Comparative structure of starches from high-amylose maize inbred lines and their hybrids

Accepted Manuscript Comparative structure of starches from high-amylose maize inbred lines and their hybrids Lingshang Lin, Dongwei Guo, Lingxiao Zhao, Xudong Zhang, Juan Wang, Fengmin Zhang, Cunxu Wei PII:

S0268-005X(15)00264-7

DOI:

10.1016/j.foodhyd.2015.06.008

Reference:

FOOHYD 3039

To appear in:

Food Hydrocolloids

Received Date: 23 November 2014 Revised Date:

30 May 2015

Accepted Date: 2 June 2015

Please cite this article as: Lin, L., Guo, D., Zhao, L., Zhang, X., Wang, J., Zhang, F., Wei, C., Comparative structure of starches from high-amylose maize inbred lines and their hybrids, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.06.008. 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.

ACCEPTED MANUSCRIPT Comparative structure of starches from high-amylose maize inbred lines and their hybrids

Lingshang Lin a,1, Dongwei Guo b,1, Lingxiao Zhao a, Xudong Zhang b, Juan Wang a, Fengmin

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Zhang c, Cunxu Wei a*

Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern

Key Laboratory of Biology and Genetic Improvement of Maize in Arid Area of Northwest

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Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Chin

Region, Ministry of Agriculture, Northwest A & F University, Yangling 712100, China Testing Center, Yangzhou University, Yangzhou 225009, China

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Lin L. S. and Guo D. W. contributed equally to this work.

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Graphical abstract

ACCEPTED MANUSCRIPT Comparative structure of starches from high-amylose maize inbred lines and their

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hybrids

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Lingshang Lin a,1, Dongwei Guo b,1, Lingxiao Zhao a, Xudong Zhang b, Juan Wang a, Fengmin

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Zhang c, Cunxu Wei a*

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a

Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center for Modern

Key Laboratory of Biology and Genetic Improvement of Maize in Arid Area of Northwest

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Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China

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Region, Ministry of Agriculture, Northwest A & F University, Yangling 712100, China

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Testing Center, Yangzhou University, Yangzhou 225009, China

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Lin L. S. and Guo D. W. contributed equally to this work.

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* Corresponding author

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Cunxu Wei: College of Bioscience and Biotechnology, Yangzhou University, Yangzhou

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225009, China; Tel.: +86 514 87997217; E-mail: [email protected]

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Running title

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Structure of high-amylose inbred and hybrid maize starches

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ACCEPTED MANUSCRIPT Abstract

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High-amylose maize starch is of interest because of its health benefits and industrial uses.

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Hybrid maize is widely grown for the practical and economical value of heterosis. However,

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starch structure of high-amylose hybrid maize has seldom been reported compared with that

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of high-amylose inbred maize. In this study, structure of starches from high-amylose maize

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inbred lines and their hybrids was investigated and compared. Starch granule became smaller

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in size and more spherical, oval or elongated in shape with increasing amylose content.

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Hybrid maize starch had lower amylose content and long branch-chain of amylopectin and

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higher short branch-chain and branching degree of amylopectin than inbred maize starch.

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Inbred maize starch had B-type crystalline structure with low relative crystallinity, but hybrid

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maize starch had CB-type crystalline structure and high relative crystallinity. Hybrid maize

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starch had lower degree of order at a short-range scale on the edge of starch granule than

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inbred maize starch. Hybrid maize starch had higher double helix content and lower

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amorphous starch than inbred maize starch. Hybrid maize starch had higher peak intensity and

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longer Bragg spacing of lamellar structure than inbred maize starch. Hybrid maize starch had

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lower gelatinization temperature and higher gelatinization enthalpy and swelling power than

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inbred maize starch. The different structural properties of starches had significant correlation.

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Keywords: Maize inbred line; Maize hybrid line; High-amylose starch; Molecular structure;

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Crystalline structure; Lamellar structure.

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1. Introduction Cereal storage starches consist of two main components: linear amylose and highly

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branched amylopectin, and exist as discrete semicrystalline granules. The granule morphology

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including shape and size is dependent on botanic origin (Jane, Kasemsuwan, Leas, Zobel, &

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Robyt, 1994). Amylose content and amylopectin structure have an important effect on

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physicochemical and functional properties of starch. The semicrystalline granule displays a

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hierarchical structural periodicity and has a layered organization with alternating

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semicrystalline and amorphous growth rings (Gallant, Bouchet, & Baldwin, 1997). The

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semicrystalline rings are formed by a lamellar structure of alternating crystalline and

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amorphous regions with a regular repeat distance of 9−10 nm. The crystalline regions of the

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lamellae are thought to be formed by double helices of amylopectin side chains packed

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laterally into a crystalline lattice (Blazek & Gilbert, 2011). The amylopectin side chains can

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form two types of helices in starch granule. Helices that are packed in the short-range order

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are defined as the double helical order, and helices that are packed in the long-range order are

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related to the packing of double helices forming crystallinity (Atichokudomchai, Varavinit, &

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Chinachoti, 2004). The crystallinity of starch had three types of A-, B- and C-type (Cheetham

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& Tao, 1998).

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Resistant starch is a portion of starch that cannot be hydrolyzed in the upper

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gastrointestinal tract and functions as a substrate for bacterial fermentation in the large

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intestine (Englyst, Kingman, & Cummings, 1992). In general, resistant starch content of

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granular starch is positively correlated with amylose content (Sang, Bean, Seib, Pedersen, &

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Shi, 2008). Therefore, high-amylose starches have a high-level of resistant starch content and

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ACCEPTED MANUSCRIPT are of interest because of their potential health benefits. Some high-amylose cereal crop lines

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have been developed using mutation or transgenic breeding approaches, their starches show

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significantly different structural and functional properties from normal starches (Carciofi et al.,

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2012; Jiang, Campbell, Blanco, & Jane, 2010; Kim et al., 2005; Regina et al., 2006; Slade et

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al., 2012; Zhu et al., 2012).

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Maize is an important food and feed crop because its seeds provide large amounts of

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starch. Maize starch (especially amylose) is also an important industrial raw material. Maize

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amylose, which is characterized by a high degree of polymerization and good film formation,

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is far superior to other amyloses in the areas of support films, foods, medical treatments,

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textiles, paper making, packaging, petroleum, environmental protection, optical fibers, printed

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circuit boards, and electronic chips (Guan et al., 2011; Jiang et al., 2013). High-amylose

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maize starch is the best material for the manufacture of photo-dissociative plastics and can

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potentially help to control serious “white pollution” (Guan et al., 2011; Jiang et al., 2013). In

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addition, high-amylose maize has health benefits through its high resistant starch content

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(Jiang et al., 2010). Therefore, many high-amylose maize inbred lines have been developed

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(Guan et al., 2011; Jiang et al., 2010; Li, Jiang, Campbell, Blanco, & Jane, 2008; Shi, Capitani,

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Trzasko, & Jeffcoat, 1998).

