Resistant starch isolated from enzymatic, physical, and acid treated pea starch: Preparation, structural characteristics, and in vitro bile acid capacity

LWT - Food Science and Technology 116 (2019) 108541

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Resistant starch isolated from enzymatic, physical, and acid treated pea starch: Preparation, structural characteristics, and in vitro bile acid capacity

T

Dingting Zhou, Zhen Ma∗, Jiangbin Xu, Xiaoping Li, Xinzhong Hu College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi'an, Shaanxi, 710062, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Pea starch Processing treatments Resistant starch Multi-scale structural order Bile acid binding capacity

The multi-scale structural order and bile acid (BA) binding capacity of resistant starch (RS) isolated from native, different processed and recrystallized pea starch samples were studied. Different treatments applied include acid hydrolysis (AHPRS), pullulanase-debranching (PDPRS), autoclaving (ACPRS), ultrasound-autoclaving (UAPRS), and microwave cooking (MCPRS). Subsequently, the native, processed and recrystallized starch were subjected to in vitro digestion using thermostable α-amylase and amyloglucosidase to obtain the isolated RS. The Mw values of RS samples ranged from 2.448 × 104−9.750 × 106 g/mol, with the highest value noticed for ACPRS, and the lowest one for NPRS. The B-type and a small amount of V-type crystalline pattern was observed in all RS samples. Based on X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) measurement, the RS isolated from native pea starch, which is of type II resistant starch, exhibited the significantly lowest crystallinity and degree of order (DO), compared with RS3 samples prepared from processed and recrystallized starch. The relative crystallinities measured by 13CNMR (16.78–18.97%) were generally higher than the values of crystallinity obtained from XRD (3.36–12.23%). The strongest absorption that is related to O-H stretching between 3000 and 3600/cm on the FT-IR spectra, the highest DO value, and the highest BA binding capacity were observed for AHPRS.

1. Introduction Pea (Pisum sativum L.) is a predominant crop in world trade and ranks second in the grain legume production globally (Majeed, Wani, Hamdani, & Bhat, 2018). Pea starch is primarily available as a byproduct from the extraction process of pea proteins. The industrial application of pea starch is very limited because of its low thermal stability, low acid and shear resistance, and high tendency to retrograde (Zhou, Ma, Yin, Hu, & Boye, 2019). Nevertheless, pea starch is good source of resistant starch with the reported values between 21% and 53.4% (Lehmann, Rössler, Schmiedl, & Jacobasch, 2003; Li et al., 2019; Lu, Belanger, Donner, & Liu, 2018), and because of its high amylose content (35–65%), pea starch also has a high tendency to form recrystallized resistant starch during processing and storage (Skrabanja, Liljeberg, Hedley, Kreft, & Björck, 1999). Resistant starch (RS) is defined as the sum of starch and its degradation products that is not absorbed in the small intestine of healthy individuals and can be fermented by microflora into short-chain fatty acids in guts that perform metabolic and colonic health benefits (Asp & Björck, 1992). Substantial progresses have been made in the preparation of resistant starch by a variety of processing methods including

∗

heat-moisture, autoclaving, microwaving, high hydrostatic pressure, extrusion, ultrasound, acid hydrolysis, and enzymatic debranching treatments (Ma, Hu, & Boye, 2018), with the purpose of modifying its structure, techno-functional, and consequently its physiological functions. The structural characterizations in these studies were generally performed directly on the native and processed starches that are abundant in RS (i.e., the processed starch before digestion). The physiological property of the processed starch, however, is more dependent on the structural characteristics of the modified starch subsequent to digestion (i.e., isolated resistant starch) rather than before digestion (Mutungi et al., 2011; Zhou, Cao, & Zhou, 2013; Zhou, Zhang, Zheng, Chen, & Yang, 2013). Therefore, this work was undertaken to study and compare the multi-scale structural characteristics and the bile acid binding capacity of resistant starch isolated from the native, enzymatic, physical, and acid treated pea starch, aiming to provide fundamental knowledge for the designing of RS3 (resistant starch type III, also known as recrystallized starch) products with targeted structure, techno-functional and physiological properties for food industrial applications.

Corresponding author. E-mail address: [email protected] (Z. Ma).

https://doi.org/10.1016/j.lwt.2019.108541 Received 27 April 2019; Received in revised form 9 July 2019; Accepted 22 August 2019 Available online 22 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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2. Materials and methods

(pullulanase debranched pea resistant starch).

