International Journal of Biological Macromolecules 87 (2016) 28–33
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Understanding how the aggregation structure of starch affects its gastrointestinal digestion rate and extent Pei Chen a,∗ , Kai Wang a , Qirong Kuang b , Sumei Zhou b , Dazheng Wang c , Xingxun Liu b,∗∗ a b c
School of Food Science, South China Agricultural University, Guangzhou 510642, China Institute of Food Science and Technology (IFST), Chinese Academy of Agricultural Science (CAAS), Beijing100193, China Zhengzhou Second People’s Hospital, Zhengzhou 450006, China
a r t i c l e
i n f o
Article history: Received 8 December 2015 Received in revised form 31 December 2015 Accepted 5 January 2016 Available online 17 February 2016 Keywords: Wheat starch Multi-scale structure In vitro digestion properties
a b s t r a c t Regulating the starch gastrointestinal digestion rate by control of its aggregation structure is an effective way, but the mechanism is still not clear. Multi-scale structure of waxy and normal wheat starches were studied by confocal laser scanning and scanning electron microscopes, as well as wide-angle and small-angle X-ray techniques in this study. In vitro digestion kinetics of those two starches and structure–digestion relationship were also discussed. Both waxy and normal starches show A-type diffraction pattern, but waxy variety shows a slightly higher crystallinity. Small-angle X-ray scattering results show that waxy wheat starch has higher scattering peak intensity (Imax ) and a larger crystallinity lamellar repeat distance (Lp ) compared with the normal wheat starch. We suggested that the higher digestion rate of waxy starch at initial stage is mainly due to more small-size particles, but the higher crystallinity and the larger crystalline lamellar size limit the digestion extent. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Glycemic index (GI) is an important parameter to evaluate nutritional properties for carbohydrate-containing foods according to Food and Agriculture Organization (FAO) and World Health Organization (WHO) [1]. Starch, a major carbohydrate source in human diets, is a key component to control the blood glucose level in human body. The starch in a high GI food is digested and absorbed rapidly, resulting in high postprandial blood glucose and insulin levels, which over the long term are enhancing the risks of diet-related diseases including type-2 diabetes and cardiovascular disease [2]. Therefore, controlling the digestion rate and extent of granular starches and understanding the factors to affect the starch digestion is important for starch-based food design and development [3]. The application of Log of Slope (LOS) analysis of digestibility curves based on the well-documented first-order reaction kinetics of amylolysis has been used to characterize the reaction rate of starch amylolysis. LOS plots could be very sensitive to changes in digestion rate constant from the conventional digestion curves,
∗ Corresponding author. Fax: +86 20 85280270. ∗∗ Corresponding author. Fax: +86 10 62813477. E-mail addresses: [email protected], [email protected] (P. Chen), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.ijbiomac.2016.01.119 0141-8130/© 2016 Elsevier B.V. All rights reserved.
and assist in understanding how starch digestion rates change deduced by structure differences [4–6]. The digestion rate (k) could be affected by the molecular level and aggregation structure of granular starches. For molecular level, it have been reported that the digestion rate tends to increase with longer chain length of long amylose branches and smaller ratios of long amylopectin to short amylopectin branches, as well as smaller relative amount of long amylose branches to short amylopectin branches [7]. The aggregation structures dominant by lamellar and crystalline structures [8], also play a key role in determining the digestion rate and extent. For example, Zhang et al. used extrusion technique to induce loworder starch with a relatively lower digestion rate [9]. However, the aggregation structures of granular starches are complicated, the mechanism to control the digestion rate and extent is still not fully understood [3]. Waxy wheat was firstly developed in Japan through traditional hybridizations genetic modification in 1995 [10], then was successfully introduced to Australia, USA, Canada and China [11,12]. Waxy wheat starch, which is biosynthesized when the three granulebound starch synthesis (GBSS) genes are absent or nonfunctional, has distinct structural characteristics such as very low amylose content and high crystallinity. The special structure leads to the desirable functional properties such as high swelling power and peak viscosity, low retrogradation level, setback viscosity and pasting temperature. With the increasing applications of waxy wheat starches in foods, a good understanding of the aggregation struc-
P. Chen et al. / International Journal of Biological Macromolecules 87 (2016) 28–33
29
Fig. 1. Typical starch digestion curves and LOS plots of cooked and raw wheat starches.
