Accepted Manuscript

NMR spectroscopy of starch systems

Fan Zhu

PII: S0268-005X(16)30555-0
DOI: 10.1016/j.foodhyd.2016.10.015
Reference: FOOHYD 3633

To appear in: Food Hydrocolloids

Received Date: 26 June 2016

Revised Date: 4 October 2016
Accepted Date: 7 October 2016

Please cite this article as: Zhu, F., NMR spectroscopy of starch systems, Food Hydrocolloids (2016), doi:
10.1016/j.foodhyd.2016.10.015.

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3 NMR spectroscopy of starch systems

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5 Fan Zhu*

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7 School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New

8 Zealand

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9 * Correspondence, e-mail: fzhu5@yahoo.com

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13 Running title: starch NMR

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15 Abstract

16 NMR spectroscopy is used to study the structure and composition of diverse food materials

17 including starch, which is utilised in food and other industries. NMR spectroscopy has been

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18 used to investigate the properties of various starch systems. These include chemical

19 composition, physical and chemical structures, gelatinization, retrogradation, enzyme

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20 hydrolysis, and modifications of starches from diverse botanical origins. The interactions of

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21 starch with other food components in complex systems have also been studied by NMR

22 spectroscopy. Both solid and liquid state NMR techniques have been used, including 1H

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23 NMR, 13C NMR, 31P NMR, and 17O NMR. These techniques can be non-destructive, and
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24 provide novel insights into the structure of starch systems. NMR spectroscopy is

25 complementary to other analytical techniques such as X-ray diffraction and calorimetry for
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26 the characterization of simple and complex starch systems.

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27 Keywords: NMR spectroscopy; starch; structure; property; modification; interaction

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28 1. Introduction

29 NMR (nuclear magnetic resonance) spectroscopy has been widely used for the analysis of

30 food materials such as dairy products, fats and oils, and wine and beverages (Spyros & Dais,

31 2012). The first use of NMR in food science was in the 1950s when the moisture of foods

32 was measured by low resolution NMR. The applications of NMR for food characterisation

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33 have become widespread in the 1980s. NMR spectroscopy is a major analytical tool of food

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34 science in recent years (Spyros & Dais, 2012; Marcone et al., 2013). This is due to the

35 development of user-friendly instrumentation, the need of effective analytical methods for

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36 quality control and food authentication, and the increasing demand from food industry to

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37 innovate the processes and products (Spyros & Dais, 2012; Marcone et al., 2013).
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38 NMR phenomena have the origins within the nucleus of certain atom types such as C and P.

39 The individual atoms have a net “nuclear” spin. The effects can be noted in a magnetic field,
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40 involving the energy exchange between two levels at least (resonance) (Gidley, 2014). For
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41 one sample, different nuclei, such as 1H, 13C, 31P, can be chosen to study different aspects of
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42 the samples and to extract the relevant information under the natural/industrial conditions

43 (Laws et al., 2002; Spyros & Dais, 2012). Some NMR experiments need no separation of
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44 diverse food components, and require relatively a small amount of efforts for sample pre-

45 treatment and preparation as compared with traditional methods (Spyros & Dais, 2012). The
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46 food samples can be lipid, semi-solid, and solid. The obtained complex NMR spectra can be
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47 further treated with multivariate statistical analysis to gain additional structural information

48 of food systems (Flanagan et al., 2015).

49 Starch is a major component of our diet. It is also an important industrial ingredient for

50 various food and non-food applications. The two types of starch molecules are the amylose

51 (linear and smaller) and the amylopectin (branched and larger). Starch molecules are simply

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52 consisted of glucose units linked by α-(1-4) linkages and branched by α-(1-6) linkages (Pérez

53 & Bertoft, 2010). The molecules are naturally assembled in the form of semi-crystalline

54 granules with sizes ranging from ~1 to 100 µm (Pérez & Bertoft, 2010). Understanding the

55 structural changes of starch during various processing (e.g., retrogradation and modification)

56 would be critical for better uses of starch in various scenarios. Different methods have been

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57 used for starch structural characterisation. An advantage of NMR over other techniques such

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58 as wide-angle X-ray diffraction (XRD) (an abbreviation list of instruments used is presented

59 in supplementary Table 1) is that the other components such as lipids, polyphenols, and

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60 protein do not interfere with the starch spectra (Flanagan et al., 2015).

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61 Numerous publications have well documented the background and principles of NMR
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62 spectroscopy (Laws et al., 2002; Spyros & Dais, 2012; Gidley, 2014). The theoretical

63 principles of starch NMR has also been detailed (Gidley, 2014). The basics of starch structure
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64 and functional properties have been systematically reviewed in detail (BeMiller & Whistler,

65 2009; Pérez & Bertoft, 2010). Therefore, the basics of NMR spectroscopy and starch are not
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66 covered in the review. This review focuses on the applications of NMR-based techniques for
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67 structural characterization of various starch systems. These include the chemical composition,
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68 molecular order, interactions, gelatinization, retrogradation, and modifications. Due to the

69 large amount of literature published, representative references have been selected to cover
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70 specific topics.
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72 2. Chemical composition

73 Chemical compositional parameters, including the contents of amylose and phosphorus-

74 containing compounds in starch, have been measured by different NMR techniques (Table 1).

75 Compared with the traditional methods for compositional analysis, NMR-based methods can

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76 be more efficient and time-saving (Tabata & Hizukuri, 1971). The anomeric protons

77 involving in the α-(1,4) and α-(1,6) linkages give rise to different signals in the 1H NMR

78 spectra. Based on this principle, Dunn and Krueger (1999) analysed the amylose contents of

79 starches from various botanical sources using 1H NMR. The results of amylose contents

80 (ranging from 12−76%) agreed well with those measured by the iodine affinity-

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81 spectrophotometry-based method. 31P NMR has been used to characterize the nature and

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82 composition of phosphorus-containing compounds in starch (Muhrbeck & Tellier, 1991; Lim

83 et al., 1994; Genkina & Kurkovskaya, 2013; Kasemsuwan & Jane, 1996). DMSO was used to

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84 increase the solubility of starch α-dextrins for better quantification. Phosphate monoester,

85 inorganic phosphate, and phospholipids of starch in DMSO solution were resolved and

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quantified (Kasemsuwan & Jane, 1996). The position and degree of phosphorylation on
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87 starch chains can be determined (Muhrbeck & Tellier, 1991). The normal cereal starches
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88 mostly possess phospholipids. The legume and potato starches mostly contain phosphate

89 monoesters. And the tuber and root starches are free of phospholipids. Phosphate monoesters
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90 of all starches were mostly found on the C-6 than on the C-3 of the glucose unit (Lim et al.,
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91 1994). Phosphorylation on the C-3 position was independent of potato variety, while that on

92 the C-6 position was found variety-dependent (Muhrbeck & Tellier, 1991). Schmieder et al.
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93 (2013) employed a combination of heteronuclear 1H, 13C double, 1H, 13C, 31P triple NMR
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94 techniques, and successfully determined the phosphorylation sites of both the starch and
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95 glycogen in heterogeneous samples. A full assignment of the resonances of carbohydrates

96 was archived by analysing a set of reference compounds.

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98 3. Structural characterization

99 3.1. Impact of hydration extent

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100 The 13C CP/MAS NMR spectrum is very sensitive to the hydration of starch (Veregin et al.,

101 1986; Cheetham & Tao, 1998b; Paris et al., 1999) (supplementary material Fig. 1). The

102 increasing moisture content of starch increased the peak sharpness to various degrees,

103 depending on the polymorph type and starch composition (Paris et al., 1999; Cheetham &

104 Tao, 1998b) (supplementary material Fig. 1). The increasing peak resolution was attributed to

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105 the increased mobility of the amorphous regions in the starch granules. The masking effects

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106 of resonances from the amorphous region are lost (Morgan et al., 1995). Therefore, the

107 moisture content of the systems should be calibrated for the correct estimation of starch

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108 structure.

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109 3.2.Starch physical structure AN
110 Solid state 13C CP/MAS NMR has been used to quantify the molecular order of granular

111 starch (Flanagan et al., 2015; Man et al., 2013) (Table 2). Each carbon of the glycosyl unit in
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112 starch is assigned for specific peak in the spectrum (supplementary material Fig. 2). 13C
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113 CP/MAS NMR spectra of A-type starches differs from those of B-type starches in some
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114 detail. The peak of C-1 with chemical shift of ~100 ppm is a triplet for A-type starches, and a

115 doublet for B-type starches (Veregin et al., 1986). This difference is readily attributed to the
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116 specific arrangement of the crystals in the granules (Gidley & Bociek, 1985). The spectrum

117 of the native starch was compared with that of the amorphous starch. The contents of the
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118 double and single helices as well as the amorphous material in the granules can be quantified,
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119 and the degree of crystallinity can be calculated (supplementary material Fig. 2) (Gidley &

120 Bociek, 1985; Flanagan et al., 2015). For example, Man et al. (2013) quantified the molecular

121 order of starches from rice mutants deficient in SBE I (SBE represents starch branching

122 enzyme) and SBE IIb genes. The mutation decreased the degree of crystallinity, the

123 proportion of double helix, while increasing the proportion of single helix. Therefore, the

124 solid state 13C CP/MAS NMR technique can readily provide information on the physical

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125 structure of starch granules as affected by various factors. Flanagan et al. (2015) employed a

126 partial least squares model to rapidly quantify the amount of ordered double helices directly

127 from the 13C CP/MAS NMR spectrum of starch. This approach removes the procedures of

128 curve fitting and does not need any amorphous standard, while giving accurate results.

129 Amorphous starch has been characterised by various NMR techniques (Kalichevsky et al.,

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130 1992; Paris et al., 2001a and 2001b). Spectral decomposition of C-1 peak of 13C CP/MAS

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131 NMR spectrum under 1H decoupling revealed five types of α-(1-4) linkages in starch (Paris et

132 al., 2001a and 2001b). The glass transition temperatures of amorphous waxy maize starches

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133 varying in moisture contents have been measured by pulsed (through rigid lattice limit) and

solid state 13C CP/MAS NMR (Kalichevsky et al., 1992) (supplementary material Fig. 3).

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135 The water plasticizing effect (10 and 22% water content) generally followed the Couchman-

136 Karasz equation, which is commonly used for predicting the glass transition temperatures of
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137 random copolymers and amorphous mixtures. The comparison between different methods

138 [DSC (differential scanning calorimetry) vs NMR] in measuring the starch glass transition is
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139 discussed in a following section (section 9: correlations between NMR and other analytical
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140 techniques).
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141 The degree of branching (DB) of starch has been studied by 1H NMR based on the anomeric

142 protons involved in α-(l-4) and α-(l-6) linkages (Gidley, 1985; Nilsson et al., 1996; Dunn &
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143 Krueger, 1999; Tizzotti et al., 2011; Syahariza et al., 2013). D2O was commonly employed as
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144 the solvent for the DB quantification (Gidley, 1985). For example, DB of potato amylose and

145 maize amylopectin were 1 and 4.77%, respectively (Nilsson et al., 1996). DB of starches

146 from 14 rice genotypes ranged from 2.7−3.6% (Syahariza et al., 2013). DB of both intact and

147 degraded starches can be measured by 1H NMR (Gidley, 1985). Tizzotti et al. (2011)

148 employed dimethyl-d6 sulfoxide with the addition of a small amount of deuterated

149 trifluoroacetic acid (TFA) as the starch solvent. This resulted in a well-defined and clear 1H

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150 NMR spectrum with improved quantification efficiency. DB by NMR analysis showed a

151 good correlation with that by a traditional method which is discussed in a following section

152 (section 9: correlations between NMR and other analytical techniques).

153 3.3.Structure of amylose crystals

154 The physical structure of the amylose crystals was studied by 13C CP/MAS NMR (Horii et al.,

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155 1987; Paris et al., 1999). A-type and B-type amylose crystals had different patterns of NMR

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156 spectra (supplementary material Fig. 4). Increasing water content increased the peak

157 resolution. The C-1 peak was a triplet for the crystals with A-type polymorph and a doublet

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158 for the B-type samples. Therefore, the native starch and amylose crystals share similar C-1

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159 peak pattern of NMR spectrum regarding the polymorphism. Compared with native starch,
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160 the amylose crystals had NMR spectra with much sharper peaks due to the much increased

161 degree of crystallinity (Horii et al., 1987).

