NMR spectroscopy of starch systems

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

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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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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NMR spectroscopy of starch systems

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Fan Zhu*

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

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Zealand

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* Correspondence, e-mail: [email protected]

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Running title: starch NMR

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Abstract

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

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

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

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

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

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

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

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NMR, 13C NMR, 31P NMR, and 17O NMR. These techniques can be non-destructive, and

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provide novel insights into the structure of starch systems. NMR spectroscopy is

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complementary to other analytical techniques such as X-ray diffraction and calorimetry for

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the characterization of simple and complex starch systems.

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

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1. Introduction NMR (nuclear magnetic resonance) spectroscopy has been widely used for the analysis of

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

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

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

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

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

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

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

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innovate the processes and products (Spyros & Dais, 2012; Marcone et al., 2013).

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NMR phenomena have the origins within the nucleus of certain atom types such as C and P.

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The individual atoms have a net “nuclear” spin. The effects can be noted in a magnetic field,

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involving the energy exchange between two levels at least (resonance) (Gidley, 2014). For

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one sample, different nuclei, such as 1H, 13C, 31P, can be chosen to study different aspects of

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the samples and to extract the relevant information under the natural/industrial conditions

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(Laws et al., 2002; Spyros & Dais, 2012). Some NMR experiments need no separation of

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diverse food components, and require relatively a small amount of efforts for sample pre-

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treatment and preparation as compared with traditional methods (Spyros & Dais, 2012). The

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food samples can be lipid, semi-solid, and solid. The obtained complex NMR spectra can be

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further treated with multivariate statistical analysis to gain additional structural information

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

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

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

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

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

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

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

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

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

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

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

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

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

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Numerous publications have well documented the background and principles of NMR

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spectroscopy (Laws et al., 2002; Spyros & Dais, 2012; Gidley, 2014). The theoretical

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principles of starch NMR has also been detailed (Gidley, 2014). The basics of starch structure

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and functional properties have been systematically reviewed in detail (BeMiller & Whistler,

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2009; Pérez & Bertoft, 2010). Therefore, the basics of NMR spectroscopy and starch are not

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covered in the review. This review focuses on the applications of NMR-based techniques for

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structural characterization of various starch systems. These include the chemical composition,

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molecular order, interactions, gelatinization, retrogradation, and modifications. Due to the

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large amount of literature published, representative references have been selected to cover

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specific topics.

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2. Chemical composition

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

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

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

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

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

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

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

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

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

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

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

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

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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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starch chains can be determined (Muhrbeck & Tellier, 1991). The normal cereal starches

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mostly possess phospholipids. The legume and potato starches mostly contain phosphate

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monoesters. And the tuber and root starches are free of phospholipids. Phosphate monoesters

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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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1994). Phosphorylation on the C-3 position was independent of potato variety, while that on

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the C-6 position was found variety-dependent (Muhrbeck & Tellier, 1991). Schmieder et al.

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(2013) employed a combination of heteronuclear 1H, 13C double, 1H, 13C, 31P triple NMR

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techniques, and successfully determined the phosphorylation sites of both the starch and

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glycogen in heterogeneous samples. A full assignment of the resonances of carbohydrates

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was archived by analysing a set of reference compounds.

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3. Structural characterization

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3.1. Impact of hydration extent

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

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

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

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

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

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

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

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

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structure.

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3.2.Starch physical structure

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Solid state 13C CP/MAS NMR has been used to quantify the molecular order of granular

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starch (Flanagan et al., 2015; Man et al., 2013) (Table 2). Each carbon of the glycosyl unit in

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starch is assigned for specific peak in the spectrum (supplementary material Fig. 2). 13C

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CP/MAS NMR spectra of A-type starches differs from those of B-type starches in some

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detail. The peak of C-1 with chemical shift of ~100 ppm is a triplet for A-type starches, and a

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doublet for B-type starches (Veregin et al., 1986). This difference is readily attributed to the

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specific arrangement of the crystals in the granules (Gidley & Bociek, 1985). The spectrum

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of the native starch was compared with that of the amorphous starch. The contents of the

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double and single helices as well as the amorphous material in the granules can be quantified,

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and the degree of crystallinity can be calculated (supplementary material Fig. 2) (Gidley &

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Karasz equation, which is commonly used for predicting the glass transition temperatures of

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random copolymers and amorphous mixtures. The comparison between different methods

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[DSC (differential scanning calorimetry) vs NMR] in measuring the starch glass transition is

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discussed in a following section (section 9: correlations between NMR and other analytical

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techniques).

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The degree of branching (DB) of starch has been studied by 1H NMR based on the anomeric

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protons involved in α-(l-4) and α-(l-6) linkages (Gidley, 1985; Nilsson et al., 1996; Dunn &

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Krueger, 1999; Tizzotti et al., 2011; Syahariza et al., 2013). D2O was commonly employed as

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the solvent for the DB quantification (Gidley, 1985). For example, DB of potato amylose and

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

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

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

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

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

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

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

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

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

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

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

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

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

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peak pattern of NMR spectrum regarding the polymorphism. Compared with native starch,

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the amylose crystals had NMR spectra with much sharper peaks due to the much increased

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

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The water mobility in starch has been studied by 17O and 2H NMR (Richardson et al., 1987),

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and 2H and 1H NMR (Li et al., 1998; Baianu et al., 1999). Three types of water were proposed

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for potato starch. The first type was related to the anisotropically bound water. The second

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type was related to the trapped water, and the third one was from the average between free

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and weakly bound water populations (Richardson et al.. 1987; Baianu et al., 1999). Li et al.