Fig. 2. Starch granules of waxy wheat starch (a1, a2, a3) and normal wheat starch (b1, b2,b3) observed by SEM and CLSM.
ture differences between waxy and normal wheat starches is required and further results in the different digestion rate and extent becoming increasingly important to the area of food processing and nutrition. Moreover, the thickness of crystalline and amorphous lamellar structure of waxy wheat starches as measured by small anger X-ray scattering (SAXS) and their relationship
with the molecular structure are not fully understood as well. In this study, the multi-scale aggregation state structure of normal and waxy wheat starches were investigated, and the digestion rate and extent were evaluated by LOS methods. In addition, the structure–digestion relationship of wheat starches were also discussed.
30
P. Chen et al. / International Journal of Biological Macromolecules 87 (2016) 28–33
2. Experimental
2.5. Small-angle X-ray scattering (SAXS)
2.1. Materials
The lamellar structure of starch was studied using a small-angle X-ray scattering system (Anton-Paar, Graz, Austria) equipped with a PW3830 X-ray generator (PANalytical), operated at 50 mA and 40 kV, using Cu K␣ radiation with a wavelength of 0.1542 nm as X-ray source. Starch slurries with similar moisture content (MC, about 65%) were prepared for this experiment and equilibrated at 20 ◦ C for 24 h before the test. The samples were filled into a sample cell and measured for 10 min. SAXS curves were plotted as a function of relative peak intensity, I, versus q, the scattering vector. 1-D correlation function were used to analyze the corresponding parameters such as long period Lp (also known as Bragg spacing d or lamellar repeat distance), hard block thickness Lc and soft block thickness La of starch lamellar structure [17].
Chinese spring (CS) wheat and its near-isogenic waxy type were used in this work. A waxy wheat line “Caiwx” and Yangmai01-2 were used as donor and recurrent parents, respectively. Starch was isolated using the dough-washing method as previously described [13]. The amylose content of waxy and normal wheat starches are 2.6% and 27% respectively, using a colorimetry method through determined by iodine binding [14]. ␣-Amylase (Sigma A-3176, from porcine pancreas, 250 U/mL), pepsin (Sigma P-6887, from porcine gastric mucosa), pancreatin (Sigma P-1750, from porcine pancreas) and amyloglucosidase (AMG, Sigma A-7420 from Aspergillus Niger) were used. All other chemicals were of analytical grade.
2.6. In vitro starch digestibility 2.2. Scanning electron microcopy A scanning electron microscope (X230, Philips, Eindhoven, Netherlands) was used to investigate the appearance and surface of the starch granules. The samples were coated with iridium in a vacuum evaporator, and viewed in the SEM at a low accelerating voltage of 2 kV. Diameters of starch granules were conducted using the Gun Image Manipulation Program. The diameter of individual granules was calculated using a sphere equal in area to the granule. More than 200 particles were calculated for each sample, and the results were based on the average of the measurement.
2.3. Confocal laser scanning microscopy About 5 g (dry basis) of starch granules was hydrolyzed by suspending in 100 mL of 2 mol/L HCl solution at 35 ◦ C for different times (days). After hydrolysis, the insoluble residues were washed several times with distilled water to neutrality. Native and hydrolyzed starch samples were prepared for CLSM as previously described [13,15]. Starch granules (∼10 mg) were dispersed in 15 L of freshly made APTS solution (10 mM APTS dissolved in 15% acetic acid) and 15 L of 1 M sodium cyanoborohydride was added. The reaction mixture was incubated at 30 ◦ C for 15–18 h, with the granules washed 5 times with 1 mL of distilled water and finally suspended in 20 L of 1:1 (v/v) glycerol/water mixture. A drop of the mixture was then mounted on a glass plate for microscopy. A confocal laser scanning microscope (TCS SP2, Leica Microsystems, Wetzlar, Germany) equipped with an Ar/Hg laser and a stand for fixed fluorescent cell samples was used to investigate the internal morphologies of wheat starches. The Leica objective lens used were: 60× plan apo/1.40 oil UV. During image acquisition, each line was scanned four times, and averaged to reduce noise.