2.1. Materials

2.2.6. Isolation of pea resistant starch Isolation of the RS samples was performed using the in vitro digestion method according to the procedure described by Zhang, Zeng, Wang, Zeng, and Zheng (2014) with slight modification. The native and different processed starches (10 g, dry weight) were mixed with 200 mL of citric acid buffer (pH 6.0). The thermostable α-amylase (5000 U/mL) was added and the suspension was incubated at 95 °C for 1 h in an orbital incubator shaker at 128 rpm. After adjusting the pH to 4.5 with a citric acid solution (4 mol/L), the mixture was incubated with amyloglucosidase (70 U/mg) at 60 °C for 1 h. After centrifugation at 4000g for 10 min, the resulting residues were washed three times with 75%, 85%, 95% ethanol, dried in an oven at 45 °C and ground before passing through a 149-μm sieve. The RS isolated from native pea starch is referred to as NPRS (native pea resistant starch).

The native pea starch was provided by Chengdongwang Food Co., Ltd. (Chengdu, Sichuan, China). Thermostable α-amylase (A3306, 5000 U/mL), amyloglucosidase (10115, 70 U/mg), and Pullulanase (E2412, ~1498 U/g) were obtained from Sigma Chemical Company (SigmaAldrich, St. Louis, Missouri, USA). All other chemicals and reagents used in this study were of analytic grade. 2.2. Preparation and isolation of pea resistant starch 2.2.1. Autoclaving 180 g of native pea starch was suspended in 900 mL distilled water. The starch suspension was heated at 121 °C for 30 min in an autoclave (LD2X–30KBS, Shen'an medical Co., Ltd. Shanghai, China) after pregelatinization in boiling water for 5 min. The obtained mixture was then cooled to 25 °C and stored at 4 °C for 24 h. The autoclaved pea starch was allowed to dry at 45 °C for 24 h before enzymatic isolation process. The ultimate products are referred to as ACPRS (autoclaved pea resistant starch).

2.3. X-ray diffraction (XRD) analysis The XRD traces of the RS samples were measured using a X-ray diffractometer (D/Max2550VB+/PC, Rigaku Corporation, Rigaku, Japan) following the method of Yin, Ma, Hu, Li, and Boye (2018) using MDI Jade 6.0 software (Materials Data Inc, Liverpool, CA). The samples were scanned in the angular range (2θ) between 5° and 45° with step intervals of 0.01° and a scanning rate of 5°/min, the operation condition was set at 40 kV and 40 mA. Before analysis, all RS samples were equilibrated in a chamber with saturated NaCl solution at room temperature for 120 h.

2.2.2. Microwave cooking Native pea starch (180 g) was dispersed in 1200 mL of distilled water. The starch suspension was heated under the microwave power of 700 W for 5 min in a microwave oven (Midea™, Beijing, China, China), cooled to room temperature, stored at 4 °C for 24 h, and dried at 45 °C for 24 h. The obtained sample was then enzymatically isolated and is denoted as MCPRS (microwave cooked pea resistant starch).

2.4. Fourier transform infrared spectroscopy (FT-IR)

2.2.3. Ultrasound-autoclaving treatment Native pea starch (180 g) was dispersed in 400 mL of distilled water (starch: water = 9:20). The starch suspension was exposed to ultrasound treatment in an ultrasonic transducer (KQ3200B-type ultrasonic bath, Kunshan Ultrasonic Instruments Co. Ltd., Kunshan, Jiangsu, China) at the power of 300 W for 50 min at 25 °C. The obtained sample was then autoclaved at 121 °C for 30 min, cooled to 25 °C, stored at 4 °C for 24 h, and dried at 45 °C for 24 h. The ultimate isolated resistant starch is denoted as UAPRS (ultrasound-autoclaved pea resistant starch).

FT-IR spectra of the RS were acquired by a Fourier transform infrared spectrometer (TENSOR27; Bruker Optics GmBH, Ettlingen, Germany) according to the method described by Zhang et al. (2014). The spectra were recorded at the wavelengths between 400 and 4000/ cm with a resolution of 4/cm. 2.5. Solid state 13C cross-polarization and magic angle spinning nuclear magnetic resonance (13C CP/MAS NMR) spectroscopy An AVANCE III 400 MHz WB spectrometer (Bruker Inc., Germany) at a13C frequency of 100.62 MHz with spin rate of 6 kHz and scan numbers of 1600 was used for the measurement of the 13C CP/MAS NMR spectra of resistant starch samples. The relative crystallinity of samples was estimated using MestReNova (v. 9.0.1) software (Mestrelab Research, Santiago de Compostela, Spain) following the method described by Ma, Yin et al. (2018).

