Properties of lotus seed starch-glycerin monostearin V-complexes after long-term retrogradation

Journal Pre-proofs Properties of lotus seed starch-glycerin monostearin V-complexes after longterm retrogradation Yixin Zheng, Bailong Wang, Zebin Guo, Yi Zhang, Baodong Zheng, Shaoxiao Zeng, Hongliang Zeng PII: DOI: Reference:

S0308-8146(19)32025-4 https://doi.org/10.1016/j.foodchem.2019.125887 FOCH 125887

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Food Chemistry

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16 September 2019 10 November 2019 10 November 2019

Please cite this article as: Zheng, Y., Wang, B., Guo, Z., Zhang, Y., Zheng, B., Zeng, S., Zeng, H., Properties of lotus seed starch-glycerin monostearin V-complexes after long-term retrogradation, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125887

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Properties of lotus seed starch-glycerin monostearin

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V-complexes after long-term retrogradation

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Running title: Properties of lotus seed starch-glycerin monostearin

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Yixin Zhenga,b, Bailong Wanga, Zebin Guoa, Yi Zhang a,b,c, Baodong Zhenga,b,c,

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Shaoxiao Zeng a,b,c*, Hongliang Zenga,b,c

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aCollege

of Food Science, Fujian Agriculture and Forestry University, Fuzhou

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350002, China

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bFujian

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Special Starch, Fujian Agriculture and Forestry University, Fuzhou 350002, China

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cChina-Ireland

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Structure Design, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Provincial Key Laboratory of Quality Science and Processing Technology in

International Cooperation Centre for Food Material Science and

15 16 17 18 19 20 21 Corresponding

author. Tel.: +86 591 83736738; fax: +86 591 83739118. E-mail address: [email protected] (S. Zeng); [email protected] (H. Zeng)

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Abstract: The properties of lotus seed starch-glycerin monostearin with V6II and

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V6I-complexes formed at 50 MPa and 100 MPa after long-term retrogradation (named

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as LS-GMS-50 and LS-GMS-100, respectively) were investigated. The results

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indicated LS-GMS-50 and LS-GMS-100 were conducive to the formation of

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crystallinity and an ordered structure of starch compared to lotus seed, lotus seed at 50

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MPa and 100 MPa (LS, LS-50 and LS-100), especially V6I-complexes. The presence

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of V6I-complexes had the superior ability to enhance water retention of starch gel

29

compared to V6II-complexes. V-complexes inhibited the aggregation of molecular

30

chain and changed the molecular chain to nanoscale, especially V6I-complexes.

31

Moreover, physicochemical properties demonstrated V-complexes lowered thermal

32

enthalpy value and heat sensitivity compared to other samples. Rheological

33

measurement showed V-complexes improved the flow behavior and viscoelasticity of

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retrograded starch. Thus, a formation mechanism was that V-complexes improved the

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internal network structure and freed up space to store water molecules.

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Keyword: Lotus seed starch; Glycerin monostearin; V-complexes; Long-term

37

retrogradation; Structural properties; Physicochemical properties

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44

1. Introduction

45

Starch is the most abundant reserve carbohydrate in plants and it is a major

46

source of energy in daily life (Kumar, Brennan, Zheng, & Brennan, 2018), while

47

starch retrogradation is a non-equilibrium thermo reversible recrystallization process

48

that occurs between glucan molecules in gelatinized starch during cooling (Wang,

49

Wang, Li, Chen, & Zhang, 2017). Based on the recrystallization of amylose and

50

amylopectin, starch retrogradation can be classified into short-term or long-term.

51

Amylose is the main component of short-term retrogradation, which can be controlled

52

by starch modification, temperature, and the addition of non-starch components. It is

53

generally believed that the long-term storage of starch from 1 to 28 days may reflect

54

the long-term retrogradation process (Niu, Zhang, Xia, Liu, & Kong, 2018). The

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long-term retrogradation of starch is attributed to amylopectin recrystallization, which

56

is difficult to control and usually occurs if starch gel is stored in long-term storage. A

57

recent study indicated that long-term retrogradation of starch was correlated with the

58

slow self-association of branched side-chains, profoundly impacting the texture, favor,

59

digestibility, and functional properties of starchy food (Ji, Liu, Zhang, Yu, Xiong, &

60

Sun, 2017).

61

It well known that lipids, acting as a common anti-staling agent, play a crucial

62

role in improving the shelf life of starchy food. When lipids are added into a

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gelatinized starch system, amylose can undergo a conformational change to form a

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single helical structure with a hydrophobic cavity that can react with numerous

65

hydrophobic ligands, forming amylose-lipid complexes (V-complexes) (Putseys,

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Lamberts, & Delcour, 2010). Many advances have indicated that the amylose–lipid

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complexes have an appreciable effect on inhibiting the short-term and long-term

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retrogradation of starch. The complexes of rice starch and ß-cyclodextrin–lipid stored

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at 4°C for 2 hours were responsible for retarding short-term retrogradation (Tian,

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Yang, Li, Xu, Zhan, & Jin, 2010). The formation of V-type complexes between

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palmitic acid and maize starch improved the amylopectin retrogradation during

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long-term storage (Mariscal ‐ Moreno, Figueroa ‐ C á rdenas, Santiago ‐ Ramos, &

73

Rayas‐Duarte, 2018). Moreover, our previous study (Chen, Zeng, Zeng, Guo, Zhang,

74

& Zheng, 2017) found that starch-glycerin monostearin complexes of crystal nuclei

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from lotus seed that were formed by different conditions had different

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anti-retrogradation characteristics. The findings revealed that the different starch-lipid

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complexes after long-term retrogradation likely impact on the properties of lotus seed

78

starch. The results presented by (Wang, Wang, Yu, & Wang, 2016) indicated that a

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small proportion of amylopectin could form V-type complexes with lipids, which was

80

mainly dependent on the short side-chain of amylopectin and steric hindrance, and the

81

interaction of amylopectin and lipids might be directly relevant to the change of starch

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properties and the inhibition of long-term retrogradation.

