Accepted Manuscript Structural Characteristics and Physicochemical Properties of Lotus Seed Resistant Starch Prepared by Different Methods Shaoxiao Zeng, Xiaoting Wu, Shan Lin, Hongliang Zeng, Xu Lu, Yi Zhang, Baodong Zheng PII: DOI: Reference:

S0308-8146(15)00524-5 http://dx.doi.org/10.1016/j.foodchem.2015.03.143 FOCH 17390

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

24 December 2014 23 March 2015 25 March 2015

Please cite this article as: Zeng, S., Wu, X., Lin, S., Zeng, H., Lu, X., Zhang, Y., Zheng, B., Structural Characteristics and Physicochemical Properties of Lotus Seed Resistant Starch Prepared by Different Methods, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.03.143

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Structural Characteristics and Physicochemical Properties of Lotus Seed Resistant Starch Prepared by Different Methods

Running title: Structure and Physicochemical Property of Lotus Seed Resistant Starch

Shaoxiao Zenga,b, Xiaoting Wua, Shan Lina, Hongliang Zenga, Xu Lua, Yi Zhanga, b*, Baodong Zheng a, b∗

a

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou,

Fujian, P. R. China 350002 b

Institute of Food Science and Technology, Fujian Agriculture and Forestry

University, Fuzhou 350002, China

Corresponding author. Tel.: +86 591 83736738; fax: +86 591 83739118. E-mail address: [email protected] (Y. Zhang), [email protected] (B. Zheng) ∗

Abstract Lotus seed resistant starch (LRS) is commonly known as resistant starch type 3 (LRS3). The objective of this study was to investigate the effect of different preparation methods on the structural characteristics and physicochemical properties of LRS3. The molar mass of LRS3 prepared by autoclaving method (GP-LRS3) and ultrasonic-autoclaving method (UP-LRS3) was mainly distributed in the range 1.0×104 ~ 2×104 g/mol while a decrease of LRS3 prepared by microwave-moisture method (MP-LRS3) was observed. The particle of MP-LRS3 was smaller and relatively smoother while UP-LRS3 was bigger and rougher compared to GP-LRS3. Among these samples, GP-LRS3 exhibited the highest degree of ordered structure and crystallinity, the amorphous region of MP-LRS3 was the biggest and UP-LRS3 displayed the highest degree of double helical structure. Additionally, MP-LRS3 displayed the strongest solubility and swelling power while UP-LRS3 exhibited the strongest iodine absorption ability and thermostability, which were affected by their structural characteristics. Keywords: Lotus seed; resistant starch; preparation method; structural characteristics; physicochemical properties

1 Introduction Resistant starch (RS) is the total of starch and its degradation products that escape digestion in the small intestine of healthy individuals and may be completely or partially fermented in the colon (Englyst, Kingman, & Cummings, 1992), which has been mentioned to have physiological benefits resembling those of soluble dietary fibers, including prevention of gastrointestinal diseases and cardiovascular disease; reducing the risk of ulcerative colitis and colon cancer; promotion of bacterial growth and mineral absorption (Zhang, Zeng, Wang, Zeng, & Zheng, 2014). There are five categories of RS in foods: RS1, physically inaccessible starch to digestion; RS2, native granule or un-gelatinized starch; RS3, retrograded or crystalline starch; RS4, chemically modified starch and RS5 is the amylose-lipid complexed starch (Fuentes Z. et al., 2011). Up to now, a great deal of studies have been carried out on RS3 because of its thermal stability (Shi & Gao, 2011). RS3 can be obtained by hydrothermal method (including Heat Moist Treatment, HMT; Annealing, ANN; Autoclaving; Educed-Pressurized), extrusion processing treatment, microwave-moisture treatment, debranching degradation treatment, ultra-high pressure treatment and ultrasonic treatment (Augustin, Sanguansri, & Htoon, 2008). Mangala et al. (1999) used autoclaving treatment to prepare cereal resistant starch. A few of studies have reported that the structural characteristics and physicochemical properties of RS3 were affected by different preparation methods (Fan et al., 2013; Mahadevamma, Harish Prashanth, & Tharanathan, 2003). Wu (Wu, 2012) studied the structure and physicochemical property of resistant starch prepared

from pea starch were different by enzyme hydrolysis method (debranching enzyme treatment), physical method (heat-moisture treatment), chemical method (cross-linking treatment) and compound modified method (heat-moisture cooperated with cross-linking treatment). Zhang et al. (2013) reported maize starch processed with a combination of α-amylase and pullulanase exhibited a considerably lower dissolution and digestibility than that treated with high pressure-processed. Therefore, different preparation methods played very important role in the structural and physicochemical properties of RS3, which further affected its biological activity. Little investigation has considered on the RS3 prepared by microwave-moisture and ultrasonic-autoclaving methods. Lotus seeds are the excellent resource that has the concomitant functionality of both medicine and foodstuff because of their special pharmacological ingredients (Wu et al., 2007). The high content of amylose (~ 40%, w/w) is beneficial for the formation of lotus seed resistant starch type 3 (LRS3) (Zhang, Zeng, Wang, Zeng, & Zheng, 2014). Our previous research indicated that LRS3 lacked the polarization cross and the irregularly shaped LRS3 granules had a rougher surface, B-type crystal structure, and great level of molecular order. Moreover, LRS3 was more effective than either glucose or HAMS in promoting the proliferation of bifidobacteria. LRS3 increased the number of bifidobacteria, achieved a brief lag phase, enhanced the bacterial tolerance under the gastrointestinal condition, and obtained a higher production of butyric acid. The prebiotic effect of LRS3 was affected by the structure (Zhang, Wang, Zheng, Lu, & Zhuang, 2013). However, information about the