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162 3.4.Water mobility in starch granules

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163 The water mobility in starch has been studied by 17O and 2H NMR (Richardson et al., 1987),
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164 and 2H and 1H NMR (Li et al., 1998; Baianu et al., 1999). Three types of water were proposed

165 for potato starch. The first type was related to the anisotropically bound water. The second
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166 type was related to the trapped water, and the third one was from the average between free

167 and weakly bound water populations (Richardson et al.. 1987; Baianu et al., 1999). Li et al.
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168 (1998) studied the molecular mobility of “freezable” and “unfreezable” water in waxy maize
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169 starch by 1H NMR coupled with DSC analysis. Decreasing the temperature decreased the

170 proportion of the mobile water. Much of the water (>50% of water present) remained highly

171 immobile when the starch was in a glassy, solid, semi-crystalline state. This suggests that the

172 glassy state of starch may not be employed to predict the molecular mobility of water in

173 relation to food stability (Li et al., 1998).

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174 3.5.V-type amylose inclusion complexes

175 Amylose can form the V-type inclusion complexes with small guest molecules both in the

176 solution and in solid states (Pérez & Bertoft, 2010). Jane et al. (1985) analysed the structure

177 of V-type complexes formed in solution by 13C NMR. The downfield shifts induced by

178 complexing agents such as iodine were larger for the signals of C-1 and C-4 than those of C-2,

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179 C-3, and C-6. This suggests the formation of V-type complexes in the solution. The physical

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180 structure of V-type complexes with different guest molecules and amyloses in solid state has

181 been probed mostly by solid state 13C CP/MAS NMR (Table 3) and some NMR spectra are

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182 shown (supplementary material Fig. 5). Le Bail et al. (2013 & 2015) assigned the peaks of

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183 different carbons of V-type amylose complexes with specific chemical shifts. The chemical
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184 shifts of 102.7, 81.4, 74.9, 71.6, 61.3, and 60 ppm were related to C-1, C-4, C-3, C2-C5, and

185 C-6, respectively. Gidley & Bociek (1988) linked the chemical shifts of C-1 and C-4 to the
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186 torsion angles (φ and ψ) of conformation. The chemical shifts of C-1 and C-4 of the amylose

187 in both solution and solid states were found to be more susceptible to the induced
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188 conformational changes as a result of guest molecule inclusion (Jane et al., 1985; Le Bail et
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189 al., 2005; Gidley & Bociek, 1988). There are differences in the NMR spectra among V6I,
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190 V6II, V6III, V7, and V8-type amylose complexes due to the differences in the size of helical

191 cavity (six-, seven-, and eight-fold helices) and the physical positioning of guest molecules
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192 (Le Bail et al., 2005; Gidley and Bociek, 1988). For example, the NMR spectra of V6- and
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193 V7-type inclusion complexes are similar. In comparison, the V8-type complexes with 1-

194 naphthol had the downward chemical shifting by 1 ppm for both the C-1 and C-4 peaks

195 (Gidley & Bociek, 1988). The un-complexed guest molecules that remained in the samples

196 may be detected by NMR. For example, the un-complexed palmitic acids were resolved in
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197 C CP/MAS NMR spectrum with the chemical shift of 33.6 ppm, while the chemical shift of

198 the complexed palmitic acids was 32.4 ppm (Lebail et al., 2000). The chemical shift (32.4

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199 ppm) of the complexes indicated the existence of both trans and gauche conformations for

200 the fatty acid chains. The structural differences between different types of V-type complexes

201 as revealed by 13C CP/MAS NMR can also be reflected by the results from other analytical

202 techniques such as DSC and XRD. The NMR method should be employed in combination

203 with other techniques (e.g., XRD and DSC) to better understand the nature of V-type

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204 amylose inclusions complexes.

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205 4. Gelatinization and retrogradation

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206 Gelatinization and retrogradation are related to starch interactions with water and are

207 fundamental for starch applications. Various NMR techniques (13C CP/MAS, time-domain 1H,

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208 O, and 31P NMR) have been used to understand the structural changes of both starch and
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209 water during these processes (Table 4). The content of double helices in starch granules as

210 measured by solid state 13C CP/MAS NMR was positively correlated with the enthalpy
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211 change of gelatinization (∆H) as measured by DSC (Cooke & Gidley, 1992). Hydration
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212 properties of potato starch were studied through the CP and SP modes of solid state 13C and
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213 P MAS NMR (Larsen et al., 2013). As expected, hydration increased the molecular order of

214 starch while gelatinization and hydrolysis reduced the immobile fractions of native starch.
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215 Phosphorus-containing compounds became completely mobile upon gelatinization (Larsen et

216 al., 2013). Proton properties were also studied by 1H NMR (Ritota et al., 2008; Rondeau-
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217 Mouro et al., 2015). Four types of water differing in their mobility were observed in starch-
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218 water systems during gelatinization (Ritota et al., 2008). Water with the highest mobility was

219 related to the bulk of water, while the others were associated with the chemical and diffusive

220 exchanges with starch components (Ritota et al., 2008). Rondeau-Mouro et al. (2015)

221 employed the time-domain 1H NMR to study the temperature-associated changes of T2

222 (spin–spin relaxation time) during starch gelatinization. Two T2 components were due to the

223 slow diffusional exchanges between different water layers in starch. The fraction with the

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224 shortest T2 was related to non-exchangeable protons of CH in both the amylopectin and

225 amylose. Low-resolution NMR measurements (proton NMR) are fast and require no sample

226 pre-treatment, while providing useful information of the starch gelatinization process (Ritota

227 et al., 2008). Cheetham and Tao (1998a) employed 17O NMR relaxation technique and

228 revealed that T2 change was related to the water content and the degree of starch

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229 gelatinization. Higher contents of amylose and phosphates were related to lower water

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230 mobility. Upon gelatinization with no/little shearing, the granules are not completely

231 dissolved and remain in fragile forms termed the starch ghost (Zhang et al., 2014). The ghost

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232 structure of gelatinized starch was probed by solid state 13C CP/MAS NMR (Zhang et al.,

233 2014). V-type inclusion complexes were found in potato and maize starch ghosts. Upon α-

234
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amylase hydrolysis, the V-type polymorph became more prominent while a small amount of
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235 B-type polymorph was recorded in the potato starch ghost. It was suggested that temporary
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236 entanglements of amylopectin and their size are related to the ghost formation (Zhang et al.,

237 2014).
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238 Starch retrogradation has been studied by 13C CP/MAS and 1H NMR (Gidley, 1989;
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239 Ambigaipalan et al., 2013; Teo & Seow, 1992; Smits et al., 1998). 13C CP/MAS NMR
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240 analysis showed that the intensity of C-4 peak decreased during the retrogradation of pulse

241 starches, while the content of double helices increased (Ambigaipalan et al., 2013). In
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242 amylose gel, the B-type polymorph was observed (Gidley, 1989). 1H NMR analysis of the re-
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243 crystallized starch showed that the rate of decay after radiofrequency pulse was different than

244 that of the amorphous starch (Teo & Seow, 1992). This could be due to the water release

245 from the hydrated starch chains during re-crystallization. Smits et al. (1998) analysed the

246 retrogradation of starch stored below and above the glass transition temperature (Tg). T1

247 relaxation time decreased before increasing when the storage temperature was above Tg,

248 while it increased before levelling off when the storage temperature was below Tg. In the

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249 former case, the initial decrease and the further increase were due to the water adsorption and

250 the recrystallization of starch, respectively. In the latter case, the increase was mostly due to

251 the decrease in free volume (Smits et al., 1998). Kulik and Haverkamp (1997) employed one-

252 and two-dimensional exchange solid-state NMR for the analysis of waxy maize starch

253 retrogradation. Slow motions with the correlation time of tens of milliseconds were detected.

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254 Starch retrogradation occurred over a wide range of correlation times.

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255 5. Enzyme susceptibility and resistant starch

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256 The structural changes of maize starches varying in amylose content during enzyme

257 hydrolysis and processing were followed by solid state 13C CP/MAS NMR spectroscopy

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258 (Htoon et al., 2009; Shrestha et al., 2012). Hydrolysis by artificial saliva α-amylase had little
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259 effect on the contents of double and single helices as well as the non-ordered proportion of

260 maize starches (Shrestha et al., 2012). Enzyme hydrolysis of the thermally-processed maize
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261 starches with high amylose contents (>50%) increased the contents of double helices while
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262 decreasing the proportion of amorphous starch (Htoon et al., 2009). The molecular structure
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263 of resistant starch after enzyme hydrolysis was characterised by various NMR techniques

264 (Colquhoun et al., 1995). Relaxation and solid state 13C CP/MAS NMR tests showed that the
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265 amylose gel had a double helical (rigid) fraction and a mobile amorphous fraction. Upon

266 hydrolysis by porcine pancreatic α-amylase, the mobile fraction was removed and the gel
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267 became rigid and fully ordered (Colquhoun et al., 1995). The gels of amylomaize V,
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268 amylomaize VII, and wheat starches after enzyme hydrolysis contained 60−70% of double

269 helical structure as revealed by solid state 13C CP/MAS NMR. Structurally-defected B-type

270 double helical aggregates were reported in the cooled gels.

271 6. Modifications

272 6.1.Physical modifications

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273 C CP/MAS and 1H NMR techniques have been used to probe the structural changes of

274 starches upon different physical treatments including high pressure, melt-process and

275 ultrasound, and microwave (Table 5). These processes gelatinized starch and increased the

276 content of amorphous material in the systems (Deng et al., 2014; Guo et al., 2015; Lima &

277 Andrade, 2010; Fan et al., 2013a and 2013b). High pressure had no effect on the chemical

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278 shifts of carbon resonance peaks, while decreasing the relative crystallinity of starch. Pressure

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279 treatment up to 600 MPa increased the proportion of the amorphous regions of starch, while

280 having no effect on the polymorph type (Deng et al., 2014; Guo et al., 2015). Rapid heating

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281 in oil bath and microwave heating had similar effects in increasing the content of amorphous

282 material and decreasing that of the double helices (Fan et al., 2013a and 2013b). 1H NMR

283
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284 than did the microwave heating (Fan et al., 2013a and 2013b). Sonicated and melt-processed
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285 high amylose maize starch had similar spectra, and the ultrasound better de-aggregated the

286 starch (Lima and Andrade, 2010).

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287 6.2.Chemical modifications

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288 The structural changes of various starches hydrolysed by acid and alkaline solutions were
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289 followed by 13C CP/MAS NMR (Table 5). The acid treatment reduced the content of

290 amorphous starch while increasing that of double helix and degree of crystallinity
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291 (Atichokudomchai et al., 2004; Cai et al., 2014a). Wang and Copeland (2012) showed that
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292 the alkali hydrolysis had no effect on the chemical shifts of various carbon peaks of pea

293 starch spectrum while broadening the resonance peak of C-2–C-5 sites. The content of double

294 helix decreased due to the alkali hydrolysis of the amorphous regions of the granules. Alkali

295 treatment had no effect on the NMR spectra of rice starches differing in amylose contents (24

296 and 58%) (Cai et al., 2014b).