83

Lotus is an economically important aquatic plant in Asia that has been used for

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as long as 1,300 years in China. Lotus seed has various bioactive components,

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including alkaloids, glycosides, flavonoids, vitamins, minerals, and dietary fiber

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(Zhang, Zeng, Wang, Zeng, & Zheng, 2014), and is a popular ingredient for local

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food and can be processed into commercial products, such as pudding, noodles, chips,

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canned food, and beverages. However, lotus seed contains high amylose starch, which

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is more likely to generate the retrogradation behavior. The staling of starch can

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destroy the starch-protein network and decrease its water retention capacity. As a

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consequence, these changes in lotus seed starch will severely increase the firmness of

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related products, resulting in the reduction of shelf life and decreased acceptance by

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consumers. According to our previous study (Chen et al., 2017), different

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homogenization pressure had no influence on the retrogradation of lotus seed starch,

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but was significantly related to the retrogradation of V-complexes. The V-complexes

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formed by low pressure homogenization exhibited some ability to retard the starch

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retrogradation, and the effects were more significant as the pressure increased, which

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were mainly due to the formation of V-complexes in different crystal forms that

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contributed to the inhibition of amylose recrystallization. Therefore, since it is

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amylopectin, not amylose, that determines the long-term storage of lotus seed

101

products, and there are few specific and detailed mechanisms for interpreting the

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V-type complex how to influence the properties of starch after long-term

103

retrogradation.

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Thus, the objective of the present study was to investigate the physicochemical

105

and structural properties of lotus seed starch-glycerin monostearin V-complexes after

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long-term retrogradation. The structural properties of starch after long-term

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retrogradation were characterized by 13C cross-polarization and magic angle spinning

108

nuclear magnetic resonance (13C CP/MAS NMR), fourier transform infrared

109

spectroscopy (FTIR), low-field nuclear magnetic resonance (1H-NMR), and atomic

110

force microscopy (AFM). Differential scanning calorimetry (DSC) and rheological

111

measurement were used to understand the change of physicochemical properties of

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starch after long-term retrogradation. Moreover, the effects of V-type complexes on

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the long-term retrogradation of starch in lotus seed were discussed.

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2. Materials and methods

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2.1 Materials

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Lotus seed starch (Green Field Fujian Food Co., Ltd., Fujian, China) was

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isolated as previously described (Zhang et al., 2014). The amylose/amylopectin

118

content of lotus seed starch was 40/60, as measured using amylose/amylopectin assay

119

kit purchased from Nanjing Chemical Co. (Nanjing, China). Glycerin monostearin

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(GMS) with a hydrophile-lipophile balance of 3.8 ± 0.1 was obtained from TNJ

121

Chemical Industry Co. Ltd. (Hehui, China). All other chemical reagents used in this

122

study were of analytical grade.

123

2.2 Preparation of lotus seed starch-glycerin monostearin complexes

124

The raw starch was defatted by a mixture of ethanol/ water and dried in an air

125

oven (DGG-9036A, Jiangdong Precision Instrument Co., Ltd., Suzhou, China).

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Glycerin monostearin (200 mg, 5%, w/w, dry starch base) was added to defatted

127

starch dispersion (4 g, 8%, w/w) before heating at 50°C for 5 min. The slurry was

128

homogenized using a microfluidization apparatus (SPCH-10, Stansted Fluid Power

129

Ltd., Harlow, UK) under pressure (50 MPa or 100 MPa) for five times (names as

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LS-GMS-50 and LS-GMS-100, respectively). Corresponding control samples were

131

prepared without GMS (names as LS-50 and LS-100, respectively). A cooling water

132

circulation device (XT5218B8, Xutemp Temptech Co.,Ltd., Hangzhou, China) was

133

used to maintain the temperature of the apparatus at 25°C. After the homogenization

134

pressure treatment, the samples were gelatinized and stored in a climatic cabinet at

135

4°C. The samples were removed from the climatic cabinet after 28 days and dried

136

using a freeze dryer (FDU-1200, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The dried

137

samples were used to analyze physicochemical and structural properties. Additionally,

138

the samples of starch gel without freeze drying were prepared the same to measure the

139

moisture distribution treatment. As a control, a sample without pressure treatment or

140

GMS addition was also prepared and named as LS.

141

2.3 13C CP/MAS NMR determination

142

Approximately 200–300 mg of starch samples were prepared for the 13C nuclear

143

magnetic resonance (13C-NMR) spectrometer (AVANCE III 500, Bruker Ltd.,

144

Karlsruhe, Germany) for scanning at measurement frequency of 100.62 MHz. The test

145

probe was 7 mm H/X CP-MAS, the number of scans was 1,500, the spin speed was 6

146

kHz, and the acquisition time was 0.013 s. The region of C1 reflected the degree of

147

crystallization of the starch crystal region, the region of C4 reflected the degree of

148

amorphousness of the starch granules, and the region of C2,3,5 reflected the degree of

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amylose freedom, which were confirmed by a previous study (Zeng et al., 2015). The

150

region of C1, C4, and C2,3,5 were fitted and calculated by Peakfit 4.0(Systat Software,

151

California, US). The formula is as follows:

152

C1 region(%) = 100 ×

C1 Ctotal

C4

153

C4 region(%) = 100 ×

154

C2,3,5 region(%) = 100 ×

Ctotal C2,3,5 Ctotal

155

where C1 is peak area of the vibration peak in the C1 region; C4 is the peak area of the

156

vibration peak in the C4 region; C2,3,5 is the peak area of the vibration peak in the C2,3,5

157

region, and Ctotal is the total area of the vibration peaks.

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2.4 FTIR measurement

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The sample was mixed with anhydrous KBr powder (the amount of KBr added

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was 30 times the sample volume) in an agate mortar and rapidly milled under an

161

infrared lamp. Then, the powdered sample was pressed into a sheet and placed in an

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FTIR (Avatar360, Thermo Nicolet Corporation Ltd, Madison, US) for measurement.

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The scanning range was 4000–400 cm-1, the number of scans was 16–32, and the

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resolution was 4 cm-1. The FTIR spectra were plotted with Origin 8.5 (Originlab,

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Northampton, US).

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2.5 LF-1H NMR determination

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The water distribution of starch gel after long-term storage was measured using a

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23-NMR analyzer (NMI20-015 V-I; Niumag, Co., Ltd., Shanghai, China). The proton

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transversal time measurements were performed by a previously-described method

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(Zeng et al., 2016). According to the results of proton transversal time, the proportion

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of water in the different region was calculated by area integration.

