Alkali-Induced Changes in Functional Properties and in Vitro

Article pubs.acs.org/JAFC

Alkali-Induced Changes in Functional Properties and in Vitro Digestibility of Wheat Starch: The Role of Surface Proteins and Lipids Shujun Wang,*,† Heyang Luo,† Jian Zhang,† Yan Zhang,‡ Zhonghu He,‡,§ and Shuo Wang*,† †

Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China ‡ Institute of Crop Science, and §International Maize and Wheat Improvement Center (CIMMYT) China Office, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China ABSTRACT: The bread wheat starch was treated with 0.025 and 0.0625 M NaOH solution for 1, 2, and 3 weeks at 30 °C, and the changes in functionality and in vitro digestibility were evaluated. NaOH treatment reduced protein and lipid contents of wheat starch from 0.46 to 0.20% and from 0.59 to 0.25%, respectively. No significant changes were observed in the amylose content, relative crystallinity, and short-range order of double helices, but there was evidence showing that morphology of some starch granules was altered. The swelling power and starch solubility of wheat starch increased from 11.4 to 14.1 g/g and from 10.9 to 22.1%, respectively. The thermal transition temperatures were increased greatly, but the enthalpy change remained largely unchanged. Alkali treatment greatly decreased the pasting temperature, but the pasting viscosities were altered in different ways. The resistant starch (RS) content of wheat starch was decreased significantly from 69.9 to 45.2%, while the starch that is digested slowly (SDS) content was increased greatly from 13.6 to 34.5%. Our results showed that alkali treatment can significantly alter the functionality and in vitro digestibility of wheat starch granules by removing the surface proteins and lipids rather than significantly altering the internal structure of starch granules. KEYWORDS: bread wheat, starch granule, alkali treatment, protein and lipid, functionality, in vitro digestibility

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INTRODUCTION Starch is the main storage polysaccharide of higher plants, including bread wheat (Triticum aestivum L.), and the main source of energy in human diets. Native starch is synthesized by the coordinated interaction of multiple biosynthetic enzymes and deposited as semi-crystalline granules with a very complex hierarchical structure.1−3 Linear amylose and highly branched amylopectin together comprise starch granules. Native starch has great variability in granular structure and functionality between and within plant species and even from the same plant cultivar grown under different conditions. The differences in the fine structure of starch granules, such as the amylose content, distribution and displacement of amylopectin-branched chains, and ways that these molecules are arranged into crystalline and amorphous regions, are the major causes for the great variability in starch functionality and digestibility. Starch functionality, particularly gelatinization and retrogradation, is the major determinant for food processing and digestion.4,5 Apart from the fine structure of granules, starch surface components play an innegligible role in starch functionality and digestibility.6−10 As the first barrier for starch granules to react with water, chemical reagents, and enzymes, starch surface characteristics (for example, associated components) determine largely the initial rate of these reactions. Generally, cereal starches bear more granule-associated proteins and lipids on the surface than other tuber, root, or legume starches.6 Removal of surface proteins and lipids can alter the functional properties of native starch7,10−13 and increase the chemical reaction activity with modifying reagents.14 As one of the most important glycaemic carbohydrates in many foods, the digestion of starch has important health © 2014 American Chemical Society

implications. Starch that is digested rapidly (RDS) in the upper gut causes a rapid elevation of glucose into the bloodstream, triggering physiological responses that over time are associated with increased risks for diabetes, cardiovascular disease, and cancer.15,16 On the other hand, starch that is digested slowly (SDS), and starch that passes largely undigested into the colon [resistant starch (RS)] are associated with health benefits through better blood glucose control and prebiotic effects from promoting the growth of beneficial colonic microflora.17 Most starch consumed by humans has undergone some form of processing or cooking, which greatly increases the digestion of starch. The digestion of starch in processed foods is largely dependent upon the extent of starch gelatinization and retrogradation.5 In contrast, the digestion of native starch is very slow and affected by many factors, such as the granular morphology, amylose content, fine structure of amylopectin, crystallinity, amylose−lipid complexes, and presence of surface proteins and lipids.5,18 Surface proteins and lipids are assumed to block the access of enzymes to starch granules, hence reducing the susceptibility of starch to enzymatic digestion.9,18−20 However, little experimental evidence is available to corroborate this assumption. Alkali reagents, such as NaOH, are used widely in the production of many traditional starchy foods, which include yellow alkaline noodle and waxy rice dumpling.11,13,14 To understand the quality of these products, the effect of alkali Received: Revised: Accepted: Published: 3636