structural characteristics and physicochemical properties of Lotus seed resistant starch prepared by different methods is not reported. Therefore, the objective of this study was to investigate the effect of different prepared methods on the structural and physicochemical properties of LRS3. LRS3 was prepared by autoclaving method (GP-LRS3), microwave-moisture method (MP-LRS3) and ultrasonic-autoclaving method (UP-LRS3), respectively. Their structure were characterized by size exclusion chromatography connected with multi-angle laser light scattering and refractive index (SEC-MALLS-RI) system, environmental scanning electron microscopy (ESEM), X-ray diffraction (XRD), fourier transform infrared (FT-IR), and solid state 13C nuclear magnetic resonance (NMR) spectroscopy. Furthermore, the solubility, swelling power, iodine absorption curve and thermal properties were studied and compared. 2 Materials and methods 2.1 Materials Fresh lotus seeds were obtained from Green acres (Fujian) food co., LTD (Fujian, China). There were three modification of starches prepared from lotus seed. α-amylase (10,000 U/ml) was purchased from ANKOM (New York, U.S.A). Glucoamylase (100,000 U/ml) was acquired from Aladdin Reagent co., LTD (Shanghai, China). All other chemicals and reagents used in the experiment were analytical grade. 2.2 Preparation and purification of lotus seed resistant starch Lotus seed native starch and GP-LRS3 were prepared using the previous method

reported by Zhang et al (2014). 250 g (dry basis) of LRS3 was suspended in 500 ml of citric acid buffer (pH 6.0) in a 1-L conical flask. The suspensions were hydrolyzed by the addition of thermostable α-amylase (10,000 U/ml, obtained from ANKOM, New York, U.S.A) at 95°C for 1 h in an orbital incubator-shaker (SHA-C, Guo Hua Electric Applicance, Changzhou, China) at 128 rpm. After adjusting the pH to 4.5 with a citric acid solution (4 mol/L), glucoamylase (300 U/mL, obtained from Sigma, St. Louis, U.S.A.) was added (5000 U/g of starch) and incubated at 60°C for 1 h in an orbital incubator-shaker at 128 rpm. The suspensions were centrifuged (L-530, Xiang Yi Laboratory Instrument Development Co., Ltd, Hunan, China) at 2,850 g for 10 min. The resulting precipitates or residues were washed three times with distilled water and ethanol solutions of different concentrations (75%, 85%, and 95%; all determined on a v/v basis). The residues were dried at 45°C, ground, and passed through a 185-µm mesh screen. MP-LRS3: lotus seed native starch (150 g) was dispersed in 1000 ml distilled water (starch: water, 3:20), and the starch suspensions were heated under 640 W of microwave power for 2 min (M700, Mei Di Co., Ltd, Shanghai, China), cooled to room temperature, and stored at 4°C for 24 h. And then the gelatinised starch was dried at 50°C in a drying oven. Thereafter, the sample was dried and ground before sieving through a 185 µm mesh screen. Then the sample were purified as described above for GP-LRS3, obtained MP-LRS3. UP-LRS3: lotus seed native starch (450 g) was dispersed in 1000 ml distilled water (starch: water, 9:20), and the starch suspensions were packed with vacuum

package machine, and then treated with an ultrasonic transducer (KQ2200DE, Ultrasonic Instrument, Kunshan, China) at the ultrasonic power of 300 W for 55 min at 25°C. After that, the mixture was pressure-cooked in an autoclave at 115°C for 15 min, cooled to room temperature, and stored at 4°C for 24 h. Finally, the recrystallizing starch was dried at 50°C, and ground using a ring sieve with an aperture size of 185 µm. Then the sample were purified as described above for GP-LRS3, obtained UP-LRS3. 2.3 Content determination of lotus seed resistant starch The content of lotus seed resistant starch prepared by autoclaving, microwave-moisture and ultrasonic-autoclaving method was calculated as Eq. (1): LRS content (%, W/W) =

Weight of resistant starch × 100 Weight of native starch

(1)

2.4 Structural characteristics 2.4.1 SEC-MALLS-RI system A Waters SEC system was equipped with Waters model 12-6 pump and a Waters WISH-01 auto sampler followed by a 18-angle MALS detector (DAWN-HELEOS II, Wyatt Technology in Santa Barbara, CA, USA) which had a laser wavelength of 664.1 nm and a refractive index detector (Shodex RI-101, Showa Denko k.k.., Japan). The mobile phase was DMSO with LiBr (50 mmol/l) filtered through a 0.22 um PTFE filter coupled with sand core filtration unit ( Jin Teng Laboratory Instrument Development Co., Ltd, Tianjin, China) and then degassed with ultrasound treatment for 15min. A certain amount of the sample (~12.5 mg) was suspended in 25 ml of the mobile phase and heated in 90 °C in a water bath for 2 h, and then stirred in a