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2.6 AFM observation

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The stored complex gel was diluted to 10 μg/mL, and 5 μL was placed into the

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center of the mica plate in the culture dish. The Petri dish was covered with a lid and

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placed at room temperature for 24 h. After the water was completely evaporated, the

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mica plate was placed under a rectangular cantilever probe for measurement with an

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atomic force microscope (5500ILM, Agilent Technologies Inc., California, USA). The

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range of the measurement spanned 4 μm × 4 μm and the scanning speed was 1.15

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frames. The topography of the sample was processed using SCANASYST-AIR

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software to obtain a 2D aggregate morphology of the molecular chains in the gel, and

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the average length and height (nm) of the surface was calculated by Nanoscope

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Software (NanoScope v 5.30r3, Bruker, Veeco, USA).

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2.7 DSC measurement

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The samples were sealed in a high-pressure stainless steel pan and then the

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enthalpy change (ΔH) of retrograded starch was measured by a DSC (DSC-200FC,

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NETZSCH, Selb, Germany) following the methods described in a previous study

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(Zhao, Jiang, Zheng, Zhuang, Zheng, & Tian, 2017). The initial temperature (To),

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peak temperature (Tp), and termination temperature (Tc) were recorded.

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2.8 Rheological properties

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The dried samples were dissolved in distilled water to configure suspensions (8%

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w/w). Subsequently, the suspensions were heated at 95°C for 30 min. After cooling to

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25°C, 1 mL of the samples was pipetted onto the rheometer (MCR302, Anton Paar,

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Graz, Austria). The experiment was measured by a 50 mm flat rotor, the plate

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clamping distance was 0.5 mm, and the rheometer plate temperature was 25°C.

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2.8.1 Apparent viscosity and thixotropy of the sample

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The gelatinized samples were investigated by linear change of the rotational speed.

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The experiment was set as follows: the rotor rotational speed increased linearly from

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1 s-1 to 300 s-1 in the range of 3 min. Then, the rotor linearly dropped from 300 s-1 to 1

199

s-1 at the same rate. The apparent viscosity and thixotropy of the sample during the

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shearing process were recorded to determine patterns.

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2.8.2 Frequency vibration scan of the sample

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The experiment determined the linear viscoelastic region of the samples by

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amplitude sweep (λ = 0.5%), maintained the vibration amplitude, and adjusted the

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vibration frequency to 1–10 Hz. The storage modulus (G') and loss modulus (G'') of

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the sample during frequency vibration were recorded, and the loss tangent was

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obtained by the modulus relationship (G''/G'= tanδ).

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2.9 Statistical analysis

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Triplicate measurements were performed for each experiment. Graphs were

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constructed using Origin 8.5 (Originlab, Northampton, US). Data were analyzed and

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significant differences were determined by DPS 9.05 (Science Press, Beijing, China).

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Statistical significance was considered at a P ≤0.05.

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3. Results and Discussion

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3.1 Structural properties by solid-state 13C CP/MAS NMR spectroscopy

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The nuclear magnetic resonance data of lotus seed starch and its V-complexes

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after long-term retrogradation are shown in the Figure 1(A). The carbon chemical

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shifts of the major regions were identified in 94-105 ppm for C1; in 68-78 ppm for

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C2,3,5; in 80-84 ppm for C4, and in 58-65 ppm for C6. Native lotus seed starch and the

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lotus seed starch treated by microfluidization showed two major peaks at 100 and 101

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ppm in the C1 region (Figure 1(A) black dotted coil), which was in line with the

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vibration characteristics of B-type resistant starch crystals (Zeng et al., 2018).

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Compared to the native retrograded starch, with the homogenization pressure

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increasing, the region of C1 and C4 both decreased to some extent (Table 1), especially

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when the homogenization pressure reached 100 MPa (LS-100). In addition, the region

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of C1 and C4 of lotus seed starch decreased by 1.78 ± 0.01 and 0.34 ± 0.01,

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respectively, suggesting that the high pressure homogenization destroyed the crystal

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structure of retrograded starch, leading to a decrease in the crystallinity and an

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increase in the degree of amylose double helixes. This was in agreement with our

228

previous study (Guo, Zeng, Lu, Zhou, Zheng & Zheng, 2015), which showed that the

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reduction of crystallinity and the presence of disordered structure were attributed to

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the destruction of ultra-high pressure treatment, meaning that the double helix

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structure of amylose leaked out of the starch structure. Furthermore, the region of

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C2,3,5 decreased with the increase of homogenization pressure, which suggested that

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the amylose of retrograded starch treated by high pressure had a higher value of

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freedom. These results revealed that the high-pressure homogenization strongly

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affected the tight gel structure of starch, causing the breakdown of amylose and

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amylopectin into shorter chain lengths, which caused difficulty in the recombination

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of the single molecular chain and the short amylose chains to be free in the starch

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system. When GMS was added to the starch system, the pattern showed a significant

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change with the two peaks of the C1 region transformed into a sharp unimodal

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structure compared with the control (Figure 1(A) pink dotted coil). A significant

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change in the chemical shift of 102.4, 81.8, 71.7, and 60.1ppm occurred in the

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complexes formed at 50 MPa (LS-GMS-50), which was the feature peak of the V6II

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complex. The complexes formed by 100 MPa (LS-GMS-100) showed strong

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vibration signal peaks at the chemical shift of 102.6, 81.4, 71.6, and 61.4 ppm,

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implying the formation of microcrystalline complexes (V6I complexes) (Gidley &

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Bociek, 1988).

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Observed in Table 1, the proportion of the C1 and C4 regions increased

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significantly with the presence of V-complexes, and the crystal proportion of V6I

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complexes was higher than V6II complexes. These results showed that a highly

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ordered structure of V-type complexes was more conducive to the formation of

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crystallinity and the reduction of amylose double helixes during long-term

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retrogradation. This was supported by the results obtained from our previous study

253

(Jia, Sun, Chen, Zheng, & Guo, 2018), where the lotus seed starch-fatty acid

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complexes were directly associated with the proportion of the crystallization region,

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and amylose spirochetes combine with fatty acids to form a complex, which reduced

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the degree of the amylose double helix. Moreover, the C2,3,5 region of V-complexes

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also increased significantly. The proportion of C2,3,5 reached the maximum of 52.32 ±

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0.02, especially with the formation of the V6I complexes, indicating that the free

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amylose chain and GMS formed a compact composite system after long-term

260

retrogradation. The V6I complexes compared to V6II complexes had a superior ability

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to make the structure more complete and uniform. These results demonstrated that the

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formation of microcrystalline V-complexes not only improved the gel structure of

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retrograded starch, but contributed to the stability of the crystal structure during

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long-term retrogradation, which was consistent with a previous study (Lu, Shi, Zhu,

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Li, & Huang, 2019) that showed that maize starch-fatty acid complexes were

266

conductive to the formation of a crystallization region and the stability of crystal

267

structure. The dense V-type crystal structure formed in starch could be used as a new

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type of resistant starch (RS5) in the cecum to be fermented by the intestinal flora, of

269

which acted as bioactive constituents in food.