2.2.4. Acid hydrolysis The starch slurry (100 g/L) was prepared by suspending the native pea starch (20 g) in 200 mL distilled water. This slurry was pre-gelatinized for 10 min in boiling water prior to the addition of 1 mol/L HCl at a proportion of 1:1 (starch:acid, g/mL). Acid hydrolysis was continued in a water bath at 45 °C for 2 h. The acidic hydrolyzed mixture was then adjusted to pH 6.0 with 2 mol/L sodium hydroxide solution immediately after acid hydrolysis and pressure cooked at 121 °C for 30 min. The processed sample was then cooled to 25 °C, retrograded at 4 °C for 24 h, and dried at 45 °C for 24 h. The resulting product was then subjected to enzymatic isolation process and is denoted as AHPRS (acid hydrolyzed pea resistant starch).

2.6. Size exclusion chromatography (SEC) The weight distribution of pea RS samples was measured by an Agilent 1260 series SEC system (Agilent Technologies, Waldbronn, Germany) equipped with a multi-angle laser light scattering (MALLS, DAWN-HELEOS II, Wyatt Technology Corp., Santa Barbara, CA, USA) following the procedure in the study of Ma, Yin et al. (2018). The samples were eluted by DMSO/LiBr at a flow rate of 0.3 mL/min, and were separated using GRAM 3000 and GRAM 10,000 Å chromatographic columns (PSS). The weight-average molar mass (Mw), numberaverage molecular mass (Mn), and polydispersity index (Mw/Mn) were estimated using Wyatt ASTRA™ software (v. 6.1.4.25).

2.2.5. Pullulanase debranching Native pea starch suspension was prepared at a concentration of 100 g/L and was heated at 85 °C for 15 min with continuous stirring. The pre-gelatinized sample was then autoclaved at 121 °C for 30 min, cooled to 50 °C and immediately adjusted to pH 5.2 with 1 mol/L sodium hydroxide solution. The obtained slurry was incubated at 46 °C for 8 h after the addition of pullulanase (30 U/g). At the end of the time period, the sample was heated for 10 min at 100 °C to inactive the enzyme and cooled to room temperature. The processed sample was then retrograded at 4 °C for 24 h and dried at 45 °C, the ultimate isolated samples is referred to as PDPRS

2.7. Small angle X-ray scattering (SAXS) The SAXS spectra of the RS samples were obtained by a GmbH SAXS system (Bruker AXS, Germany) operated at 50 kV and 600 μA, with Nifiltered Cu-Kα radiation (λ = 0.154 nm). The SAXS pattern was 2

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ACPRS, PDPRS, and AHPRS than in NPRS (Mutungi et al., 2011). The observation was consistent with those reported by Leong, Karim, and Norziah (2007) that the transformation of the C- to B- type crystalline pattern was normally accompanied during the process of obtaining RS from the acid/enzyme hydrolyzed and autoclaved starch. A small amount of the V-type crystalline polymorph, resulting from the peak appeared at 20°, was also shown for pea RS samples, especially for MCPRS, ACPRS, PDPRS, and AHPRS. The previous studies suggested that changes in RS polymorphism could induce an ecological shift in the colonic microbiota and thus resulted in different effects in bowel health (Lesmes, Beards, Gibson, Tuohy, & Shimoni, 2008). The relatively crystallinities (C1), which were calculated according to method described by Yin et al. (2018), are displayed in Table 1. It could be clearly seen that the C1 value for RS from native pea starch (NPRS) was significantly lower (P < 0.05) than those for RS isolated from processed pea starch. The above observation indicated that different processing treatments applied seemed to have a positive effect on the formation of a more ordered double helix structure within the longrange crystalline domain of resistant starch. RS1 and RS2, the predominant type of resistant starch found in native peas, are either embedded in raw granules or being protected from digestion because of physical encapsulation. RS3, the major fractions of resistant starch in processed pea starch, can be formed in retrograded starch that require heating/degradation and storage. Having included the milling process, the resistant starch samples obtained from the native pea starch eliminated the contribution of RS1 (Perera, Meda, & Tyler, 2010), the NPRS thus was presented in the form of RS2. Based on the obtained XRD results, it can be inferred that RS2 from native peas exhibited lower crystallinity compared with the RS3 samples from retrograded pea starch. The isolated RS2 and RS3 have demonstrated to be differentiated in their fermentation behavior due to the variations in multi-scale structural features, particularly the polymer chain distribution in the resistant residues (Ma & Boye, 2018). It was also noticed that AHPRS exhibited the significantly highest C1 value, followed by UAPRS, suggesting that a significantly higher amount of double helical chain was arranged into large crystal entities capable of diffracting X-rays in AHPRS and UAPRS (Mutungi et al., 2011). The C1 value of PDPRS was significantly lower than that of ACPRS and MCPRS (P < 0.05), suggesting that the combined treatment of autoclaving-debranching showed a less pronounced effect on the formation of long-range ordered structure in RS compared with autoclaving and microwave cooking of peas.