magnetic stirring apparatus at 25 °C for 24 h to ensure the full dissolution in the mobile phase. Then the sample solutions were centrifuged at 13,500 g at 20 °C for 20 min and filtered through a 0.45 nm PTFE filter film (Jin Teng Laboratory Instrument Development Co., Ltd, Tianjin, China) before injected into the SEC column (Shodex P8514-805, Showa Denko k.k.., Japan). The column temperature was controlled at 50 °C and RI detector temperatures were maintained at 35 °C. The flow rate and total injected volume were 0.3 ml/min and 1 ml, respectively. Before injection of resistant starch sample, the molecular weight (Mw) of dextran was analyzed to test the chromatography system. The Mw of tested dextrans was (3.8×105 g/mol) in agreement with the reported value by the manufacturer (SigmaeAldrich Canada Ltd. in Oakville, ON, Canada). Two injections were completed for each sample. Mw and Mn were calculated using the Astra V software (Version 5.3.4.20, Wyatt Technology in Santa Barbara, CA, USA) . A dn/dc value of 0.074 ml/g for starch was applied in calculations using the Zimm extrapolation model (Naguleswaran, Vasanthan, Hoover, Chen, & Bressler, 2014). 2.4.2 Environment scanning electron microscopy ESEM was conducted on a microscope (PHILIPS-XL30 ESEM, Philips-FEI, Netherlands) at an accelerating voltage of 15 keV. The samples were deposited on the copper stubs using double-sided adhesive tape and then sputter-coated with gold in a sputter coater (Cressington Scientific Instruments, Watford, UK). 2.4.3 X-ray diffraction X-ray diffractogram was obtained with X-ray diffractometer (X’Pert Pro MPD,

Philips, Netherlands) equipped with a θ-θ goniometer. Samples were scanned with Cu-Kα radiation (λ=0.25nm) in the angular range 2θ =5°-45° with step intervals of 0.0128916° and a scanning rate of 0.02° /min for continuous scanning (Blazek, Gilbert, & Copeland, 2011). The X-ray generator operating conditions were 40 kV and 30 mA. Diffractograms were processed using PeakFit software version 4.12 (SeaSolve Software Inc., Framingham, U.S.A.).

CCL =

SC × 100(%) ST

SCCL =

S SC × 100(%) ST

C L = CCL + SCCL

(2)

(3) (4)

where CCL is the proportion of crystalline region, SC is the crystallization area, ST is the total area, SCCL is the proportion of sub-crystalline region, SSC is the sub-crystallization area, and CL is the degree of crystallinity. 2.4.4 Fourier transform infrared spectroscopy Samples were diluted with potassium bromide (1:100, v/v) and dried under an infrared lamp in an agate mortar to eliminate the effect of both the free and crystalline water with the absorption peaks. The mixed powder was put into a vacuum compression and pressured into a sheet. FT-IR analyses of the samples were carried out with fourier transform infrared spectrometer (VERTEX70, Bruker Co., Ltd, U.S.A.) following the method of Chung (2009) with minor modification. The spectra, recorded against an empty cell as the background, were acquired at wavelength from 400 to 4000 cm–1with 4 cm–1 resolution at room temperature.

2.4.5 13C nuclear magnetic resonance spectroscopy The solid state 13C CP/MAS (Cross-Polarization and Magic Angle Spinning) NMR experiment was scanned using solid-state 13C CP/MAS NMR (AVANCE Ⅲ 500, Bruker Co., Ltd, Switzerland) at 125.7 MHz for the 13C nucleus according to the method of Mahadevamma et al. (2003). About 200–300 mg of sample was accumulated with a spectral width of 40 kHz at a magnetic field of Ultra Shield Plus SB (54mm) 11.7467T. The relative degrees of crystallinity of sample were processed using PeakFit software version 4.12. RC L =

SF × 100(%) ST

(5)

Where RCL is the relative degree of crystallinity, SF is fitting peak area of C1, and ST is the total peak area of C1. 2.5 Physicochemical properties

2.5.1 Solubility and swelling power About 2.000 g of sample was dispersed in 20 ml distilled water and cooked at different temperatures of 50, 60, 70, 80, and 90°C for 40 min, respectively. The solution was mixtured by urbine mixer (QL-866, Kylin-bell Instrument manufacturing co., LTD, Jiangsu, China) every 5 minutes. After that it was cooled to room temperature, centrifuged at 2,850×g for 10 min, and then supernate was collected into weighing bottles, dried at 105°C until the weight was no change to measure the dissolved resistant starch. Residues in the bottom were weighed to determinate the swelling power (SP) value. The solubility (S) (%) and SP (g/g on dry weight basis) values were calculated by Equations (6) and (7) as follows:

Solubility(S ) = ( A / W ) ×100%

(6)

Swelling Power(SP) = P / W (1 − S )

(7)

