An insight into starch slowly digestible features enhanced by microwave treatment

Journal Pre-proof An insight into starch slowly digestible features enhanced by microwave treatment Nannan Li, Lili Wang, Siming Zhao, Dongling Qiao, Caihua Jia, Meng Niu, Qinlu Lin, Binjia Zhang PII:

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DOI:

https://doi.org/10.1016/j.foodhyd.2020.105690

Reference:

FOOHYD 105690

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Food Hydrocolloids

Received Date: 9 November 2019 Revised Date:

19 January 2020

Accepted Date: 19 January 2020

Please cite this article as: Li, N., Wang, L., Zhao, S., Qiao, D., Jia, C., Niu, M., Lin, Q., Zhang, B., An insight into starch slowly digestible features enhanced by microwave treatment, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2020.105690. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.

An Insight into Starch Slowly Digestible Features Enhanced by Microwave Treatment

Nannan Li a,1, Lili Wanga,1, Siming Zhao a, Dongling Qiao b, Caihua Jia a, Meng Niu a, Qinlu Lin c, Binjia Zhang a*

a

Group for Cereals and Oils Processing, College of Food Science and Technology, Key Laboratory of

Environment Correlative Dietology (Ministry of Education), Huazhong Agricultural University, Wuhan 430070, China b

Glyn O. Phillips Hydrocolloid Research Centre at HBUT, School of Food and Biological Engineering,

Hubei University of Technology, Wuhan 430068, China c

National Engineering Laboratory for Rice and By-product Deep Processing, College of Food Science and

Engineering, Central South University of Forestry and Technology, Changsha 410004, China

1

These authors contributed equally to this work.

*

Corresponding author. Email address: [email protected] (B. Zhang).

1

An insight into starch slowly digestible features enhanced by

2

microwave treatment

3 4

Nannan Li a,1, Lili Wanga,1, Siming Zhao a, Dongling Qiao b, Caihua Jia a, Meng Niu a, Qinlu Lin c,

5

Binjia Zhang a*

6 7

a

8

Environment Correlative Dietology (Ministry of Education), Huazhong Agricultural University, Wuhan

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430070, China

Group for Cereals and Oils Processing, College of Food Science and Technology, Key Laboratory of

10

b

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Hubei University of Technology, Wuhan 430068, China

12

c

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Engineering, Central South University of Forestry and Technology, Changsha 410004, China

Glyn O. Phillips Hydrocolloid Research Centre at HBUT, School of Food and Biological Engineering,

National Engineering Laboratory for Rice and By-product Deep Processing, College of Food Science and

14

1

These authors contributed equally to this work.

*

Corresponding author. Email address: [email protected] (B. Zhang). 1

15

Abstract: The rice starch following microwave cooking with storage showed more slowly digestible

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starch and a lower digestion rate than did the conventionally treated counterpart. The underlying

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mechanism was interpreted by inspecting starch multi-level structural evolutions during digestion.

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Accompanying digestion, not only were starch matrices digested, leading to porous substrate and

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probably less polymorphs and nanoscale orders, but also starch chain reassembly occurred, causing A

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to B crystalline transform for untreated starch and formation of new molecular organization (repeat

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length: 5 nm) for treated starches. Hence, the digestion for native and treated starches was governed

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by concurrent matrix hydrolysis and molecular reassembly during digestion. The ultimate digestion

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of a specific structure was affected by the state of structural system. Unlike common views, the

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polymorphs in processed starches without native architecture were preferentially digested. Also,

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compared to conventionally treated counterpart, the microwave treated starch exhibited enhanced

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molecular reassembly during digestion, eventually displaying stronger slowly digestible features.

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Keywords: starch; microwave cooking; digestion feature; multi-scale structural evolution

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2

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1. Introduction Starch is naturally a major storage carbohydrate in green plants, and is consumed widely as an

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ingredient of foods supplying energy for humans. The digestion of food starch matter generates

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glucose component utilized by human body, and thus shows links to risks of diet-related diseases

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such as diabetes, obesity and cardiovascular disease (Robertson, Currie, Morgan, Jewell, & Frayn,

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2003). The diet starch contains resistant starch (RS), slowly digestible starch (SDS), and rapidly

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digestible starch (RDS) (Englyst, Kingman, & Cummings, 1992). The higher SDS/RS level and

36

lower digestion rate could slow the glucose release, decreasing food glycemic index and being

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beneficial to human health (Fuentes-Zaragoza, Riquelme-Navarrete, Sánchez-Zapata, &

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Pérez-Álvarez, 2010; Lehmann & Robin, 2007). Hence, to develop food products with improved

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health benefits, increasing efforts have been practiced to enhance slowly digestible features of starch

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ingredient and reduce its digestion rate.