270

3.2 Structural properties by FT-IR spectroscopy

271

The infrared spectra of lotus seed starch and its V-complexes treated by different

272

pressures are shown in Figure 1(B) and the corresponding data were summarized in

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Table 1. The infrared peak position of 850cm-1 and 928cm-1 represent the vibration of

274

side chain branch of amylopectin; 1083cm-1 represents the vibration of amylose single

275

helix, and 950 cm-1–1065 cm-1 is the fingerprint region of the double helix

276

conformation and ordered structure of starch (Figure 1(B) black dotted coil), which

277

was obtained from previous studies (Zhang et al., 2014; Wiercigroch et al., 2017).

278

Such studies showed that 995 cm-1/1022 cm-1 used to be the degree of the amylose

279

double helix, 991 cm-1 was to be the degree of amylopectin double helix, and 1047

280

cm-1/1022 cm-1 was the ordered degree of starch structure. With the increase in the

281

homogenization pressure to 50 MPa, 995 cm-1/1022 cm-1 had no significant change

282

compared to native retrograded starch after long-term retrogradation, but when the

283

homogenization pressure reached 100 MPa, the value of 995 cm-1/1022cm-1 decreased

284

from 1.080 ± 0.008 to 1.058 ± 0.015. These results suggested that low

285

homogenization pressure could destroy the structure of lotus seed starch, only leading

286

to the leakage of amylose and breakage of the double helix, which still had the ability

287

to re-form the double helical structure during long-term retrogradation. When the

288

starch was treated with a high homogenization pressure (100 MPa), the high-pressure

289

shear caused the leakage of amylose and the further break down of amylose into

290

smaller and shorter starch chains. Such processes resulted in a difficulty re-forming

291

the amylose chain into a double helix, which was consistent with the results of the

292

NMR that indicated that the amylose chain had a higher degree of freedom and

293

disorder.

294

Furthermore, measurements at 991 cm-1 and 1047 cm-1/1022 cm-1 were not

295

affected by the low homogenization pressure (50 MPa), but changed significantly

296

with high homogenization pressure (100 MPa) (Table 1). Those findings suggested

297

that high homogenization pressure could degrade the double helix of amylopectin and

298

destroy the gel structure, resulting in an extremely disordered state of starch.

299

Interestingly, with the formation of V6II complexes (LS-GMS-50), the values of 991

300

cm-1 and 995 cm-1/1022 cm-1 both further decreased by 0.017 ± 0.002 and 0.079 ±

301

0.008, and when the homogenization pressure was 100 MPa, the value of 991 cm-1

302

and 995 cm-1/1022 cm-1 were reduced by the presence of V6I complexes

303

(LS-GMS-100) to a minimum of 1.032 ± 0.004 and 1.185 ± 0.026, respectively (Table

304

1). This indicated that the reduction of the amylose double helix was due to the

305

combination of the amylose single helix and GMS, but the reduction of the

306

amylopectin double helix might be primarily affected by the content of the amylose

307

double helix. A previous study (Zhang et al., 2019) also supported this, where the

308

double helix structure of potato amylose could be used as the seed nucleus of

309

amylopectin to accelerate the recrystallization and retrogradation of amylopectin.

310

With the formation of LS-GMS, the value of 1047 cm-1/1022 cm-1 increased greatly

311

(Table 1) and the value of LS-GMS-100 was markedly higher than LS-GMS-50,

312

indicating that the formation of V-type complexes contributed to the ordering of the

313

starch structure. Additionally, this phenomenon further suggested that V6I complexes

314

had a more compact and stable crystal structure, compared to V6II complexes, and the

315

V- complexes were conducive to the inhibition of the recrystallization of amylopectin

316

and amylose, which was directly related to the retardation of long-term retrogradation.

317

Such findings were in agreement with a previous study (Yu, Wang, Chen, Li, & Wang,

318

2018). Specifically, Yu et al. (2018) showed that the retrogradation of wheat starch

319

would be inhibited by V-complexes between stearic acid and amylose or the

320

side-chain of amylopectin, and the formation of microcrystalline V-complexes was

321

beneficial for the stability of starch structure, while was not sensitive to the digestion

322

of amylase.

323

3.3 Water distribution of LS-GMS complexes

324

The water distribution of lotus seed starch and its V-complexes are shown in

325

Figure 2(A), and the corresponding values for different relaxation periods obtained

326

from our previous study (Zeng et al., 2016) for A21 (bound water), A22 (immobile

327

water), and A23 (free water) are summarized in Table 1. After long-term retrogradation,

328

the pattern of native starch showed a large area of water distribution during the high

329

relaxation period, indicating that the gel structure of lotus seed starch shrunk and

330

precipitated a large amount of free water, which was due to the recrystallization of

331

amylopectin. When the starch was treated by microfluidization, the bound water

332

distribution of LS-50 migrated to the high relaxation period, but the proportion of

333

water had no significant change compared with native retrograded starch. With the

334

pressure further increased to 100 MPa, the free water (A23) of retrograded starch

335

increased by 4.38 ± 0.01 compared to LS-50, and the content of immobile water

336

decreased by 6.36 ± 0.01 (Table 1), indicating that high homogenization pressure

337

could destroyed the molecular structure of the unit cell leading to the presence of

338

more free water in the starch system, which was in accordance with our previous

339

study (Chen et al., 2017). Specifically, we previously showed that high pressure

340

homogenization would promote the migration of moisture of lotus seed starch,

341

causing more immobile water to become free water. Furthermore, it was worth noting