January 15, 2014 March 24, 2014 March 26, 2014 March 26, 2014 dx.doi.org/10.1021/jf500249w | J. Agric. Food Chem. 2014, 62, 3636−3643

Journal of Agricultural and Food Chemistry

Article

Table 1. Protein, Lipid, and Amylose Contents, Average Particle Size, Relative Crystallinity (XRD), and Short-Range Order (FTIR) of Native and Alkali-Treated Starchesa samples

protein content (%)

lipid content (%)

amylose content (%)

average particle size (μm)

XRD crystallinity (%)

FTIR 1045/1022 ratio

native starch 0.1% 1W 0.1% 2W 0.1% 3W 0.25% 1W 0.25% 2W 0.25% 3W

0.46 ± 0.02 d 0.34 ± 0.07 c 0.30 ± 0.02 bc 0.31 ± 0.04 bc 0.26 ± 0.06 ab 0.25 ± 0.05 ab 0.20 ± 0.02 a

0.59 ± 0.03 c 0.38 ± 0.02 b 0.36 ± 0.05 b 0.35 ± 0.04 b 0.28 ± 0.01 a 0.26 ± 0.02 a 0.25 ± 0.03 a

27.1 ± 0.8 a 27.9 ± 0.7 a 27.7 ± 0.5 a 27.4 ± 0.7 a 26.9 ± 0.6 a 27.4 ± 1.0 a 26.8 ± 0.6 a

21.1 ± 0.02 a 21.1 ± 0.02 a 21.1 ± 0.10 a 21.2 ± 0.10 a 24.6 ± 0.03 b 25.8 ± 0.04 c 25.5 ± 0.04 c

32.7 31.6 31.4 31.5 31.4 31.4 31.5

0.658 0.650 0.651 0.649 0.648 0.649 0.647

XRD and FTIR calculations are within SD of 2%. Values are the mean ± SD. Values with the same letters in the same column are not significantly different (p < 0.05). a