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In fact, starch is a semicrystalline biopolymer having sophisticated multi-scale structures

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resulting from amylose and amylopectin chain assembly. The structures on multiple scales in native

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starch involve the single/double helices, the polymorphs (crystallites), the periodic semicrystalline

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lamellae, the growth rings, and the whole granule (Doutch & Gilbert, 2013; Luengwilai & Beckles,

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2009; Perez & Bertoft, 2010; Pikus, 2005). Typically, pristine starches are processed into usable

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forms for human consumption. While processed by specific methods such as cooking, the packed

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starch chains can be disassociated from the multi-level structures and are converted into mainly

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nonordered forms; then, during storage, these nonordered chains reassemble to generate a new

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multi-scale structural system. Research has revealed that the multi-scale structural characteristics,

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e.g., the penetrability of structures and polymorphic type, show influences on starch functions and 3

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properties such as digestion behaviors (Blazek & Gilbert, 2010b; Qiao, et al., 2017a). In this regard,

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disclosing the structure-digestibility links for starches following specific processing would allow an

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in-depth understanding of how this processing tailors starch digestion features.

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To regulate starch functions and properties including digestibility, series of processing methods

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such as conductive heating, extrusion, autoclaving, microwave heating, heat-moisture treatment and

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high pressures are used to alter starch structural characteristics (Dundar & Gocmen, 2013; Hasjim &

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Jane, 2009; Li, et al., 2019b; Linsberger-Martin, Lukasch, & Berghofer, 2012; Liu, Zhang, Chen, Li,

58

& Zheng, 2019). In particular, microwave heating shows a rapid heat generation rate and is

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recognized as an effective and efficient method for processing of food products (Chandrasekaran,

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Ramanathan, & Basak, 2013). It was reported that the heating by microwave exhibits influences on

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the ordered, nanoscale and morphological characteristics of starches and their practical properties

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involving gelatinization and enzyme hydrolysis (Fan, et al., 2013; Guo, et al., 2019; Li, et al., 2019a).

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For starch digestion, one hypothesis is that the bulk density of starch structure matrices governs their

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digestion rate by affecting the diffusion of enzymes and thus the absorption and catalysis.

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Consistently, the native starch has complicated structures containing densely packed glucan chains

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and thus an enzyme digestion rate constant several times lower than that of sufficiently processed

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starch by cooking (Bertoft & Manelius, 1992; Noda, et al., 2008). Also, the starches from mainly

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cereals exhibit numerous surface pores, enhancing enzyme penetrability toward starch substrate and

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accelerating starch digestion (Blazek, et al., 2010b). On the other hand, the liquid crystal nature of

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starch (Daniels & Donald, 2004; Qiao, et al., 2017b) may allow the occurrence of starch chain

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reassembly events in water-containing digestion medium, other than the concurrent hydrolysis of

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glucan matrices. Therefore, one can hypothesize that the digestion of not only native starch but also 4

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its treated form following a processing method is very likely to be governed by simultaneously

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occurred hydrolysis and chain reorganization events during digestion.

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Our results affirm that the microwave treatment could endowed the starch with enhanced slowly

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digestible characteristics (increased SDS and reduced digestion rate), relative to the treatment with

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conventional heating and storage. However, it is still unclear that how microwave cooking followed

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by storage regulates starch SDS content and digestion rate from above hypothesized view of

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concurrent hydrolysis and chain reassembly during digestion. Hence, a starch from indica rice, one

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important cereal consumed worldwide, was as the raw material for treatment of microwave or

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conventional heating with storage. Then, combined techniques were used to inspect the starch

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multi-scale structural evolutions during digestion; and the results clearly confirmed the present of

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concurrent matrix hydrolysis and chain reassembly as digestion proceeded. The two concurrent

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events were discussed to understand how microwave cooking with storage enhances the slowly

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digestible features of starch.

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2. Materials and methods

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2.1 Materials

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An indica rice was commerially supplied by Xiangyang Saiya Rice Co., Ltd. (Xiangyang, China).

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Two kinds of enzymes were used for the digestion of starch, including 10115 Aspergillus niger

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amyloglucosidase (activity: 64 unit/mg) and A3176 pancreatic α-amylase (activity: 25 unit/mg)

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supplied by Sigma-Aldrich. A kind of glucose Assay Kit was purchased from Shanghai Mind

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Bioengineering Co., Ltd. (Shanghai, China). The other chemical reagents were of analytical grade.

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2.2 Starch isolation 5

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To isolate indica rice starch (viz., IRS), approximately 1000 g of the rice was added in a beaker

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having 3000 g of water, which was stored under ambient conditions for three hours. Then, the soaked

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rice was treated using a GM-WZ150 colloid mill (Shishou, China) and centrifuged at 3000 g for 15

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min to acquire precipitate of milled rice, followed by drying at 40 °C for 24 h. Thereafter, 800 g of

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rice powder after drying was added into three times in weight of NaOH solution (0.2% w/v), and

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stored at room temperature (26 °C) for two hours; the slurry was centrifuged at 3000 g for 10 min.

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This process was repeated for another three times using the same NaOH solution and two times using

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distilled water, accompanied with neutralization by 0.1 mol/L HCl and washing using 95% ethanol.