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(1998) studied the molecular mobility of “freezable” and “unfreezable” water in waxy maize

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starch by 1H NMR coupled with DSC analysis. Decreasing the temperature decreased the

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

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

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

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

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3.5.V-type amylose inclusion complexes Amylose can form the V-type inclusion complexes with small guest molecules both in the

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

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

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

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

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

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

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different carbons of V-type amylose complexes with specific chemical shifts. The chemical

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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

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C-6, respectively. Gidley & Bociek (1988) linked the chemical shifts of C-1 and C-4 to the

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torsion angles (φ and ψ) of conformation. The chemical shifts of C-1 and C-4 of the amylose

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in both solution and solid states were found to be more susceptible to the induced

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conformational changes as a result of guest molecule inclusion (Jane et al., 1985; Le Bail et

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al., 2005; Gidley & Bociek, 1988). There are differences in the NMR spectra among V6I,

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V6II, V6III, V7, and V8-type amylose complexes due to the differences in the size of helical

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cavity (six-, seven-, and eight-fold helices) and the physical positioning of guest molecules

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(Le Bail et al., 2005; Gidley and Bociek, 1988). For example, the NMR spectra of V6- and

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V7-type inclusion complexes are similar. In comparison, the V8-type complexes with 1-

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

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

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may be detected by NMR. For example, the un-complexed palmitic acids were resolved in

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

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C CP/MAS NMR spectrum with the chemical shift of 33.6 ppm, while the chemical shift of

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

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

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

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

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amylose inclusions complexes.

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

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

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water during these processes (Table 4). The content of double helices in starch granules as

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measured by solid state 13C CP/MAS NMR was positively correlated with the enthalpy

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change of gelatinization (∆H) as measured by DSC (Cooke & Gidley, 1992). Hydration

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properties of potato starch were studied through the CP and SP modes of solid state 13C and

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starch while gelatinization and hydrolysis reduced the immobile fractions of native starch.

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Phosphorus-containing compounds became completely mobile upon gelatinization (Larsen et

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al., 2013). Proton properties were also studied by 1H NMR (Ritota et al., 2008; Rondeau-

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Mouro et al., 2015). Four types of water differing in their mobility were observed in starch-

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water systems during gelatinization (Ritota et al., 2008). Water with the highest mobility was

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

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

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

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

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

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O, and 31P NMR) have been used to understand the structural changes of both starch and

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P MAS NMR (Larsen et al., 2013). As expected, hydration increased the molecular order of

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

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

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

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

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

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

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

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

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

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amylase hydrolysis, the V-type polymorph became more prominent while a small amount of

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B-type polymorph was recorded in the potato starch ghost. It was suggested that temporary

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entanglements of amylopectin and their size are related to the ghost formation (Zhang et al.,

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2014).

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Starch retrogradation has been studied by 13C CP/MAS and 1H NMR (Gidley, 1989;

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Ambigaipalan et al., 2013; Teo & Seow, 1992; Smits et al., 1998). 13C CP/MAS NMR

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analysis showed that the intensity of C-4 peak decreased during the retrogradation of pulse

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starches, while the content of double helices increased (Ambigaipalan et al., 2013). In

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amylose gel, the B-type polymorph was observed (Gidley, 1989). 1H NMR analysis of the re-

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crystallized starch showed that the rate of decay after radiofrequency pulse was different than

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

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

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

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

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

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

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

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

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

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Starch retrogradation occurred over a wide range of correlation times. 5. Enzyme susceptibility and resistant starch

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

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

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(Htoon et al., 2009; Shrestha et al., 2012). Hydrolysis by artificial saliva α-amylase had little

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effect on the contents of double and single helices as well as the non-ordered proportion of

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maize starches (Shrestha et al., 2012). Enzyme hydrolysis of the thermally-processed maize

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starches with high amylose contents (>50%) increased the contents of double helices while

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decreasing the proportion of amorphous starch (Htoon et al., 2009). The molecular structure

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of resistant starch after enzyme hydrolysis was characterised by various NMR techniques

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(Colquhoun et al., 1995). Relaxation and solid state 13C CP/MAS NMR tests showed that the

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amylose gel had a double helical (rigid) fraction and a mobile amorphous fraction. Upon

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hydrolysis by porcine pancreatic α-amylase, the mobile fraction was removed and the gel

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became rigid and fully ordered (Colquhoun et al., 1995). The gels of amylomaize V,

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amylomaize VII, and wheat starches after enzyme hydrolysis contained 60−70% of double

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

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double helical aggregates were reported in the cooled gels.

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6. Modifications

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6.1.Physical modifications

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

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

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

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

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

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

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

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

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

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

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analysis showed that rapid conventional heating gave a lower water mobility of starch system

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than did the microwave heating (Fan et al., 2013a and 2013b). Sonicated and melt-processed

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high amylose maize starch had similar spectra, and the ultrasound better de-aggregated the

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starch (Lima and Andrade, 2010).

