscosities of the dilute polymer solution and the pure solvent, respectively, and c is the polymer concentration. Additionally, [η] was calculated using the Flory-Fox and Ptitsyn-Eizner equations (Flory and Fox, 1951; Ptitsyn and Eizner, 1960), which could be applied under the non-theta condition solutions:
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[η], 〈R〉, and M were the intrinsic viscosity (100 mL/g), the radius of gyration (cm), and the molecular weight (g/mol), respectively. Φ is the corrected Flory universal constant for non-theta conditions and Φ0=2.86×1021 is applied for theta conditions (ε=0). The value a is the exponent of the Mark-Houwink equation (Podzimek, 2011), which is expressed as the relation between [η] and M:
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Eq. 15 indicates the relation between a and the slope of conformation plot ν:
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3.1 Hydrodynamic radius from DLS The dissociation of water into a hydrogen ion H+ and a hydroxide ion OH− is induced by increasing the temperature of the solution and the pressure in the compressed hot water reaction field. Therefore, the ion product of water Kw increases and the reaction field becomes more intense (Marshall and Franck, 1981). Yoshioka and Shimizu reported that the average Rh of fine starch was dependent on the preparation conditions of the compressed hot water process, and the hydrolysis of starch was further refined. The same tendency was confirmed here; that is, Rh of the starch decreased with temperature as the reaction field increased (Yoshioka and Shimizu, 2014). Figure 1 shows the average Rh for a pre-filtration fine starch solution and for a filtered fine starch solution. The average Rh of 164 nm, 151 nm, 109 nm, 42.3 nm and 34.9 nm were obtained from the compressed hot water process at 160 °C, 165 °C, 170 °C, 175 °C and 180 °C for the pre-filtration fine starch solution. The RSF values were derived from the fine starch prepared at 160 °C (2.69), 165 °C (2.46), 170 °C (3.41), 175 °C (1.40) and 180 °C (2.99).
Hydrodynamic radius Rh obtained from DLS with different compressed hot water process temperature at 160 °C (○), 165 °C (□), 170 °C (△), 175 °C (●) and 180 °C (■).
The average Rh decreased in fine starch prepared at 160 °C and 165 °C after filtration, and the RSF values decreased by filtration: 160 °C (1.09), 165 °C (1.44), 170 °C (1.23), 175 °C (1.23), 180 °C (2.06). The average Rh of 98.9 nm, 101 nm, 95.5 nm, 41.2 nm and 26.8 nm were obtained from the 160 °C, 165 °C, 170 °C, 175 °C and 180 °C compressed hot water process for the filtrated fine starch solution.
3.2 Particle conformation data from SEC-MALS A single peak was confirmed in the MALS signal for all of the samples. The tail at high volumes indicated that the MALS signal was detected after the peak. It appeared in fine starch prepared at 160 °C, 165 °C, and 170 °C [Fig. 2 (A)]. Fine starch prepared at 175 °C and 180 °C exhibited a symmetric peak because of the reduced branching structures in the amylopectin and the decreasing particle sizes via hydrolysis reduced the tails [Fig. 2 (B)]. In this experiment, 〈Rg〉 and M decreased with temperature. Also, the radius of gyration 〈Rg〉 in the range of 60–80 nm was detected after the peak in the same samples [Fig. 3 (A, B)]. The amount of branching linkage in the waxy rice starch was 4.0–5.5% (Hizukuri, 1993). The tails occurred because the fine starch particles with branched amylopectin structures, large particle sizes, and particle aggregation were caught in the column filter (Podzimek, 2011). Table 1 lists the results obtained from SEC-MALS, including the average M (Mn, Mw, Mz, Mz+1), the polydispersity (Mw / Mn, Mz / Mn), the average 〈Rg〉 (〈Rg〉n, 〈Rg〉w, 〈Rg〉z), the slope of the conformation plots, and [η]. The weight-average molecular weight Mw and weight-average radius of gyration 〈Rg〉w decreased by 0.729×106 g/mol and 14.6 nm, respectively, at 180 °C. In general, amylopectin has a large molecular weight of 108–109 g/mol (Buléon et al., 1998). The compressed hot water process is one of the most powerful hydrolysis reaction fields for polymers, and the molecular weight of amylopectin decreased here by 3–4 orders of magnitude.