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After centrifugation at 3000 g for 10 min, the precipitate was dried at 35 °C overnight to acquire the

104

dried starch before treatment. The amylose content for this starch was 14.56±0.39%, which as

105

measured based on an iodine colorimetric method (Ihwa Tan, 2007).

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2.3 Preparation of cooked starch followed by storage

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According to a recent method with modifications (Guo, et al., 2019), the starch slurry (50 g) at

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starch concentration of 20% was prepared and added into a triangle bottle. This bottle was placed

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into boiling water for 30 min to acquire conventionally cooked starch. The starch with microwave

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cooking was prepared using a microwave oven (MKX-J1A, Qingdao Microwave Creative

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Technology Co., Ltd., China) operated at 8 W per gram of starch slurry for 3 min. Then, the starches

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after cooking were cooled to 25 ± 2 °C, and stored at 4 °C for 72 h in a refrigerator. The starches

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following cooking and storage were dried in an oven at 40 °C overnight. The samples after drying

114

were ground to obtain the samples subjected to the treatment of conventional or microwave cooking

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followed by storage. In this article, sample codes such as “IRS-C-20” will emerge; “IRS” indicate the

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type of starch, “C” means the conventional cooking, and “20” shows the digestion time. Moreover, 6

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“IRS” represents the untreated starch.

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2.4 Scanning electron microscopy (SEM)

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A scanning electron microscope system (JSM-6390, NTC, Japan) was applied to inspect the

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microscopic features of the starches digested for different time periods. The equipment was operated

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at a voltage of 15 kV. To observe, each of the starches were mounted onto metal sample stages

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covered with carbon tapes, followed by coating with a thin gold layer using a coater.

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2.5 X-ray diffraction (XRD)

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Using a method reported recently (Miao, et al., 2018), the crystalline characteristics for the

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starches were evaluated through an inhouse X-ray diffractometer (JDX-10P3A, Tokyo, Japan). The

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diffractometer was equipped with Cu Kα X-ray source having wavelength of 0.1542 nm. For the

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starch samples, the XRD patterns at angle ranges (2θ) of 5–40° were recorded for analysis. A

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previously stablished method (Lopez-Rubio, Flanagan, Gilbert, & Gidley, 2008) was applied to

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generate starch relative crystallinity degree (Xc) by using the PeakFit software (Version 4.12).

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2.6 Small-angle X-ray scattering (SAXS)

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The starch SAXS measurements were performed on the BL19U2 BioSAXS beamline at the

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Shanghai Synchrotron Radiation Facility (Shanghai, China). For measurements, the starch slurries

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having 20% starch concentration were prepared and stored at ambient conditions for two hours. Then,

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the slurries were placed on the sample stages, and the scattering data of the starches were collected

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through a Pilatus 1M detector. The testing time for each starch was 10 s. Moreover, the sample stage

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with pure water was used as the background, and the sample data were background subtracted. The

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data of each sample was recorded at q values of ca. 0.01 to 0.20 Å−1. Here, q is the scattering vector

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equal to 4πsinθ/λ where λ is the wavelength of X-ray and 2θ is the scattering angle. 7

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2.7 In vitro digestion

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A recent method (Qiao, et al., 2019b) was used to acquire the starch digestion plots, digested

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starch amount against time. Briefly, 90 mg of starch was added into a tube having 6 mL of deionized

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water, and 10 mL of pH 6.0 sodium acetate buffer was added. The tube was incubated at 37 °C for 10

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min. Then, 5 mL of buffer solution containing 42 unit/mL amyloglucosidase and 42 unit/mL

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α-amylase was pipetted into the tube to be digested. A glucose oxidase/peroxidase reagent from

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Megazyme was applied to obtain the glucose content in the digestion medium. The glucose

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concentration of the standard solution was 1.0 mg/mL. In addition, the amounts for digested starch

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(RDS) within 20 min, digested starch (SDS) within 20-120 min, and undigested starch (RS) within

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120 min were calculated (Englyst, et al., 1992).

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2.8 First-order kinetics

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The typical digestion data collected in section 2.7 were further analyzed using first-order kinetic

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function (Eq. (1)) and its transformed function, i.e., the logarithm of slope (LOS) plot (Eq. (2))

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(Butterworth, Warren, Grassby, Patel, & Ellis, 2012; Edwards, Warren, Milligan, Butterworth, &

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Ellis, 2014). Inspecting the changes in the slope of LOS plots (ln(dCt/dt)) against time (t), one would

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acquire the number of digestion periods shown by different rate coefficients. Then, the accurate rate

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coefficient, k, at a starch digestion stage could be calculated via non-linear fitting based on Eq. (1).

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= ln

1−

= − × + ln

×

1 ×

2

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Here, Ct (%) indicates the digested starch amount at a given time t (min); C∞ (%) means the 8

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estimated percentage of starch digested while a digestion stage was finished; k (min−1) represents the

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starch digestion rate coefficient. In the present work, the LOS plots indicated the converted digestion

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data (ln[(Ci+2−Ci)/(ti+2− ti)]) versus ((ti+2+ ti)/2) except the last two points.

