Structural changes of waxy and normal maize starches modified by heat moisture treatment and their relationship with starch digestibility

Accepted Manuscript Title: Structure Changes of Waxy and Normal Maize Starches Modified by Heat Moisture Treatment and Their Relationship with Starch Digestibility Authors: Yongzhi Chen, Qingyu Yang, Xiaojuan Xu, Liang Qi, Zhihong Dong, Zhigang Luo, Xuanxuan Lu, Xichun Peng PII: DOI: Reference:

S0144-8617(17)31006-8 http://dx.doi.org/10.1016/j.carbpol.2017.08.121 CARP 12728

To appear in: Received date: Revised date: Accepted date:

4-8-2017 23-8-2017 28-8-2017

Please cite this article as: Chen, Yongzhi., Yang, Qingyu., Xu, Xiaojuan., Qi, Liang., Dong, Zhihong., Luo, Zhigang., Lu, Xuanxuan., & Peng, Xichun., Structure Changes of Waxy and Normal Maize Starches Modified by Heat Moisture Treatment and Their Relationship with Starch Digestibility.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.08.121 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Structure Changes of Waxy and Normal Maize Starches Modified by Heat Moisture Treatment and Their Relationship with Starch Digestibility Yongzhi Chen1, Qingyu Yang1, 2, Xiaojuan Xu1, Liang Qi1, Zhihong Dong1, Zhigang Luo1*, Xuanxuan Lu3*, Xichun Peng4 1. School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China; 2. College of Grain Science and Technology, Shenyang Normal University, Shenyang, 110034, China; 3. Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Rd, New Brunswick, NJ 08901, USA; 4.Department of Food Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou, 510632, China; *Corresponding

author:

Luo, Z. G. Tel: +86-20-87113845, Fax: +86-20-87113848, E-mail address: [email protected] Lu, X. X. Tel: +1 848 565 5791, Fax: +1 732 932 6776, E-mail address: [email protected].

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Research Highlights 

> HMT decreased the degree of branching of waxy and normal maize starches.



> Three transformation patterns were observed in starch granules during HMT.



> Part of ordered phases were transformed into amorphous phases during HMT.



> The more ordered phase was destroyed, the higher content of SDS was obtained.



> The digestibilities of native and HMT modified starches were varied.

Abstract In the present study, the variations in structure of waxy and normal maize starches modified by heat-moisture treatment (HMT) for different treating time (3 h and 9 h) were investigated. HMT caused the destruction of starch granules. The 1H NMR confirmed that glycosidic bonds were broken during HMT. The 13C NMR result suggested that HMT caused the transformation of starch granules from double and single helical components into amorphous components. Heat-moisture treated starches exhibited higher gelatinization temperature (To, Tp and Tc), narrower gelatinization temperature range (Tc-To) and lower gelatinization enthalpy (ΔH). HMT caused the rearrangement of starch molecules, degeneration of double helices and formation of new single helix. In addition, in vitro digestibility assessment indicated that the contents of rapidly digestible starch (RDS) and slowly digestible starch (SDS) were improved and resistant starch (RS) was reduced after HMT, which was related to the decrease of single and double helical components. Keywords: Structure changes; Maize starches; Heat-moisture treatment; Digestibility.

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1. Introduction As one of the main components of the cereals and tubers, starches supply the metabolic energy to enable people’s daily activities. Recent studies have suggested that the nutritional physiology properties of starch, the rate of digestion and speed of absorption, significantly affect human health and relate to a series of health problems, such as diabetes and cardiovascular diseases (Xu et al., 2017). According to Englyst method, starch is classified into three portions based on the rate of glucose release and extent of digestibility, which are rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) (Englyst, Kingman, & Cummings, 1992). RDS digested within the first 20 min of enzyme incubation is able to induce sharp increase in the postprandial blood glucose and insulin level within a short time. SDS with digestion time between 20 to 120 min, provides a comparatively slow and prolonged release of glucose and prevents large variations in blood glucose concentrations after meals. RS, to some extent, is similar to dietary fiber, which resists the hydrolysis of enzyme and is hardly digested within 120 min (Miao, Jiang, & Zhang, 2009). Starches containing considerable RDS are regarded as high glycemic index foods. Byrnes et al. (1995) found that long-term consumption of high GI foods would induce nonreversible insulin resistance in rats, which possibly led to non-insulin dependent diabetes mellitus. On the contrary, consumption of foods rich in SDS results in a diet with low GI, which can help improve glycemic control, decrease the incidence and prevalence of obesity, diabetes, cardiovascular disease and even the occurrence of 3