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6.2.Chemical modifications

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The structural changes of various starches hydrolysed by acid and alkaline solutions were

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followed by 13C CP/MAS NMR (Table 5). The acid treatment reduced the content of

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amorphous starch while increasing that of double helix and degree of crystallinity

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(Atichokudomchai et al., 2004; Cai et al., 2014a). Wang and Copeland (2012) showed that

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the alkali hydrolysis had no effect on the chemical shifts of various carbon peaks of pea

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

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

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

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

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

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

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

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

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

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

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

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

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

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starch samples in dimethyl-d6 sulfoxide with deuterated trifluoroacetic acid (TFA) addition,

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and analysed the degree of substitution of octenyl succinic anhydride (OSA)-modified

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starches by 1H-NMR. The starch degradation by TFA had no effect on the detection. This

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method gave an improved spectrum resolution and accuracy of quantification (Tizzotti et al.,

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2011). The introduction of new functional groups has been verified by 13C CP/MAS, 13C, and

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OSA-modified soluble maize starch. The modified starch had the extra peaks at 0.8−3.0 ppm

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and a shoulder at 5.56 ppm in the 1H NMR spectrum due to the OSA group. The intensity of

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these peaks increased with the increasing degree of substitution. Changes were also observed

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in the 13C spectrum. C-1 signal of the internal glucosyl units of the modified starch became

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broader. A shoulder peak of C-4 was observed. These suggest that the substitution occurred at

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

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

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

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

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H NMR analysis. For example, Ye et al. (2014) used both 13C and 1H NMR to analyse the

14

ACCEPTED MANUSCRIPT 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

327

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

328

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

329

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

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

group (170–175 ppm) were recorded in the spectrum of modified starch. This suggests the

333

successful esterification of starch with the methyl laurate (Geng et al., 2010).

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

336

(Salomonsson et al., 1991; Sandhu et al., 2012). In bromine-oxidised potato starch, the level

337

and position of keto and carboxylic groups were determined (Salomonsson et al., 1991). 1H

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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6.3.Starch-grafted copolymers

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

15

ACCEPTED MANUSCRIPT 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

351

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

352

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

353

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

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

Zhang et al. (2008) analysed the structure of starch-g-poly(sodium acrylate) by solid state 13C

357

CP/MAS NMR. The spectrum of the copolymer was compared with that of the pure starch

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

360

involved in the grafting process.

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

363

incorporation of montmorillonite clay and silica (nanoparticles) (Brito et al., 2015). Proton

364

spin-lattice relaxation time of the blends was between that of starch and PLA, suggesting the

365

interactions and miscibility. Nanoparticle addition increased the relaxation time, suggesting

366

the occurrence of new interactions and the reduced molecular mobility.

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7. Interactions with other components

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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 16

ACCEPTED MANUSCRIPT 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

376

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

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

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

formation of the helical structure of amylopectin external chains. Some of the resulting

382

products of starch interactions with other components (e.g., protein) in the solid form have

383

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

384

et al., 2013). For example, Luo et al. (2013) analysed the structure of enzyme-modified

385

starch-zinc complexes. Compared with the control, the C-6 chemical shift of the complexes

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

388

et al. (2013) studied the interactions between rice starch and 1-butanol in an HCl solution. In

389

comparison with the control, an extra peak for C-1 at 100.3 ppm was observed in the sample.

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.

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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 17

ACCEPTED MANUSCRIPT 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.

401

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

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.,

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

spectra of the wheat and rye starches were similar. The 31P NMR spectra of wheat and rye

407

starches were different due to the difference in lipid composition. 13C CP/MAS NMR

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

410

the presence of the non-starch components (Hoover, 1995). Bosmans et al. (2012) employed

411

1

412

material Fig. 8). The proton peaks were assigned according to simple model systems. This

413

analysis helped better understand the role of water with different molecular mobility in bread

414

properties. The structure of starch in extruded films was analysed by 13C CP/MAS NMR

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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H NMR relaxometry to study the water status in bread and model systems (supplementary

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ACCEPTED MANUSCRIPT 421

9. Correlations between NMR and other analytical techniques 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

426

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

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

429

1

430

these two assays agreed well with each other. The degree of branching (DB) of various

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

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

435

double helices of starch measured by 13C CP/MAS NMR were compared with the degree of

436

crystallinity of starch measured by XRD (Lopez-Rubio et al., 2008; Witt & Gilbert, 2014;

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

439

al. (2008) noted that the contents of double helices in starch granules are not equal to that of

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

19

ACCEPTED MANUSCRIPT (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

451

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

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

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

amylose–amylopectin chains. Therefore, a comprehensive approach employing different

457

instruments should be used to gain a holistic picture of the starch retrogradation. Kalichevsky

458

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

459

DSC, Instron texture analyser, and dynamic mechanical thermal analysis (DMTA). The glass

460

transition temperatures of starch measured by NMR were lower than those by DSC and other

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

463

transition with an increased mobility for a large proportion of the starch chains (Kalichevsky

464

et al., 1992). The degree of substitution (acetylation and hydroxypropylation) as well as the

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

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10. Conclusions 20

ACCEPTED MANUSCRIPT 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

476

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

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

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

physicochemical properties of starch and food systems. The composition and molecular

482

structure of starch obtained from NMR analysis correlate well with the results of other

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-

485

saving, while the solid state NMR method can be non-destructive. Besides NMR, most of the

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

488

physicochemical properties of starch. It should be stressed that some structural parameters of

489

starch are sensitive to the nature of the measurement technique. The knowledge obtained

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.