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297 The position and level of substitution in starch (e.g., acetylated, phosphorylated,

298 hydroxylpropylated, octenyl succinic anhydride (OSA)-modified) have been determined by

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299 H NMR (Table 5). The principles of the determination are similar to that of measuring the

300 degree of branching of starch (Tizzotti et al., 2011). For the measurement, the starch was

301 either intact or in the forms of limit dextrins from enzyme hydrolysis (de Graaf et al., 1995;

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302 Xu & Seib, 1997; Zhao et al., 2015). For example, Xu and Seib (1997) determined the

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303 hydroxypropyl (HP) levels of modified maize, wheat, and cassava starches based on the

304 intensity of HP methyl signal in comparison with that of the methine and methylene (HCO)

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305 multiplet. The position of hydroxypropylation was determined from the anomeric proton

306 signals of the spectrum of α-limit dextrins (Xu & Seib, 1997). Tizzotti et al. (2011) dissolved

307
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starch samples in dimethyl-d6 sulfoxide with deuterated trifluoroacetic acid (TFA) addition,
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308 and analysed the degree of substitution of octenyl succinic anhydride (OSA)-modified

starches by 1H-NMR. The starch degradation by TFA had no effect on the detection. This
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310 method gave an improved spectrum resolution and accuracy of quantification (Tizzotti et al.,
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311 2011). The introduction of new functional groups has been verified by 13C CP/MAS, 13C, and
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312 H NMR analysis. For example, Ye et al. (2014) used both 13C and 1H NMR to analyse the

313 OSA-modified soluble maize starch. The modified starch had the extra peaks at 0.8−3.0 ppm
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314 and a shoulder at 5.56 ppm in the 1H NMR spectrum due to the OSA group. The intensity of
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315 these peaks increased with the increasing degree of substitution. Changes were also observed

in the 13C spectrum. C-1 signal of the internal glucosyl units of the modified starch became
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317 broader. A shoulder peak of C-4 was observed. These suggest that the substitution occurred at

318 the O-2 and O-3 positions (Ye et al., 2014). Chauhan et al. (2015) analysed the potato starch

319 by both 1H and 13C NMR. Thiolated starch had an additional peak at 2.1 ppm (SH group) and

320 another peak between 3 and 4 ppm (–CH2 in the –OC–CH2SH). The extra signals of CH2 at

321 3–3.5 ppm and of CH3 at 1.8–2.5 ppm for the S−ethyl groups were noted for the sulphonium

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322 structure. Differences in the 13C NMR spectra were observed (Chauhan et al., 2015). New

323 peaks in the range of 175–180 ppm were noted for the thiolated starch. The new peak at ~50

324 ppm was due to the carbon in the chloroacetyl group. These peaks of both 1H and 13C NMR

325 spectra verified the successful sulfur-functionalization of starch (Chauhan et al., 2015). Solid

326 state 13C CP/MAS NMR has been used to verify the successful modification of starch (Rashid

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327 et al., 2012; Wei et al., 2008; Geng et al., 2010). Wei et al. (2008) analysed the cationic maize

starch by 13C CP/MAS NMR. Compared with the control, extra peaks at around 31 ppm were

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329 due to the fragments of methylene of the long cationic chain, indicating the successful

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330 production of cationic starch. Geng et al. (2010) analysed the maize starch laurate by 13C

331 CP/MAS NMR. Carbon resonances of the fatty ester chains (10–35 ppm) and that of the ester

332
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group (170–175 ppm) were recorded in the spectrum of modified starch. This suggests the
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333 successful esterification of starch with the methyl laurate (Geng et al., 2010).
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334 Starch was degraded by oxidation and ozone treatment (Salomonsson et al., 1991; Sandhu et

335 al., 2012). Structure of the degraded products was studied by 1H NMR and/or 13C NMR
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336 (Salomonsson et al., 1991; Sandhu et al., 2012). In bromine-oxidised potato starch, the level
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337 and position of keto and carboxylic groups were determined (Salomonsson et al., 1991). 1H
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338 NMR analysis of ozone-treated wheat starch revealed that the β-glucuronic acid group was at

339 the C-1 position and keto group at the C-2 position (Sandhu et al., 2012).
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340 6.3.Starch-grafted copolymers

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341 Starch-grafted copolymers have many applications such as superabsorbent and thermoplastics.

342 Structures of starch-grafted copolymers were probed by solution 1H and 13C NMR as well as

343 solid state 13C CP/MAS NMR (Table 6). For example, Zou et al. (2012) analysed starch-g-

344 polyacrylamide by solution 13C NMR to reveal the grafting positions and ratios on specific

345 carbons (supplementary material Fig. 6). The peaks at 180–190 ppm were related to the

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346 amide carbonyl, and those at 35–50 ppm corresponded to the hybridised carbon atoms (–

347 (CH2–CH)n) units in the copolymers. A new peak at 61.8 ppm was related to the grafting of

348 C-6 (Zou et al., 2012). The intensity ratio of peaks at 63.5 ppm and 61.8 ppm reflected the

349 ratio of C-6 grafting. Yang et al. (2015) verified the grafting of maleic anhydride (MA) and

350 epoxidized cardanol (epicard) onto maize starch by 1H NMR. A new peak at 13 ppm in the

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351 spectrum of the starch graft was related to the carboxyl group and the peaks at 6.1–6.6 ppm

were of the double bonds. In contrast, the 1H NMR spectrum of the epicard-grafted starch had

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352

353 no peak at 13 ppm, suggesting the interactions of carboxyl groups with the epoxy groups. The

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354 peaks at 6.7–7.2 ppm were due to the benzene groups of the epicard. These structural features

355 obtained from the NMR spectra confirmed the successful production of the copolymers.

356
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Zhang et al. (2008) analysed the structure of starch-g-poly(sodium acrylate) by solid state 13C
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357 CP/MAS NMR. The spectrum of the copolymer was compared with that of the pure starch
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358 and poly(sodium acrylate). The spectra shared a similarity with some differences. An extra

359 shoulder of the spectrum was recorded on the C-6 peak, indicating the OH group of C-6 was
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360 involved in the grafting process.

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361 Physical mixtures of starch and other polymers were also studied by NMR. 1H NMR

362 relaxometry was used to study the starch and poly(lactic acid) (PLA) blend with the
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363 incorporation of montmorillonite clay and silica (nanoparticles) (Brito et al., 2015). Proton
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364 spin-lattice relaxation time of the blends was between that of starch and PLA, suggesting the
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365 interactions and miscibility. Nanoparticle addition increased the relaxation time, suggesting

366 the occurrence of new interactions and the reduced molecular mobility.

367 7. Interactions with other components

368 In food systems, multiple ingredients co-exist. The interactions of starch with various non-

369 starch components may greatly impact the quality of food products. Some of the starch

370 interactions with various food ingredients have been examined by both 1H and solid state 13C

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371 CP/MAS NMR (Table 7). For example, the interactions of rice starch with tea polyphenols

372 during starch gelatinization were probed by 1H NMR (Wu et al., 2011). The non-covalent

373 interactions were revealed when compared to the 1H spectra of physical mixtures of starch

374 and polyphenols. The altered coupling constants suggest stronger interactions in the samples

375 during gelatinization. Li et al. (2014) analysed the interactions between amylose and metal

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376 ion (Cu2+). Compared with the 1H spectrum of amylose in D2O, the presence of Cu2+

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377 broadened the carbon peaks (supplementary material Fig. 7). This suggests the binding of

378 Cu2+ ions by the OH groups of starch (Li et al., 2014). Beeren & Hindsgaul (2013) studied

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379 the binding interactions between potato amylopectin and HPTS-C16H33 (an amphiphilic

380 molecule) by 1H NMR. Upon binding, the proton signals shifted downfield due to the

381
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formation of the helical structure of amylopectin external chains. Some of the resulting
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382 products of starch interactions with other components (e.g., protein) in the solid form have

been studied by 13C CP/MAS NMR (Pizzoferrato et al., 1998 and 1999; Hu et al., 2013; Luo
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383

384 et al., 2013). For example, Luo et al. (2013) analysed the structure of enzyme-modified
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385 starch-zinc complexes. Compared with the control, the C-6 chemical shift of the complexes
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386 moved downfield by 0.96 ppm, while the chemical shifts of the other carbons had no change.

387 This suggests that the interactions were mainly on the OH group of C-6 (Luo et al., 2013). Hu
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388 et al. (2013) studied the interactions between rice starch and 1-butanol in an HCl solution. In
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389 comparison with the control, an extra peak for C-1 at 100.3 ppm was observed in the sample.
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390 This suggests the formation of V-type complexes between amylose and butanol. Therefore, it

391 is possible to better study the nature of both the process and resulting products of starch

392 interactions by NMR techniques.

393 8. Starch in food and complex systems

394 The structural changes of starch and water in some food systems (bread and film) have been

395 studied by various NMR techniques (Table 8). Primo-Martín et al. (2007) studied the

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396 structural changes of starch in flour, bread crumb, and bread crust by solid state 13C CP/MAS

397 NMR. The triplet of C-1 peak of the starch in flour was lost in the bread due to baking and

398 gelatinization. The increasing intensity of peaks at 82 and 102 ppm suggests an increasing

399 proportion of amorphous region. An upward displacement of the C-1 peak position in the

400 bread crust and crumb suggests the formation of V-type inclusion complexes. Sivam et al.

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401 (2013) used 13C CP/MAS NMR to study the starch properties of bread as affected by pectin

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402 and polyphenol addition. Polyphenols and pectins decreased the intensity of C-6 peak by

403 35−40%. The reasons remain to be illustrated by employing simple model systems (e.g.,

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404 starch-pectin mixture). Mihhalevski et al. (2012) employed various NMR techniques to study

405 the structure and composition of the starches in wheat and rye sourdough breads. 13C NMR

406
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spectra of the wheat and rye starches were similar. The 31P NMR spectra of wheat and rye
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407 starches were different due to the difference in lipid composition. 13C CP/MAS NMR
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408 analysis revealed the differences in the structures of retrograded starches in the breads. Starch

409 retrogradation can be affected by various factors such as starch structure, composition, and
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410 the presence of the non-starch components (Hoover, 1995). Bosmans et al. (2012) employed
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1
411 H NMR relaxometry to study the water status in bread and model systems (supplementary

412 material Fig. 8). The proton peaks were assigned according to simple model systems. This
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413 analysis helped better understand the role of water with different molecular mobility in bread

properties. The structure of starch in extruded films was analysed by 13C CP/MAS NMR
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414
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415 (Pushpadass et al., 2009). The extrusion process induced the changes in the NMR spectrum.

416 The C1 peak in the spectrum of starch in the film was a duplet (indicating B-type polymorph),

417 while that of the native starch was a triplet (indicating A-type polymorph). This suggests the

418 occurrence of re-crystallization of the gelatinized starch after the extrusion. The applications

419 of NMR spectroscopy in characterising the pharmaceutical tablets of starch have been

420 reviewed (Thérien-Aubin & Zhu, 2009), and thus is not related in the present review.

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421 9. Correlations between NMR and other analytical techniques

422 The results of some specific parameters of starch obtained from the NMR analysis were

423 compared with those measured by other techniques (Table 9). The studied aspects of starch

424 included chemical composition, structures of granules and components (e.g., degree of

425 branching), gelatinization and retrogradation, glass transition temperatures, and degree of

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426 substitution in modified starch. Kasemsuwan and Jane (1996) analysed the total phosphorus

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427 content of different starches by both 31P NMR and colorimetry-based methods. The results of

428 these two methods agreed well. Dunn and Krueger (1999) analysed the amylose contents by

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1
429 H NMR and also by the iodine-binding spectrophotometry-based assay. The results from

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430 these two assays agreed well with each other. The degree of branching (DB) of various
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431 starches were measured by 1H NMR and high-performance size-exclusion chromatography

432 (HPSEC), and was also reflected by the blue value obtained from starch-iodine interaction
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433 (Nilsson et al., 1996; Syahariza et al., 2013). Both 1H NMR and HPSEC methods gave

434 similar DB values which were highly correlated with the blue value of starch. The contents of
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435 double helices of starch measured by 13C CP/MAS NMR were compared with the degree of
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436 crystallinity of starch measured by XRD (Lopez-Rubio et al., 2008; Witt & Gilbert, 2014;
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437 Mutungi et al., 2012). Contents of double helices of different types of starches were

438 positively correlated with the degree of crystallinity from the XRD analysis. Lopez-Rubio et
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439 al. (2008) noted that the contents of double helices in starch granules are not equal to that of
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440 degree of crystallinity as some of the double helices do not crystallize. This discrepancy was

441 also observed when monitoring the structural changes of crystalline parts of starch during

442 acid hydrolysis by two different methods (i.e., NMR and XRD) (Atichokudomchai et al.,

443 2004) (supplementary material Fig. 9). The degree of crystallinity was also correlated with

444 the data from Fourier transform-Raman spectroscopy and gelatinization parameters

445 (temperatures and ∆H) by DSC. The results were highly correlated with the NMR results

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446 (Witt & Gilbert, 2014; Mutungi et al., 2012; Cooke and Gidley, 1992). Starch retrogradation

447 reflected by 1H NMR was positively correlated with that by the Instron texture analyser

448 (Seow & Teo, 1996). Retrogradation of pulse starches quantified by 13C CP/MAS NMR was

449 compared with that by FTIR–ATR, XRD, DSC, and the enzyme susceptibility of α-amylase

450 (Ambigaipalan et al., 2013). The extents of retrogradation measured by DSC, XRD, and

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451 NMR were similar, and those by NMR, FTIR-ATR, and enzyme susceptibility methods were

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452 different. The similarity and discrepancy indicate that different methods may reflect different

453 aspects of the retrogradation. Ambigaipalan et al. (2013) suggested that 13C CP/MAS NMR is

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454 sensitive to the conformational changes related with amylopectin crystallization while FTIR

455 more reflects the crystallization and re-association of both amylopectin-amylopectin and

456
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amylose–amylopectin chains. Therefore, a comprehensive approach employing different
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457 instruments should be used to gain a holistic picture of the starch retrogradation. Kalichevsky

et al. (1992) studied the glass transition temperature of amorphous starch by pulsed 1H NMR,
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458

459 DSC, Instron texture analyser, and dynamic mechanical thermal analysis (DMTA). The glass
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460 transition temperatures of starch measured by NMR were lower than those by DSC and other
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461 techniques by 20−30 oC (supplementary material Fig. 3). The NMR technique probes a lower

462 mobility distance scale and determines proton mobility, while DSC and DMTA relate to a
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463 transition with an increased mobility for a large proportion of the starch chains (Kalichevsky
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464 et al., 1992). The degree of substitution (acetylation and hydroxypropylation) as well as the
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465 degree of grafting of starch copolymers were analysed by NMR techniques. The results from

466 NMR analysis were compared with that of wet chemistry-method (e.g., Johnson and titration

467 methods) (de Graaf et al., 1995; Gurruchaga et al., 1992). NMR-based methods (1H and 13C)

468 agreed well with the traditional wet chemistry methods for the modification analyis, while

469 being time-saving.

