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Effects of debranching and repeated heat-moisture treatments on structure, physicochemical properties and in vitro digestibility of wheat starch
Food Chemistry 294 (2019) 440–447
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Effects of debranching and repeated heat-moisture treatments on structure, physicochemical properties and in vitro digestibility of wheat starch Meng-Na Lia,b, Bao Zhanga,b, Ying Xiea,b, Han-Qing Chena,b, a b
T
⁎
Engineering Research Center of Bio-Process, Ministry of Education, Hefei University of Technology, Hefei, Anhui 230009, PR China School of Food and Biological Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China
ARTICLE INFO
ABSTRACT
Keywords: Wheat starch Debranching Repeated heat-moisture treatment Structure Physicochemical properties In vitro digestibility
In this study, wheat starch (WS) was firstly debranched with pullulanase (PUL) and then subjected to repeated heat-moisture treatments (RHMT). The effects of PUL and RHMT on the structure, physicochemical properties and in vitro digestibility of WS were investigated. The proton nuclear magnetic resonance spectroscopy confirmed that the ratio of α-1,6 glycosidic linkage decreased. Raman and Fourier transform-infrared spectroscopy demonstrated that more ordered structure of starch was formed. Differential scanning calorimetry and X-ray diffraction analysis revealed that RHMT could enhance thermal stability and degree of crystal perfection of PULWS sample. Scanning electron microscopy results showed that more agglomerates appeared on the surfaces of RHMT starch granules. The swelling power and solubility significantly decreased after HMT. Additionally, the resistant starch (RS) content of RHMT samples significantly increased. These results suggest that debranching and RHMT can significantly change the physicochemical properties and digestibility of WS, and it’s beneficial to the RS formation.
1. Introduction Starch, which is composed of linear amylose and branched amylopectin as the structural unit, is the major dietary source of energy in human diets (Zhang, Sofyan, & Hamaker, 2008). From nutritional perspective, starch is commonly classified into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) according to the rate and extent of starch digestion in vitro (Englyst, Kingman, & Cummings, 1992). Starches rich in SDS and RS are considered as low glycemic index (GI) foods, which is commonly associated with positive health effects including the reduction of blood lipid level, the improvement of glucose tolerance, the prevention of coronary heart disease in diabetic and healthy subjects (Rizkalla, Bellisle, & Slama, 2002). Therefore, foods containing high levels of SDS and RS have drawn much attention due to their benefits for human health. The rate of starch digestion is affected by many factors, such as the amylose/amylopectin ratio (Tharanathan & Mahadevamma, 2003), interactions of starch with proteins and lipids (Ai, Hasjim, & Jane, 2013), and various modification methods including physical, chemical and enzymatic modifications. Among the modification methods, debranching treatment can produce the large amount of linear starch chains, which are resistant to digestive enzyme hydrolysis due to the formation of tightly packed crystals (Li, Gao, & Ward, 2015). The SDS ⁎
and RS levels in waxy corn starch and oat starch significantly increased after hydrolysis by pullulanase (PUL) (Liu et al., 2016; Li, Wang, Lee, & Li, 2018). Moreover, PUL hydrolysis reduced the viscosity of oat starch paste (Li et al., 2018). Some combined methods of physical modification and debranching treatment have been used to modify starch in order to enhance SDS and RS fractions in recent years. A synergistic treatment of pullulanase debranching and ultrasonication was applied to enhance SDS and RS contents of debranched pea starch (Lu, Belanger, Donner, & Liu, 2018). Debranching followed by heat-moisture treatment (D-HMT) was utilized to modify the structure of sweet potato starch, and SDS content was as high as 31.60% (Huang, Zhou, Jin, Xu, & Chen, 2015). Moreover, debranching and temperature-cycled crystallization followed by heatmoisture treatment (HMT) and annealing treatments significantly increased the RS content of cassava starch (Boonna & Tongta, 2018). Zeng et al. (2014) also mentioned that the SDS fraction of waxy rice starch significantly increased after debranching treatment and subsequent recrystallization under isothermal and temperature-cycled conditions. These studies suggested that a combination of physical modification and debranching treatment exhibited significant impacts on the formation of SDS and RS in starches from different botanical sources. In recent years, some studies have also explored the effects of HMT or debranching treatment on the structure, physicochemical properties
Corresponding author at: School of Food and Biological Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China. E-mail address: [email protected] (H.-Q. Chen).
https://doi.org/10.1016/j.foodchem.2019.05.040 Received 30 November 2018; Received in revised form 22 March 2019; Accepted 7 May 2019 Available online 08 May 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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and in vitro digestibility of wheat starch or waxy wheat starch (Cai & Shi, 2010; Wang, Yang, Su, & Chen, 2012). HMT involves treatment of starch samples at a low moisture level (< 35%, w/w) and temperature of 84–120 °C for a certain period of time (15 min to 16 h) (Chung, Liu, & Hoover, 2009). During HMT, the balance point between the treatment conditions (temperature and moisture content) and the extent of starch modification could be reached in a relatively short time, thus the degree of starch modification was limited. However, repeated heat-moisture treatments (RHMT) is a process of repeated heating and cooling, which may exert great effect on the properties of starch samples. Therefore, we presume that the starch samples are firstly debranched with PUL to improve the motion of starch molecules and then subjected to RHMT, the modification extent will be improved significantly. To test the above hypothesis, in the present study, we explored the impacts of a combination of debranching and RHMT on the structure, physicochemical properties and in vitro digestibility of wheat starch.
perform three cycles of heat-moisture treatment (HMT-3). The wheat starch samples treated with pullulanase debranching and followed by RHMT for 1, 2, and 3 times were designated as PUL-WS-HMT1, PULWS-HMT2, and PUL-WS-HMT3, respectively. 2.4. Glycosidic bond analysis The proton magnetic resonance (1H NMR) spectroscopy (VNMRS 600 MHz, Agilent, USA) was utilized to analyze the glycosidic linkage ratio of starch samples according to the procedure described by ByungHoo et al. (2013). Starch sample (50 mg) was dissolved in 1 mL deuterium oxide (D2O), and heated in a boiling water bath for 30 min followed by freezing-drying. Then freezing-dried starch sample was redissolved in 1 mL D2O. The 1H NMR spectrum of starch sample was obtained at 60 °C. The α-1,4 and α-1,6 glycosidic bonds were determined from peaks at 5.45 ppm and 5.06 ppm, respectively. The ratio of α-1,6 glycosidic bond was calculated by dividing the peak area of α1,6 glycosidic bond by the total peak area of α-1,4 glycosidic bond and α-1,6 glycosidic bond in the 1H NMR spectra.
2. Materials and methods 2.1. Materials
2.5. Laser confocal micro-Raman (LCM-Raman) spectroscopy
Wheat starch (WS) was provided by Anhui Ante Food Co., Ltd. (Suzhou, China); The amylose, protein, lipids and ash contents of WS were 24.45%, 0.52%, 0.67%, and 0.25%, respectively. Type VI-B αamylase from porcine pancreas (EC 3.2.1.1, A3176) and amyloglucosidase (EC 3.2.1.3) were obtained by Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China), respectively. Pullulanase was purchased from Amano Enzyme Manufacturing Co., Ltd. (Tianjin, China). All other reagents utilized were of analytical grade.
The LCM-Raman spectra of starch samples were determined using a LabRam HR Evolution system (Horiba scientific, France) based on the previous study (Zheng et al., 2018). A 785 nm green diode laser source was used. The spectra were scanned in the wavenumber range of 50–2000 cm−1 with a spectral resolution of 7 cm−1. The full width at half maximum (FWHM) of peak at 480 cm−1 was obtained by the software of Origin 8.5. 2.6. Fourier transform-infrared (FT-IR) spectroscopy
2.2. Preparation of debranched starch sample
FT-IR spectra of starch samples were recorded using a spectrometer (Nicolet 6700, Thermo Electric Corporation, Waltham, MA, USA) based on the method described by Xie, Li, Chen, and Zhang (2019). Briefly, starch sample was mixed evenly with KBr powder and pressed into a transparent round tablet. All the spectra were recorded in wavenumber range of 500–4000 cm−1 at a resolution of 4 cm−1 by 64 scans.
The preparation of debranched starch sample was carried out based on the method of Li et al. (2018) with slight modifications. Briefly, 10 g starch was suspended in 100 mL phosphate buffer solution (0.2 M, pH 5.8), and incubated at 100 °C with constant stirring for 30 min in a water bath to complete gelatinization. The starch paste was then incubated at 55 °C and debranched with pullulanase (60 U/g starch) in a shaking water bath for 6 h. The enzyme was inactivated by adding 300 mL absolute ethanol. After centrifugation at 3369 g for 10 min, the precipitate was washed with absolute ethanol three times, dried in an oven at 45 °C and then sieved through 100-mesh sieve, thus the debranched starch sample was obtained, and it was designated as PUL-WS sample.