recorded in the vector of q range from 0.07 to 2.3/nm (Ma, Yin et al., 2018). The temperature was kept at 25.0 ± 0.1 °C during the measurement. 2.8. Scanning electron microscopy (SEM) The RS samples were placed on a circular stub using the doublesided adhesive tape and were sputter coated with 50 nm thickness of gold layer. The morphological images of RS were recorded by a Quanta 200 SEM (FEI Company, Hillsboro, Oregon, USA) at an acceleration voltage of 20 kV with the magnification of 2000✕. 2.9. In vitro bile acid (BA) binding capacity The BA binding capacity measurement was conducted by the methodology reported by Kim and White (2010). The unbound bile acid content in the supernatant was measured by TBA ELISA kit (Shanghai Enzyme-linked Biotechnology Company, Shanghai, China). 2.10. Statistical analysis At least duplicate measurements were carried out. Statistical significance of difference was tested by one-way analysis of variance using PRISM (V3.02, GraphPad Software, Inc., CA, USA) by Duncan's multiple comparison test at 5% significance level. 3. Result and discussion 3.1. Crystalline structure characteristics by XRD Fig. 1 presents the XRD patterns of RS isolated from native and processed pea starch. The strong reflections at 2θ = 17.34°, 22.1°, and 23.7° indicated that the type B crystalline structure was formed in all RS samples (Lopez-Rubio, Flanagan, Gilbert, & Gidley, 2008a; Ma & Boye, 2018). Particularly, the intensities of the diffraction peak between 22.1° and 23.7° for RS isolated from processed starch were relatively stronger than that for NPRS, suggesting that due to the processing treatments applied, larger amount of the type B crystalline arrangement was evident with an increase in the size of crystallites in UAPRS, MCPRS,

3.2. FT-IR spectroscopy analysis As shown in Fig. 2, the intensity of absorption peak between 800 and 1200/cm, which was attributed to the stretching vibration of C-C, C-OH, and C-H, and the absorption peak at 2929 /cm, which reflected the C-H2 stretching, were both relatively higher for RS samples from processed pea starch than NPRS. Particularly, the FT-IR absorptions at 1047/cm and 995/cm are associated with molecular order of starch polymers, whereas the absorption peak at ~1022/cm is linked to the amorphous or disorder region. The proportions of the integrated peak area at 995/1022 cm−1 and 1047/1022 cm−1 were adopted to estimate the variations in degree of double helix (DD), and degree of order (DO), respectively (Ma & Boye, 2018). The DO values of RS samples from native and processed pea starch was in the following order: AHPRS > UAPRS > ACPRS > MCPRS > PDPRS > NPRS. This ranking was consistent with those values observed for relative crystallinity by XRD. It was also noticed from Fig. 2 that the intensity of the broad absorption between 3000 and 3600/cm, which was linked with the stretching vibration of free and hydrogen bonded hydroxyl groups, was comparatively lower for NPRS compared with other RS samples, whereas the strongest intensity at 3000–3600/cm was observed for AHPRS. This finding was consistent with the data obtained for C1 and DO values, suggesting that the higher degree of degradation caused by

Fig. 1. X-ray diffraction patterns of resistant starch isolated from native and processed pea starch, where (A) NPRS (native pea resistant starch); (B) UAPRS (ultrasound-autoclaved pea resistant starch); (C) MCPRS (microwave cooked pea resistant starch); (D) ACPRS (autoclaved pea resistant starch); (E) PDPRS (pullulanase debranched pea resistant starch); (F) AHPRS (acid hydrolyzed pea resistant starch). 3

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Table 1 Multi-scale structural order, molecular weight distribution, and in vitro bile acid binding capacity of resistant starch isolated from native and processed pea starch. Samples

AHPRS UAPRS ACPRS MCPRS PDPRS NPRS

1047/1022 cm−1 ratio by FT-IR (DO value) 1.2222 1.1349 1.1311 1.1298 1.1128 1.0284

± ± ± ± ± ±

0.0006a 0.0008b 0.0013c 0.0003c 0.0004d 0.0004e

995/1022 cm−1 ratio by FT-IR (DD value)

Crystallinity by XRD (C1, %)

13

0.0020a 0.0001d 0.0004b 0.0009b 0.0001e 0.0004c

12.23 ± 0.01a 11.13 ± 0.26b 5.56 ± 0.03c 5.39 ± 0.08c 4.61 ± 0.14d 3.59 ± 0.01e

18.97 17.84 17.59 16.78 18.09 17.78

1.0788 0.9963 1.0426 1.0403 0.9906 1.0264

± ± ± ± ± ±

Crystallinity by C NMR (C2, %)

