Revisiting Mechanisms Underlying Digestion of Starches - American

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Revisiting Mechanisms Underlying Digestion of Starches Yanhua Wang,†,‡ Chen Chao,†,‡ Hongjie Huang,†,‡ Shaokang Wang,‡,§ Shuo Wang,∥ Shujun Wang,*,†,‡ and Les Copeland⊥

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State Key Laboratory of Food Nutrition and Safety and ‡School of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, People’s Republic of China ∥ Tianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai University, Tianjin 300071, People’s Republic of China ⊥ Sydney Institute of Agriculture, School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia ABSTRACT: The factors that determine the digestion rate of starches were revealed using different forms of starches and a mixture of α-amylase and amyloglucosidase. Gelatinized starch samples with a degree of gelatinization (DG) from 12.2 to 100% for potato starch and from 7.1 to 100% for lotus seed starch were obtained. With an increasing DG, the short- and long-range molecular orders of both starches were disrupted progressively. The first-order digestion rate constant (k) of both starches increased with an increasing DG, although the positive linear relationships between DG and k differed (R2 = 0.87 for potato starch, and R2 = 0.74 for lotus seed starch). The mean fluorescence intensity showed a positive linear correlation with DG, which was strong for potato starch (R2 = 0.99) and relatively weaker for lotus seed starch (R2 = 0.54). These results indicated that DG is a major determinant for the digestion rate of potato starch and lotus seed starch and that the access/binding of enzymes to starch was the main rate-limiting factor for digestion of starches. KEYWORDS: potato starch, lotus seed starch, degree of gelatinization, digestion rate, enzyme binding, fluorescence intensity

1. INTRODUCTION Starch is the main energy source for both humans and monogastric mammals (excluding carnivores). The rate and extent of starch digestion affects the postprandial blood glucose concentrations and corresponding insulin release, which is of considerable nutritional interest in relation to the increasing incidence of obesity and diet-related chronic diseases.1−3 The digestion of starch is dependent upon many factors that influence the initial enzymatic binding to and subsequent catalytic action on starch.4,5 The digestibility of isolated starch is dependent upon a multiplicity of factors, including botanical origin, granular morphology structure (shape, size, and porosity), degree of crystallinity, amylose content, fine structure of amylose and amylopectin, and surface-associated proteins and lipids.3,6−8 Incompletely gelatinized starch granules occur in raw and lightly processed foods, and their enzymatic breakdown is of greater interest for animal feeds and industrial fermentations than for human digestion. When heated in excess water, starch granules are gelatinized with the disruption of multi-scale ordered structure. The structural changes of starch during cooking or processing are considered to be a major determinant of starch functionality for food processing and human nutrition.9−11 The enzymatic hydrolysis of cooked starch is affected by the degree of gelatinization (DG)3,8,12 or the quantity of flexible α-glucan chains protruding from the gelatinized granule surface.13 Some studies have shown that amylolysis of starch is increased with increasing DG.14−18 However, other studies showed that cooking conditions19,20 or degree of structural order in gelatinized starch21,22 has little effect on starch digestion. These discrepancies, which are likely to reflect the particular experimental conditions, suggest the © XXXX American Chemical Society

need to further study the mechanisms underlying the digestibility of cooked starches, especially from different botanical sources. In a previous study, we investigated comprehensively the relationship between DG varying from 0 to 100% and in vitro digestibility of wheat starch (WS). Our results showed that DG, as measured by the extent of disruption of the crystalline structure, was not the major determinant of the rate and extent of starch digestion. We proposed that the digestion rate of cooked WS was determined predominantly by enzyme access/ binding to starch rather than the subsequent catalytic reaction.22 Although similar proposals have been discussed by others,5 direct supporting experimental evidence is still limited. Therefore, to test this hypothesis, the effects of DG on the digestibility of B-type potato starch and C-type lotus seed starch were investigated, so that the general principles that determine the digestion rate of cooked starches may be better understood.

2. MATERIALS AND METHODS 2.1. Materials. Potato starch (PS), α-amylase (PPA, A3176, EC 3.2.1.1, type VI-B from porcine pancreas, 13 units/mg), α-amylase (AA, A6255), and fluorescein isothiocyanate (FITC, F7250) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Lotus seed starch (LSS) was isolated from lotus seeds (Green Field Fujian Food Co., Ltd., Fujian, China) according to a method described previously.23 The amylose contents of PS and LSS were 33.2 and 50.8%, respectively, as determined by the method of Chrastil.24 The moisture, lipid, and crude Received: April 26, 2019 Revised: June 30, 2019 Accepted: July 3, 2019

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DOI: 10.1021/acs.jafc.9b02615 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