2.7. X-ray diffraction The crystallinity of starch samples were analyzed using an X-ray diffractometer (D/MAX 2500 V, Rigaku Corporation, Japan) based on the method described by Li, Xie, Chen, and Zhang (2019). The starch sample was equilibrated in a desiccator containing saturated sodium chloride solution for one week before X-ray diffraction analysis. The diffractometer was worked at 40 kV and 40 mA with Cu-Kα radiation. The scanning region of diffraction angle (2θ) ranged from 5° to 35° at a step size of 0.02° and a scanning speed of 2°/min. The software Jade 6.0 was applied to calculate the degree of relative crystallinity.
2.3. Preparation of starch sample by repeated heat-moisture treatments (RHMT) The preparation of starch sample by RHMT was carried out according to the method described by Huang, Zhou, Jin, Xu, and Chen (2016). Briefly, PUL-WS sample (10 g) was accurately weighed into in a screw capped container, and the moisture content of PUL-WS sample was adjusted to 30% by adding a certain amount of distilled water with constant stirring. After fully mixing, the sealed container was equilibrated at room temperature for 24 h and heated at 100 °C for 3 h in an air oven. After heating, the heat-moisture treated starch sample was cooled to room temperature and dried in an air oven at 45 °C for 24 h, ground and sieved, thus starch sample was obtained by one cycle of heat-moisture treatment (HMT-1). HMT-1 starch sample was weighed and placed into a screw capped container. After the moisture content of the starch sample was adjusted to 30%, the container was sealed, equilibrated and heated as described above. After heating, the starch sample was cooled, dried, ground, sieved to perform two cycles of heatmoisture treatment (HMT-2). According to the same procedure as mentioned above, HMT-2 starch sample was treated successively to
2.8. Determination of amylose content and iodine binding analysis The iodine colorimetric method was used to determine amylose content of starch samples as described by Martinez and Prodolliet (2010). Briefly, the iodine reagent was prepared by dissolving 0.1 g I2 and 1 g KI into 100 mL distilled water. Starch sample (0.1 g) was mixed with 1 mL absolute ethanol and 9 mL NaOH solution (1 M). Then the mixture was heated in a boiling water bath for 30 min. After cooling, 5.0 mL of mixture was taken and mixed with 1 mL HCl (1 M) and 2 mL iodine reagent, and then adjusted to a final volume of 100 mL with distilled water. The amylose content was obtained by determining the absorbance of final solution at 620 nm. The iodine binding curves was obtained using a UV/visible spectrophotometer (UV-2600, Shimadzu Corporation, Japan). Concisely, Starch sample (80 mg) was dissolved in 90% dimethyl sulphoxide 441
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(DMSO) solution (20 mL) and heated in a boiling water bath for 20 min. 1.0 mL of solution was taken and mixed with iodine reagent (5 mL) and distilled water (40 mL), and then adjusted to a final volume of 50 mL with distilled water. The mixture was equilibrated at room temperature for 30 min. The absorbance values were determined by scanning wavelength range of 400–800 nm to obtain iodine binding curves. 2.9. Scanning electron microscopy (SEM) analysis The morphological characteristics of starch samples were observed using a scanning electron microscopy (SEM) (SU8020, Hitach Int., Japan) as described by Xie et al. (2019). The starch sample was mounted on an aluminum plate using double-sided adhesive tape before being coated with a thin film of gold under vacuum condition. The images of starch samples were taken with magnification of 500 × at an accelerating voltage of 1.0 kV. 2.10. Differential scanning calorimetry (DSC) analysis The thermal properties of starch samples were analyzed by DSC (Q200, TA Instruments, New Castle, DE, USA) according to the method described by Li et al. (2019). Briefly, 3 mg starch sample was accurately weighed into an aluminum pan, and an appropriate volume of distilled water was added to obtain a ratio of 1:3 (starch/water). Then the aluminum pan was hermetically sealed and equilibrated at 4 °C for 24 h. The pan was scanned from 30 °C to 120 °C at a heating rate of 10 °C /min. An empty pan was utilized as a reference during scanning. The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and gelatinization enthalpy (ΔH) were determined by the DSC software (TA Instruments, New Castle, DE, USA).
Fig. 1. 1H NMR spectra of native and modified wheat starch samples. WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively.
RS (%) = [(TS
RDS
SDS )/TS ] × 100
where G20 and G120 represent the amounts of glucose released in 20 and 120 min of hydrolysis, respectively; GF is the free glucose content; TS is the weight of total starch.
2.11. Swelling power (SP) and solubility (S)
2.13. Statistical analysis
The swelling power and solubility of starch samples were determined based on the method of Tao et al. (2018). Briefly, 0.4 g starch sample was dispersed into 20 mL distilled water, and the dispersion was placed in water bath at 80 °C with continuous shaking. After 30 min, the gelatinized starch sample was centrifuged at 2915 g for 15 min. The supernatant was collected and dried at 105 °C to constant weight and weighed. The sediment was weighed to calculate swelling power. The swelling power and solubility were calculated according to the following equations:
All experiments were performed at least in triplicate, and the data were expressed as the mean values ± standard deviations and analyzed using the analysis of variance (ANOVA), and then subjected to Tukey’s test (p < 0.05) using SPSS version 19.0 statistical software (SPSS Inc., Chicago, IL, USA).
SP(g/g) = sediment weight/[dry weight of starch× (1 S)]
The 1H NMR spectra and degree of branching of native and modified starch samples are presented in Fig. 1. Hernández et al. (2008) showed that the chemical shift of anomeric protons in α-1,4 and α-1,6 glycosidic bonds was distinct from those in the free reducing ends. As shown in Fig. 1, the chemical shift of anomeric protons in α-1,4 glycosidic bond was 5.45 ppm, while the chemical shift of anomeric protons in α1,6 glycosidic bond was 5.06 ppm. These results were similar to a previous study (Liu et al., 2018). The ratio of α-1,6 glycosidic linkage of native wheat starch was 6.32%, whereas the α-1,6 glycosidic linkage ratio decreased to 4.17% after debranching treatment. Similar result has been reported by Liu et al. (2018), who investigated the molecular interactions in debranched waxy maize starch, and indicated that pullulanase hydrolyzed α-1,6 glycosidic linkages, thus producing more linear chains. After HMT, the ratio of α-1,6 glycosidic linkage further decreased, suggesting that HMT caused the degradation of starch molecules. The ratios of α1,6 glycosidic linkage of PUL-WS-HMT1, PUL-WS-HMT2 and PUL-WSHMT3 samples decreased to 3.89%, 3.27% and 2.51%, respectively. Chen et al. (2017) mentioned that the degree of branching in waxy and normal maize starches decreased as the cycling times of HMT increased, indicating that the α-1,6 glycosidic linkage was easily destroyed by HMT due to the weaker steric hindrance around the α-1,6 glycosidic linkage. Wang, Zhang, Chen, and Li (2016) also reported that HMT
3. Results and discussion 3.1. Glycosidic bond analysis
S(%) = (dried supernatant weight/dry weight of starch) × 100 2.12. Determination of in vitro digestibility of starch samples In vitro digestibility of starch sample was analyzed based on the method of Englyst et al. (1992) with slight modifications. Briefly, 200 mg starch sample was suspended in 15 mL phosphate buffer (pH 5.2), and then five glass beads were added. 5 mL of mixed enzyme solution (290 U/mL pancreatic α-amylase and 50 U/mL amyloglucosidase) was added, followed by shaking at 37 °C in a water bath. At specific time intervals (from 0 to 240 min), 0.5 mL of enzymatic hydrolysis solution of starch sample was taken, and 4.5 mL absolute ethanol was added to deactivate the enzymes. After centrifugation at 2915 g for 10 min, the glucose content in the supernatant was analyzed by 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The glucose content was multiplied with the factor of 0.9 to calculate the amount of hydrolyzed starch. The RDS, SDS, and RS fractions in starch sample were measured by the following formulas:
RDS (%) = (G20
GF ) × 0.9 × 100
SDS (%) = (G120
G20) × 0.9 × 100 442
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Relative intensity
A
WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3 200
400
600
800
1000
1200
1400
1600
1800
-1
Wavenumbers (cm )
B
WS
Transmittance intensity
PUL-WS
4000
PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
3500
3000
2500
2000
1500
1000
500
-1
Wavenumbers (cm ) Fig. 2. LCM-Raman spectra (A) and FT-IR spectra (B) of native and modified wheat starch samples. WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively.