470 10. Conclusions

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471 NMR techniques have been used to probe diverse aspects of starch systems. These techniques

472 include liquid state 1H NMR, 13C NMR, 31P NMR, and 17O NMR and solid state 13C CP/MAS

473 NMR. Various aspects/parameters of starch that have been studied by NMR include chemical

474 composition, degree of molecular order, degree of branching, degree of gelatinization and

475 retrogradation, glass transition temperature, physical structure of V-type amylose inclusion

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476 complexes, and extents and position of modification. NMR techniques have also been used to

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477 characterise starch interactions with various non-starch food components such as polyphenols,

478 proteins, and minerals. The baking and staling processes of bread have been monitored, and

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479 the physical structure of starch in edible films has been followed. The structural information

480 obtained from NMR analysis provides a strong basis for better understanding the

481
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physicochemical properties of starch and food systems. The composition and molecular
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482 structure of starch obtained from NMR analysis correlate well with the results of other
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483 techniques such as XRD and wet chemistry-based methods. Compared with traditional

484 methods for starch characterisation, the NMR approach can be simple, efficient, and time-
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485 saving, while the solid state NMR method can be non-destructive. Besides NMR, most of the
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486 studies also employed some other techniques to characterise starch systems. NMR-based

487 techniques complement the others for a comprehensive understanding of the structural and
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488 physicochemical properties of starch. It should be stressed that some structural parameters of
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489 starch are sensitive to the nature of the measurement technique. The knowledge obtained
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490 from NMR analysis will be of great importance for a better understanding of the starch

491 functionalities and the role of starch in food and non-food applications.

492

493 References

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 ACCEPTED MANUSCRIPT
494 Ai, Y., Hasjim, J., & Jane, J. L. (2013). Effects of lipids on enzymatic hydrolysis and

495 physical properties of starch. Carbohydrate Polymers, 92, 120–127.

496 Ambigaipalan, P., Hoover, R., Donner, E., & Liu, Q. (2013). Retrogradation characteristics of

497 pulse starches. Food Research International, 54, 203–212.

PT
498 Atichokudomchai, N., Varavinit, S., & Chinachoti, P. (2004). A study of ordered structure in

499 acid-modified tapioca starch by 13C CP/MAS solid-state NMR. Carbohydrate Polymers, 58,

RI
500 383–389.

SC
501 Baianu, I. C., Yakubu, P. I., & Ozu, E. (1999). Structural and hydration studies of waxy and

502 mealy potato starch cultivars by deuterium, carbon-13 CP-MAS/MASS NMR, and electron

503
U
Microscopy. Macromolecular Symposia, 140, 187−195.
AN
504 Beeren, S. R., & Hindsgaul, O. (2013). Nature’s dendrimer: characterizing amylopectin as a
M

505 multivalent host. Angewandte Chemie International Edition, 52, 11265–11268.

506 BeMiller, J., & Whistler, R. (2009). Starch: Chemistry and Technology. Academic Press.
D
TE

507 Bosmans, G. M., Lagrain, B., Deleu, L. J., Fierens, E., Hills, B. P., & Delcour, J. A. (2012).

508 Assignments of proton populations in dough and bread using NMR relaxometry of starch,
EP

509 gluten, and flour model systems. Journal of Agricultural and Food Chemistry, 60,

510 5461−5470.
C
AC

511 Bosmans, G. M., Lagrain, B., Ooms, N., Fierens, E., & Delcour, J. A. (2013). Biopolymer

512 interactions, water dynamics, and bread crumb firming. Journal of Agricultural and Food

513 Chemistry, 61, 4646−4654.

514 Brito, L. M., Sebastião, P. J. O., & Tavares, M. I. B. (2015). NMR relaxometry evaluation of

515 nanostructured starch-PLA blends. Polymer Testing, 45, 161−167.

22
 ACCEPTED MANUSCRIPT
516 Cai, J., Cai, C., Man, J., Yang, Y., Zhang, F., & Wei, C. (2014a). Crystalline and structural

517 properties of acid-modified lotus rhizome C-type starch. Carbohydrate Polymers, 102, 799–

518 807.

519 Cai, J., Yang, Y., Man, J., Huang, J., Wang, Z., Zhang, C., Gu, M., Liu, Q., & Wei, C.

520 (2014b). Structural and functional properties of alkali-treated high-amylose rice starch. Food

PT
521 Chemistry, 145, 245–253.

RI
522 Cappai, M. G., Alesso, G. A., Nieddu, G., Sanna, M., & Pinna, W. (2013). Electron

SC
523 microscopy and composition of raw acorn starch in relation to in vivo starch digestibility.

524 Food & Function, 4, 917–922.

525
U
Chauhan, K., Priya, V., Singh, P., Chauhan, G. S., Kumari, S., & Singhal, R. K. (2015). A
AN
526 green and highly efficient sulfur functionalization of starch. RSC Advances, 5, 51762–51772.
M

527 Cheetham, N. W. H., & Tao, L. (1998a). Oxygen-17 NMR relaxation studies on

528 gelatinization temperature and water mobility in maize starches. Carbohydrate Polymers, 35,
D

529 279−286.
TE

530 Cheetham, N. W. H., & Tao, L. (1998b). Solid state NMR studies on the structural and
EP

531 conformational properties of natural maize starches. Carbohydrate Polymers, 36, 285–292.

532 Cheng, W., Luo, Z., Li, L., & Fu, X. (2015). Preparation and characterization of debranched-
C

533 starch/phosphatidylcholine inclusion complexes. Journal of Agricultural and Food Chemistry,

AC

534 63, 634−641.

535 Cioica, N., Fechete, R., Cota, C., Nagy, E.M., David, L., & Cozar, O. (2013). NMR

536 relaxation investigation of the native corn starch structure with plasticizers. Journal of

537 Molecular Structure, 1044, 128–133.

23
 ACCEPTED MANUSCRIPT
538 Colquhoun, I. J., Parker, R., Ring, S.G., Sun, L., & Tang, H. R. (1995). An NMR

539 spectroscopic characterisation of the enzyme-resistant residue from α-amylolysis of an

540 amylose gel. Carbohydrate Polymers, 27, 255−259.

541 Cooke, D., & Gidley, M. J. (1992). Loss of crystalline and molecular order during starch

542 gelatinisation: origin of the enthalpic transition. Carbohydrate Research, 227, 103−112.

PT
543 de Graaf, R.A., Lammers, G., Janssen, L. P. B. M., & Beenackers, A. A. C. M. (1995).

RI
544 Quantitative analysis of chemically modified starches by 1H-NMR spectroscopy.

SC
545 Starch/Stärke, 47, 469−475.

546 Deng, Y., Jin, Y., Luo, Y., Zhong, Y., Yue, J., Song, X., & Zhao, Y. (2014). Impact of

547
U
continuous or cycle high hydrostatic pressure on the ultrastructure and digestibility of rice
AN
548 starch granules. Journal of Cereal Science, 60, 302−310.
M

549 Dunn, Jr. L. B., & Krueger, W. J. (1999). Branching ratios of starch via proton nuclear

550 magnetic resonance and their use in determining amylose/amylopectin content: evidence for
D

551 three types of amylopectin. Macromolecular Symposia, 140, 179−186.

TE

552 Fan, D., Ma, S., Wang, L., Zhao, H., Zhao, J., Zhang, H., & Chen, W. (2013b). 1H NMR
EP

553 studies of starch–water interactions during microwave heating. Carbohydrate Polymers, 97,

554 406– 412.

C

555 Fan, D., Ma, W., Wang, L., Huang, J., Zhang, F., Zhao, J., Zhang, H., & Chen, W. (2013a).
AC

556 Determining the effects of microwave heating on the ordered structures of rice starch by

557 NMR. Carbohydrate Polymers, 92, 1395−1401.

558 Flanagan, B. M., Gidley, M. J., & Warren, F. J. (2015). Rapid quantification of starch

559 molecular order through multivariate modelling of 13C CP/MAS NMR spectra. Chemical

560 Communications, 51, 14856−14858.

24
 ACCEPTED MANUSCRIPT
561 Geng, F., Chang, P. R., Yu, J., & Ma, X. (2010). The fabrication and the properties of

562 pretreated corn starch laurate. Carbohydrate Polymers, 80, 360–365.

563 Genkina, N. K., & Kurkovskaya, L. N. (2013). A novel method for the determination of

564 phospholipids in starch matrixes. Journal of Analytical Chemistry, 68, 170–172.

PT
565 Gidley, M. J. (1985). Quantification of the structural features of starch polysaccharides by

566 N.M.R. spectroscopy. Carbohydrate Research, 139, 85−93.

RI
567 Gidley, M. J. (1989). Molecular mechanisms underlying amylose aggregation and gelation.

SC
568 Macromolecules, 22, 351−358.

569 Gidley, M. J. (2014). Starch NMR in Starch Polymers: From Genetic Engineering to Green

570
U
Applications edited by P. Halley, L. Averous. (pp, 243−253). Elsevier.
AN
571 Gidley, M. J., & Bociek, S. M. (1985). Molecular organization in starches: a 13C CP/MAS
M

572 NMR study. Journal of the American Chemical Society, 107, 7040−7044.
D

573 Gidley, M. J., & Bociek, S. M. (1988). 13C CP/MAS NMR studies of amylose inclusion
TE

574 complexes, cyclodextrins, and the amorphous phase of starch granules: relationships between

575 glycosidic linkage conformation and solid-state 13C chemical shifts. Journal of the American
EP

576 Chemical Society, 110, 3820−3829.

C

577 Gidley, M. J., Cooke, D., Darke, A.H., Hoffmann, R.A., Russell, A.L., & Greenwell, P.
AC

578 (1995). Molecular order and structure in enzyme-resistant retrograded starch. Carbohydrate

579 Polymers, 28, 23−31.

580 Guo, Z., Zeng, S., Zhang, Y., Lu, X., Tian, Y., & Zheng, B. (2015). The effects of ultra-high

581 pressure on the structural, rheological and retrogradation properties of lotus seed starch. Food

582 Hydrocolloids, 44, 285−291.

25
 ACCEPTED MANUSCRIPT
583 Gurruchaga, M., Goñi, I., Vazquez, B., Valero, M., & Guzmán, G. M. (1992). Analysis of

584 graft copolymers onto starch by 13C NMR spectroscopy. Macromolecules, 25, 3009−3014.

585 Thérien-Aubin, H., & Zhu, X. X. (2009). NMR spectroscopy and imaging studies of

586 pharmaceutical tablets made of starch. Carbohydrate Polymers, 75, 369−379.

PT
587 Hoover, R. (1995). Starch retrogradation. Food Reviews International, 11, 331−346.

588 Horii, F., Yamamoto, H., Hirai, A., & Kitamaru, R. (1987). Structural study of amylose

RI
589 polymorphs by cross-polarization-magic-angle spinning, 13C-N.M.R. spectroscopy.

SC
590 Carbohydrate Research, 160, 29−40.

591 Htoon, A., Shrestha, A. K., Flanagan, B. M., Lopez-Rubio, A., Bird, A. R., Gilbert, E.P., &

592
U
Gidley, M. J. (2009). Effects of processing high amylose maize starches under controlled
AN
593 conditions on structural organisation and amylase digestibility. Carbohydrate Polymers, 75,
M

594 236–245.

595 Hu, X., Wei, B., Zhang, B., Li, H., Xu, X., Jin, Z., & Tian, Y. (2013). Interaction between
D

596 amylose and 1-butanol during 1-butanol-hydrochloric acid hydrolysis of normal rice starch.
TE

597 International Journal of Biological Macromolecules, 61, 329−332.

EP

598 Hu, Y., & Tang, M. (2015). Synthesis of starch-g-lactic acid copolymer with high grafting

599 degree catalyzed by ammonia water. Carbohydrate Polymers, 118, 79–82.

C

Jane, J. L., Robyt, J. F., & Huang, D. H. (1985). 13C-N.M.R study of the conformation of
AC

600

601 helical complexes of amylodextrin and of amylose in solution. Carbohydrate Research, 140,

602 21−35.