protein contents were 12.6, 0.1, and 0.29% for PS and 10.9, 0.2, and 0.27% for LSS, respectively, as measured by the methods of AOAC Method 925.10, AOAC Method 920.39, and AOAC Method 979.09.25 WS used was described by Wang et al.22 Glucose oxidase/peroxidase kit (GOPOD format) and Aspergillus niger amyloglucosidase (AMG, 3260 units/mL) were purchased from Megazyme International Ireland, Ltd. (Bray Co., Wicklow, Ireland). All other chemical reagents were of analytical grade. 2.2. Preparation of Gelatinized Starch Samples. PS and LSS were weighed accurately into polypropylene bags, and deionized water was added to obtain moisture contents of 40, 50, and 60% for PS and 35, 40, 45, 48, 50, 52, and 55% for LSS, respectively (w/w, wet basis). The bags were sealed and heated in a water bath at the specified temperatures for 10 min for PS and 5 min for LSS. After heating, the samples were immersed immediately in liquid nitrogen, freeze-dried, ground into a powder, and passed through a 150 μm sieve. Freeze drying has been shown to have a significant effect on the structure and functionality of PS.26 Hence, native PS was also freeze-dried according to a method described in ref 27 and used as a control. 2.3. Differential Scanning Calorimetry (DSC). Thermal properties of starch samples were analyzed using a differential scanning calorimeter (200 F3, Netzsch, Selb, Germany) equipped with a thermal analysis data station. Starch samples (approximately 3 mg) were weighed accurately into a 40 μL aluminum pan. Distilled water was added with a pipet to obtain a starch/water ratio of 1:3 (w/v) in the DSC pans. The pans were sealed and allowed to stand overnight at room temperature before recording the DSC profile. 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 onset (To), peak (Tp), and conclusion (Tc) temperatures and enthalpy of gelatinization (ΔH) were obtained through data recording software. All measurements were performed in triplicate. The DG of each sample was calculated according to the formula22 DG (%) = (1 − ΔHgelatinized starch /ΔHisolated starch) × 100 where ΔHgelatinized starch is the enthalpy change of gelatinized starch and ΔHisolated starch is the enthalpy change of isolated starch. 2.4. Scanning Electron Microscopy (SEM). The morphology of starch samples was imaged using a scanning electron microscope (JSMIT300LV, JEOL, Tokyo, Japan). The samples were mounted on the stub with double-sided adhesive tape and coated with gold prior to imaging.22 An accelerating voltage of 5 kV was used during imaging. 2.5. Laser Confocal Micro-Raman (LCM-Raman) Spectroscopy. A Renishaw Invia Raman microscope system (Renishaw, Wottonunder-Edge, U.K.) equipped with a Leica microscope (Leica Biosystems, Wetzlar, Germany) and a 785 nm green diode laser source was used. The Raman system was calibrated at 520 cm−1 using a silicon

Figure 1. DSC thermograms of (a) PS and (a′) LSS samples with different DG (PS, potato starch; LSS, lotus seed starch; and DG, degree of gelatinization).

Table 1. Thermal Properties of Native and Heat-Treated Starch Samplesa treatment

To (°C)

Tp (°C)

NPS freeze-dried PS PS−50%−51 °C PS−40%−52 °C PS−60%−51 °C PS−50%−52 °C PS−50%−53 °C PS−40%−56 °C PS−40%−61 °C PS−40%−66 °C PS−50%−61 °C PS−50%−71 °C

61.4 54.0 56.5 56.3 56.0 56.8 56.8 58.2 58.3 59.2 58.9

± 0.0 ± 0.2 ± 0.1 ± 0.1 ± 0.1 ± 1.4 ± 0.2 ± 0.1 ± 0.2 ± 0.8 ± 0.4 ND

f a bc bc b bc c d d e de

65.9 58.7 60.2 60.3 59.9 60.2 61.1 62.3 62.9 62.2 62.3

LSS LSS−35%−58 °C LSS−40%−58 °C LSS−35%−63 °C

67.8 67.6 67.9 70.6

± ± ± ±

b a c d

75.7 75.3 75.2 75.3

0.1 0.1 0.1 0.1

ΔH (J/g)

Tc (°C)

Potato Starch ± 0.2 e ± 0.1 a ± 0.1 b ± 0.1 bc ± 0.1 b ± 0.1 b ± 0.2 c ± 0.1 d ± 0.2 d ± 0.5 d ± 2.1 d ND Lotus Seed Starch ± 0.1 c ± 0.2 ab ± 0.1 a ± 0.0 ab B

DG (%)

71.7 65.6 66.1 66.2 66.2 65.9 67.1 67.4 68.3 70.0 70.6

± 0.3 ± 0.1 ± 0.2 ± 0.1 ± 0.1 ± 0.1 ± 0.4 ± 0.3 ± 0.2 ± 0.8 ± 1.4 ND

e a a a a a b b c d d

14.9 13.2 13.1 10.6 9.8 8.6 7.6 5.7 4.2 2.8 1.0

± 0.6 ± 0.2 ± 0.3 ± 0.2 ± 0.1 ± 0.3 ± 0.1 ± 0.4 ± 0.1 ± 0.0 ± 0.1 ND

j i i h g f e d c b a

82.0 81.6 82.0 81.6

± ± ± ±

a a a a

16.8 15.6 15.1 13.8

± ± ± ±

j i i h

0.5 0.2 0.3 0.1

0.4 0.1 0.3 0.5

0.0 12.2 28.9 34.0 41.8 48.6 61.8 71.8 80.3 93.3 100.0 0 7.1 10.1 17.9


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Journal of Agricultural and Food Chemistry Table 1. continued To (°C)

treatment LSS−40%−63 LSS−45%−68 LSS−48%−68 LSS−50%−70 LSS−55%−70 LSS−48%−73 LSS−50%−73 LSS−55%−75

°C °C °C °C °C °C °C °C

71.3 74.6 75.2 76.3 76.5 76.6 76.5

± 0.1 ± 0.1 ± 0.0 ± 0.1 ±0i ± 0.1 ± 0.1 ND

Tp (°C) e f g h j ijk

75.5 77.6 78.1 79.4 79.8 80.2 80.2

ΔH (J/g)

Tc (°C)