to an aluminum stub. The mounted starch samples were coated with gold prior to imaging in a scanning electron microscope (SU1510, Hitachi High-Technologies Corporation, Tokyo, Japan). The accelerating voltage was 10.0 kV. Particle Size Distribution. The particle size distribution of native and alkali-treated starch granules was measured using laser light scattering (Mastersizer X, Malvern, U.K.). A polydisperse mode of analysis and a 300 mm lens were used. The starch was evenly dispersed in deionized water with magnetic agitation to attain an obscuration of 20−30%. The measurements were performed in duplicate. The median volume-based diameter was used to represent the average particle size. Swelling Power and Starch Solubility. Swelling power (SP) and starch solubility (SS) of native and alkali-treated starches were determined in triplicate according to the method described elsewhere.26 Thermal Analysis. Thermal transition analysis of starch samples was made using a differential scanning calorimeter (DSC 200 F3, NETZSCH, Selb, Germany) equipped with a thermal analysis data station and data recording software. The operating conditions and definitions of thermal transition parameters were described elsewhere.27 Pasting Properties. The pasting profiles were analyzed using a Newport Scientific rapid visco analyzer 4 (RVA-4) (Newport Scientific, Warriewood, New South Wales, Australia). Starch slurries containing 8% (w/w) starch (dry weight) in a total weight of 28 g were held at 50 °C for 1 min before heating at a rate of 6 °C/min to 95 °C, holding at 95 °C for 5 min, then cooling at a rate of 6 °C/min to 50 °C, and held at 50 °C for 2 min. The speed of the mixing paddle was 960 rpm for the first 10 s and then 160 rpm for the remainder of the experiment. Peak viscosity (PV), viscosity at trough [also known as minimum viscosity (MV)], and final viscosity (FV) were recorded, and breakdown (BD, which is PV minus MV) and setback (SB, which is FV minus MV) were calculated using the Thermocline software provided with the instrument. In Vitro Starch Digestibility. In vitro starch digestibility was determined by a modified Englyst et al. procedure.28 Amylase solution was prepared by suspending 1.3 g of porcine pancreatic α-amylase (28 units/mg, Sigma A3176) in 11.9 mL of water at 37 °C with magnetic stirring for 10 min. The mixture was centrifuged (1500g for 10 min) and 0.1 mL of amyloglucosidase (3260 units/mL, Megazyme) was added to 8 mL of the supernatant. Starch (100 mg) was dispersed in 4 mL of 0.1 M sodium acetate buffer (pH 5.2), and after adding 1 mL of the freshly prepared enzyme solution, the mixture was incubated at 37 °C with magnetic stirring (260 rpm). Aliquots (0.1 mL) were taken at intervals and mixed with 0.9 mL of 95% ethanol. The glucose released was measured by the glucose oxidase−peroxidase reagent (Megazyme). Starch classifications based on the rate of hydrolysis were rapidly digested starch (RDS, digested within 20 min), slowly digested starch (SDS, digested between 20 and 120 min), and resistant starch (RS, undigested starch after 120 min). Statistical Analysis. All experiments were conducted at least twice, and the results were reported as the mean values and standard deviations (SDs). In the case of X-ray diffraction (XRD) and FTIR, only one measurement was performed. Analysis of variance (ANOVA) by Duncan’s test (p < 0.05) was conducted using the SPSS 10.0 statistical software program (SPSS, Inc., Chicago, IL).

addition on the properties of starch seems to be very important. In this study, we aimed at determining the effect of alkali treatment on functionality and in vitro enzymic digestibility of bread wheat starch. The information obtained will be very useful in understanding the effect of NaOH addition on the quality and nutrition of starchy food products.

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EXPERIMENTAL SECTION

Materials. Beijing 0045 is a leading hard wheat variety in the northern China plain region. Sound grains harvested in 2011−2012 season were provided by the Institute of Crop Science, Chinese Academy of Agricultural Science. Dried wheat grains were tempered and milled into flour using a Buhler Experimental Mill (Buhler Bros, Uzwil, Switzerland), with the flour yield of 75%. Starch was isolated from wheat flour using a dough ball method.21 Preparation of Alkali-Treated Starch. Alkali-treated starch was prepared according to Wang and Copeland,22 with minor modifications. Three lots of wheat starch (10 g, dry weight) were each suspended in 100 mL of 0.025 and 0.0625 M NaOH solution. After incubation at 30 °C for 1, 2, and 3 weeks with intermittent shaking by hand to resuspend the starch granules, the slurry was filtered through Whatman No. 1 filter paper with suction. The starch cake was washed with distilled water until the pH value of the filtrate was 7 and then washed twice with water-free ethanol. The resulting alkali-treated starch was dried overnight at room temperature. Protein and Lipid Contents. The nitrogen content of starch samples was determined by standard Kjeldahl methodology. The protein content (%) was then calculated from protein (%) = nitrogen (%) × 6.25. The lipid content of starches was determined gravimetrically after extraction with ether at 60−70 °C for 6−8 h. Apparent Amylose Content. The apparent amylose content was determined by the iodine binding colorimetric method.23 Starch Crystallinity. Starch crystallinity was measured using a Panalytical X’Pert Pro X-ray diffractometer (PANalytical, Eindhoven, Netherlands) with a Co Kα source (λ = 0.1789 nm) operating at 45 kV and 35 mA. The detailed operating conditions and sample treatment before measurement were described elsewhere.24 The relative crystallinity was quantitatively estimated as a ratio of the crystalline area to the total area between 4 and 40° (2θ) using the Origin software (version 7.5, Microcal, Inc., Northampton, MA). Fourier Transform Infrared (FTIR) Spectroscopy. The infrared (IR) spectra were obtained with a Bruker Tensor 27 FTIR scanner (Bruker, Germany). The starch samples were mixed with KBr powder in a weight ratio of 1:150 and then ground finely. The resulting fine powders were pressed into the transparent round tablets and tested by the transmission method. The sample holder with KBr was used for the background spectra, and 64 co-added scans were taken for each sample from 4000 to 400 cm−1 at a resolution of 4 cm−1. The samples were dried at 105 °C for 12 h before analysis to avoid interference by moisture. The ratio of absorbance at 1045 to that at 1022 cm−1 was calculated to represent the short-range ordered structure of starch.25 Granule Morphology. Native and alkali-treated starches were fixed onto the surface of double-sided, carbon-coated adhesive tape attached 3637

