Structural Orders of Wheat Starch Do Not Determine the In Vitro

Article pubs.acs.org/JAFC

Structural Orders of Wheat Starch Do Not Determine the In Vitro Enzymatic Digestibility Shujun Wang,*,† Shaokang Wang,† Lu Liu,† Shuo Wang,*,†,‡ and Les Copeland§ †

Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China ‡ Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100048, People’s Republic of China § School of Life and Environmental Sciences, The University of Sydney, Camperdown, New South Wales 2006, Australia ABSTRACT: In this study, we elucidated the underlying mechanisms that are responsible for the rate-limiting step for wheat starch digestion. Wheat starch samples with a degree of gelatinization (DG) ranging from 0 to 100% were prepared. As DG increased, the ordered structures of the starch were disrupted increasingly. In contrast, almost all of the increase in the rate and extent of in vitro enzymatic digestion coincided with a DG of only 6% and a minor loss of structural order. As DG increased beyond 6%, digestibility of the starch increased only slightly. We propose that the access and binding of enzymes to starch is greatly increased with only a small DG, which is followed by the simultaneous hydrolysis of crystalline and amorphous areas in gelatinized starch. In vitro enzymatic digestibility of starch was determined predominantly by enzyme binding to starch rather than the ordered structures of starch. KEYWORDS: starch digestion, kinetic constant, enzyme binding, degree of gelatinization, multiscale structure

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action.5 Local molecular density in starch as a result of crystallinity or dense packing of amorphous regions is considered to have a major influence on amylase digestion kinetics, by limiting enzyme binding and/or slowing catalysis.6 Most starch consumed by humans has undergone some form of cooking or hydrothermal treatment, which leads to varying degrees of starch gelatinization in processed starchy food products.7−12 The effect of gelatinization on in vitro digestibility of starch has been wellstudied previously.13−16 Gelatinization increases the susceptibility of starch to enzymatic hydrolysis, with many studies showing that a higher degree of gelatinization (DG) leads to greater digestibility of the starch.14,15,17,18 However, some studies showed that there were only small differences in starch digestibility between partially gelatinized and fully gelatinized starch.16,19,20 Hence, there is a need to resolve the relationship between starch structural organization and the digestion kinetics to further our understanding of the mechanism of starch amylolysis. In the present study, we prepared gelatinized starch samples under controlled conditions with DG ranging from 0 to 100%. The in vitro enzymatic digestibility of these starch samples was evaluated and correlated with the changes in the multiscale starch structures as measured using differential scanning calorimetry (DSC), X-ray diffraction (XRD), attenuated total reflectance− Fourier transform infrared (ATR−FTIR) and Raman spectroscopies, scanning electron microcopy (SEM), and light microscopy (LM). To the best of our knowledge, this is the

INTRODUCTION The enzymatic degradation of biomacromolecules is often complex and involves a combination of enzymes, which act on the substrate with different specificities and at different rates. Identifying the factors that determine the rate and extent of breakdown of biomacromolecules (such as starch and protein) is important for human and animal nutrition, food and beverage processing, and industrial fermentations. Using starch as an example substrate to identify the key factors determining the enzymatic degradation of biomacromolecules is highly recommended given the wide application of starch in industries.1 The complex, multiscale structural organization within starch granules arises during starch biosynthesis. The arrangement of double helices, crystalline and amorphous lamellae, super helices, blocklets, and growth rings into the semi-crystalline granules is the major determinant of starch functionality, including susceptibility to enzymic breakdown.2 Upon heating in water, starch granules undergo an irreversible phase transition, referred to as gelatinization, during which the multiscale structures of the granules are disrupted progressively.3 The rate and extent of starch gelatinization are dependent upon a multiplicity of factors, including the origin and type of starch, the water availability, the temperature, the rate and duration of heating, and the magnitude of shear forces.4 The extent of structural changes that starch undergoes during gelatinization is the major determinant of starch functionality for food processing and human nutrition. Because starch is the most important glycemic carbohydrate in foods, starch digestibility is of considerable interest nutritionally in relation to the increasing incidence of obesity and diet-related chronic diseases.4 The digestibility of starch has been proposed to be determined predominantly by two types of factors: (i) barriers that slow or prevent access/binding of the enzyme to starch and (ii) structural features that slow or prevent amylase © 2017 American Chemical Society