± ± ± ± ± ±

0.01a 0.00ab 0.00b 0.00b 0.01ab 0.01b

Double helix content by 13C NMR (%) 61.00 68.23 67.96 68.02 66.59 66.38

± ± ± ± ± ±

0.01b 0.00a 0.01a 0.00a 0.01a 0.01a

In vitro bile binding capacity

Dm by SAXS

Mw (g/mol)

Mn (g/mol)

Mw/Mn

83.41 ± 0.17a 14.77 ± 0.41d 19.68 ± 0.72c 33.10 ± 1.79b 5.63 ± 0.48e 17.93 ± 0.14cd

1.59 1.82 1.60 1.65 1.71 1.77

2.654 × 106 6.974 × 106 9.750 × 106 4.535 × 106 1.838 × 105 2.448 × 104

1.148 × 106 2.675 × 106 6.675 × 106 1.059 × 106 1.789 × 105 2.101 × 104

2.311 2.607 1.477 4.282 1.028 1.165

NPRS (native pea resistant starch); UAPRS (ultrasound-autoclaved pea resistant starch); MCPRS (microwave cooked pea resistant starch); ACPRS (autoclaved pea resistant starch); PDPRS (pullulanase debranched pea resistant starch); AHPRS (acid hydrolyzed pea resistant starch). *For a given parameter, mean values bearing different lower case letters within the same column are significantly different (P < 0.05) based on Duncan's multiple comparison test.

Fig. 2. FT-IR spectra of resistant starch isolated from native and processed pea starch, where (A) NPRS (native pea resistant starch); (B) UAPRS (ultrasoundautoclaved pea resistant starch); (C) MCPRS (microwave cooked pea resistant starch); (D) ACPRS (autoclaved pea resistant starch); (E) PDPRS (pullulanase debranched pea resistant starch); (F) AHPRS (acid hydrolyzed pea resistant starch).

Fig. 3. Solid-state 13C CP/MAS of resistant starch isolated from native and processed pea starch, where (A) NPRS (native pea resistant starch); (B) UAPRS (ultrasound-autoclaved pea resistant starch); (C) MCPRS (microwave cooked pea resistant starch); (D) ACPRS (autoclaved pea resistant starch); (E) PDPRS (pullulanase debranched pea resistant starch); (F) AHPRS (acid hydrolyzed pea resistant starch).

different processing conditions could lead to a higher chance of molecular chain arrangement towards the evolution into coil-to-helix transition in resistant starch, especially for AHPRS. The metabolic activity studies by Zhou, Cao et al. (2013) and Zhou, Zhang et al. (2013) suggested that the starch samples with higher values of DO tended to produce greater amount of butyrate at a faster metabolic rate, whereas the samples with lower ratio of 1047/1022 cm−1 yielded greater levels of lactate and acetate upon fermentation of their corresponding starches.

3.3. Ordered structure by

Table 2 Chemical shifts of the major peaks corresponding to different carbon atoms of glucose for resistant starch samples by 13C CP/MAS NMR. Samples

Chemical shifts (ppm) of carbon atom of glucose C2,3,5

C1 AHPRS UAPRS ACPRS MCPRS PDPRS NPRS

13

C CP/MAS NMR

The 13C CP/MAS NMR spectra and their fitted peak profiles of resistant starch from native and different processed pea starches are shown in Fig. 3. The chemical shifts at 94–105 ppm, 80–84 ppm, and 58–65 ppm are linked with C1, C4, and C6 of hexapyranoses, respectively, whereas the overlapping resonance centered between 68 and 79 ppm are attributed to C2,3,5 (Table 2) (Atichokudomchai, Varavinit, & Chinachoti, 2004). The multiplicity of the signals at C1 region in the 13 C CP NMR spectra provides insights into the crystalline packing nature and the amorphous structures the starch granule. In type B crystalline structure, maltose is the repeating unit which forms the three-folded double helical structure. The doublet peak appeared in the C1 region suggested the presence of type B crystalline polymorph in all RS samples. The existence of the broad peak centered at 82 ppm for C4