Isolated starch granules (200 mg, dry weight basis) were heated in distilled water with a starch-to-water ratio of 1:5 in a tube for 15 min then cooled in −20 ◦ C refrigerator and freeze dried for in vitro experiment. Both raw and cooked starch was incubated with 1.0 mL artificial saliva solution containing 250 U/mL pancreatic ␣-amylase in carbonate buffer (14.4 mM, pH 7.0, containing 21.1 mM potassium chloride, 1.59 mM calcium chloride and 0.2 mM magnesium chloride) at ambient temperature for 1 min. Procine pepsin (1 mg/mL) in HCl solution (5 mL, 0.02 M) was then added to the mixture and the mixture was stirred at 80 rpm in a water bath at 37 ◦ C for 30 min. The digesta were then neuturalized with 5 mL NaOH (0.02 M) and mixed with 25 mL sodium acetate buffer (pH 6, 0.2 M, containing 200 mM CaCl2 and 0.49 M MgCl2 ). Pancreatin (2 mg/mL) and 58 U/mL amyloglucosidase in the same sodium acetate buffer solution (5 mL) was added to the digesta, and the mixture was incubated at 37 ◦ C and at 85 rpm. Aliquots (0.2 mL) were collected at different time points up to 180 min, and the digestion was stopped by adding 0.75 mL of 95% ethanol. The digestibility of freshly gelatinized starch (g/100 g of dry starch) was determined from the amount of glucose released in the supernatant, which was converted to the mass of digested starch by a factor of 0.9. The starch digestion data can be fitted to a piecewise first-order equation following Zou’s method [18]. A logarithm of slope (LOS) plot is obtained by expressing the first derivate of the first order equation in logarithmic form: ln(dCt /dt) = −kt + ln(C∞ k)
(1)
k and C∞ are calculated from the slope and intercept respectively. 2.7. Statistical analysis The mean values of digestion rate and relative crystallites of starch were analyzed with SPSS (version 22.0, IBM SPSS Statistics) using Tukey’s Pairwise Comparisons with confidence levels of 95% and 90% in performing an analysis of variance (ANOVA) test. 3. Results and discussion
2.4. Wide-angle X-ray diffraction 3.1. The digestibility of raw and cooked starch Wide-angle X-ray diffraction (WAXD) analysis was studied by a Bruker D8 Diffractometer operating at 40 kV and 40 mA, Cu K␣ radiation monochromatized with a graphite sample monochromator. The moisture content of all samples was adjusted to ∼10% before test to reduce the water effect. Relative crystallinity (RC) of the samples was quantitatively estimated by measuring the peak area of crystallinity following the method described before [16]. The intensity was measured from 2 to 40◦ as a function of 2 and at a scanning speed of 2◦ /min.
In vitro digestion kinetic profiles of cooked and raw starches are shown in Fig. 1. The digestion rate and extent of starch or starchcontaining food vary depending on the enzyme type/concentration [19], and rheology properties [20,21]. Therefore, the same physical state of starch was used in this kinetic study. In order to obtain a logarithmic digestion curves, both ␣-amylase and amyloglucosidase (AMG) were used to convert sufficient starch substrate to glucose products over the time course [4,7].