dx.doi.org/10.1021/jf500249w | J. Agric. Food Chem. 2014, 62, 3636−3643


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Figure 1. continued

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Figure 1. SEM photographs of native and alkali-treated wheat starch granules: (A1 and A2) native starch, (B1 and B2) 0.1% NaOH for 1 week, (C1 and C2) 0.1% NaOH for 2 weeks, (D1 and D2) 0.1% NaOH for 3 weeks, (E1 and E2) 0.25% NaOH for 1 week, (F1 and F2) 0.25% NaOH for 2 weeks, and (G1 and G2) 0.25% NaOH for 3 weeks.

Figure 2. FTIR spectra of native and alkali-treated wheat starches.

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RESULTS

content decreased from 0.59% for native starch to 0.25% for starch treated with 0.25% NaOH for 3 weeks. Similarly, no significant differences were noted in the lipid content of starches treated for different times. The amylose content of native wheat starch was 27.1%, which remained essentially unchanged after alkali treatment. Starch Granule Morphology and Particle Size Distribution. Native wheat starch was observed to be composed of

Protein, Lipid, and Amylose Contents. Native wheat starch had a protein content of 0.46%, which was decreased significantly by alkali treatments (Table 1). After 0.1% NaOH treatment, the protein content decreased to 0.30−0.34%. The protein content decreased further when starch was treated with 0.25% NaOH. No significant differences were observed in the protein content of starches treated for different times. The lipid 3639



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significantly to 13.0−14.0 g/g after alkali treatment (Table 2). No significant differences were observed between alkali-treated starches. Likewise, alkali treatment resulted in the significant increase in starch solubility from 10.9% for native starch to 22.1% for starch treated with 0.25% NaOH for 3 weeks. The degree of increase varied with treatment time and NaOH concentration. Thermal Transition. The thermal transition profiles of native and alkali-treated starches are presented in Figure 4, and the corresponding parameters are summarized in Table 2. The onset (To), peak (Tp), and conclusion (Tc) temperatures of wheat starch were 57.3, 61.7, and 66.1 °C, respectively. The enthalpy change (ΔH) was 9.3 J/g (Table 2). Alkali treatment resulted in the significant increase in To, Tp, and Tc of wheat starch. For example, To, Tp, and Tc increased to 61.7, 64.9, and 68.7 °C for starch treated with 0.25% NaOH for 3 weeks. Starch treated with 0.25% NaOH showed a greater increase in thermal transition temperatures than starch treated with 0.1% NaOH. For starch treated with 0.1% NaOH, longer treatment resulted in the greater increase in the thermal transition temperatures. However, no significant differences were observed between starches treated with 0.25% NaOH. The thermal transition temperature range (Tc − To) decreased from 8.8 °C for native starch to 7.0 °C for starch treated with 0.25% NaOH for 3 weeks, but the enthalpy change did not change significantly after alkali treatment. Pasting Properties. The pasting profiles and parameters of wheat starch before and after alkali treatment are summarized in Figure 5 and Table 3, respectively. Alkali treatment increased breakdown viscosity and setback viscosity but decreased trough viscosity and pasting temperature. The 0.25% NaOH treatment decreased trough viscosity of wheat starch more greatly than the 0.1% NaOH treatment. However, the 0.1% NaOH treatment increased breakdown viscosity and setback viscosity of wheat starch more significantly than the 0.25% NaOH treatment. Alkali treatment significantly decreased the pasting temperature from 89.6 to 67.4 °C, with no significant differences being observed between alkali-treated starches. In contrast to the above pasting parameters, the peak and final viscosities were affected by alkali treatment in different ways. For example, the peak and final viscosities were increased after 0.1% NaOH treatment but decreased after 0.25% NaOH treatment. In Vitro Enzymatic Digestibility. The proportions of RDS, SDS, and RS in wheat starch were changed after alkali treatment (Table 4). Alkali treatment increased the RDS and SDS contents of starch granules significantly, with the latter being increased more greatly from 13.6% in native starch to 34.5% in starch treated with 0.25% NaOH for 2 weeks. Significant differences were noted in RDS and SDS contents between alkali-treated starches. In contrast to RDS and SDS, the RS content was decreased significantly from 69.9% in native starch to 45.2% in starch treated with 0.25% NaOH for 2 weeks. Likewise, significant differences were observed in the RS content between alkali-treated starches.