Received: Revised: Accepted: Published: 1697

September 9, 2016 February 4, 2017 February 4, 2017 February 4, 2017 DOI: 10.1021/acs.jafc.6b04044 J. Agric. Food Chem. 2017, 65, 1697−1706

Article

Journal of Agricultural and Food Chemistry Table 1. Thermal Properties of Starch after Heating at Different Temperatures and Water Contentsa

a

treatment

To (°C)

Tp (°C)

Tc (°C)

ΔH (J/g)

DG (%)

NWS NWS-30%-50 °C NWS-40%-50 °C NWS-50%-50 °C NWS-60%-50 °C NWS-62.5%-50 °C NWS-30%-55 °C NWS-30%-60 °C NWS-40%-55 °C NWS-30%-65 °C NWS-50%-55 °C NWS-40%-60 °C NWS-60%-55 °C NWS-40%-65 °C NWS-50%-60 °C NWS-60%-60 °C NWS-50%-65 °C NWS-60%-65 °C

56.9 ± 0.2 a 57.4 ± 0.1 b 58.6 ± 0.1 c 58.8 ± 0.1 cd 59.1 ± 0.2 d 58.7 ± 0.1 c 59.9 ± 0.0 e 60.7 ± 0.1 f 61.3 ± 0.1 g 62.0 ± 0.0 hi 61.7 ± 0.1 h 61.9 ± 0.1 hi 62.1 ± 0.2 i 62.1 ± 0.2 hi 63.0 ± 0.4 j 64.1 ± 0.4 k 64.0 ± 0.3 k NDb

61.6 ± 0.1 a 61.4 ± 0.3 a 61.9 ± 0.1 b 62.1 ± 0.0 bc 62.5 ± 0.2 d 62.2 ± 0.1 cd 62.9 ± 0.1 e 63.9 ± 0.2 f 64.4 ± 0.2 g 65.4 ± 0.1 h 65.2 ± 0.1 h 65.5 ± 0.2 h 66.1 ± 0.1 i 66.2 ± 0.3 i 66.9 ± 0.4 j 68.0 ± 0.3 k 67.7 ± 0.1 k ND

66.8 ± 0.4 bc 65.9 ± 0.3 a 66.1 ± 0.0 a 66.4 ± 0.2 ab 67.0 ± 0.2 bc 66.9 ± 0.2 bc 67.3 ± 0.3 c 68.1 ± 0.3 d 69.1 ± 0.2 e 70.0 ± 0.1 h 70.1 ± 0.1 h 69.9 ± 0.2 h 70.7 ± 0.1 i 71.1 ± 0.1 i 71.9 ± 0.2 j 72.7 ± 0.8 k 73.8 ± 0.8 l ND

10.8 ± 0.2 n 10.1 ± 0.2 m 9.8 ± 0.2 l 9.7 ± 0.1 l 9.1 ± 0.2 k 8.9 ± 0.2 k 8.9 ± 0.1 k 8.2 ± 0.4 j 7.0 ± 0.3 i 6.4 ± 0.0 h 4.8 ± 0.2 g 4.2 ± 0.3 f 3.0 ± 0.1 e 2.5 ± 0.0 d 2.1 ± 0.1 c 1.1 ± 0.0 b 0.5 ± 0.0 a ND

0.0 6.0 9.1 9.7 15.3 16.9 17.2 23.6 35.2 40.8 55.5 60.7 71.8 76.4 80.3 89.8 95.4 100.0