800 cm−1 were attributed to CeC skeletal modes of α-glucose of α-1,4 glycosidic linkage starches (Flores-Morales et al., 2012). As shown in Table 1, the FWHM of the band at 480 cm−1 of PUL-WS sample slightly decreased in comparison with native wheat starch. HMT had significant impact on the structural changes in starch. The values of FWHM at 480 cm−1 of PUL-WS sample significantly decreased from 15.38 to 14.97, 14.73 and 14.88 after RHMT from one time to three times, respectively. These results indicated that more ordered structure of starch was formed during HMT. The further enhanced interactions of starch chains and the rearrangements of the dissociated double helices after RHMT would contribute to an increase in crystal perfection, thus forming more ordered structure of starch (Jacobs & Delcour, 1998). FT-IR spectra of starch samples are presented in Fig. 2B. And the absorbance ratios of 1047/1022 cm−1 and 1022/995 cm−1 in FT-IR spectrum are associated with short-range ordering of starch (Soest,
resulted in starch degradation when high-amylose maize starches were modified by HMT. 3.2. LCM-Raman and FT-IR spectroscopy The structural changes in starch could be probed by Raman spectroscopy (Flores-Morales, Jiménez-Estrada, & Mora-Escobedo, 2012). The full width at half maximum (FWHM) of the band at 480 cm−1 was sensitive to the molecular order/crystallinity of starch (Wang, Wang, Yu, & Wang, 2016). The LCM-Raman spectroscopy and the FWHM of the band at 480 cm−1 of native and modified starch are presented in Fig. 2A and Table 1, respectively. The band at 1127 cm−1 was associated with the contribution of two main vibrational modes CeO stretching and CeOeH deformation. The band at 1459 cm−1 was assigned to the CH2 deformations, while the bands between 1100 and 443
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Table 1 The solubility, swelling power, amylose content, FWHM at 480 cm−1, infrared (IR) absorbance ratios and relative crystallinity (RC) of native and modified starch samples. Samples
Solubility (%)
Swelling power (g/g)
WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
5.15 ± 0.05a 31.60 ± 0.49d 12.68 ± 0.22c 8.40 ± 0.69b 6.68 ± 0.14b
8.77 3.27 1.55 1.52 1.53
± ± ± ± ±
0.04c 0.15b 0.02a 0.09a 0.05a
Amylose content (%)
FWHM at 480 cm−1
24.45 ± 0.94d 38.56 ± 0.75e 20.69 ± 1.63c 18.16 ± 1.94bc 7.86 ± 1.34a
16.53 15.38 14.97 14.73 14.88
± ± ± ± ±
IR ratio of 1047/1022 cm−1
0.47b 0.61b 0.75ab 0.42a 0.31a
0.895 0.939 0.948 0.954 0.955
± ± ± ± ±
IR ratio of 1022/995 cm−1
0.003a 0.004b 0.001c 0.002d 0.005d
1.058 1.020 0.996 0.998 0.986
± ± ± ± ±
RC (%)
0.002c 0.002b 0.009a 0.007a 0.009a
24.45 20.61 30.17 33.00 35.19
± ± ± ± ±
0.38b 1.42a 0.52c 0.13d 0.20e
WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively. FWHM at 480 cm−1 indicates the full width at half maximum of the band at 480 cm−1. Values with different letters in the same column are significantly different (p < 0.05).
Tournois, Wit, & Vliegenthart, 1995). The higher values of the absorbance ratios of 1047/1022 cm−1 and the lower values of the absorbance ratios of 1022/995 cm−1 indicated the more ordered structure of starch. As shown in Table 1, for the PUL-WS sample, the absorbance ratio of 1047/1022 cm−1 increased, while the ratio of 1022/995 cm−1 decreased compared to native starch, indicating that the short-range molecular order of the PUL-WS sample was increased, which was consistent with the results of the values of FWHM at 480 cm−1. Moreover, the absorbance ratios of 1047/1022 cm−1 of PUL-WS sample significantly increased from 0.939 to 0.948, 0.954 after RHMT from one time to two times, respectively. However, there were no significant differences in the absorbance ratios of 1022/995 cm−1 of RHMT starch samples. Huang et al. (2016) reported that the absorbance ratio of 1047/1022 cm−1 of sweet potato starch decreased gradually as the cycling times of RHMT increased from one time to three times, while it increased gradually when cycling times of RHMT was beyond three times. The increased absorbance ratio might be attributed to further interactions between starch chains and the rearrangements of the amylose and amylopectin molecules.
A
Relative intensity
WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
5
10
15
20
25
30
35
Diffraction angle 1.2
3.3. X-ray diffraction and relative crystallinity
B
WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
1.0
The X-ray diffraction patterns and relative crystallinity of native and modified starch samples are presented in Fig. 3A and Table 1, respectively. Native wheat starch exhibited a typical A-type X-ray diffraction pattern with diffraction peaks at 15°, 17°, 18° and 23°. This result was consistent with the previous study (Shevkani, Singh, Bajaj, & Kaur, 2017). Debranching and heat-moisture treatment remained the original crystalline pattern of starch sample, which was in agreement with the previous studies (Li, He, Dhital, Zhang, & Huang, 2017; Wang et al., 2018). Meanwhile, an obvious increase in peak intensity at 20° was observed in modified starch samples, indicating the formation of V-type crystal structure. The peak intensity of PUL-WS sample at 17° became stronger, whereas the peak intensity at 18° was weaker in comparison with native starch. Liu et al. (2016) also mentioned that debranched waxy corn starch showed a major peak at 2θ = 17°, which was similar to B-type crystal structure. As shown in Table 1, the relative crystallinity of PUL-WS sample was lower than that of native wheat starch. This result was consistent with the results reported by Huang et al. (2015), who mentioned that numerous double helices were destroyed during debranching treatment. However, the relative crystallinity increased as cycling times of HMT increased. Boonna and Tongta (2018) reported that debranched cassava starch prepared by temperature cycling treatment and then subjected to HMT may form co-crystallites of A- and B-type. This could result in an increase of the relative crystallinity. Furthermore, HMT promoted the rearrangements of debranched starch chains and then developed into more ordered crystalline structure (Chung, Liu, & Hoover, 2010). The higher relative crystallinities observed in PUL-WSHMT2 and PUL-WS-HMT3 samples could be attributed to an increase in
Absorbance
0.8 0.6 0.4 0.2 0.0 450
500
550
600
650
700
750
800
Wavelength (nm) Fig. 3. X-ray diffraction patterns (A) and wavelength scanning profiles of iodine binding (B) of native and modified wheat starch samples. WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively.
crystal perfection or the formation of new crystallites during HMT. Huang et al. (2015) also reported that the relative crystallinity of sweet potato starch modified by four and five cycling times of repeated heatmoisture treatment increased because of the enhanced interactions between starch chains and the rearrangement of the dissociation of double helices. 444
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Fig. 4. Scanning electron microscopy (SEM) of native and modified wheat starch samples. WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively.
3.4. Amylose content and iodine binding analysis
temperature, which was so-called resistant starch type 3 (RS3). Li et al. (2017) also reported that maize and potato granular starches modified by partial debranching treatment exhibited higher melting temperatures and lower ΔH. HMT had a significant impact on thermal properties of PUL-WS sample. After HMT, the To and Tp of PUL-WS-HMT1 sample were shifted to higher temperatures in comparison to PUL-WS sample. Moreover, ΔH significantly increased as the cycling times of HMT increased from one time to three times, which was attributed to the increase in relative crystallinity. PUL-WS-HMT3 sample exhibited the highest gelatinization temperatures. Huang et al. (2016) reported that the gelatinization temperatures increased as the cycling times of HMT increased, which was attributed to the fact that starch chains were disrupted and rearranged during HMT, and the perfection of double helical order increased as the cycling times of HMT increased. In the present study, short linear chains produced by debranching treatment had better mobility and formed more perfect crystalline structure. Therefore, HMT could enhance the degree of crystal perfection and thermal stability of debranched wheat starch.