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When maize plants are self-pollinated (i.e., inbred) in successive generations, their vigor

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and grain yield rapidly deteriorate. However, when two inbred lines from unrelated

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populations are crossed, both vigor and grain yield of the F1 hybrid often exceed that

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observed for the original source populations. This phenomenon, termed heterosis, refers to the

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superiority in performance of the F1 hybrid over either one of its parents (Tollenaar,

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ACCEPTED MANUSCRIPT Ahmadzadeh, & Lee, 2004). For the practical and economical value of hybrid vigor and grain

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yield, hybrid maize is widely grown by farmer, and relatively few maize inbred lines are

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grown. Many literatures have reported the physicochemical properties of high-amylose crop

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starches from transgenic or mutant inbred lines (Huang et al., 2015; Jiang et al., 2010; Li et al.,

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2008; Man et al., 2012; Wang, White, Pollak, & Jane, 1993). According to our knowledge,

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little work has reported the properties of high-amylose crop starches from hybrid lines.

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A high-amylose maize mutant was found in our previous study (Li et al., 2014). We had

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utilized this mutant to develop some high-amylose maize inbred lines and their hybrids

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through crossing, backcrossing, and self-crossing methods. In this study, starches were

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isolated from one normal maize, four high-amylose maize inbred lines, and two high-amylose

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maize hybrids. Their physicochemical structures were investigated. The objective of this

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study was to compare the structure of starches from high-amylose maize inbred lines and their

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hybrids.

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2. Materials and methods

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2.1. Plant materials

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One normal maize cultivar Xianyu 335, four high-amylose maize inbred lines (Zae28,

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Zae35, Zae49 and Zae50), and two high-amylose maize hybrids (Zae35×Zae28 and

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Zae49×Zae50) were used in this study. The inbred lines were developed from a native

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high-amylose amylose-extender (ae) mutant of Xianyu 335. The mutant as male parent

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crossed with four inbred lines of PH4CV, PH6WC, male parent and female parent of Linao to

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obtain the first filial generation (F1). The F1 backcrossed with the four inbred lines for four

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generations, then self-crossed four generations to obtain the four inbred lines of Zae28, Zae35,

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ACCEPTED MANUSCRIPT Zae49, and Zae50, respectively. The agronomic traits of Zae28, Zae35, Zae49, and Zae50

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could be stably inherited, and were very similar to those of PH4CV, PH6WC, male parent of

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Linao, and female parent of Linao. The Zae35 as female parent crossed with Zae28 to obtain

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the F1, then the F1 massed pollination to obtain the Zae35×Zae28. Similarly, Zae49 as female

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parent crossed with Zae50 to obtain the F1, then the F1 massed pollination to obtain the

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Zae49×Zae50. Plants were grown in the experiment field of Northwest A&F University,

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Yangling, China.

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2.2. Starch isolation

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Starch was isolated from mature seeds according to the methods of Li et al. (2008) and

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Ketthaisong, Suriharn, Tangwongchai, and Lertrat (2013) with some modifications. Maize

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seeds were steeped in an aqueous solution of 0.45% sodium metabisulfite at 4 °C overnight.

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After that, the steep water was drained off, kernels were washed with distilled water and kept

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soaked in water while removing the germ and the husk with the help of forceps. After removal,

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the grains were ground and homogenized in a mortar with a pestle. The homogenate was

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squeezed through four layers of cotton cloth. The residue was ground and homogenized again

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with additional water until no more starch was released. The combined extract was filtered

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with 100, 200, 300 and 400 mesh sieves. Starch was collected by centrifugation at 6000 g for

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5 min, suspended in an aqueous solution of 0.1 M NaCl, and stirred for one hour using

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magnetic stirrer. This treatment was repeated 3 times. Then the sediment was resuspended in

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0.2% (w/v) sodium hydroxide aqueous solution and stirred for one hour. The treatment was

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repeated 3 times. The purified starch was washed 5 times with water. During washing, the

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upper non-white layer was carefully scraped off. Finally, the white starch sediment was

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further washed twice with anhydrous ethanol, dried at 40 °C, ground into powders, and passed

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through a 100 mesh sieve.

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2.3. Granule size analysis Granule size analysis was carried out using a laser diffraction particle size analyzer

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(Mastersizer 2000, Malvern, England). The starches were suspended in distilled water and

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stirred at 2000 rpm. The obscuration in all measurements was >10%. The instrument output

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had a volume distribution as the fundamental measurement and median of diameter. Particle

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size is defined in terms of the 10th percentile [d(0.1)], median [d(0.5)], 90th percentile

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[d(0.9)], surface-weighted mean [D(3,2)], and volume-weighted mean [D(4,3)].

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2.4. Scanning electron microscope observation

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Starch powder was suspended in anhydrous ethanol. Starch-ethanol suspension (20 µL)

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was applied to an aluminum stub using double-sided adhesive tape and dried in a vacuum

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oven overnight at 40 °C. The starch samples were coated with gold using a sputter coater, and

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were observed and photographed using an environmental scanning electron microscope

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(ESEM, Philips XL-30).

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2.5. Measurements of iodine absorption spectrum, iodine blue value, and apparent

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amylose content

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Starch was defatted with 85% (v/v) methanol and dissolved in urea dimethyl sulphoxide

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(UDMSO) solution. The starch-UDMSO solution was treated with iodine solution according

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to the method of Man et al. (2012). The iodine absorption spectrum was scanned from 400 to

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900 nm with a spectrophotometer (Ultrospec 6300 pro, Amershan Biosciences). Iodine blue

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value was measured at 680 nm. Apparent amylose content was evaluated from absorbance at

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ACCEPTED MANUSCRIPT 620 nm. The recorded values were converted to amylose content by reference to a standard

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curve prepared with amylopectin from maize and amylose from potato. The experiments were

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carried out three times.

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2.6. Gel permeation chromatography analysis

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Starch was deproteinized with protease and sodium bisulfite, and debranched with

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isoamylase following the methods of Tran et al. (2011) and Li, Hasjim, Dhital, Godwin, and

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Gilbert (2011). The molecular weight distribution of debranched starch was analyzed using a

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PL-GPC 220 high temperature chromatograph (Agilent Technologies UK Limited, Shropshire,

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UK) with three columns (PL110-6100, 6300, 6525) and a differential refractive index detector

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according to the method of Cai, Cai, Man, Zhou and Wei (2014). The eluent system was

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dimethyl sulphoxide (DMSO) containing 0.5 mM NaNO3 at a flow rate of 0.8 mL/min. The

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column oven temperature was controlled at 80°C. Standard dextrans of known molecular

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weights (2800, 18500, 111900, 410000, 1050000, 2900000 and 6300000) were used for

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column calibration. The experiments were performed two times.

