Accepted Manuscript

Characterization and comparative study of starches from seven purple sweet

potatoes

Long Zhang, Linglong Zhao, Xiaofeng Bian, Ke Guo, Lu Zhou, Cunxu Wei

PII: S0268-005X(17)31945-8
DOI: 10.1016/j.foodhyd.2018.02.006
Reference: FOOHYD 4265

To appear in: Food Hydrocolloids

Received Date: 20 November 2017

Revised Date: 3 February 2018
Accepted Date: 6 February 2018

Please cite this article as: Zhang, L., Zhao, L., Bian, X., Guo, K., Zhou, L., Wei, C., Characterization
and comparative study of starches from seven purple sweet potatoes, Food Hydrocolloids (2018), doi:
10.1016/j.foodhyd.2018.02.006.

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1 Characterization and comparative study of starches from seven purple sweet potatoes

2 Long Zhanga,b,1, Linglong Zhaoa,b,1, Xiaofeng Bianc,1, Ke Guoa,b, Lu Zhoua,b, Cunxu Weia,b,*

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3 Key Laboratory of Crop Genetics and Physiology of Jiangsu Province / Key Laboratory of

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4 Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou

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5 225009, China

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6 Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu

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7 Province / Joint International Research Laboratory of Agriculture & Agri-Product Safety of

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8 the Ministry of Education, Yangzhou University, Yangzhou 225009, China
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9 Institute of grain crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

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10 These authors contributed equally to this work.
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11 Corresponding Author: C. Wei, E-mail: cxwei@yzu.edu.cn
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13 Abstract

14 Sweet potato starch has great edible and economic value and has been widely studied.

15 Starches from different varieties have significantly different properties. Previous studies are

16 focused on white, yellow and orange sweet potato. In this study, starches were isolated from

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17 purple sweet potato variety Ningzi 1 and six advanced breeding lines with different genotype,

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18 their structural and functional properties were investigated and compared. Seven starches all

19 had round, polygonal, oval and semi-oval shapes with central hila, but showed different

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20 granule sizes. The starch content in dry root tubers varied from 40.1 to 55.1%. The apparent

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21 amylose content ranged from 24.6 to 31.0%. The seven sweet potatoes all had CA-type starch.
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22 The swelling power and water solubility at 95 °C ranged from 22.5 to 29.0 g/g and 13.1 to

23 16.9%, respectively. The starches displayed a two peak gelatinization thermogram, the first
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24 peak temperature ranged from 64.1 to 69.0 °C and the second peak temperature ranged from
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25 73.5 to 80.8 °C. The peak viscosity, hot viscosity, breakdown viscosity and setback viscosity
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26 varied from 2535 to 3750 mPa s, 1517 to 2450 mPa s, 1018 to 1622 mPa s, and 474 to 1036

27 mPa s, respectively. The seven starches had different hydrolysis degrees to porcine pancreatic
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28 α-amylase and Aspergillus niger amyloglucosidase. All the starches had high resistance to the
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29 in vitro digestion. This study could provide important information for the utilization of purple
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30 advanced breeding lines in breeding and food and nonfood industries.

31 Keywords: purple sweet potato; starch; structural properties; functional properties.

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33 1. Introduction

34 Sweet potato [Ipomoea batatas (L.) Lam.], a dicotyledonous plant in the family

35 Convolvulaceae, originates in Latin America and nowadays has become an important

36 economic crop in many Asia, Africa and Latin America countries, especially in China. Sweet

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37 potato provides huge food for developing countries to prevent malnutrition and enhance food

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38 security, as its extensive adaptability and relatively rich nutrition (Bovell-Benjamin, 2007).

39 Therefore, a large number of sweet potato varieties have been cultivated, for example, more

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40 than 2000 varieties are grown in China. Starch is the main component of sweet potato, and its

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41 content in dry root tuber varies from 50 to 80% in different varieties (Abegunde, Mu, Chen, &
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42 Deng, 2013; Zhu, Yang, Cai, Bertoft, & Corke, 2011). In sweet potato, starch is synthesized

43 through a complex pathway regulated by multiple starch-synthesizing enzymes, including,

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44 ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble

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45 starch synthase (SSS), starch branching enzyme (SBE), starch de-branching enzyme (DBE),
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46 and starch phosphorylase (SP). Starch properties depend on the coordinated action of all these

47 starch-synthesizing enzymes rather than on individual enzyme (Zhang et al., 2017).

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48 Sweet potato starch is widely used in food and nonfood industries, such as noodles,
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49 baking foods, fast food, candy products, textile industry, and alcohol production
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50 (Bovell-Benjamin, 2007; Chen, Schols, & Voragen, 2010). The structural and functional

51 properties of starch determine its application. Therefore, in the past few decades, a lot of

52 studies have been carried out on the properties of sweet potato starch, including morphology

53 (Lee & Lee, 2016; Tian, Rickard, & Blanshard, 1991; Zhu et al., 2011), granule size (Tian et

54 al., 1991; Zhang & Oates, 1999), amylose content (Chen et al., 2010), crystalline structure

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55 (Kim, Ren, & Shin, 2013; Takeda, Tokunaga, Takeda, & Hizukuri, 1986), water binding

56 capacity and swelling volume (Chen et al., 2010; Osundahunsi, Fagbemi, Kesselman, &