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2.9 Statistical analysis

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The results were expressed as means ± standard deviations. The statistical analysis was carried

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out using the version 20.0 IBM SPSS software (Chicago, IL, USA). A statistical difference at P <

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0.05 was considered to be significant.

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3. Results and discussion

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3.1 Digestion characteristics

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Fig. 1 presents the digestion plots (digested amount versus time) and the related LOS plots and

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fit curves for the starches. For the untreated starch (IRS), only one linear range could be observed for

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the LOS plots, indicative of a monophasic digestion manner obeying the first order kinetics

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(Butterworth, et al., 2012; Qiao, et al., 2019b). More exactly, the enzyme digestion of starch substrate

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is in fact a pseudo first order course, since the rate constant of starch hydrolysis could be varied by

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the concentration of enzymes presenting in the digestion system (Butterworth, et al., 2012). With

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cooking followed by storage, both of conventionally- and microwave-treated starch clearly exhibited

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two consecutive linear regions for the LOS plots with apparently different slopes. This result

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revealed a typical dual-stage digestion pattern for the two starches after cooking and storage.

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Similarly, earlier findings report that starch substrate can display multiple (dual or triple) stage

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hydrolysis manner during the enzyme digestion (Kim, Choi, Park, & Moon, 2017; Qiao, et al.,

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2017a). Note that the starch digestion rate constant derived the LOS plots is inherent inaccurate;

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therefore, a non-linear fitting method based on the first-order kinetic function was used to fit the 9

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original digestion plots, and the LOS results were only used to show the number of associated

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digestion stages (Guo, et al., 2019).

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Table 1 lists the digestion results for the starches before and after cooking followed by storage.

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Compared to the pristine starch, the conventional treatment made the digestion rate (k1) at first stage

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11 times higher. The microwave treated starch showed a less effective increase in the k1 value (about

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9 times higher than that for native starch), together with a similar digestion rate (k2) at the second

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phase relative to that for the conventionally processed starch. Thus, the substrate matrices within the

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multi-level structures of the microwave treated starch could be more resistant to the permeation and

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hydrolysis of enzymes. In addition, the starch after microwave treatment had more SDS than did the

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counterpart with the conventional treatment, followed by comparative less RDS and RS. These

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results clearly indicate that the microwave cooking with storage could slow the digestion, and more

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apparently enhance the RDS formation than did the conventional cooking and storage. Consistently,

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our recent findings confirmed the microwave-enhanced formation of SDS fractions (Li, et al., 2019a).

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The enhancement of SDS formation here was less effective, presumably due to the longer storage

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time and the higher enzyme concentration used in the present work.

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3.2 Morphologic evolutions during digestion

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To inspect the changes in micron scale morphological features during digestion, Fig. 2 presents

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the SEM graphs for the untreated and treated starches at different digestion time points. For the

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pristine starch before digestion, there were predominantly pentagonal granules with a radius close to

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5 µm as reflected by the scale bar and dominantly a dense surface. The digestion after 20 min led to

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multiple micropores on the granule surfaces, resulting from a notable amount of starch hydrolysis

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(approximately 3.43%) via an inside-out manner by enzymes (Blazek, et al., 2010b). No observed 10

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ratios of broken granules emerged and the sharp edges on the granule surface became less visible

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after this short time digestion by enzymes. A longer digestion time period (120 min) resulted in more

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prominently changes in the granule morphology. All the granules show much more apparent porous

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morphologic features on the granule surfaces with enlarged pore sizes and even channels into the

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granule interior. Moreover, this longer digestion time could lead to the emergence of several granule

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fragments attached onto the surfaces of relatively intact granules.

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The treated starches, regardless of the cooking manner, were irregular in shape with a compact

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surface. After the digestion of 20 min, the surface for these two treated starched became fragmented

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and rougher, due to the enzyme hydrolysis events of rapidly digestible fractions (26%-27% in

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content). Evidently larger holes could be seen for the treated starches than for the native counterpart.

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That is, the starches after cooking and storage should have structure matrices less robust than those

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for native state starch with sophisticated semicrystalline architecture. This has been shown by the

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more intact granules without apparent grooves on the surface. While being digested for a longer time

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(120 min), the two starches after the cooking followed by storage could be more intensely

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hydrolyzed by enzyme molecules, thus evidently broken starch substrates (into small fragments)

218

could be seen. No substantial differences in the morphologic evolutions could be observed for the

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two kinds of processed starches in the course of digestion.

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3.3 Crystalline structural evolutions during digestion

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XRD can be applied to clearly evaluate the changes in the starch polymorphic characteristics

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such as the type and amount of polymorphs. Fig. 3 displays the XRD curves for untreated and treated

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starches with different digestion times. The pristine starch in Fig. 3a showed characteristics from

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A-type polymorphs as reflected by a doublet at 2θ  of about 17° and 18°, with strong diffraction 11

225

signals at around 15° and 23° (Li, et al., 2017; Zhang, Li, Liu, Xie, & Chen, 2013). Unlike other

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cereal starches such as maize starch (Blazek, et al., 2010b), there were no notable V-type crystallites

227

assembled from single helices, as shown by undetectable V-type diffractions such as that at about

228

19.8°. Undergoing 20 min of enzyme digestion did not substantially alter the positions and intensities

229

of the diffraction peaks. This phenomenon could be also observed even after 120 min of digestion.