Values are means ± SD. Values with the same letters in the same column are not significantly different (p < 0.05). bND = not determined. where ΔHgelatinized starch is the enthalpy change of gelatinized starch and ΔHnative starch is the enthalpy change of native starch. In Vitro Enzymatic Digestibility of Starch Samples. Starch digestibility was analyzed according to the procedure of Wang et al.,26 which was modified from Englyst et al.27 Starch (100 mg, dry weight basis) was dispersed in 4 mL of 0.1 M sodium acetate buffer (pH 5.2) containing 6.67 mmol/L CaCl2, and 1 mL of freshly prepared enzyme solution containing 1645 units of amylase and 41 units of amyloglucosidase was added. The starch/enzyme mixtures were incubated at 37 °C with stirring at 260 rpm for 2 h. At specific time points (from 5 to 120 min), an aliquot (0.05 mL) of the hydrolysate was withdrawn and mixed with 0.95 mL of 95% ethanol to deactivate the enzymes. The amount of glucose released was determined using the Megazyme GOPOD kit. The percentage of hydrolyzed starch was calculated by multiplying the glucose content with a factor of 0.9. The digestograms of starch hydrolysis were fitted to the first-order rate equation5,28−30

first study to reveal digestion mechanisms of gelatinized starches with such a wide range of DG.

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MATERIALS AND METHODS

Materials. Wheat grains (Triticum aestivum L. var. Yangmai) were provided by Lixiahe Agricultural Research Institute, Yangzhou, Jiangsu, China. Starch was isolated from the grains according to the method of Wang et al.21 The apparent amylose content of the starch was 27.7%, as determined according to the method of Chrastil.22 The moisture, lipid, and protein contents were 8.7, 0.4, and 0.22%, respectively, as measured by the methods of AOAC International.23 The glucose oxidase/ peroxidase kit (GOPOD format) and Aspergillus niger amyloglucosidase (3260 units/mL) were purchased from Megazyme International Ireland, Ltd. (Bray Co., Wicklow, Ireland). α-Amylase (Sigma, EC 3.2.1.1, type VI-B from porcine pancreas, 13 units/mg) was purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other chemical reagents were of analytical grade. Preparation of Gelatinized Starch Samples. Gelatinized starch samples were prepared by heating starch−water mixtures with moisture contents of 30, 40, 50, 60, and 62.5% (w/w, wet basis) at temperatures of 50, 55, 60, and 65 °C for 5 min, giving a total of 18 samples. The native starch was weighed accurately into polypropylene bags, and the correct amount of distilled water was added to give the desired moisture contents. The bags were sealed and heated in a water bath at the specified temperatures, after which the samples were immersed immediately in liquid nitrogen, freeze-dried, ground into a powder, and passed through a 100 μm sieve for further use. Thermal Properties of Starch Samples. Thermal properties of the starch samples were examined using a differential scanning calorimeter (200 F3, Netzsch, Germany) equipped with a thermal analysis data station. Starch samples (approximately 3 mg, 8.7% moisture) were weighed accurately into a 40 μL aluminum pan. The DSC enthalpy change of wheat starch was found previously to be constant at a starch/water ratio of 1:5.24 Hence, a starch/water ratio of 1:5 (w/v) was used in the present study to determine the thermal properties of the heated starches. The starch−water mixtures were allowed to stand overnight at room temperature before DSC measurement. The samples were heated from 20 to 100 °C at a heating rate of 10 °C/min. An empty aluminum pan was used as the reference. The DG, which is a measure of the amount of crystallites that melted during heating,14,15,25 was calculated according to the formula

Ct = C∞(1 − e−kt ) where Ct is the amount of starch digested at time t (min), C∞ is the estimated amount of starch digested at the reaction end point, and k (min−1) is the first-order rate coefficient. For ease of interpretation, Ct was expressed as the percentage of starch hydrolyzed. The value of k can be calculated from the slope of a linear least squares fit of a plot of ln(1 − C/C∞) against t. Crystallinity of Starch Samples. Crystallinity of starch samples was analyzed using a D/Max-2500VK/PC X-ray diffractometer (Shimadzu, Japan) operating at 40 kV and 40 mA. Starch samples were equilibrated over a saturated NaCl solution for 1 week before measurement. The starch powder was packed tightly in a round glass cell and scanned from 5° to 35° (2θ) at a rate of 1°/min and a step size of 0.02°. The relative crystallinity was calculated as the ratio of the crystalline area to the total area between 5° and 35° (2θ) using the Jade 6.0 software. ATR−FTIR Spectroscopy. The infrared spectra of starch samples were obtained using a Thermo Scientific Nicolet IS50 spectrometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Starch (150 mg) was pressed into round tablets and scanned between 4000 and 400 cm−1. Each spectrum was recorded against air as the background. Spectra were baseline-corrected automatically by OMNIC 8.0 and deconvoluted from 1200 to 800 cm−1 with a half bandwidth of 19 cm−1 and an enhancement factor of 1.9. The ratio of absorbances at 1047/1022 cm−1 was obtained from the deconvoluted spectra to characterize the short-range molecular order of starch samples.