57 Shimoni, 2003), thermal and pasting properties (Tian et al., 1991), and hydrolysis and

58 digestion properties (Liu, Donner, Yin, Huang, & Fan, 2006; Zhang & Oates, 1999). The

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59 properties of flours and/or starches from different varieties in China (Abegunde et al., 2013;

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60 Chen et al., 2010; Zhu et al., 2011), Korea (Kim et al., 2011, 2013), Japan (Kohyama &

61 Nishinari, 1991; Noda, Takahata, Sato, Ikoma, & Mochida, 1996), Sri Lanka (Senanayake,

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62 Ranaweera, Gunaratne, & Bamunuarachchi, 2013a, b), Philippines (Collado et al., 1999),

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63 Papua New Guinean and Australian (Waramboi, Dennien, Gidley, & Sopade, 2011), Nigeria
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64 (Osundahunsi et al., 2003), and Caribbean (Aina, Falade, Akingbala, & Titus, 2009) have

65 been investigated and compared. These studies showed that starches from different varieties
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66 have significantly different properties. Sweet potato has white, yellow, orange, and purple
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67 root tuber (Tang, Cai, & Xu, 2015). The previous studies are focused on white, yellow, and
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68 orange sweet potato. To our knowledge, there is a lack of documentation for starch properties

69 of purple sweet potato with different genotypes. Purple sweet potato is rich in anthocyanins
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70 and starch. The starch degradation provides abundant substrates for anthocyanin biosynthesis
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71 in root tuber of purple sweet potato. The generated anthocyanin in the cytosol is transported
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72 into and stored in the vacuoles (Wang et al., 2016). Purple sweet potato is becoming more

73 favored by consumers due to the function of scavenging free radical, antimutagenicity,

74 anticarcinogen, and antihypertensive. Therefore, its variety selection and improvement have

75 become one of the breeding strategies of sweet potato in China (Zhang, Wang, Liu, & Wang,

76 2009).

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77 Recently, we have developed some purple sweet potato advanced breeding lines by

78 hybridization. In this study, starches were isolated from the purple sweet potato root tubers (a

79 variety and six advanced breeding lines), their physicochemical properties were investigated

80 and compared. Our objective was to reveal the structural and functional properties of the

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81 purple sweet starches and provide some information for the further utilization of these

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82 advanced breeding lines in breeding and food and nonfood industries.

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83 2. Materials and methods

84 2.1. Plant materials

85
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Purple sweet potato variety Ningzi 1 and six advanced breeding lines were used in this
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86 study (Table 1). The advanced breeding lines were developed through different parental
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87 hybridization. These materials were grown under normal agronomic practices in the

88 experimental field of Jiangsu Academy of Agricultural Sciences, Nanjing, China in 2016. The
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89 root tubers after harvest were stored at 12 °C before use.

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90 2.2. Measurement of water, starch, soluble sugar and protein contents in root tuber
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91 Fresh root tubers were photographed with a Nikon Coolpix L22 camera. The water

92 content of fresh root tuber was determined by oven-drying method. The contents of soluble
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sugar and starch were measured following the method of Gao et al. (2014). Briefly, the fresh
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94 root tubers were washed, peeled and sliced into small pieces. The pieces were immediately

95 put in oven at 105 °C for 2 h to inactivate the enzymes including α and β amylases and at

96 80 °C for 2 d to obtain the dry sample. The sample was extensively ground and passed

97 through a 100-mesh sieve to obtain the flour. The soluble sugar in the flour was extracted

98 three times using 80% ethanol, and the ethanol-insoluble starch was hydrolyzed using HClO4.

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99 Both the soluble sugar and hydrolyzed starch were determined using anthrone-H2SO4 method.

100 The protein content (N×6.25) in dry flour was measured using an Elementar Analysensysteme

101 Gmbh Vario EL cube CHN-Nitrogen analyser.

102 2.3. Starch isolation

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103 Starch was isolated from fresh root tubers following the method described by Gao et al.

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104 (2014) with slight modifications. The root tubers were washed and cut into small pieces. The

105 pieces were homogenized in deionized water using a home blender. The homogenate was

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106 filtered with 100-, 200-, 300-, and 400-mesh sieves, successively. The starch was treated with

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107 0.2% NaOH to remove the surface protein and anthocyanin many times until that the
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108 supernatant was clear and colorless. The starch precipitate was washed with deionized water

109 and dehydrated with anhydrous ethanol. Finally, the samples were dried at 40 °C, ground into
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110 powders, and passed through a 100-mesh sieve.

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111 2.4. Morphology observation of starch

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112 For micromorphology and Maltese cross observation, the starch suspension in 50%

113 glycerol was viewed with an Olympus BX53 polarized light microscope under normal and
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114 polarized light. For surface structure observation, starch granules were coated with gold and
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115 viewed using a scanning electron microscope (Hitachi S-4800).

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116 2.5. Granule size analysis of starch

117 Granule size of starch was measured using a laser diffraction instrument (Mastersizer

118 2000, Malvern) following the method of Cai et al. (2014b).

119 2.6. Measurements of iodine absorption spectrum, iodine blue value, and apparent amylose

120 content

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121 The starch-iodine absorption spectrum was determined using an Ultrospec 6300 pro

122 spectrophotometer (Amersham Bioscience, Cambridge, UK). The maximum absorption

123 wavelength (λmax), iodine blue value, and apparent amylose content were calculated

124 according to the method of Man et al. (2014).