some cancer (Jenkins, 2007; Rizkalla, Bellisle, & Slama, 2002). Björck et al. (2000) suggested that foods with high proportion of SDS may be beneficial for satiety, physical performance, as well as decrease the blood lipid levels and insulin resistant through reducing the stress on regulatory systems related to glucose homoeostasis. According to some published papers (T.-T. Huang, Zhou, Jin, Xu, & Chen, 2015; Tufvesson, Skrabanja, Björck, Elmståhl, & Eliasson, 2001; Xie, Hu, Jin, Xu, & Chen, 2014), RS is usually fermented by microorganisms in human’s colon and produces short-chain fatty acids which have positive influence on colonic health. In order to meet the requirements of health, foods with higher content of SDS and RS are needed to be developed. As commercial products, waxy and normal maize starches are widely used in food industry and usually applied to produce starch derivatives and food additives. In the past few decades, a variety of methods, including enzymatic modification, chemical modification and physical modification, have been applied to modify starch to increase the content of SDS and RS. As one of the physical methods, heat-moisture treatment (HMT), only adopting water and heat during process, is regarded as a relative safe means compared with the enzymatic and chemical methods. And now, an increasing number of researchers have focused on the effects of HMT on starch digestibility and physicochemical properties. Ambigaipalan et al. (2014) reported the digestibility of bean starches subjected to HMT at different temperatures, and found that HMT decreased the content of SDS at all temperatures but increased RS content at 80 and 100 ℃. Sun et al. (2014) studied the swelling power, solubility, gelatinization, crystallinity and morphology of heat4

moisture treated sorghum starch and flour. Ye et al. (2016) reported the effect of reaction conditions on nutritional properties of normal maize starch. Although plenty of work has been conducted to study the variations in starch digestibility and physicochemical properties before and after HMT, few studies have investigated the effect of HMT on starch structure and digestibility, as well as their relationship. Therefore, the objective of present study was to investigate the structure changes of waxy and normal maize starches modified by HMT for different treating time. Moreover, waxy and normal maize starches have different amylose content, which may influence the structure changes during HMT. And it’s necessary to understand the role of amylose in starch structure changes during HMT. The structural changes, including degree of branching (DB) of starch molecules, single and double helical components, amorphous and crystalline phase, were analyzed. The effect of structure changes on starch digestibility were also investigated, which may have some guiding significances in future study to understand the variations in relative content of RDS, SDS and RS in starches after HMT.

2. Materials and methods 2.1. Materials Waxy maize starch (WMS) and normal maize starch (NMS) were obtained from Lihua Starch Co., Ltd (Qinghuangdao of Shandong, China) and Tiancheng Corn development Co., Ltd (Changchun of Jilin, China), respectively. Amyloglucosidase (EC3.2.1.3, Cat. No. A7095, activity 260 U/mL) from Aspergillus niger and α-amylase

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type VI-B from porcine pancreas (EC3.2.1.1, Cat. No. A3176, activity 10 U/mg) were purchased from Sigma-Aldrich Chemical Co.(St. Louis, MO, USA). Glucose oxidaseperoxidase (GOPOD) assay kits were purchased from Megazyme International Ireland Ltd.(Wicklow, Ireland). Other chemicals and solvents were all of analytical grade. 2.2. Apparent amylose content and lipid content Apparent amylose content of samples was estimated by measuring iodine affinities of defatted whole starch using a potentiometric autotitrator (888 Titrando, Brinkmann Instrument, Westbury, NY, USA) according to the method reported by Li et al. (2017). Starch samples were dissolved and defatted in 90% dimethylsulfoxide (DMSO) solution, and followed by alcohol precipitation. A certain amount of precipitated sample (100 mg) is weighed and transferred into a dry beaker. The starch samples were suspended by adding distilled water (1 mL) and KOH solution (1 M, 5 mL) slowly with stirring. The HCl solution (0.5 M) was used to neutralize the suspension and then KI (0.5 M, 10 mL) was added. An appropriate amount of distilled water is added to obtain a total weight of 100.9 g over the weight of the empty beaker. The suspension is potentiometrically titrated with iodine at 30 ℃with continuous stirring. Apparent amylose content was calculated by dividing the iodine affinity of the starch by 19.0%, the typical value of iodine affinity for purified maize amylose. Lipid content of starch samples was determined according to the standard AOAC methods (2000).

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2.3. Heat-moisture treatment Starch samples (45 g, dry basis) were accurately weighed into glass containers, and the moisture content was adjusted to 25% by adding an appropriate amount of distilled water slowly with stirring, then, sealed and equilibrated for 48 h at room temperature. The samples were placed in a stainless steel reactor with cover and then heated in an electric blast oven at 120 ℃for 3 h and 9 h, respectively. After heating treatment, the HMT modified starches were cooled to room temperature, subsequently air dried at 40 ℃overnight, then crushed and passed through 100-mesh sieve for further analysis. Native starch samples without processing were used as controls. 2.4. Preparation of amorphous samples Amorphous standards were prepared according to the method reported by Tan et al. (2007) with some modifications. Starches (2 g, dry basis) were weighed and dispersed into distilled water to obtain 1% (w/v) suspensions. Then, the suspensions were heated at 100 ℃for 1 h with continuously stirring, and after quick-freeze to solid state, the solutions were lyophilized. 2.5. Light microscopy The granule morphologies and birefringences of native and HMT modified starch were observed by using an Olympus BX-51 light microscope (Tokyo, Japan) under the brightfield and polarized light, respectively. A drop of native and HMT modified