Where A is the dried supernatant weight, W is the dried sample weight, and P is the mass of Residue. 2.5.2 Iodine absorption curve Iodine absorption spectra of resistant starch were measured according to the method of Takeda (1983) with a minor modification. The starch (20 mg) was suspended in 1.0 ml of absolute ethanol. Then 10.0 ml distilled water was added and adjusted the pH 6.0-7.0 with HCl (1.0 M), and then diluted to 50 ml with distilled water. An aliquot (10 ml) of the solution was added to 2 ml of 0.2% iodine solution and made up to 100 ml with distilled water. Then the absorbance was determined from 500 to 800 nm with a spectrophotometer. 2.5.3 Thermal properties Thermal properties of the samples were tested by Differential Scanning Calorimetry (DSC 200 F3 Maria, NETZSCH Corporation, Germany). Starch (2.5 mg, dry weight) was loaded into a capacity aluminium pan (Jin Heng Instrument co., LTD, Shanghai, China) and 7.5 µl distilled water was added with the help of microsyringe. The pans were sealed hermetically to prevent moisture loss and kept overnight at 25°C before heating in the DSC. A sealed empty aluminum pan was used as a reference. Sample pans were heated at a rate of 10°C/min from 30 to 250°C. Onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and gelatinisation enthalpy (∆H) (J/g of dry starch) were determined. As the endotherms

are essentially symmetrical, the total gelatinisation temperature range (Tr) and peak height index (PHI) can be established by Equations (8) and (9) (Krueger, Knutson, Inglett, & Walker, 1987): Tr = [2(Tp − To )]

(8)

PHI = △H /(Tp − To)

(9)

2.6 Statistical Analysis

Data were evaluated by one-way analysis of variance. Significant differences were determined using the DPS 9.50 system (Science Press, Beijing, China). Statistical significance was set to p < 0.05. 3 Results and discussion 3.1 Content determination of LRS

The content of LRS prepared by autoclaving, microwave-moisture and ultrasonic-autoclaving method were 41.89±1.23%, 39.53±0.57% and 56.12±0.32%, respectively. The LRS content treated by microwave-moisture was lower than that by autoclaving method. This may be attributed to the rapid heating mechanism caused by microwave during gelatinization and the amylose could not absolutely outflow from native starch, which reduced the content of LRS formation. The result was consistent with the report by Palav and Seetharaman (2007). Meanwhile, the LRS content treated by ultrasonic-autoclaving was greater than that by autoclaving method. Ultrasonic pretreatment induced high pressure gradients and high local velocities, which caused shear forces that contributed to damaging granules and cutting long chains into appropriate length ones (Jambrak et al., 2010). The result led to the increase of LRS

content. 3.2 Molar mass distribution of GP-LRS3, MP-LRS3 and UP-LRS3

The cumulative weight fractions at different molar mass ranges of the three samples are shown in Fig. 1. GP-LRS3, MP-LRS3 and UP-LRS3 presented molar mass distributions with a fraction of 86.61- 89.82% in the ranges lower than 2×104 g/mol, respectively. They were distributed in the range of 1×104 to 2×104 g/mol and accounted for 67.69%, 47.87% and 69.26% of the total LRS3, respectively. They were distributed in the range of 0.8×104 to 1×104 g/mol with 12.08%, 29.95% and 16.24%, respectively. Comparatively, the range of molar mass from 2×104 to 3×104 g/mol only accounted for 5.59-7.27%, and less 7% of molecules was distributed in the range higher than 3×104g/mol. The molar mass of GP-LRS3 and UP-LRS3 were mainly distributed in the range 1.0×104 ~ 2×104 g/mol while a decrease of MP-LRS3 was observed, indicating the molecular weight of LRS3 was affected by the microwave method while the ultrasonic method had no obvious influence upon it. As the molecular weight of glucose units is 162, the degree of polymerization (DP) = Mw/162 (Luo, Gao, & Yang, 2004). The Mw, Mn, Mw/Mn and DP of GP-LRS3, MP-LRS3, UP-LRS3 are shown in Table 1(A). As it shows, the Mw value of UP-LRS3 displayed the highest (1.595×104g/mol), GP-LRS3 was 1.522×104g/mol and MP-LRS3 had the lowest (1.442×104 g/mol), indicating the a decrease of MP-LRS3 was observed, which was consistent with the result of molar mass distribution. M n and M w values of GP-LRS3, MP-LRS3 and UP-LRS3 were lower than those of native starch (3.173×105 g/mol, 1.307×106 g/mol) indicated that native starch

might have higher amylose content than the other types of starch analyzed. Moreover, the Mn and M w values of high amylose maize starch (HAMS) (2.239×106 g/mol, 4.097×106 g/mol) were significantly higher than those of lotus seed starch and resistant starches. This indicated that HAMS was composed of highly polymerized amylose (Zhang, Zeng, Wang, Zeng, & Zheng, 2014). The polydispersity coefficient values of GP-LRS3, MP-LRS3 and UP-LRS3 were 1.249, 1.298 and 1.247, respectively. This indicated that all samples had narrower molecular weight distribution. Luckett (2012) reported the relatively appropriate degree of polymerization (DP) was benefit for the formation of double helices and crystallization. In this study, the DP values of GP-LRS3, MP-LRS3, UP-LRS3 were 94, 89, and 98, respectively. The double helices and crystallization were formed smoothly with these DP. 3.3 Morphological characteristics