342

that the bound water distribution of LS-100 migrated towards the low relaxation

343

period, and its content increased by 1.98 ± 0.01 compared to LS-50. A previous study

344

(Miles, Morris, Orford, & Ring, 1985) interpreted this as more free water participated

345

in the recrystallization process of the amylopectin side chain, and the formation of

346

each unit required the conversion of two molecules of free water into 1 molecule of

347

bound water. When the GMS was mixed with the starch, the V6II complexes formed

348

by low homogenization pressure (50MPa) showed the lower proportion of A23 and the

349

higher proportion of A22 compared to LS, indicating that the formation of V-type

350

complexes were beneficial for binding more moisture in the gel network structure

351

after long-term retrogradation. Interestingly, the bound water of V6II-complexes

352

decreased by 1.08 ± 0.01 compared to LS-50, which was possibly due to the weak

353

starch network structure, indicating that the V6II-complex formed by low

354

homogenization pressure (50 MPa) was an unstable and non-uniform composite

355

starch gel system, resulting in the conversion of bound water to immobile water

356

during long-term storage. When the homogenization pressure reached 100 MPa, the

357

complexes (LS-GMS-100) transformed into a steady state system (V6I complexes) and

358

displayed the lowest proportion of free water and the highest proportion of bound

359

water and immobile water (Table 1) among these samples. According to our previous

360

study (Chen, Fu, Chang, Zheng, Zhang, & Zeng, 2019), high intermediate water

361

content of LS-GMS-100 was very beneficial to the formation of a starch

362

microcrystalline region and the enhancement of the water holding capacity. This was

363

consistent with the results of NMR and FTIR, which showed that V-complexes had a

364

higher proportion of crystalline region and a more ordered structure. Furthermore, our

365

findings were confirmed by a previous study (Cheng et al., 2018), which showed that

366

the presence of amylose-linoleic acid V-complexes enhanced the water holding

367

capacity of starch structure, which was due to the hydrogen bond between hydrophilic

368

hydroxyl groups of the complex and starch.

369

3.4 Microstructure of molecular chains

370

In order to further explore the association of molecular chains in the starch gel,

371

the atomic force morphological images of lotus seed starch and its V-complexes are

372

shown in Figure 2(B). The molecular chains of native retrograded starch gel were

373

aggregated (Figure 2 B1, blue arrow), which formed a random sheet-like structure

374

with a length of 281.2 ± 15.3. When the starch gel was treated with a low

375

homogenization pressure (50 MPa) (Figure 2 B2, blue arrow), the average length of

376

the molecular chain aggregation increased by 42.8 ± 1.8 nm, compared to native

377

retrograded starch. Those data implied that the shearing force of low homogenization

378

pressure degraded amylopectin into a longer amylose chain, resulting in an increase in

379

the length of the aggregation. This observation was supported by a previous study

380

(Wei, Cai, Jin, & Tian, 2016), which showed that homogenization pressure treatment

381

degraded amylopectin into longer amylose chains due to the destruction of

382

amylopectin α-1,4 glycosidic bonds. With the homogenization pressure further

383

increased to 100 MPa, the average length of the molecular chain aggregation greatly

384

decreased by 88.2 ± 1.5 compared to LS-50 (Figure 2 B3, blue arrow), indicating that

385

a high homogenization pressure further decomposed the length of amylose into

386

shorter chains, which led to a reduction in the size of the aggregation. This result was

387

consistent with the results of the NMR and FTIR, indicating that amylose chains that

388

were too short would lead to a decrease in the crystallinity of the starch, making it

389

difficult to reorganize the starch structure after long-term retrogradation. When the

390

GMS was composited with the lotus seed starch, the degree of molecular chain

391

aggregation of the V6II complex (LS-GMS-50) significantly decreased, only a few dot

392

structures were observed (Figure 2 B4, the red arrow). Furthermore, the size of the

393

molecular chain aggregation was significantly reduced with the increase of

394

homogenization pressure. When the homogenization pressure reached 100 MPa, the

395

average length of the aggregated V6I complex (LS-GMS-100) molecular chains were

396

less than 10 nm long (Figure 2 B5, red arrow). These results all demonstrated that the

397

shorter amylose molecular chains treated by high homogenization pressure were

398

composite with the GMS to form the nanoscale of the molecular chain, and the V6I

399

complexes could effectively inhibit the aggregation of molecular chains. Those

400

findings were in accordance with a previous study (Ocloo, Ray, & Emmambux, 2019)

401

showed that maize starch could form V-complexes with stearic acid at a nanoscale.

402

Additionally, a study published by Tian, Yang, Li, Xu, Zhan, & Jin (2010) also

403

showed that the formation of V-complexes between β-cyclodextrin and rice starch

404

could significantly decrease the size of starch aggregation and retard the

405

retrogradation of starch.

406

3.5 Thermal properties

407

The enthalpy absorption peak spectrum of retrograded starch and its

408

V-complexes after long-term retrogradation are shown in Figure 3. The retrograded

409

starch without GMS exhibited a prominent enthalpy absorption peak in the

410

temperature range of 71.6–81.8°C (PeakI), which was the thermal peak of retrograded

411

amylopectin (Silverio, Fredriksson, Andersson, Eliasson, & Polymers, 2000).

412

Observed from the Figure 3, the homogenization pressure could not cause the

413

appearance of other enthalpy absorption peaks, indicating no new substances were

414

produced in samples without GMS during the homogenization process. However,

415

when the GMS was mixed with the starch, a new thermal absorption peak appeared in

416

the temperature range of 90.9–100.8°C (PeakII), which was considered to be the

417

absorption peak of the amylose-lipid complex (Zhang, Huang, Luo, & Fu, 2012),

418

suggesting that V-complexes were formed in samples and LS-GMS has a higher

419

absorption temperature than other samples without GMS. This result was in

420

accordance with the studies reported by Chang, He, & Huang (2013) that the

421

V-complexes consisting of corn starch and lauric acid played an important role in

422

improving the thermal sensitivity of starch. Moreover, the measurement of ΔH was

423

always used to determine the degree of recrystallization of starch samples. The data

424

presented in Table 1 illustrated that the enthalpy of the starch samples treated by

425

microfluidization (50 MPa and 100 MPa) were both lower than native retrograded

426

starch, and the trend of the decrease in the enthalpy value mainly depended on the