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2.7. X-ray diffraction analysis

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X-ray diffraction analysis of starch was carried out on an X-ray powder diffractometer

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(D8, Bruker, Germany). The samples were exposed to the X-ray beam at 200 mA and 40 kV.

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The scanning region of the diffraction angle (2θ) was from 3° to 40° with a step size of 0.02°

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and a count time of 0.8 s. Before measurements, the specimens were stored in a moist

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chamber where a saturated solution of NaCl maintained a constant humidity (relative

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humidity = 75%) for 1 week. The relative crystallinity was measured following the method

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described by Wei, Qin, Zhou et al. (2010), and quantitatively analyzed in triplicate.

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2.8. Attenuated total reflectance-Fourier transforms infrared analysis Attenuated total reflectance-Fourier transforms infrared analysis of starch was carried out

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on a Varian 7000 Fourier transforms infrared spectrometer with a DTGS detector equipped

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with a attenuated total reflectance single-reflectance cell containing a germanium crystal (45°

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incidence angle) (PIKE Technologies, USA) as previously described by Wei, Qin, Zhou et al.

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(2010). Original spectra were corrected by subtraction of the baseline in the region from 1200

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to 800 cm−1 before deconvolution was applied using Varian Resolutions Pro software. The

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assumed line shape was Lorentzian with a half-width of 19 cm−1 and a resolution

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enhancement factor of 1.9. The experiments were performed three times.

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2.9. Solid-state 13C CP/MAS NMR analysis

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B0=9.4T on a Bruker AVANCE III 400 WB spectrometer as described previously by Cai et al.

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(2014). Amorphous starch was prepared by gelatinizing native starch following the method of

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Atichokudomchai et al. (2004). The quantitative analysis of single-helix, double-helix, and

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amorphous component within starch was carried out according to the method described by

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Tan, Flanagan, Halley, Whittaker, and Gidley (2007). The spectrum of amorphous starch was

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matched to the intensity of native starch at 84 ppm and subtracted to produce the ordered

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subspectrum. The areas of the amorphous and ordered subspectrum relative to the total area of

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the original spectrum at C1 region yielded the percentage of amorphous and ordered

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components, respectively. The ordered subspectrum at C1 region was peak fitted by using

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PeakFit version 4.12, which yielded 4 peaks at about 99.5, 100.5, 101.5, and 103.0 ppm. The

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peak at 103 ppm was coincident with the C1 chemical shifts of the single helical V-type

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ACCEPTED MANUSCRIPT conformation, and the other peaks were double helix components. Comparing the areas of

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single helix and double helix peaks at C1 region in the ordered subspectrum, the relative

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proportions of single helix and double helix were obtained. The spectra were quantitatively

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analyzed in triplicate.

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2.10. Small-angle X-ray scattering analysis

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Small-angle X-ray scattering measurement of starch was performed according to the

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method of Yuryev et al. (2004) with some modifications. Starch was dispersed in an excess of

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distilled water to form slurries. Small-angle X-ray scattering measurement was obtained using

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a Bruker NanoStar small-angle X-ray scattering instrument equipped with Vantec 2000

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detector and pin-hole collimation for point focus geometry. The X-ray source was a copper

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rotating anode (0.1 mm filament) operating at 50 kV and 30 W, fitted with cross coupled

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Göbel mirrors, resulting in Cu Kα radiation wavelength of 1.5418 Å. The optics and sample

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chamber were under vacuum to minimize air scattering. During X-ray exposure, the starch

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slurries were kept in sealed cells to prevent dehydration. The small-angle X-ray scattering

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data sets were analyzed using DIFFRACplus NanoFit software. Parameters of the small-angle

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X-ray scattering spectrum were determined according to the simple graphical method (Yuryev

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et al., 2004). The experiment was performed two times.

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2.11. Thermal property of starch

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Thermal property of starch was conducted according to Zaidul et al. (2008) with some

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modifications. Five milligrams of starch was mixed with 15 µL of water and hermetically

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sealed in an aluminum pan. The sample was equilibrated for 2 h at room temperature, then

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heated from room temperature to 130 °C at a heating rate of 2 °C/min using a differential

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scanning calorimetry (200-F3, NETZSCH, Germany). The experiment was performed three

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times.

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2.12. Swelling power and water solubility of starch Swelling power and water solubility of starch were determined according to the

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small-scale method of Konik-Rose et al. (2001) with some modifications. Starch-water slurry

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(2%, w/v) was heated in a water bath at 95 °C for 30 min with regular gentle inversions (20

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times over the first minute, then twice at 1.5, 2, 3, 4, 5, 7.5, 10, 15, 25 min). The sample was

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cooled to room temperature in cool water, and centrifuged at 8000 g for 20 min. The

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supernatant was removed for determining soluble carbohydrate with anthrone-H2SO4 method.

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The swelling power was determined by measuring the amount of original precipitate from the

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centrifugation and calculating the amount of water absorbed by the starch (percent weight

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increase) after subtraction of the amount of soluble carbohydrate. The water solubility was

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obtained by calculating the amount of soluble carbohydrate by the starch. The experiments

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were performed in triplicate.

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2.13. Statistical analysis

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One-way analysis of variance (ANOVA) by Tukey’s test and Pearson correlation analysis

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were evaluated using the SPSS 16.0 Statistical Software Program.

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3. Results and discussion

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3.1. Morphological structure of starch

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Isolated starch was observed using scanning electron microscope (Fig. 1). Most of

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normal maize starch granules were large polygonal (Fig. 1A), which was in agreement with

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the previous report (Wang et al., 1993). However, these large polygonal starch granules were 11

ACCEPTED MANUSCRIPT seldom observed in high-amylose maize inbred lines. They became more spherical. Some

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small spherical granules and elongated granules appeared in inbred lines, especially for Zae49

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and Zae50 (Fig. 1B-1E, 1H). The high-amylose hybrid maize starches had large polygonal

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granules, small spherical granules, and short rod granules (Fig. 1F, 1G). Many literatures

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report that high-amylose crop starches contain different morphology granules. The elongated,

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internal hollow, and aggregate starch granules are observed in high-amylose maize (Jiang et

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al., 2010), wheat (Regina et al., 2006; Slade et al., 2012), and rice (Kim et al., 2005; Wei, Qin,

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Zhu et al., 2010), respectively. The proportion of elongated starch granule increases with

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increasing amylose content in high-amylose maize (Jiang et al., 2010).

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The laser diffraction instrument for granule size measurement can provide the derived

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outputs of a volume distribution, distribution information and standard mean diameter by

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assuming spherical particles. The volume distribution indicated that starch granules were

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unimodal size distributions in normal maize and high-amylose maize inbred and hybrid lines

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(data not shown). The granule size of starch was listed in Table 1. The starch granule from

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normal maize was significantly larger than those from high-amylose maize inbred and hybrid

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lines. Among the high-amylose maize lines, except the Zae28, the inbred lines had

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significantly smaller starch granule size than the hybrid lines. The starch from Zae49 had the

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smallest size among the maize starches. Many literatures report that the granule size of starch

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becomes small in high-amylose crops (Karlsson, Leeman, Björck, & Eliasson, 2007; Wang et

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al., 1993).