two populations of granules, namely, large A-type granules and small B-type granules (Figure 1). The surface of granules was largely smooth, with some grooves or indentations. After 0.1% NaOH treatment, some starch granules were observed to be deformed or roughened on the surface (panel D1 of Figure 1). A very small proportion of granules was observed to be disrupted (panels C2 and D2 of Figure 1). These changes became more obvious after longer exposure to the alkali or after treatment with 0.25% NaOH. Some starch granules appeared to adhere to form some aggregates or agglomerates (panel G1 of Figure 1). In contrast, the large A-type granules were subjected to more severe damage than the small B-type granules. Native wheat starch had an average granule size of 21.1 μm. The 0.1% NaOH treatment did not cause significant changes in the average granule size. However, the average granule size increased significantly to about 25.0 μm after 0.25% NaOH treatment (Table 1). FTIR of Native and Alkali-Treated Starches. The FTIR spectrum of native wheat starch is presented in Figure 2. The characteristic peaks at 3380 and 2930 cm−1 were attributed to the vibration of O−H stretching and the C−H deformation of the glucose unit, respectively. The absorbance at 1643 cm−1 was assigned to the vibration of O−H stretching of water absorbed in the amorphous regions of starch. There were three characteristic peaks occurring between 1019 and 1065 cm−1, which were assigned to the vibration of C−O stretching. The peak at 1019 cm−1 was ascribed to the vibration of C−O−H deformation. Absorbances at 1080 and 1156 cm−1 were assigned to the coupling of C−O, C−C, and O−H bond stretching and bending and asymmetrical stretching of the C−O−C glycosidic bond.25,29 Alkali treatment did not greatly alter the FTIR spectra of starch samples. However, there was a small decrease in the absorbance ratio at 1045 and 1022 cm−1 after alkali treatment (Table 1), from 0.658 for native starch to 0.647 for starch treated with 0.25% NaOH for 3 week. XRD. Native wheat starch presented a characteristic XRD pattern of A-type polymorphs (Figure 3), with the diffraction

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Figure 3. XRD patterns of native and alkali-treated wheat starches.