The amylose content and the iodine binding curves are shown in Table 1 and Fig. 3B, respectively. The amylose content of PUL-WS sample significantly increased compared to native starch, corresponding to the maximum absorbance (λmax) values at about 600 nm. Li et al. (2018) also reported that the amylose content of oat starch significantly increased during debranching treatment. These results indicated that pullulanase hydrolysis could cleave the branching points in amylopectin, thus generating larger amount of linear chains. However, the λmax values sharply decreased as the cycling times of RHMT increased. Zavareze and Dias (2011) reported that the formation of amylose-lipid complex and the interactions of amylose-amylose and/or amylose-amylopectin were responsible for the decrease of the amylose content, thus resulting in the low iodine affinity. 3.5. SEM analysis The morphologies of native and modified starch granules are presented in Fig. 4. WS was composed of large A-type starch granules and small B-type starch granules, and showed spherical and oval shapes. PUL-WS sample exhibited irregular shapes and compact structures, and the surface of starch sample was smooth. Zeng, Zhu, Chen, Gao, and Yu (2016) reported that the compact and rigid structures of starch had higher resistance to enzyme. However, as shown in Fig. 4, more and more agglomerates occurred as the cycling times of RHMT increased, and the surfaces of RHMT starch granules became rougher with densely packed structure, which was possibly attributed to partial gelatinization of the starch granules. In this study, the morphology changes of starch granules could be associated with starch digestibility.
3.7. Swelling power and solubility The swelling power and solubility of native and modified starch samples are shown in Table 1. The swelling power of native wheat starch decreased from 8.77 g/g to 3.27 g/g, while the solubility increased from 5.15% to 31.60% after debranching treatment. The increased solubility was attributed to the fact that more short linear chains generated by pullulanase hydrolysis may have good solubility. Debranched starch is partially soluble in water and can hold more water to form hydrogel due to its hydrophilicity (Liu, Gu, Hong, Cheng, & Li, 2017). Debranched starch has potential applications in food and pharmaceutical industries, and it can be utilized as fat/protein replacer and tableting excipient in drug formulations (Liu et al., 2017). Chung et al. (2009) reported that the higher extent of amylose leaching in pea and lentil starches could be ascribed to their higher amylose contents. In this study, the amylose content of PUL-WS sample was much higher than that of native starch, which could result in the increase of solubility. The swelling power and solubility of PUL-WS sample significantly decreased after HMT. Similar results were reported by Chung et al. (2009), who mentioned that the decrease in swelling power and
3.6. Thermal properties The thermal transition temperatures and enthalpy changes (ΔH) of native and modified wheat starch samples are shown in Table 2. Compared with native starch, PUL-WS sample showed higher onset gelatinization temperature (To), peak gelatinization temperature (Tp) and conclusion gelatinization temperature (Tc) and a broader gelatinization temperature range (ΔT), but lower ΔH. The results were attributed to the fact that linear starch chains produced by pullulanase debranching could easily retrograde and exhibit a high melting peak 445
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Table 2 Thermal properties and in vitro digestibility of native and modified starch samples. Samples WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
To 57.28 88.74 94.97 95.53 96.35
Tp ± ± ± ± ±
a
0.26 0.21b 0.41c 0.24 cd 0.49d
Tc a
63.06 ± 0.34 100.45 ± 0.36b 101.59 ± 0.16c 102.43 ± 0.20c 103.57 ± 0.13d
ΔT a
72.05 ± 0.25 108.72 ± 0.51b 109.79 ± 0.40b 113.98 ± 0.70c 114.79 ± 0.12c
ΔH
14.77 19.98 14.82 18.45 18.44
± ± ± ± ±
a
0.63 0.46b 0.14a 0.90b 0.43b
RDS (%) b
8.15 ± 0.43 5.11 ± 0.14a 7.85 ± 1.02b 10.78 ± 0.76c 13.60 ± 1.52d
28.22 22.27 19.13 18.80 18.78
± ± ± ± ±
SDS (%) c
0.54 0.68b 0.88ab 0.74a 0.59a
RS (%) a
22.57 ± 0.67 7.05 ± 0.35b 3.99 ± 0.59b 0.96 ± 1.25a 0.72 ± 0.38a
49.21 70.68 76.88 80.24 80.50
± ± ± ± ±
0.42a 0.65b 0.62c 0.84d 0.58d
WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively. To: onset temperature; Tp: peak temperature; Tc: conclusion temperature; ΔT: gelatinization temperature range; ΔH: gelatinization enthalpy. RDS indicates rapidly digestible starch; SDS indicates slowly digestible starch; RS indicates resistant starch. Values with different letters in the same column are significantly different (p < 0.05).
solubility after HMT could be attributed to increased crystallite perfection and additional interactions between amylose-amylose and/or amylose-amylopectin chains. In addition, the increase in the amounts of amylose-lipid complexes could also decrease solubility. As the cycling times of HMT increased from one time to three times, the solubility further decreased, while no significant difference was observed in the swelling power. Short linear chains produced after debranching treatment exhibited better mobility, which performed the interaction better during HMT. The interaction between short linear chains could strengthen with the increasing cycling times of HMT, thus resulting in the decrease in solubility. Huang et al. (2016) also reported that the swelling power and solubility of sweet potato starch decreased gradually as the cycling times of RHMT increased, but there was no significant difference in swelling power between the RHMT-5 starch sample and the RHMT-4 starch sample. The available hydroxyl groups for hydration and the diffusion of amylose and amylopectin molecules decreased due to the formation of more ordered double-helical structure as the cycling times of RHMT increased.
4. Conclusions The debranching and RHMT exerted significant effects on the structure and physicochemical properties and in vitro digestibility of wheat starch. HMT could enhance the thermal stability and degree of crystal perfection of debranched wheat starch, which was confirmed by the results of DSC and XRD. HMT induced the degradation of starch molecules, which was demonstrated by 1H NMR analysis. SEM results revealed that more densely packed structures was formed as the cycling times of RHMT increased. The FWHM of the band at 480 cm−1 and the absorbance ratios of 1047/1022 cm−1 and 1022/995 cm−1 gave sufficient evidences that more ordered structure of starch was formed during HMT. Compared with PUL-WS sample, the swelling power and solubility significantly decreased after HMT, which corresponded to the decrease in amylose content and low iodine affinity. The contents of RDS and SDS in PUL-HMT starch samples decreased, while the content of RS increased and reached the maximum (80.50%) after RHMT for 3 times. These results suggest that a combination of debranching and RHMT is beneficial to the formation of RS. RS has a large number of potential applications in food and pharmaceutical industries, and it can ameliorate the postprandial glycemic response and reduce the incidence of obesity. It is ideal for utilization in ready-to-eat cereals, noodles and baked foods. In conclusion, a combination of debranching and RHMT is a promising method for food processors to design functional starchy food with low digestibility.
3.8. In vitro digestibility analysis The in vitro digestibility of native and modified starch samples are shown in Table 2. Compared with native wheat starch, the RDS and SDS contents of PUL-WS sample decreased significantly, while the RS content increased dramatically. Similar results of low digestibility have been reported in PUL modified oat starch (Li et al., 2018). Ozturk, Koksel, Kahraman, and Ng (2009) reported that the debranching and drying conditions were suitable for RS formation. The more short starch chains, the more difficult for the enzymes to digest (Zhang et al., 2008). Liu et al. (2017) also mentioned that the gel network formed by the rearrangement and aggregation of short linear chains could resist to erosion and degradation by enzymes, thereby resulting in low enzymatic hydrolysis of debranched starch. As shown in Table 2, after HMT, the RS content further increased, which was attributed to the enhanced interactions of short linear starch chains. According to XRD and FTIR results, it could be seen clearly that the realignment between debranched starch chains was enhanced, and more ordered structure of starch was formed after HMT. And when the cycling times of RHMT were from one time to two times, the content of RS increased from 76.88% to 80.24%, while the contents of RDS and SDS decreased from 19.13% and 3.99% to 18.80% and 0.96%, respectively. There were no significant differences in in vitro digestibility between PUL-WS-HMT2 and PUL-WS-HMT3 samples. The interactions between debranched starch chains further increased as the cycling times of RHMT increased and the level of RS reached the maximum (80.50%) when cycling time of RHMT was three times. The low susceptibility of starch to digestive enzymes was attributed to the formation of more perfect crystalline structure.