P. Chen et al. / International Journal of Biological Macromolecules 87 (2016) 28–33
50
percentage(%)
45 40
normal wheat starch
35
waxy wheat starch
30 25 20 15 10 5 0 2~5
5~8 8~12 12~15 15~18 18~21 21~24 24~27 27~30 30~33
Granule diameter(um) Fig. 3. Size distributions of waxy and normal wheat starch granules (in number).
Fig. 1 shows the typical starch digestion curve and LOS fit plots from cooked and raw normal and waxy wheat starches. For raw starches, it can be seen that the waxy starch was hydrolyzed rapidly at the early stage, but digestion extent of the waxy starch was lower after 6 h hydrolysis, comparing with the normal starch. However, it should be noted that there is more starch left after hydrolysis (such as after 24 h hydrolysis). For cooked starches, the digestion rate was increased significantly due to the gelatinization process which enhances the enzyme access to the starch molecules (substrates) [22]. LOS fitting analysis was applied to the starch digestion kinetic profiles to obtain first-order coefficients k (k values are listed in Fig. 1), showing that all digestion profiles can be described by a single-phase pseudo-first order process (R2 > 0.90). The digestion rate coefficient of cooked starches is markedly higher than that of raw starches. It should be noticed that the k value for cooked waxy wheat starch is much higher than cooked the wheat starch, which is very unusual and may be due to the quick retrogradation of high amylose content starch. 3.2. Multi-scale structure of starch The morphological and micro-structural features of waxy and normal wheat starches were revealed by SEM and CLSM (Fig. 2). SEM photos (Fig. 2) show that both waxy and normal wheat starches have a bimodal size distribution, comprising of small and
large granules. For wheat starches, small spherical granules (Bgranules) range in size from approximately 1 to 10 M, whereas larger lenticular-shaped granules range from about 15 to 40 M, known as A-granules. The A- and B-granules were smooth with no cracks on the surface, suggesting the low level of starch damage during fractionation. The waxy and normal wheat starch granules were ellipse and spherule shapes with size distribution between 2 and 35 M (Fig. 3) [23]. No significant differences were observed in the granule morphology of waxy and normal wheat starches. More smaller particles (
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Understanding how the aggregation structure of starch affects its gastrointestinal digestion rate and extent Pei Chen a,∗ , Kai Wang a , Qirong Kuang b , Sumei Zhou b , Dazheng Wang c , Xingxun Liu b,∗∗ a b c
School of Food Science, South China Agricultural University, Guangzhou 510642, China Institute of Food Science and Technology (IFST), Chinese Academy of Agricultural Science (CAAS), Beijing100193, China Zhengzhou Second People’s Hospital, Zhengzhou 450006, China
a r t i c l e
i n f o
Article history: Received 8 December 2015 Received in revised form 31 December 2015 Accepted 5 January 2016 Available online 17 February 2016 Keywords: Wheat starch Multi-scale structure In vitro digestion properties
a b s t r a c t Regulating the starch gastrointestinal digestion rate by control of its aggregation structure is an effective way, but the mechanism is still not clear. Multi-scale structure of waxy and normal wheat starches were studied by confocal laser scanning and scanning electron microscopes, as well as wide-angle and small-angle X-ray techniques in this study. In vitro digestion kinetics of those two starches and structure–digestion relationship were also discussed. Both waxy and normal starches show A-type diffraction pattern, but waxy variety shows a slightly higher crystallinity. Small-angle X-ray scattering results show that waxy wheat starch has higher scattering peak intensity (Imax ) and a larger crystallinity lamellar repeat distance (Lp ) compared with the normal wheat starch. We suggested that the higher digestion rate of waxy starch at initial stage is mainly due to more small-size particles, but the higher crystallinity and the larger crystalline lamellar size limit the digestion extent. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Glycemic index (GI) is an important parameter to evaluate nutritional properties for carbohydrate-containing foods according to Food and Agriculture Organization (FAO) and World Health Organization (WHO) [1]. Starch, a major carbohydrate source in human diets, is a key component to control the blood glucose level in human body. The starch in a high GI food is digested and absorbed rapidly, resulting in high postprandial blood glucose and insulin levels, which over the long term are enhancing the risks of diet-related diseases including type-2 diabetes and cardiovascular disease [2]. Therefore, controlling the digestion rate and extent of granular starches and understanding the factors to affect the starch digestion is important for starch-based food design and development [3]. The application of Log of Slope (LOS) analysis of digestibility curves based on the well-documented first-order reaction kinetics of amylolysis has been used to characterize the reaction rate of starch amylolysis. LOS plots could be very sensitive to changes in digestion rate constant from the conventional digestion curves,
∗ Corresponding author. Fax: +86 20 85280270. ∗∗ Corresponding author. Fax: +86 10 62813477. E-mail addresses: [email protected], [email protected] (P. Chen), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.ijbiomac.2016.01.119 0141-8130/© 2016 Elsevier B.V. All rights reserved.