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3.2. Molecular structure of starch

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The absorption spectra of iodine-starch complexes were clearly different among normal

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ACCEPTED MANUSCRIPT maize and high-amylose maize inbred and hybrid lines (data not shown). The maximum

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absorption wavelength, iodine blue value, and apparent amylose content of starch by

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determining the absorption spectra of iodine-starch complexes are summarized in Table 2.

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The apparent amylose content of high-amylose maize starches ranged from 57.4% to 76.4%,

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which was significantly higher than that of the normal maize starch (32.1%). Among

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high-amylose maize lines, starches from hybrid lines had lower apparent amylose content

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than those from inbred lines. The higher apparent amylose contents of the high-amylose

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maize starches were illustrated by the higher iodine blue values. In contrast, the maximum

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absorption wavelength of the high-amylose maize starch was significantly lower than that of

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the normal maize starch. This result agreed with the high-amylose wheat starches isolated

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from Australian wheat cultivars as previously reported by Hung, Maeda, Miskelly, Tsumori,

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and Morita (2008).

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The molecular weight distribution of isoamylase-debranched starch is shown in Fig. 2.

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Clear three peaks were observed in gel permeation chromatography profiles. The peaks 1 and

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2 consist of short (A and short B chains) and long (long B chains) branch-chains of

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amylopectin, respectively, and peak 3 includes amylose (Song & Jane, 2000; Wang et al.,

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1993). The starches from normal maize and high-amylose maize inbred and hybrid lines had

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significantly different gel permeation chromatography profiles, their parameters were listed in

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Table 2. Starch from normal maize had the highest short branch-chain content and the lowest

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long branch-chain content of amylopectin, and starches from maize inbred lines Zae49 and

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Zae50 had the lowest short branch-chain content and the highest long branch-chain content of

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amylopectin. The starches from maize hybrid lines had higher short branch-chain and lower

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peak 1 to peak 2 might be used as an index of the extent of branching of amylopectin; the

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higher the ratio, the higher the branching degree (Wang et al., 1993). The branching degree is

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negatively relative with the average chain length of amylopectin (Song & Jane, 2000; Wang et

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al., 1993). In this study, normal maize starch had the highest branching degree, and Zae49 and

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Zae50 starches had the lowest branching degree, indicating that the average chain length of

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amylopectin was the shortest in normal maize starch, and the longest in Zae49 and Zae50

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starches. The starches from maize hybrid lines had higher branching degree of amylopectin

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than starches from maize inbred lines. The starch with high amylose content has been

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reported to have low branching degree of amylopectin in high-amylose potato and maize

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(Hofvander, Andersson, Larsson, & Larsson, 2004; Wang et al., 1993).

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The amylose content determined by gel permeation chromatography was the lowest in

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normal maize starch, and the highest in Zae49 and Zae50 starches. The amylose content was

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significantly lower than apparent amylose content determined by iodine colorimetry. The

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apparent amylose content overestimates the amylose content because the long branch-chain of

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amylopectin can also bind iodine (Shi et al., 1998). The difference between amylose content

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and apparent amylose content was markedly significant in high-amylose maize starches,

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especially for Zae49 and Zae50, indicating that high-amylose starches had more long

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branch-chains of amylopectin than normal starch. The difference between amylose and

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apparent amylose content also showed that starch from maize hybrid line had lower long

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branch-chains of amylopectin than starch from maize inbred line. These results were in

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accordance with the branching degree of amylopectin.

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3.3. Crystalline structure of starch The X-ray diffraction pattern of starch is shown in Fig. 3. Normal maize starch had

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typical A-type X-ray diffraction pattern with strong reflection at about 15° and 23° 2θ, and an

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unresolved doublet at 17° and 18° 2θ, and was similar to that of normal cereal starches

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(Cheetham & Tao, 1998). High-amylose maize starches showed B-type X-ray diffraction

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pattern with reflections at about 5.6°, 15°, 17°, 20°, 22° and 24° 2θ. Many high-amylose

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cereal starches were reported to have B-type crystallinity (Jiang et al., 2010; Li et al., 2008). It

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is noteworthy that the changes of peak intensity at 5.6°, 20°, 22° and 24° 2θ. The peak of 20°

309

2θ is a typical amylose−lipid complex diffraction peak, and its intensity is positively relative

310

with amylose content (Cheetham & Tao, 1998). In the present study, the change of peak

311

intensity at 20° 2θ was in agreement with amylose content of starch. The peak at 5.6° 2θ is the

312

characteristic of B-type crystallinity. The C-type starch, which is the mixture of A- and B-type

313

allomorph, shows different X-ray diffraction patterns according to the proportion of A- and

314

B-type allomorph. When the content of B-type allomorph is significantly higher than that of

315

A-type allomorph, the C-type starch shows a CB-type (closer to B-type) X-ray diffraction

316

pattern with weak reflection at about 5.6°, 22° and 24° 2θ (Cheetham & Tao, 1998). In the

317

present study, the X-ray diffraction patterns of two hybrid maize starches showed weak

318

reflections at about 5.6°, 22° and 24° 2θ, indicating that A-type allomorph might exist in

319

hybrid maize starches. Cheetham and Tao (1998) thought that the crystalline type of maize

320

starch could change from A- to B- via C-type when amylose content increased, and the maize

321

starch with about 40% apparent amylose content had C-type crystallinity. In the present study,

322

the starches from two maize hybrids had 57.4% and 59.9% apparent amylose content, and

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showed CB-type crystallinity. The relative crystallinity of starch is listed in Table 3. Normal maize starch had the

325

highest relative crystallinity, and high-amylose Zae49 and Zae50 starch had the lowest

326

relative crystallinity. High-amylose hybrid maize starch had lower relative crystallinity than

327

normal maize starch and higher relative crystallinity than high-amylose inbred maize starch.

328

These results indicated that the relative crystallinity was negatively relative with the amylose

329

content (Cheetham & Tao, 1998).