Lotus Seed Starch ± 0.2 bc ± 0.1 d ± 0.1 e ±0f ± 0.3 g ± 0.2 h ± 0.2 h ND

81.6 82.0 81.9 83.8 83.9 84.8 84.4

± 0.2 ± 0.1 ± 0.1 ± 0.2 ± 0.5 ± 0.6 ± 0.4 ND

a a a b bc cd d

12.4 9.6 8.3 6.0 3.8 2.7 1.4

± 0.7 ± 0.0 ± 0.4 ± 0.6 ± 0.5 ± 0.2 ± 0.2 ND

DG (%) g f e d c b a

26.2 42.9 50.6 64.3 77.4 83.9 91.6 100

The samples are designated according to percent moisture and temperature used in the treatment. Values are means ± SD. Values with the same letters in the same column are not significantly different (p < 0.05). To, Tp, Tc, and ΔH are the onset temperature, peak temperature, conclusion temperature, and enthalpy change, respectively. DG, degree of gelatinization; NPS, native potato starch; PS, potato starch; LSS, lotus seed starch; and ND, not determined.

a

Figure 2. continued C


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Figure 2. SEM images of PS and LSS samples with different DG: (a) NPS, (b) freeze-dried PS, (c) DG = 12.2%, (d) DG = 28.9%, (e) DG = 34.0%, (f) DG = 41.8%, (g) DG = 48.6%, (h) DG = 61.8%, (i) DG = 71.8%, (j) DG = 80.3%, (k) DG = 93.3%, (l) DG = 100%, (a′) LSS, (b′) DG = 7.1%, (c′) DG = 10.1%, (d′) DG = 17.9%, (e′) DG = 26.2%, (f′) DG = 42.9%, (g′) DG = 50.6%, (h′) DG = 64.3%, (i′) DG = 77.4%, (j′) DG = 83.9%, (k′) DG = 91.6%, and (l′) DG = 100% (SEM, scanning electron microscopy; DG, degree of gelatinization; NPS, native potato starch; PS, potato starch; and LSS, lotus seed starch). semiconductor. Spectra of each sample were taken from at least six positions in the range of 3500−100 cm−1, at a resolution of approximately 7 cm−1. The full width at half maximum (fwhm) of the band at 480 cm−1, which is usually used to characterize the short-range molecular order of starch,28 was obtained using the WIRE 2.0 software. 2.6. Crystallinity. X-ray diffraction (XRD) analysis was performed using a D8 Advance X-ray diffractometer (Bruker, Karlsruhe, Germany) operating at 40 kV and 40 mA. All of the starch samples were equilibrated to constant humidity before measurement by storage for 1 week in a desiccator over a saturated NaCl solution. The starch powder was packed tightly in a round glass cell and scanned from 4° 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 4°

and 35° (2θ) using the software of TOPAS 5.0 (Bruker, Karlsruhe, Germany). 2.7. In Vitro Enzymatic Digestibility. The in vitro enzymatic hydrolysis of freeze-dried starch samples was performed according to the procedure described elsewhere.29 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 PPA and 41 units of AMG was added. The starch−enzyme mixtures were incubated at 37 °C with stirring at 260 rpm for 2 h. At specified time points during the digestion, an aliquot of the hydrolysate was withdrawn and mixed with 95% ethanol to deactivate the enzymes. After centrifugation at 14500g for 3 min, the glucose content in the supernatant was determined using the D


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Megazyme GOPOD kit. The digestograms, obtained by plotting the percentage of digested starch as a function of the hydrolysis time, were fitted to the first-order rate equation 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 (i.e., the amount of starch available for hydrolysis), and k (min−1) is the firstorder rate coefficient. The amount of glucose released after 3 h (not shown) was taken as the value of C∞ to confirm that the reaction had leveled off. 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 − Ct/C∞) against t. The degree of hydrolysis (DH, %) of starch samples was defined as the percentages of starch hydrolyzed at 120 min. 2.8. Confocal Laser Scanning Microscopy (CLSM). Conjugation of AA (A6255, Sigma) with FITC (F7250, Sigma) was performed in carbonate buffer (0.1 M, pH 9) according to the method described,30,31 with some modifications. The unbound dye was removed by Sephadex G 25 gel filtration with phosphate-buffered saline (PBS, pH 7.2). The fluorescein/protein molar ratio (F/P) is defined as the ratio of moles of FITC to moles of protein in the conjugate according to the formula molar F/P =

A495 /195 MW 389 [A 280 − (0.35A495)] /E 0.1%

where MW is the molecular weight of the protein, 389 is the molecular weight of FITC, and E0.1% is the absorption at 280 nm of a protein at 1.0 mg/mL. For the FITC−AA conjugate, F/P was 0.93 and activity was 1364 units/mL, where 1 unit will liberate 1.0 mg of maltose from starch in 3 min at pH 6.9 at 37 °C. The maltose content was measured by the PAHBAH assay.32,33 The protein concentrations of the FITC− AA conjugate and unlabeled enzyme stock solutions were 0.76 and 42 mg/mL, respectively. Starch samples (20 mg, dry weight basis) were dispersed in 800 μL of 0.1 M sodium acetate buffer (pH 5.2), and 200 units of FITC−AA conjugate was added. The suspensions were magnetically stirred in an ice water bath for exactly 3 min before adding 95% ethanol (5 mL) to deactivate the enzymes. The suspensions were centrifuged (5000g for 3 min) and washed 3 times using ultrapure water (5 mL, first 2 times) and ethanol (5 mL, the last time) to remove the unbound FITC−AA conjugates. The precipitate was suspended in 50% glycerol for observation using the confocal laser scanning microscope (Nikon A1, Nikon, Japan) with a Plan-Apo 40× lens and NIS-Elements software. Starch images were taken using a frame size of 1024 × 1024 with an optical slice of 8 μm thickness. The mean fluorescence intensity (FI) of FITC−AA−starch complexes was calculated using the software of NIS-Elements Basic Research.