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That is, the digestion enzymes did not show preferential attack to the amorphous or crystalline

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regions, causing almost no visible changes to the related diffraction peaks. This agrees with an earlier

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finding that the enzymes led to the even hydrolysis of amorphous and crystalline materials in native

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cereal starches (Zhang, Ao, & Hamaker, 2006).

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In Fig. 3b and 3c, the two starch samples contained mainly B-type polymorphs identified by the

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typical crystalline diffraction peaks at 2θ of 15°, 17°, 22° and 24° (Qiao, et al., 2019a), as well as

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notable amounts of V-type polymorphs affirmed by the diffractions at around 19.8° (Tan, Flanagan,

237

Halley, Whittaker, & Gidley, 2007). Accounting for this, the chains of native starch assembled into

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its monoclinic crystal units were sufficiently disrupted by the hydrothermal effects during cooking;

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then, the non-ordered starch chains re-organized into helical components during storage to construct

240

B- (hexagonal crystal units) or V-type crystallites. However, much different from the case for

241

untreated starch, the digestion just after the short time (20 min) induced prominent reductions in the

242

diffraction intensities for not only B-crystallites but also V-crystallites. An increase in the digestion

243

time to 120 min further weakened the crystalline features, which was confirmed by a less-resolved

244

peak at 17° and a merge of three diffractions at 19.8°, 23° and 24°. The microwave-treated starch

245

showed similar evolutions in the diffraction pattern compared to the conventionally treated starch,

246

with slightly stronger diffractions for the former. 12

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Upon such results, we summarize that the crystalline regions (both B- and V-type polymorphs)

248

within the treated starch matrices were less resistant to the enzyme hydrolysis effects than the

249

counterparts packed within untreated starch having fantastic multi-scale structured matrices. The

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polymorphs in the treated starch could be classified as RDS (digested within 20 min), SDS (digested

251

between 20-120 min), and RS (residual fractions after 120 min). Also, the present findings deepen

252

the current understanding of V-type polymorphs (organized single helical components) that have

253

been extensively and commonly recognized as type-5 resistant starch fractions (Raigond, Ezekiel, &

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Raigond, 2015).

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3.4 Nano-structural evolutions during digestion

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The crystallites of starch can be packed into lamellar or nonlamellar regions, with amorphous

257

materials, to form starch nanoscale structures such as periodic semicrystalline lamellae. The

258

characteristics of such as nano-structures could be unambiguously inspected by analytical methods

259

such as SAXS and small angle neutron scattering (Blazek, et al., 2010b; Blazek & Gilbert, 2011; Li,

260

et al., 2019a; Zhang, et al., 2019a). To interrogate the changes in starch nano-structure following

261

enzyme digestion, Fig. 4 includes the SAXS plots for pristine and treated starches with different

262

digestion times. Typically, the starch without treatment possessed a scattering peak at q of around

263

0.071 Å-1, ascribed to the periodic amorphous-crystalline lamellar stacking of starch. The average

264

thickness (d), i.e., inter-lamellar repeat distance, of the semicrystalline lamellae was about 8.85 nm

265

as calculated using Woolf-Bragg equation, d = 2π/q (Zhang, et al., 2019b). In Fig. 4a, when the

266

digestion time reached 20 min, there were a drastic decrease in the scattering intensity of the whole

267

profile including the peak range. In the paracrystalline model for finite semi-crystalline lamellae in

268

an amorphous background (amorphous growth rings) (Cameron & Donald, 1992), the scattering 13

269

intensities of lamellar peak and low-q (below the position of peak maximum) ranges are governed by

270

related structural parameters, e.g., the electron density difference (∆ρ = ρc - ρa) between crystalline

271

(ρc) and amorphous (ρa) lamellae and that (∆ρu = ρu - ρa) between background materials (ρu) and

272

amorphous lamellae (ρa). A higher ∆ρ causes an overall increase in the intensity of scattering profile;

273

and a higher ∆ρu induces an increase the low q range but a reduction in the definition the lamellar

274

peak. Hence, the reduced overall scattering intensity including the peak and the lower q values (Fig.

275

4a) revealed reductions in ∆ρ and probably ∆ρu. To explain this, (i) the digestion with 20 min more

276

effectively loosened the assembly of helical components in the crystalline lamella space rather than

277

the aggregation of nonordered chains in the amorphous lamella space; (ii) the enzyme molecules

278

enter the background region first for granular starch (Blazek, et al., 2010b), and the amorphous

279

growth rings suffered greater hydrolysis effects than did the amorphous lamellar regions.