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125 2.7. Crystalline structure analysis of starch

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126 The crystalline structure of starch was analyzed using an X-ray powder diffractometer

127 (XRD) (D8, Bruker, Germany) according to the method of Wei et al. (2010a). The relative

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128 crystallinity was the ratio of the crystallinity area to the total diffraction area.

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129 2.8. Short-range ordered structure analysis of starch
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130 The ordered structure of starch external region was analyzed using an attenuated total

131 reflectance-Fourier transforms infrared (ATR-FTIR) spectrometer (Cary 610/670, Varian,

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132 USA) following the method of Wei et al. (2010b).

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133 2.9. Swelling power and water solubility determination of starch

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134 The swelling power and water solubility of starch were measured at 75, 85 and 95 °C

135 using the method of Konik-Rose et al. (2001).

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136 2.10. Thermal property analysis of starch

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137 Three mg of starch was mixed with 9 µL distilled water and sealed in an aluminium pan.
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138 After equilibrating for 2 h at room temperature, samples were heated from 25 to 130 °C at a

139 rate of 10 °C/min using a differential scanning calorimetry (DSC, 200-F3, Netzsch,

140 Germany).

141 2.11. Pasting property analysis of starch

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142 The pasting properties of starch were determined using a rapid visco analyzer (RVA, 3D,

143 Newport Scientific, Narrabeen, Australia). Starch suspension (8% solids) was first held at

144 50 °C for 1 min and then heated to 95 °C at a rate of 12 °C/min. The temperature was held at

145 95 °C for 2.5 min, and then the starch was cooled down to 50 °C at a rate of 12 °C/min and

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146 held at 50 °C for 1.4 min.

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147 2.12. Enzyme hydrolysis analysis of starch

148 The starch was hydrolyzed by porcine pancreatic α-amylase (PPA, Sigma A3176) or

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149 Aspergillus niger amyloglucosidase (AAG, Megazyme E-AMGDF) according to the method

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150 of Fan et al. (2016). AN
151 2.13. In vitro digestion analysis of starch

152 In vitro digestion of native, gelatinized and retrograded starches was analyzed following
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153 the method of Fan et al. (2016). For briefly, 1% (w/v) starch suspension was heated at 98 ºC
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154 for 12 min to obtain the gelatinized starch, and the gelatinized starch recrystallized at 4 ºC for
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155 36 h to prepare the retrograded starch. The starch was digested by both PPA (Sigma A3176)

156 and AAG (Megazyme E-AMGDF) at 37 ºC for 20 or 120 min. The released glucose was
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157 measured using the glucose assay kit (Megazyme, K-GLUC). The rapidly digestible starch
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158 (RDS, digested within 20 min), slowly digestible starch (SDS, digested between 20 and 120
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159 min) and resistant starch (RS, undigested after 120 min) were determined according to the

160 digestion rate.

161 2.14. Statistical analysis

162 The data reported in all the tables were means ± standard deviation (means ± SD). The

163 one-way analysis of variance (ANOVA) by Tukey’s test was evaluated using the SPSS 16.0

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164 Statistical Software Program.

165 3. Results and discussion

166 3.1. Water, starch, soluble sugar, and protein contents of root tuber

167 The fresh root tuber photos of seven purple sweet potatoes are shown in Fig. 1. The

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168 transverse section of root tuber showed that Ningzi 1, X66-1, X83-1, X85-15, and Y79-4 had

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169 mixed purple and white color, while Y16-5 and Y46-4 were purple completely. The contents

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170 of water, starch, soluble sugar, and protein are summarized in Table 1. All the fresh root tubers

171 contained high water content, and ranged from 62.6 to 73.6% with the lowest in X85-15 and

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the highest in X66-1. The result was similar to that (65.0-74.6%) reported by Senanayake et al.
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173 (2013a). For starch content, Y79-4 was the lowest (40.1%) and X85-15 was the highest
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174 (55.1%). The starch content is one important trait of high agronomic importance in sweet

175 potato, and is controlled by genetic and environmental factors. The starch content varies
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176 greatly among genotypes, but for each genotype, it is relatively stable among different
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177 planting years or environments, indicating that the genotype has a larger effect than does the
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178 environment on starch content (Lu et al., 2015). In the present study, both X83-1 and X85-15

179 had significantly higher starch content than the other breeding lines, suggesting that starch
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biosynthesis was more efficient in X83-1 and X85-15 than in the others. Therefore, the use of
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181 X83-1 and X85-15 should be encouraged. A recent study shows that both water content and

182 starch accumulation in developing root tuber of sweet potato affect the starch content in

183 different genotypes (Zhang et al., 2017). For soluble sugar content, X85-15 was the lowest

184 (11.6%) and Ningzi 1 was the highest (21.7%). The result was slightly higher than a few

185 Japanese varieties, which contained high starch content (Kohyama & Nishinari, 1991). The

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186 protein content was low in all the purple sweet potatoes and ranged from 1.91 to 3.84%,

187 which was similar to previous reports in five Sri Lanka sweet potato varieties (Senanayake et

188 al., 2013a).