Figure 3. Raman spectra of (a) PS and (a′) LSS samples with different DG (PS, potato starch; LSS, lotus seed starch; and DG, degree of gelatinization).

Table 2. Crystallinity, fwhm of the Band at 480 cm−1, and Kinetic Constant (k) of Starch Samples with Different DGa DG (%)

crystallinity (%)

NPS freeze-dried PS 12.2 28.9 34.0 41.8 48.6 61.8 71.8 80.3 93.3 100

26.6 21.9 21.3 19.9 18.6 15.6 14.1 11.8 10.9 10.2 0 0

LSS 7.1 10.1

41.20 40.30 40.50

fwhm of the band at 480 cm−1 Potato Starch 15.58 ± 0.19 a 18.13 ± 0.10 b 18.12 ± 0.30 b 18.47 ± 0.80 bc 19.01 ± 0.98 cd 19.12 ± 0.57 d 19.32 ± 0.98 de 19.47 ± 1.03 def 19.74 ± 0.60 ef 20.04 ± 0.86 f 21.11 ± 0.49 g 21.66 ± 0.59 g Lotus Seed Starch 16.47 ± 0.31 a 17.34 ± 0.24 b 17.43 ± 0.15 b E

k (min−1) 0.020 0.024 0.024 0.028 0.029 0.032 0.032 0.032 0.034 0.035 0.038 0.036

± ± ± ± ± ± ± ± ± ± ± ±

0.003 0.004 0.002 0.001 0.001 0.001 0.001 0.001 0.002 0.002 0.001 0.001

R2 a a b c cd de de ef ef fg h gh

0.007 ± 0.002 a 0.016 ± 0.002 b 0.020 ± 0.001 c

0.9958 0.9972 0.9975 0.9983 0.9942 0.9990 0.9991 0.9980 0.9991 0.9986 0.9986 0.9933 0.9981 0.9988 0.9987


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Journal of Agricultural and Food Chemistry Table 2. continued DG (%)

crystallinity (%)

17.9 26.2 42.9 50.6 64.3 77.4 83.9 91.6 100

39.50 38.10 28.80 23.80 19.20 16.10 12.70 9.70 0

fwhm of the band at 480 cm−1 Lotus Seed 17.47 ± 17.57 ± 18.49 ± 18.59 ± 19.08 ± 19.67 ± 20.02 ± 21.08 ± 22.37 ±

Starch 0.23 b 0.18 b 0.23 c 0.29 c 0.19 d 0.22 e 0.18 e 0.40 f 0.54 g

k (min−1) 0.028 0.027 0.028 0.028 0.028 0.033 0.034 0.036 0.036

± ± ± ± ± ± ± ± ±

0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001

R2 d d d d d e e f f

0.9993 0.9988 0.9957 0.9961 0.9959 0.9992 0.9981 0.9964 0.9965

Values are means ± SD. Values with the same letters in the same column are not significantly different (p < 0.05). DG, degree of gelatinization; fwhm, full width at half maximum; PS, potato starch; NPS, native potato starch; and LSS, lotus seed starch. a

Figure 5. Relationship between DG and structures of (a) PS and (a′) LSS (PS, potato starch; LSS, lotus seed starch; and DG, degree of gelatinization). performed. One-way analysis of variance (ANOVA) followed by post hoc Duncan’s multiple range tests (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.).

Figure 4. XRD patterns of (a) PS and (a′) LSS samples with different DG (PS, potato starch; LSS, lotus seed starch; and DG, degree of gelatinization).

3. RESULTS AND DISCUSSION 3.1. Thermal Properties of Starch Samples. The DSC thermograms and thermal transition properties of native and

2.9. Statistical Analysis. All analyses were performed at least in triplicate, and the results are reported as the mean values and standard deviation (SD). In the case of XRD, only one measurement was F


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Figure 6. continued

G


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Figure 6. Digestograms of (a) PS and (a′) LSS samples with different DG, fit of digestion data to first-order kinetics of (b) PS and (b′) LSS samples, and kinetic constants of (c) PS and (c′) LSS samples (PS, potato starch; LSS, lotus seed starch; and DG, degree of gelatinization).

preheated starch samples are shown in Figure 1 and Table 1, respectively. Freeze drying significantly decreased gelatinization temperatures and ΔH of B-type PS (Table 1) but had little effect on LSS. Hence, the freeze-dried PS and native LSS were used as the control. With the control of the heating conditions, starch samples were prepared with a wide range of DG from 12.2 to 100% for PS and from 7.1 to 100% for LSS. With an increasing DG, the gelatinization temperatures of both starches increased and ΔH decreased (Table 1). The gradual decreases in ΔH and increases in thermal transition temperatures with increasing DG were indicative of gradual disruption of less stable starch crystallites during simulated cooking prior to DSC analysis, in general agreement with previous studies.20,22 3.2. Granular Morphology. Native potato starch (NPS) granules appeared to be smooth with oval, irregular, or ellipsoidal shapes (Figure 2a). The surface of freeze-dried starch granules appeared to be slightly damaged, as evidenced by the occurrence of a few scratches or concavities (Figure 2b). For starch samples with DG of 12.2%, the surface of a small proportion of large starch granules appeared roughened and showed signs of disruption (Figure 2c). With an increasing DG, a greater number of starch granules was more visibly disrupted, such that, at DG of 71.8%, only the rough contours of granules were observed (Figure 2i). At DG of 90%, all of the starch granules were disrupted completely. Similar changes in granular