DISCUSSION Effect of the Alkali Treatment on the Structure of Wheat Starch Granules. Alkali treatment decreased the protein and lipid contents of wheat starch significantly. Similar observations have also been reported with starches treated with alkali solution13 and SDS buffer7 or washed with alkali solution in starch extraction.30−33 The decreased protein and lipid contents of alkali-treated starch could be attributed to the solubilization of surface starch granule-associated proteins and lipids as internal

peaks occurring at 2θ 17.7°, 20.1°, 21.1°, and 26.9°. Alkali treatment did not change the polymorphic forms of wheat starch. The relative crystallinity of native wheat starch granules was about 32.7%, which slightly decreased to about 31.4−31.6% after alkali treatment (Table 1). Swelling Power and Starch Solubility. Native wheat starch granules had a swelling power of 11.4 g/g, which increased 3640



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Table 2. Swelling Power, Starch Solubility, and Thermal Transition Parameters of Native and Alkali-Treated Starchesa

a

samples

SP (g/g)

SS (%)

To (°C)

Tp (°C)

Tc (°C)

Tc − To (°C)

ΔH (J/g)

native starch 0.1% 1W 0.1% 2W 0.1% 3W 0.25% W 0.25% 2W 0.25% 3W

11.4 ± 0.1 a 13.6 ± 0.1 c 14.1 ± 0.5 d 13.4 ± 0.2 bc 13.1 ± 0.1 bc 13.0 ± 0.8 b 13.1 ± 0.1 bc

10.9 ± 0.9 a 21.5 ± 0.6 d 19.2 ± 0.2 b 20.8 ± 1.2 cd 19.9 ± 0.1 bc 21.4 ± 0.6 d 22.1 ± 0.3 d

57.3 ± 0.1 a 58.7 ± 0.2 b 59.2 ± 0.1 b 60.0 ± 0.1 c 61.2 ± 0.1 d 61.5 ± 0.5 d 61.7 ± 0.1 d

61.7 ± 0.2 a 62.8 ± 0.0 b 63.4 ± 0.1 c 63.8 ± 0.1 c 64.5 ± 0.2 d 64.8 ± 0.5 d 64.9 ± 0.1 d

66.1 ± 0.1 a 67.4 ± 0.1 b 67.5 ± 0.1 b 67.9 ± 0.2 bc 68.6 ± 0.5 c 68.9 ± 0.9 c 68.7 ± 0.2 c

8.8 ± 0.2 a 8.7 ± 0.1 a 8.3 ± 0.3 ab 7.9 ± 0.2 b 7.4 ± 0.1 bc 7.4 ± 0.4 bc 7.0 ± 0.1 c

9.3 ± 0.6 a 10.7 ± 0.7 a 9.8 ± 0.8 a 10.4 ± 1.5 a 9.8 ± 0.3 a 9.6 ± 0.6 a 10.5 ± 0.3 a

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

Table 4. Proportion of RDS, SDS, and RS in Native and AlkaliTreated Starchesa samples

RDS (%)

SDS (%)

RS (%)


16.5 ± 1.6 a 19.9 ± 1.0 b 19.3 ± 1.6 b 19.2 ± 2.0 b 19.7 ± 1.4 b 20.3 ± 2.4 bc 21.2 ± 1.3 c

13.6 ± 1.7 a 28.8 ± 0.5 b 28.9 ± 2.4 b 31.2 ± 0.7 bc 32.9 ± 3.0 c 34.5 ± 2.1 d 33.3 ± 0.2 c

69.9 ± 9.1 d 51.3 ± 0.5 c 51.8 ± 1.2 c 49.6 ± 1.2 bc 47.4 ± 0.6 b 45.2 ± 0.3 a 45.5 ± 0.1 a