330

3.4. Short-range ordered structure of starch

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The attenuated total reflectance-Fourier transforms infrared spectrum of starch is shown

332

in Fig. 4. Normal maize starch had significantly different attenuated total reflectance-Fourier

333

transforms infrared spectrum from high-amylose maize starches. Though high-amylose maize

334

starches had similar attenuated total reflectance-Fourier transforms infrared spectra, the ratios

335

of 1045/1022 and 1022/995 cm−1 were significantly different among them (Table 3). The

336

attenuated total reflectance-Fourier transforms infrared spectrum of starch is sensitive to the

337

short-range ordered structure in the external region of starch granule (Sevenou, Hill, Farhat, &

338

Mitchell, 2002). The ratio of absorbance 1045/1022 cm−1 is used to measure the amount of the

339

ordered starch to the amorphous starch at the starch granular surface and quantify the degree

340

of order, and that of 1022/995 cm−1 can be used as a measure of the proportion of amorphous

341

to ordered carbohydrate structure in the starch (Sevenou et al., 2002). The different ratios of

342

1045/1022 and 1022/995 cm-1 indicated that these starches had significantly different

343

short-range ordered structure in the external region of starch granule. High-amylose starch

344

showed higher degree of organization at a short-range scale on the edge of starch granule than

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ACCEPTED MANUSCRIPT normal starch, and hybrid maize starch had lower degree of order than inbred maize starch.

346

Sevenou et al. (2002) reported that potato and amylomaize starches with B-type crystallinity

347

had higher values for the ratio 1045/1022 cm−1 and the lower values for the ratio 1022/995

348

cm−1 than normal wheat and maize starches with A-type crystallinity. The present results

349

confirmed that a high degree of organization at a short-range scale existed on the edge of

350

high-amylose maize starches with B-type crystallinity, even though they had low relative

351

crystallinity.

352

3.5. Helix structure of starch The solid-state

13

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C CP/MAS NMR patterns for starches are shown in Fig. 5. The

354

resonances at different ppm were assigned according to data reported in the literature

355

(Atichokudomchai et al., 2004; Tan et al., 2007). The use of

356

structure provides information on the molecular organization at short distance scales (Tan et

357

al., 2007). The C1 resonance contains information both on the crystalline nature as well as the

358

noncrystalline (but rigid) chains. The multiplicity of the C1 resonance corresponds to the

359

packing type of the starch granules. A-type starch shows a triplet at the C1 region, while

360

B-type starch shows a doublet (Atichokudomchai et al., 2004). In this study, amorphous starch

361

had one peak at 103 ppm, which arises from the amorphous domain for C1 (Atichokudomchai

362

et al., 2004). Normal maize starch showed triplets at about 99.5, 100.5, and 101.5 ppm and a

363

shoulder peak at 103 pm, and high-amylose maize starches had an inconspicuous doublet at

364

about 100.0 and 101.0 ppm and a strong shoulder peak at 103 ppm (Fig. 5), indicating that

365

normal maize starch was A-type crystallinity and high-amylose maize starch was B-type

366

crystallinity. The intensity of peak at 103 ppm is positively relative with amylose content (Tan

C CP/MAS NMR in starch

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et al., 2007). The intensity variation of peak at 103 ppm showed that normal maize starch had

368

the low amylose content and inbred maize starch the high amylose content.

369

Tan et al. (2007) proposed a method for analyzing the

13

C CP/MAS NMR spectra of

starch samples to estimate the relative proportions of amorphous, single helix, and double

371

helix components in starch. In this analysis, starch spectra were separated into amorphous and

372

ordered subspectra, using intensity at 84 ppm as a reference point. The ordered subspectra

373

were peak-fitted. A combination (50/50) of Lorentzian and Gaussian profiles gave an

374

acceptable fit (data not shown). The proportions of single helix, double helix, and amorphous

375

components are listed in Table 3, and showed difference among normal maize and

376

high-amylose inbred and hybrid maize lines. The normal maize starch had high double helix

377

content and low amorphous component, and inbred maize starch had low double helix content

378

and high amorphous component. The double helices in the short- and long-range distance can

379

both be detected by 13C CP/MAS NMR, but only the double helices in the long-range distance

380

can be detected by X-ray diffraction (Atichokudomchai et al., 2004). The higher value for

381

double helix content compared with X-ray crystallinity suggested that not all double helix

382

chain segments were within crystalline arrays (Tan et al., 2007).

383

3.6. Lamellar structure of starch

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384

The lamellar structure of starch granule can be analyzed using small-angle X-ray

385

scattering spectra. The small-angle X-ray scattering spectra of starches are shown in Fig. 6,

386

their parameters are listed in Table 4. The scattering peak position and Bragg spacing were

387

similar in the starches from high-amylose inbred lines and their hybrids, but significantly

388

different among the starches from normal maize, high-amylose maize inbred line, and

18

ACCEPTED MANUSCRIPT high-amylose maize hybrid. The peak intensity also showed significant difference among

390

maize starches. Normal maize starch had the highest peak intensity and peak full width at half

391

maximum, and high-amylose inbred maize starch showed the lowest peak intensity and peak

392

full width at half maximum. Yuryev et al. (2004) reported that an increase in amylose content

393

in wheat starch was accompanied by the decreasing peak scattering intensity. The scattering

394

peak position is thought to arise from the periodic arrangement of alternating crystalline and

395

amorphous lamellae of amylopectin, and corresponds to the lamellar repeat distance or Bragg

396

spacing. The location of the peak depends on the size of lamella, and may differ among

397

starches from different plants (Blazek & Gilbert, 2011). The scattering peak intensity results

398

from the electron density difference between crystalline and amorphous regions of the

399

lamellae (Blazek & Gilbert, 2011). In this study, the low peak intensity of high-amylose starch

400

might be caused by several factors: co-crystallization of amylose macromolecules with

401

amylopectin side-chains within crystalline lamellae, accumulation of amylose chains oriented

402

transverse to the lamellar stack within amorphous lamellae, and accumulation of amylose

403

tie-chains both inside crystalline and amorphous lamellae (Blazek & Gilbert, 2011; Yuryev et

404

al., 2004).

405

3.7. Thermal property of starch

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406

Thermal parameters of starch are presented in Table 5. The high-amylose maize starches

407

showed higher gelatinization temperatures, wider gelatinization temperature range, and lower

408

gelatinization enthalpy than normal maize starch. Among high-amylose maize lines, starches

409

from hybrid lines had lower gelatinization peak temperature than those from inbred lines. The

410

present results agreed with the previous reports that the amylose double helices and B-type

19

ACCEPTED MANUSCRIPT crystallinity in high-amylose starch required high temperature and energy to disorder and the

412

gelatinization enthalpy decreased with increasing amylose content (Matveev et al., 2001;

413

Richardson, Jeffcoat, & Shi, 2000).

414

3.8. Swelling power and water solubility of starch

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The swelling power and water solubility of starch at 95 °C are presented in Table 5. The

416

high-amylose maize starches showed lower swelling power and water solubility than normal

417

maize starch. Among high-amylose maize lines, starches from hybrid lines had higher

418

swelling power than those from inbred lines. Swelling power and water solubility provide

419

measures of the magnitude of interaction between starch chains within the amorphous and

420

crystalline domains. The extent of this interaction is influenced by the contents and

421

characteristics of amylose and amylopectin (Kaur, Singh, McCarthy, & Singh, 2007).