Declaration of Competing Interest All authors declare that they have no conflict of interests. Acknowledgements This study was supported by the Key Research and Development Program of Anhui Province, China (No. 16030701079) and the National Key R&D Program of China (No. 2017YFD0400405). References Ai, Y., Hasjim, J., & Jane, J. L. (2013). Effects of lipids on enzymatic hydrolysis and physical properties of starch. Carbohydrate Polymers, 92(1), 120–127. Boonna, S., & Tongta, S. (2018). Structural transformation of crystallized debranched cassava starch during dual hydrothermal treatment in relation to enzyme digestibility. Carbohydrate Polymers, 191, 1–7. Byung-Hoo, L., Yan, L., Phillips, R. J., Reuhs, B. L., Kyra, J., Rose, D. R., ... Hamaker, B. R. (2013). Enzyme-synthesized highly branched maltodextrins have Slow glucose generation at the mucosal α-glucosidase level and are slowly digestibleIn vivo. PLoS One, 8(4), e59745. Cai, L., & Shi, Y. C. (2010). Structure and digestibility of crystalline short-chain amylose from debranched waxy wheat, waxy maize, and waxy potato starches. Carbohydrate Polymers, 79(4), 1117–1123. Chen, Y., Yang, Q., Xu, X., Qi, L., Dong, Z., Luo, Z., ... Peng, X. (2017). Structural changes
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Effects of debranching and repeated heat-moisture treatments on structure, physicochemical properties and in vitro digestibility of wheat starch Meng-Na Lia,b, Bao Zhanga,b, Ying Xiea,b, Han-Qing Chena,b, a b
T
⁎
Engineering Research Center of Bio-Process, Ministry of Education, Hefei University of Technology, Hefei, Anhui 230009, PR China School of Food and Biological Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China
ARTICLE INFO
ABSTRACT
Keywords: Wheat starch Debranching Repeated heat-moisture treatment Structure Physicochemical properties In vitro digestibility
In this study, wheat starch (WS) was firstly debranched with pullulanase (PUL) and then subjected to repeated heat-moisture treatments (RHMT). The effects of PUL and RHMT on the structure, physicochemical properties and in vitro digestibility of WS were investigated. The proton nuclear magnetic resonance spectroscopy confirmed that the ratio of α-1,6 glycosidic linkage decreased. Raman and Fourier transform-infrared spectroscopy demonstrated that more ordered structure of starch was formed. Differential scanning calorimetry and X-ray diffraction analysis revealed that RHMT could enhance thermal stability and degree of crystal perfection of PULWS sample. Scanning electron microscopy results showed that more agglomerates appeared on the surfaces of RHMT starch granules. The swelling power and solubility significantly decreased after HMT. Additionally, the resistant starch (RS) content of RHMT samples significantly increased. These results suggest that debranching and RHMT can significantly change the physicochemical properties and digestibility of WS, and it’s beneficial to the RS formation.
1. Introduction Starch, which is composed of linear amylose and branched amylopectin as the structural unit, is the major dietary source of energy in human diets (Zhang, Sofyan, & Hamaker, 2008). From nutritional perspective, starch is commonly classified into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) according to the rate and extent of starch digestion in vitro (Englyst, Kingman, & Cummings, 1992). Starches rich in SDS and RS are considered as low glycemic index (GI) foods, which is commonly associated with positive health effects including the reduction of blood lipid level, the improvement of glucose tolerance, the prevention of coronary heart disease in diabetic and healthy subjects (Rizkalla, Bellisle, & Slama, 2002). Therefore, foods containing high levels of SDS and RS have drawn much attention due to their benefits for human health. The rate of starch digestion is affected by many factors, such as the amylose/amylopectin ratio (Tharanathan & Mahadevamma, 2003), interactions of starch with proteins and lipids (Ai, Hasjim, & Jane, 2013), and various modification methods including physical, chemical and enzymatic modifications. Among the modification methods, debranching treatment can produce the large amount of linear starch chains, which are resistant to digestive enzyme hydrolysis due to the formation of tightly packed crystals (Li, Gao, & Ward, 2015). The SDS ⁎
and RS levels in waxy corn starch and oat starch significantly increased after hydrolysis by pullulanase (PUL) (Liu et al., 2016; Li, Wang, Lee, & Li, 2018). Moreover, PUL hydrolysis reduced the viscosity of oat starch paste (Li et al., 2018). Some combined methods of physical modification and debranching treatment have been used to modify starch in order to enhance SDS and RS fractions in recent years. A synergistic treatment of pullulanase debranching and ultrasonication was applied to enhance SDS and RS contents of debranched pea starch (Lu, Belanger, Donner, & Liu, 2018). Debranching followed by heat-moisture treatment (D-HMT) was utilized to modify the structure of sweet potato starch, and SDS content was as high as 31.60% (Huang, Zhou, Jin, Xu, & Chen, 2015). Moreover, debranching and temperature-cycled crystallization followed by heatmoisture treatment (HMT) and annealing treatments significantly increased the RS content of cassava starch (Boonna & Tongta, 2018). Zeng et al. (2014) also mentioned that the SDS fraction of waxy rice starch significantly increased after debranching treatment and subsequent recrystallization under isothermal and temperature-cycled conditions. These studies suggested that a combination of physical modification and debranching treatment exhibited significant impacts on the formation of SDS and RS in starches from different botanical sources. In recent years, some studies have also explored the effects of HMT or debranching treatment on the structure, physicochemical properties
Corresponding author at: School of Food and Biological Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China. E-mail address: [email protected] (H.-Q. Chen).
https://doi.org/10.1016/j.foodchem.2019.05.040 Received 30 November 2018; Received in revised form 22 March 2019; Accepted 7 May 2019 Available online 08 May 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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and in vitro digestibility of wheat starch or waxy wheat starch (Cai & Shi, 2010; Wang, Yang, Su, & Chen, 2012). HMT involves treatment of starch samples at a low moisture level (< 35%, w/w) and temperature of 84–120 °C for a certain period of time (15 min to 16 h) (Chung, Liu, & Hoover, 2009). During HMT, the balance point between the treatment conditions (temperature and moisture content) and the extent of starch modification could be reached in a relatively short time, thus the degree of starch modification was limited. However, repeated heat-moisture treatments (RHMT) is a process of repeated heating and cooling, which may exert great effect on the properties of starch samples. Therefore, we presume that the starch samples are firstly debranched with PUL to improve the motion of starch molecules and then subjected to RHMT, the modification extent will be improved significantly. To test the above hypothesis, in the present study, we explored the impacts of a combination of debranching and RHMT on the structure, physicochemical properties and in vitro digestibility of wheat starch.
perform three cycles of heat-moisture treatment (HMT-3). The wheat starch samples treated with pullulanase debranching and followed by RHMT for 1, 2, and 3 times were designated as PUL-WS-HMT1, PULWS-HMT2, and PUL-WS-HMT3, respectively. 2.4. Glycosidic bond analysis The proton magnetic resonance (1H NMR) spectroscopy (VNMRS 600 MHz, Agilent, USA) was utilized to analyze the glycosidic linkage ratio of starch samples according to the procedure described by ByungHoo et al. (2013). Starch sample (50 mg) was dissolved in 1 mL deuterium oxide (D2O), and heated in a boiling water bath for 30 min followed by freezing-drying. Then freezing-dried starch sample was redissolved in 1 mL D2O. The 1H NMR spectrum of starch sample was obtained at 60 °C. The α-1,4 and α-1,6 glycosidic bonds were determined from peaks at 5.45 ppm and 5.06 ppm, respectively. The ratio of α-1,6 glycosidic bond was calculated by dividing the peak area of α1,6 glycosidic bond by the total peak area of α-1,4 glycosidic bond and α-1,6 glycosidic bond in the 1H NMR spectra.
2. Materials and methods 2.1. Materials
2.5. Laser confocal micro-Raman (LCM-Raman) spectroscopy
Wheat starch (WS) was provided by Anhui Ante Food Co., Ltd. (Suzhou, China); The amylose, protein, lipids and ash contents of WS were 24.45%, 0.52%, 0.67%, and 0.25%, respectively. Type VI-B αamylase from porcine pancreas (EC 3.2.1.1, A3176) and amyloglucosidase (EC 3.2.1.3) were obtained by Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China), respectively. Pullulanase was purchased from Amano Enzyme Manufacturing Co., Ltd. (Tianjin, China). All other reagents utilized were of analytical grade.
The LCM-Raman spectra of starch samples were determined using a LabRam HR Evolution system (Horiba scientific, France) based on the previous study (Zheng et al., 2018). A 785 nm green diode laser source was used. The spectra were scanned in the wavenumber range of 50–2000 cm−1 with a spectral resolution of 7 cm−1. The full width at half maximum (FWHM) of peak at 480 cm−1 was obtained by the software of Origin 8.5. 2.6. Fourier transform-infrared (FT-IR) spectroscopy
2.2. Preparation of debranched starch sample
FT-IR spectra of starch samples were recorded using a spectrometer (Nicolet 6700, Thermo Electric Corporation, Waltham, MA, USA) based on the method described by Xie, Li, Chen, and Zhang (2019). Briefly, starch sample was mixed evenly with KBr powder and pressed into a transparent round tablet. All the spectra were recorded in wavenumber range of 500–4000 cm−1 at a resolution of 4 cm−1 by 64 scans.