ESEM images of GP-LRS3, MP-LRS3 and UP-LRS3 at different magnifications are presented in Fig. 2. At 100 magnification, all samples showed characteristic block and irregular structure. The particle of MP-LRS3 was smaller while UP-LRS3 was bigger compared to GP-LRS3, which may be because that the molecular weight of amylose was reduced by the microwave method while ultrasonic increased the dissolving out of the appropriate length amylose and the quality of recrystallization . Jambrak et al. (2010) reported that ultrasonic would induce high pressure gradients and high local velocities of liquid layers in their vicinity, which caused shear forces that were capable of damaging granules and cutting long chains into appropriate

length ones. At 1000 magnification, it was apparent that all samples had a rather uneven surface, and there were numerous layered strips (Red arrows in Fig. 2) on granule surface. This could be attributed to the leaching of amylose from starch, the loss of the amylopectin crystalline region during heating, and re-association of the starch chains within the granules (Naguleswaran, Vasanthan, Hoover, & Bressler, 2014). However, MP-LRS3 behaved a relative smooth surface, indicating the rapid heating effect on starch caused by microwave during gelatinization, amylose could not absolutely outflow from starch granule, which would reduce the appropriate numbers of amylose for RS formation. Cavities (Blue arrows in Fig. 2) were observed on the rough surfaces of UP-LRS3, which could be attributed to the cavitation of the ultrasonic. 3.4 X-ray diffraction pattern and crystallinity

The XRD patterns of MP-LRS3 and UP-LRS3 are shown in Fig. 3(A) and the pattern of GP-LRS3 was quoted from our previous report (Zhang, Zeng, Wang, Zeng, & Zheng, 2014). They were high similarity, suggested that resistant starch prepared from different methods had the same crystalline pattern. All the samples exhibited B-type crystalline arrangements with three strong peaks at 2θ of 19.77º, 25.60º and 27.94º. Our previous research has shown that the appearance of a peak at 2θ of 19.77º was attributed to the retrogradation that occurs at 4 °C (Zhang, Zeng, Wang, Zeng, & Zheng, 2014). Lotus seed native starch displayed a typical C-type crystalline structural pattern with peaks at 2θ of 17.63º, 19.72º, 21.11º, 26.91º and two broad peaks at 25.49º and 28.20º. When the native starch was treated with microwave, the

crystalline pattern turned from C-type to B-type, which was consistent with the impact of autoclaving treatment on crystalline pattern. By contrast, ultrasonic also did not change the B-type crystalline pattern of LRS3. The result indicated that there were no differences in the crystalline pattern of LRS3 prepared by autoclaving, microwave-moisture and ultrasonic-autoclaving methods. The difference in crystalline pattern was just only depend on the retrogradation way (Sajilata, Singhal, & Kulkarni, 2006). The X-ray diffraction parameters and crystallinity level calculated from the ratio of diffraction peak area and total diffraction area are given in Table 1(B). The data of GP-LRS3 was quoted from our previous study (Zhang, Zeng, Wang, Zeng, & Zheng, 2014).The proportion of crystalline region was stronger than that of sub-crystalline region. Among these three samples, GP-LRS3 (46.47%) had the highest proportion of crystalline region and the MP-LRS3 (40.30%) had the lowest one. This was because high temperature and pressure was more efficient than microwave-moisture in the formation of crystalline region. However, compared with GP-LRS3 (46.47%), UP-LRS3 (42.71%) had a lower level of crystalline region as the effect of ultrasonic, this suggested that the large crystal in UP-LRS3 was less than GP-LRS3. The sub-crystalline region of MP-LRS3 (37.79%) and UP-LRS3 (37.92%) were higher than GP-LRS3 (35.63%), indicated that microwave-moisture was more beneficial than autoclaving in primary crystallite formation, and ultrasonic pretreatment can improving the sub-crystalline region of autoclaving resistant starch. The crystallinity of GP-LRS3 (81.65%) was greatest while MP-LRS3 (78.09%) was weakest, which

was consistent with the result of the crystalline region. 3.5 Fourier-transform infrared spectroscopy

The short-order range, such as chain conformation and double helical order, played very important role in FT-IR spectra (Bernazzani, Peyyavula, Agarwal, & Tatikonda, 2008). The FT-IR spectra of MP-LRS3 and UP-LRS3 shown Fig. 3(B) and GP-LRS3 quoted from the previous study (Zhang, Zeng, Wang, Zeng, & Zheng, 2014) showed similar, this indicated that there were no different in chemical groups of LRS3 prepared by the different methods. Howener, the absorption intensity of LRS in the band from 800 to 1,200 cm–1 was weaker than that of native starch, indicating that certain conformation changes had occurred in LRS. The IR spectrum in this region was characterized by three main modes with maximum absorbance at 1047, 1022 and 995 cm-1 (Zhang, Chen, Liu, & Wang, 2010). The absorption peaks at 1047 and 995 cm-1 were associated to the ordered structures and hydrated crystalline of resistant starch, respectively. The peak at 1022 cm-1 was related to amorphous structures. and the ratio of intensity at 1047/1022 cm-1 (R1047/1022) could be used to descript the degree of order (DO), and the ratio of intensity at 995/1022 cm−1 (R995/1022) characterized the internal changes in the degree of the double helix (DD) (Van Soest, Tournois, de Wit, & Vliegenthart, 1995), which were both calculated as shown in Table 1(C). As shown in Table 1(C), the DO values of GP-LRS3, MP-LRS3 and UP-LRS3 were 1.007, 0.997 and 1.000, respectively. And it had the same trend with crystallinity (Table 1(B)), indicating the crystallinity structure was built by orderly starch chains. The DD values of GP-LRS3, MP-LRS3 and UP-LRS3 were 1.026, 1.018 and 1.032,

respectively. The result showed ultrasonic pretreatment could promote the formation of the double helix, and microwave treatment had the weakest effect on enhancing the number of double helix structure. Furthermore, it was clearly observed that the levels of 995/1022 ratios of each sample were higher than the 1047/1022 ratios, suggesting the amount of double helices were higher than those of crystallinity. Because not all of the double helical structure were arranged into crystals (Htoon et al., 2009). 3.6 Solid state 13C nuclear magnetic resonance spectroscopy 13