603 Kalichevsky, M. T., Jaroszkiewicz, E. M., Ablett, S., Blanshard, J. M. V., & Lillford, P. J.

604 (1992). The glass transition of amylopectin measured by DSC, DMTA and NMR.

605 Carbohydrate Polymers, 18, 77−88.

26
 ACCEPTED MANUSCRIPT
606 Kasemsuwan, T., & Jane, J. (1994). Location of amylose in normal starch granules. II.

607 Locations of phosphodiester cross-linking revealed by phosphorus-31 nuclear magnetic

608 resonance. Cereal Chemistry, 71, 282−287.

609 Kasemsuwan, T., & Jane, J. L. (1996). Quantitative method for the survey of starch

610 phosphate derivatives and starch phospholipids by 31P nuclear magnetic resonance

PT
611 spectroscopy. Cereal Chemistry, 73, 702−707.

RI
612 Kulik, A. S., & Haverkamp, J. (1997). Molecular mobility of polysaccharide chains in starch

SC
613 investigated by two-dimensional solid-state NMR spectroscopy. Carbohydrate Polymers, 34,

614 49−54.

615
U
Larsen, F. H., Kasprzak, M. M., Lærke, H. N., Knudsen, K. E. B., Pedersen, S., Jørgensen, A.
AN
616 S., & Blennow, A. (2013). Hydration properties and phosphorous speciation in native,

617 gelatinized and enzymatically modified potato starch analyzed by solid-state MAS NMR.
M

618 Carbohydrate Polymers, 97, 502−511.

D

619 Laws, D. D., Bitter, H. M. L., and Jerschow, A. (2002). Solid-state NMR spectroscopic
TE

620 methods in chemistry. Angewandte Chemie International Edition, 41, 3096−3129.

EP

621 Le Bail, P., Chauvet, B., Simonin, H., Rondeau-Mouro, C., Pontoire, B., de Carvalho, M., &

622 Le-Bail, A. (2013). Formation and stability of amylose ligand complexes formed by high
C

623 pressure treatment. Innovative Food Science and Emerging Technologies, 18, 1–6.
AC

624 Le Bail, P., Rondeau, C., & Buléon, A. (2005). Structural investigation of amylose complexes

625 with small ligands: Helical conformation, crystalline structure and thermostability.

626 International Journal of Biological Macromolecules, 35, 1–7.

27
 ACCEPTED MANUSCRIPT
627 Lebail, P., Buleon, A., Shiftan, D., & Marchessault, R. H. (2000). Mobility of lipid in

628 complexes of amylose–fatty acids by deuterium and 13C solid state NMR. Carbohydrate

629 Polymers, 43, 317–326.

630 Le-Bail, P., Lorentz, C., Pencreac’h, G., Soultani-Vigneron, S., Pontoire, B., López Giraldo,

631 L. J., Villeneuve, P., Hendrickx, J., & Tran, V. (2015). Trapping by amylose of the aliphatic

PT
632 chain grafted onto chlorogenic acid: Importance of the graft position. Carbohydrate Polymers,

RI
633 117, 910–916.

SC
634 Li, S., Dickinson, L. C., & Chinachoti, P. (1998). Mobility of “unfreezable” and “freezable”

635 water in waxy corn starch by 2H and 1H NMR. Journal of Agricultural and Food Chemistry,

U
636 46, 62−71.
AN
637 Li, Y., Lin, S., Hu, J., Liu, G., Zhang, G., Tu, Y., Luo, H., & Li, W. (2014). Metal ion

638 induced-assembly of amylose in aqueous solution. Carbohydrate Polymers, 102, 489–496.

M

639 Lim, S. T., Kasemsuwan, T., & Jane, J. L. (1994). Characterization of phosphorus in starch
D

640 by 31P-nuclear magnetic resonance spectroscopy. Cereal Chemistry, 71, 488−493.

TE

641 Lim, Y. M., Hoobin, P., Ying, D. Y., Burgar, I., Gooley, P. R., & Augustin, M. A. (2015).
EP

642 Physical characterisation of high amylose maize starch and acylated high amylose maize

643 starches. Carbohydrate Polymers, 117, 279–285.

C

644 Lima, F. F., & Andrade, C. T. (2010). Effect of melt-processing and ultrasonic treatment on
AC

645 physical properties of high-amylose maize starch. Ultrasonics Sonochemistry, 17, 637–641.

646 Lopez-Rubio, A., Flanagan, B. M., Gilbert, E. P., & Gidley, M. J. (2008). A novel approach

647 for calculating starch crystallinity and its correlation with double helix content: a combined

648 XRD and NMR study. Biopolymers, 89, 761−768.

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 ACCEPTED MANUSCRIPT
649 Lu, X., Luo, Z., Fu, X., & Xiao, Z. (2013). Two-step method of enzymatic synthesis of starch

650 laurate in ionic liquids. Journal of Agricultural and Food Chemistry, 61, 9882−9891.

651 Luo, Z., Cheng, W., Chen, H., Fu, X., Peng, X., Luo, F., & Nie, L. (2013). Preparation and

652 properties of enzyme-modified cassava starch−zinc complexes. Journal of Agricultural and

653 Food Chemistry, 61, 4631−4638.

PT
654 Man, J., Yang, Y., Huang, J., Zhang, C., Chen, Y., Wang, Y., Gu, M., Liu, Q., & Wei, C.

RI
655 (2013). Effect of simultaneous inhibition of starch branching enzymes I and IIb on the

SC
656 crystalline structure of rice starches with different amylose contents. Journal of Agricultural

657 and Food Chemistry, 61, 9930−9937.

658
U
Marchessault, R. H., & Taylor, M. G. (1985). Solid-state 13C-c.p.-m.a.s. n.m.r. of starches.
AN
659 Carbohydrate Research, 144, C1−C5.
M

660 Mihhalevski, A., Heinmaa, I., Traksmaa, R., Pehk, T., Mere, A., & Paalme, T. (2012).

661 Structural changes of starch during baking and staling of rye bread. Journal of Agricultural
D

662 and Food Chemistry, 60, 8492−8500.

TE

663 Morgan, K. R., Furneaux, R. H., & Larsen, N. G. (1995). Solid-state NMR studies on the
EP

664 structure of starch granules. Carbohydrate Research, 276, 387−399.

665 Muhrbeck, P., & Tellier, C. (1991). Determination of the posphorylation of starch from
C

666 native potato varieties by 31P NMR. Starch/Stärke, 43, 25−27.

AC

667 Mutungi, C., Passauer, L., Onyango, C., Jaros, D., & Rohm, H. (2012). Debranched cassava

668 starch crystallinity determination by Raman spectroscopy: Correlation of features in Raman

669 spectra with X-ray diffraction and 13C CP/MAS NMR spectroscopy. Carbohydrate Polymers,

670 87, 598−606.

29
 ACCEPTED MANUSCRIPT
671 Nilsson, G. S., Bergquist, K. E., Nilsson, U., & Gorton, L. (1996). Determination of the

672 degree of branching in normal and amylopectin type potato starch with H-NMR spectroscopy.

673 Improved resolution and two-dimensional spectroscopy. Starch/Stärke, 48, 352−357.

674 Paris, M., Bizot, H., Emery, J., Buzaré, J. Y., & Buléon, A. (2001a). NMR local range

675 investigations in amorphous starchy substrates I. Structural heterogeneity probed by 13C CP–

PT
676 MAS NMR. International Journal of Biological Macromolecules, 29, 127–136.

RI
677 Paris, M., Bizot, H., Emery, J., Buzaré, J.Y., & Buléon, A. (1999). Crystallinity and

SC
678 structuring role of water in native and recrystallized starches by 13C CP-MAS NMR

679 spectroscopy 1: Spectral decomposition. Carbohydrate Polymers, 39, 327–339.

680
U
Paris, M., Bizot, H., Emery, J., Buzaré, J.Y., & Buléon, A. (2001b). NMR local range
AN
681 investigations in amorphous starchy substrates: II-Dynamical heterogeneity probed by 1H/13C

682 magnetization transfer and 2D WISE solid state NMR. International Journal of Biological
M

683 Macromolecules, 29, 137–143.

D

684 Pizzoferrato, L., Paci, M., & Rotilio, G. (1998). Structural modification and bioavailability of
TE

685 starch components as related to the extent of Maillard reaction: an enzymatic degradation and

686 a solid-state 13C CPMAS NMR study. Journal of Agricultural and Food Chemistry, 46,
EP

687 438−441.
C

688 Pizzoferrato, L., Rotilio, G., & Paci, M. (1999). Modification of structure and digestibility of
AC

689 chestnut starch upon cooking: a solid state 13C CP MAS NMR and enzymatic degradation

690 study. Journal of Agricultural and Food Chemistry, 47, 4060−4063.

691 Primo-Martín, C., van Nieuwenhuijzen, N. H., Hamer, R. J., & van Vliet, T. (2007).

692 Crystallinity changes in wheat starch during the bread-making process: Starch crystallinity in

693 the bread crust. Journal of Cereal Science, 45, 219–226.

30
 ACCEPTED MANUSCRIPT
694 Pushpadass, H. A., Kumar, A., Jackson, D. S., Wehling, R. L., Dumais, J. J., & Hanna, M. A.

695 (2009). Macromolecular changes in extruded starch-films plasticized with glycerol, water and

696 stearic acid. Starch/Stärke, 61, 256–266.

697 Rashid, I., Omari, M. H. A., Leharne, S. A., Chowdhry, B. Z. and Badwan, A. (2012). Starch

698 gelatinization using sodium silicate: FTIR, DSC, XRPD, and NMR studies. Starch/Stärke, 64,

PT
699 713–728.

RI
700 Richardson, S. J., Baianu, I. C., & Steinberg, M. P. (1987). Mobility of water in corn starch

SC
701 suspensions determined by nuclear magnetic resonance. Starch/Stärke, 39, 79−83.

702 Ritota, M., Gianferri, R., Bucci, R., & Brosio, E. (2008). Proton NMR relaxation study of

703
U
swelling and gelatinisation process in rice starch–water samples. Food Chemistry, 110, 14–22.
AN
704 Rondeau-Mouro, C., Cambert, M., Kovrlija, R., Musse, M., Lucas, T., & Mariette, F. (2015).
M

705 Temperature-associated proton dynamics in wheat starch-based model systems and wheat

706 flour dough evaluated by NMR. Food and Bioprocess Technology, 8, 777–790.
D

707 Salomonsson, A. C. B., Andersson, R. E., Torneport, L. J. & Theander, O. (1991). A 1H- and
TE

13
708 C-n.m.r. study of bromine-oxidised potato starch. Carbohydrate Research, 217, 221−225.
EP

709 Sandhu, H. P. S., Manthey, F. A., & Simsek, S. (2012). Ozone gas affects physical and

710 chemical properties of wheat (Triticum aestivum L.) starch. Carbohydrate Polymers, 87,
C

711 1261– 1268.

AC

712 Sasmal, D., Singh, R.P., & Tripathy, T. (2015). Synthesis and flocculation characteristics of a

713 novel biodegradable flocculating agent amylopectin-g-poly(acrylamide-co-N-

714 methylacrylamide). Colloids and Surfaces A: Physicochemical and Engineering Aspects, 482,

715 575–584.

31
 ACCEPTED MANUSCRIPT
716 Schmieder, P., Nitschke, F., Steup, M., Mallow, K., & Specker, E. (2013). Determination of

717 glucan phosphorylation using heteronuclear 1H,13C double and 1H, 13C, 31P triple-resonance

718 NMR spectra. Magnetic Resonance in Chemistry, 51, 655–661.

719 Seow, C. C., & Teo, C. H. (1996). Staling of starch-based products: a comparative study by

720 firmness and pulsed NMR measurements. Starch/Stärke, 48, 90−93.

PT
721 Shrestha, A. K., Blazek, J., Flanagan, B. M., Dhital, S., Larroque, O., Morell, M. K., Gilbert,

RI
722 E. P., & Gidley, M. J. (2012). Molecular, mesoscopic and microscopic structure evolution

SC
723 during amylase digestion of maize starch granules. Carbohydrate Polymers, 90, 23–33.