The preparation of debranched starch sample was carried out based on the method of Li et al. (2018) with slight modifications. Briefly, 10 g starch was suspended in 100 mL phosphate buffer solution (0.2 M, pH 5.8), and incubated at 100 °C with constant stirring for 30 min in a water bath to complete gelatinization. The starch paste was then incubated at 55 °C and debranched with pullulanase (60 U/g starch) in a shaking water bath for 6 h. The enzyme was inactivated by adding 300 mL absolute ethanol. After centrifugation at 3369 g for 10 min, the precipitate was washed with absolute ethanol three times, dried in an oven at 45 °C and then sieved through 100-mesh sieve, thus the debranched starch sample was obtained, and it was designated as PUL-WS sample.
2.7. X-ray diffraction The crystallinity of starch samples were analyzed using an X-ray diffractometer (D/MAX 2500 V, Rigaku Corporation, Japan) based on the method described by Li, Xie, Chen, and Zhang (2019). The starch sample was equilibrated in a desiccator containing saturated sodium chloride solution for one week before X-ray diffraction analysis. The diffractometer was worked at 40 kV and 40 mA with Cu-Kα radiation. The scanning region of diffraction angle (2θ) ranged from 5° to 35° at a step size of 0.02° and a scanning speed of 2°/min. The software Jade 6.0 was applied to calculate the degree of relative crystallinity.
2.3. Preparation of starch sample by repeated heat-moisture treatments (RHMT) The preparation of starch sample by RHMT was carried out according to the method described by Huang, Zhou, Jin, Xu, and Chen (2016). Briefly, PUL-WS sample (10 g) was accurately weighed into in a screw capped container, and the moisture content of PUL-WS sample was adjusted to 30% by adding a certain amount of distilled water with constant stirring. After fully mixing, the sealed container was equilibrated at room temperature for 24 h and heated at 100 °C for 3 h in an air oven. After heating, the heat-moisture treated starch sample was cooled to room temperature and dried in an air oven at 45 °C for 24 h, ground and sieved, thus starch sample was obtained by one cycle of heat-moisture treatment (HMT-1). HMT-1 starch sample was weighed and placed into a screw capped container. After the moisture content of the starch sample was adjusted to 30%, the container was sealed, equilibrated and heated as described above. After heating, the starch sample was cooled, dried, ground, sieved to perform two cycles of heatmoisture treatment (HMT-2). According to the same procedure as mentioned above, HMT-2 starch sample was treated successively to
2.8. Determination of amylose content and iodine binding analysis The iodine colorimetric method was used to determine amylose content of starch samples as described by Martinez and Prodolliet (2010). Briefly, the iodine reagent was prepared by dissolving 0.1 g I2 and 1 g KI into 100 mL distilled water. Starch sample (0.1 g) was mixed with 1 mL absolute ethanol and 9 mL NaOH solution (1 M). Then the mixture was heated in a boiling water bath for 30 min. After cooling, 5.0 mL of mixture was taken and mixed with 1 mL HCl (1 M) and 2 mL iodine reagent, and then adjusted to a final volume of 100 mL with distilled water. The amylose content was obtained by determining the absorbance of final solution at 620 nm. The iodine binding curves was obtained using a UV/visible spectrophotometer (UV-2600, Shimadzu Corporation, Japan). Concisely, Starch sample (80 mg) was dissolved in 90% dimethyl sulphoxide 441
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(DMSO) solution (20 mL) and heated in a boiling water bath for 20 min. 1.0 mL of solution was taken and mixed with iodine reagent (5 mL) and distilled water (40 mL), and then adjusted to a final volume of 50 mL with distilled water. The mixture was equilibrated at room temperature for 30 min. The absorbance values were determined by scanning wavelength range of 400–800 nm to obtain iodine binding curves. 2.9. Scanning electron microscopy (SEM) analysis The morphological characteristics of starch samples were observed using a scanning electron microscopy (SEM) (SU8020, Hitach Int., Japan) as described by Xie et al. (2019). The starch sample was mounted on an aluminum plate using double-sided adhesive tape before being coated with a thin film of gold under vacuum condition. The images of starch samples were taken with magnification of 500 × at an accelerating voltage of 1.0 kV. 2.10. Differential scanning calorimetry (DSC) analysis The thermal properties of starch samples were analyzed by DSC (Q200, TA Instruments, New Castle, DE, USA) according to the method described by Li et al. (2019). Briefly, 3 mg starch sample was accurately weighed into an aluminum pan, and an appropriate volume of distilled water was added to obtain a ratio of 1:3 (starch/water). Then the aluminum pan was hermetically sealed and equilibrated at 4 °C for 24 h. The pan was scanned from 30 °C to 120 °C at a heating rate of 10 °C /min. An empty pan was utilized as a reference during scanning. The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and gelatinization enthalpy (ΔH) were determined by the DSC software (TA Instruments, New Castle, DE, USA).
Fig. 1. 1H NMR spectra of native and modified wheat starch samples. WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively.
RS (%) = [(TS
RDS
SDS )/TS ] × 100
where G20 and G120 represent the amounts of glucose released in 20 and 120 min of hydrolysis, respectively; GF is the free glucose content; TS is the weight of total starch.
2.11. Swelling power (SP) and solubility (S)
2.13. Statistical analysis
The swelling power and solubility of starch samples were determined based on the method of Tao et al. (2018). Briefly, 0.4 g starch sample was dispersed into 20 mL distilled water, and the dispersion was placed in water bath at 80 °C with continuous shaking. After 30 min, the gelatinized starch sample was centrifuged at 2915 g for 15 min. The supernatant was collected and dried at 105 °C to constant weight and weighed. The sediment was weighed to calculate swelling power. The swelling power and solubility were calculated according to the following equations:
All experiments were performed at least in triplicate, and the data were expressed as the mean values ± standard deviations and analyzed using the analysis of variance (ANOVA), and then subjected to Tukey’s test (p < 0.05) using SPSS version 19.0 statistical software (SPSS Inc., Chicago, IL, USA).
SP(g/g) = sediment weight/[dry weight of starch× (1 S)]
The 1H NMR spectra and degree of branching of native and modified starch samples are presented in Fig. 1. Hernández et al. (2008) showed that the chemical shift of anomeric protons in α-1,4 and α-1,6 glycosidic bonds was distinct from those in the free reducing ends. As shown in Fig. 1, the chemical shift of anomeric protons in α-1,4 glycosidic bond was 5.45 ppm, while the chemical shift of anomeric protons in α1,6 glycosidic bond was 5.06 ppm. These results were similar to a previous study (Liu et al., 2018). The ratio of α-1,6 glycosidic linkage of native wheat starch was 6.32%, whereas the α-1,6 glycosidic linkage ratio decreased to 4.17% after debranching treatment. Similar result has been reported by Liu et al. (2018), who investigated the molecular interactions in debranched waxy maize starch, and indicated that pullulanase hydrolyzed α-1,6 glycosidic linkages, thus producing more linear chains. After HMT, the ratio of α-1,6 glycosidic linkage further decreased, suggesting that HMT caused the degradation of starch molecules. The ratios of α1,6 glycosidic linkage of PUL-WS-HMT1, PUL-WS-HMT2 and PUL-WSHMT3 samples decreased to 3.89%, 3.27% and 2.51%, respectively. Chen et al. (2017) mentioned that the degree of branching in waxy and normal maize starches decreased as the cycling times of HMT increased, indicating that the α-1,6 glycosidic linkage was easily destroyed by HMT due to the weaker steric hindrance around the α-1,6 glycosidic linkage. Wang, Zhang, Chen, and Li (2016) also reported that HMT
3. Results and discussion 3.1. Glycosidic bond analysis
S(%) = (dried supernatant weight/dry weight of starch) × 100 2.12. Determination of in vitro digestibility of starch samples In vitro digestibility of starch sample was analyzed based on the method of Englyst et al. (1992) with slight modifications. Briefly, 200 mg starch sample was suspended in 15 mL phosphate buffer (pH 5.2), and then five glass beads were added. 5 mL of mixed enzyme solution (290 U/mL pancreatic α-amylase and 50 U/mL amyloglucosidase) was added, followed by shaking at 37 °C in a water bath. At specific time intervals (from 0 to 240 min), 0.5 mL of enzymatic hydrolysis solution of starch sample was taken, and 4.5 mL absolute ethanol was added to deactivate the enzymes. After centrifugation at 2915 g for 10 min, the glucose content in the supernatant was analyzed by 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The glucose content was multiplied with the factor of 0.9 to calculate the amount of hydrolyzed starch. The RDS, SDS, and RS fractions in starch sample were measured by the following formulas:
RDS (%) = (G20
GF ) × 0.9 × 100
SDS (%) = (G120
G20) × 0.9 × 100 442
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Relative intensity
A
WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3 200
400
600
800
1000
1200
1400
1600
1800
-1
Wavenumbers (cm )
B
WS
Transmittance intensity
PUL-WS
4000
PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
3500
3000
2500
2000
1500
1000
500
-1
Wavenumbers (cm ) Fig. 2. LCM-Raman spectra (A) and FT-IR spectra (B) of native and modified wheat starch samples. WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively.