427

intensity of the homogenization pressure. These changes indicated that the

428

homogenization treatment influenced the recrystallization of starch during long-term

429

retrogradation, which led to a reduction of the retrograded starch gel strength and a

430

weakening of the starch heat resistance. The results were supported by a study

431

conducted by Hu, Zhang, Jin, Xu, & Chen (2017) that suggested that high pressure

432

treatment would destroy the crystallization region of wheat starch, leading to the

433

crystallization zone to be more susceptible to thermal damage and a low enthalpy of

434

starch. When the GMS was added to the starch system, the thermal enthalpy value of

435

V6II complexes further decreased by 1.628 ± 0.061, compared to LS-50. When the

436

homogenization pressure reached 100 MPa, the thermal enthalpy value of

437

V6I-complexes (LS-GMS-100) reached 3.067 ± 0.042, suggesting that the formation

438

of the V6I complexes lowered the thermal sensitivity of starch while improved the

439

sturdy gel structure of starch, which were possibly attributed to the effects of

440

V-complexes on the inhibition of the recrystallization of amylose and amylopectin

441

during long-term retrogradation. These results were in accordance with the study

442

reported by Chen, Ren, Zhang, Tong, & Rashed (2015), which showed that the

443

reduction of thermal enthalpy value on resistant starch was mainly associated with the

444

inhibition of amylopectin retrogradation. The other research findings obtained from

445

Okumus, Tacer-Caba, Kahraman, & Nilufer-Erdil (2018) have also indicated that the

446

V-complexes of brown lentil starch and fatty acids have higher heat absorption

447

temperature, which was due to the fact that V-complexes transformed the original

448

structure into a more compact granular structure and improved the properties of the

449

starch structure, implying the decent resistance of V-complex on external interference.

450

3.6 Rheological properties

451

3.6.1 Flow behavior of apparent viscosity

452

Flow behavior of lotus seed starch and its complexes after long-term

453

retrogradation are shown in Figure 4 A. The apparent viscosity of the retrograded

454

starch showed a downward trend with the increase of shear rate, indicating that all the

455

samples conformed to shear-thinning behavior. Furthermore, with the increasing of

456

homogenization pressure, the retrograded starch had a significant change on the

457

rheological properties. When the homogenization pressure approached a higher level

458

(100 MPa), the pseudoplasticity of retrograded starch was weakened. This finding

459

was in accordance with previous research (Wang, Li, Wang, Liu, & Adhikari, 2012),

460

which showed that high homogenization treatment would fracture the amylose chains

461

and the branches of amylopectin, resulting in the flexible movement of the degraded

462

macromolecules and the weakening of starch flow properties. The results of our FTIR

463

experiments were also in agreement with those findings, as severely sheared starch

464

chains were difficult to reconstitute to form a stable gel structure after long-term

465

retrogradation, causing the change of flow properties. When GMS was added into the

466

starch system, the formation of V6II complexes (LS-GMS-50) further weakened the

467

flow properties of the retrograded starch. This was especially evident when the

468

homogenization pressure was further increased and the flow behavior of

469

V6I-complexes formed by 100 MPa had a lower pseudoplasticity than the

470

V6II-complexes relatively. These results revealed that the presence of V-complexes

471

would significantly change the flow properties of the retrograded starch, making it

472

closer to the fluidity of the water during long-term retrogradation. In addition, the V6I

473

complexes had superior improvement compared to V6II complexes, which was

474

consistent with a previous study (Zia ud, Xiong, Wang, Chen, & Ullah, 2019) that

475

showed a high complex index of the corn starch-sucrose fatty acid complexes had

476

positive effects on the flow properties of corn starch.

477

3.6.2 In-shear structural recovery analysis

478

The thixotropy of lotus seed starch and its V-complexes after long-term

479

retrogradation are shown in Figure 4 B. The thixotropic ring was considered to be an

480

important indicator of the recovery of starch gel structure. Figure 4 B shows that the

481

native retrograded starch exhibited a small thixotropic ring area, which indicated that

482

the starch gel had better structural elasticity after long-term retrogradation. This result

483

was interpreted by the studies reported by Colussi, Kaur, Zavareze, Dias, Stewart, &

484

Singh (2018), in which the compact structure of retrograded starch was primarily

485

connected with the re-associated and ordered structure of amylose and amylopectin

486

chains during retrogradation. However, as the homogenization pressure gradually

487

increased, the thixotropic ring area of the starch gel was larger than before, implying

488

that high homogenization pressure would make the starch gel structure difficult to

489

return to the original structure, which was directly related to the inhibition of amylose

490

and amylopectin recrystallization. This was supported by the NMR results that the

491

weakening of the starch network strength and the reduction of the crystallization area

492

were attributed to the force of high homogenization pressure. In addition, a study

493

conducted by Li, Zhu, Mo, & Hemar (2019) showed that high hydrostatic pressure

494

contributed to the reduction of the maize starch crystallization region and the

495

formation of a loose structure. This trend as strongly inhibited by the formation of

496

LS-GMS. The V-type complexes were very effective in suppressing the increase in

497

the area of the starch thixotropic ring under the homogenization pressure treatment,

498

and the V6I complexes compared to other samples exhibited a lower thixotropic level.

499

These results demonstrated that V-type complexes would greatly improve the

500

structural elasticity of the starch gel. Compared to other samples, V6I complexes had

501

the predominant shear resistance to prevent shear thinning, which was consistent with

502

a previous study (Liu, Chi, Huang, Li, & Chen, 2019), which showed that type II

503

V-complexes (V6I complexes) between high amylose corn starch and lauric acid

504

formed by high pressure treatment had more intact and ordered structures than

505

V-complexes formed by ordinary pressure.

506

3.6.3 Dynamic rheological properties

507

The curves of the storage modulus and loss modulus, which were used to

508

characterize viscoelasticity, are shown in Figure 4 C. The storage modulus value (G')

509

of native retrograded starch was much higher than the loss modulus value (G''), which

510

was a feature of rigid structure. When the starch gel was homogenized by

511

microfluidization, the storage modulus (G') and the loss modulus (G'') decreased with

512

the increase of homogenization pressure (Figure 6 C), indicating that the starch gel

513

gradually developed towards a weak and amorphous gel. This observation was in

514

accordance with a previous study (Hussain, Vatankhah, Singh, & Ramaswamy, 2016),

515

where high-pressure treatment would strongly destroy the non-covalent bonds of corn

516

starch gel, thus weakening the viscoelastic properties of starch. Importantly, the

517

formation of the V-type complex further promoted the continuation of this trend

518

compared with the control (Figure 4 C). A study conducted by Meng, Sun, Fang,

519

Chen, & Li (2014) suggested that rice starch-sucrose fatty acid V-complexes

520

improved the rigid structure of the starch and weakened the colloidal joint of starch to

521

some extent, resulting in the retardation of amylopectin recrystallization by reducing

522

the mobility of molecular chains during long-term retrogradation.