morphology with increasing DG were observed for LSS samples (panels a′−l′ of Figure 2). 3.3. Short-Range Molecular Order Measured by LCMRaman Spectroscopy. Five strong Raman bands were observed at 2900, 1264, 940, 865, and 480 cm−1 (Figure 3), which are related to ν (C−H) modes, skeletal (C−C−O), νs (C1−O−C5), νs (C1−O−C4), and δ (CH2), respectively.34−36 The fwhm values of the band at 480 cm−1 (Table 2), which indicate the degree of short-range molecular order in starch, increased significantly from 15.58 for native PS to 18.13 for freeze-dried PS, confirming the disruption of the short-range ordered structure of PS by freeze drying. With an increasing DG, the intensity of the major bands at 2900, 1264, 943, 865, and 480 cm−1 in the spectra of both starches decreased gradually (Figure 3) and the fwhm of the band at 480 cm−1 increased from 18.13 to 21.66 for PS and from 16.47 to 22.37 for LSS (Table 2), respectively. These observations indicated the gradual disruption of the short-range ordered structure of starch with increasing DG. 3.4. Long-Range Ordered Structure Measured by XRD. Native PS exhibited the typical B-type X-ray pattern, with reflections at 5.5°, 14.9°, 17.1°, 22°, and 24° (2θ) (Figure 4a). Freeze drying disrupted the crystalline structure of PS, as shown by the decrease in relative crystallinity (from 26.6 to 21.9%) and intensity of diffraction peaks. With an increasing DG, the intensity of diffraction peaks and the relative crystallinity decreased H


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Journal of Agricultural and Food Chemistry gradually (Table 2). At 100% DG, the XRD pattern indicated that the starch was completely amorphous. Native LSS exhibited a C-type diffraction pattern, with reflections at about 5.6°, 15.0°, 17.0°, 17.9°, and 23.1° (2θ) (Figure 4a′). The peak at 5.6° (2θ), being the characteristic peak of C-type starch, almost disappeared at DG of 7.1%, indicating the preferential disruption of B-type crystallites upon heating. With increases in DG, the XRD peaks weakened and the relative crystallinity decreased (Table 2). The preceding XRD and Raman studies have shown that the long-range crystallinity and short-range double helical molecular order of both PS and LSS were disrupted progressively with increasing DG. Crystallinity showed a strong negative, linear correlation with DG (R2 = 0.92 for PS, and R2 = 0.97 for LSS), whereas the correlation between DG and the fwhm of the band at 480 cm−1 was linear and positive (R2 = 0.94 for PS, and R2 = 0.93 for LSS) (Figure 5). Similar results were also reported in a previous study with WS.22 3.5. In Vitro Digestibility of Starch Samples. Panels a and a′ of Figure 6 display the in vitro enzymatic digestograms of PS and LSS samples, respectively. To clearly show the fit of the data points to the first-order rate equation, data from representative digestograms were plotted against the theoretical curves (panels b and b′ of Figures 6). These data fitted well to the firstorder rate plots, and correlation coefficients (R2 values) greater than 0.99 for the values of the first-order rate constant k were obtained (Table 2). As expected, native PS and LSS were digested most slowly and gave the lowest k values (0.020 and 0.007 min−1, respectively). The maximum extent of hydrolysis at 120 min was close to 70% for PS and 85−90% for LSS, which occurred at or above about 50 and 43% DG, respectively. The k value for freeze-dried PS was 0.024 min−1, and this increase in susceptibility to enzymatic digestion is attributed to the damage caused by freeze drying to the granule surface and ordered structures. The rate constant for PS digestion increased gradually with increasing DG, leveling off at 0.032 min−1 at 42% DG, and then increasing only slowly to a maximum value of 0.035−0.038 min−1 at 80% DG. For LSS, the value of k increased sharply at low DG (e.g., 0.017 and 0.028 min−1 at 7 and 18% DG, respectively) and then increased more gradually to a maximum value of 0.036 min−1 at 90% DG. Strong linear positive correlations were observed between DG and k for PS (R2 = 0.87) (Figure 7a) and LSS (R2 = 0.74) (Figure 7a′), indicating that the susceptibility of both starches to enzymic breakdown increased with increasing DG and that the susceptibility of PS to amylolysis was more dependent upon DG than LSS. It is interesting to note that the maximum value of k was very similar for the enzymatic hydrolysis of highly gelatinized PS and LSS. The strong relationship between susceptibility to hydrolysis and DG found for PS and LSS was not consistent with the previous finding that the rate and extent of WS amylolysis are not determined by the DG.22 No correlations were observed between DG and DH and between DG and k for WS when data from the previous study22 were plotted (Figure 7a″). The access/binding of enzymes to starch is considered to be the main rate-limiting factor for starch digestion,5 whereas structural orders may play a lesser role, as proposed from the studies with WS.22 3.6. CLSM. To test the hypothesis that access/binding of enzymes to starch is the main rate-limiting factor for starch digestion and, thus, reveal the mechanisms underlying the digestibility of starches, the ability of AA to bind to PS, LSS, and WS with different DG was studied by CLSM imaging and the FI