800 cm−1 were attributed to CeC skeletal modes of α-glucose of α-1,4 glycosidic linkage starches (Flores-Morales et al., 2012). As shown in Table 1, the FWHM of the band at 480 cm−1 of PUL-WS sample slightly decreased in comparison with native wheat starch. HMT had significant impact on the structural changes in starch. The values of FWHM at 480 cm−1 of PUL-WS sample significantly decreased from 15.38 to 14.97, 14.73 and 14.88 after RHMT from one time to three times, respectively. These results indicated that more ordered structure of starch was formed during HMT. The further enhanced interactions of starch chains and the rearrangements of the dissociated double helices after RHMT would contribute to an increase in crystal perfection, thus forming more ordered structure of starch (Jacobs & Delcour, 1998). FT-IR spectra of starch samples are presented in Fig. 2B. And the absorbance ratios of 1047/1022 cm−1 and 1022/995 cm−1 in FT-IR spectrum are associated with short-range ordering of starch (Soest,
resulted in starch degradation when high-amylose maize starches were modified by HMT. 3.2. LCM-Raman and FT-IR spectroscopy The structural changes in starch could be probed by Raman spectroscopy (Flores-Morales, Jiménez-Estrada, & Mora-Escobedo, 2012). The full width at half maximum (FWHM) of the band at 480 cm−1 was sensitive to the molecular order/crystallinity of starch (Wang, Wang, Yu, & Wang, 2016). The LCM-Raman spectroscopy and the FWHM of the band at 480 cm−1 of native and modified starch are presented in Fig. 2A and Table 1, respectively. The band at 1127 cm−1 was associated with the contribution of two main vibrational modes CeO stretching and CeOeH deformation. The band at 1459 cm−1 was assigned to the CH2 deformations, while the bands between 1100 and 443
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Table 1 The solubility, swelling power, amylose content, FWHM at 480 cm−1, infrared (IR) absorbance ratios and relative crystallinity (RC) of native and modified starch samples. Samples
Solubility (%)
Swelling power (g/g)
WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
5.15 ± 0.05a 31.60 ± 0.49d 12.68 ± 0.22c 8.40 ± 0.69b 6.68 ± 0.14b
8.77 3.27 1.55 1.52 1.53
± ± ± ± ±
0.04c 0.15b 0.02a 0.09a 0.05a
Amylose content (%)
FWHM at 480 cm−1
24.45 ± 0.94d 38.56 ± 0.75e 20.69 ± 1.63c 18.16 ± 1.94bc 7.86 ± 1.34a
16.53 15.38 14.97 14.73 14.88
± ± ± ± ±
IR ratio of 1047/1022 cm−1
0.47b 0.61b 0.75ab 0.42a 0.31a
0.895 0.939 0.948 0.954 0.955
± ± ± ± ±
IR ratio of 1022/995 cm−1
0.003a 0.004b 0.001c 0.002d 0.005d
1.058 1.020 0.996 0.998 0.986
± ± ± ± ±
RC (%)
0.002c 0.002b 0.009a 0.007a 0.009a
24.45 20.61 30.17 33.00 35.19
± ± ± ± ±
0.38b 1.42a 0.52c 0.13d 0.20e
WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively. FWHM at 480 cm−1 indicates the full width at half maximum of the band at 480 cm−1. Values with different letters in the same column are significantly different (p < 0.05).
Tournois, Wit, & Vliegenthart, 1995). The higher values of the absorbance ratios of 1047/1022 cm−1 and the lower values of the absorbance ratios of 1022/995 cm−1 indicated the more ordered structure of starch. As shown in Table 1, for the PUL-WS sample, the absorbance ratio of 1047/1022 cm−1 increased, while the ratio of 1022/995 cm−1 decreased compared to native starch, indicating that the short-range molecular order of the PUL-WS sample was increased, which was consistent with the results of the values of FWHM at 480 cm−1. Moreover, the absorbance ratios of 1047/1022 cm−1 of PUL-WS sample significantly increased from 0.939 to 0.948, 0.954 after RHMT from one time to two times, respectively. However, there were no significant differences in the absorbance ratios of 1022/995 cm−1 of RHMT starch samples. Huang et al. (2016) reported that the absorbance ratio of 1047/1022 cm−1 of sweet potato starch decreased gradually as the cycling times of RHMT increased from one time to three times, while it increased gradually when cycling times of RHMT was beyond three times. The increased absorbance ratio might be attributed to further interactions between starch chains and the rearrangements of the amylose and amylopectin molecules.
A
Relative intensity
WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
5
10
15
20
25
30
35
Diffraction angle 1.2
3.3. X-ray diffraction and relative crystallinity
B
WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
1.0
The X-ray diffraction patterns and relative crystallinity of native and modified starch samples are presented in Fig. 3A and Table 1, respectively. Native wheat starch exhibited a typical A-type X-ray diffraction pattern with diffraction peaks at 15°, 17°, 18° and 23°. This result was consistent with the previous study (Shevkani, Singh, Bajaj, & Kaur, 2017). Debranching and heat-moisture treatment remained the original crystalline pattern of starch sample, which was in agreement with the previous studies (Li, He, Dhital, Zhang, & Huang, 2017; Wang et al., 2018). Meanwhile, an obvious increase in peak intensity at 20° was observed in modified starch samples, indicating the formation of V-type crystal structure. The peak intensity of PUL-WS sample at 17° became stronger, whereas the peak intensity at 18° was weaker in comparison with native starch. Liu et al. (2016) also mentioned that debranched waxy corn starch showed a major peak at 2θ = 17°, which was similar to B-type crystal structure. As shown in Table 1, the relative crystallinity of PUL-WS sample was lower than that of native wheat starch. This result was consistent with the results reported by Huang et al. (2015), who mentioned that numerous double helices were destroyed during debranching treatment. However, the relative crystallinity increased as cycling times of HMT increased. Boonna and Tongta (2018) reported that debranched cassava starch prepared by temperature cycling treatment and then subjected to HMT may form co-crystallites of A- and B-type. This could result in an increase of the relative crystallinity. Furthermore, HMT promoted the rearrangements of debranched starch chains and then developed into more ordered crystalline structure (Chung, Liu, & Hoover, 2010). The higher relative crystallinities observed in PUL-WSHMT2 and PUL-WS-HMT3 samples could be attributed to an increase in
Absorbance
0.8 0.6 0.4 0.2 0.0 450
500
550
600
650
700
750
800
Wavelength (nm) Fig. 3. X-ray diffraction patterns (A) and wavelength scanning profiles of iodine binding (B) of native and modified wheat starch samples. WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively.
crystal perfection or the formation of new crystallites during HMT. Huang et al. (2015) also reported that the relative crystallinity of sweet potato starch modified by four and five cycling times of repeated heatmoisture treatment increased because of the enhanced interactions between starch chains and the rearrangement of the dissociation of double helices. 444
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Fig. 4. Scanning electron microscopy (SEM) of native and modified wheat starch samples. WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively.
3.4. Amylose content and iodine binding analysis
temperature, which was so-called resistant starch type 3 (RS3). Li et al. (2017) also reported that maize and potato granular starches modified by partial debranching treatment exhibited higher melting temperatures and lower ΔH. HMT had a significant impact on thermal properties of PUL-WS sample. After HMT, the To and Tp of PUL-WS-HMT1 sample were shifted to higher temperatures in comparison to PUL-WS sample. Moreover, ΔH significantly increased as the cycling times of HMT increased from one time to three times, which was attributed to the increase in relative crystallinity. PUL-WS-HMT3 sample exhibited the highest gelatinization temperatures. Huang et al. (2016) reported that the gelatinization temperatures increased as the cycling times of HMT increased, which was attributed to the fact that starch chains were disrupted and rearranged during HMT, and the perfection of double helical order increased as the cycling times of HMT increased. In the present study, short linear chains produced by debranching treatment had better mobility and formed more perfect crystalline structure. Therefore, HMT could enhance the degree of crystal perfection and thermal stability of debranched wheat starch.