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492 493

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Table captions

2

Table 1 Chemical composition of starch quantified by NMR spectroscopy

3

Table 2 Structural characterisation of starch studied by NMR spectroscopy

4

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

5

Table 4 Gelatinization and retrogradation processes monitored by NMR spectroscopy

6

Table 5 Structure of modified starches as studied by NMR spectroscopy

7

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

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

SC

M AN U

TE D

EP

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

AC C

10

RI PT

1

1

ACCEPTED MANUSCRIPT

Table 1

Parameter

Starch

NMR type

Major findings

31

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

RI PT

11

source Potato

P NMR

SC

Degree of phosphorylation on

on C-6 position varied greatly among different genotypes

Phosphate

Various

31

P NMR

M AN U

the C-3 and C-6

Normal cereal starches contained mostly phospholipids. Tuber and root starches were free of phospholipids. Legume and potato starches mostly contained phosphate monoesters.

phosphate,

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

phospholipids

units Various

31

P NMR

Potato starch mostly contained phosphate monoester (0.086%). Wheat starch contained

Tellier, 1991

Lim et al., 1994

Kasemsuwan and

EP

Phosphate

TE D

monoester, inorganic sources

Reference

mostly phospholipids (0.058%), high-amylose (50% amylose content) maize starch mostly Jane, 1996

phosphate,

contained phosphate monoesters (0.0049%) and phospholipids (0.015%). Waxy maize

phospholipids

starch had trace amounts of phosphate monoesters

phosphorylation site Curcuma

Heteronuclear

on glucan chains

1

rhizome

AC C

monoester, inorganic sources

A combination of heteronuclear 1H, 13C and 1H, 13C, 31P NMR spectra efficiently

H, 13C double, determined the phosphorylation sites. Monophosphate esters are on the C-3 and C-6

Schmieder et al., 2013 2

ACCEPTED MANUSCRIPT

H NMR

RI PT

1

Phosphatidylcholine was mixed with starch in DMSO. Phospholipid content was

Genkina &

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

Kurkovskaya,

quantification of phospholipids without the regards of sample forms

2013

Branching ratios of starch were obtained from 1H NMR. This ratio was then used for the

Dunn and

determination of amylose content

Krueger, 1999

SC

sources

P NMR

M AN U

Various

31

TE D

Amylose content

Wheat

EP

Phospholipids

H,13C, 31P triple positions of the glucose unit

AC C

1

3

ACCEPTED MANUSCRIPT

12

Table 2 NMR type

Major findings

Molecular order

Solid state 13C CP/MAS

13

NMR

starch for the first time

Solid state 13C CP/MAS

13

NMR

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

SC

Various sources

C NMR spectrum was interpreted in relation to the crystallinity of

C NMR spectrum was interpreted in relation to the crystallinity of

Reference Marchessault & Taylor, 1985 Gidley &

M AN U

Molecular order

Maize, potato

RI PT

Targeted structure Starch source

of starch

Rice

Solid state 13C CP/MAS

13

NMR

triplet for A-type polymorph starches, and a doublet for B-type starches 1986 The relative degree of crystallinity (37.2−58.9%), relative proportion of Man et al., 2013

NMR

single helices (1−7.9%), double helices (39.6−61.8%), and amorphous

Various sources

AC C

Molecular order

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

Solid state 13C CP/MAS

EP

Molecular order

Various sources

TE D

Molecular order

(37.2−52.5%) material were quantified. Deficiency of starch branching enzymes I and IIb decreased degree of crystallinity, portion of double helices, and increased the portion of single helices of starch

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 4

ACCEPTED MANUSCRIPT

Waxy maize

temperature

Pulsed NMR, 13C CP/MAS Pulsed NMR and 13C CP/MAS NMR successfully characterised the

Kalichevsky et

NMR

al., 1992

glass transition of amorphous starch with water contents of 10−22%

RI PT

Glass transition

Potato starch, amylose,

13

C CP/MAS NMR, 1H/13C Five types of α(1–4) linkages were found by decompositions of C1

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

Branching ratio

Various sources

processing 1

Paris et al.,

M AN U

semi-crystalline status

SC

Structure of

H NMR, 13C NMR

Ratios of α(1–4) to α(1–6) linkages of native and degraded starches were Gidley, 1985 determined by 1H NMR and ranged from 17.5 to 26. Glucose and the

1

H NMR

average unit chain length Maize starches varying in 1H NMR

AC C

Branching ratio

amylose content, potato, rice, pea, cassava, wheat Branching ratio

Maize, rice

EP

Branching ratio and Various sources

TE D

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

1

H NMR

DB ranged from 1 (potato amylose) to 4.77% (maize amylopectin).

Nilsson et al.,

Average unit chain length (CL) ranged from 21 (maize amylopectin) to

1996

100 glucosyl residues (potato amylose) Ratios of α(1–4) to α(1–6) linkages were determined by 1H NMR. Maize Dunn and starches had the ratios ranging from 21.7 to 83.9%

Krueger, 1999

NMR was used to efficiently determine the degree of branching (DB) of Tizzotti et al.,

5

ACCEPTED MANUSCRIPT

2011; Syahariza

from 2.7−3.6%

et al., 2013

RI PT

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

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)

14

glycosidic linkages

SC

13

M AN U

15 16

AC C

EP

TE D

17

6

ACCEPTED MANUSCRIPT

Table 3

RI PT

18

Targeted feature

Amylose type

Complexing agent

NMR type

Major findings

References

Formation of

Potato

DMSO, KOH, iodine,

13

Soluble V-type inclusion complexes in solution were

Jane et al., 1985

C NMR

α-naphthol, methyl

characterised. Increasing concentration of complexing agents

inclusion complexes in

alcohol, tert-butyl

induced downfield shift of the signals of C-1 and C-4 (larger shift)

solution

alcohol, n-butyl

M AN U

SC

amylose/amylodextrin

and C-2, C-3, and C-6 (smaller shift). The differences in the shifts

alcohol, cyclohexanol

for C-1 and C-4 indicate the differences in the compactness of the helical structure Gidley and

hexanoic acid, tert-butyl CP/MAS

not affected by the type of complexing agents. V8-type complex

Bociek, 1988

complexes

alcohol, n-butanol, 1-

with 1-naphthol had chemical shifts of C-1 and C-4 peaks

NMR spectra of solid V- Synthetic type amylose inclusion complexes, A- and B-

amylose

AC C

naphthol

TE D

amylose

Ethanol

C

NMR

EP

type amylose inclusion

13

NMR spectra of V6 and V7-type complexes are similar, and are

NMR spectra of solid V- Potato, synthetic Sodium palmitate,

downward by 1 ppm (supplementary material Fig. 5)