100.9 101.54, 101.04, 101.02, 100.93, 101.21,

100 94.45 94.25 94.32 94.49

75.38, 75.43, 75.43, 75.32, 75.35, 75.38,

72.35 72.57, 71.08 72.39 72.38 72.36 72.29

C4

C6

82.19

62.08 62.13 62.07 62.02 62.02 65.21, 61.77

82.82

was resulted from the amorphous region (Fan et al., 2013). The appearance of the tiny peak at 94 ppm suggested the existence of V-type crystalline structure. The observation was in accordance with the XRD observation and those reported by Mahadevamma, Harish Prashanth, and Tharanathan (2003). The relative crystallinities (C2) and contents of double helix, which were estimated from 13C CP/MAS NMR spectra following the method reported by Yin et al. (2018), are presented in Table 1. The C2 values from 13C CP/MAS NMR was in the range of 16.78–18.97%, which was generally higher than the values of relative crystallinity (C1) quantified 4

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Particularly, the surface fractal dimension (−3 < α < −4) and mass fractal dimension (−1 < α < −3) were adopted to evaluate the degree of smoothness and compactness of the scattering object, respectively (Liu et al., 2017). The α values for all RS samples were in the range between −1.59 and −1.82, indicating that all samples were classified as the mass fractal structure and the density characteristic of the scattering object had a self-similar attribute (Suzuki et al., 1997). The mass fractal dimension, Dm, which gives insights into the physical arrangement of the polymer segments (i.e., the degree of compactness), is expressed as follows: Dm = −α (−1 < α < −3). The higher Dm value of UAPRS suggested the more compact interior structure of the RS obtained from ultrasonic-autoclaved pea starch. The scattering intensity is linked with the difference of electron density between the ordered crystalline area and the amorphous phase of the lamellar structure of starch granules (Blazek & Gilbert, 2011). The highest scattering intensity at Imax was observed for MCPRS, and the lowest one was obtained for PDPRS. This finding was consistent with the results obtained for the polydispersity index measured by SECMALLS, suggesting a correlation between the chain length distribution and the electron density contrast of the amorphous and crystalline regions.

by X-ray diffraction. This confirmed the earlier findings that in contrast to the XRD measurement, which could only provide information on the regularly repeating ordering of double helices, the 13C CP/MAS NMR is capable of measuring the ordered structure on the short-range scale including the irregularly packed structures and small chain aggregates that are arranged in amorphous phase (Mutungi et al., 2011). The double helix content of AHPRS (61.00%) was significantly lower than other RS samples (66.38–68.23%) obtained in this study (Table 1), suggesting that double helical structure that were either resistant from the beginning of processing or developed upon processing and enzymatic isolation process were arranged in a relatively crystalline pattern. 3.4. Mw by SEC-MALLS The Mw, Mn, and Mw/Mn of RS samples isolated from native and pea starch processed by different methods are displayed in Table 1. The Mw values of RS samples ranged from 2.448 × 104−9.750 × 106 g/mol, with the highest value noticed for ACPRS, and the lowest one for NPRS. The results suggested that during the processing of pea starch, the occurrence of gelatinization/degradation and retrogradation induced the formation of a more compact network structure, which protected the starch molecules being attacked by enzymatic hydrolysis during isolation, thus leading to the relatively higher Mw values observed for AHPRS, UAPRS, ACPRS, MCPRS, PDPRS compared with NPRS. The significantly lowest Mw value of NPRS was in accordance with its lowest values of DO and C1 measured by FTIR and XRD, indicating that the chain length with low Mw value was not in favor for the development of perfect crystalline structure in NPRS. Similar findings have also been reported by Yin et al. (2018), who found that the processed lentil starch chains with lower Mw values tended to have a negative impact on the development of ordered crystalline structure. Kiatponglarp, Tongta, Rolland-Sabaté, and Buléon (2015) also reported that the amount of the crystalline structure decreased with the continuous decrease in Mw. The polydispersity index (PDI), known as the ratio of Mw to Mn, generally reflects the variety of molecular shapes and the range of molecular weight distribution. A value of PDI close to 1.0 represents for a homogeneous molecular weight distribution in starch polymers. The relatively higher PDI value for MCPRS suggested its wider molecular weight distribution than other RS samples. This could be explained by the mechanism of microwave cooking that resulted in a rapid rise in temperature which restricted granular swelling and rupture of granules, thereby causing the heterogeneous degradation and the broader molecular weight distribution in MCPRS (Palav & Seetharaman, 2007). The observation was further corroborated by its lowest C2 value measured by 13CNMR. Comparatively, it was noticed that PDPRS exhibited the lowest PDI value, indicating that pullulanase debranching had a pronounced effect on the formation of a homogeneous chain length distribution in RS.