DG (%) = (1 − ΔHgelatinized starch /ΔHnative starch) × 100 1698

DOI: 10.1021/acs.jafc.6b04044 J. Agric. Food Chem. 2017, 65, 1697−1706

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Journal of Agricultural and Food Chemistry

Figure 1. (a) Digestograms of starch samples with different DG, (b) fit of digestion data to first-order kinetics, and (c) kinetic constants of some representative samples. Laser Confocal Micro (LCM)-Raman Spectroscopy. Raman spectra were recorded using a Renishaw Invia Raman microscope system (Renishaw, Gloucestershire, U.K.) equipped with a Leica microscope (Leica Biosystems, Wetzlar, Germany) and a 785 nm green diode laser source. Each spectrum of starch samples (4000−400 cm−1) was collected from at least five different spots with a resolution of approximately 7 cm−1. The full width at half maximum (fwhm) of the band at 480 cm−1 was calculated using the software of WIRE 2.0, which can be taken as an indicator of the ordered structure in starch.20,31 SEM. The morphology of starch samples was imaged using a scanning electron microscope (JSM-IT300LV, JEOL, Japan). The freeze-dried samples were mounted on the stub double-sided adhesive tape and coated with gold prior to imaging. An accelerating voltage of 5 kV was used during imaging. LM. A light microscope (DM-400M-LED, Leica, Germany) was used to observe the changes in granule birefringence during gelatinization. Approximately 0.1 g of starch samples was weighed into the plastic tubes, and 5 mL of ethanol was added and mixed homogeneously. One drop of suspension was applied onto a microscope slide, covered with a coverslip, and dried in a horizontal position for 5 min. A polarized light mode was used for imaging. Statistical Analysis. All analyses were performed at least in triplicate, and the results are reported as the mean values and standard deviations (SDs). In the case of XRD, only one measurement was

performed. One-way analysis of variance (ANOVA) followed by posthoc Duncan’s multiple range test (p < 0.05) was conducted to determine the significant differences between mean values using the SPSS 19.0 Statistical Software Program (SPSS, Inc. Chicago, IL, U.S.A.).

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RESULTS

Thermal Properties of Starch Samples with Different DG. The onset (To), peak (Tp), and conclusion (Tc) temperatures, enthalpy change (ΔH), and DG are given in Table 1. As DG increased from 0 to 95.4%, To, Tp, and Tc increased gradually from 56.9, 61.6, and 66.8 °C, respectively, to corresponding values of 64.0, 67.7, and 73.8 °C. Over the same range of DG, ΔH decreased from 10.8 to 0.5 J/g (Table 1). The increased thermal transition temperatures of the preheated starch samples are attributed to the preferential melting of less stable crystallites, leaving behind more stable residual crystallites with a higher melting temperature.3 The decrease in ΔH is due to fewer crystallites remaining as DG increased. In Vitro Enzymatic Digestibility. The in vitro enzymatic digestograms of starch samples with different DG are shown in Figure 1a. Native starch presented a more or less linear digestion profile over 2 h. In contrast, all of the digestograms of starches 1699

DOI: 10.1021/acs.jafc.6b04044 J. Agric. Food Chem. 2017, 65, 1697−1706

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Journal of Agricultural and Food Chemistry Table 2. Crystallinity, Infrared (IR) Ratios, fwhm, and Kinetic Constant (k) of Starch Samples with Different DGa

a

DG (%)

crystallinity (%)

IR ratio of 1047/1022 cm−1

IR ratio of 1022/998 cm−1

fwhm at 480 cm−1

k (min−1)

0 6.0 9.1 9.7 15.3 16.9 17.2 23.6 35.2 40.8 55.5 60.7 71.8 76.4 80.3 89.8 95.4 100