13

A triplet C-1 peak for the A-type crystalline form and a doublet C- Horii et al.,

CP/MAS

1 peak for B-type crystals were recorded (supplementary material 1987

NMR, 13C

Fig. 4). Spectrum of V-type amylose is rather different from that of

C

7

ACCEPTED MANUSCRIPT

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

RI PT

type amylose crystals

amyloses are shorter than that of cellulose (suggesting more mobility) Stearic, palmitic, oleic,

13

Chemical shift of the mid-chain methylenes had a change by ~2−3 Snape et al.,

type amylose inclusion

linoleic, linolenic, and

CP/MAS

ppm, suggesting the complexation process. The mid-chain

complexes

docosahexaenoic acids, NMR

1998

M AN U

C

SC

NMR spectra of solid V- Potato

methylenes of lipids in the V-type complexes had the same chemical shifts, except for docosahexaenoic acid. Analysis of

glycerol monooleate,

cross-polarisation dynamics showed that the bulky polar groups

TE D

glycerol monopalmitate, lysophosphatidylcholine

situated outside the V-helical segments and was adjacent to the amorphous regions

Menthone, decanal, 1-

13

Differences in NMR spectra were observed among V6I, V6II,

type amylose inclusion

naphthol, 1-butanol

CP/MAS

V6III, and V8-type amylose complexes. Rehydration increased the 2005

NMR

sharpness of carbon resonance peak, and desorption induced the

AC C

complexes

EP

NMR spectra of solid V- Potato

C

Le Bail et al.,

transition from V6II to V6I-type

8

ACCEPTED MANUSCRIPT

amylose, pea

complexes

13

A single resonance peak at 32.4 ppm was assigned to the

Lebail et al.,

CP/MAS

complexed fatty acids. The uncomplexed palmitic acid was also

2000

NMR,

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

deuterium

relaxation tests revealed a difference between the uncomplexed

NMR

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

C

RI PT

type amylose inclusion

Palmitic, lauric acids

SC

NMR spectra of solid V- Synthetic

M AN U

deuterium NMR analysis revealed partially disassociated complexes of amylose-palmitic acid and a full complexation for the amylose–lauric acid

Decanoic acid,

type amylose inclusion

bean (Vicia

carvacrol

complexes

faba), pea,

C

Peaks of 102.7, 81.4, 74.9, 71.6, and 61.3 ppm were related to C1, Le Bail et al.,

C4, C3, C2-C5, and C6, respectively, and these were of V6I form. 2013

NMR

High pressure treatment induced the formation of V-type

EP

CP/MAS

complexes at a low temperature (40 oC)

AC C

tapioca

13

TE D

NMR spectra of solid V- Potato, broad

9

ACCEPTED MANUSCRIPT

NMR spectra of solid V- Potato

3-O-palmitoyl

13

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

NMR

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

complexes

RI PT

C

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

type amylose inclusion

13

Phosphatidylcholine

C

potato

complexes

In comparison with the physical mixture of phosphatidylcholine

M AN U

NMR spectra of solid V- Debranched

SC

V-type complexes depended on the graft position

CP/MAS

and debranched maize starch, C-4 peak at 86.46 ppm was better

NMR

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

Cheng et al., 2015

EP

result of inclusion complex formation

AC C

19

TE D

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

10

ACCEPTED MANUSCRIPT

20

Table 4 NMR type

Major findings

Wheat, maize,

13

The enthalpy change of gelatinisation measured by DSC mostly reflects the loss of molecular (double-

Cooke and

cassava, potato

NMR

helical) order which is measured by solid state NMR

Gidley, 1992

Maize starches

17

17

varying in amylose

relaxation

RI PT

Starch source

O NMR

O spin-spin relaxation time (T2) measurements were used to monitor the starch gelatinization as

affected by various factors. The changes in T2 were related to the degree of gelatinization and water

Cheetham and Tao, 1998a

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

TE D

content

M AN U

C CP/MAS

SC

Gelatinization

Reference

the water mobility during gelatinization 1

H NMR

Four water populations were observed in starch-water systems. One of them was related to the bulk of

EP

Rice

water, while the others were related to the chemical and diffusive exchanges of water with starch

Ritota et al., 2008

AC C

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

13

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

11

ACCEPTED MANUSCRIPT

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

RI PT

fractions of starch. Carbons of the α(1–4) linkages needed high hydration rates for uniform chemical shifts. Immobile phosphorus-containing chains were only found in the suspension of native starch 13

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

C CP/MAS

SC

Potato, maize

M AN U

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

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

Rondeau-Mouro

NMR

et al., 2015

amylopectin and amylose. Two T2 components were due to slow diffusional exchanges between different water layers

Retrogradation

Amylose gel structure was studied. B-type polymorph was observed from the gel of diluted amylose

and 1H NMR

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

C CP/MAS

EP

13

Gidley, 1989

AC C

Synthetic, potato

TE D

Wheat

mobile amorphous single chains

12

ACCEPTED MANUSCRIPT

Various sources

Pulsed 1H NMR Pulsed 1H NMR was used to monitor the retrogradation of starch. Proton signals from re-crystallized