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starch suspensions (1:1 water/glycerol) were taken spreading on a glass slide and covered with a coverslip. The micrographs were recorded at 500× magnification. 2.6. 1H NMR spectroscopy The 1H NMR spectra were measured by using the Bruker Avance II 600 MHz spectrometer. The electron distribution in starch molecules was altered along with the changes of chemical environment of hydrogen. These changes resulted in the variation of peak position obtained by the proton NMR measurement. Starch samples were prepared according to the method described by Yang et al. (2017) with some slight modifications. Approximately 10 mg of starch samples were thoroughly dissolved into 1 ml DMSO-d6 in a water bath at 50 ℃for 60 min. The NMR data was collected for 16 scans at 25 ℃. The chemical shift scale was calibrated using the residual DMSO-d6 signal at 2.549 ppm. The NMR data were processed using MestReNova software V. 9.0 (Mestrelab Research Co.; Spain). Degree of branching (DB) was calculated from the results of 1H NMR using the integral measurements from the following peaks: α-(1,4)-glycosidic bonds (Iα-(1,4)) at approximately 5.11 ppm and α-(1,6)-glycosidic bonds (Iα-(1,6)) around 4.75 ppm. The DB was calculated using the following equation:

(1) 2.7. 13C CP/MAS NMR spectroscopy

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The solid-state 13C NMR experiments were performed on a Bruker Avance III HD 400 spectrometer at a 13C frequency of 75.46 MHz. Approximately 200 mg of samples were packed in a 4 mm diameter, cylindrical, PSZ (partially stabilized zirconium oxide) rotor with a KelF end cap. The rotor was spun at 5-6 kHz at the magic angle (54.7°). The 90° pulse width with 5 µs, a contact time of 1 ms and a recycle delay of 3 s were used for all samples. The spectral width was 38 kHz, acquisition time 50 ms, time domain points 2k, transform size 4k, and line broadening 50 Hz. At least 2400 scans were accumulated for each spectrum. Spectra were referenced to external adamantane. 2.8. Thermal properties The thermal properties of native and modified starch samples were assessed via differential scanning calorimetry (DSC-8000, Perkin Elmer, Norwalk, CT, USA). Starch sample (3 mg, dry basis) was accurately weighed in high-pressure stainless steel pan with gold-plated copper cover, and an appropriate of distilled water was carefully added to obtain a ratio of 1:2 (starch/water). The pans were hermetically sealed, then kept at room temperature for 24 h to make sure a complete equilibration of water and sample. After equilibration, the pans were scanned from 30 to 140 ℃at a rate of 10 ℃/min. An empty pan was used as a reference during scanning. The DSC software was applied to analyze onset temperature (To), peak temperature (Tp), conclusion temperature, gelatinization temperature range (Tc-To) and gelatinization enthalpy (ΔH).

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2.9. X-ray diffraction Native and heat-moisture treated starches were equilibrated in a chamber with 100% relative humidity at 25 ℃for 24 h before X-ray analysis. The X-ray patterns were performed on X-ray diffractometer (D8 Advance, Bruker, Germany). The analyzer was operated at 40 kV and 40 mA with Cu Kα radiation (λ=0.154 nm). The sample powder was packed tightly in a rectangular glass cell, and then scanned over the range of 5-35° 2θ angles at a rate of 2°/min at 25 ℃. The relative crystallinity of the sample was calculated using the following equation: Relative crystallinity (%) = 100Ac / (Aa + Ac)

(2)

where, Aa was the area of the amorphous peak and Ac was the area of the crystalline peak. 2.10. Measurement of in vitro digestibility The starch digestibility was analyzed based on the procedure of Englyst et al. (1992) with some slight modifications. Starch sample (200 mg, dry basis) was accurately weighed into centrifuge tube (50 ml) with screw cap, and one magnetic stirrer and ten glass beads (4 mm diameter) were added into the tubes, followed by adding 15 ml of sodium acetate buffer (0.2 mol/L, pH 5.2). After completely vortexing and mixing, the tubes were equilibrated in a water bath at 37 ℃for 10 min, and then 5 ml sodium acetate buffer containing amyloglucosidase (15 U/ml) and porcine pancreatic α-amylase (290 U/ml) were added into each tube, followed by rotating (350 rpm) on constant temperature magnetic stirrer in a water bath at 37 ℃. 10