280

Again, for the untreated starch, the prolonged digestion time (120 min) made the overall

281

decrease in the scattering pattern less evident than did the shorter digestion period (20 min). This

282

revealed less prominently reduced ∆ρ and ∆ρu, related to enhanced digestions of amorphous starch

283

especially in the lamellae, since the enzyme molecules took sufficient time to enter and hydrolyze the

284

amorphous lamellar phases. More interestingly, the SAXS results in Fig. S1a (supplementary

285

material) showed a defined 100 inter-helix peak at about 0.39 Å-1 (B-polymorphs) for the untreated

286

starch after 120 min digestion. This is interpreted in terms of the reassembly of double helices

287

(non-crystalline or those from monoclinic units of A-polymorphs) into hexagonal units of

288

B-polymorphs. To be specific, the digestion with prolonged time gradually hydrolyzed the

289

amorphous background and lamellae, weakening their constraint on the crystalline and helical

290

regions. This encouraged the movement of helices and the subsequent realignment into B-type units 14

291

via encapsulation of increased water molecules (up to thirty-six) during starch digestion. Note that

292

the XRD curves of lyophilized starch (IRS-120) did not display observed diffractions of B-type

293

crystallites, related to the dehydration-induced transition of crystalline regions from smectic to

294

nematic state (Vermeylen, et al., 2006). But, the hydrated starch as characterized by SAXS clearly

295

presented the signal from B-type crystallites (100 inter-helix peak). In addition, a less resolved

296

lamellar peak occurred after 20 and 120 min, confirming a reduced amount of lamellar state starch

297

after the enzyme digestion. Agreeing earlier findings (Blazek, et al., 2010b), a gentle shift of lamellar

298

peak position (from 0.071 to 0.069 Å-1 here) occurred as the digestion proceeded.

299

For the processed starches in Fig. 4b and 4c, no lamellar peak occurred and instead a less

300

defined, broad shoulder emerged at around 0.03-0.04 Å-1, indicating the present of molecular orders

301

in an amorphous matrix (Lopez-Rubio, Flanagan, Shrestha, Gidley, & Gilbert, 2008). That is, this

302

much broader peak indicates the existence of a kind of nonperiodic organization constituted by

303

amorphous and crystalline materials, largely different from the typically found periodic

304

semicrystalline lamellae. Upon digestion after 20 min, slightly reduced scattering intensity in the

305

shoulder range was seen for the starch with conventional treatment. This implies that the enzymes

306

during initial 20 min preferentially digested the polymorphs (see XRD above), lowering the

307

ordered-nonordered density contrast. However, the microwave treated starch showed a more

308

inflected shoulder after the 20 min of digestion, with a slightly enhanced scattering intensity

309

throughout the shoulder. This suggests the occurrence of nanoscale molecular reorganization other

310

than the digestion of ordered and amorphous starches, strengthening the nonperiodic structure

311

constructed by orders and amorphous phases in the partially digested starch residues and expanding

312

the nonordered-ordered density difference. Furthermore, the digestion induced starch chain 15

313

reassembly was also affirmed by the emergence of a scattering peak at ca. 0.137 Å-1 (Fig. 4b-c and

314

Fig. S1b-c in supplementary material), indicative of the formation of new molecular organization

315

having a repeat length of ca. 5 nm. This is the first time that such characteristic molecular

316

organization was clearly shown for the full hydrated starch residues (with much fewer amylose

317

chains) containing SDS plus RS fractions.

318

If the digestion sustained to 120 min, one would observe a much less defined shoulder

319

irrespective of the cooking manner used, and more intensely reduced scattering intensity of the

320

overall pattern for the conventionally treated starch than for the microwave treated counterpart (see

321

Fig. 4b and 4c). Hence, the digestion between 20-120 time also preferentially hydrolyzed the

322

molecular orders accompanying the proceeded hydrolysis of amorphous and ordered regions, which

323

weakened the nonperiodic ordered-amorphous structure and reduced the amorphous-ordered density

324

difference. Yet, relative to the starch with conventional processing, the starch following microwave

325

processing underwent a less apparent reduction in the density difference between the amorphous and

326

ordered phases. This means that the latter had a higher compactness of the ordered regions after the

327

long time (120 min) digestion. Again, for the new molecular organizations formed via molecular

328

reassembly during 20 min of digestion, the enzyme hydrolysis between 20-120 min could make the

329

associated reflection peak at around 0.137 Å-1 much less visible (only a tiny peak observed) (Fig.

330

S1b and S1c in supplementary material). That is, the new kind of molecular organization suffered

331

gradual erosions during the digestion of 20-120 min, again unlike the previous case that the starch

332

chain organizations in digested extruded high-amylose starch mainly existed as enzyme resistant

333

fractions (Lopez-Rubio, Htoon, & Gilbert, 2007).

334

3.5 Interpretation of Microwave-Enhanced SDS Formation 16

335

In the course of starch digestion, normally two types of enzyme molecules, α-amylase and

336

amyloglucosidase, take part in the hydrolysis of starch matrices containing assembled glucan chains.