523

The loss tangent (tanδ) was used as an analytical indicator of gel structure. As

524

shown in Figure 4 D, the overall trend of loss tangent constantly increased with the

525

increased of homogeneous pressure and reached the highest value at 100 MPa,

526

indicating that the rigid gel structure of starch was weakened by the high

527

homogenization pressure, which was in agreement with results obtained from our

528

previous study (Guo et al., 2015) revealed that ultra-high pressure treatment could

529

significantly weaken the intensity of the gel network structure of starch, resulting in

530

the change of viscoelasticity and the retardation of lotus seed starch retrogradation.

531

When the GMS was added to the starch system, the loss tangent of the V-type

532

complexes was obviously lower than other samples without GMS, and the effects of

533

V6I-complexes

534

(LS-GMS-50) (Figure 4 D). Those findings suggested that the formation of V6I

535

complexes not only protect the starch network structure from the high shear force

536

from microfluidization, but also greatly improved rigid gel structure and balanced the

537

viscoelasticity of starch. From the perspective of flow plasticity, the amylose-lipid

538

complex was more advantageous as a resistant starch. This finding was in accordance

539

with a study conducted by Oyeyinka, Singh, Ma, & Amonsou (2016) that the bambara

540

starch-lysophosphatidylcholine complexes blocked the interaction between starch

541

molecules and inhibited the formation of double helixes of amylose and amylopectin,

542

as well as impacted the junction zones and gel network during retrogradation, leading

543

to the stability of gel structure and the improvement of viscoelasticity.

(LS-GMS-100)

were

more

significant

than

V6II-complexes

544 545

3.7 Insight into the mechanism of lotus seed starch and its V-complexes after

546

long-term retrogradation

547

The starch component (amylose and amylopectin) and water molecules of native

548

starch are evenly distributed in the starch system. After long-term retrogradation (28

549

d), the recrystallization of amylopectin with amylose as the seed nucleus is formed

550

into a rigid gel structure, and the mass of water molecules are precipitated from the

551

starch gel, which is caused by the shrinkage of the starch gel structure. When the

552

GMS is added into the starch system, the single helix of amylose decomposed by

553

homogenization pressure treatment can be combined with GMS, but the complex

554

index of V6II-complexes formed at 50 MPa is at a low level. Despite this situation,

555

after long-term retrogradation (28 d), V6II-complexes still have the ability to inhibit

556

the recrystallization of amylopectin and improve the water retention of starch gel,

557

which is due to the reduction of the amylose double helix and the formation of

558

hydrogen bonds between hydrophilic hydroxyl groups of the V6II-complexes and

559

starch. When the homogenization pressure reaches 100 MPa, high homogenization

560

pressure promotes the formation of V6I-complexes, leading to a high complex index.

561

After the long-term retrogradation (28 d), V6I-complexes improve the gel structure of

562

the starch and make the structure more ordered and stretchier, which enhances the

563

water retention of starch gel to a large extent and inhibits the long-term retrogradation

564

of lotus seed starch.

565

4. Conclusions Our objective was to investigate physicochemical and structural properties of

566 567

lotus

seed

starch-glycerin

monostearin

V-type

568

retrogradation. The results determined by

569

formation of microcrystalline V-complexes could protect the gel structure from high

13C-NMR

complexes

after

long-term

and FTIR revealed that the

570

shear force while contributed to the stability of the crystal structure during long-term

571

retrogradation, and the V6I-complexes had the superior ability to make the starch

572

structure more compact and uniform than V6II complexes. Additionally, 1H-NMR

573

indicated that high homogenization pressure could cause more free water to

574

precipitate from the gel structure, but the presence of V-complexes enhanced the

575

water holding capacity of starch gel due to the hydrogen bond between hydrophilic

576

hydroxyl groups of the complex and starch. AFM showed that the shearing force of

577

high homogenization pressure decomposed the length of amylose into shorter chains

578

and made it difficult to reorganize the starch structure after long-term retrogradation,

579

leading to the reduction of the aggregation size. Nevertheless, the formation of

580

V-complexes further inhibited the aggregation of the molecular chain and converted

581

the molecular chain to the nanoscale. As determined with DSC, V-complexes had

582

lower enthalpy value and thermal sensitivity than other samples without GMS,

583

meaning that V-complexes increased the heat absorption temperature of starch and

584

decreased the heat resistance of starch, which was due to the inhibition of amylopectin

585

recrystallization. The results of the rheological measurement showed that simple

586

homogenization pressure could weaken the rigid gel structure and viscoelasticity of

587

lotus seed starch, but with the formation of V-complexes, the gel intensity and

588

viscoelasticity of starch would be greatly improved and balanced. In general, there is

589

a close relationship between the water holding capacity and structural properties of

590

starch. Based on the results of structural and physicochemical determination, the

591

formation mechanism was established as the precipitation of free water from native

592

retrograded starch was due to the shrinkage of starch gel structure, but the formation

593

of V-complexes improved and optimized the internal network structure, led to the

594

high viscoelasticity of starch branches to hold more water molecules in the starch

595

granules, and convert free water into bound water after long-term retrogradation. The

596

results provided a theoretical basis for the storage and processing of lotus seed starch

597

as well as prompted V6I complex as a new type of resistant starch.

598

Conflict of interest

599 600 601

The authors declare no conflict of interest. Acknowledgements This work was supported by the Project of International Cooperation and

602

Exchanges in Science and Technology of Fujian Agriculture and Forestry University

603

(grant number KXGH17001), the National Natural Science Foundation of China

604

(grant number 31871820 and 31701552), the Support Project for Distinguished

605

Young Scholars of Fujian Agriculture and Forestry University (grant number

606

xjq201714), the Program for Leading Talent in Fujian Provincial University (grant

607

number 660160190) and Program for New Century Excellent Talents in Fujian

608

Province University (grant number KLA18058A).