C CP/MAS NMR is used to evaluate the short-range ordered structures in

starch granules. Fig. 3(C) shows the 13C CP/MAS NMR spectra of MP-LRS3 and UP-LRS3, the spectra of GP-LRS3 was quoted from the study by Zhang et al. (2014). The chemical shifts of each major peak and relative crystallinity of the samples are presented in Table 1(D). From the Fig. 3(C), the double peaks were observed in the C1 signal region, indicating that all of LRS3 were B-type starch, which was consistent with the results of XRD. The signal at 99-102 ppm in the C1 region represented information on the double helices, however, signal at 102 and 104 ppm showed the simple helix (Morrison, Law, & Snape, 1993). No Chemical shifts at 102 and 104 ppm were found, suggesting that the simple helix was not existed in the samples. The broad peak shoulder near 81 ppm in the C4 region provided information on the amorphous components. The C4 region of MP-LRS3 was bigger than UP-LRS3, suggested that the amorphous components in the MP-LRS3 was higher that UP-LRS3, indicated that microwave-moisture had a weak influence on amylose dissolution during

gelatinization and crystallization. This result was consistent with the result of FT-IR. The proportion of the amorphous phase (PPA) could be measured by the proportion of C4 peak fitting area relative to the total area of the spectrum. The double helix structure increased with the decrease of the amorphous phase (Zhang, Zeng, Wang, Zeng, & Zheng, 2014). In Table 1(D), it was found that the PPA of UP-LRS3 was significantly lower (p < 0.05) than other samples. This means that UP-LRS3 contained the greatest proportion of double helix structure among these samples, which was consistent with the analysis result by FT-IR. The C2, C3, and C5 regions mainly represented the B-type double helices resulting from the residues of free amylose. In addition, the relative crystallinity by NMR was accordant with the results by XRD. Additionally, LRS3 displayed a higher relative crystallinity compared with that of native starch, indicating that recrystallization was an important step to obtain a more stable structure during the formation of resistant starch (Zhang, Zeng, Wang, Zeng, & Zheng, 2014). 3.7 Solubility

Solubility properties of samples shown in Fig. 4(A) increased as the temperature increase from 55 °C to 95 °C, attributing to the fact that the crystal structure was gradually destroyed in the process of heating, and the hydrogen bonding of water was easy to associate with the hydroxyl of amylose. At the beginning of heating (55-75°C), the solubility of MP-LRS3 was higher than GP-LRS3 and UP-LRS3. This was because that MP-LRS3 contained the most amorphous starch region (2.86%) by NMR, which was firstly intruded during moisture heating, and the remaining un-associated

starch chains could solubilize into water and amylose leaching mainly from the amorphous region, which would result in the high solubility value (Lawal, 2011). A similar increase in solubility after heat-moisture treatment was observed for Bambarra groundnut starch (Adebowale & Lawal, 2002) and white sorghum starch (Olayinka, Adebowale, & Olu-Owolabi, 2008). These studies reported that the undefined structure was easier destroyed than orderly crystalline structure. In the latter stages of the heating (85-95°C), UP-LRS3 had the lowest solubility values comparing to GP-LRS3 and MP-LRS3, which maybe attributed to the biggest particle size of UP-LRS3 concluded from ESEM and the smallest polydispersity value (Mw/Mn) by SEC-MALLS-RI system. The result was in accordance with the report by Li et al (2011). The solubility of resistant starch from mung bean was affected by its particle structure and the polydispersity in solution. Therefore, the solubility of LRS3 was directly linked to amorphous s region and particle structure. 3.8 Swelling power

The swelling power of starch granules presented the combination extent of starch chains within the amorphous and crystalline domains. The swell of the starch granules began from a relatively loose amorphous region, and then the amorphous region closing to crystallization region, and crystallization region last (Adebowale & Lawal, 2002). The swelling power of samples is shown in Fig. 4(B). Within the range temperature (55-75°C), the swelling power of GP-LRS3, MP-LRS3 and UP-LRS3 were not different, indicating the amorphous region of resistant starch could not be gelatinized at the low temperature. As temperature increased from 85 to 95°C, the

swelling power of MP-LRS3 improved the most rapidly comparing to GP-LRS3 and UP-LRS3. At the end of heating, MP-LRS3 showed the highest swelling power value while UP-LRS3 was the weakest. This was because the structure of amorphous starch (2.86%) analyzed by NMR in MP-LRS3 was intruded and dissolved in the function of water and heating, and then it absorbed heat energy to gelatinize. The swelling power of resistant starch was dependent absorption of water by the starch granules particularly in the amorphous starch regions , which was related to the channels and crystal structure or hydrogen bonding (Yu, Ma, Menager, & Sun, 2012). Li et al (2011) reported that the presence of strong bonding forces, such as double helix, played an important role in limiting swell. The most double helix structure of UP-LRS3 analyzed by FT-IR and NMR surrounded the amorphous regions, which made the swelling power of UP-LRS3 significantly lower than that of MP-LRS3. 3.9 Iodine absorption curve