26.7 22.8 22.3 22.0 20.9 20.8 20.1 19.3 17.6 16.9 16.1 15.2 11.3 11.7 10.6 7.1 5.2 4.1

0.703 ± 0.007 h 0.669 ± 0.002 fg 0.669 ± 0.000 fg 0.669 ± 0.000 fg 0.666 ± 0.002 fg 0.668 ± 0.002 fg 0.668 ± 0.001 fg 0.662 ± 0.003 ef 0.658 ± 0.001 de 0.652 ± 0.005 cd 0.649 ± 0.004 c 0.647 ± 0.003 bc 0.639 ± 0.004 a 0.647 ± 0.003 bc 0.642 ± 0.003 ab 0.651 ± 0.004 c 0.653 ± 0.000 cd 0.648 ± 0.004 bc

1.157 ± 0.014 a 1.168 ± 0.005 abcd 1.164 ± 0.011 abc 1.160 ± 0.001 ab 1.169 ± 0.019 abcd 1.171 ± 0.015 abcde 1.173 ± 0.011 abcdef 1.198 ± 0.018 abcdef 1.211 ± 0.012 cdefg 1.201 ± 0.027 abcdef 1.209 ± 0.039 bcdefg 1.216 ± 0.037 defg 1.220 ± 0.024 efg 1.203 ± 0.008 abcdef 1.220 ± 0.003 efg 1.220 ± 0.006 efg 1.221 ± 0.023 fg 1.257 ± 0.042 g

16.17 ± 0.39 a 17.16 ± 0.08 b 17.25 ± 0.34 b 17.34 ± 0.23 b 17.57 ± 0.14 bcd 17.45 ± 0.24 bc 17.48 ± 0.22 bc 17.95 ± 0.38 de 17.87 ± 0.37 cde 18.20 ± 0.60 ef 18.26 ± 0.44 ef 18.44 ± 0.48 f 18.51 ± 0.54 f 18.65 ± 0.53 f 19.21 ± 0.57 g 19.69 ± 0.51 h 21.20 ± 0.88 i 21.05 ± 0.45 i

0.008 ± 0.001 a 0.020 ± 0.000 b 0.021 ± 0.002 bc 0.020 ± 0.002 b 0.024 ± 0.001 cde 0.023 ± 0.003 bcd 0.025 ± 0.002 cde 0.025 ± 0.003 cde 0.026 ± 0.002 de 0.026 ± 0.000 de 0.026 ± 0.002 de 0.027 ± 0.002 e 0.025 ± 0.000 cde 0.026 ± 0.001 de 0.027 ± 0.004 e 0.027 ± 0.001 e 0.026 ± 0.003 de 0.026 ± 0.001 de

Values are means ± SD. Values with the same letters in the same column are not significantly different (p < 0.05).

Long-Range Ordered Structure of Starch Samples Measured by XRD. The XRD patterns of native starch and starch samples with different DG are shown in Figure 2. Native

that had been preheated were characterized by a rapid digestion phase during the initial 40 min, followed by a slower digestion phase (Figure 1a). The amounts of native starch hydrolyzed at 5 min and 2 h were 2.4 and 54.1%, respectively. For starch samples with DG from 6.0 to 100%, there were no significant differences in the percent hydrolysis at each time point. For example, the final digestion percentages at 2 h were 88.1, 90.3, 85.0, 88.2, and 87.3% for starch samples with DG of 6.0, 16.9, 40.8, 76.4, and 100%, respectively. To gain a better mechanistic understanding of the enzymic digestion of starch samples with different DG, the digestograms were fitted to the first-order kinetic equation. Because starch samples with DG from 6 to 100% all showed similar digestion profiles, only the fits of digestograms of the samples with DG of 9.1, 40.8, 71.8, and 95.4% are presented in panels b and c of Figure 1. The values of k derived from the fit of the data to the first-order kinetic equation are given in Table 2. All of the correlation coefficients derived from Figure 1a were above 0.99, confirming that the enzymic digestion of the starches fitted well to first-order kinetics and is a single-phase process. The plot of ln(1 − C/C∞) against t was linear, with all R2 values above 0.98. The values of k obtained from the slopes of the linear plots ranged from 0.008 min−1 for native starch to 0.026 min−1 for fully gelatinized starch (Table 2). The k value has been suggested to indicate the susceptibility of starch or starchy foods toward amylolysis.32 Higher k values indicate faster digestion rates. As expected, the lowest k value in this experiment (0.008 min−1) was obtained for native starch, which had the slowest digestion rate. Starch with the lowest DG (6.0%) had a 2.5-fold increase in the k value (0.020 min−1) over that for native starch. Interestingly, an increase in DG to 15.3% led to a further increase of only 20% in the k value to 0.024 min−1. No significant increases in k occurred as DG continued to increase (Table 2 and Figure 1c). In summary, these results show that almost all of the increase in the rate and extent of amylolysis occurred as the DG was raised from 0 to 6.0% and that further increases in DG had little effect on the rate and extent of enzymic hydrolysis. To the best of our knowledge, this is the first study that starch samples with such a wide range of DG did not show significant differences in in vitro enzymatic digestibility.