422

Amylose restrains swelling and maintains the integrity of swollen granules, and the

423

lipid-complexed amylose chains restrict both granular swelling and amylose leaching (Tester

424

& Morrison, 1992). The different molecular weight distribution of high-amylose starches

425

from maize hybrid and inbred lines resulted in their different swelling powers and water

426

solubilities.

427

3.9. Correlation between different structural properties of starch

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428

Pearson correlation coefficients for the relationships between different structural

429

properties are presented in Table 6. These structural properties had significant correlation.

430

The iodine blue value, apparent amylose content, long branch-chain of amylopectin, amylose

431

content, IR ratio of 1045/1022 cm−1, amorphous starch content, and the scattering peak

432

position of small-angle X-ray scattering had significantly positive correlations. They were

20

ACCEPTED MANUSCRIPT significant negatively correlated with the granule size, short branch-chain of amylopectin, the

434

ratio of short and long branch-chain of amylopectin, relative crystallinity, IR ratio of

435

1022/995 cm−1, double helix content, and the Bragg spacing, scattering peak intensity and

436

peak full width at half maximum of small-angle X-ray scattering, which had significantly

437

positive correlations. Hofvander et al. (2004) reported that there was a significantly positive

438

correlation between branching degree of amylopectin and granule size in high-amylose potato.

439

The amylose content was negatively correlative with branching degree of amylopectin and

440

granule size (Hofvander et al., 2004; Wang et al., 1993). The amylose content and IR ratio of

441

1045/1022 cm−1 were significant positively correlated with gelatinization temperature and

442

negatively correlated with gelatinization enthalpy, swelling power and water solubility. The

443

amylopectin short branch-chain, relative crystallinity and IR ratio of 1022/995 cm−1 were

444

significant negatively correlated with gelatinization temperature and positively correlated with

445

gelatinization enthalpy, swelling power and water solubility. Similar correlations between

446

molecular structure and thermal property and swelling power have been reported in

447

high-amylose rice and maize starches (Huang et al., 2015).

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Two major hypotheses have been proposed regarding the underlying heterosis. The

449

dominance hypothesis attributes heterosis to the accumulation of favorable dominant genes or

450

masking of deleterious recessives in the hybrid. The overdominance hypothesis argues that

451

the heterozygous combination of the alleles at a single locus is superior to either of the

452

homozygous combinations (Tollenaar et al., 2004). The high-amylose crop mutant lines have

453

low starch content and weight in grain (Slade et al., 2012; Regina et al., 2006), which is

454

disadvantageous characters for plants. The present results showed that the high-amylose

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ACCEPTED MANUSCRIPT maize hybrid lines had lower amylose content than their parental inbred lines, but their grains

456

had higher starch content and kernel weight than inbred lines, which was the important

457

character of hybrid. The results agreed with that the grain yield of hybrid line often exceeded

458

that of inbred line (Tollenaar et al., 2004). The high-amylose starches from maize hybrid lines

459

had higher content of amylopectin short branch-chain and lower contents of amylopectin long

460

branch-chain and amylose than the starches from maize inbred lines. The different molecular

461

weight distributions of starches resulted in the different physicochemical properties between

462

maize inbred and hybrid lines. Although the maize hybrid lines had lower amylose content

463

than the inbred lines, they had higher amylose content than normal maize and higher starch

464

content and weight in grain than the inbred lines. Therefore, it was very useful and important

465

to breed high-amylose maize hybrid materials for the point of amylose content and weight in

466

grains.

467

4. Conclusion

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The morphological, molecular, crystal, short- and long-range ordered, helical, and

469

lamellar structures of starch were investigated and compared between high-amylose maize

470

inbred and hybrid lines. Starch granule became smaller in size and more spherical, oval or

471

elongated in shape with increasing amylose content. Inbred maize starch had B-type

472

crystallinity and hybrid maize starch had CB-type crystallinity. Hybrid maize starch had lower

473

amylose content, long branch-chain of amylopectin, degree of order at a short-range scale on

474

the edge of starch granule, amorphous starch, and gelatinization temperature, but higher short

475

branch-chain of amylopectin, branching degree of amylopectin, relative crystallinity, double

476

helix content, lamellar peak intensity, gelatinization enthalpy, and swelling power than inbred

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ACCEPTED MANUSCRIPT maize starch. The above results could provide the structural information for utilizing starches

478

from high-amylose maize hybrids in food and nonfood industries.

479

Acknowledgments

480

We are grateful to Prof. Qiaoquan Liu (Yangzhou University, China) for kindly providing the

481

apparatus and technical assistance in GPC analysis. This study was financially supported by

482

grants from the National Natural Science Foundation of China (31270221), the Talent Project

483

of Yangzhou University, the Innovation Program for Graduates of Jiangsu Province, and the

484

Priority Academic Program Development of Jiangsu Higher Education Institutions.

485

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611

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612

27

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Tables and figures

614

Table 1 Diameter of starch granule.

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d(0.1) (µm)

d(0.5) (µm)

d(0.9) (µm)

D(3,2) (µm)

D(4,3) (µm)

Xianyu 335

8.774±0.002 e

14.935±0.004 f

22.103±0.005 g

7.605±0.002 e

14.924±0.004 g

Zae28

6.122±0.002 c

12.525±0.001 e

21.133±0.002 f

7.210±0.002 d

13.013±0.001 f

Zae35

5.672±0.002 b

11.101±0.005 c

18.549±0.009 c

6.828±0.002 c

11.536±0.005 c

Zae49

4.574±0.002 a

8.860±0.004 a

13.920±0.008 a

5.288±0.001 a

8.968±0.004 a

Zae50

4.678±0.002 a

9.193±0.004 b

15.281±0.008 b

5.790±0.002 b

9.526±0.005 b

Zae35×Zae28

6.556±0.001 d

12.579±0.002 e

19.768±0.005 e

7.147±0.089 d

12.740±0.005 e

Zae49×Zae50

6.280±0.195 c

19.221±0.240 d

6.882±0.033 c

12.327±0.002 d

EP

TE D

M AN U

SC

Starch

AC C

12.153±0.070 d

615

Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (p<0.05).

616

The d(0.1), d(0.5), and d(0.9) are the granule sizes at which 10, 50, and 90% of all the granules by volume are smaller, respectively; D(3,2) is the

617

surface area weighted mean diameter; D(4,3) is the volume weight mean diameter.