Aliquots of hydrolyzed solution (0.5 ml) were taken at 20 and 120 min intervals and immediately placed into centrifuge tube containing 4 ml of absolute ethanol to deactivate the enzymes. Then the tubes were centrifuged at 4000 × g for 10 min. The glucose content of supernatant was determined using glucose oxidase-peroxidase (GOPOD method) assay kits (Wicklow, Ireland). The percentages of RDS, SDS and RS in the starch samples were analyzed according to their definition by Englyst et al. (1992). The equations were as follows: RDS (%) = (G20 - GF) × 0.9 × 100

(3)

SDS (%) = (G120 - G20) × 0.9 × 100

(4)

RS (%) = [(TS - RDS - SDS)/TS] × 100

(5)

where, G20 and G120 were the contents of glucose released after 20 and 120 min, respectively; GF was the content of free glucose and TS was total starch weight. The percentage of hydrolyzed starch was calculated by multiplying a factor of 0.9 with the glucose content. Each sample was analyzed in triplicate. 2.11. Statistical analysis Analytical experiments were performed at least in triplicate for each sample. All results were reported as mean ± standard deviation (SD) using analysis of variance (ANOVA) with Tukey’s pairwise comparison. Significant differences between the natives and modified samples were determined at a 5% level using SPSS V. 17.0 software (SPSS Inc. Chicago, IL). The 13C NMR spectra were analyzed using the PeakFit V. 4.12 software (SYSTAT Software Inc., CA) to fit all spectra. 11

3. Results and discussion 3.1. Morphological Structure The micrographs of native and HMT modified starches are presented in Fig. S1 (Supplementary data). HMT caused the destruction of starch granules, which could be observed in all modified starches. Moreover, such phenomenon became more serious with the increase of treating time. Besides, HMT resulted in the agglomeration of starch granules. Similar finding was reported by Zavareze et al. (2010), who reported the agglomeration in HMT modified high-amylose and medium-amylose rice starch granules. Huang et al. (2015) studied the sweet potato starch treated with heat and moisture and concluded that partial gelatinization appearing in HMT process resulted in the agglomeration of starches. The birefringence patterns of native and heat-moisture treated starches were viewed under the polarized light. Both of the native and HMT modified starches performed Maltese cross centered at the hilum. Compared to the native starches, the heat-moisture treated samples exhibited a weaker and fuzzier Maltese cross (signed with arrows in Fig. S1). Chung et al. (2009) reported similar findings occurred in maize starch and pea starch subjected to HMT. The Maltese cross pattern reflected the ordered and disordered components with an anisotropic phenomenon in starch granule. With the increase of treating time, Maltese cross became increasingly weak and disappeared eventually, which indicated the destruction of ordered structure in the starch granules. 3.2. 1H NMR spectroscopy 12

The spectra and DB of waxy and normal maize starches are presented in Fig. 1. The 1

H NMR spectroscopy could provide information on destruction degree of molecular

structure branching under HMT. According to the report by Cheng et al. (2015), the 1H NMR could distinguish α-(1,6) from α-(1,4)-linkages of starch based on chemical shifts of 4.7-5.0 ppm for α-(1,6)-linkages and 5.1-5.4 ppm for α-(1,4)-linkages. The changes of DB in starch samples after HMT could be obtained by measuring the ratio of their peak area. Compared to the 1H NMR of native waxy and normal maize starches, the proton chemical environments of heat-moisture treated starches varied. As shown in Fig. 1A, the DB of WMS-Native, WMS-HMT3h and WMS-HMT9h were 7.00%, 6.71% and 5.40%, respectively, indicating that more α-(1,6)-glycosidic bonds than α-(1,4)glycosidic bonds were destroyed during HMT. Yang et al. (2017) suggested that the steric hindrance around the α-(1,4)-glycosidic bonds was stronger than that around α(1,6)-glycosidic bonds in starch. Therefore, the α-(1,6)-glycosidic bonds were more susceptible to heat-moisture treatment. Compared to the native normal maize starch, the DB of NMS-HMT3h and NMS-HMT9h decreased to 4.43% and 1.11%, respectively, suggesting HMT resulted in the degradation of starch molecules. Similar phenomenon has been reported by Wang et al. (2016), who studied the structure of heatmoisture modified starch and found that HMT induced the molecular degradation. Watcharatewinkul et al. (2010) also reported that HMT caused starch degradation with degraded areas represented by shallow indentations along the growth ring and longitudinal grooves originating from the hilum.