The amylose content and the iodine binding curves are shown in Table 1 and Fig. 3B, respectively. The amylose content of PUL-WS sample significantly increased compared to native starch, corresponding to the maximum absorbance (λmax) values at about 600 nm. Li et al. (2018) also reported that the amylose content of oat starch significantly increased during debranching treatment. These results indicated that pullulanase hydrolysis could cleave the branching points in amylopectin, thus generating larger amount of linear chains. However, the λmax values sharply decreased as the cycling times of RHMT increased. Zavareze and Dias (2011) reported that the formation of amylose-lipid complex and the interactions of amylose-amylose and/or amylose-amylopectin were responsible for the decrease of the amylose content, thus resulting in the low iodine affinity. 3.5. SEM analysis The morphologies of native and modified starch granules are presented in Fig. 4. WS was composed of large A-type starch granules and small B-type starch granules, and showed spherical and oval shapes. PUL-WS sample exhibited irregular shapes and compact structures, and the surface of starch sample was smooth. Zeng, Zhu, Chen, Gao, and Yu (2016) reported that the compact and rigid structures of starch had higher resistance to enzyme. However, as shown in Fig. 4, more and more agglomerates occurred as the cycling times of RHMT increased, and the surfaces of RHMT starch granules became rougher with densely packed structure, which was possibly attributed to partial gelatinization of the starch granules. In this study, the morphology changes of starch granules could be associated with starch digestibility.
3.7. Swelling power and solubility The swelling power and solubility of native and modified starch samples are shown in Table 1. The swelling power of native wheat starch decreased from 8.77 g/g to 3.27 g/g, while the solubility increased from 5.15% to 31.60% after debranching treatment. The increased solubility was attributed to the fact that more short linear chains generated by pullulanase hydrolysis may have good solubility. Debranched starch is partially soluble in water and can hold more water to form hydrogel due to its hydrophilicity (Liu, Gu, Hong, Cheng, & Li, 2017). Debranched starch has potential applications in food and pharmaceutical industries, and it can be utilized as fat/protein replacer and tableting excipient in drug formulations (Liu et al., 2017). Chung et al. (2009) reported that the higher extent of amylose leaching in pea and lentil starches could be ascribed to their higher amylose contents. In this study, the amylose content of PUL-WS sample was much higher than that of native starch, which could result in the increase of solubility. The swelling power and solubility of PUL-WS sample significantly decreased after HMT. Similar results were reported by Chung et al. (2009), who mentioned that the decrease in swelling power and
3.6. Thermal properties The thermal transition temperatures and enthalpy changes (ΔH) of native and modified wheat starch samples are shown in Table 2. Compared with native starch, PUL-WS sample showed higher onset gelatinization temperature (To), peak gelatinization temperature (Tp) and conclusion gelatinization temperature (Tc) and a broader gelatinization temperature range (ΔT), but lower ΔH. The results were attributed to the fact that linear starch chains produced by pullulanase debranching could easily retrograde and exhibit a high melting peak 445
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Table 2 Thermal properties and in vitro digestibility of native and modified starch samples. Samples WS PUL-WS PUL-WS-HMT1 PUL-WS-HMT2 PUL-WS-HMT3
To 57.28 88.74 94.97 95.53 96.35
Tp ± ± ± ± ±
a
0.26 0.21b 0.41c 0.24 cd 0.49d
Tc a
63.06 ± 0.34 100.45 ± 0.36b 101.59 ± 0.16c 102.43 ± 0.20c 103.57 ± 0.13d
ΔT a
72.05 ± 0.25 108.72 ± 0.51b 109.79 ± 0.40b 113.98 ± 0.70c 114.79 ± 0.12c
ΔH
14.77 19.98 14.82 18.45 18.44
± ± ± ± ±
a
0.63 0.46b 0.14a 0.90b 0.43b
RDS (%) b
8.15 ± 0.43 5.11 ± 0.14a 7.85 ± 1.02b 10.78 ± 0.76c 13.60 ± 1.52d
28.22 22.27 19.13 18.80 18.78
± ± ± ± ±
SDS (%) c
0.54 0.68b 0.88ab 0.74a 0.59a
RS (%) a
22.57 ± 0.67 7.05 ± 0.35b 3.99 ± 0.59b 0.96 ± 1.25a 0.72 ± 0.38a
49.21 70.68 76.88 80.24 80.50
± ± ± ± ±
0.42a 0.65b 0.62c 0.84d 0.58d
WS indicates native wheat starch; PUL-WS indicates that wheat starch is debranched with pullulanase; PUL-WS-HMT1, PUL-WS-HMT2, and PUL-WS-HMT3 indicate that wheat starch is firstly debranched with pullulanase and then subjected to repeated heat-moisture treatment for 1, 2, 3 times, respectively. To: onset temperature; Tp: peak temperature; Tc: conclusion temperature; ΔT: gelatinization temperature range; ΔH: gelatinization enthalpy. RDS indicates rapidly digestible starch; SDS indicates slowly digestible starch; RS indicates resistant starch. Values with different letters in the same column are significantly different (p < 0.05).
solubility after HMT could be attributed to increased crystallite perfection and additional interactions between amylose-amylose and/or amylose-amylopectin chains. In addition, the increase in the amounts of amylose-lipid complexes could also decrease solubility. As the cycling times of HMT increased from one time to three times, the solubility further decreased, while no significant difference was observed in the swelling power. Short linear chains produced after debranching treatment exhibited better mobility, which performed the interaction better during HMT. The interaction between short linear chains could strengthen with the increasing cycling times of HMT, thus resulting in the decrease in solubility. Huang et al. (2016) also reported that the swelling power and solubility of sweet potato starch decreased gradually as the cycling times of RHMT increased, but there was no significant difference in swelling power between the RHMT-5 starch sample and the RHMT-4 starch sample. The available hydroxyl groups for hydration and the diffusion of amylose and amylopectin molecules decreased due to the formation of more ordered double-helical structure as the cycling times of RHMT increased.
4. Conclusions The debranching and RHMT exerted significant effects on the structure and physicochemical properties and in vitro digestibility of wheat starch. HMT could enhance the thermal stability and degree of crystal perfection of debranched wheat starch, which was confirmed by the results of DSC and XRD. HMT induced the degradation of starch molecules, which was demonstrated by 1H NMR analysis. SEM results revealed that more densely packed structures was formed as the cycling times of RHMT increased. The FWHM of the band at 480 cm−1 and the absorbance ratios of 1047/1022 cm−1 and 1022/995 cm−1 gave sufficient evidences that more ordered structure of starch was formed during HMT. Compared with PUL-WS sample, the swelling power and solubility significantly decreased after HMT, which corresponded to the decrease in amylose content and low iodine affinity. The contents of RDS and SDS in PUL-HMT starch samples decreased, while the content of RS increased and reached the maximum (80.50%) after RHMT for 3 times. These results suggest that a combination of debranching and RHMT is beneficial to the formation of RS. RS has a large number of potential applications in food and pharmaceutical industries, and it can ameliorate the postprandial glycemic response and reduce the incidence of obesity. It is ideal for utilization in ready-to-eat cereals, noodles and baked foods. In conclusion, a combination of debranching and RHMT is a promising method for food processors to design functional starchy food with low digestibility.
3.8. In vitro digestibility analysis The in vitro digestibility of native and modified starch samples are shown in Table 2. Compared with native wheat starch, the RDS and SDS contents of PUL-WS sample decreased significantly, while the RS content increased dramatically. Similar results of low digestibility have been reported in PUL modified oat starch (Li et al., 2018). Ozturk, Koksel, Kahraman, and Ng (2009) reported that the debranching and drying conditions were suitable for RS formation. The more short starch chains, the more difficult for the enzymes to digest (Zhang et al., 2008). Liu et al. (2017) also mentioned that the gel network formed by the rearrangement and aggregation of short linear chains could resist to erosion and degradation by enzymes, thereby resulting in low enzymatic hydrolysis of debranched starch. As shown in Table 2, after HMT, the RS content further increased, which was attributed to the enhanced interactions of short linear starch chains. According to XRD and FTIR results, it could be seen clearly that the realignment between debranched starch chains was enhanced, and more ordered structure of starch was formed after HMT. And when the cycling times of RHMT were from one time to two times, the content of RS increased from 76.88% to 80.24%, while the contents of RDS and SDS decreased from 19.13% and 3.99% to 18.80% and 0.96%, respectively. There were no significant differences in in vitro digestibility between PUL-WS-HMT2 and PUL-WS-HMT3 samples. The interactions between debranched starch chains further increased as the cycling times of RHMT increased and the level of RS reached the maximum (80.50%) when cycling time of RHMT was three times. The low susceptibility of starch to digestive enzymes was attributed to the formation of more perfect crystalline structure.
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