One of the most characteristic features of starch is the formation of the blue complex with iodine which can be very useful for analysis of changes in its degree of polymerization (DP) caused by enzymatic, chemical or physical modifications (Sujka & Jamroz, 2013). The absorbance of the resistant starch from 500-800 nm was shown in Fig. 4(C). All of GP-LRS3, MP-LRS3 and UP-LRS3 displayed a maximum absorption peak (560-610 nm). All samples had sharp maximum absorption peaks, suggesting that resistant starch had a relatively centralized distribution of molecular weight. These results were consistent with the results by SEC-MALLS-RI system. Among the three samples, the DP value of UP-LRS3 was greatest while MP-LRS3

was the smallest. UP-LRS3 exhibited the strongest iodine absorption ability while MP-LRS3 was weakest, which was consistent with the results of their degree of polymerization. Therefore, the iodine absorption ability of LRS3 was affected by the degree of polymerization. 3.10 Thermal properties

To gain further information on the change in the helical arrangement structure related to the crystalline structure, the DSC thermograms of the samples were collected. The thermograms are shown in Fig. 4(D), and thermal characteristic parameters are shown in Table 2. Gelatinization occurs initially in the amorphous regions, as opposed to the crystalline regions, of the granule, for the hydrogen bonding was weakened in these areas (Singh, Singh, Kaur, Singh Sodhi, & Singh Gill, 2003). The starch transition temperatures and gelatinization enthalpies by DSC were related to characteristics of the starch granule, such as degree of crystallinity, which provided structural stability and made the granule more resistant towards gelatinization (Barichello, Yada, Coffin, & Stanlet, 1990). Tp was related to the length of double helix structure, and enthalpy of gelatinization was related to crystallinity and the loss of molecular order within the granule (Gernat, Radosta, Anger, & Damaschun, 1993). However, it had been shown that the enthalpic transition was primarily due to the loss of double helical order rather than crystallinity (Cooke & Gidley, 1992). The enthalpy was an indicator of uncoiling and dissociation energy of the double helices. In Table 2, UP-LRS3 showed the highest Tp (117.9°C) and ∆H

(1346J/g), while the Tp (117.1°C) and ∆H (565J/g) was lowest for MP-LRS3. These results were attributed to the proportion of double helical structures of LRS3 through hydrogen bonds and other intermolecular forces, which was consistent with the DD value by FT-IR and PPA value by NMR. Among three samples, the Tr value of MP-LRS3 (24.6°C) was the highest and UP-LRS3 (20.6°C) was the smallest. Difference in Tr reflected the extent of heterogeneity of crystallites within the granules of GP-LRS3, MP-LRS3 and UP-LRS3 (Li, Ward, & Gao, 2011). PHI was the ratio of ∆H for gelatinization to the gelatinization temperature range and a measure of uniformity in gelatinization. The PHI value of UP-LRS3 (130.68) was for the greatest while MP-LRS3 was the smallest, which was a result of greater amounts of double helices or stronger interaction between starch chains within the crystalline domains of UP-LRS3 compared to MP-LRS3. Therefore, the thermostability of LRS3 was mainly affected by the double helical structure. 4 Conclusions

The structural characteristics and physicochemical properties of lotus seed resistant starch prepared by different methods were firstly investigated and compared. The LRS content prepared by autoclaving, microwave-moisture and ultrasonic-autoclaving method were 41.89±1.23%, 39.53±0.57% and 56.12±0.32%, respectively. The structure of LRS3 showed that the molar mass of GP-LRS3 and UP-LRS3 were mainly distributed in the range 1.0×104 ~ 2×104 g/mol and they accounted for 67.69% and 69.26% of total LRS3, while a decrease of MP-LRS3 was observed. The particle of MP-LRS3 was smaller and relatively smoother while

UP-LRS3 was bigger and rougher compared to GP-LRS3 by ESEM. Among these samples, the crystallinity of GP-LRS3 was the greatest observed by X-ray diffraction. GP-LRS3 exhibited the highest the degree of ordered structure and the amorphous region of MP-LRS3 was biggest by FT-IR and 13C NMR as well as UP-LRS3 displayed the highest degree of double helical structure. These results indicated that the structure of LRS3 was influenced by the preparation methods. In addition, MP-LRS3 displayed the greatest solubility and swelling power, which were affected by the amorphous region, particle structure. UP-LRS3 exhibited the strongest iodine absorption ability and thermostability, which were affected by the degree of polymerization and degree of double helical structure, respectively. The different physicochemical properties of GP-LRS3, MP-LRS3 and UP-LRS3 were attributed to their different structure. Interestingly, the property was a result of a combination of multiple factors. The result will provide the background and practical knowledge for the application of lotus seed resistant starch. MP-LRS3 could be explored as a prebiotic and UP-LRS3 was a potential thermal stabilizer in food industries. Acknowledgements

This study is financially supported by the National Natural Science Fund (No: 31301441) and the Fujian agriculture and forestry university outstanding youth fund (No: xjp201204). Abbreviations

RS, resistant starch; RS3, resistant starch type 3; LRS3, Lotus seed resistant starch; GP-LRS3, autoclaving treatment purified lotus seed resistant starch; MP-LRS3,

microwave-moisture treatment purified lotus seed resistant starch; UP-LRS3, ultrasonic-autoclaving treatment purified lotus seed resistant starch; SEC-MALLS-RI, size exclusion chromatography coupled with multi-angle light scattering and refractive index detector; ESEM, scanning electron microscopy; XRD, X-ray diffraction; FT-IR, Fourier transform infrared spectroscopy; 13C NMR, solid state 13C nuclear magnetic resonance spectroscopy; DSC, Differential scanning calorimetry; DP, degree of polymerization; DD, degree of double helical structure; DO, Degree of ordered structure.