618 28

ACCEPTED MANUSCRIPT

619

Table 2 Molecular structure of starch. λmax (nm)

BV

AAC (%) SBC (%)

621

RSL

LBC (%)

AC (%)

18.9±0.5 a

28.4±0.8 a

2.79±0.06 d

609.3±0.6 a

0.354±0.006 a

32.1±0.6 a

52.7±0.3 e

Zae28

605.0±1.0 b

0.584±0.013 b

61.5±0.3 d

30.6±0.3 c

32.9±0.4 b

36.6±0.1 b

0.93±0.02 c

Zae35

605.0±1.0 b

0.625±0.008 c

65.5±0.7 e

26.2±0.6 b

32.3±0.2 b

41.5±0.5 c

0.81±0.02 b

Zae49

606.7±0.6 b

0.718±0.003 d

76.4±0.5 f

19.4±0.1 a

34.1±0.4 c

46.5±0.5 d

0.57±0.01 a

Zae50

606.3±0.6 b

0.705±0.008 d

75.3±0.5 f

18.6±0.2 a

35.2±0.2 c

46.2±0.1 d

0.53±0.01 a

Zae35×Zae28

604.7±0.6 b

0.564±0.002 b

57.4±0.6 b

32.2±0.1 d

31.6±0.5 b

36.2±0.5 b

1.02±0.02 c

Zae49×Zae50

605.0±1.0 b

0.579±0.011 b

59.9±0.4 c

31.9±0.8 cd

31.8±0.0 b

36.4±0.8 b

1.00±0.02 c

EP

TE D

M AN U

SC

Xianyu 335

Data are means ± standard deviations, n = 3 for λmax, BV and AAC, n = 2 for SBC, LBC, AC and RSL. Values in the same column with different

AC C

620

RI PT

Molecular weight distribution Starch

letters are significantly different (p<0.05).

622

λmax, maximum absorption wavelength of starch-iodine complex; BV, iodine blue value of starch-iodine complex at 680 nm; AAC, apparent

623

amylose content; SBC, short branch-chain of amylopectin; LBC, long branch-chain of amylopectin; AC, amylose content, RSL, ratio of SBC

624

to LBC.

625 29

ACCEPTED MANUSCRIPT

Table 3 Relative crystallinity, IR ratio, and relative proportions of single helix, double helix, and amorphous components of starch. IR ratio Starch

628

Relative proportion (%)

1045/1022 cm−1

1022/995 cm−1

Single helix

Double helix

Amorphous component

5.3±0.1 a

41.9±1.1 d

52.8±1.3 a

26.9±0.6 d

0.592±0.003 a

0.882±0.007 e

Zae28

23.1±0.9 c

0.707±0.009 b

0.711±0.011 d

5.8±0.1 a

31.8±0.7 ab

62.5±0.9 d

Zae35

21.3±0.6 b

0.717±0.002 c

0.669±0.011 c

7.6±0.2 c

33.4±0.7 bc

59.1±0.9 bc

Zae49

18.4±0.7 a

0.782±0.003 e

0.576±0.006 b

9.0±0.3 d

30.4±0.9 a

60.6±1.2 cd

Zae50

18.8±0.8 a

0.823±0.005 f

0.538±0.008 a

6.9±0.2 b

31.8±0.8 ab

61.3±1.0 cd

Zae35×Zae28

23.6±0.5 c

0.711±0.000 bc

0.694±0.008 d

10.7±0.3 e

32.0±0.8 ab

57.3±1.1 b

Zae49×Zae50

23.3±0.6 c

0.742±0.002 d

0.659±0.004 c

5.6±0.2 a

34.9±0.9 c

59.5±1.1 bc

EP

TE D

M AN U

SC

Xianyu 335

Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (p<0.05).

AC C

627

Relative crystallinity (%)

RI PT

626

30

ACCEPTED MANUSCRIPT

Table 4 Small-angle X-ray scattering parameters of starch. Imax (counts)

Smax (Å−1)

∆S (Å−1)

D (nm)

Xianyu 335

204.17±3.48 f

0.062±0.000 a

0.019±0.000 c

10.1±0.0 c

Zae28

40.41±0.01 c

0.067±0.000 c

0.014±0.001 a

9.4±0.0 a

Zae35

29.89±0.02 b

0.067±0.000 c

0.014±0.001 a

9.4±0.0 a

Zae49

20.70±0.93 a

0.068±0.000 c

0.014±0.001 a

9.2±0.0 a

Zae50

23.16±0.70 ab

0.067±0.001 c

0.015±0.000 a

9.4±0.1 a

Zae35×Zae28

61.10±4.18 d

0.064±0.001 b

0.017±0.000 b

9.9±0.1 b

Zae49×Zae50

81.80±2.79 e

0.017±0.000 b

9.7±0.1 b

M AN U

SC

RI PT

Starch

TE D

629

0.065±0.001 b

Data are means ± standard deviations, n = 2. Values in the same column with different letters are significantly different (p<0.05).

631

Imax, peak intensity; Smax, peak position; ∆S, peak full width at half maximum; D, Bragg spacing (2π/Smax).

AC C

632

EP

630

31

ACCEPTED MANUSCRIPT

Table 5 Thermal property, swelling power and water solubility of starch. Thermal parameter Starch ∆T (°C)

To (°C)

Tp (°C)

Tc (°C)

Xianyu 335

60.6±0.1a

67.8±0.1a

73.0±0.1a

12.4±0.2a

Zae28

64.3±0.4bc

74.5±0.7c

82.8±0.6b

Zae35

62.6±0.4ab

76.7±1.1d

Zae49

66.9±0.8d

Zae50

RI PT

633

SP (g/g)

WS (%)

∆H (J/g)

21.9±0.1d

20.3±0.7e

18.5±0.8b

9.5±0.1c

9.4±0.5c

20.7±0.3e

84.4±1.1b

21.9±0.8c

7.3±0.2b

8.2±0.3b

16.0±0.7d

85.1±1.1f

89.8±0.2c

22.8±0.6c

4.6±0.5a

6.2±0.2a

13.1±0.2b

64.9±1.1cd

79.2±1.7e

84.0±1.1b

19.1±0.4b

4.6±0.4a

5.6±0.3a

10.5±0.7a

Zae35×Zae28

65.0±1.9cd

70.6±0.1b

82.6±1.4b

17.7±0.9b

8.3±0.5b

9.8±0.2c

15.8±0.4cd

Zae49×Zae50

65.9±0.4cd

71.6±0.2b

84.7±0.9b

18.8±1.1b

7.7±0.5b

11.2±0.1e

14.8±0.3c

EP

TE D

M AN U

SC

13.5±0.6d

Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (p<0.05).

635

To, gelatinization onset temperature; Tp, gelatinization peak temperature; Tc, gelatinization conclusion temperature; ∆T, gelatinization

636

AC C

634

temperature range (Tc-To); ∆H, gelatinization enthalpy; SP, swelling power at 95 °C; WS, water solubility at 95 °C.