RI PT

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

Teo and Seow, 1992

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

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

dimensional

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

SC

One- and two-

M AN U

Waxy maize

exchange Solid state NMR Potato

13

C CP/MAS

Kulik and Haverkamp, 1997

The retrogradation of starch was monitored below and above the glass transition temperature (Tg). Below Smits et al.,

TE D

NMR, 1H NMR Tg, the relaxation time of 1H NMR increased due to the decreasing free volume before reaching a plateau. 1998 Above Tg, water absorption decreased the relaxation time before an increase due to recrystallization. 13C

13

The retrogradation of starches from various pulses (faba bean, black bean, pinto bean). Intensity of C-4

NMR

peak decreased during retrogradation, while the content of double helices increased

C CP/MAS

AC C

Pulse starches

EP

CP/MAS NMR was not an efficient technique for retrogradation detection Ambigaipalan et al., 2013

13

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Table 5

Modification

Starch

NMR type

Rice, lotus

13

seed

NMR

Uses

RI PT

21

Physical

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

SC

High pressure

References

CP/MAS NMR. 600 MPa increased the proportion of amorphous material of starch, and had no

Guo et al., 2015

M AN U

effect on the polymorph type. High pressure had no effect on the chemical shifts of various carbon peaks, while decreasing the relative crystallinity High-amylose 1H NMR

Starch was melt-processed (100 oC, 40 rpm, 8 min) and ultrasonicated (750 W, 20 kHz) in the

and ultrasound

maize starch

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

TE D

Melt-processing

Lima and

NMR analysis indicated the de-aggregating of starch induced by ultrasound 13

C CP/MAS Microwave heating increased the proportion of amorphous material and decreased that of both

Fan et al., 2013a

EP

Rice

NMR, 1H

single and double helices. The effect of rapid heating in oil bath was similar to that of microwave and 2013b

NMR

heating. Water mobility of starch-water system was also monitored by 1H NMR. Water mobility

AC C

Microwave

of samples subjected to rapid conventional heating was lower and that of microwave heating was higher Chemical 14

ACCEPTED MANUSCRIPT

Lotus

13

rhizome,

NMR

C CP/MAS Acid hydrolysis increased the double helix content and decreased the amorphous portion of starch. Atichokudomchai The relative crystallinity increased sharply during the initial hydrolysis before levelling off

RI PT

Acid hydrolysis

cassava 13

C CP/MAS Alkali hydrolysis had no effect on chemical shifts, broadened the resonance peak of C-2–C-5

NMR Alkali hydrolysis

Rice

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

SC

Pea

13

NMR

and 58%)

1

Molar substitution of modified starches was quantified by 1H NMR

hydroxypropylated Hydroxypropylated Maize, wheat, 1H NMR

Copeland, 2012

de Graaf et al., 1995

of α-limit dextrins

cassava Hydroxypropylated Sweet potato

Wang and

Both the level and position of hydroxypropylation of starch were determined by 1H NMR analysis Xu and Seib, 1997

EP

H NMR

31

P, 1H NMR Molar substitution was quantified by 1H NMR, and hydroxypropylation mostly occurred at O-2

AC C

Potato

TE D

Substitution Acetylated,

al., 2014a;

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

M AN U

Alkali hydrolysis

et al., 2004; Cai et

Zhao et al., 2015

(61%), followed by O-6 (21%) and O-3 (17%) Phosphorylation

Canna edulis

31

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

15

ACCEPTED MANUSCRIPT

Laurate

Maize

Solid state 13C 13C NMR analysis verified the esterification of starch with methyl laurate

Geng et al., 2010

Laurate

RI PT

NMR High-amylose 1H NMR

Four extra peaks with chemical shifts of 1, 1.2, 1.4, 2.25 ppm were observed in the NMR

maize

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

Maize

modified Starch sulfide

13

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

NMR Potato

M AN U

C=O–O–SiO2Na

SC

of substitution

Lu et al., 2013

of C=O–O–SiO2Na moieties in starch

13

C, 1H NMR In 1H NMR spectrum, thiolated starch has additional peaks at 2.1 ppm (SH group) and between 3 Chauhan et al., 2015

TE D

and 4 ppm (–CH2 in the –OC–CH2SH group). In 13C NMR spectrum, thiolated starch has new signals between 175–180 ppm. Intense peaks round 30 ppm were noted for CH2 and 17 ppm for

Acylated

Octenyl succinic

1

Degrees of substitution of various acylated starches (phthalate, cinnamate, benzoate, and

Thakore et al.,

nanoparticles

palmiate) were measured by 1H NMR

2013

High amylose 1H NMR

Starch acetate, propionate, and butyrate were produced. Acylation reduced the molecular mobility Lim et al., 2015

maize

of starch (reduced T2). Drying and storage further reduced the molecular mobility

Starch

Maize

1

H NMR

H NMR

AC C

Acylated

EP

CH3 from di-ethylamine

Rapid determination of degree of substitution and degree of branching of the modified starch was Tizzotti et al.,

16

ACCEPTED MANUSCRIPT

developed by dissolving starch in dimethyl-d6 sulfoxide with deuterated trifluoroacetic acid

modified

addition

OSA

Rice

RI PT

anhydride (OSA)-

13

C, 1H NMR 1H and 13C NMR analysis confirmed the formation of octenyl succinic esters. The esterification

2011

Zhang et al., 2013

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

Sugary maize

13

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

M AN U

OSA

SC

position

peaks at 0.8−3.0 ppm and a shoulder at 5.56 ppm in 1H NMR spectrum. Modified starch had increased ratio of α(1,6) linkages. OSA modification broadened C-1 peak. A shoulder of C-4 peak

Cross-linking

Sweet potato

31

TE D

at 78.1 ppm was observed. The substitution occurred at the O-2 position P, 1H NMR The position and level of phosphorus in cross-linked starch were determined by NMR.