189 3.2. Morphology and size distribution of starch

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190 The observation of polarized light microscope and scanning electron microscope showed

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191 that the starch granules in seven purple sweet potatoes were similar, had round, polygonal,

192 oval, and semi-oval shapes, and contained small and large granules (Fig 1). The result was in

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193 agreement with the previous report in different sweet potato varieties (Chen et al., 2010; Kim

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194 et al., 2013; Lee & Lee, 2016; Senanayake et al., 2013b). All the starch granules had the
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195 typical “Maltese crosses” with the hila in the center of granules (Fig 1). Though the volume

196 distributions of seven starches were significantly different, they all showed bimodal size
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197 distributions. Most of starch granules ranged from 5 to 30 µm. Y16-5 and Ningzi 1 starches
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198 had the smallest and the largest granule size among seven starches, respectively (Table 2).
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199 The difference in granule size could be due to different genotype backgrounds. The median

200 diameters ranging from 12 to 22 µm and granule sizes ranging from 3 to 60 µm were also
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201 reported in sweet potato starch using Shimadzu centrifugal particle size analyzer (Walter,
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202 Truong, Wiesenborn, & Carvajal, 2000) and scanning electron microscopy (Osundahunsi et
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203 al., 2003). The significant difference of starch granule size in different papers was mainly due

204 to the different assay method.

205 3.3. Iodine absorption spectrum and amylose content of starch

206 The absorbance spectra of starch-iodine complex were significantly different among

207 seven purple sweet potato starches (Fig. 2A). The maximum absorption wavelength, iodine

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208 blue value, and apparent amylose content are summarized in Table 2. The apparent amylose

209 content ranged from 24.6 to 31.0%. Y46-4 had the lowest apparent amylose content and

210 Ningzi 1 had the highest apparent amylose content. Many papers report that the amylose

211 content ranged from 15.3 to 21.1% in four Japanese sweet potatoes (Noda et al., 1996), from

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212 23.3 to 26.5% in eleven China sweet potatoes (Zhu et al., 2011), and from 14.7 to 30.5% in

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213 eight Korean sweet potatoes (Kim et al., 2013). The significantly different amylose content in

214 different varieties seemed to stem mainly from the different genotype backgrounds. The

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215 amylose content and amylose to amylopectin ratio are important factors affecting starch

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216 structure and properties, which determine the applications of starch and the characteristics of
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217 final products. Therefore, starch with variable amylose content is of interest. The biosynthesis

218 of amylose mainly involves GBSS and SBE for the small number of long chain branches,
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219 while three types of enzymes are essential for amylopectin: SSS, SBE and DBE (Lai et al.,
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220 2016). The sweet potato plants with significantly different amylose contents have been
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221 produced by changing the expression of genes encoding sweet potato GBSS and SBE

222 (Kitahara et al., 2007). The variation in the expression of GBSS and SSS genes may directly
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223 influence the amylose to amylopectin ratio among different sweet potato genotypes, but the
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224 expression of genes encoding other starch-synthesizing enzymes shows no direct correlation
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225 with starch composition (Zhang et al., 2017).

226 3.4. Crystalline structure of starch

227 The XRD spectra of seven purple sweet potato starches are showed in Fig. 2B. Plant

228 starches are usually divided into A-, B-, and C-type according to their XRD spectra. The

229 branch-chains of amylopectin form A- and B-type crystallinity, with amylopectin short

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230 branch-chains and closed branching points forming A-type crystallinity and amylopectin long

231 branch-chains and distant branching points forming B-type crystallinity. A- and B-type

232 starches contain only A- and B-type crystallinity, respectively, while C-type starch is a

233 mixture of both A- and B-type crystallinity (He & Wei, 2017). According to the proportion of

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234 A- and B-type crystallinity, C-type starch can further be divided into CA-type (closer to

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235 A-type), typical C-type, and CB-type (closer to B-type). The typical C-type starch shows

236 strong diffraction peaks at about 17° and 23° 2θ, and small peaks at about 5.6° and 15° 2θ.

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237 Compared with typical C-type starch, CA-type starch has a shoulder peak at about 18° 2θ, and

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238 CB-type starch has two shoulder peaks at about 22° and 24° 2θ (Cai, Cai, Man, Zhou, & Wei,
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239 2014a; He & Wei, 2017). According to XRD patterns, all the seven sweet potatoes had

240 CA-type starch (Fig.2B), but their relative crystallinities were different (Table 2). The about 30%
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241 relative crystallinity is also reported in sweet potato starch (Waramboi et al., 2011). A-, CA-,
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242 CB- and C-type starches are all reported in sweet potato in previous papers. For examples,
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243 Takeda et al. (1986) and Waramboi et al. (2011) reported that some sweet potato varieties

244 from Japan and Australia had A-type starch. Lee and Lee (2016) reported that the orange,
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245 white, and purple sweet potatoes contained CA-type starch. Tian et al. (1991) reported that the
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246 native sweet potato had CA-type or typical C-type starch, which depended on the specific
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247 samples. Kim et al. (2013) reported the eight Korean sweet potatoes had A- and CB-type

248 starches. The crystalline type of sweet potato starch is not only affected by genotype, but also

249 affected by growth temperature. The sweet potato grown at 15 °C soil has C-type starch,

250 while that at 33 °C soil has A-type starch (Genkina et al., 2003). In addition, suppressing

251 SBEII expression in sweet potato can decrease branch-chains with more than DP 100 and

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252 around DP 6-11 and increase branch-chains around DP 12-15 and DP 24-33 in amylopectin,

253 and change crystalline structure from C-type to B-type (Kitahara et al., 2007).