337

α-Amylase molecules randomly cleave α-1,4 linkages of glucan chains; amyloglucosidase molecules

338

degrade next-to-terminal or terminal linkages of starch molecules from their non-reducing ends. The

339

starch digestion course by enzymes involves three core events on the enzyme diffusion toward starch

340

matrices, the enzyme-chain complex formation (binding to glucan chains) and the followed

341

degradation of glycosidic bonds (catalytic events) (Colonna, Leloup, & Buléon, 1992). The digestion

342

behaviors, e.g., rate constant, of starch can be varied by factors, such as the type of polymorphs, the

343

surface pores and the molecular structure (Blazek & Copeland, 2010a; Syahariza, Sar, Hasjim,

344

Tizzotti, & Gilbert, 2013). Such theory and structural evolutions caused by digestion could help us in

345

visiting the mechanism of starch digestion (Fig. 5).

346

For the native starch, the enzymes molecules tended to diffuse toward and simultaneously

347

corrode not only the amorphous background and lamella regions but also the crystalline lamella

348

phases via an inside-out manner (see SAXS and SEM), throughout the digestion of RDS within

349

initial 20 min and SDS within 20-120 min. The gradual removal of starch materials on different

350

scales led to increased numbers of pores in the granule substrate; the hydrolyzed content of

351

crystallites was proportionable with that of amorphous regions at a ratio close to the original

352

crystallinity degree (see negligibly changed XRD pattern with digestion). Such changes to the

353

crystallites in lamellae were stronger relative to previous investigations where mainly amorphous

354

growth rings were preferentially digested (Blazek, et al., 2010b). This greater change was confirmed

355

by an evident reduction in the peak intensity. Also, accompanying starch digestion, the helices

356

originally in the A-crystallites and the non-crystalline forms could encapsulate water molecules to 17

357

form hexagonal units of B-crystallites. This molecule reassembly in untreated starch was associated

358

with the liquid crystal nature of starch chains (Daniels, et al., 2004; Qiao, et al., 2017b). Thus, in

359

untreated starch having sophisticated multi-scale architecture, the SDS formation was not determined

360

by a specific structure but by the enzyme availability of starch structure matrices resulting from

361

concurrent hydrolysis and molecular reassembly during the digestion.

362

In contrast, the treated starches did not contain any native architecture, and showed primarily

363

irregular morphology, B plus V polymorphs and nanoscale nonperiodic organizations. The digestion

364

simultaneously hydrolyzed the ordered and amorphous regions, resulting from the diffusion of

365

enzyme molecules within the matrices and subsequently their absorption and catalysis events. The

366

crystallites (B and V) were more available to the enzymes than were the amorphous regions

367

containing nonordered chains (see weakened diffractions above). This deepens the understanding of

368

native starch that B-type crystallites are less susceptible to enzyme digestion relative to A-type forms

369

(Blazek, et al., 2010b), and again confirmed that the enzyme availability of a specific structure are

370

not determined by isolated features such as crystalline type. Also, the digestion resulted in removal of

371

bulk matrices from starch substrate (see occurred porosity and breakage) and nano-structural

372

evolutions. Especially, the molecular reassembly of two treated starches were affirmed by the

373

formation of new molecular organization (repeat distance: about 5 nm) after 20 min of digestion. The

374

microwave treated starch displayed stronger molecular reorganizations than did the counterpart with

375

conventional treatment, as shown by the improved shoulder peak for the former. This relatively

376

intense molecular reassembly, accompanying starch hydrolysis, played roles in reducing the fast

377

availability of starch structures to enzymes within 20 min, but strengthening the slow availability of

378

starch matrices to enzymes within 20-120 min. Consistently, more SDS with a lower digestion rate 18

379

could be seen for the microwave treated starch than for that following conventional treatment.

380

4. Conclusions

381

The multi-level structural evolutions of starch during digestion were inspected to better

382

understand how microwave cooking with storage enhances the slowly digestible features of starch.

383

During digestion, not only were starch matrices ultimately digested, leading to porous substrate and

384

reduced polymorphs and nanoscale orders, but also starch chain reassembly occurred, causing A to B

385

crystalline transform for untreated starch and formation of new molecular organization for the

386

processed starches with storage. This occurrence of concurrent matrix hydrolysis and molecular

387

reassembly during digestion played roles in determining the digestion features of native and treated

388

starches.

389

The ultimate digestion of a specific structure matrix, actual availability by enzymes under

390

competed hydrolysis and reassembly, were affected by the state of whole structural system.

391

Consistently, the polymorphs in native sophisticated architecture were similarly available by

392

enzymes as compared to the nonordered background and lamellar phases; however, the polymorphs

393

in processed starches without native architecture were preferentially disrupted, being different earlier

394

views. Compared to conventionally treated starch, the microwave treated sample showed stronger

395

molecular reassembly during digestion, confirmed by the strengthened nanoscale orders after 20 min

396

and the more intense crystalline diffractions after 120 min. Such stronger chain reassembly tended to

397

enhance the slowly digestible features for microwave treated starch, causing an increased SDS level

398

and a lowered digestion rate. Our results here enable a better understanding of starch before and after

399

cooking with storage, and thus are of value for rational control of starch slowly digestible

400

characteristics. 19

401

Acknowledgment

402

The authors would like to acknowledge the National Natural Science Foundation of China

403

(31701637), and the Project funded by China Postdoctoral Science Foundation (2018M642865 and

404

2019T120708). We thank the staffs from BL19U2 beamline of National Facility for Protein Science

405

in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility, for assistance during data collection.