Figure 7. Relationships between (●) DG and the first-order kinetic constant (k), (■) DG and DH, and (▲) DG and the mean FI of (a) potato, (a′) lotus seed, and (a″) wheat starch samples (DG, degree of gelatinization; DH, degree of hydrolysis; and FI, fluorescence intensity).

of the FITC−AA−starch complexes. The FTIC−AA conjugate bound mainly to the outer surfaces of native PS and LSS (marked by the yellow dotted arrows in panels a and a′ of Figure 8), although some seemed to bind to the inner part of granules as well (red arrows in panels a and a′ of Figure 8). In contrast, the fluorescence seemed to distribute evenly on the granule surface and in the inner part of native WS (shown by the red arrows in Figure 8a″), indicating that binding of AA to PS I


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Figure 8. continued

J


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Figure 8. continued

K


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Figure 8. CLSM images of starch−FITC−AA complexes at different DG: (a) NPS, (b) freeze-dried PS, (c) DG = 28.9%, (d) DG = 34.0%, (e) DG = 41.8%, (f) DG = 61.8%, (g) DG = 71.8%, (h) DG = 93.3%, (i) DG = 100%, (a′) LSS, (b′) DG = 10.1%, (c′) DG = 17.9%, (d′) DG = 26.2%, (e′) DG = 42.9%, (f′) DG = 50.6%, (g′) DG = 64.3%, (h′) DG = 77.4%, (i′) DG = 91.6%, (a″) WS, (b″) DG = 9.1%, (c″) DG = 23.6%, (d″) DG = 40.8%, (e″) DG = 55.5%, (f″) DG = 71.8%, (g″) DG = 80.3%, (h″) DG = 89.8%, and (i″) DG = 100% (CLSM, confocal laser scanning microscopy; FITC, fluorescein isothiocyanate; AA, α-amylase of A6255; NPS, native potato starch; PS, potato starch; LSS, lotus seed starch; WS, wheat starch; and DG, degree of gelatinization).

Table 3. Mean FI of Starch Samples with Different DGa DG (%) PS 0 12.2 28.9 34.0 41.8 61.8 71.8 93.3 100

FI

DG (%)

325.42 ± 13.34 a 346.64 ± 7.74 b 361.36 ± 15.1 bc 371.44 ± 8.5 cd 386.60 ± 9.86 de 402.07 ± 11.52 e 424.26 ± 10.97 f 442.38 ± 11.78 fg 455.18 ± 10.27 g

LSS 0 10.1 17.9 26.2 42.9 50.6 64.3 77.4 91.6

FI

DG (%)

FI

307.78 ± 9.58 a 328.18 ± 11.96 b 343.83 ± 9.93 c 362.57 ± 8.52 d 361.79 ± 9.66 d 365.84 ± 5.18 d 363.50 ± 8.91 d 366.43 ± 7.10 d 368.39 ± 4.79 d

WS 0 9.1 23.6 40.8 55.5 71.8 80.3 91.6 100

204.47 ± 8.16 a 270.41 ± 8.55 b 269.86 ± 19.58 b 265.42 ± 14.23 b 270.07 ± 15.59 b 257.88 ± 12.01 b 270.19 ± 11.64 b 257.81 ± 21.16 b 270.14 ± 11.65 b

Values are means ± SD. Values with the same letters in the same column are not significantly different (p < 0.05). DG, degree of gelatinization; FI, fluorescence intensity; PS, potato starch; LSS, lotus seed starch; and WS, wheat starch.

a

and LSS is more heterogeneous than binding of AA to WS. Dhital et al. also found that the FTIC−AA conjugate binds differently to maize and potato starches.30 Freeze drying damaged some PS granules (Figure 2b), which increased the binding of FTIC−AA and, hence, a stronger fluorescence signal (shown by solid arrows in Figure 8b). As the PS granules were disrupted increasingly by gelatinization, more

binding sites would have become available for the FTIC−AA conjugate, resulting in a stronger fluorescence signal. Similar results were also observed for the LSS samples, although the fluorescence signal seemed to reach a maximum at 26% DG (panels a′−i′ of Figure 8). WS granules, with pinholes, cavities, and channels on the surface differ in morphology compared to PS and LSS. These structural features are likely to favor access of L


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Figure 9. Schematic diagram of starch digestion as affected by DG: (A) enzyme access/binding to substrate (rate limiting) and (B) enzymatic catalysis for (a) isolated starch, (b) starch with low DG, (c) starch with intermediate DG, and (d) starch with high DG (DG, degree of gelatinization).

study,22 we proposed a different mechanistic explanation for digestion of PS and LSS with different DG, as illustrated in Figure 9. Native PS and LSS present granules of smooth surface with dense molecular packing, which does not facilitate the diffusion of enzymes into the granules. As a result, the enzymes can only bind to the granule surface and attack from the surface (panel a of Figure 9A), thus leading to slow digestion of raw starches. When starch was gelatinized to a low degree (for example, DG of 12.2−41.8% for PS and 7.1−17.9% for LSS), the granule was disrupted to some extent, leading to the increased access/binding of enzymes to starch (panel b of Figure 9A) and a higher rate of starch digestion (Table 2). With gradual increases in DG, starch granules were disrupted increasingly severely. As a result, the access/binding of enzymes to starch was progressively increased, resulting in an increasing digestion rate of starches (panels c and d of Figure 9A). After binding to form the enzyme−starch complexes, the catalytic hydrolysis of glucosidic bonds was not the main rate-limiting factor for the digestion rate of starch (Figure 9B). This study has shown that the digestibility of starches as affected by DG is species-related. The digestion rate of both PS and LSS increased with increasing DG. PS presented a stronger linear relationship between DG and k compared to LSS. The results showed that DG is a greater rate-limiting factor for in vitro digestibility of PS and LSS than for in vitro digestibility of WS.