254 3.5. Ordered structure of starch

255 The ATR-FTIR spectrum can reflect the short ranged ordered structure in starch external

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256 region. The ratio of absorbance 1045/1022 cm-1 is usually used to quantify the ordered degree,

and that of 1022/995 cm-1 can be used as a measure of the proportion of amorphous to ordered

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258 carbohydrate structure in the starch (Sevenou, Hill, Farhat, & Mitchell, 2002). The

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259 deconvoluted ATR-FTIR spectra of seven purple sweet potato starches are presented in Fig.

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260 2C, and their ratios of 1045/1022 cm-1 and 1022/995 cm-1 are shown in Table 2. The IR ratio
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261 of 1045/1022 cm-1 ranged from 0.631 to 0.671 with the lowest for Y16-5 and the highest for

262 Y79-4. The ratio of 1022/995 cm-1 ranged from 0.769 to 0.797, and displayed no significant
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263 differences among the seven purple sweet potato starches. The correlation analysis between
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264 granule size and IR ratio of 1045/1022 cm-1 indicated that the short-range degree was
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265 significantly positively correlated with granule size (P < 0.01), which was in agreement with

266 the results of Cai et al. (2014a).

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267 3.6. Swelling power and water solubility of starch

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268 The swelling power and water solubility of starches at different temperatures (75, 85, and
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269 95 °C) are summarized in Table 3. In general, swelling power and water solubility increased

270 with temperatures and the great variation was observed at 75 and 85 °C. Similar trend was

271 observed by previous researchers on diverse sweet potato starches (Zhu et al., 2011). The

272 swelling power of starches at 95 °C ranged from 22.5 to 29.0 g/g and water solubility from

273 13.1 to 16.9%. The swelling power is a measure of the water-holding capacity of starch after

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274 being heated in water, cooled, and centrifuged, while the water solubility reflects the degree of

275 dissolution during the starch swelling procedure (Carcea & Acquistucci, 1997). The hydration

276 and swelling of starch during heating reflects the magnitude of interaction between starch

277 chains. The amylose to amylopectin ratio may affect the extent of this interaction, resulting in

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278 variation in the swelling power and solubility of starch. The amylose can act as an inhibitor of

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279 starch granule swelling and hinder the disruption of amylopectin double helices (Lai et al.,

280 2016). The swelling power in eleven sweet potato starches ranged from 13.3 to 20.4 g/g (Zhu

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281 et al., 2011), which was slightly lower than the results of the present study. The starches from

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282 forty-four sweet potato genotypes in the Philippines showed that the swelling power ranged
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283 from 24.5 to 32.7 g/g and water solubility ranged from 12.1 to 24.1% (Osundahunsi et al.,

284 2003), which were similar to the results of the present study.
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285 3.7. Thermal properties of starch

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286 The thermal properties of starch samples were determined by DSC, and their
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287 thermograms are given in Fig. 3A. The starches displayed two separate peaks in the DSC

288 thermogram, especially in Ningzi 1, X66-1, Y16-5 and Y79-4. Similar result was also
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289 reported in sweet potato starch, and the first and the second gelatinization peak corresponded
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290 to B- and A-type crystallinity, respectively (Genkina et al., 2003; Genkina, Wasserman, Noda,
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291 Tester, & Yuryev, 2004). In the present study, the two peak DSC thermograms further showed

292 that sweet potato starch contained both A- and B-type crystallinity. The second peak was

293 stronger than the first peak, indicating that the proportion of A-type crystallinity was higher

294 than that of B-type crystallinity. This result agreed with XRD analysis (CA-type starch). The

295 thermogram was peak fitted by using PeakFit version 4.12, and the gelatinization

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296 temperatures are shown in Table 4. The onset temperature ranged from 53.0 to 57.6 °C, the

297 first gelatinization peak temperature ranged from 64.1 to 69.0 °C, the second gelatinization

298 peak temperature ranged from 73.5 to 80.8 °C, and the conclusion temperature ranged from

299 85.0 to 89.7 °C among seven starches. The similar two peak DSC thermogram of starch has

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300 been reported in sweet potato grown in low soil temperature (Genkina et al., 2003; 2004).

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301 Some studies also showed that the gelatinization temperatures ranged from 61 to 84 °C in

302 twenty-five Papua New Guinean and Australian sweet potato varieties (Waramboi et al. 2011),

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303 from 58 to 82 °C in three Chinese sweet potato varieties (Chen et al., 2010), and from 66 to

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304 76 °C in red and white sweet potato varieties (Osundahunsi et al., 2003).
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305 3.8. Pasting properties of starch

306 The pasting properties of starch are presented in Fig. 3B and Table 4, and were
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307 significantly different among seven purple sweet potato starches. Peak viscosity ranged from
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308 2535 to 3750 mPa s with the highest for Y16-5 and the lowest for Ningzi 1. Hot viscosity
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309 ranged from 1517 to 2450 mPa s. Breakdown viscosity ranged from 1018 to 1622 mPa s with

310 highest for X85-15 and the lowest for Ningzi 1. Breakdown viscosity is used to measure the
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311 starch pasting resistance to heat. The lower breakdown viscosity value means higher ability to
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312 withstand heating (Abegunde et al., 2013). Therefore, X85-15 contained lower resistance to
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313 heat and shear than other starches. Final viscosity ranged from 1991 to 3486 mPa s,

314 corresponding to Ningzi 1 and Y16-5, respectively. Final viscosity indicates the stability to