609

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610 611 612 613 614 615 616 617 618 619 620

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different methods. Food Chemistry, 186, 213-222. Zhang, B., Huang, Q., Luo, F. X., & Fu, X (2012). Structural characterizations and digestibility of debranched high-amylose maize starch complexed with lauric acid. Food Hydrocolloids, 28, 174-181. Zhang, W. H., Wang, J., Guo, P. P., Dai, S. S., Zhang, X. Y., Meng, M., Shen, S. G., Zhang, A. X., & Dou, H. Y (2019). Study on the retrogradation behavior of starch by asymmetrical flow field-flow fractionation coupled with multiple detectors. Food Chemistry, 277, 674-681. Zhang, Y., Zeng, H. L., Wang, Y., Zeng, S. X., & Zheng, B. D (2014). Structural characteristics and crystalline properties of lotus seed resistant starch and its prebiotic effects. Food Chemistry, 155, 311-318. Zhao, Y. T., Jiang, Y. J., Zheng, B. D., Zhuang, W. J., Zheng, Y. F., & Tian, Y. T (2017). Influence of microwave vacuum drying on glass transition temperature, gelatinization temperature, physical and chemical qualities of lotus seeds. Food Chemistry, 228, 167-176. Zia, U. D., Xiong, H. G., Wang, Z. J., Chen, L., & Ullah, I (2019). Effects of sucrose fatty acid ester addition on the structural, rheological and retrogradation behavior of high amylose starch-based wood adhesive. International Journal of Adhesion and Adhesives, 89, 51-58.

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure captions:

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Figure 1 Ordered structural properties of lotus seed starch- glycerin monostearin

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complexes after long-term retrogradation by (A) 13C CP/MAS NMR and (B) FTIR

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Figure 2 Water distributions and microscopic images of lotus seed starch-glycerin

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monostearin complexes after long-term retrogradation by (A) LF-1H NMR and (B)

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AFM

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Figure 3 Thermal properties of lotus seed starch-glycerin monostearin complexes after

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long-term retrogradation by DSC

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Figure 4 Rheological properties of lotus seed starch-glycerin monostearin complexes

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after long-term retrogradation by rheometer

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Figure 5 Machanism of lotus seed starch-glycerin monostearin complexes on

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long-term retrogradation

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Table 1 Structural parameters of lotus seed starch- glycerin monostearin complexes LS

LS-50

LS-100

LS-GMS-50

LS-GMS-1 00

C1

16.75±0.02a

15.67±0.05d

14.97±0.02e

15.93±0.02c

16.12±0.02b

C4

2.78±0.03c 51.62±0.05

2.62±0.02d

2.44±0.02e

4.12±0.01b

4.56±0.02a

C2,3,5

b

51.01±0.02d 1.080±0.008

50.88±0.03e 1.058±0.01 5c 1.304±0.01 1c 1.253±0.00 2d

51.46±0.02c 1.063±0.006

52.32±0.02a 1.032±0.004

bc

d

1.342±0.027

1.185±0.026

c

d

1.268±0.001

1.275±0.002

b

a

995cm-1/1022cm-1 991cm-1 1047cm-1/1022cm-1

1.082±0.01 2a 1.435±0.02 1a 1.262±0.00 3c

ab

1.421±0.017 b

1.261±0.002 c

4.22±0.01c 13.21±0.03

4.21±0.02c

6.19±0.03b

3.13±0.02d

8.75±0.02a

d

12.03±0.03c

5.67±0.03e

35.75±0.02b

43.10±0.02a

b

83.76±0.03c

88.14±0.04a

61.12±0.04d

48.15±0.04e

72.2±0.1c

71.6±0.1d

72.5±0.3bc

75.1±0.2a

72.8±0.3b

Tp (℃)

77.1±0.2c

76.2±0.1e

77.5±0.1b

78.6±0.2a

76.7±0.2d

Tc (℃)

81.6±0.2b

81.8±0.1b

81.3±0.1c

82.6±0.2a

80.4±0.1d

90.9±0.1b

91.5±0.1a

Tp (℃)

95.8±0.1b

96.7±0.1a

Tc (℃)

99.6±0.1b 3.249±0.085

100.8±0.1a 3.067±0.042

d

e

A21(bound water)(%) A22(immobile water)(%)

82.57±0.03 A23(free water)(%) PeakI

PeakII

T0 (℃)

T0 (℃)

ΔH(J/g)

6.796±0.01 7a

4.877±0.024 b

3.829±0.04 3c

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Different letters in the same row represent significant differences between different treatments (p < 0.05). Where C1 is peak area of the vibration peak in the C1 region, C4 is peak area of the vibration peak in the C4 region, C2,3,5 is peak area of the vibration peak in the C2,3,5 region, 995cm-1/1022cm-1 is the degree of amylose double helix, 991cm-1 is the degree of amylopectin double helix, 1047cm-1/1022cm-1 is the ordered degree of starch structure, A21 is the content of bound water of retrograded starch gel, A22 is the content of immobile water of retrograded starch gel, A23 is the content of free water of retrograded starch gel, To is the initial temperature of retrograded sample, Tp is the peak temperature of retrograded sample, Tc is the termination temperature of retrograded sample, ΔH is the enthalpy value of retrograded sample.

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Author Contributions： 37

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Yixin Zheng: Carrying out the experiments, Performing the data analyses, WritingOriginal draft preparation, Editing. Bailong Wang: Collecting test data, Performing the data analyses. Zebin Guo: Interpreting the results and Revising the paper. Yi Zhang: Revising the paper. Baodong Zheng: Helping perform the analysis with constructive discussions, Supervision. Shaoxiao Zeng: Leading the relevant project, Designing the study, Writing- Reviewing and Editing. Hongliang Zeng: Leading the relevant project, Designing the study, Writing- Reviewing and Editing

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 LS with GMS improved formation of crystallinity and an ordered structure of

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starch.

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 V6I-complexes had superior ability to enhance water retention of starch than V6II.

811

 V-complexes inhibited aggregation of molecular chain and changed it to

812 813 814 815

nanoscale.  V-complexes improved heat sensitivity and flow behavior than other crystal samples.  V-complexes with great water retention improved physicochemical properties.

816

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