enzymes to the starch and penetration into the granule interior, which could explain the distribution of the fluorescence signal more evenly throughout the whole granule (Figure 8a″). Nevertheless, even a small degree of disruption of WS granules led to an increase in the fluorescence signal (panels b″−i″ of Figure 8). The FI was first calculated to assess the degree of binding of the FTIC−AA conjugate to the starch samples (Table 3), and the correlation of FI with DG was tested (Figure 7). For PS and LSS, the FI of FTIC−AA−starch complexes increased with increasing DG, giving a strong positive linear relationship for PS (R2 = 0.99; Figure 7a) and a weaker correlation between FI and DG for LSS (R2 = 0.54; Figure 7a′). No correlation was found between FI and DG of WS samples (R2 = 0.22; Figure 7a″). The strength of these correlations was in line with the extent of the respective correlations between DG and k and DG and degree of starch hydrolysis for the three starches (Figure 7), providing evidence in favor of the hypothesis that the digestion rate of starch is mainly determined by the access/binding of enzymes to starch rather than the subsequent catalytic hydrolysis.22 Increasing DG provided greater access/binding of AA to the starch, especially for PS and LSS. Even a low DG of WS increased access of enzymes and the starch digestion rate. Higher DG did not increase enzyme binding and the digestion rate of starch. 3.7. Proposed Mechanism for In Vitro Digestion of PS and LSS. On the basis of the present study and our previous M


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(12) Slaughter, S. L.; Ellis, P. R.; Butterworth, P. J. An investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches. Biochim. Biophys. Acta, Gen. Subj. 2001, 1525 (1−2), 29−36. (13) Baldwin, A. J.; Egan, D. L.; Warren, F. J.; Barker, P. D.; Dobson, C. M.; Butterworth, P. J.; Ellis, P. R. Investigating the mechanisms of amylolysis of starch granules by solution-state NMR. Biomacromolecules 2015, 16, 1614−1621. (14) Holm, J.; Lundquist, I.; Bjorck, I.; Eliasson, A. C.; Asp, N. G. Degree of starch gelatinization, digestion rate of starch in vitro, and metabolic response in rats. Am. J. Clin. Nutr. 1988, 47, 1010−1016. (15) Juansang, J.; Puttanlek, C.; Rungsardthong, V.; Puncha-arnon, S.; Uttapap, D. Effect of gelatinisation on slowly digestible starch and resistant starch of heat-moisture treated and chemically modified canna starches. Food Chem. 2012, 131, 500−507. (16) Miao, M.; Zhang, T.; Mu, W.; Jiang, B. Effect of controlled gelatinization in excess water on digestibility of waxy maize starch. Food Chem. 2010, 119 (1), 41−48. (17) Parada, J.; Aguilera, J. M. In vitro digestibility and glycemic response of potato starch is related to granule size and degree of gelatinization. J. Food Sci. 2009, 74 (1), E34−E38. (18) Chung, H. J.; Lim, H. S.; Lim, S. T. Effect of partial gelatinization and retrogradation on the enzymatic digestion of waxy rice starch. J. Cereal Sci. 2006, 43, 353−359. (19) Tamura, M.; Singh, J.; Kaur, L.; Ogawa, Y. Impact of the degree of cooking on starch digestibility of riceAn in vitro study. Food Chem. 2016, 191, 98−104. (20) Wang, S.; Li, P.; Zhang, T.; Yu, J.; Wang, S.; Copeland, L. In vitro starch digestibility of rice flour is not affected by method of cooking. LWTFood Sci. Technol. 2017, 84, 536−543. (21) Xiong, W.; Zhang, B.; Huang, Q.; Li, C.; Pletsch, E. A.; Fu, X. Variation in the rate and extent of starch digestion is not determined by the starch structural features of cooked whole pulses. Food Hydrocolloids 2018, 83, 340−347. (22) Wang, S.; Wang, S.; Liu, L.; Wang, S.; Copeland, L. Structural orders of wheat starch do not determine the in vitro enzymatic digestibility. J. Agric. Food Chem. 2017, 65, 1697−1706. (23) Guo, Z.; Zeng, S.; Lu, X.; Zhou, M.; Zheng, M.; Zheng, B. Structural and physicochemical properties of lotus seed starch treated with ultra-high pressure. Food Chem. 2015, 186, 223−230. (24) Chrastil, J. Improved colorimetric determination of amylose in starches or flours. Carbohydr. Res. 1987, 159 (1), 154−158. (25) AOAC International. Official Methods of Analysis of AOAC International, 17th ed.; AOAC International: Gaithersburg, MD, 2000. (26) Zhang, B.; Wang, K.; Hasjim, J.; Li, E.; Flanagan, B. M.; Gidley, M. J.; Dhital, S. Freeze-drying changes the structure and digestibility of B-polymorphic starches. J. Agric. Food Chem. 2014, 62, 1482−1491. (27) Wang, S.; Liu, C.; Wang, S. Drying methods used in starch isolation change properties of C-type chestnut (Castanea mollissima) starches. LWTFood Sci. Technol. 2016, 73, 663−669. (28) Wang, S.; Li, P.; Yu, J.; Guo, P.; Wang, S. Multi-scale structures and functional properties of starches from Indica hybrid, Japonica and waxy rice. Int. J. Biol. Macromol. 2017, 102, 136−143. (29) Wang, S.; Luo, H.; Zhang, J.; Zhang, Y.; He, Z.; Wang, S. Alkaliinduced changes in functional properties and in vitro digestibility of wheat starch: The role of surface proteins and lipids. J. Agric. Food Chem. 2014, 62, 3636−3643. (30) Dhital, S.; Warren, F. J.; Zhang, B.; Gidley, M. J. Amylase binding to starch granules under hydrolysing and non-hydrolysing conditions. Carbohydr. Polym. 2014, 113, 97−107. (31) The, T. H.; Feltkamp, T. E. Conjugation of fluorescein isothiocyanate to antibodies. I. Experiments on the conditions of conjugation. Immunology 1970, 18, 865−873. (32) Moretti, R.; Thorson, J. S. A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay. Anal. Biochem. 2008, 377, 251−258. (33) Bhattarai, R. R.; Dhital, S.; Gidley, M. J. Interactions among macronutrients in wheat flour determine their enzymic susceptibility. Food Hydrocolloids 2016, 61, 415−425.