315 the swollen granule structure. Setback viscosity ranged from 474 to 1036 mPa s,

316 corresponding to Ningzi 1 and Y16-5, respectively. Setback viscosity shows the tendency of

317 starch paste to retrogradation. Y16-5 showed higher retrogradation tendency due to the higher

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318 setback viscosity. Peak temperature varied significantly from 79.7 to 84.7 °C in starches of

319 Ningzi 1 and X83-1, respectively. The above values were in accordance with the peak

320 temperature range (67.2-86.6 °C) reported in previous studies (Abegunde et al., 2013; Kim et

321 al., 2013; Osundahunsi et al., 2003). Pasting properties are influenced by the size and shape of

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322 starch granule, amylose content, and branch-chain length distribution of amylopectin. The

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323 amylose leached from swollen granules during heating is re-associated to form a network

324 during the cooling process and increase the final viscosity (Kim et al., 2013).

SC
325 3.9. Enzymatic hydrolysis properties of starch

U
326 The hydrolysis of seven starches by PPA or AAG is shown in Fig. 3C and 3D. A biphasic
AN
327 hydrolysis trend by PPA or AAG was observed in seven starches with an initial rapid

328 hydrolysis of the amorphous region followed by a decreased hydrolysis (Li, Vasanthan,
M

329 Hoover, & Rossnagel, 2004). For PPA hydrolysis, Y16-5 starch was hydrolyzed faster than
D

330 the other starches (Fig. 3C). Zhang and Oates (1999) reported that the degree of hydrolysis in
TE

331 26 h ranged from 48.8 to 63.4% among five sweet potato starches, which was comparable to

332 the present result at the same hydrolysis time. Granule size is a critical factor in determining
EP

333 the hydrolysis of starch by amylase, and small granules show higher hydrolysis rate than large
C

334 granules (Noda et al., 2008). The starch with low ordered degree decreases the resistance to
AC

335 hydrolysis (Sevenou et al., 2002). The present results showed that Y16-5 starch had low

336 apparent amylose content, small granule size, and low ordered structure among seven starches,

337 leading to that it was hydrolyzed faster than the other starches. For AAG hydrolysis, seven

338 starches showed significantly different hydrolysis degrees (Fig. 3D). AAG hydrolyses starch

339 from the outer surface of the granule, while PPA hydrolyzes starch firstly from granule

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340 surface and then from inside to outside (Li et al., 2004). The different hydrolysis patterns

341 resulted in different susceptibility of seven starches to PPA and AAG hydrolysis.

342 3.10. In vitro digestive properties of starch

343 In vitro digestion of starch by both PPA and AAG is employed to simulate the effects of

PT
344 small intestine hydrolysis and subsequent glycemic response of starch. According to the rate

RI
345 and extent of starch digestion, starch is generally classified into RDS, SDS, and RS (Englyst,

346 Kingman, & Cummings, 1992). The in vitro digestion properties of seven starches were

SC
347 determined in native, gelatinized, and retrograded starches and are shown in Table 5. The

U
348 gelatinization disrupts the inter- and intra-molecular hydrogen bonds between starch chains,
AN
349 which results in that the starch is easily degraded. When gelatinized starch retrogrades, the

350 amylose chains associate to form the double helices structure and the amylopectins
M

351 recrystallize to form the crystallites, which increase the resistance to digestive enzymes
D

352 (Chung, Lim, & Lim, 2006). Therefore, native starches had extremely lower RDS and higher
TE

353 RS than gelatinized and retrograded starches, and gelatinized starches had higher RDS and

354 lower RS than retrograded starches. Starch morphology, size, and crystalline structure affect
EP

355 digestive properties of native starch, but they are disrupted during gelatinization and have no
C

356 effect on digestive properties of gelatinized starch (Wang & Copeland, 2013). In the present
AC

357 study, the RS in native starches ranged from 84.5 to 86.4%, which was significantly higher

358 than in rice (59.4%) and maize (43.6%) (Lin et al., 2016; Wang, Huang, Zhao, Chen, & Wei,

359 2015). The result was also in agreement with the previous report that tuber starch contained a

360 large amount of RS and was less digestible than cereal starches (Liu et al., 2006). Gelatinized

361 starch and retrograded starches also contained higher RS and ranged from 10.2 to 14.7% and

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362 12.8 to 22.4%, respectively. Therefore, sweet starches were more suitable for obesity and

363 cardiovascular disease patients than rice and maize starches.

364 4. Conclusion

365 Starches were isolated from purple sweet potato root tubers of a variety Ningzi 1 and six

PT
366 advanced breeding lines. They all had round, polygonal, oval and semi-oval shapes with

RI
367 central hila, but showed significantly different granule sizes. The seven starches showed

SC
368 CA-type crystallinity, but had some differences in amylose content, thermal properties, pasting

369 properties, and hydrolysis properties. The amylose was high in Ningzi 1 and low in Y46-4.

370
U
The Ningzi 1 and Y16-5 starch had high and low swelling power at 95 ºC, respectively. The
AN
371 thermal temperature was low in Y16-5 and high in X66-1. Ningzi 1 starch had low breakdown
M

372 and setback viscosities, and Y79-4 had high breakdown and setback viscosities. All the

373 starches had high resistance to in vitro digestion. The gelatinized and retrograded starch from
D

374 X83-1 had high resistant starch among 7 purple sweet potatoes. This study could provide
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375 useful information for the further utilization of the advanced breeding lines in breeding and
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376 food and nonfood industries.