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

a

proteins, and lipids are hardly extracted from the ungelatinized starch granules.34 Alkali treatment did not change the amylose content of wheat starch significantly, indicating that leaching or degradation of amylose molecules did not occur during treatment. In previous studies, alkali treatment has been shown to decrease the amylose content of pea starch granules22 or sago, corn, and potato starch granules.13 The effect of alkali treatment on pea starch granules was regarded as being analogous to a limited form of gelatinization.22 In this study, the alkali-induced gelatinization of wheat starch granules could be too limited to render the leaching or degradation of amylose molecules. Alkali treatment resulted in the deformation, disruption, or coalescence of wheat starch granules, as was also noted for alkalitreated pea, sago, corn, and potato starches13,22,35 or alkaliwashed rice and pinhão starches.30−32 The obvious morphological changes, such as disruption and coalescence, of starch granules after 0.25% NaOH treatment could result from the small swelling of wheat starch granules during alkali treatment, as evidenced by the small increase in the average particle size. The swelling of starch granules was proposed to mainly occur at amorphous growth rings and lamellae, with little effect on highdensity crystalline lamellae.32 As a result, the relative proportion of amorphous to crystalline regions increased, consistent with the observed small decrease in relative crystallinity and absorbance ratios from 1045 to 1022 cm−1 of alkali-treated starches. The

Figure 4. DSC thermograms of native and alkali-treated starches.

Figure 5. Pasting profiles of native and alkali-treated starches.

Table 3. Pasting Parameters of Native and Alkali-Treated Starchesa

a

samples

PV (cP)

TV (cP)

BD (cP)

FV (cP)

SB (cP)

PT (°C)


1610 ± 42 bc 1858 ± 50 d 1666 ± 54 c 1683 ± 55 c 1514 ± 86 b 1363 ± 43 a 1304 ± 45 a

1314 ± 63 d 1085 ± 18 c 1061 ± 29 bc 1059 ± 37 bc 980 ± 47 b 886 ± 30 a 814 ± 22 a

297 ± 21 a 773 ± 32 d 606 ± 25 c 625 ± 18 c 535 ± 39 b 477 ± 13 b 491 ± 23 b

1814 ± 79 b 2406 ± 8 d 2085 ± 23 c 1990 ± 51 c 1788 ± 82 b 1616 ± 38 a 1500 ± 41 a

501 ± 16 a 1321 ± 11 g 1024 ± 6 f 932 ± 13 e 809 ± 35 d 730 ± 8 c 687 ± 19 b

89.6 ± 1.2 b 67.5 ± 0.8 a 67.4 ± 0.6 a 68.2 ± 0.5 a 68.3 ± 0.5 a 68.7 ± 0.8 a 68.7 ± 0.9 a

Values are the mean ± SD. Values with the same letters in the same column are not significantly different (p < 0.05). 3641



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influence the pasting properties of starches in different ways, depending upon the alkali concentration, treatment period, temperature, and starch sources.11,13,22,35 Effect of the Alkali Treatment on Starch in Vitro Digestibility. Alkali treatment greatly decreased the RS content and increased the RDS and SDS contents, indicating the increased digestibility of wheat starch after alkali treatment. The increased digestibility of alkali-treated starch could be attributed to factors such as the removal of some surface proteins, roughening on the granule surface, disruption of starch granules, or increased amorphous regions in starch granules. The digestion of native starch granules is a very complex process consisting of the diffusion of the enzymes into the substrate, adsorption of enzymes to the substrate, and hydrolytic event.38−40 Of these processes, the diffusion of α-amylase into the substrate is considered as a key step of hydrolysis. The presence of some surface non-starchy substances, such as proteins and lipids, can limit the rate of enzymatic hydrolysis by reducing the diffusion of enzymes into the granules and blocking the adsorption site of the substrate.9,19 Removal of surface proteins and lipids after alkali treatment could greatly accelerate the penetration of enzymes into the granules and absorption of enzymes to its substrate, therefore increasing the digestibility of alkali-treated starch. In addition, roughening on the granule surface or granule disruption could also enhance the accessibility of enzymes to starch, hence increase the enzymatic digestibility. With the easy penetration of enzymes into alkali-treated starch granules, the resistant part of starch is more accessible for amylase to digest than that of native starch granules, leading to the decreased RS content. In summary, alkali treatment can remove the proteins and lipids (mostly from the surface) from starch granules without significantly affecting the internal crystalline structure of starch. In contrast, the functionality of starch granules was altered greatly by alkali treatment. The swelling power and starch solubility were increased greatly by alkali treatment. However, the alkali treatment effect on properties of thermal transition and pasting varied with the concentration of NaOH and treatment period. The in vitro digestibility of starch granules was increased significantly by alkali treatment, as evidenced by the increased RDS and SDS contents and decreased RS content. The alkaliinduced changes in functionality and in vitro digestibility of wheat starch granules could be predominantly attributed to the removal of surface proteins and lipids, but the alterations to granular morphology and small swelling of amorphous regions may also be taken into account. Our results clearly indicated that alkali treatment can be taken to be an important modification method to modify the quality and nutritional properties of starchy food products.