724 Silva, A., Sousa, E., Palmeira, A., Amorim, P., de Pinho, P. G., & Ferreira, D. A. (2014).

725
U
Interaction between hydroxyethyl starch and propofol: computational and laboratorial study.
AN
726 Journal of Biomolecular Structure and Dynamics, 32, 1864–1875.
M

727 Sivam, A. S., Waterhouse, G. I. N., Zujovic, Z. D., Perera, C. O., & Sun-Waterhouse, D.

728 (2013). Structure and dynamics of wheat starch in breads fortified with polyphenols and
D

729 pectin: an ESEM and solid-state CP/MAS 13C NMR spectroscopic study. Food and
TE

730 Bioprocess Technology, 6, 110−123.

EP

731 Smits, A. L. M., Ruhnau, F. C., Vliegenthart, J. F. G., & van Soest, J. J. G. (1998). Ageing of

732 starch based systems as observed with FT-IR and solid state NMR spectroscopy.
C

733 Starch/Stärke, 50, 478–483.

AC

734 Snape, C. E., Morrison, W. R., Maroto-Valer, M. M., Karkalas, J., & Pethrick, R. A. (1998).

735 Solid state 13C NMR investigation of lipid ligands in V-amylose inclusion complexes.

736 Carbohydrate Polymers, 36, 225−237.

737 Spyros, A., & Dais, P. (2012). NMR spectroscopy in food analysis. London: Royal Society of

738 Chemistry.

32
 ACCEPTED MANUSCRIPT
739 Syahariza, Z. A., Sar, S., Hasjim, J., Tizzotti, M. J., & Gilbert, R. G. (2013). The importance

740 of amylose and amylopectin fine structures for starch digestibility in cooked rice grains. Food

741 Chemistry, 136, 742–749.

742 Tabata, S., & Hizukuri, S. (1971). Studies on starch phosphate. Part 2. Isolation of glucose 3-

743 phosphate and maltose phosphate by acid hydrolysis of potato starch. Starch/Stärke, 23, 267–

PT
744 272.

RI
745 Teo, C. H., & Seow, C. C. (1992). A pulsed NMR method for the study of starch

SC
746 retrogradation. Starch/Stärke, 44, 288−292.

747 Thakore, S., Valodkar, M., Soni, J. Y., Vyas, K., Jadeja, R. N., Devkar, R. V., & Rathore, P.

748
U
S. (2013). Synthesis and cytotoxicity evaluation of novel acylated starch nanoparticles.
AN
749 Bioorganic Chemistry, 46, 26–30.
M

750 Tizzotti, M. J., Sweedman, M. C., Tang, D., Schaefer, C., & Gilbert, R. G. (2011). New 1H

751 NMR procedure for the characterization of native and modified food-grade starches. Journal
D

752 of Agricultural and Food Chemistry, 59, 6913–6919.

TE

753 Veregin, R. P., Fife, C. A., Marchessault, R. H., & Taylor, G. M. (1987). Correlation of 13C
EP

754 chemical shifts with torsional angles from high-resolution, 13C-C.P.-M..A.S. N. M. R. studies

755 of crystalline cyclomalto-oligosaccharide complexes, and their relation to the structures of the
C

756 starch polymorphs. Carbohydrate Research, 160, 41−56.

AC

757 Veregin, R. P., Fyfe, C. A., Marchessault, R. H., & Taylor, M. G. (1986). Characterization of

758 the crystalline A and B starch polymorphs and investigation of starch crystallization by high-

759 resolution 13C CP/MAS NMR. Macromolecules, 19, 1030−1034.

760 Wang, S., & Copeland, L. (2012). Effect of alkali treatment on structure and function of pea

761 starch granules. Food Chemistry, 135, 1635–1642.

33
 ACCEPTED MANUSCRIPT
762 Warren, F. J., Gidley, M. J., & Flanagan, B. M. (2016). Infrared spectroscopy as a tool to

763 characterise starch ordered structure—a joint FTIR–ATR, NMR, XRD and DSC study.

764 Carbohydrate Polymers, 139, 35–42.

765 Wei, Y., Cheng, F., & Zheng, H. (2008). Synthesis and flocculating properties of cationic

766 starch derivatives. Carbohydrate Polymers, 74, 673–679.

PT
767 Witt, T., & Gilbert, R. G. (2014). Causal relations between structural features of amylopectin,

RI
768 a semicrystalline hyperbranched polymer. Biomacromolecules, 15, 2501−2511.

SC
769 Wu, Y., Lin, Q., Chen, Z., & Xiao, H. (2011). The interaction between tea polyphenols and

770 rice starch during gelatinization. Food Science and Technology International, 17, 569−577.

771
U
Xu, A., & Seib, P. A. (1997). Determination of the level and position of substitution in
AN
772 hydroxypropylated starch by high-resolution 1H-NMR spectroscopy of alpha-limit dextrins.
M

773 Journal of Cereal Science, 25, 17–26.

774 Yang, Y., Tang, Z., Xiong, Z., & Zhu, J. (2015). Preparation and characterization of
D

775 thermoplastic starches and their blends with poly(lactic acid). International Journal of
TE

776 Biological Macromolecules, 77, 273–279.

EP

777 Ye, F., Miao, M., Huang, C., Lu, K., Jiang, B., & Zhang, T. (2014). Elucidation of substituted

778 ester group position in octenyl succinic anhydride modified sugary maize soluble starch.
C

779 Journal of Agricultural and Food Chemistry, 62, 11696−11705.

AC

780 Zhang, B., Dhital, S., Flanagan, B. M., & Gidley, M. J. (2014). Mechanism for starch granule

781 ghost formation deduced from structural and enzyme digestion properties. Journal of

782 Agricultural and Food Chemistry, 62, 760−771.

34
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783 Zhang, J., & Wang, Z. W. (2009). Optimization of reaction conditions for resistant Canna

784 edulis Ker starch phosphorylation and its structural characterization. Industrial Crops and

785 Products, 30, 105–113.

786 Zhang, Q., Xu, K., & Wang, P. (2008). Study on structure and molecular dynamics of starch/

787 superabsorbent by 13C solid state NMR. Fibers and Polymers, 9, 271−275.

PT
788 Zhang, Z., Zhao, S., & Xiong, S. (2013). Molecular properties of octenyl succinic esters of

RI
789 mechanically activated Indica rice starch. Starch/Stärke, 65, 453–460.

SC
790 Zhao, J., Chen, Z., Jin, Z., de Waard, P., Buwalda, P., Gruppen, H., & Schols, H. A. (2015).

791 Level and position of substituents in cross-linked and hydroxypropylated sweet potato

792
U
starches using nuclear magnetic resonance spectroscopy. Carbohydrate Polymers, 131, 424–
AN
793 431.
M

794 Zou, W., Yu, L., Liu, X., Chen, L., Zhang, X., Qiao, D., & Zhang, R. (2012). Effects of

795 amylose/amylopectin ratio on starch-based superabsorbent polymers. Carbohydrate Polymers,

D

796 87, 1583–1588.

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1 Table captions

2 Table 1 Chemical composition of starch quantified by NMR spectroscopy

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3 Table 2 Structural characterisation of starch studied by NMR spectroscopy

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4 Table 3 Structure of V-type inclusion complexes revealed by NMR spectroscopy

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5 Table 4 Gelatinization and retrogradation processes monitored by NMR spectroscopy

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6 Table 5 Structure of modified starches as studied by NMR spectroscopy

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7 Table 6 Structure of starch graft copolymers as studied by NMR spectroscopy

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8 Table 7 Interactions of starch with non-starch components as monitored by NMR spectroscopy

9 Table 8 NMR characterization of bread and starch films

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10 Table 9 Correlations between NMR and other analytical techniques in structural characterization of starch systems
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11 Table 1

Parameter Starch NMR type Major findings Reference

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source

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31
Degree of Potato P NMR Degree of phosphorylation on the C-3 position was independent of potato variety, while that Muhrbeck and

SC
phosphorylation on on C-6 position varied greatly among different genotypes Tellier, 1991

the C-3 and C-6

U
31

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Phosphate Various P NMR Normal cereal starches contained mostly phospholipids. Tuber and root starches were free Lim et al., 1994

monoester, inorganic sources of phospholipids. Legume and potato starches mostly contained phosphate monoesters.

M
phosphate, Phosphate monoesters of all starches were more on the C-6 than on the C-3 of the glucose

D
phospholipids units

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31
Phosphate Various P NMR Potato starch mostly contained phosphate monoester (0.086%). Wheat starch contained Kasemsuwan and
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monoester, inorganic sources mostly phospholipids (0.058%), high-amylose (50% amylose content) maize starch mostly Jane, 1996
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phosphate, contained phosphate monoesters (0.0049%) and phospholipids (0.015%). Waxy maize
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phospholipids starch had trace amounts of phosphate monoesters

phosphorylation site Curcuma Heteronuclear A combination of heteronuclear 1H, 13C and 1H, 13C, 31P NMR spectra efficiently Schmieder et al.,
1
on glucan chains rhizome H, 13C double, determined the phosphorylation sites. Monophosphate esters are on the C-3 and C-6 2013

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1
H,13C, 31P triple positions of the glucose unit
31
Phospholipids Wheat P NMR Phosphatidylcholine was mixed with starch in DMSO. Phospholipid content was Genkina &

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determined by 31P NMR with tributyl phosphate as the standard. This method allows the Kurkovskaya,

RI
quantification of phospholipids without the regards of sample forms 2013
1
Amylose content Various H NMR Branching ratios of starch were obtained from 1H NMR. This ratio was then used for the Dunn and

SC
sources determination of amylose content Krueger, 1999

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12 Table 2

Targeted structure Starch source NMR type Major findings Reference

PT
Molecular order Maize, potato Solid state 13C CP/MAS 13
C NMR spectrum was interpreted in relation to the crystallinity of Marchessault &

RI
NMR starch for the first time Taylor, 1985

SC
Molecular order Various sources Solid state 13C CP/MAS 13
C NMR spectrum was interpreted in relation to the crystallinity of Gidley &

NMR starch for the first time. The spectrum was related to the polymorph type Bociek, 1985

U
AN
of starch

Molecular order Various sources Solid state 13C CP/MAS 13

C NMR spectrum was related to the starch polymorph. C-1 peak was a Veregin et al.,

M
NMR triplet for A-type polymorph starches, and a doublet for B-type starches 1986

D
Molecular order Rice Solid state 13C CP/MAS The relative degree of crystallinity (37.2−58.9%), relative proportion of Man et al., 2013

NMR
TE single helices (1−7.9%), double helices (39.6−61.8%), and amorphous
EP
(37.2−52.5%) material were quantified. Deficiency of starch branching
C

enzymes I and IIb decreased degree of crystallinity, portion of double

AC

helices, and increased the portion of single helices of starch

Molecular order Various sources Solid state 13C CP/MAS A partial least squares model was produced to rapidly quantify the Flanagan et al.,

NMR ordered structure in starch granules from the NMR spectrum 2015

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Glass transition Waxy maize Pulsed NMR, 13C CP/MAS Pulsed NMR and 13C CP/MAS NMR successfully characterised the Kalichevsky et

temperature NMR glass transition of amorphous starch with water contents of 10−22% al., 1992

PT
13
Structure of Potato starch, amylose, C CP/MAS NMR, 1H/13C Five types of α(1–4) linkages were found by decompositions of C1 Paris et al.,

RI
amorphous starch amylopectin processed magnetization transfer and resonance spectrum. Distributions of average glycosidic linkages 2001a and

into amorphous and 2D WISE solid state NMR dihedral angles (ϕ, Ψ) were related to the structures resulted from the 2001b

SC
semi-crystalline status processing

U
1
Branching ratio Various sources H NMR, 13C NMR Ratios of α(1–4) to α(1–6) linkages of native and degraded starches were Gidley, 1985

AN
determined by 1H NMR and ranged from 17.5 to 26. Glucose and the

M
reducing residues of larger glucans can be differentiated by 13C NMR

D
1
Branching ratio and Various sources H NMR DB ranged from 1 (potato amylose) to 4.77% (maize amylopectin). Nilsson et al.,

TE
average unit chain Average unit chain length (CL) ranged from 21 (maize amylopectin) to 1996
EP
length 100 glucosyl residues (potato amylose)

Branching ratio Maize starches varying in 1H NMR Ratios of α(1–4) to α(1–6) linkages were determined by 1H NMR. Maize Dunn and
C

amylose content, potato, starches had the ratios ranging from 21.7 to 83.9% Krueger, 1999
AC

rice, pea, cassava, wheat

1
Branching ratio Maize, rice H NMR NMR was used to efficiently determine the degree of branching (DB) of Tizzotti et al.,

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maize starch as 3.34%. DB of rice starches from 14 genotypes ranged 2011; Syahariza

from 2.7−3.6% et al., 2013

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13 DB of whole starch molecules is defined as the percentage of α(1–6) glycosidic linkages (branching points) to the total of α-(1–4) and α-(1–6)

RI
14 glycosidic linkages

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15

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16

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17

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18 Table 3

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Targeted feature Amylose type Complexing agent NMR type Major findings References
13
Formation of Potato DMSO, KOH, iodine, C NMR Soluble V-type inclusion complexes in solution were Jane et al.,