C

377 Acknowledgements
AC

378 This study was financially supported by grants from the National Natural Science

379 Foundation of China (31570324), the Qing Lan Project of Jiangsu Province, the Talent Project

380 of Yangzhou University, and the Priority Academic Program Development of Jiangsu Higher

381 Education Institutions.

382 References

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515 physico-chemical properties of sweet potato starches. Food Chemistry, 65, 157-163.

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517 sweetpotato starch. Starch, 63, 249-259.

518

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519 Tables

520 Table 1 Water content of fresh root tuber and contents of starch, soluble sugar and protein of dry root tuber from purple sweet potato. a

PT
Genotype Category Water content (%) Starch content (%) Soluble sugar content (%) Protein (%)

RI
Ningzi 1 Variety 73.0±0.9cd 43.6±0.3c 21.7±0.1f 2.33±0.04c

SC
X66-1 Breeding line 73.6±0.8d 46.2±0.4d 17.3±0.1c 1.92±0.02a

U
X83-1 Breeding line 68.5±0.2b 53.6±0.4e 11.7±0.1a 3.05±0.00d

AN
X85-15 Breeding line 62.6±0.2a 55.1±0.3f 11.6±0.2a 1.91±0.03a

M
Y16-5 Breeding line 71.8±0.1c 44.2±0.5c 16.8±0.1b 3.84±0.01e

D
Y46-4 Breeding line 72.3±0.4cd 42.1±0.5b 19.4±0.2d 2.22±0.02b

Y79-4 Breeding line 73.0±0.3cd

TE 40.1±0.4a 19.9±0.0e 3.80±0.04e
EP
a
521 Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (p < 0.05).
C

522
AC

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523 Table 2 Granule size, iodine absorption parameter, relative crystallinity, and IR ratio of starch. a

Granule size b Iodine absorption parameter c Relative IR ratio d

Genotype

PT
d(0.5) D[3,2] D[4,3] λmax (nm) BV (OD680) AAC (%) crystallinity (%) 1045/1022 cm-1 1022/995 cm-1

Ningzi 1 18.24±0.01g 9.06±0.00g 18.78±0.01f 585±2ab 0.30±0.01c 31.0±0.7d 27.7±0.1abc 0.666±0.007b 0.769±0.003a

RI
X66-1 17.26±0.01f 8.50±0.00f 17.86±0.01e 581±4a 0.26±0.01a 24.8±0.4a 25.7±0.2a 0.676±0.011b 0.770±0.013a

SC
X83-1 15.74±0.01c 7.79±0.00c 16.43±0.01c 587±3abc 0.30±0.01c 30.7±0.1cd 28.5±0.7c 0.652±0.007ab 0.767±0.029a

X85-15 16.54±0.01e 8.11±0.00d 17.39±0.01d 590±3bc 0.30±0.01c 28.6±1.1bc 25.8±0.9ab 0.657±0.001ab 0.797±0.021a

U
Y16-5 12.32±0.02a 6.52±0.01a 12.80±0.02a 593±2c 0.29±0.01bc 27.6±0.8b 27.3±0.5abc 0.631±0.006a 0.775±0.009a

AN
Y46-4 14.63±0.00b 7.40±0.00b 15.03±0.00b 588±3abc 0.27±0.01ab 24.6±0.9a 28.1±0.8bc 0.653±0.006ab 0.770±0.006a

M
Y79-4 16.21±0.01d 8.30±0.00e 16.45±0.01c 588±2abc 0.29±0.01bc 27.5±0.9b 25.6±0.5a 0.671±0.002b 0.789±0.031a

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

TE
b
525 Granule size is measured by laser diffraction instrument. The d(0.5) is the granule size at which 50% of all the granules by volume are smaller.
EP
526 The D[3,2] and D[4,3] are the surface-weighted and volume-weighted mean diameter, respectively.

c
527 The parameters are obtained from the absorption spectrum of starch-iodine complex. λmax, maximum absorption wavelength; BV, iodine blue
C
AC

528 value; AAC, apparent amylose content.

529

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530 Table 3 Swelling power and water solubility of starch. a

Swelling power (g/g) Water solubility (%)

PT
Genotype
75 ºC 85 ºC 95 ºC 75 ºC 85 ºC 95 ºC

RI
Ningzi 1 11.4±0.5c 22.5±0.3cd 29.0±0.8d 9.0±0.2e 11.6±0.3cd 15.3±0.4ab

SC
X66-1 9.2±0.1b 23.1±0.7d 26.9±1.0bc 4.6±0.2ab 9.3±0.3a 14.1±1.5a

U
X83-1 7.3±0.7a 21.5±0.3bc 26.0±0.6b 4.6±0.7ab 10.6±0.3b 14.2±0.5a

AN
X85-15 7.9±0.6ab 23.5±0.7d 27.9±0.3cd 5.8±0.8bc 11.8±0.5d 16.9±0.1b

M
Y16-5 11.3±0.7c 18.6±0.3a 22.5±0.4a 7.8±0.7de 10.8±0.2bc 15.2±1.1ab

D
Y46-4 8.0±0.2ab 21.0±0.5b 25.3±0.1b 4.2±0.2a 9.9±0.4ab 13.1±1.0a

TE
Y79-4 12.4±0.3c 21.7±0.0bc 26.3±0.0bc 6.7±0.3cd 9.6±0.3a 13.9±0.7a
EP
a
531 Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (p < 0.05).
532
C
AC