decreased crystallinity of alkali-treated starch was also observed with pea,22 pinhão,32 and rice30 starches. Effect of the Alkali Treatment on Starch Functionality. Swelling power and starch solubility of the wheat starch were increased greatly by the alkali treatment. The considerable increase in swelling power of alkali-treated wheat starch could be attributed to the partial removal of surface proteins and lipids, which were considered to inhibit the swelling of starch granules. Similar results were also reported by other researchers.7,10,11 In other studies, the observed increase in swelling power of alkalitreated corn and potato starches was attributed to the reduction of restraining effects of amylose in the amorphous regions, allowing for the granules to swell more freely.13,35 Upon heating in excess water, alkali-treated starch granules were visually observed to form a more transparent and softer paste, which was poured out in a small amount with supernatant during the swelling power test. The separated paste in the supernatant was taken as solubilized starch, hence resulting in the large increase in starch solubility. Alkali treatment increased the thermal transition temperatures of wheat starch but decreased the temperature range. Higher DSC transition temperatures are thought to be associated with a higher degree of crystallinity or more ordered crystalline structure, which makes the granules more resistant to be gelatinized. Because there was a small decrease in relative crystallinity, the increased gelatinization temperature and decreased temperature range indicated that the stability and homogeneity of the crystal structure of alkali-treated starch was enhanced in comparison to that of native starch. In addition to the solubilization of surface proteins and lipids, the annealing of starch granules during alkali treatment may also occur, which leads to the increased stability and homogeneity of the crystal structure.36 The enthalpy change was little changed by alkali treatment, consistent with the slight decrease in the relative crystallinity of starch granules. Previous studies have shown that the effect of alkali treatment on the starch thermal transition varies with the alkali concentration, treatment period, and starch sources.13,22,35 Alkali treatment with 0.1% NaOH significantly increased the peak viscosity, with 1 week treatment leading to the greatest increase. The increase in peak viscosity is consistent with the increased swelling power of alkali-treated starches. In contrast, 0.25% NaOH treatment resulted in the decreased peak viscosity of wheat starch, with three week treatment causing the greatest decrease. The decrease in peak viscosity of alkali-treated starches with 0.25% NaOH seemed to be inconsistent with the increased swelling power. After treatment with 0.25% NaOH, starch granules were assumed to be more fragile to destruction (or amylopectin molecules more vulnerable to degradation), especially when heated under the shear forces.22 The decreased peak viscosity after 0.25% NaOH treatment was indicative of the degradation of amylopectin molecules during RVA heating under shear forces. The degradation of amylopectin molecules was further corroborated by the decreased trough viscosity and increased breakdown viscosity of alkali-treated starches. The setback viscosity is thought to result from the rearrangement of amylose molecules that have leached from swollen starch granules during cooling and is indicative of the retrogradation tendency of starch.37 The increased setback viscosity of alkalitreated starch indicated the faster retrogradation of starch paste. Alkali treatment decreased the pasting temperature by about 12 °C, indicating that alkali-treated starch has a more rapid swelling tendency than native starch. Alkali treatment has been shown to

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Funding

Shujun Wang gratefully acknowledges the financial support from the National Natural Science Foundation of China (31371720) and t he Natural Science Foundation of Tianjin (13JCYBJC38100). Shujun Wang also greatly appreciates the financial support of Haihe River Scholar Program (000050401) from Tianjin University of Science and Technology. Notes

The authors declare no competing financial interest. 3642



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