RI
amylose/amylodextrin α-naphthol, methyl characterised. Increasing concentration of complexing agents 1985

SC
inclusion complexes in alcohol, tert-butyl induced downfield shift of the signals of C-1 and C-4 (larger shift)

U
solution alcohol, n-butyl and C-2, C-3, and C-6 (smaller shift). The differences in the shifts

AN
alcohol, cyclohexanol for C-1 and C-4 indicate the differences in the compactness of the

M
helical structure
13
NMR spectra of solid V- Potato, synthetic Sodium palmitate, C NMR spectra of V6 and V7-type complexes are similar, and are Gidley and

D
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type amylose inclusion amylose hexanoic acid, tert-butyl CP/MAS not affected by the type of complexing agents. V8-type complex Bociek, 1988

complexes alcohol, n-butanol, 1- NMR with 1-naphthol had chemical shifts of C-1 and C-4 peaks
EP
naphthol downward by 1 ppm (supplementary material Fig. 5)
C

13
NMR spectra of solid V- Synthetic Ethanol C A triplet C-1 peak for the A-type crystalline form and a doublet C- Horii et al.,
AC

type amylose inclusion amylose CP/MAS 1 peak for B-type crystals were recorded (supplementary material 1987

complexes, A- and B- NMR, 13C Fig. 4). Spectrum of V-type amylose is rather different from that of

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type amylose crystals spin-lattice A- and B-type in that the C-4 line is separated from C-2, C-3, and

relaxation C-5 atoms. 13C T1 values of the crystalline components of

PT
amyloses are shorter than that of cellulose (suggesting more

RI
mobility)
13
NMR spectra of solid V- Potato Stearic, palmitic, oleic, C Chemical shift of the mid-chain methylenes had a change by ~2−3 Snape et al.,

SC
type amylose inclusion linoleic, linolenic, and CP/MAS ppm, suggesting the complexation process. The mid-chain 1998

U
complexes docosahexaenoic acids, NMR methylenes of lipids in the V-type complexes had the same

AN
glycerol monooleate, chemical shifts, except for docosahexaenoic acid. Analysis of

M
glycerol monopalmitate, cross-polarisation dynamics showed that the bulky polar groups

D
lysophosphatidylcholine situated outside the V-helical segments and was adjacent to the

TE
amorphous regions
13
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NMR spectra of solid V- Potato Menthone, decanal, 1- C Differences in NMR spectra were observed among V6I, V6II, Le Bail et al.,

type amylose inclusion naphthol, 1-butanol CP/MAS V6III, and V8-type amylose complexes. Rehydration increased the 2005
C

complexes NMR sharpness of carbon resonance peak, and desorption induced the
AC

transition from V6II to V6I-type

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13
NMR spectra of solid V- Synthetic Palmitic, lauric acids C A single resonance peak at 32.4 ppm was assigned to the Lebail et al.,

type amylose inclusion amylose, pea CP/MAS complexed fatty acids. The uncomplexed palmitic acid was also 2000

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complexes NMR, detected in the samples with a chemical shift of 33.6 ppm. T1

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deuterium relaxation tests revealed a difference between the uncomplexed

NMR and complexed fatty acids (by 1.1−1.2 s). Temperature-dependent

SC
deuterium NMR analysis revealed partially disassociated

U
complexes of amylose-palmitic acid and a full complexation for

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the amylose–lauric acid

M
13
NMR spectra of solid V- Potato, broad Decanoic acid, C Peaks of 102.7, 81.4, 74.9, 71.6, and 61.3 ppm were related to C1, Le Bail et al.,

D
type amylose inclusion bean (Vicia carvacrol CP/MAS C4, C3, C2-C5, and C6, respectively, and these were of V6I form. 2013

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complexes faba), pea, NMR High pressure treatment induced the formation of V-type

complexes at a low temperature (40 oC)

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tapioca
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13
NMR spectra of solid V- Potato 3-O-palmitoyl C Spectrum had peaks with chemical shifts of 102.7, 81.4, 74.9, Le-Bail et al.,

type amylose inclusion chlorogenic acid CP/MAS 71.6, 61.3, and 60 ppm, and they were related to C1, C4, C3, C2- 2015

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complexes NMR C5, and C6 carbons, respectively. Only the grafted part (palmitoyl

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moiety) was included in the helical cavity, and the formation of the

V-type complexes depended on the graft position

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13
NMR spectra of solid V- Debranched Phosphatidylcholine C In comparison with the physical mixture of phosphatidylcholine Cheng et al.,

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type amylose inclusion potato CP/MAS and debranched maize starch, C-4 peak at 86.46 ppm was better 2015

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complexes NMR separated from the combined C-2, C-3, C-5 peak by 1 ppm.

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Chemical shifts of C1- and C-4 peaks moved up by 1−1.5 ppm as a

D
result of inclusion complex formation

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20 Table 4

Starch source NMR type Major findings Reference

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Gelatinization

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13
Wheat, maize, C CP/MAS The enthalpy change of gelatinisation measured by DSC mostly reflects the loss of molecular (double- Cooke and

SC
cassava, potato NMR helical) order which is measured by solid state NMR Gidley, 1992

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17 17
Maize starches O NMR O spin-spin relaxation time (T2) measurements were used to monitor the starch gelatinization as Cheetham and

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varying in amylose relaxation affected by various factors. The changes in T2 were related to the degree of gelatinization and water Tao, 1998a

M
content content. Higher contents of amylose and phosphates were related to lower water mobility. KI decreased

D
the water mobility during gelatinization

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1
Rice H NMR Four water populations were observed in starch-water systems. One of them was related to the bulk of Ritota et al.,
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water, while the others were related to the chemical and diffusive exchanges of water with starch 2008

components. The solid-to-liquid ratios from single–pulse experiment followed the water uptake of starch
C
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during swelling and gelatinisation processes

13
Potato, wheat, maize C and 31P solid CP and SP modes of 13C and 31P solid state MAS NMR were combined to probe the hydration properties Larsen et al.,

state MAS NMR of potato starch in native, gelatinized, and enzyme-modified status. Hydration increased the molecular 2013

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order. Gelatinization and enzyme treatments (branching enzyme and/or β-amylase) reduced the immobile

fractions of starch. Carbons of the α(1–4) linkages needed high hydration rates for uniform chemical

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shifts. Immobile phosphorus-containing chains were only found in the suspension of native starch
13

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Potato, maize C CP/MAS The structure of starch ghosts after gelatinization was probed. V-type amylose helices were present in Zhang et al.,

NMR maize and potato starch ghosts before enzyme digestion. After α-amylase hydrolysis, a small amount of 2014

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B-type polymorph was observed in potato starch ghost, while the V-type structure became more obvious

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in maize starch ghost

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Wheat Time-domain 1H Fraction of the shortest T2 relaxation time was related to the nonexchangeable protons in CH of both Rondeau-Mouro

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NMR amylopectin and amylose. Two T2 components were due to slow diffusional exchanges between et al., 2015

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different water layers

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Retrogradation
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Synthetic, potato C CP/MAS Amylose gel structure was studied. B-type polymorph was observed from the gel of diluted amylose Gidley, 1989

and 1H NMR solution. Gel from concentrated amylose solution (10%) contained rigid B-type double helices and more
C

mobile amorphous single chains

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Various sources Pulsed 1H NMR Pulsed 1H NMR was used to monitor the retrogradation of starch. Proton signals from re-crystallized Teo and Seow,

starch had a different rate of decay after radiofrequency pulse as compared with that of the amorphous 1992

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starch. NMR-based technique is simple, non-destructive, and has little requirement of sample size

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Waxy maize One- and two- Slow motions with correlation time of tens of milliseconds were detected by stimulated echo and two- Kulik and

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dimensional dimensional exchange NMR. Retrogradation occurs over a wide range of correlation times Haverkamp,

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exchange 1997

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Solid state NMR
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Potato C CP/MAS The retrogradation of starch was monitored below and above the glass transition temperature (Tg). Below Smits et al.,

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NMR, 1H NMR Tg, the relaxation time of 1H NMR increased due to the decreasing free volume before reaching a plateau. 1998

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Above Tg, water absorption decreased the relaxation time before an increase due to recrystallization. 13C

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CP/MAS NMR was not an efficient technique for retrogradation detection
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Pulse starches C CP/MAS The retrogradation of starches from various pulses (faba bean, black bean, pinto bean). Intensity of C-4 Ambigaipalan et
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NMR peak decreased during retrogradation, while the content of double helices increased al., 2013
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21 Table 5

Modification Starch NMR type Uses References

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Physical

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High pressure Rice, lotus C CP/MAS The structural changes of rice starch as affected by high pressure treatment were followed by 13C Deng et al., 2014;

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seed NMR CP/MAS NMR. 600 MPa increased the proportion of amorphous material of starch, and had no Guo et al., 2015

effect on the polymorph type. High pressure had no effect on the chemical shifts of various carbon

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peaks, while decreasing the relative crystallinity

Melt-processing High-amylose 1H NMR Starch was melt-processed (100 oC, 40 rpm, 8 min) and ultrasonicated (750 W, 20 kHz) in the Lima and

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and ultrasound maize starch presence of glycerol. 1H NMR spectra of sonicated and melt-processed samples were similar. 1H Andrade, 2010

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NMR analysis indicated the de-aggregating of starch induced by ultrasound

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Microwave Rice C CP/MAS Microwave heating increased the proportion of amorphous material and decreased that of both Fan et al., 2013a
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NMR, 1H single and double helices. The effect of rapid heating in oil bath was similar to that of microwave and 2013b

heating. Water mobility of starch-water system was also monitored by 1H NMR. Water mobility
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NMR
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of samples subjected to rapid conventional heating was lower and that of microwave heating was

higher

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Acid hydrolysis Lotus C CP/MAS Acid hydrolysis increased the double helix content and decreased the amorphous portion of starch. Atichokudomchai

rhizome, NMR The relative crystallinity increased sharply during the initial hydrolysis before levelling off et al., 2004; Cai et

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cassava al., 2014a;
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Alkali hydrolysis Pea C CP/MAS Alkali hydrolysis had no effect on chemical shifts, broadened the resonance peak of C-2–C-5 Wang and

NMR sites, and decreased the content of double helices from 35 to 30% Copeland, 2012

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Alkali hydrolysis Rice C CP/MAS Alkali hydrolysis had no effect on NMR spectra of rice starches varying in amylose contents (24 Cai et al., 2014b

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NMR and 58%)

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Substitution

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Acetylated, Potato H NMR Molar substitution of modified starches was quantified by 1H NMR de Graaf et al.,

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hydroxypropylated 1995

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Hydroxypropylated Maize, wheat, 1H NMR Both the level and position of hydroxypropylation of starch were determined by 1H NMR analysis Xu and Seib, 1997
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cassava of α-limit dextrins
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Hydroxypropylated Sweet potato P, 1H NMR Molar substitution was quantified by 1H NMR, and hydroxypropylation mostly occurred at O-2 Zhao et al., 2015
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(61%), followed by O-6 (21%) and O-3 (17%)

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Phosphorylation Canna edulis P NMR Starch phosphate monoesters had chemical shifts of 4.076 and 4.564 ppm. Phosphorylation Zhang and Wang,

occurred at C-2 or C-3, and C-6 positions of starch 2009

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Laurate Maize Solid state 13C 13C NMR analysis verified the esterification of starch with methyl laurate Geng et al., 2010

NMR

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Laurate High-amylose 1H NMR Four extra peaks with chemical shifts of 1, 1.2, 1.4, 2.25 ppm were observed in the NMR Lu et al., 2013

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maize spectrum of starch laurate. The peak intensity of these peaks increased with the increasing degree

of substitution

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C=O–O–SiO2Na Maize C CP/MAS A carboxyl group with chemical shift at 168 ppm in the NMR spectrum confirmed the formation Rashid et al., 2012

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modified NMR of C=O–O–SiO2Na moieties in starch

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Starch sulfide Potato C, 1H NMR In 1H NMR spectrum, thiolated starch has additional peaks at 2.1 ppm (SH group) and between 3 Chauhan et al.,

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and 4 ppm (–CH2 in the –OC–CH2SH group). In 13C NMR spectrum, thiolated starch has new 2015

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signals between 175–180 ppm. Intense peaks round 30 ppm were noted for CH2 and 17 ppm for