This study provides direct evidence that the initial access/ binding of digestive enzymes to starch was the major ratelimiting factor for the first-order kinetics of starch digestion.

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AUTHOR INFORMATION

Corresponding Author

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

Shujun Wang: 0000-0003-4501-8047 Present Address

§ Shaokang Wang: School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong 510640, People’s Republic of China.

Funding

The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (31871796) and the Natural Science Foundation of Tianjin City (17JCJQJC45600 and 18ZYPTJC00020). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED DG, degree of gelatinization; PS, potato starch; NPS, native potato starch; LSS, lotus seed starch; WS, wheat starch; FITC, fluorescein isothiocyanate; PPA, α-amylase of A3176; AA, αamylase of A6255; AMG, amyloglucosidase; DSC, differential scanning calorimetry; SEM, scanning electron microscopy; XRD, X-ray diffraction; CLSM, confocal laser scanning microscopy; fwhm, full width at half maximum; DH, degree of hydrolysis; FI, fluorescence intensity

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REFERENCES

(1) Dona, A. C.; Pages, G.; Gilbert, R. G.; Kuchel, P. W. Digestion of starch: In vivo and in vitro kinetic models used to characterise oligosaccharide or glucose release. Carbohydr. Polym. 2010, 80, 599− 617. (2) Lehmann, U.; Robin, F. Slowly digestible starchIts structure and health implications: A review. Trends Food Sci. Technol. 2007, 18, 346− 355. (3) Wang, S.; Copeland, L. Molecular disassembly of starch granules during gelatinization and its effect on starch digestibility: A review. Food Funct. 2013, 4, 1564−1580. (4) Butterworth, P. J.; Warren, F. J.; Ellis, P. R. Human α-amylase and starch digestion: An interesting marriage. Starch-Stärke 2011, 63, 395− 405. (5) Dhital, S.; Warren, F. J.; Butterworth, P. J.; Ellis, P. R.; Gidley, M. J. Mechanisms of starch digestion by alpha-amylase-structural basis for kinetic properties. Crit. Rev. Food Sci. Nutr. 2017, 57, 875−892. (6) Copeland, L.; Blazek, J.; Salman, H.; Tang, M. C. Form and functionality of starch. Food Hydrocolloids 2009, 23, 1527−1534. (7) Pérez, S.; Bertoft, E. The molecular structures of starch components and their contribution to the architecture of starch granules: A comprehensive review. Starch-Stärke 2010, 62, 389−420. (8) Singh, J.; Dartois, A.; Kaur, L. Starch digestibility in food matrix: A review. Trends Food Sci. Technol. 2010, 21, 168−180. (9) Wang, S.; Li, C.; Copeland, L.; Niu, Q.; Wang, S. Starch retrogradation: A comprehensive review. Compr. Rev. Food Sci. Food Saf. 2015, 14, 568−585. (10) Zavareze, E. R.; Guerra Dias, A. R. Impact of heat-moisture treatment and annealing in starches: A review. Carbohydr. Polym. 2011, 83, 317−328. (11) Rehman, Z.-u.; Shah, W. Thermal heat processing effects on antinutrients, protein and starch digestibility of food legumes. Food Chem. 2005, 91, 327−331. N


Article

Journal of Agricultural and Food Chemistry (34) Fechner, P. M.; Wartewig, S.; Kleinebudde, P.; Neubert, R. H. H. Studies of the retrogradation process for various starch gels using Raman spectroscopy. Carbohydr. Res. 2005, 340, 2563−2568. (35) Flores-Morales, A.; Jimenez-Estrada, M.; Mora-Escobedo, R. Determination of the structural changes by FT-IR, Raman, and CP/ MAS C-13 NMR spectroscopy on retrograded starch of maize tortillas. Carbohydr. Polym. 2012, 87 (1), 61−68. (36) Wang, S.; Wang, J.; Zhang, W.; Li, C.; Yu, J.; Wang, S. Molecular order and functional properties of starches from three waxy wheat varieties grown in China. Food Chem. 2015, 181, 43−50.

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Revisiting Mechanisms Underlying Digestion of Starches - American

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