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3.3. 13C CP/MAS NMR spectroscopy The 13C CP/MAS NMR provided the elucidation of starch molecular organization at short distance scales. The spectra of samples were typically regarded as the composite of spectra from amorphous, single and double helical components (Gidley & Bociek, 1985; Tan et al., 2007). The signals from different spectral positions were classified into several C regions, which were the C1 region (90-110 ppm), C2, C3 and C5 regions (68-77 ppm), C4 region (80-85 ppm) and C6 region (60-65 ppm) (Gidley & Bociek, 1985; Fan et al., 2013). According to the report by Gidley et al. (1985), C1 and C4 carbons involved in glycosidic linkages and would therefore be expected to be the most sensitive to polysaccharide conformation. Those two carbon sites showed large chemical shift displacements between crystalline and amorphous phase, indicating substantial conformational differences. The analysis of 13C NMR spectra involved the decomposition of the spectra using a substraction technique. Before the substraction, a scaling factor was applied to adjust the intensity of amorphous subspectrum thus the zero intensity at a certain chemical shift could be obtained (Tan et al., 2007). Tan et al. (2007) suggested that the position of this zero intensity point was chosen to be within C4 region since it was relatively independent from other resonances and didn’t overlap with other C regions. Furthermore, it was widely accepted that amorphous starch contributed to most of the intensity in C4 region (Gidley & Bociek, 1988; Veregin, Fyfe, & Marchessault, 1987; Veregin, Fyfe, Marchessault, & Taylor, 1986). Tan et al. (2007) studied the nature and proportions of double helices, single helix and amorphous 14

phase in starch granules and concluded a test criterion to check whether a proper amorphous subspectrum was obtained. The test criterion required the absence of negative peak in ordered subspectrum after substraction. Taking native waxy maize starch as an example, the decomposition of the 13C NMR spectra of WMS-Native is presented in Fig. 2A. The ordered subspectra were acquired by subtracting the amorphous subspectra from the original spectra of the samples. At the present work, the chemical shift at 84 ppm was used as the reference position, and negative intensity seldom appeared in ordered subspectra. Taking native waxy maize starch as an example, the results of fitted spectra peak using PeakFit software, including original spectra, amorphous subspectra and ordered subspectra, are shown in Fig. 2B-D. Each subspectrum yielded 8-9 peaks with Gaussian profiles, and similar result could be found in the report by Bogracheva et al. (2001). The substraction technique provided an accessible way to calculate the proportions of ordered and disordered components within the starch granules. That was to calculate the ratios of different component areas to total area of the samples spectra. And the ordered subspectra were supposed to be a combination of single helix and double helices. The relative contents of single helix and double helices in starch samples were analyzed using the method reported by Tan et al. (2007). As presented in Table 1, HMT caused drastic changes in waxy maize starch granules. The relative content of amorphous phase and double helices in native waxy maize starch were 56.96% and 43.04%, respectively. When waxy maize starch was treated for 3h, approximate 3.18% of double helices unwound and transformed into 15

single helix, while approximate 1.55% of double helices were destroyed and transformed into amorphous components. Compared to native waxy maize starch, the single helix in WMS-HMT3h increased to 3.18%, but decreased to 2.58% in WMSHMT9h. This variation suggested that the transformation of double helices into single helix and single helix into amorphous phase occurred simultaneously during HMT. The relative contents of single helix in WMS-HMT9h were lower than in WMSHMT3h, which indicated that more single helices were destroyed than transformed when waxy maize starch was treated for 9 h. As for normal maize starch, the variations in amorphous phase and double helices performed a similar trend as waxy maize starch. The relative content of amorphous phase in NMS-Native, NMS-HMT3h and NMS-HMT9h were 59.72%, 60.98% and 66.17%, respectively. Compared to native normal maize starch, the double helices in NMS-HMT3h and NMS-HMT9h decreased by 1.67% and 7.33%, respectively, while the single helix increased by 0.41% and 0.88%, respectively. The percentage changes of single helix were not consistent with the double helices, which might be attributed to the function that HMT caused the double helical components transformed into single helix and amorphous phase.

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Table 1. The relative contents of different components in waxy and normal maize starches before and after HMT Samples

Amorphous phase/%

Single helix/%

Double helices/%

Apparent amylose content/%

Lipid content/%

WMS-Native

56.96

0

43.04

0.5

0.08

WMS-HMT3h

58.51

3.18

38.31

-

-

WMS-HMT9h

60.78

2.58

36.64

-

-

NMS-Native

59.72

2.33

37.95

26.8

0.10

NMS-HMT3h

60.98

2.74

36.28

-

-

NMS-HMT9h

66.17

3.21

30.62

-

-

The changes in relative contents of double helices, single helix and amorphous phase indicated the occurrence of transformation among them during HMT. According to the analysis of 1H NMR and