3.6. Morphological characteristics The morphological properties of the RS samples were examined by scanning electron microscopy and are shown in Fig. 5. All resistant starch exhibited a characteristic irregular and block structure in flaky shapes. This could be explained by the disassembling of granular structure and the formation of short linear amylose chains which were then aggregated and developed into the irregularly shaped particles. The surface topography was characterized by the appearance of layered strips which could have been left by the degradation after different processing treatments and enzymatic hydrolysis process. The SEM images observed in this study resembled those structures of resistant starch that have been reported previously (Lopez-Rubio et al., 2008b; Zhang et al., 2014). Compared with RS samples obtained from processed starch (RS3), NPRS (RS2) showed less amounts of residual fragments with smaller granular size. There were still some large aggregates remaining for RS3 samples, though most of the digestive residues were present as small clumps. According to Lopez-Rubio et al. (2008b), during the in vitro enzymatic digestion of processed starch, a significant reduction in particle size and the formation of small clusters was observed. The observation in this study could thus be attributed to a higher degree of hydrolysis in AHPRS, ACPRS, PDPRS, MCPRS, and UAPRS during enzymatic isolation, leading to a more pronounced reduction in particle size of the aggregates. 3.7. Bile acid binding capacity Bile acids (BA) are synthesized in the liver from cholesterol. Majority of the BA is actively reabsorbed by terminal ileum after emulsification with fat in small intestine. Meanwhile, the other possible pathway is the entrapment of bile by resistant starch in the large intestine, which is then excreted with feces and thus leads to a reduction the blood cholesterol level. To calculate the remaining bile acid concentration of the supernatant of the RS samples, a calibration curve of y = 0.1301x + 0.2812 (R2 = 0.9696) was established between the absorbance (y) and solution concentration (x). The relative bile acid binding capacities of the RS samples were found to be in the range between 5.63 and 83.41% (Table 1). The significant differences among the results of in vitro BA binding capacities may be attributed to many factors. The FT-IR results revealed that AHPRS showed the significantly highest DD value among all RS samples, whereas the PDPRS exhibited the lowest one (Table 1). This observation corresponded well with the BA binding capacity results, indicating that the ability of RS to form inclusion complexes with bile acid was directly related to its double

3.5. Molecular order by SAXS analysis The typical peak at 0.06–0.07/nm, which corresponded to a Bragg distance of ~9–10 nm periodicity occurring form the alternating crystalline and amorphous structure of starch granule, could no longer be found in all RS samples (Fig. 4). The observation suggested the destruction of the granule's long-range lamellar order and the further molecular rearrangement accompanied by the growth of new and incomplete crystal structures in the amorphous matrix during different processing and enzymatic isolation process. Suzuki, Chiba, and Yarno (1997) suggested that the retrograded starch could form a different lamellar stacking structure in comparison with that of the native starch. Similar findings have also been reported by Ma, Yin et al. (2018) and Lopez-Rubio, Flanagan, Shrestha, Gidley, and Gilbert (2008b). The curve was fitted to a power law model in the low q-region (I(q) ~qα), where the exponent α is used to describe the surface/mass fractal dimensions and the self-similar structure of starch granules. 5

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Fig. 4. SAXS patterns of resistant starch isolated from native and processed pea starch, where (A) NPRS (native pea resistant starch); (B) UAPRS (ultrasound-autoclaved pea resistant starch); (C) MCPRS (microwave cooked pea resistant starch); (D) ACPRS (autoclaved pea resistant starch); (E) PDPRS (pullulanase debranched pea resistant starch); (F) AHPRS (acid hydrolyzed pea resistant starch).

the deoxycholic acid molecule, the interaction with the hydroxyl group in starch is stronger. Comparison of the BA binding capacity results obtained in this study suggested that AHPRS had the highest health promoting effect because of its conceivable cholesterol lowering potential.

helical structure. Researches have also shown that the bile acid binding capacity of RS due to its helical structure was one of the mechanisms that lead to the decreased BA levels in the supernatant (Soral-Śmietana & Wronkowska, 2004; Verbeek, De, Tijburg, Van, & Beynen, 1995). According to Abadie, Hug, Kübli, and Gains (1994), and Hinrichs et al. (1987), the most likely binding site for amphiphilic ligand with undigested starch is in the hydrophobic interior of the helical structures that are formed by the 1,4-linked glucose units of amylose and amylopectin. In addition, the degree of affinity of such interaction depends on both the number of binding sites per unit and the stability of starch helical complex. The relatively loose interior structure of AHPRS, as evidenced by its lowest Dm value measured by SAXS (Table 1), may also contribute to the most pronounced enhancement in bile acid binding capacity of AHPRS compared with other RS samples. This could be explained by the assumption that bile acids, especially dihydroxy-BA, may enter through the surface cracks of AHPRS and resulted in a stronger interaction between the RS and BA. As reported by Zhou, Xia, Zhang, and Yu (2006), due to the presence of two hydroxyl groups in