and assist in understanding how starch digestion rates change deduced by structure differences [4–6]. The digestion rate (k) could be affected by the molecular level and aggregation structure of granular starches. For molecular level, it have been reported that the digestion rate tends to increase with longer chain length of long amylose branches and smaller ratios of long amylopectin to short amylopectin branches, as well as smaller relative amount of long amylose branches to short amylopectin branches [7]. The aggregation structures dominant by lamellar and crystalline structures [8], also play a key role in determining the digestion rate and extent. For example, Zhang et al. used extrusion technique to induce loworder starch with a relatively lower digestion rate [9]. However, the aggregation structures of granular starches are complicated, the mechanism to control the digestion rate and extent is still not fully understood [3]. Waxy wheat was firstly developed in Japan through traditional hybridizations genetic modification in 1995 [10], then was successfully introduced to Australia, USA, Canada and China [11,12]. Waxy wheat starch, which is biosynthesized when the three granulebound starch synthesis (GBSS) genes are absent or nonfunctional, has distinct structural characteristics such as very low amylose content and high crystallinity. The special structure leads to the desirable functional properties such as high swelling power and peak viscosity, low retrogradation level, setback viscosity and pasting temperature. With the increasing applications of waxy wheat starches in foods, a good understanding of the aggregation struc-
P. Chen et al. / International Journal of Biological Macromolecules 87 (2016) 28–33
29
Fig. 1. Typical starch digestion curves and LOS plots of cooked and raw wheat starches.
Fig. 2. Starch granules of waxy wheat starch (a1, a2, a3) and normal wheat starch (b1, b2,b3) observed by SEM and CLSM.
ture differences between waxy and normal wheat starches is required and further results in the different digestion rate and extent becoming increasingly important to the area of food processing and nutrition. Moreover, the thickness of crystalline and amorphous lamellar structure of waxy wheat starches as measured by small anger X-ray scattering (SAXS) and their relationship
with the molecular structure are not fully understood as well. In this study, the multi-scale aggregation state structure of normal and waxy wheat starches were investigated, and the digestion rate and extent were evaluated by LOS methods. In addition, the structure–digestion relationship of wheat starches were also discussed.
30
P. Chen et al. / International Journal of Biological Macromolecules 87 (2016) 28–33
2. Experimental
2.5. Small-angle X-ray scattering (SAXS)
2.1. Materials
The lamellar structure of starch was studied using a small-angle X-ray scattering system (Anton-Paar, Graz, Austria) equipped with a PW3830 X-ray generator (PANalytical), operated at 50 mA and 40 kV, using Cu K␣ radiation with a wavelength of 0.1542 nm as X-ray source. Starch slurries with similar moisture content (MC, about 65%) were prepared for this experiment and equilibrated at 20 ◦ C for 24 h before the test. The samples were filled into a sample cell and measured for 10 min. SAXS curves were plotted as a function of relative peak intensity, I, versus q, the scattering vector. 1-D correlation function were used to analyze the corresponding parameters such as long period Lp (also known as Bragg spacing d or lamellar repeat distance), hard block thickness Lc and soft block thickness La of starch lamellar structure [17].