Figure 2. XRD patterns of starch samples with different DG.

wheat starch exhibited a typical A-type XRD pattern, with strong reflections at about 15°, 17°, 18°, and 23° (2θ).21 As seen in Figure 2, the XRD peaks decreased markedly as DG increased, almost disappearing as the DG reached 95.4%, with the exception of the amylose−lipid complex peak at 20.0° (2θ).20 The relative crystallinity decreased gradually from 26.7% for native starch to 4.1% for fully gelatinized starch (Table 2). These observations were consistent with the DSC results. The residual crystallinity of fully gelatinized wheat starch could be attributed to the presence of the crystalline amylose−lipid complex, which melts at about 115−125 °C.33 Short-Range Molecular Order Measured by ATR−FTIR and LCM-Raman Spectroscopies. The ATR−FTIR spectra of starch samples with different DG are presented in Figure 3. The ratios of absorbances at 1047/1022 cm−1 showed a decreasing trend, and those at 1022/998 cm−1 showed an 1700

DOI: 10.1021/acs.jafc.6b04044 J. Agric. Food Chem. 2017, 65, 1697−1706

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Journal of Agricultural and Food Chemistry

during gelatinization or retrogradation.2,3 Band narrowing is an indicator for a narrower distribution of band energy in the more ordered structure; thus, fwhm can reflect the degree of ordered structure in starch. With increasing DG, the Raman bands became decreasingly pronounced and the fwhm of the band at 480 cm−1 increased from 16.17 for native starch to 21.05 for fully gelatinized starch. These observations indicated that the shortrange ordered structure of starch decreased with increasing DG, consistent with XRD and FTIR results. With increasing DG, the fwhm at 940 and 865 cm−1 showed a similar change as observed for the band at 480 cm−1. Granular Morphology Observed by SEM and LM. The SEM micrographs of starch samples with different DG are shown in Figure 5. Native wheat starch is composed of large disk-like granules (A type) and small spherical granules (B type). Grooves, indentations, or pinholes were observed on the surface of some starch granules, as reported in other studies.26,41,42 No obvious changes in granular morphology, except for coalescence of some starch granules, were observed at DG below 16.9% (Figure 5f). However, at greater DG, the starch granules became increasingly disrupted (panels g−r of Figure 5). At a DG of 80.3% (Figure 5o), a few intact starch granules were still evident, but as the DG reached 90%, no granules were observed, indicative of the complete disruption of granular morphology. The series of images in Figure 6 is consistent with the polydispersity of starch granule morphology and other evidence showing that starch gelatinization is not a homogeneous process.43 Polarized light microscopy (PLM) images showed native wheat starch to produce a clear “Maltese cross” pattern (Figure 6). As DG increased, the central regions of the “Maltese cross” became more blurred (panels c−f of Figure 6), leading to a complete loss of birefringence when the DG was 76.4% (Figure 6o), consistent with the complete disruption of starch crystallites, in agreement with SEM results.