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C NMR, we concluded that at least three

transformation patterns may happen in starch granules during HMT. As presented in Fig. 3, the three transformation patterns were, a) part of double helical components were destroyed and transformed into amorphous components; b) some double helices were unwound and transformed into single helix and then further transformed into amorphous phase; c) plenty of covalent bonds were destroyed and starches were degraded with the produce of short-chain molecules. 3.4. Thermal properties The thermal properties of waxy and normal maize starches are summarized in Table 2. Starch gelatinization is an endothermic transition relevant to the dissociation of 17

amylopectin double helices from the ordered structure to disordered structure (Li et al., 2017; Zhang, & Huang, 2017). HMT significantly influenced the thermal properties of starches, and waxy and normal maize starches exhibited some similar changes after HMT. As shown in Table 2, the gelatinization temperatures (To, Tp and Tc) of heatmoisture treated waxy and normal maize starches were shifted to higher temperatures. According to Luo et al. (2008), the onset temperature (To) was referred as the melting temperature of weakest crystalline in starch granules, while the conclusion temperature was the melting temperature of high-perfection crystalline. With the increase of treating time, the onset temperatures of both waxy and normal maize starches were significantly increased (HMT9h > HMT3h > Native), which suggested that HMT may destroy the weak crystalline in starch, and the high-perfection crystalline may survive. The increase of conclusion temperature may be attributed to formation of more stable crystalline during HMT. Similar results could be found in the study of Huang et al. (2016). Compared to native waxy and normal maize starches, the gelatinization temperature range (Tc-To) were decreased by 5.40 ℃ in WMS-HMT3h, 7.70 ℃ in WMS-HMT9h, 2.45 ℃ in NMS-HMT3h, and 2.84 ℃ in NMS-HMT9h, respectively. Luo et al. (2006) suggested that the value of Tc-To represented the degree of heterogeneity of crystallites within the starch granules. The Tc-To in all HMT modified samples were narrower than those of native counterparts, indicating that HMT destroyed the crystalline structure in starch granules and decreased the crystallite heterogeneity.

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1All

data are averages of three determinations with standard deviation. Means ± standard

deviation. Means in a column with different letters are significantly different (p < 0.05) by the least significant difference (LSD) test.

The gelatinization enthalpy (ΔH) primarily represented the loss of double helical components within starch granules (Cooke & Gidley, 1992; Singh, Singh, Kaur, Sodhi, & Gill, 2003). The values of ΔH in WMS-Native, WMS-HMT3h and WMS-HMT9h were 12.73 J/g, 3.11 J/g and 2.73 J/g, respectively, suggesting that HMT destroyed the double helical structure and resulted in reduction of double helices. This trend was consistent with the result determined by 13C NMR (presented in Table 1). Normal maize starch showed the similar variation in content of double helices. The gelatinization enthalpy of native waxy maize starch (12.73 J/g) was higher than native normal maize starch (11.40 J/g), which could be attributed to the higher content of double helices in waxy maize starch confirmed by the 13C NMR experiments. 3.5. X-ray diffraction The X-ray diffractograms and relative crystallinity of samples are presented in Fig. 4. Waxy maize starch exhibited the crystalline pattern of A-type polymorph, which had the strong diffraction at 2θ values of 15.0°, 17.1°, 18.0° and 23.0°. Although the crystalline pattern of waxy maize starch remained unchanged after HMT, there were drastic changes occurred in internal structure, which could be proved by the variations (enclosed with a box in Fig. 4A) of the unresolved doublet with the peaks at 17.1° and 18.0°. With the increasing of treating time, the 19

diffraction at 17.1° was gradually weakened, while the diffraction at 18.0° was increasingly strengthened. This could be ascribed to the fact that HMT increased the mobility of starch chains and the rearrangement of starch molecules occurred in starch granules, especially in crystalline structure. The X-ray diffraction provided information on the long-range molecular order, generally regarded as crystallinity, which was linked with ordered arrays of double helices formed by the amylopectin side chains. The relative crystallinity of WMS-Native, WMS-HMT3h and WMS-HMT9h were 38.13%, 34.20% and 31.23%, respectively, indicating that HMT destroyed the double helices in starch granules. The destruction of double helices was also proved by 13C CP/MAS NMR spectrum technology and DSC gelatinization enthalpy (ΔH). Compared to native normal maize starch, the crystalline pattern of heat-moisture treated starch changed from A to A + V pattern with a noticeable increase of peak intensity at 2θ values of 7.5°, 13.0° and 19.9° (signed with arrows in Fig. 4B). This finding was similar with the results reported by Chen et al. (2015), who found HMT changed the crystallization structure of wheat starch from A to A + V pattern and increased the peak intensity at 2θ values of 7.6°, 13.0° and 20.0°. Normal maize starch had a higher amylose content compared to waxy maize starch; therefore, more V-type polymorphs were formed in normal maize starch during HMT. Compared to native normal maize starch, the relative crystallinity of NMS-HMT3h and NMS-HMT9h were decreased by 1.78% and 6.60%, respectively. HMT disrupted the hydrogen bonds and even covalent bonds in starch molecules, and induced the change of crystallite orientation (Deka & Sit, 2016; Gunaratne & Hoover, 2002; Klein 20