SEC-MALS results for fine starch prepared by the compressed hot water process at 160 °C 
, 165 °C 
, 170 °C 
, 175 °C 
, and 180 °C 
. (A): volume vs. detector signals obtained from MALS photometer at 90 °, (B): conformation plot and enlarged conformation plot for 160 °C, 165 °C, and 170 °C.
SEC-MALS results for fine starch prepared by the compressed hot water process at 160 °C , 165 °C , 170 °C , 175 °C , and 180 °C . (A): volume vs. molecular weight, (B): volume vs. radius of gyration.
| Compressed hot water process [°C] | Mn [× 106 g/mol] | Mw [× 106 g/mol] | Mz [× 106 g/mol] | Polydispersity (Mw/Mn) | Polydispersity (Mz/Mn) | 〈Rg〉n [nm] | 〈Rg〉w [nm] | 〈Rg〉z [nm] | Slop of conformation plot | Intrinsic viscosity [100mL/g] |
|---|---|---|---|---|---|---|---|---|---|---|
| 160 °C | 16.0 | 16.3 | 16.7 | 1.02 | 1.02 | 677 | 68.2 | 68.9 | 0.40 | 1.34 |
| 165 °C | 13.1 | 13.4 | 13.7 | 1.02 | 1.02 | 61.2 | 61.3 | 61.6 | 0.38 | 1.30 |
| 170 °C | 12.0 | 12.6 | 13.3 | 1.05 | 1.05 | 60.5 | 61.2 | 62.2 | 0.32 | 1.77 |
| 175 °C | 4.49 | 5.18 | 5.89 | 1.15 | 1.15 | 34.8 | 36.4 | 38.1 | 0.35 | 0.801 |
| 180 °C | 0.562 | 0.729 | 0.954 | 1.30 | 1.30 | 13.2 | 14.6 | 16.2 | 0.40 | 0294 |
The two polydispersity values Mw / Mn and Mz / Mn increased at 170–180 °C. Fine starch with a wide range of molecular weights was prepared from the intense hydrolysis. Also, the removal of large particles by 0.45-µm filtration was affected.
From the conformation plots, fine starch had a slope of 0.32–0.40, which indicated that it was close to a spherical structure, or a tightly aggregated random coil. Figure 2 (B) is the enlarged conformation plot of fine starch prepared at 160 °C, 165 °C, and 170 °C. The upswing of the conformation plot at low molecular weight was caused by the branching structure of the fine starch, which also affected column fractionation (Podzimek, 2011).
A pathway for waxy rice starch hydrolysis by compressed hot water is suggested in Scheme 1. Natural amylopectin is a randomly branched structure with α(1–6) linkages with 4.0–5.5% branching linkages. The branching and its long chain that affected column fractionation in SEC gradually decreased in compressed hot water. Thus, precise column fractionation was obtained by amylopectin hydrolysis (intermolecular dehydration) at 175 °C and 180 °C.
Waxy rice starch hydrolysis pathway in the compressed hot water process. (a) fine starch prepared at 160 °C with an average Rh=98.9 nm, and 〈Rg〉w=68.2 nm, (b) fine starch at 175 °C with an average Rh=41.2 nm, and 〈Rg〉w=36.4 nm, (c) fine starch at 180 °C with an average Rh=26.8 nm, and 〈Rg〉w=14.6 nm. The solid-line and broken-line circles indicate hydrodynamic particle size and radius of gyration.
The macromolecular structure of fine starch prepared by a compressed hot water process was analyzed with DLS and SEC-MALS. The branching structure and the long chain of amylopectin were diminished by the compressed hot water process via hydrolysis. In fine starch prepared at 160 °C, 165 °C, and 170 °C, tails in the multi-angle light scattering peaks, upswings in the conformation plots, and upturns in the plots of gyration radii and elution volumes were all the result of branching structures. At 175 °C and 180 °C, amylopectin branching was diminished and symmetric scattering peaks were observed. Finally, the weight-average molecular weight Mw and the weight-average radius of gyration 〈Rg〉w were decreased by 0.729×106 g/mol and 14.6 nm, respectively, at 180 °C. The average hydrodynamic radius decreased by 34.9 nm in the DLS measurement. We propose a pathway for waxy rice starch hydrolysis by a compressed hot water process. In conclusion, fine starch prepared via compressed hot water processes at 175 °C and 180 °C were well fitted to the adaptation theory.
Acknowledgements Fiber dynamic light scattering and SEC-MALS were provided by the instrumental analysis services of the Global Facility Center of Hokkaido University.