Figure 3. ATR−FTIR spectra of starch samples with different DG.

increasing trend, as DG increased (Table 2). The IR spectrum of starch, especially the region at 1300−800 cm−1 assigned to C−C and C−O stretching and C−H−O bending modes, is sensitive to the changes in short-range molecular order of starch.34,35 The bands at 1047 and 1022 cm−1 are sensitive to the changes in the crystalline and amorphous structure of starch, respectively,34 allowing the ratios of absorbances at 1047/1022 and 1022/998 cm−1 to be used to characterize the ordered structure of starch. Higher ratios of absorbances at 1047/1022 cm−1 and lower ratios of absorbances at 1022/998 cm−1 indicate higher relative crystallinity.36−38 Hence, our observations indicated that the short-range molecular order of starch was disrupted increasingly with increasing DG, consistent with DSC and XRD results. The Raman spectra of starch samples with different DG and the corresponding fwhm of the band at 480 cm−1 are presented in Figure 4 and Table 2, respectively. Several clear bands were observed at 2900, 1264, 940, 865, and 480 cm−1 (Figure 4), which are related to C−H stretching, CH2OH (side chain) related mode, skeletal mode vibrations of α-1,4 glycosidic linkages (C−O−C), CH2 deformation, and skeletal modes of the pyranose ring, respectively.31,39,40 Of these, the band at 480 cm−1 is usually used to characterize the change of molecular order

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DISCUSSION Effect of DG and Structural Orders on In Vitro Starch Digestibility. In the present study, we studied the multiscale structures and in vitro enzymatic digestibility of starch samples with DG ranging from 0 to 100%. Disruption of the multiscale structures in granules occurred progressively with increasing DG, although the degree of disruption was different (Figures 7a). Of these multiscale structures, crystallinity and the ratios of absorbances at 1047/1022 cm−1 showed strong and weak negative linear correlations, respectively, with DG (R2 = 0.96 and 0.59), whereas the ratios of absorbances at 1022/998 cm−1 and the fwhm of the band at 480 cm−1 showed positive linear correlations with DG (R2 = 0.84 and 0.84). The above results indicated that ΔH reflects primarily the disruption of long-range molecular orders rather than short-range molecular orders in starch granules.3 The effect of DG on degree of hydrolysis (DH), which is defined as the percentages of starch hydrolysis after 2 h of incubation, and the first-order kinetic constant (k) is shown in Figure 7b. It can be clearly seen that no linear correlations were observed between DG and DH and between DG and k. All of the increase in susceptibility to enzymic breakdown, as indicated by the value of k and DH, occurred when the DG was less than 10% (Figure 7b). The values of DH and k did not change significantly as DG values were greater than 10%. These results indicate that the extent of structural order is not the key determinant of the rate of in vitro enzymatic digestibility of cooked starch. The small differences in enzymic digestibility between starches with DG

Figure 4. Raman spectra of starch samples with different DG. 1701

DOI: 10.1021/acs.jafc.6b04044 J. Agric. Food Chem. 2017, 65, 1697−1706

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Journal of Agricultural and Food Chemistry

Figure 5. SEM images of starch samples with different DG: (a) DG = 0, (b) DG = 6.0, (c) DG = 9.1, (d) DG = 9.7, (e) DG = 15.3, (f) DG = 16.9, (g) DG = 17.2, (h) DG = 23.6, (i) DG = 35.2, (j) DG = 40.8, (k) DG = 55.5, (l) DG = 60.7, (m) DG = 71.8, (n) DG = 76.4, (o) DG = 80.3, (p) DG = 89.8, (q) DG = 95.4, and (r) DG = 100.

starch increased with increasing DG of the starch14,15,17,19,44,45 or how fully gelatinized starch had higher in vitro enzymatic digestibility than partially gelatinized starch.19 The slower digestion rate of starch with a lower DG was attributed to a greater amount of residual crystallites.19 Starch crystallites are

ranging from 6 to 100% indicated that structural order in gelatinized starch is not the major factor in determining the rate of enzymic digestion of starch. Reports from several previous studies have described how the in vitro enzymatic digestibility or in vivo glycemic response of 1702

DOI: 10.1021/acs.jafc.6b04044 J. Agric. Food Chem. 2017, 65, 1697−1706

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Journal of Agricultural and Food Chemistry

Figure 6. PLM images of starch samples with different DG: (a) DG = 0, (b) DG = 6.0, (c) DG = 9.1, (d) DG = 9.7, (e) DG = 15.3, (f) DG = 16.9, (g) DG = 17.2, (h) DG = 23.6, (i) DG = 35.2, (j) DG = 40.8, (k) DG = 55.5, (l) DG = 60.7, (m) DG = 71.8, (n) DG = 76.4, (o) DG = 80.3, (p) DG = 89.8, (q) DG = 95.4, and (r) DG = 100.