4. Conclusion The RS2 fraction from native pea starch (NPRS) exhibited lower values of crystallinity (C1) and DO compared with RS3 samples obtained from the processed starch. AHPRS exhibited the significantly highest values of C1 and DO, as well as the strongest absorption between 3000 and 3600/cm on the FT-IR spectra. The Mw values of RS samples ranged from 2.448 × 104−9.750 × 106 g/mol, with the highest value noticed for ACPRS, and the lowest one for NPRS. The significantly lowest Mw value of NPRS was in accordance with its lowest crystallinity and degree of order measured by XRD and FT-IR, indicating that the chain length with low Mw value was not in favor for Fig. 5. Scanning electron microscopy of resistant starch isolated from native and processed pea starch, where (A) ACPRS (autoclaved pea resistant starch); (B) PDPRS (pullulanase debranched pea resistant starch); (C) UAPRS (ultrasound-autoclaved pea resistant starch); (D) MCPRS (microwave cooked pea resistant starch); (E) AHPRS (acid hydrolyzed pea resistant starch); (F) NPRS (native pea resistant starch).

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the development of perfect crystalline structure in NPRS. The α values estimated from the SAXS measurement suggested that all RS samples were classified as the mass fractal structure and the density characteristic of the scattering object had a self-similar attribute. Comparison of the BA binding capacity results obtained in this study suggested that AHPRS had the highest health promoting effect because of its conceivable cholesterol lowering potential.

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Declarations of interest None. Acknowledgement The authors would like to acknowledge the research funds from the National Natural Science Foundation of China (31501405); the Funded Projects for the Academic Leaders and Academic Backbones, Shaanxi Normal University (18QNGG010); and the Fundamental Research Funds for the Central Universities of China (GK201702012). References Abadie, C., Hug, M., Kübli, C., & Gains, N. (1994). Effect of cyclodextrins and undigested starch on the loss of chenodeoxycholate in the faeces. Biochemical Journal, 299, 725–730. Asp, N.-G., & Björck, I. (1992). Resistant starch. Trends in Food Science & Technology, 3, 111–114. Atichokudomchai, N., Varavinit, S., & Chinachoti, P. (2004). A study of ordered structure in acid-modified tapioca starch by 13C CP/MAS solid-state NMR. Carbohydrate Polymers, 58, 383–389. Blazek, J., & Gilbert, E. P. (2011). Application of small-angle X-ray and neutron scattering techniques to the characterisation of starch structure: A review. Carbohydrate Polymers, 85, 281–293. Fan, D., Ma, W., Wang, L., Huang, J., Zhang, F., Zhao, J., et al. (2013). Determining the effects of microwave heating on the ordered structures of rice starch by NMR. Carbohydrate Polymers, 92, 1395–1401. Hinrichs, W., Buttner, G., Steifa, M., Betzel, C., Zabel, V., Pfannemuller, B., et al. (1987). An amylose antiparallel double helix at atomic resolution. Science, 238, 205–208. Kiatponglarp, W., Tongta, S., Rolland-Sabaté, A., & Buléon, A. (2015). Crystallization and chain reorganization of debranched rice starches in relation to resistant starch formation. Carbohydrate Polymers, 122, 108–114. Kim, H. J., & White, P. J. (2010). In vitro bile-acid binding and fermentation of high, medium, and low molecular weight β-glucan. Journal of Agricultural and Food Chemistry, 58, 628–634. Lehmann, U., Rössler, C., Schmiedl, D., & Jacobasch, G. (2003). Production and physicochemical characterization of resistant starch type III derived from pea starch. Food/ Nahrung, 47, 60–63. Leong, Y. H., Karim, A. A., & Norziah, M. H. (2007). Effect of pullulanase debranching of sago (Metroxylon sagu) starch at subgelatinization temperature on the yield of resistant starch. Starch - Stärke, 59, 21–32. Lesmes, U., Beards, E. J., Gibson, G. R., Tuohy, K. M., & Shimoni, E. (2008). Effects of resistant starch type III polymorphs on human colon microbiota and short chain fatty acids in human gut models. Journal of Agricultural and Food Chemistry, 56, 5415–5421. Liu, X., Xiao, X., Liu, P., Yu, L., Li, M., Zhou, S., et al. (2017). Shear degradation of corn starches with different amylose contents. Food Hydrocolloids, 66, 199–205. Li, L., Yuan, T. Z., Setia, R., Raja, R. B., Zhang, B., & Ai, Y. (2019). Characteristics of pea, lentil and faba bean starches isolated from air-classified flours in comparison with

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