et al., 2013), which could influenced the whole structure of starch granules whether in ordered or disordered phase. These may be the reasons for the reduction of the relative crystallinity. Structure changes of waxy and normal maize starches during HMT were confirmed by 1H NMR, 13C NMR, DCS and XRD. The schematic model of native normal maize starch (Ⅰ) and heat-moisture treated starch (Ⅱ) are presented in Fig. 5, which clearly illustrated the starch structure variations during HMT. Starch samples were sealed and heated in the stainless steel reactor, and the water molecules continuously converted the thermal energy into the kinetic energy at the beginning of procedure. The starch granules underwent the bombardment from water molecules. Moreover, some water molecules with high speed and energy entered into the internal structure of granules through the surface pores and channels, causing the degradation of starch granules. As shown in Fig. 5, some starch granules were broken down into several parts, and the semi-crystalline layers were destroyed after HMT, which caused the decrease of crystallinity as confirmed by XRD. The blocklets were not dense packing as before and whole structure tended to be cracked and misplaced, which resulted in the decrease of crystallite heterogeneity as determined by DSC. There were breakage of glycosidic bonds (decrease of DB) and rearrangement of molecules (increase of V-type single helix) occurring during HMT, which were confirmed by 1H NMR and 13C NMR, respectively.

21

3.6. In vitro digestibility of native and modified starches The digestion properties of native and modified starches are presented in Fig. 6. Two amylolytic enzymes, amyloglucosidase and porcine pancreatic α-amylase, were applied to evaluate the starch digestibility. The amyloglucosidase could cleave the α(1,4)-glycosidic bonds and α-(1,6)-glycosidic bonds, while the α-amylase resulted in the breakage of α-(1,4)-glycosidic bonds. As shown in Fig. 6, the heat-moisture treatment had a major influence on starch digestibility. Compared to native waxy maize starch, the SDS contents in WMS-HMT3h and WMS-HMT9h increased to 31.92% and 34.63%, respectively, while the RS decreased to 53.33% and 48.02%, respectively. Similar trends were also found in normal maize starch. The RDS and SDS contents were largely increased (NMS-HMT9h>NMS-HMT3h>NMS-Native) and RS content was decreased severely. According to the “Induced Fit Theory” (Jr, 1995), the enzyme and substrate combined gradually at the beginning of enzymatic hydrolysis, and the active site of enzyme gradually changed to fit the substrate. Therefore, at least two factors were supposed to influence the starch digestibility. One is the binding capacity between amylolytic enzymes and starch granules; the other is the number of binding sites and their surroundings. Enzymatic hydrolysis requires the binding of amylolytic enzymes to starch molecules. Zhang et al. (2006) found that α-amylase needed to bind substrate from the side of starch molecules before the hydrolysis. The side-binding pattern (versus a head-on orientation) means that α-amylase needs to bind starch molecules in a direction parallel to double helices. For native waxy and normal maize starch

22

granules, the digestion mechanism belongs to “inside-out” digestion pattern (Zhang et al., 2006). Although there are many surface pores and channels for enzymatic hydrolysis from the hilum region toward the outside of the granules (Gallant, Bouchet, Buléon, & Pérez, 1992), the digestion rate and degree are still limited. For heat-moisture treated starches, the content of double helices and the relative crystallinity were decreased, which meant the heat-moisture modified starches no longer retained the dense packing as their native counterparts. During HMT, starch granules were destroyed and the structure of starch granules was loosened, which led to easier combination between amylolytic enzymes and starch granules. The number of binding sites and their surroundings played important roles in starch digestibility. According to the report by Gilles et al. (1996), porcine pancreatic α-amylase has five subsites to bind substrate, and each subsite could bind a glucose unit. And its hydrolysis product profile (maltose, maltotriose, and other dextrins) indicates that α-amylase requires binding to at least three glucose units before cleaving an α-(1,4)-glycosidic bond. In starch granules, the double helices formed by two parallel, left-handed helices (Imberty, Chanzy, Pérez, Bulèon, & Tran, 1988; Imberty & Perez, 1988), while the single helix formed by one left-handed helix and often with a complex agent being included in the helical channel (Snape, Morrison, Marotovaler, Karkalas, & Pethrick, 1998). Both of these structures allowed complex and dense packing between starch chains. The dense packing structure could protect glycosidic linkages from the attack of amylolytic enzymes. Ambigaipalan et al. (2014) also suggested that double helices would restrict the accessibility of amylolytic 23

enzymes towards the glycosidic bonds. Furthermore, the double helices would pack into crystalline lamella and then form a denser super-helix structure (Pérez & Bertoft, 2010), which, to some extent, let many glycosidic bonds hiding in the dense structure away from the enzymatic hydrolysis. Lots of hydrogen bonds were formed around the glycosidic bonds, to maintain the helical configuration in starch granules. According to the previous studies (Goto, Kuwano, Kanlayakrit, & Hayashida, 1995; Williamson, Belshaw, Noel, Ring, & Williamson, 1992), the hydrogen bonds were destroyed, and further the glycosidic bonds were cleaved by amylolytic enzymes. After HMT, many hydrogen bonds were destroyed and the helical structure was not compact as before, thus, the glycosidic bonds were available for enzymatic hydrolysis.