Chinese spring (CS) wheat and its near-isogenic waxy type were used in this work. A waxy wheat line “Caiwx” and Yangmai01-2 were used as donor and recurrent parents, respectively. Starch was isolated using the dough-washing method as previously described [13]. The amylose content of waxy and normal wheat starches are 2.6% and 27% respectively, using a colorimetry method through determined by iodine binding [14]. ␣-Amylase (Sigma A-3176, from porcine pancreas, 250 U/mL), pepsin (Sigma P-6887, from porcine gastric mucosa), pancreatin (Sigma P-1750, from porcine pancreas) and amyloglucosidase (AMG, Sigma A-7420 from Aspergillus Niger) were used. All other chemicals were of analytical grade.
2.6. In vitro starch digestibility 2.2. Scanning electron microcopy A scanning electron microscope (X230, Philips, Eindhoven, Netherlands) was used to investigate the appearance and surface of the starch granules. The samples were coated with iridium in a vacuum evaporator, and viewed in the SEM at a low accelerating voltage of 2 kV. Diameters of starch granules were conducted using the Gun Image Manipulation Program. The diameter of individual granules was calculated using a sphere equal in area to the granule. More than 200 particles were calculated for each sample, and the results were based on the average of the measurement.
2.3. Confocal laser scanning microscopy About 5 g (dry basis) of starch granules was hydrolyzed by suspending in 100 mL of 2 mol/L HCl solution at 35 ◦ C for different times (days). After hydrolysis, the insoluble residues were washed several times with distilled water to neutrality. Native and hydrolyzed starch samples were prepared for CLSM as previously described [13,15]. Starch granules (∼10 mg) were dispersed in 15 L of freshly made APTS solution (10 mM APTS dissolved in 15% acetic acid) and 15 L of 1 M sodium cyanoborohydride was added. The reaction mixture was incubated at 30 ◦ C for 15–18 h, with the granules washed 5 times with 1 mL of distilled water and finally suspended in 20 L of 1:1 (v/v) glycerol/water mixture. A drop of the mixture was then mounted on a glass plate for microscopy. A confocal laser scanning microscope (TCS SP2, Leica Microsystems, Wetzlar, Germany) equipped with an Ar/Hg laser and a stand for fixed fluorescent cell samples was used to investigate the internal morphologies of wheat starches. The Leica objective lens used were: 60× plan apo/1.40 oil UV. During image acquisition, each line was scanned four times, and averaged to reduce noise.
Isolated starch granules (200 mg, dry weight basis) were heated in distilled water with a starch-to-water ratio of 1:5 in a tube for 15 min then cooled in −20 ◦ C refrigerator and freeze dried for in vitro experiment. Both raw and cooked starch was incubated with 1.0 mL artificial saliva solution containing 250 U/mL pancreatic ␣-amylase in carbonate buffer (14.4 mM, pH 7.0, containing 21.1 mM potassium chloride, 1.59 mM calcium chloride and 0.2 mM magnesium chloride) at ambient temperature for 1 min. Procine pepsin (1 mg/mL) in HCl solution (5 mL, 0.02 M) was then added to the mixture and the mixture was stirred at 80 rpm in a water bath at 37 ◦ C for 30 min. The digesta were then neuturalized with 5 mL NaOH (0.02 M) and mixed with 25 mL sodium acetate buffer (pH 6, 0.2 M, containing 200 mM CaCl2 and 0.49 M MgCl2 ). Pancreatin (2 mg/mL) and 58 U/mL amyloglucosidase in the same sodium acetate buffer solution (5 mL) was added to the digesta, and the mixture was incubated at 37 ◦ C and at 85 rpm. Aliquots (0.2 mL) were collected at different time points up to 180 min, and the digestion was stopped by adding 0.75 mL of 95% ethanol. The digestibility of freshly gelatinized starch (g/100 g of dry starch) was determined from the amount of glucose released in the supernatant, which was converted to the mass of digested starch by a factor of 0.9. The starch digestion data can be fitted to a piecewise first-order equation following Zou’s method [18]. A logarithm of slope (LOS) plot is obtained by expressing the first derivate of the first order equation in logarithmic form: ln(dCt /dt) = −kt + ln(C∞ k)