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Fig. 1

Fig. 2

Fig. 3

Fig. 4

Figure captions Fig. 1 Cumulative weight fraction of GP-LRS3, MP-LRS3 and UP-LRS3 in different molecular weight ranges. Fig. 2 ESEM images of LRS3: (A) GP-LRS3, magnification 100×; (B) MP-LRS3, magnification 100×; (C) UP-LRS3, magnification 100×; (D) GP-LRS3, magnification 1000×; (E) MP-LRS3, magnification 1000×; and (F) UP-LRS3, magnification 1000×. Fig. 3 X-ray diffraction patterns, FT-IR spectra and

13

C CP/MPS NMR spectra of

MP-LRS3 and UP-LRS3: (A) X-ray diffraction patterns; (B) FT-IR spectra; and (C) 13

C CP/MPS NMR spectra.

Fig. 4 Solubility, swelling power, Iodine absorption curve and DSC thermograms of GP-LRS3, MP-LRS3 and UP-LRS3: (A) Solubility at different temperature: (B) Swelling power at different temperature; (C) Iodine absorption curve; and (D) DSC thermograms. Data are presented as mean ± standard deviation (n = 3). Lower case letters within the same temperature are significantly different (p < 0.05).

Table 1 Molecular structure characteristics, crystallinity, molecular orders and chemical shifts of GP-LRS3, MP-LRS3 and UP-LRS3 (A) Molecular structure characteristics of GP-LRS3, MP-LRS3, UP-LRS3 by SEC-MALLS-RI system Sample

Mw (g/mol)

Mn (g/mol)

Mw/Mn

DP (Mw/162)

GP-LRS3

4

1.522×10 (0.596%)

4

1.218×10 (0.608%)

1.249 (0.851%)

94

MP-LRS3

4

1.442×10 (0.787%)

4

1.111×10 (0.849%)

1.298 (1.158%)

89

UP-LRS3

1.595×104 (0.695%)

1.279×104 (0.766%)

1.247 (2.448%)

98

(B) Crystallinity characteristics of GP-LRS3, MP-LRS3 and UP-LRS3 by XRD Samples

Crystalline

Proportion of crystalline region

Proportion of sub-crystalline region

Crystallinity

pattern

（%）

（%）

（%）

a

b

81.65±0.09a

GP-LRS3

B-type

46.47±0.13

35.63±0.12

MP-LRS3

B-type

40.30±0.08c

37.79±0.07a

78.09±0.14c

B-type

b

a

80.63±0.08b

UP-LRS3

42.71±0.11

37.92±0.04

(C) Molecular orders of GP-LRS3, MP-LRS3 and UP-LRS3 by FT-IR Samples

1047/1022 ratio (DO)

995/1022 ratio (DD)

GP-LRS3

1.007±0.001a

1.026±0.003b

MP-LRS3

0.997±0.002c

1.018±0.000c

UP-LRS3

1.000±0.000b

1.032±0.003a

(D) Chemical shifts of GP-LRS3, MP-LRS3 and UP-LRS3 by Samples

GP-LRS3

MP-LRS3

UP-LRS3

99.35 101.09

99.87 101.40

99.81 101.40

C4

C-2,3,5

C NMR

Relative

Chemical shifts (ppm) C1

13

crystallinity（%）

PPA*（%）

C6

71.22 82.10

73.88

61.52

89.91±0.09a

2.52±0.04b

61.00

83.09±0.14c

2.86±0.03a

61.16

85.63±0.08

76.19 70.91 81.68

72.14 74.09 70.65

81.49

72.21 74.61

Results are expressed as mean ± standard deviation; Lower case letters within the same column are significantly different (p < 0.05).

b

c

2.37±0.02

Table 2 Thermal properties of GP-LRS3, MP-LRS3 and UP-LRS3 Samples

To (°C)

Tp (°C)

Tc (°C)

∆H(J/g)

Tc -To (°C)

Tr (°C)

PHI (J/g°C)

GP-LRS3

106.3

117.4

128.5

1284

22.2

22.2

115.68

MP-LRS3

104.8

117.1

132.0

565

27.2

24.6

45.94

UP-LRS3

107.6

117.9

127.8

1346

20.2

20.6

130.68

To, Tp, Tc indicate the temperature of the onset, peak, conclusion of gelatinization, respectively. ∆H indicates enthalpy of gelatinization. Tc -To and Tr indicate the gelatinization temperature range. PHI indicates the peak height index.

Highlights ►The structural properties of GP-LRS3, MP-LRS3 and UP-LRS3 were

characterized. ► The physicochemical properties of GP-LRS3, MP-LRS3 and UP-LRS3 were

investigated. ►The structural difference of LRS3 was attributed to the preparation methods. ► The physicochemical properties of LRS3 were affected by their structural

properties.