4. Conclusion The present study showed that HMT caused the transformation of starch granules from ordered phase into disordered phase as proved by 13C NMR spectrum technology. Relative content of double helices reduced was also confirmed by DSC which revealed gelatinization enthalpy with a downtrend. XRD showed that starch crystallinity gradually decreased with the increase of treating time. The 1H NMR spectroscopy gave sufficient evidence that the DB of heat-moisture treated starches decreased compared to their native counterparts. All of these changes resulted in accessibility to binding sites and were beneficial to the combination between amylolytic enzymes and starch granules. These may be the reasons why the decrease of ordered phase caused the increase of digestibility. Furthermore, the more ordered 24

components were destroyed, the higher content of RDS and SDS were obtained. The relationship between starch structure and digestibility provides a valuable thinking to future studies aiming at improving the contents of SDS and RS, which is to make sure that the adopted method could decrease the binding capacity between amylolytic enzymes and starch granules. That is to say, the modified methods need to decrease accessibility of hydrolysis sites, improve the steric hindrance of glycosidic linkages and strengthen the interaction of starch chains.

Acknowledgments This research was supported by the National Natural Science Foundation of China (21576098, 21376097), the Project funded by China Postdoctoral Science Foundation (2016M590787, 2017T100616), the Key Project of Science and Technology of Guangdong Province (2017B090901002, 2016A050502005, 2015A020209015), the Key Project of Science and Technology of Guangzhou City (201508020082).

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α-(1,4)-linkage

(A)

Degree of branching

α-(1,6)-linkage WMS-HMT9h

5.40%

WMS-HMT3h

6.71%

WMS-Native

7.00%

4.2

4.4

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

Chemical shift (ppm) α-(1,4)-linkage

(B)

Degree of brabching

α-(1,6)-linkage NMS-HMT9h

1.11%

NMS-HMT3h

4.43%

NMS-Native

5.05%

4.2

4.4

4.6

4.8

5.0

5.2

5.4

Chemical shift (ppm)

32

5.6

5.8

6.0

Fig. 1. 1H NMR spectra of waxy (A) and normal (B) maize starches. (Waxy maize starches with different treatment time (0h, 3h, 9h) were denoted as WMS-Native (without treatment), WMS-HMT3h and WMS-HMT9h, respectively. Normal maize starches with same treating time were denoted as NMS-Native, NMS-HMT3h and NMS-HMT9h.)

Fig. 2. Decomposition of 13C NMR spectra of WMS-Native (A) into amorphous and ordered phase and peak fitting of the original spectrum(B), amorphous subspectrum (C) and ordered subspectrum (D) from native waxy maize starch.

33

Fig. 3. Three transformation patterns in starch granules during HMT

34

A Relative Crystallinity WMS-HMT9h

31.43%

WMS-HMT3h

34.20%

WMS-Native

38.13%

5

10

15

20

25

30

35

Diffraction angle (2θ)

B Relative Crystallinity

NMS-HMT9h

26.55%

NMS-HMT3h 31.37%

NMS-Native 33.15%

5

10

15

20

25

30

35

Diffraction angle (2θ)

Fig. 4. X-ray diffraction spectra and relative crystallinity of waxy (A) and normal (B) maize starches

35

Fig. 5. The schematic model of native (Ⅰ ) and heat-moisture treated (Ⅱ ) normal maize starches

36

Fig. 6. Digestibility of waxy and normal maize starches before and after HMT

Table 2. Thermal properties of native and modified waxy and normal maize starches1

Samples

To/℃

Tp/℃

Tc/℃

Tc-To/℃

ΔH/J·g-1

WMS-Native

66.95±1.54c

77.18±0.58c

88.08±2.94b

21.13±1.40a

12.73±1.84a

WMS-HMT3h

75.02±1.72b

81.50±0.37b

90.76±1.81ab

15.73±2.87b

3.11±0.15b

37

WMS-HMT9h

82.02±1.23a

89.22±1.21a

95.44±4.03a

13.43±4.30b

2.73±1.47b

NMS-Native

68.70±0.85C

73.12±0.61C

82.20±1.39C

13.50±0.86A

11.40±1.41A

NMS-HMT3h

75.52±0.99B

79.85±0.20B

86.57±1.21B

11.05±0.82B

2.83±0.24B

NMS-HMT9h

83.78±2.23A

89.72±2.85A

96.77±0.41A

10.66±1.23B

2.77±1.01B

38