(1)
k and C∞ are calculated from the slope and intercept respectively. 2.7. Statistical analysis The mean values of digestion rate and relative crystallites of starch were analyzed with SPSS (version 22.0, IBM SPSS Statistics) using Tukey’s Pairwise Comparisons with confidence levels of 95% and 90% in performing an analysis of variance (ANOVA) test. 3. Results and discussion
2.4. Wide-angle X-ray diffraction 3.1. The digestibility of raw and cooked starch Wide-angle X-ray diffraction (WAXD) analysis was studied by a Bruker D8 Diffractometer operating at 40 kV and 40 mA, Cu K␣ radiation monochromatized with a graphite sample monochromator. The moisture content of all samples was adjusted to ∼10% before test to reduce the water effect. Relative crystallinity (RC) of the samples was quantitatively estimated by measuring the peak area of crystallinity following the method described before [16]. The intensity was measured from 2 to 40◦ as a function of 2 and at a scanning speed of 2◦ /min.
In vitro digestion kinetic profiles of cooked and raw starches are shown in Fig. 1. The digestion rate and extent of starch or starchcontaining food vary depending on the enzyme type/concentration [19], and rheology properties [20,21]. Therefore, the same physical state of starch was used in this kinetic study. In order to obtain a logarithmic digestion curves, both ␣-amylase and amyloglucosidase (AMG) were used to convert sufficient starch substrate to glucose products over the time course [4,7].
P. Chen et al. / International Journal of Biological Macromolecules 87 (2016) 28–33
50
percentage(%)
45 40
normal wheat starch
35
waxy wheat starch
30 25 20 15 10 5 0 2~5
5~8 8~12 12~15 15~18 18~21 21~24 24~27 27~30 30~33
Granule diameter(um) Fig. 3. Size distributions of waxy and normal wheat starch granules (in number).
Fig. 1 shows the typical starch digestion curve and LOS fit plots from cooked and raw normal and waxy wheat starches. For raw starches, it can be seen that the waxy starch was hydrolyzed rapidly at the early stage, but digestion extent of the waxy starch was lower after 6 h hydrolysis, comparing with the normal starch. However, it should be noted that there is more starch left after hydrolysis (such as after 24 h hydrolysis). For cooked starches, the digestion rate was increased significantly due to the gelatinization process which enhances the enzyme access to the starch molecules (substrates) [22]. LOS fitting analysis was applied to the starch digestion kinetic profiles to obtain first-order coefficients k (k values are listed in Fig. 1), showing that all digestion profiles can be described by a single-phase pseudo-first order process (R2 > 0.90). The digestion rate coefficient of cooked starches is markedly higher than that of raw starches. It should be noticed that the k value for cooked waxy wheat starch is much higher than cooked the wheat starch, which is very unusual and may be due to the quick retrogradation of high amylose content starch. 3.2. Multi-scale structure of starch The morphological and micro-structural features of waxy and normal wheat starches were revealed by SEM and CLSM (Fig. 2). SEM photos (Fig. 2) show that both waxy and normal wheat starches have a bimodal size distribution, comprising of small and
large granules. For wheat starches, small spherical granules (Bgranules) range in size from approximately 1 to 10 M, whereas larger lenticular-shaped granules range from about 15 to 40 M, known as A-granules. The A- and B-granules were smooth with no cracks on the surface, suggesting the low level of starch damage during fractionation. The waxy and normal wheat starch granules were ellipse and spherule shapes with size distribution between 2 and 35 M (Fig. 3) [23]. No significant differences were observed in the granule morphology of waxy and normal wheat starches. More smaller particles (