Zhao et al., 2015

EP

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

Cationization

Maize

13

AC C

occurred at O-3 and O-6 positions. There is 1 cross-link per 2900 glucosyl residues C CP/MAS NMR spectra of three cationic starch derivatives (cationic groups varying in chain length) were

NMR

Wei et al., 2008

similar. Additional peaks near 31 ppm were related to the methylene of the cationic group, confirming the formation of starch ethers

17

ACCEPTED MANUSCRIPT

Oxidised starch

Potato

13

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

Ozone treatment

Wheat

1

H NMR

1

al., 1991

RI PT

cleavage occurred between C-2 and C-3 H NMR analysis showed that a keto group formed at C-2 position, and β-glucuronic acid was

formed at C-1 position

2012

AC C

EP

TE D

M AN U

SC

22

Sandhu et al.,

18

ACCEPTED MANUSCRIPT

23

Starch

Table 6

RI PT

24

Graft group

NMR

Use

Various types

Solution 13C NMR

Confirmation of the grafting and measurement of the degree of grafting

Maize

Sodium acrylate

13

C CP/MAS NMR

1

M AN U

Maize

SC

type

Reference

Gurruchaga et al., 1992

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

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

Maize

Acrylamide

Solution 13C NMR

TE D

acrylate and starch

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

EP

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

Maize

Lactic acid

13

AC C

Increasing amylose content of starch increased the length of grafted segment C NMR, heteronuclear NMR analysis confirmed the formation of starch-g-lactic acid copolymer

multiple bond coherence

Hu and Tang, 2015

(HMBC)

19

ACCEPTED MANUSCRIPT

Maize

Maleic anhydride,

1

H NMR

1

H NMR analysis confirmed the formation of starch copolymers

1

H and 13C NMR

1

H and 13C NMR analysis confirmed the formation of amylopectin-g-

Yang et al., 2015

n.a.

Acrylamide-co-Nmethylacrylamide

RI PT

epoxidized cardanol

poly(acrylamide-co-N-methylacrylamide)

M AN U

26 27

32 33

EP

31

AC C

30

TE D

28 29

2015

SC

25

Sasmal et al.,

34 20

ACCEPTED MANUSCRIPT

35

Table 7 Reference

Glycerol and

1

H NMR

Native starch granules as affected by glycerol and water were studied by relaxation NMR. The

Cioica et al.,

water

relaxation

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

2013

SC

Maize

RI PT

Starch source Other component NMR technique Major observations

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

Potato and

Protein

Solid state 13C

M AN U

distributions were related to the degree of crystallinity of starch Impact of Maillard reactions between starch and amino acid (lysine) on starch structure was

CP/MAS NMR followed by solid state 13C CP/MAS NMR. Intensity of the C1 resonance peak decreased with

chestnut

TE D

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

Pizzoferrato et al., 1998 and 1999

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

Solid state 13C

Starch modified by α-amylase and glucoamylase was reacted with zinc. The chemical shift of C-6

Luo et al.,

EP

Cassava

AC C

CP/MAS NMR peak of the complexes moved downfield by 0.96 ppm, while that of the other peaks had little change, 2013 as compared with the control. The results suggest that the zinc mostly interacted with OH group of

C-6 of the glucose units

Potato amylose

Metal ion

1

H NMR

The interactions between amylose and Cu2+ broadened the peaks of 1H NMR spectrum of amylose

Li et al.,

(supplementary material Fig. 7). The ratio of signal areas of protons H(2–6) to H(1) decreased upon 2014 21

ACCEPTED MANUSCRIPT

Cu2+ addition. This suggests the binding of Cu2+ with OH groups of amylose

HPTS-C16H33

After 1-butanol-HCl hydrolysis of starch, an additional peak at 100.3 ppm was detected for C1,

NMR

indicating the formation of V-type amylose inclusion complexes

2013

1

Upon binding to amylopectin, the amphiphile of HPTS-C16H33 unfolds, and proton signals of 1H

Beeren &

NMR spectrum shifted downfield

Hindsgaul,

C CP/MAS

H NMR

amylopectin

Rice

Tea polyphenols

1

H NMR

RI PT

Potato

13

SC

1-butanol

M AN U

rice

Hu et al.,

2013

Starch and tea polyphenols were mixed and gelatinized. Compared with physical mixtures of

Wu et al.,

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

2011

TE D

sample were identical. The coupling constants were different, suggesting stronger interactions in the sample than the physical mixture 1

H NMR

Compared with individual component, 1H NMR spectrum of the propofol-hydroxyethyl starch

Silva et al.,

EP

Hydroxyethyl Propofol

complexes had broadened peak at 8.00 ppm and downfield shifts of proton signals of glucose units. 2014

starch

36 37

Table 8

AC C

This suggests the occurrence of the interactions between the hydroxyethyl starch and propofol

22

ACCEPTED MANUSCRIPT

Targeted property

NMR technique Major observations

Bread

Crystallinity of

13

Compared with the spectrum of starch in flour, that of the starch in bread crumb showed a narrowing of Primo-Martín

starch in bread

NMR

the C-1 peak, an upward chemical shift to 102.1 ppm, and the loss of the triplet characteristics. Bread