637

32

ACCEPTED MANUSCRIPT

Table 6 Pearson correlation coefficients between different structural properties of starch. d(0.5)

639 640 641

AAC

SBC

LBC

AC

RSL

RC

IR1

IR2

−0.948**

0.999**

0.953** −0.998** −0.999** 0.946**

0.957** −0.952**

−0.994**

0.967**

0.958** −0.965**

−0.962** −0.972**

0.997** −0.964** −0.953** −0.928**

0.954**

−0.375

0.408

0.782*

0.966** −0.996** −0.868* 0.959** −0.829*

0.948** −0.953**

0.954** −0.975** −0.970** 0.425

−0.909** −0.923**

0.839*

0.909**

−0.998**

0.856*

0.392

Am

0.392

0.928** −0.425

0.911** −0.957** −0.803*

−0.371

0.954**

0.312

0.799*

−0.823*

0.843*

−0.559

0.848*

−0.844*

0.910**

0.724

−0.886** −0.732

0.799*

−0.788*

0.054

−0.858*

−0.818*

0.858*

0.857*

−0.854*

0.791*

0.843*

−0.808*

0.708

−0.744

0.148

−0.786*

0.856*

0.820*

−0.864*

−0.863*

0.861*

−0.803*

−0.845*

−0.148

0.794*

−0.865*

0.835*

−0.948** −0.961**

0.957** −0.976** −0.869*

0.776*

−0.865*

0.875** −0.859*

−0.934

**

0.771 0.870

*

0.767 0.855

*

−0.741

∆S

−0.823* 0.645

0.819*

0.850*

−0.717

0.752

0.983**

0.855*

−0.855*

0.881** −0.448

0.965** −0.885** −0.863*

0.872*

0.870*

0.813*

−0.714

0.749

−0.244

0.851*

0.966**

0.419

*

−0.783

*

**

−0.725

−0.666

0.795 0.763

*

−0.789

*

−0.804

*

−0.791

−0.874*

−0.962**

0.693

0.524

−0.531 **

−0.694

−0.483

−0.753

−0.790*

0.689

0.928

0.320

−0.713

0.660

0.889

0.931**

−0.911**

0.902**

0.850*

−0.924** −0.852*

0.843*

−0.878**

0.459

−0.890**

0.788*

0.813*

−0.818*

−0.846*

0.903**

0.907**

−0.894**

0.849*

0.864*

−0.887** −0.853*

0.758*

−0.813*

0.422

−0.831*

0.740

0.884**

−0.887** −0.899** −0.881**

−0.975** −0.969**

0.970**

0.989**

−0.443

0.830*

−0.726

0.911**

0.961**

−0.970**

−0.883

**

0.934**

0.930**

−0.888** −0.965**

−0.944

**

*

−0.000**

−0.858* 0.973**

−0.853

0.789 *

*

−0.849*

TE D

−0.691

*

Imax

−0.368

0.842*

−0.874*

D

0.945** −0.994**

−0.709

*

Smax

0.918** −0.912** −0.918**

0.973** −0.917** −0.947** −0.409

M AN U

0.849*

DH

SC

−0.821*

SH

RI PT

−0.960**

EP

BV AAC SBC LBC AC RSL RC IR1 IR2 SH DH Am Smax D Imax ∆S To Tp Tc ∆T ∆H SP WS

BV

AC C

638

−0.895** −0.793*

−0.747

0.753

0.870*

0.742

0.886** −0.976** −0.985**

0.983** −0.984** −0.908**

0.991**

0.897** −0.918**

0.938** −0.465

0.959** −0.867*

−0.830*

0.839*

0.989**

0.890**

0.840*

0.763*

0.663

0.805*

0.870*

0.529

−0.396

0.403

0.577

0.387

−0.765*

−0.751

−0.636

−0.813*

−0.867*

−0.386

−0.397

* and ** indicate the significant difference at p<0.05 and p<0.01 level, respectively (n=7). The abbreviations of d(0.5), BV, AAC, SBC, LBC, AC, RSL, Smax, D, Imax, ∆S, To, Tp, Tc, ∆T, ∆H, SP and WS are shown in Table 1, 2, 4 and 5. RC, relative crystallinity; IR1: IR ratio of 1045/1022 cm−1; IR2: IR ratio of 1022/995 cm−1; SH, single helix; DH, double helix; Am, amorphous component. 33

ACCEPTED MANUSCRIPT 642

Figure captions

643

Fig. 1.

644

Zae35; D, Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50; H, Zae50. Scale bar = 10 µm.

645

Fig. 2.

646

Xianyu 335; B, Zae28; C, Zae35; D, Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50.

647

Fig. 3.

648

E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50.

649

Fig. 4.

650

Xianyu 335; B, Zae28; C, Zae35; D, Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50.

651

Fig. 5.

652

Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50; H, amorphous starch.

653

Fig. 6.

654

Zae35; D, Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50.

RI PT

SC

Gel permeation chromatography profiles of isoamylase-debranched starches. A,

M AN U

X-ray diffraction spectra of starches. A, Xianyu 335; B, Zae28; C, Zae35; D, Zae49;

Attenuated total reflectance-Fourier transforms infrared spectra of starches. A,

C CP/MAS NMR spectra of starches. A, Xianyu 335; B, Zae28; C, Zae35; D,

TE D

13

EP

Small-angle X-ray scattering patterns of starches. A, Xianyu 335; B, Zae28; C,

AC C

655

Scanning electron micrographs of starch granules. A, Xianyu 335; B, Zae28; C,

34

656

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

657

Fig. 1.

Scanning electron micrographs of starch granules. A, Xianyu 335; B, Zae28; C,

658

Zae35; D, Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50; H, Zae50. Scale bar = 10 µm.

659

35

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

660

Fig. 2.

662

Xianyu 335; B, Zae28; C, Zae35; D, Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50.

EP AC C

663

Gel permeation chromatography profiles of isoamylase-debranched starches. A,

TE D

661

36

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

664

Fig. 3.

666

E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50.

EP AC C

667

X-ray diffraction spectra of starches. A, Xianyu 335; B, Zae28; C, Zae35; D, Zae49;

TE D

665

37

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

668

Fig. 4.

670

Xianyu 335; B, Zae28; C, Zae35; D, Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50.

EP AC C

671

Attenuated total reflectance-Fourier transforms infrared spectra of starches. A,

TE D

669

38

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

672 13

Fig. 5.

674

Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50; H, amorphous starch.

EP AC C

675

C CP/MAS NMR spectra of starches. A, Xianyu 335; B, Zae28; C, Zae35; D,

TE D

673

39

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

676

Fig. 6.

Small-angle X-ray scattering patterns of starches. A, Xianyu 335; B, Zae28; C,

678

Zae35; D, Zae49; E, Zae50; F, Zae35×Zae28; G, Zae49×Zae50.

AC C

EP

TE D

677

40

ACCEPTED MANUSCRIPT Starch structure was compared between high-amylose maize inbred and hybrid lines.




Starch granules became smaller and more spherical with increasing amylose content.




Hybrid maize had lower amylose content and amorphous starch than inbred maize.




Hybrid maize had CB-type crystallinity and inbred maize had B-type crystallinity.




Hybrid maize had higher double helix and lamellar peak intensity than inbred maize.

AC C

EP

TE D

M AN U

SC

RI PT