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CH3 from di-ethylamine
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Acylated Starch H NMR Degrees of substitution of various acylated starches (phthalate, cinnamate, benzoate, and Thakore et al.,

nanoparticles palmiate) were measured by 1H NMR 2013

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Acylated High amylose 1H NMR Starch acetate, propionate, and butyrate were produced. Acylation reduced the molecular mobility Lim et al., 2015
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maize of starch (reduced T2). Drying and storage further reduced the molecular mobility
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Octenyl succinic Maize H NMR Rapid determination of degree of substitution and degree of branching of the modified starch was Tizzotti et al.,

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anhydride (OSA)- developed by dissolving starch in dimethyl-d6 sulfoxide with deuterated trifluoroacetic acid 2011

modified addition

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OSA Rice C, 1H NMR 1H and 13C NMR analysis confirmed the formation of octenyl succinic esters. The esterification Zhang et al., 2013

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occurred at 2-OH, 3-OH, and 6-OH of glucosyl residue with 2-OH being the main substitution

position

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OSA Sugary maize C, 1H NMR Soluble starch from sugary maize was modified by OSA. The modified starch showed additional Ye et al., 2014

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peaks at 0.8−3.0 ppm and a shoulder at 5.56 ppm in 1H NMR spectrum. Modified starch had

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increased ratio of α(1,6) linkages. OSA modification broadened C-1 peak. A shoulder of C-4 peak

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at 78.1 ppm was observed. The substitution occurred at the O-2 position

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Cross-linking Sweet potato P, 1H NMR The position and level of phosphorus in cross-linked starch were determined by NMR. Zhao et al., 2015

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Monostarch monophosphate and distarch monophosphate (molar ratio of ~1:1) were produced
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from the reaction with sodium trimetaphosphate. Phosphorylation of monostarch monophosphate

occurred at O-3 and O-6 positions. There is 1 cross-link per 2900 glucosyl residues
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Cationization Maize C CP/MAS NMR spectra of three cationic starch derivatives (cationic groups varying in chain length) were Wei et al., 2008
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NMR similar. Additional peaks near 31 ppm were related to the methylene of the cationic group,

confirming the formation of starch ethers

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Oxidised starch Potato C, 1H NMR Positions and contents of the keto and carboxylic groups were determined in oxidised starch. Ring Salomonsson et

cleavage occurred between C-2 and C-3 al., 1991

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Ozone treatment Wheat H NMR H NMR analysis showed that a keto group formed at C-2 position, and β-glucuronic acid was Sandhu et al.,

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formed at C-1 position 2012

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24 Table 6

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Starch Graft group NMR Use Reference

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type

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Maize Various types Solution 13C NMR Confirmation of the grafting and measurement of the degree of grafting Gurruchaga et al.,

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1992

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Maize Sodium acrylate C CP/MAS NMR H relaxation times and solid state 13C CP/MAS NMR spectrum were used to study Zhang et al., 2008

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the compatibility of the grafted group and starch as well as the blends of sodium

acrylate and starch

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Solution 13C NMR Graft position and grafted segment length were determined by 13C NMR. Grafting Zou et al., 2012

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Maize Acrylamide

of acrylamide group mostly occurred at C-6 (supplementary material Fig. 6).

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Increasing amylose content of starch increased the length of grafted segment
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Maize Lactic acid C NMR, heteronuclear NMR analysis confirmed the formation of starch-g-lactic acid copolymer Hu and Tang,
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multiple bond coherence 2015

(HMBC)

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1 1
Maize Maleic anhydride, H NMR H NMR analysis confirmed the formation of starch copolymers Yang et al., 2015

epoxidized cardanol

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n.a. Acrylamide-co-N- H and 13C NMR 1
H and 13C NMR analysis confirmed the formation of amylopectin-g- Sasmal et al.,

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methylacrylamide poly(acrylamide-co-N-methylacrylamide) 2015

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35 Table 7

Starch source Other component NMR technique Major observations Reference

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Maize Glycerol and H NMR Native starch granules as affected by glycerol and water were studied by relaxation NMR. The Cioica et al.,

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water relaxation presence of glycerol and water increased the molecular mobility of the starch. T2 of starch with 2013

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water and glycerol had four dynamic components, while that of starch without glycerol had three. T1

distributions were related to the degree of crystallinity of starch

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Solid state 13C

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Potato and Protein Impact of Maillard reactions between starch and amino acid (lysine) on starch structure was Pizzoferrato

chestnut CP/MAS NMR followed by solid state 13C CP/MAS NMR. Intensity of the C1 resonance peak decreased with et al., 1998

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increasing lysine concentration. The Maillard reaction due to roasting induced the formation of V- and 1999

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type complexes and reduced the proportion of B-type polymorph in starch

Cassava zinc Solid state 13C

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Starch modified by α-amylase and glucoamylase was reacted with zinc. The chemical shift of C-6 Luo et al.,
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CP/MAS NMR peak of the complexes moved downfield by 0.96 ppm, while that of the other peaks had little change, 2013
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as compared with the control. The results suggest that the zinc mostly interacted with OH group of
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C-6 of the glucose units

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Potato Metal ion H NMR The interactions between amylose and Cu2+ broadened the peaks of 1H NMR spectrum of amylose Li et al.,

amylose (supplementary material Fig. 7). The ratio of signal areas of protons H(2–6) to H(1) decreased upon 2014

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Cu2+ addition. This suggests the binding of Cu2+ with OH groups of amylose
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rice 1-butanol C CP/MAS After 1-butanol-HCl hydrolysis of starch, an additional peak at 100.3 ppm was detected for C1, Hu et al.,

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NMR indicating the formation of V-type amylose inclusion complexes 2013
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Upon binding to amylopectin, the amphiphile of HPTS-C16H33 unfolds, and proton signals of 1H

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Potato HPTS-C16H33 H NMR Beeren &

amylopectin NMR spectrum shifted downfield Hindsgaul,

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2013

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Rice Tea polyphenols H NMR Starch and tea polyphenols were mixed and gelatinized. Compared with physical mixtures of Wu et al.,

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gelatinized starch and tea polyphenols, the chemical shifts of protons of 1H NMR spectrum of the 2011

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sample were identical. The coupling constants were different, suggesting stronger interactions in the

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sample than the physical mixture

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Hydroxyethyl Propofol H NMR Compared with individual component, 1H NMR spectrum of the propofol-hydroxyethyl starch Silva et al.,
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starch complexes had broadened peak at 8.00 ppm and downfield shifts of proton signals of glucose units. 2014

This suggests the occurrence of the interactions between the hydroxyethyl starch and propofol
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37 Table 8

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Product Targeted property NMR technique Major observations Reference

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Bread Crystallinity of C CP/MAS Compared with the spectrum of starch in flour, that of the starch in bread crumb showed a narrowing of Primo-Martín

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starch in bread NMR the C-1 peak, an upward chemical shift to 102.1 ppm, and the loss of the triplet characteristics. Bread et al., 2007

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crumb and crust crust had a similar trend with a peak at 102.3 ppm for rusk rolls and one at 101.9 ppm for crispy rolls.

This indicated the formation of V-type complexes. Increase in peak intensity at 82 and 102 ppm

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indicated the increasing amounts of amorphous material. Starch of bread crumb gelatinized to a larger

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extent that that of the crust

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Bread Water mobility H NMR The proton mobility of bread crumb and crust was analysed by 1H NMR relaxometry. The assignment Bosmans et

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relaxometry of proton peaks was made by using starch-water and gluten-water model systems (supplementary al., 2012

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material Fig. 8). This analysis may help better understand the physical changes of bread during

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production and storage

Starch structure and 13C CP/MAS,

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Bread Structural changes of starch in rye sourdough and wheat breads during staling were compared by NMR Mihhalevski
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composition C, 1H, 31P techniques. 13C NMR spectra of rye and wheat starches are similar. 31P NMR analysis showed that et al., 2012
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phospholipid fraction differed in these two starches. 13C CP/MAS NMR analysis showed that the
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NMR

retrograded rye starch had different polymorph composition than the wheat starch
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Bread Starch structure C CP/MAS Addition of polyphenols and pectins on starch properties in bread was probed by 13C CP/MAS NMR. Sivam et al.,

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NMR Polyphenols and pectins decreased the intensity of C-6 peak by 35−40%. V-type complexes and 2013

amorphous starch existed in the bread

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Film Starch structure C CP/MAS C CP/MAS NMR spectrum of extruded maize starch films with glycerol and water was compared Pushpadass et

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NMR with that of the physical mixture. Difference in C1 peak was observed. C1 peak of extruded films was a al., 2009

duplet, suggesting the existence of B-type polymorph, while that of the physical mixture had A-type

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polymorph

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39 Table 9

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Starch sample Targeted feature NMR type Other techniques Major findings Reference

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Various types Total phosphorus P NMR Traditional Total phosphorus contents of starch measured by 31P NMR agreed with Kasemsuwan

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content colorimetric the results obtained from colorimetric chemical method & Jane, 1996

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chemical method

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Various types Amylose content H NMR Iodine-binding The amylose contents obtained from 1H NMR were comparable to those Dunn and

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spectrophotometry measured by iodine-binding spectrophotometry-based assay Krueger, 1999

assay

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Various sources Degree of branching 1H NMR The unit chain length of starch measured by 1H NMR agreed well with

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HPSEC and blue Nilsson et al.,

and unit chain length value that by HPSEC. The degree of branching of starch was positively 1996
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correlated with blue value
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Rice starch Degree of branching 1H NMR HPSEC Both methods gave similar DB values (2.7−3.6%) for 14 rice varieties Syahariza et
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al., 2013
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8 starches from Crystalline region C XRD Contents of double helices are not equal to the degree of crystallinity Lopez-Rubio et

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various sources CP/MAS obtained from XRD analysis, though the results of these two methods are al., 2008

NMR highly correlated

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11 waxy starches Crystalline region C XRD, DSC Contents of double helices of both native and annealed starches are Witt & Gilbert,

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from various CP/MAS positively correlated with the degree of crystallinity (XRD) and 2014

sources (native NMR gelatinization parameters (DSC)

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and annealed)

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Cassava Degree of C Fourier transform- Samples varying in the degree of crystallinity were produced from Mutungi et al.,

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crystallinity CP/MAS Raman debranched cassava starch subjected to hydrothermal processing. Degree 2012

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NMR spectroscopy, of molecular order by NMR was positively correlated with that derived

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XRD from XRD and Fourier transform-Raman spectroscopy

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Maize, waxy Gelatinization C XRD, DSC Molecular (double-helical) order of native starches was higher than Cooke and
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maize, wheat, CP/MAS degree of crystallinity measured by XRD. Molecular order is positively Gidley, 1992

potato, tapioca NMR correlated with ∆H (enthalpy change) of gelatinization

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Maize starch, Retrogradation Pulsed 1H Instron texture The results of starch retrogradation obtained from two methods had a high Seow and Teo,
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bread, rice cup NMR analyser and positive correlation 1996

cake

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Pulse starches Retrogradation C FTIR–ATR, XRD, DSC, XRD, and NMR similarly reflected the extent of retrogradation of Ambigaipalan

CP/MAS DSC, enzyme pulse starches, while NMR, FTIR-ATR, and enzyme susceptibility et al., 2013

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susceptibility (α- methods had a different pattern

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amylase)

Waxy maize Glass transition Pulsed H DSC, Instron The glass transition temperatures measured by NMR were lower than Kalichevsky et

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temperature NMR texture analyser, those by DSC and other techniques by 20−30 oC (supplementary material al., 1992

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DMTA Fig. 3)

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Cassava Degree of C XRD During initial stage of acid hydrolysis, double helix content from NMR Atichokudomc

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crystallinity of acid- CP/MAS analysis was higher than the degree of crystallinity by XRD analysis. hai et al., 2004

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hydrolysed starch NMR These two values became similar at later stages of hydrolysis

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(supplementary material Fig. 9)
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Potato Degree of H NMR Traditional Degrees of substitution for acetylated and hydroxypropylated starches de Graaf et al.,

substitution spectrophotometry measured by NMR agreed well with those measured from traditional 1995
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-based method a methods. 1H-NMR-based method required much less time

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Maize Degree of grafting of C NMR Hydrolytic Degree of grafting of starch measured by a hydrolytic method agreed well Gurruchaga et

copolymers Method with that of the NMR method al., 1992

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40 HPSEC, High-performance size-exclusion chromatography; XRD, wide-angle X-ray diffraction; DSC, differential scanning calorimetry; FTIR–

41 ATR, Fourier transform infrared spectroscopy-attenuated total reflectance; a, Johnson method and a titration-based method

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• Applications of NMR spectroscopy in starch research are summarized

• NMR spectroscopy probes structural basis of physical behaviours of starch systems

• Various types of NMR techniques reveal different aspects of starch structures

• Results of NMR spectroscopy correlate well with those of other analytical methods

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