C CP/MAS

crumb and crust

Reference

RI PT

Product

et al., 2007

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

SC

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

extent that that of the crust Bread

Water mobility

1

H NMR

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

Bosmans et

of proton peaks was made by using starch-water and gluten-water model systems (supplementary

al., 2012

TE D

relaxometry

M AN U

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

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

Starch structure and 13C CP/MAS, composition

C, 1H, 31P

Structural changes of starch in rye sourdough and wheat breads during staling were compared by NMR Mihhalevski

13

techniques. 13C NMR spectra of rye and wheat starches are similar. 31P NMR analysis showed that

NMR

phospholipid fraction differed in these two starches. 13C CP/MAS NMR analysis showed that the

AC C

Bread

EP

production and storage

et al., 2012

retrograded rye starch had different polymorph composition than the wheat starch Bread

Starch structure

13

C CP/MAS

Addition of polyphenols and pectins on starch properties in bread was probed by 13C CP/MAS NMR.

Sivam et al.,

23

ACCEPTED MANUSCRIPT

NMR

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

2013

13

13

NMR

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

C CP/MAS NMR spectrum of extruded maize starch films with glycerol and water was compared

Pushpadass et

SC

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

M AN U

polymorph

TE D

C CP/MAS

EP

Starch structure

AC C

Film

RI PT

amorphous starch existed in the bread

24

ACCEPTED MANUSCRIPT

38

Table 9

RI PT

39

Targeted feature

NMR type

Other techniques

Major findings

Various types

Total phosphorus

31

Traditional

Total phosphorus contents of starch measured by 31P NMR agreed with

colorimetric

the results obtained from colorimetric chemical method

content

chemical method Various types

Amylose content

1

H NMR

Iodine-binding

M AN U

P NMR

SC

Starch sample

The amylose contents obtained from 1H NMR were comparable to those

spectrophotometry measured by iodine-binding spectrophotometry-based assay

Rice starch

Degree of branching 1H NMR

8 starches from Crystalline region

13

C

& Jane, 1996

Dunn and Krueger, 1999

The unit chain length of starch measured by 1H NMR agreed well with

Nilsson et al.,

value

that by HPSEC. The degree of branching of starch was positively

1996

EP

and unit chain length

Kasemsuwan

HPSEC and blue

AC C

Various sources Degree of branching 1H NMR

TE D

assay

Reference

HPSEC

XRD

correlated with blue value Both methods gave similar DB values (2.7−3.6%) for 14 rice varieties

Syahariza et al., 2013

Contents of double helices are not equal to the degree of crystallinity

Lopez-Rubio et

25

ACCEPTED MANUSCRIPT

obtained from XRD analysis, though the results of these two methods are al., 2008

NMR

highly correlated

11 waxy starches Crystalline region

13

from various sources (native

C

RI PT

CP/MAS

XRD, DSC

Contents of double helices of both native and annealed starches are

Witt & Gilbert,

CP/MAS

positively correlated with the degree of crystallinity (XRD) and

2014

NMR

gelatinization parameters (DSC)

SC

various sources

Degree of

13

Fourier transform- Samples varying in the degree of crystallinity were produced from

crystallinity

CP/MAS

Raman

debranched cassava starch subjected to hydrothermal processing. Degree 2012

NMR

spectroscopy,

of molecular order by NMR was positively correlated with that derived

C

XRD 13

C

maize, wheat,

CP/MAS

potato, tapioca

NMR

Maize starch, bread, rice cup

Retrogradation

Pulsed 1H NMR

XRD, DSC

EP

Gelatinization

Mutungi et al.,

from XRD and Fourier transform-Raman spectroscopy

AC C

Maize, waxy

TE D

Cassava

M AN U

and annealed)

Molecular (double-helical) order of native starches was higher than

Cooke and

degree of crystallinity measured by XRD. Molecular order is positively

Gidley, 1992

correlated with ∆H (enthalpy change) of gelatinization

Instron texture

The results of starch retrogradation obtained from two methods had a high Seow and Teo,

analyser

and positive correlation

1996

cake

26

ACCEPTED MANUSCRIPT

Retrogradation

13

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

susceptibility (α-

methods had a different pattern

C

RI PT

Pulse starches

amylase)

Cassava

Pulsed H

DSC, Instron

The glass transition temperatures measured by NMR were lower than

temperature

NMR

texture analyser,

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

DMTA

Fig. 3)

XRD

During initial stage of acid hydrolysis, double helix content from NMR

Atichokudomc

analysis was higher than the degree of crystallinity by XRD analysis.

hai et al., 2004

Degree of

13

C

Kalichevsky et

TE D

crystallinity of acid- CP/MAS hydrolysed starch

SC

Glass transition

M AN U

Waxy maize

NMR

These two values became similar at later stages of hydrolysis

Degree of

1

H NMR

Degree of grafting of copolymers

Degrees of substitution for acetylated and hydroxypropylated starches

spectrophotometry measured by NMR agreed well with those measured from traditional

substitution

Maize

Traditional

13

C NMR

AC C

Potato

EP

(supplementary material Fig. 9) de Graaf et al., 1995

-based method a

methods. 1H-NMR-based method required much less time

Hydrolytic

Degree of grafting of starch measured by a hydrolytic method agreed well Gurruchaga et

Method

with that of the NMR method

al., 1992

27

ACCEPTED MANUSCRIPT

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

RI PT

40

42

AC C

EP

TE D

M AN U

SC

43

28

ACCEPTED MANUSCRIPT 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

AC C

EP

TE D

M AN U

SC

RI PT

•