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533 Table 4 Thermal and pasting properties of starch. a

Thermal parameters b Pasting parameters c

PT
Genotype
To (°C) Tp1 (°C) Tp2 (°C) Tc (°C) PV (mPa s) HV (mPa s) BV (mPa s) FV (mPa s) SV (mPa s) PT (°C)

RI
Ningzi 1 53.0±0.1a 67.1±0.5bc 79.7±0.3cd 89.0±0.2bc 2535±16a 1517±4a 1018±11a 1991±1a 474±6a 79.7±0.1a

X66-1 55.4±0.4b 69.0±0.4d 80.8±0.1d 89.7±0.3c 3464±17b 2380±18d 1083±1a 3104±18cd 723±0b 82.9±0.1b

SC
X83-1 57.6±0.9c 68.1±0.8cd 79.4±0.2c 88.5±0.8bc 3457±4b 2089±37b 1368±33bc 2749±11b 659±27b 84.7±0.1c

U
X85-15 56.0±0.3bc 66.5±0.1bc 78.1±0.1b 87.7±0.0b 3655±18c 2139±43bc 1515±25d 2815±32b 676±11b 83.5±0.6bc

AN
Y16-5 55.7±0.0bc 64.1±0.2a 73.5±0.1a 85.0±0.2a 3750±9d 2450±40d 1300±30b 3486±54e 1036±15d 80.6±0.0a

Y46-4 56.0±0.7bc 65.6±0.2ab 77.3±0.3b 89.6±0.4c 3664±26c 2247±32c 1417±6c 3137±20d 891±52c 83.5±0.6bc

M
Y79-4 55.6±0.5bc 66.2±0.5b 77.0±0.6b 87.7±0.6b 3668±4c 2046±28b 1622±33e 2989±43c 943±71cd 80.2±0.6a

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

TE
b
535 To, onset temperature; Tp1, gelatinization peak temperature of the first peak; Tp2, gelatinization peak temperature of the second peak; Tc
EP
536 conclusion temperature.
C

c
537 PV, peak viscosity; HV, hot viscosity; BV, breakdown viscosity (PV-HV); FV, final viscosity; SV, setback viscosity (FV-HV); PT, peak
AC

538 temperature.
539

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540 Table 5 In vitro digestion properties of starch. a

Native starch b Gelatinized starch Retrograded starch

PT
Genotype
RDS (%) SDS (%) RS (%) RDS (%) SDS (%) RS (%) RDS (%) SDS (%) RS (%)

RI
Ningzi 1 4.4±0.5a 7.6±0.6a 88.0±0.2b 82.3±0.2c 3.0±0.4a 14.7±0.6d 77.7±0.2d 4.3±0.6b 18.0±0.4d

SC
X66-1 4.1±0.3a 9.7±0.7b 86.2±0.4ab 84.5±0.2d 4.5±0.8ab 11.0±0.7ab 84.7±0.5g 2.5±0.4a 12.8±0.1a

U
X83-1 4.6±0.5a 10.5±0.8b 85.0±0.4a 76.6±0.3a 9.0±0.6e 14.4±0.6d 64.0±0.5a 13.6±0.8d 22.4±0.5f

AN
X85-15 4.1±0.6a 9.5±0.3b 86.4±0.4ab 79.9±0.4b 7.9±0.4de 12.2±0.4bc 73.3±0.2b 7.0±0.8c 19.8±0.7e

M
Y16-5 4.2±0.2a 10.9±0.1b 84.9±0.3a 81.0±0.7b 6.3±0.4cd 12.8±0.4c 79.7±0.2e 4.5±0.5b 15.8±0.3c

D
Y46-4 4.8±0.3a 9.5±0.3b 85.7±0.5a 80.4±0.9b 5.1±0.8bc 14.5±0.5d 83.5±0.5f 2.3±0.5a 14.3±0.2b

TE
Y79-4 4.3±0.4a 9.6±1.3b 86.1±1.6ab 85.5±0.3d 4.3±0.4ab 10.2±0.1a 76.0±0.3c 6.5±0.5c 17.6±0.2d
EP
a
541 Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (p < 0.05).

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C

542 RDS, rapidly digestible starch; SDS, slowly digestible starch; RS, resistant starch.
AC

543

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Figure captions

Fig. 1. The photos of purple sweet potato root tubers, the morphologies of starch granules

under normal light microscope (NLM), polarized light microscope (PLM) and scanning

electron microscope (SEM), and the granule size distribution of starches. Scale bar = 20 µm.

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Fig. 2. Spectra of iodine absorbance (A), XRD (B), and ATR-FTIR (C) of starches. The arrow

in (B) shows the shoulder peak at about 18° 2θ.

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Fig. 3. DSC thermograms (A), pasting profiles (B), PPA hydrolyses (C), and AAG hydrolyses

(D) of starches.
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Figure 1
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Figure 2
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Figure 3
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Highlights

Starches were isolated from one purple sweet potato variety and six breeding lines.

Seven starches showed similar shapes with central hila, but had different sizes.

The starches had different amylose contents, but all exhibited CA-type crystallinity.

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The starches had different swelling power, thermal, and pasting properties.

RI
The purple sweet potato starches had high resistance to hydrolysis and digestion.

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