Proposed Mechanism for In Vitro Digestibility of Gelatinized Starch. The limiting factors for enzymic hydrolysis of granular starch or gelatinized starch have been proposed to involve structural features that reduce access or binding of

considered to be a key structural barrier that slows the action of digestive enzymes.6 However, there is evidence from other studies that the in vitro enzymatic digestibility of cooked starch is not greatly affected by the extent of gelatinization.16,20 1703

DOI: 10.1021/acs.jafc.6b04044 J. Agric. Food Chem. 2017, 65, 1697−1706

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Journal of Agricultural and Food Chemistry

enzymes to the substrate and/or slow amylase action.5 On the basis of our results and those of previous studies, we offer a mechanistic explanation for residual structural order not being the major determinant of the rate of enzymic digestion of cooked starch (Figure 8). We propose that the outer layer of native starch granules contains glucan chains packed in tight semi-crystalline structures, forming a shell-like barrier that hinders binding of enzymes (Figure 8Aa). Disruption of this barrier at very low DG could greatly increase the access/binding of enzymes to the starch, even though most of the structural organization within the granule remains intact (Figure 8Ab). Further increases in DG or disruption of ordered structures do not increase the access/ binding of enzymes to the starch (panels Ac and Ad of Figure 8). Our results are consistent with the hypothesis that the binding rate of amylase to starch is strongly dependent upon the degree of order of the α-glucan chains of starch45,46 and that the formation of the enzyme−substrate complex is the rate-limiting step for the first-order kinetics of starch digestion.1 Moreover, the results illustrate the basic concept of enzyme kinetics that binding and catalysis are separate aspects of enzyme-catalyzed reactions, with separate terms to describe binding (e.g., Michaelis−Menten constant) and catalysis (maximum velocity). As proposed by many researchers and highlighted by Zhang et al.,47 the crystalline and amorphous structures are digested evenly by amylases. Once the enzymes bind to the substrate, subsequent catalysis is not rate-limiting (Figure 8B). The in vitro enzymatic digestibility of wheat starch with a wide range of DG from 0 to 100% was evaluated, and the mechanisms involved in the rate-determining step of starch digestion were revealed. Gelatinization, despite at a low degree, greatly increased the in vitro digestibility of starch. In comparison of enzyme digestibility to DG and loss of structural order, we conclude that DG or degree of ordered structure in starches is not the major rate-limiting factor for in vitro digestibility. We propose that the access/binding of enzymes to starch dominates the digestion

Figure 7. (a) Relationships between DG and multiscale structures and (b) DG and DH (upper line) and DG and the first-order kinetic constant (k) (lower line).

Figure 8. Schematic diagram to show the process of two steps of digestion: (A) enzyme access/binding to substrate (rate limiting) and (B) enzymatic catalysis (even digestion pattern of starch chains) for (a) native starch, (b) starch with low DG, (c) starch with intermediate DG, and (d) fully gelatinized starch. 1704

DOI: 10.1021/acs.jafc.6b04044 J. Agric. Food Chem. 2017, 65, 1697−1706

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Journal of Agricultural and Food Chemistry

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process and structural features are not the rate-limiting factor for starch digestion. Taken together, we conclude that the access/ binding of enzymes to starch rather than the catalytic hydrolysis is the rate-determining step for starch digestion.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: 86-22-60912486. E-mail: [email protected]. *Telephone: 86-22-60912486. E-mail: [email protected]. ORCID

Shujun Wang: 0000-0003-4501-8047 Shuo Wang: 0000-0003-0910-6146 Funding

The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (31522043 and 31430068). Shujun Wang also greatly appreciates the financial support by the Program for Innovative Research Team in University (IRT IRT_15R49). Notes

The authors declare no competing financial interest.

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