406

B. Zhang thank the Young Elite Scientists Sponsorship Program by China Association for Science

407

and Technology (2018QNRC001).

408

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523 524 525 526 527

25

556

Table 1 Digestion parameters for the starch (IRS) with conventional (C) or microwave (MC)

557

cooking followed by storage IRS

IRS-C

IRS-MC

k1 (min-1)

(2.34±0.33)×10-3 c

(2.53±0.05)×10-2 a

(2.15±0.05)×10-2 b

k2 (min-1)

－

(1.06±0.02)×10-2 a

(1.05±0.00)×10-2 a

RDS

3.43±0.15 c

27.44±0.06 a

26.01±0.42 b

SDS

16.82±1.10 c

36.86±0.62 b

41.01±0.31 a

RS

79.75±1.25 a

35.71±0.56 b

32.99±0.12 b

558

32

527

Figure Captions

528

Fig. 1 Digestion plots and LOS plots as well as their fit curves for the starch (IRS) subjected to

529

conventional (C) or microwave (MC) cooking followed by storage. ○, experimental data; ×, LOS

530

plot data;

531

kinetic model.

532

Fig. 2 SEM micrographs of undigested and digested starch (IRS) samples for 20 or 120 min. ‘C’

533

indicates conventional cooking with storage; ‘MC’ represents microwave cooking with storage.

534

Fig. 3 XRD patterns of undigested and digested starch (IRS) samples for 20 or 120 min. ‘C’ indicates

535

conventional cooking with storage; ‘MC’ represents microwave cooking with storage.

536

Fig. 4 SAXS patterns of undigested and digested starch (IRS) samples for 20 or 120 min. ‘C’

537

indicates conventional cooking with storage; ‘MC’ represents microwave cooking with storage.

538

Fig. 5 Schematic representation for how microwave treatment enhances starch slowly digestible

539

features.

or

, linear fit curve for LOS plot data;

26

or

, fit curve based on first-order

60

80

-1

40

-2

20

-3

0

100

200

300

400

500

Second stage

0

100

200

300

-1

-2

IRS-MC

20

0

400

500

ln (dc/dt)

Starch Digested Ratio (%)

Experimental data First phase model-fit Second phase model-fit LOS plat data

40

-3

-4 600

Time (min)

541

60 40 20

100

200

300

Fig. 1

27

400

-1

-2

Second stage

Time (min)

First stage

60

0 Experimental data First phase model-fit Second phase model-fit LOS plat data

0

1

80

First stage

IRS-C

-3

500

-4 600

0

-4 600

Time (min)

c 100

0

542

0

1

IRS

0

540

b 100 Starch Digested Ratio (%)

80

1

ln (dc/dt)

Experimental data Model-fit LOS plot data

ln (dc/dt)

Starch Digested Ratio (%)

a 100

IRS

IRS-20

IRS-120

IRS-C

IRS-C-20

IRS-C-120

IRS-MC

IRS-MC-20

IRS-MC-120

543

544

545 546

Fig. 2

28

b IRS-120

IRS-20

IRS

10

30

IRS-MC-120

IRS-MC-20

IRS-MC

20

30

40

2θ ( °)

548

IRS-C-20

IRS-C

10

20

30 2θ (°)

c

10

549

40

2θ (°)

Relative intensity (a.u.)

547

20

IRS-C-120

Relative intensity (a.u.)

Relative intensity (a.u.)

a

Fig. 3

29

40

a

IRS IRS-20 IRS-120

4

3

10

2

10

0.01

4

3

10

0.01

0.1

Intensity (a.u.)

10

10

3

10

2

q (A ) IRS-MC IRS-MC-20 IRS-MC-120

4

0.01

0.1 -1

q (A )

551 552

0.1 -1

q (A )

c

2

10

-1

550

IRS-C IRS-C-20 IRS-C-120

10

Intensity (a.u.)

Intensity (a.u.)

10

b

Fig. 4

553

30

554 555

Fig. 5

31

Highlights Starch digestion was governed by concurrent matrix hydrolysis and chain reassembly. Polymorphs in processed starch could be preferentially digested. Microwave treated starch showed enhanced molecular reassembly during digestion.

Author Statement

Nannan Li: Data curation, Investigation, Writing-Original draft preparation. curation, Writing-Original draft preparation. Methodology, Formal analysis. Qinlu Lin: Resources.

Lili Wang: Data

Siming Zhao: Conceptualization.

Caihua Jia: Data curation.

Dongling Qiao:

Meng Niu: Resources, Methodology.

Binjia Zhang: Conceptualization, Supervision, Writing- Review & Editing

Declaration of interests ◼ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: