Structural characterization of novel cassava starches with low and high-amylose contents in comparison with other commercial sources

Structural characterization of novel cassava starches with low and high-amylose contents in comparison with other commercial sources

Food Hydrocolloids 27 (2012) 161e174 Contents lists available at ScienceDirect Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd ...

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Food Hydrocolloids 27 (2012) 161e174

Contents lists available at ScienceDirect

Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Structural characterization of novel cassava starches with low and high-amylose contents in comparison with other commercial sources Agnès Rolland-Sabaté a, *, Teresa Sánchez b, Alain Buléon a, Paul Colonna a, Benoît Jaillais a, Hernán Ceballos b, c, Dominique Dufour b, d, e a

UR1268 Biopolymères Interactions Assemblages, INRA, F-44300 Nantes, France CIAT, Cali AA6713, Colombia Universidad Nacional de Colombia, Carrera 32 Chapinero, Palmira, Colombia d CIRAD, UMR QUALISUD, Cali, Colombia e CIRAD, UMR QUALISUD, F-34398 Montpellier, France b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 July 2011 Accepted 23 July 2011

Two new mutant cassava starches with extreme amylose contents (0 and 30e31%) have been recently reported. These mutants are drastically different from normal cassava starch whose amylose content typically ranges between 15 and 25%. The new mutants were compared with five normal cassava starches (ranging from 16.8 to 21.5% amylose) and commercial versions of amylose-free or normal potato and maize starch. Macromolecular features, crystallinity, granule sizes, and thermal properties of these starches were compared. The structure of cassava amylopectin was not modified by the waxy mutation and waxy cassava starch exhibited properties similar to the ones of waxy maize starch. Waxy cassava and maize show similar Mw and RG of amylopectin (between 408  106 g mol1 and 520  106 g mol1; 277e285 nm, respectively), whereas waxy potato amylopectin has lower M w and RG . On the contrary, the higher-amylose mutations induced by gamma rays radiation in cassava, modified deeply the branching pattern of amylopectin as well as the starch characteristics and properties: Mw and RG decreased, while branching degree increased. These modifications resulted in changes in starch granule ultrastructure (e.g. decreased starch crystallinity), a weak organized structure, and increased susceptibility to mild acid hydrolysis. The distinctive properties of the new cassava starches demonstrated in this article suggest new opportunities and commercial applications for tropical sources of starch. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Amylose Waxy Genetic variation Structural characterization Cassava

1. Introduction Starch is the most abundant storage reserve carbohydrate in plants. It is found in many different plant organs including seeds, fruits and many roots and tubers. Starch is made up of a mixture of two a-glucans built upon mainly a-(1,4) linkages. Amylose is essentially linear, whereas amylopectin has a branched structure with 5e6% a-(1,6) linkages (Buléon, Colonna, Planchot, & Ball, 1998; Pérez & Bertoft, 2010). Different scales of organization are detected from the granular shape (0.1e200 mm), the growth rings (120e400 nm), down to the crystalline lamellae (repeat distance of 9e10 nm). They * Corresponding author. Unité de Recherche sur les Biopolymères, Interactions et Assemblages, INRA, BP 71627, 44316 Nantes Cedex 03, France. Tel.: þ33 (0) 240 67 51 48; fax: þ33 (0) 240 67 51 67. E-mail addresses: [email protected], [email protected] (A. RollandSabaté), [email protected] (T. Sánchez), [email protected] (A. Buléon), [email protected] (P. Colonna), [email protected] (B. Jaillais), [email protected] (H. Ceballos), [email protected] (D. Dufour). 0268-005X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2011.07.008

are based on the packing of double helices formed from short, clustered branches of amylopectin molecules (French, 1984; Pérez & Bertoft, 2010) within thin lamella. Average branch chain length and branch chain length distribution of amylopectins are highly correlated to the crystalline allomorph (Hizukuri, 1985; Pérez & Bertoft, 2010). Amylopectin of A-type starches contains shorter average branch chains whereas amylopectin of B-type starches contains longer average branch chains. C-type starches contain amylopectins with both long and short branch chains. The actual paradigm is that amylopectin support the framework of the crystalline domains in the starch granule (French, 1984; Pérez & Bertoft, 2010; Robin, Mercier, Charbonnière, & Guilbot, 1974). The structure of starch results from the action of different biosynthetic enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (DBE) (Buléon et al., 1998), working in a precise sequential mode (Ball et al., 1996). Starch is widely employed in many applications for which the first step is generally a thermal dispersion, carried out in non-degradative

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conditions. The resulting pastes can be used for their thickening, gelling, and/or stabilizing properties. Starch owes much of its functionality to the characteristics and the conformation in solution of its constitutive polymers, as well as to their physical organization into the native semi-crystalline granules (Colonna & Buléon, 2010). Impacting biosynthetic pathways yields starches with different morphologies, amylose:amylopectin ratios, and amylopectin structures. The amylose:amylopectin ratio greatly impacts the starch functional properties. Cassava (Manihot esculenta Crantz) is one of the most important sources of commercial production of starch along with potato, maize and wheat (Davis, Supatcharee, Khandelwal, & Chibbar, 2003) particularly for tropical and subtropical regions of the world (Moorthy, 2002). It is the third most important source of calories in tropics, after rice and maize. Natural mutation (Ceballos et al., 2007), and induced ones (Ceballos et al., 2008) in cassava starch have recently been reported leading to new starches with low and high-amylose contents. The International Center for Tropical Agriculture (CIAT) is a nonprofit organization devoted to the development of eco-efficient agriculture based in Cali, Colombia. It targets resource-poor farmers based in tropical and subtropical regions of the world working with different crops, including cassava. CIAT holds in trust from FAO (Food and Agriculture Organization) the largest germplasm collection of cassava with more than 6500 accessions, including hundreds of wild relatives. Developing high-value cassava has been a key strategy followed by the cassava-breeding project at CIAT (Ceballos et al., 2006). High-value clones would increase income for farmers and strengthen markets for this crop. As indicated by Davis et al. (2003) and many other authors, identifying starches with unique physico-chemical properties which are particularly suitable to specific end uses, would be a significant scientific and economic contribution to the cassava community. The objectives of this study were to have an overview of the structural variability among the recently discovered cassava mutants (Ceballos et al., 2007, 2008) comparatively to normal and amylose-free potato and maize starches. An important output will be to relate the structural features at different scales of these starches to the different functional properties. 2. Experimental

varieties (waxy and 5G160) were obtained from two normal cassava varieties called parental waxy and 5G160 parental. Potato starches (waxy transgenic and normal potato) were a gift from AVEBE (Veendam, Netherlands). Normal and waxy maize starches were from Roquette Frères (Lestrem, France). Isoamylase from Pseudomonas amyloderamosa (glycogen 6-glucanohydrolase; EC 3.2.1.68) with an activity of 59,000 U mL1 was from Hayashibara Shoji Inc. (Okayama, Japan). The water employed during experiments was produced using a RiOsÔ5 and Synergy purification system (Millipore, Bedford, MA, U.S.A.). The moisture content was determined by thermogravimetric analysis at a heating rate of 10  C min1 until 130  C and then 130  C for 20 min. 2.1.1. Clones, roots production and cassava starch isolation The origin of the spontaneous (waxy) and induced (small granule) cassava starch mutations were described by Ceballos et al. (2007, 2008). In both cases the recessive mutations were identified after self-pollinating a heterozygous parental. Starch from the parental genotypes, where both mutations were identified, was extracted along with the mutant starches. Cloned cassava plants were grown in CIAT’s Experimental Station at Palmira. Plants were grown following the standard recommended irrigation and fertilization procedures and roots harvested 11 months after planting (standard age for harvesting cassava at Palmira). The starches were purified according to the previously described procedure (Ceballos et al., 2007, 2008). 2.2. Macromolecular features, amylose content and complexation with iodine Macromolecular features (including branching) were obtained using asymmetrical flow field flow fractionation (A4F) and highperformance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS) using the same procedures and set ups as described by Pérez et al. (2011). M n , M w , the dispersity index M w =M n and RG (nm) were established using ASTRAÒ software from Wyatt Technology Corporation (version 5.3.2.13 for PC), as previously described (Rolland-Sabaté, Amani, Dufour, Guilois, & Colonna, 2003; Rolland-Sabaté, Colonna, Mendez-Montealvo, & Planchot, 2007). The average 2

2

shrinking factor gM (gM ¼ RGwðbrÞ =RGwðlinÞ , where RGwðbrÞ is the

2.1. Materials 12 Starches from different sources were studied (Table 1). Cassava roots were obtained from CIAT in Colombia. Mutant cassava

radius of gyration of the branched molecule and RGwðlinÞ , the radius of gyration of its linear equivalent at the same molar mass) was also determined. The equation linking molar mass and radius of gyration for strictly linear amyloses was used (Rolland-Sabaté,

Table 1 Samples description, amylose content and lmax values.a Sample name

Starch

Species

Dry matter (%)

Amylose content b (%)

Amylose content c (%)

lmax (nm)

WXCS-1 WTCS-2 AMYCS-3 AMYCS-4 WTCS-5 WTCS-6 WTCS-7 WTCS-8 WTNPS-9 WXTPS-10 WXMS-11 WTMS-12

Waxy cassava Parental (wild type) waxy cassava 5G160-13 cassava 5G160-16 cassava Parental (wild type) 5G160 cassava Normal (wild type) cassava MCOL1505 Normal (wild type) cassava MTAI8 Normal (wild type) cassava HMC-1 Normal (wild type) potato Waxy potato transgenic Waxy maize Normal (wild type) maize

Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Manihot esculenta Solanum tuberosum Solanum tuberosum Zea mais Zea mais

88.9 94.8 84.7 88.5 88.6 88.4 88.4 88.3 86.7 86.7 87.6 87.3

0.0 (0.0) 18.6 (0.3) 30.1 (1.4) 30.3 (1.3) 21.5 (0.5) 16.8 (0.8) 16.8 (0.2) 19.0 (N.A.) 22.1 (0.8) 0.0 (0.0) 0.13 (0.2) 28.7 (0.5)

0.0 (N.A.) 18.5 (0.4) 27.5 (0.8) 28.4 (0.4) 21.4 (0.1) 17.7 (0.1) 17.0 (0.1) 16.8 (0.6) 22.5 (0.1) 0.0 (N.A.) 0.0 (N.A.) 25.0 (0.0)

528 590 601 606 595 578 579 579 566 535 529 609

N.A.: not available. a Standard deviations are given within parenthesis. b Values obtained from DSC measurements. c Values obtained from IBC measurements.

(0.0) (7.1) (9.9) (1.4) (7.1) (0.0) (0.0) (0.0) (0.0) (2.8) (0.7) (0.7)

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Colonna, Mendez-Montealvo, & Planchot, 2008). gM and the 3

apparent particle density ðdGappw ¼ M w =ð4p=3ÞRGw Þ were deduced from the A4FeMALLS data, as well as the average number of branching points per macromolecule ðBÞ and the average number of glucosyl units in a linear chain per branching point ðDPw =BÞ (Pérez et al., 2011). Size distributions (hydrodynamic radius distributions) were determined using the hydrodynamic radius (RH) versus elution time calibration curves previously determined with latex spheres in A4F and the hydrodynamic radius versus elution volume calibration curves previously determined using quasi-elastic laser light scattering in HPSEC (Rolland-Sabaté, Guilois, Jaillais, & Colonna, 2011). Amylose contents were determined by two different ways: differential scanning calorimetry (DSC) (Ceballos et al., 2007, 2008) and determination of iodine binding capacity (IBC) (Pérez et al., 2011). The wavelength at maximum absorption (lmax) of the iodine complexes with native starches was determined after solubilization in 1 N KOH for 3 days at 4  C under stirring (RollandSabaté, Colonna, Potocki-Véronèse, Monsan, & Planchot, 2004). 2.3. Debranched chain length distribution Starches (25 mg) were solubilized in 1 M KOH (0.5 mL) for 3 days at 4  C under gentle magnetic stirring, and 4.5 mL pure water and 0.1 M HCl (5 mL) were added. To 4 mL of this resulting solution (2.5 g L1), 40 mL of acetate buffer (100 mM, pH 3.6) were added, and debranching was performed by addition of 5 mL of undiluted isoamylase and 28 h incubation at 40  C in a water bath. The chain distributions of debranched samples were determined using highperformance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Model DX-120, Dionex, Sunnyvale, CA), using Carbopac PA100 (4 mm  250 mm) column. The pulsed potentials and durations were E1 ¼ 0.05 (t1 ¼ 400 ms), E2 ¼ 0.75 (t2 ¼ 200 ms) and E3 ¼ 0.15 (t3 ¼ 400 ms). The eluent was a gradient made from 150 mM NaOH (eluent A) and 1 M sodium acetate (NaOAc) (eluent B) at 1 mL min1 flow rate. The elution gradient was (i) 0e6 min with a linear gradient from 0% to 8% eluent B, (ii) 6e56 min with a linear gradient from 8% to 18% eluent B, (iii) 56e256 min with a second linear gradient from 18% to 38% eluent B, (iv) 256e260 min with a third linear gradient from 38% to 70% eluent B. It should be noticed that as in HPAEC-PAD response coefficients decrease with increasing DP, % area of long chains are not representative of the exact weight fraction of each DP. 2.4. Starch granule general structure and properties DSC analyses were performed on a TA Instruments DSC Q100 device (TA Instruments, Norwalk, CT, USA) using sealed stainless steel pans. The sample pans (10 mg starch and 40 mL of water) were heated twice from 0 to 120  C at a scanning rate of 3  C min1 and cooled to 0  C. The DSC runs were performed against a reference pan containing 50 mL of water. The instrument was calibrated using indium (Tm ¼ 156.6  C, DHm ¼ 28.55 J g1). Gelatinization enthalpy (DH), gelatinization onset temperature (To) and gelatinization peak temperature (Tp) of each sample were then determined on the thermograms using the Universal Analysis (TA Instruments) software and normalized to the mass of dry matter. Crystallinity information and starch granule sizes were determined as previously described (Pérez et al., 2011). The morphology of the native granules were observed using an LEICA DMLB light microscope (Leica Microsystems GmbH, Wetzlar, Germany) using a 40 magnification lens and scanning electron microscopy (SEM). The granules were deposited onto copper stubs and allowed to dry.

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The specimens were coated with Au-Pd, examined at 10.0 kV, and photographed using a JEOL JSM 5800LV scanning electron microscope. 2.5. Starch granule ultrastructure The starch granules were lintnerized as previously described (Gérard, Colonna, Buléon, & Planchot, 2002). The extent of reaction (hydrolysis %) was expressed as the percentage of dry substrate solubilized. The chain distributions of lintnerized samples were determined using HPAEC-PAD as described in Section 2.4. The crystallinity of lintnerized starch granules was determined using X-ray diffraction and their morphology observed by SEM. 2.6. Statistical analysis Principal component analysis (PCA) was performed using MatLabÒ software (The MathWorks, Inc., Natick, MA, USA). The variables were the amylose content (amylose %), M w (Mw), RGz (RG), nG, the slope of the logelog plot of the relation linking molar mass and radius of gyration, gM, DP=B (DP/B), dGappw, the crystallinity (cryst %), the proportion of B-type crystallites (B-type %), the average granule diameter (B), To, Tp and DH obtained by DSC; the observations were the starch samples. 3. Results and discussion 3.1. Amylose content Starch amylose contents ranged from 0% in waxy starches to 30% in starches from the self-pollinated progenies of 5G160 (AMYCS-3 and AMYCS-4), while most were in the range 16.8e21.5% for normal cassava cultivars, 22e28% for normal potato and normal maize starches, respectively (Table 1). Amylose content of the starch from genotype 5G160 (WTCS-5), fell within the range for wild-type cassava (21%, Table 1). Amylose contents in normal and waxy starches were in agreement with those determined by other authors (Buléon et al., 1998; Gérard, Barron, Colonna, & Planchot, 2001; Gomand et al., 2010; Sánchez et al., 2009; Tetchi, RollandSabaté, Amani, & Colonna, 2007). The values obtained using DSC measurements were equivalent with the values obtained using IBC. The values of 30% amylose are the highest reported in the literature for cassava starches (Charles, Chang, Ko, Sriroth, & Huang, 2004; Jane et al., 1999; Sánchez et al., 2009) and illustrate the limited work so far conducted to generate high-value cassava starches. lmax values ranged from 528e529 nm (WXCS-1 and WXMS-11) to 609 nm (WTMS-12) (Table 1). lmax values are influenced by the ratio amylose:amylopectin. 3.2. Macromolecular features The solubilization rates of the twelve starches (Table 2) ranged between 79% for WTCS-2 and 100% for AMYCS-3, AMYCS-4 and WTCS-6. Elution recoveries, which represent the percentage of macromolecules percolated through the HPSEC and A4F systems, were 100% using A4F and ranged from 71% to 100% (average 86%) using HPSEC. These high recovery rates indicated that the fractionation response was quantitative for all starch samples. Thus, this solubilization mode was considered to allow complete structural characterization of the starches and do not have a clear association with amylose contents (Table 2). A4F and HPSEC elugrams of all starches revealed two DRI peaks (corresponding to concentration variation) (Fig. 1), except for waxy starches which displayed a single peak. With both techniques, the light scattering (LS) signal consisted of a single peak, corresponding

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Table 2 Macromolecular characteristics of cassava, potato and maize starches determined by A4FeMALLSeQELS. Solubilization and elution recoveries, weight average molar mass ðM w Þ, z-average radius of gyration ðRGz Þ, weight average radius of gyration ðRGw Þ and dispersity index ðM w =M n Þ for the amylopectin population. Sample name

Solubilization recovery (%)

Amylopectin populationa,b,c M w  106 ðg mol1 Þ

RGz ðnmÞ

RGw ðnmÞ

M w =M n

WXCS-1 WTCS-2 AMYCS-3 AMYCS-4 WTCS-5 WTCS-6 WTCS-7 WTCS-8 WTNPS-9 WXTPS-10 WXMS-11 WTMS-12

90 79 100 100 90 100 85 97 92 92 96 100

408.2 255.3 50.4 50.8 253.0 417.9 370.2 305.9 186.3 115.8 520.7 334.5

276.7 242.0 122.5 119.5 230.4 276.2 262.5 246.5 237.3 197.8 285.4 252.2

253.9 219.5 99.3 98.7 212.7 254.1 243.5 224.1 212.6 174.5 270.1 228.3

1.25 1.47 2.23 2.03 1.32 1.37 1.26 1.34 1.88 1.33 1.17 1.33

(62.2) (64.3) (3.34) (N.A.) (22.6) (N.A.) (N.A.) (0.28) (31.7) (16.9) (46.3) (17.9)

(11.1) (10.8) (8.0) (N.A.) (3.1) (N.A.) (N.A.) (3.2) (1.4) (4.2) (14.3) (7.5)

(8.5) (3.9) (5.4) (N.A.) (1.6) (N.A.) (N.A.) (0.8) (0.7) (4.8) (10.1) (4.5)

(0.03) (0.21) (0.10) (N.A.) (0.15) (N.A.) (N.A.) (0.04) (0.37) (0.02) (0.09) (0.06)

N.A.: not available. a Weight average molar mass ðM w Þ, z-average radius of gyration ðRGz Þ, weight average radius of gyration ðRGw Þ, and dispersity index ðM w =M n Þ for the amylopectin population. b These values were taken over the whole amylopectin peak. c Standard deviations are given within parenthesis.

8.0 7.5 7.0

1

5.5

5.0

1.E+10

WXCS-1 1.E+09

0.6 1.E+08

WTCS-2

0.4

1.E+07

0.2

-1

0.8

Molar mass (g.mol )

Normalized concentration (A.U.)

a

Elution volume (mL) 6.5 6.0

AMYCS-3

0 1

10

1.E+06 1000

100

Hydrodynamic radius (nm)

250

50

1

Elution time (s) 500

750

1000

1000

WXCS-1 0.8

AMYCS-3

0.6

100 0.4

WTCS-2

0.2

0 1

10

100

Radius of gyration (nm)

Normalized concentration (A.U.)

b

10 1000

Hydrodynamic radius (nm)

Fig. 1. Size distributions and molar masses of WTCS-2, WXCS-1 and AMYCS-3 obtained by HPSEC (a) and A4F (b). Waxy cassava, WXCS-1; parental waxy cassava, WTCS-2; 5G160-13 cassava, AMYCS-3. The correspondence of elution volume and elution time to hydrodynamic radius shown on the top of the plots was made using hydrodynamic radius calibration curves according to Rolland-Sabaté et al. (2011). The thick lines represent the normalized concentrations (obtained from DRI answers) in black and in light green for WXCS-1 and WTCS-2, respectively. The thin blue line represents the normalized concentrations for AMYCS-3. The light green squares, the black triangles and the blue crosses represent the molar masses (a) and the radii of gyration (b) of WTCS-2, WXCS-1 and AMYCS-3, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

to the biggest fraction (i.e. generally amylopectin fraction) (results not shown). The fractionation techniques separate by hydrodynamic radius. For linear polymers, there is a direct relation between hydrodynamic radius and molar mass and radius of gyration. For branched polymers, it is not the case at all. Each fraction of a given hydrodynamic radius contains molecules exhibiting different molar masses and radii of gyration, i.e. different structures then the radius of gyration and the molar mass distributions couldn’t be determined using SEC or A4F for branched polymers (Gidley et al., 2010). The obtained distributions are hydrodynamic radius distribution. A4F and HPSEC elugrams were transformed to size distributions, using hydrodynamic radius (RH) versus elution volume (or elution time) calibration curves (Rolland-Sabaté et al., 2011) (Fig. 1). The size distributions of waxy starches showed one peak at RH w 120e176 nm (for WXTPS-10 and WXMS-11, respectively) corresponding to amylopectin population. The size distributions of non waxy starches generally exhibited two populations: (i) amylopectin fraction (corresponding to the main fraction) which peaked at RH w 120e176 nm (for WTCS-2 and WTMS-12, respectively) and, (ii) amylose population which peaked at RH w 20e35 nm (for WTMS-11 and WTNPS-9, and WTCS-6 and WTCS-8, respectively) with HPSEC (a shoulder rather than a peak was observed in A4F excepted for maize and potato starches). Amylose population corresponded to RH smaller than 40 nm with both methods. The exception was made by AMYCS-3, which showed in A4F a main population peaking at a RH w 65 nm, with a broad shoulder at RH smaller than 30 nm, and in HPSEC a main population peaking at a RH w 25 nm, with a shoulder at a RH w 70 nm (this behavior was also shown by AMYCS-4, but data are not presented in Fig. 1). On the first hand, this different behavior indicated that the amylopectin size and the size difference between amylose and amylopectin were lower for the AMYCS-3 and AMYCS4 compared to the other starches studied. On the other hand, the different patterns observed with A4F and HPSEC for 5G160 cultivars (Fig. 1) was due to an incomplete fractionation of the large and the small fractions (particularly using HPSEC). Both techniques provided an important overlapping of the two constitutive peaks for these starches. This was probably because an important fraction of amylose eluted with the amylopectin fraction. Alternatively, that could be indicative of the presence of an intermediate material. The results obtained with HPSEC and A4F were consistent for all the starches studied. A4F allows a better fractionation between amylose and amylopectin and a better characterization of the large amylopectin fractions (Fig. 1, Rolland-Sabaté et al., 2011). However

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the determination of M w and RG corresponding to the amylose fraction were frequently impossible due to very small LS and DRI signals for the amylose fraction linked to the extremely small amount of sample injected. For starches containing amylose it is then interesting to study both A4F and HPSEC traces. However, it is important to notice that in HPSEC the separation between amylose and amylopectin is not complete then, the DRI peak attributed to amylose (Fig. 1a) can be regarded as the sum of the DRI of amylopectin tail with the lowest hydrodynamic volumes and the DRI of amylose. With the same hydrodynamic volume, amylopectin molecules, due to their branching, were much larger than amylose molecules, so that minor amounts of amylopectin shifted the M w and RG values calculated for amylose to higher values. Then, the characteristics of amylose (M w and RG ) couldn’t be reported accurately with both techniques. Thereafter, amylopectin characteristics obtained by A4FeMALLS were reported (Table 2). Amylopectin Mw , RG , and the Mw =Mn values ranged from 50  106 g mol1 to 520  106 g mol1; 119 to 285 nm and 1.17 to 2.23, respectively (Table 2). The M w and RG values were of the same order (but slightly higher) as those reported in the literature for cassava, yam, potato and maize starches (Pérez et al., 2011; Rolland-Sabaté et al., 2003, 2007; Tetchi et al., 2007). The differences may be linked to the fractionation technique since A4F allowed the characterization of large amylopectin fractions, but may also resulted from biological variations. Waxy cassava and maize amylopectins exhibited high Mw values (408.2  106 g mol1 and 521 106 g mol1) compared to waxy potato amylopectin (116  106 g mol1). Waxy potato amylopectin exhibits slightly lower M w and RG values than the normal one whereas waxy cassava and maize amylopectins show higher M w and RG values than normal ones. Among other cassava starches the highest M w was obtained for the WTCS-6 (418  106 g mol1), whereas the smallest values were obtained for AMYCS-3 and AMYCS-4 (50  106 g mol1), which presented a higher-amylose content. By plotting the radius of gyration and molar mass of the same fraction, structural data could be determined from the exponent nG (hydrodynamic coefficient) using the equation: RG ¼ KG MnG . The inverse of nG could be considered as the fractal dimension of the global structure, dfg (Rolland-Sabaté et al., 2007). The values for nG depend on polymer shape, temperature and polymeresolvent interactions. Basically, nG ¼ 0.33 for a sphere, nG ¼ 0.5e0.6 for

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a linear random coil and nG ¼ 1 for a rod. nG-values ranged from 0.35 to 0.45 (giving dfg between 2.86 and 2.22) for studied amylopectins (Table 3). These values were in good agreement with data in the literature (Roger, Bello-Perez, & Colonna, 1999; Rolland-Sabaté et al., 2003, 2007; Tetchi et al., 2007) and indicated that these amylopectins adopted a spherical conformation, with branched chains not swollen by the solvent (dfg between 2.9 and 2.5) and moderately swollen by the solvent (dfg between 2.5 and 2.0). WTCS-5, WTCS-6, and WTCS-2 (0.35e0.36) showed the smallest nG-values (and the highest dfg values) which could indicate that these amylopectins were the densest. Nevertheless, there were no significative differences between the nG-values reported in Table 3, excepted for WXTPS-10 which showed the highest nG-value (0.45), and the lowest dfg value (2.22). These nG and dfg values obtained for WXTPS10 amylopectin in pure water were probably due to a polyelectrolyte effect because of the presence of phosphate groups bound covalently on potato amylopectin (Buléon et al., 1998). The determination of apparent particle density, dGappw could provide another mean to approach the conformation of the molecule and give then additional indication on branching. dGappw reported in Table 3 ranged from 4.6 for WTNPS-9 to 12.6 for AMYCS-4. Among waxy starches, the lowest dGappw was obtained for potato and the highest for maize (5.2 and 6.3 for WXTPS-10 and WXMS-11, respectively) while intermediate density values between 6.0 and 6.5 g mol1 nm3 were obtained for normal starches, except for potato which shows the lowest density (4.6). AMYCS-3 and AMYCS-4 showed the highest dGappw values (12.3 and 12.6, respectively). These latter could then be the densest and probably the most branched amylopectins. On the opposite, potato amylopectins would be the less branched ones. Table 3 also reports the branching characteristics of amylopectins. gM values ranged from 0.021 for WXMS-11 to 0.053e0.059 for AMYCS-4 and WXTPS-10, respectively; the number of branches ðBÞ determined using the ABC model were between 2.1e2.5  103 for WXTPS-10 and AMYCS-3, respectively and 17.6  103 for WXMS-11. The average number of glucosyl units per branching point ðDPw =BÞ was then between 120.5e125.2 for AMYCS-3 and AMYCS-4, respectively and 353.1 for WTNPS-9. The corrected average number of glucosyl units per branching point ðDPw =BmH Þ ranged between 14.5 and 15.0 for AMYCS-3 and AMYCS-4 and 42.4 for WTNPS-9 while the corrected branching degree ðBDmH ¼ 100  BmH =DPw Þ ranged from 2.4e2.5 for potato starches to 6.7e6.9 for the small granule cassava

Table 3 Structural characteristics of cassava, potato and maize amylopectins determined by A4FeMALLSeQELS.a Sample name

WXCS-1 WTCS-2 AMYCS-3 AMYCS-4 WTCS-5 WTCS-6 WTCS-7 WTCS-8 WTNPS-9 WXTPS-10 WXMS-11 WTMS-12

dGappw (g mol1 nm3)

nG

6.0 5.8 12.3 12.6 6.3 6.1 6.1 6.5 4.6 5.2 6.3 6.7

0.38 0.36 0.40 0.41 0.35 0.35 0.37 0.40 0.41 0.45 0.40 0.42

dfg

(0.02) (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) (0.01) (0.02) (0.02)

2.63 2.78 2.50 2.44 2.86 2.86 2.70 2.50 2.44 2.22 2.50 2.38

gM

0.025 0.034 0.054 0.053 0.032 0.025 0.026 0.028 0.048 0.059 0.021 0.026

ABC model

Modified ABC model

B  103

DPw =B

BmH  103

DPw =BmH

BDmH (%)

12.0 6.5 2.5 2.6 7.2 12.8 11.1 9.5 3.3 2.1 17.6 11.1

209.2 243.1 125.2 120.5 216.8 202.1 205.9 198.6 353.1 335.5 182.8 185.8

100.3 54.0 20.7 21.7 60.0 10.6 92.5 79.2 27.1 17.8 146.5 92.6

25.1 29.2 15.0 14.5 26.0 24.3 24.7 23.8 42.4 40.3 21.9 22.3

4.0 3.4 6.7 6.9 3.8 4.1 4.0 4.2 2.4 2.5 4.6 4.5

Branching parameters: average number of branching points ðBÞ, average number of glucosyl units in a linear chain per branching point ðDPw =BÞ and branching degree ðBD ¼ 100B=DPw Þ determined from the simple ABC model according to Burchard (1983), using the following equation: gM ¼ 4f½ð1 þ 2BÞ1=2 =½1 þ ð1 þ 2BÞ1=2 2 g; or following the modified ABC model according to Hizukuri model (Hizukuri, 1986; Rolland-Sabaté et al., 2007). Standard deviations are given within parenthesis. N.A.: not available. 3 2 2 a DensitiesðdGappw ¼ Mw =ð4p=3ÞRGw Þ , hydrodynamic coefficient (nG), global fractal dimension (dfg) and average shrinking factor (gM ¼ RGwðbrÞ =RGwðlinÞ , where RGwðbrÞ is the radius of gyration of the branched molecule and RGwðlinÞ , the radius of gyration of its linear equivalent at the same molar mass. The equation linking molar mass and radius of gyration for strictly linear amyloses was used (Rolland-Sabaté et al., 2008)).

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mutants AMYCS-3 and AMYCS-4 (Table 3). Waxy cassava and maize amylopectins appeared to be the most branched, maize being slightly more branched than cassava, whereas waxy potato appeared to be the least branched, with longer chain lengths. Normal varieties ranged in the same way as waxy ones and exhibited similar branching characteristics. The values obtained in this study were in agreement with the values of the average chain length ðCLÞ 18e34, determined elsewhere for potato, maize and normal cassava starches after debranching of the samples (Jane et al., 1999) or NMR (Nilsson, Bergquist, Nilsson, & Gorton, 1996) or by A4FeMALLS (RollandSabaté et al., 2007). When gM value decreases the number of branching point (B and BmH ) increases and, for the same molar mass the average chain length per branching point (DPw =B and DPw =BmH ) decreases. The opposition of the behavior of gM and B values versus chain lengths observed for AMYCS-3 and AMYCS-4 was then due to their particularly low M w compared to the other starches and indicated that they have probably a completely different branching pattern. Moreover, the radii of gyration of AMYCS-3 were clearly lower than the ones of WTCS-2 and WXCS-1 at the same hydrodynamic radii (Fig. 1b), this was another confirmation of a more compact conformation of AMYCS-3 compared to WTCS-2 and WXCS1. These results were in line with the apparent density values. 3.3. Debranched chain length distribution The distributions of debranched chains for cassava, maize and potato starches displayed two maxima: a major peak: (peak I) corresponding to DP 11e13 and a minor peak: (peak II) corresponding to DP 43e44, except for AMYCS-3 which showed no peak at high DP (Table 4). For each species crop, waxy and normal starches showed similar chain length distributions but differences were observed regarding botanical origin (Table 4). In general, cassava and maize starches had larger proportions (25.8e33.5%) of short chains (DP 6e12) and smaller proportions (6.6e9.2%) of long chains (DP > 37) than potato starches, which showed 21.0% and 10.4e11.8% for the short and long chains respectively (Table 4). These distributions were classical and had already been described for potato, maize, sweet potato and yam starches (Jane et al., 1999; McPherson & Jane, 1999). Starches from WXCS-1 and WTCS-2 showed intermediate proportions of long chains (DP > 37), higher proportions of short chains (DP 6e12) and lower proportions of DP 13e24 compared to potato and maize starches. AMYCS-3 exhibited a particularly small amount (6.6%) of long chains (DP > 37), and high amount (33.5%) of short chains (DP 6e12). The lack of peak at DP 43e44 and the low value of the highest detectable DP (Table 4) indicated that the amylopectin of AMYCS-3 contained a particularly small amount of long chains. The chain length distribution observed for AMYCS-3 was similar to those previously obtained for rice amylopectins (Jane et al., 1999) and might be related to a high branching degree of the native molecule compared to the WTCS-2 and WXCS-1 amylopectins. The average chain length ðCLÞ

calculated from HPAEC-PAD distributions reflected quite perfectly the previous observations. AMYCS-3 exhibited the lowest CL (18.4) and potato starches the highest CL (20.5e20.6), whereas maize and other cassava starches exhibited intermediate values (18.9e19.3) (Table 4). These values could be related to the branching degree of the amylopectin ð1=CLÞ, indicating that AMYCS-3 amylopectin was probably more branched than maize and other cassava ones and that potato amylopectins were the least branched. The distributions of debranched chains obtained by HPAEC-PAD were consistent with the branching characteristics determined by A4FeMALLS. In particular, a positive linear correlation (R2 ¼ 0.8666) was found between the DPw =B values and the relative amount of B3 chains (obtained by HPAEC-PAD of debranched chains), thus confirming that the DPw =B values were linked to the internal structure of amylopectin macromolecules. On the opposite, a negative linear correlation (R2 ¼ 0.7392) was found between the DPw =B values and the relative amount of A chains, thus indicate that the DPw =B values were also influenced by the external structure of amylopectin macromolecules. Among studied amylopectins, waxy and normal cassava ones exhibited high M w and RG and medium branching degree, as well as maize. AMYCS-3 and AMYCS-4 amylopectins were the smallest ones and seemed to be the most branched ones, whereas potato amylopectins the least branched ones. The branching pattern was not modified by the waxy mutation in cassava, maize and potato. On the contrary, the mutation induced by gamma rays radiation in cassava (AMYCS-3 and AMYCS-4) produces an important change in the branching characteristics of the amylopectin.

3.4. General starch granule structure and properties Laser granulometry (Fig. 2a) allowed to classify native starches in three groups as a function of their average granule size (diameter in mm): (i) starches exhibiting large granules with an average size of more than 30 mm (potato starches) (group 1), (ii) starches with medium-sized granules of 14e17 mm (normal and waxy cassava and maize starches) (group 2), (iii) starches showing small granules with an average size of 7e10 mm, represented by the AMYCS-3 and AMYCS-4 and their parental clone (group 3). In the first group, WTNPS-9 exhibited a majority of granules corresponding with size higher than 40 mm whereas WXTPS-10 showed a major population of granules between 20 and 40 mm. In the medium-sized group the fraction corresponding to starch granules sizes smaller than 7 mm was very limited (around 10%) and starches exhibited a majority of granules corresponding with size between 7 and 20 mm. Among these starches, WXCS-1 showed the highest proportion of granules sized between 20 and 40 mm (Fig. 2b). In the third group, AMYCS-3 showed (as reported by Ceballos et al., 2008) a majority of granules smaller than 7 mm (around 53%) whereas WTCS-5 and AMYCS-4 cassava starches presented a majority of medium-sized granules

Table 4 Chain length distributions of debranched starches obtained from HPAEC-PAD.a Type of starch

WXCS-1 WTCS-2 AMYCS-3 WTNPS-9 WXTPS-10 WXMS-11 WTMS-12

Peak DP

% Distribution

I

II

DP 6e9

DP 6e12

DP 13e24

DP 25e36

DP  37

12 11 11 13 13 12 12

44 44 N.A. 43 43 44 44

9.3 9.7 11.6 6.8 6.6 7.1 7.1

28.8 29.7 33.5 21.4 21.0 25.8 26.9

49.0 48.9 46.0 55.6 55.0 53.4 53.7

13.0 12.5 14.0 12.7 12.8 13.5 12.1

9.2 8.9 6.6 10.4 11.1 7.4 7.3

(0.17) (0.24) (0.06) (0.03) (0.01) (0.07) (0.05)

N.A.: not available because of the absence of a peak. a Standard deviations are given in parenthesis.

(0.61) (0.19) (0.09) (0.05) (0.11) (0.37) (0.36)

(0.22) (0.39) (0.04) (0.09) (0.02) (0.96) (0.00)

(0.58) (0.07) (0.01) (0.07) (0.14) (0.36) (0.34)

(0.20) (0.27) (0.03) (0.23) (0.05) (0.97) (0.04)

Average CL

Highest detectable DP

19.5 19.3 18.4 20.5 20.7 19.2 18.9

83 86 74 83 83 71 80

(0.04) (0.08) (0.02) (0.1) (0.03) (0.41) (0.09)

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Mean diameter of starch granules (µm)

a 50 45 40 35 30 25 20 15 10 5

W XC S1 W TC S2 AM YC S AM -3 YC S4 W TC S5 W TC S6 W TC S7 W TC S8 W TN PS -9 W XT PS -1 0 W XM S11 W TM S12

0

Relative level of granules (%)

b 100 90

Ø < 7 µm

Ø 7-20 µm

80

Ø 20-40 µm

Ø > 40 µm

70 60 50 40 30 20 10

W XC S1 W TC S AM -2 YC S3 AM YC S4 W TC S5 W TC S6 W TC S7 W TC S8 W TN PS -9 W XT PS -1 0 W XM S11 W TM S12

0

Fig. 2. Granule size distributions (diameter, B) obtained by laser granulometry. a. Mean diameter of the granules; b. Distributions of the granules size according to four categories: B < 7 mm, B between 7 and 20 mm, B between 20 and 40 mm, and B > 40 mm. Waxy cassava, WXCS-1; parental waxy cassava, WTCS-2; 5G160-13 cassava, AMYCS-3; 5G160-16 cassava, AMYCS-4; parental (wild type) 5G160 cassava, WTCS-5; normal (wild type) cassava MCOL1505, WTCS-6; normal (wild type) cassava MTAI8, WTCS-7; normal (wild type) cassava HMC-1, WTCS-8; normal (wild type) potato, WTNPS-9; waxy potato transgenic, WXTPS-10; waxy maize, WXMS-11; normal (wild type) maize, WTMS-12.

corresponding to sizes between 7 and 20 mm (70e77%) and a very small amount (5.5%) of granules larger than 20 mm. Among cassava, normal species exhibited similar granule sizes, except the parental clone of AMYCSs. The waxy mutation induced a clear decrease of granule’s size in case of potato (group 1) whereas a slight increase of the starch granule size was observed for waxy maize and cassava (WXMS-11 and WXCS-1), compared to the normal varieties (group 2). It is interesting to note that in potato, maize and cassava starches, the waxy mutation induced an increase in the proportion of granule sized between 20 and 40 mm. The 5G160 mutation (group 3) resulted in an important decrease of the starch granule size and an important change in the distribution of granules diameters (Fig. 2). In particular, contrary to waxy starches, starches from group 3 showed a very small proportion of granules sized between 20 and 40 mm. These observations were in agreement with the results reported previously on potato and cassava starches showing various amylose content (Ceballos et al., 2007, 2008; Gomand et al., 2010). The WXCS-1 granules were spherical and truncated and some of them showed facets typical of normal cassava starch (same as WTCS-2). All the observed granules were birefringent and seemed to be intact (Fig. 3a). For AMYCS-3 cassava, the granules were polyhedral and truncated, and some of them showed facets and weak birefringence (Fig. 3b). The outlines of the granules were

167

rough and there were a lot of non birefringent granules especially among the smallest ones. Small granules are known to be non birefringent, nevertheless, for AMYCS-3, even larger granules showed a blurred Maltese cross. That could be the result of fragile starch granules. Smaller granules stained dark blue after iodine treatment, meaning that they possess considerable higher proportion of amylose compared to larger ones which stained less intensely (results not shown). However, external long chains of amylopectin can interact with iodine molecules. Native starches crystallinity ranged from 43% for potato starches and WXMS-11 to 25% for AMYCS-3 and AMYCS-4 (Table 5). As expected, maize and potato starches showed an A and a B pattern respectively, whereas all cassava starches exhibited a mixture of A and B-type crystallites with a majority of A-type. These results were in line with debranched chain length distributions and data reported in literature for maize (Gérard et al., 2002), potato and tuber starches (McPherson & Jane, 1999). Generally, waxy starches showed a stable crystallinity (40e43%) higher than normal ones (30e35%), except for waxy potato which exhibited the same crystallinity as normal one. DH values for the different starches ranged from 8.9 to 18.8 J g1 (Table 5). The highest values (namely 18.8e16.9 J g1) were obtained for waxy starches, WTNPS-9 and WTCS-7. DH were generally higher for waxy starches (Table 5) in line with results already reported for maize (Perera, Lu, Sell, & Jane, 2001). DH is known to be linked to the fusion of the crystalline structure and then increases with crystallinity (Zobel, Young, & Rocca, 1988). DH reflects disorganization of double helices of amylopectin. High DH values are due to more stable double helices that can be related with chain length. These results were in line with crystallinity determinations: potato starches and WXCS-1 were also the more crystalline starches (43%), WTCS-7 starch being the exception. The lowest values obtained for AMYCS-3 and AMYCS-4 (namely 8.9, 10.0 J g1) were in line with their low crystallinity (25%) and the high amount of short chains in their amylopectin (Table 4). Tp values ranged from 56.2 to 70.6  C for AMYCS-3 and WXMS-11, respectively (Table 5). Potato, maize and cassava waxy starches (WXTPS-10, WXMS-11 and WXCS-1) had the highest Tp (69.5, 67.6 and 70.6  C, respectively). Intermediate values of DH and Tp were found for normal cassava starches. These data confirmed the conclusions reached in the literature (McPherson & Jane, 1999; Moorthy, 2002; Perera et al., 2001; Tetchi et al., 2007). WXCS-1 and potato starches displayed similar thermal properties and their resistance toward thermal breakdown was higher than those of normal and 5G160 (parental and the two progenies) cassava starches (higher values of Tp and DH). Crystallinity and DSC results from waxy species could then suggest that they have a more organized structure than normal starches. Whereas higher level of amylose in cassava and maize leads to less organized structure: normal cassava and maize exhibited generally lower values of Tp, DH and crystallinity (Table 5). Starches from AMYCS-3 and AMYCS-4 on the other hand, probably have a weak organized structure. With their low Tp and DH (Table 5), they showed the classical behavior of weak crystalline starch, unlike starches exhibiting equivalent amylose content, such as smooth pea, which exhibit generally higher Tp and DH and a higher extent of B-type crystallites (Ratnayake, Hoover, & Warkentin, 2002). AMYCS-3 and AMYCS-4 showed in addition a very low onset temperature, which is in agreement with a weakly organized structure. 3.5. Changes after mild acid hydrolysis Starches were subjected to mild acid hydrolysis (lintnerization) (Robin et al., 1974). Scanning electron microphotographs of 2 days

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Fig. 3. Photographs from light microscope (40) on polarized light and scanning electron microphotographs (1500) of starches from WXCS-1 (waxy cassava) (a) and AMYCS-3 (5G160-13 cassava) (b).

lintnerized cassava starches showed weakly hydrolyzed granules for all starches with small and big holes on their surface area (Fig. 4). WXCS-1 lintnerized granules showed the same shape as native ones, but had distinctive softer edges, stamped areas on the granules and “peeled orange” like zones on the surface area (Fig. 4a) as observed for WTCS-2. AMYCS-3 lintnerized granules differed considerably from native ones: they had a smoother surface and displayed more spherical shapes. Many of these lintnerized granules displayed a ghost pattern (Fig. 4b). There was a lower frequency of very small granules (<3e4 mm) in the 2 days lintnerized AMYCS-3 (Fig. 4b). After 10 days hydrolysis (results not shown), the residues were constituted by very smooth granules, with diameter larger than 10 mm. The longer the hydrolysis time is,

the higher the size of the remaining granules. AMYCS-3 was easily degraded by acid hydrolysis (small granules particularly) and the external surface was hydrolyzed first (lintnerized granules are smoother than native ones). Amylopectins from A-type and C-type starches had larger proportions of short chains (25.8e28.8%) (DP 6e12) and smaller proportions of long chains (7.4e9.2%) (DP > 37) than B-type starches; amylopectin from C-type starch showed intermediate proportions of long chains (DP > 37) compared to A and B-type amylopectins in line with previous observations (Hanashiro, Abe, & Hizukuri, 1996; Hizukuri, 1985; Jane et al., 1999). In particular, waxy cassava amylopectin exhibited higher proportions of short chains (DP 6e9 and 6e12) and lower proportions of DP 13e24 compared

Table 5 Thermal properties and crystallinity of native starches. Sample name

Thermal propertiesa

WXCS-1 WTCS-2 AMYCS-3 AMYCS-4 WTCS-5 WTCS-6 WTCS-7 WTCS-8 WTNPS-9 WXTPS-10 WXMS-11 WTMS-12

Crystallinityb 1

To ( C)

Tp ( C)

Tc ( C)

DH (J g )

DT ( C)

Crystallinity (%)

A-type (%)

B-type (%)

Water content (%)

61.2 58.9 50.3 53.7 55.1 58.0 57.2 56.1 60.0 63.3 63.8 60.0

68.6 64.1 56.2 57.4 60.1 62.5 62.4 61.6 65.3 69.5 70.6 67.6

75.7 72.7 68.5 66.6 69.8 72.0 71.3 69.9 71.7 74.8 79.0 76.1

16.9 13.6 10.0 8.9 14.3 15.8 16.9 15.0 18.6 18.8 15.8 11.5

14.5 13.9 18.2 13.0 14.7 14.0 14.1 13.8 11.7 11.5 15.2 16.1

40 33 25 25 33 35 35 35 43 43 43 30

85 90 90 90 85 85 85 85 0 0 100 100

15 10 10 10 15 15 15 15 100 100 0 0

19 18 19 19 18 18 18 19 22 23 17 17



(0.4) (0.2) (1.1) (1.1) (0.6) (0.2) (0.5) (0.3) (1.1) (2.4) (N.A.) (0.1)



(0.7) (0.3) (0.4) (0.8) (0.2) (0.4) (0.3) (0.1) (1.1) (1.6) (N.A.) (0.1)



(1.9) (2.0) (3.1) (2.2) (2.7) (0.6) (0.0) (0.0) (2.1) (1.1) (N.A.) (0.2)

(1.4) (0.2) (1.6) (1.0) (1.6) (1.9) (2.1) (0.8) (0.7) (1.7) (N.A.) (0.1)



To: onset gelatinization temperature; Tp: peak gelatinization temperature; Tc: conclusion temperature. N.A.: not available. a Values obtained from DSC; standard deviations are given within parenthesis. b Values obtained from X-ray diffraction; standard deviations were 5% for A-type or B-type content and 3% for crystallinity.

A. Rolland-Sabaté et al. / Food Hydrocolloids 27 (2012) 161e174

169

Fig. 4. Scanning electron microphotographs (3000) of native (left) and lintnerized (2 days) (right) starches from WXCS-1 (waxy cassava) (a) and AMYCS-3 (5G160-13 cassava) (b).

to potato and maize. The slightly lower crystallinity and the higher solubilization extent by mild acid hydrolysis of waxy cassava starch compared to potato and maize could be explained by the level of short chains (DP 6e9) unable to form double helices (Pérez & Bertoft, 2010), and thereby responsible of structural defects in the crystalline lamellae and less organized granular structure. After 2 days hydrolysis, 15% (WTNPS-9) to 43% (AMYCS-3) of the starches were solubilized (Table 6) and the residual solid of waxy starches exhibited higher hydrolysis extents than starches containing amylose in line with previous results (Bertoft, 2004; Biliaderis, Grant, & Vose, 1981; Jane, Wong, & McPherson, 1997). On the opposite, AMYCS-3 (43% solubilization) was highly susceptible to mild acid hydrolysis compared to the other cassava samples studied (20e26% solubilization, Table 6). Considering 10 days hydrolysis, WXCS-1, WTCS-6 and WTCS-7 showed the highest solubilization after mild acid hydrolysis (78, 79 and 82%, respectively) whereas AMYCS-3, AMYCS-4 and WTMS12 showed lower solubilization extents (74%, 67% and 69%, respectively). No clear relation was found between the amylose content and 10 days mild acid hydrolysis extent in agreement with previous results obtained in maize (Gérard et al., 2002). As expected, lintnerization process induced an increase in crystallinity for all the samples even after only 2 days of hydrolysis (Table 6). After mild acid hydrolysis (2 days or 10 days), the crystalline patterns remained unchanged for starches residues except for WXCS-1, WTMS-12, AMYCS-3 and AMYCS-4. Lintnerized WXCS1 displayed a 100% A-type crystalline pattern and a transition from A-type to B-type was observed for AMYCS-3 and AMYCS-4 and to a lesser extent for WTMS-12 (Table 6). Considering first the polymorphic transition from A-type to B-type, Gérard et al. (2002) suggested for maize that B-type crystallites could be intrinsically more resistant to acid than A ones. AMYCS-3 and AMYCS-4, which had a low crystallinity level in native starches, exhibited probably

an important proportion of defective crystals in agreement with crystallinity determinations and with their high proportion of very short chains (Table 3). In the case of WTMS-12, this transition could be due preferentially to a reordering phenomenon of the amylose chains in B-type crystallites, as observed for wheat starch (Hizukuri, 1961). The allomorphic transition from B-type to A-type observed for WXCS-1 had already been described by McPherson and Jane (1999) for yam and sweet potato starches after 12 days hydrolysis. They suggested that the B polymorph was preferentially hydrolyzed in these starches. This would suggest that the transition from A-type to B-type in AMYCS-3 and AMYCS-4 would be the result of a reordering phenomenon of amylose chains in B-type crystals. Normal maize starch, WTMS-12, which probably have its external surface hydrolyzed first as suggested by Bertoft (2004), showed an A to B-type polymorphic transition as well (Table 6). The HPAEC-PAD profiles of cassava, maize and potato lintnerized 10 days (Fig. 5) displayed generally two maxima: a first major peak corresponding to DP 13e15 and a second minor peak corresponding to DP 23e32, except for WTCS-2, AMYCS-3 and WTMS-12 which displayed one (very small) and two additional maxima, respectively (Fig. 5a, c and d). These two groups of chains had already been described for acid hydrolyzed starches (Bertoft, 2004; Gérard et al., 2002; Jane et al., 1997), the peak at DP 12e16 was attributed to the linear fraction and the peak at DP > 20 to the single-branched fraction, identified initially by Robin et al. (1974). The second maximum corresponded to DP 23e26 for cassava and maize lintners (mainly A-type samples) and to DP 30e32 for potato lintners (B-type samples). Considering waxy starches, WXCS-1 and WXMS-11 lintners distributions were similar whereas WXTPS-10 lintner displayed larger short chains (linear) as well as larger long chains (DP > 20) (Table 6, Fig. 5b, e and g). In addition, when for WXCS-1 and WXMS-11 lintners (A-type) two peaks clearly defined were observed, for WXTPS-10 lintner (B-type) the branched

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Table 6 Lintnerized starches hydrolysis rates, crystallinity characteristics and chain distribution. Sample name

Days of hydrolysis

Hydrolysis (%)

WXCS-1 WXCS-1 WTCS-2 WTCS-2 AMYCS-3 AMYCS-3 AMYCS-4 WTCS-5 WTCS-6 WTCS-7 WTCS-8 WTNPS-9 WTNPS-9 WXTPS-10 WXTPS-10 WXMS-11 WTMS-12

2 10 2 10 2 10 10 10 10 10 10 2 10 2 10 10 10

26 78 20 77 43 74 67 75 79 82 73 15 76 23 76 73 69

Crystallinitya

Chain distributionb

Crystallinity (%)

A-type (%)

B-type (%)

Major group DP

Minor group DP

Highest detectable DP

Average DP

Dextrins of DP > 20

þþþ þþþþ þþ þþ þþ þþ þþ þ þ þþ þþ þþ þþ þþþ þþþ þþþþ þþþ

100 100 90 90 5 10 10 90 90 90 85 0 0 0 0 100 85

0 0 10 10 95 90 90 10 10 10 15 100 100 100 100 0 15

ND 14 ND 14 ND 14 ND ND ND ND ND ND 14 ND 15 13 13

ND 25e26 ND 26/39 ND 24/33/46 ND ND ND ND ND ND 30e31 ND 31e32 25e26 24/30/38

ND 43e44 ND 56 ND 75 ND ND ND ND ND ND 43 ND 42 40 57

ND 19 ND 21 ND 26 ND ND ND ND ND ND 18 ND 18 18 20

ND 40 ND 47 ND 63 ND ND ND ND ND ND 28 ND 24 33 38

ND: not determined. a Values obtained from X-ray diffraction, (þ), (þþ), (þþþ) and (þþþþ) correspond with small, medium, high and very high increase in crystallinity compared to the native starches respectively; standard deviations were 5% for A-type or B-type content. b Values obtained from HPAEC-PAD, standard deviations were 3%.

fraction possessed no peak, but had the form of a shoulder starting from DP25. These differences in chain distributions between A and B-type lintners have already been reported (Bertoft, 2004; Gérard et al., 2002; Jane et al., 1997; McPherson & Jane, 1999). The smallest quantities of DP > 20 were observed for potato starches lintners (Table 6). HPAEC-PAD profiles of normal and waxy lintners were very similar for potato but exhibited different patterns in the case of cassava and maize (Fig. 5). HPAEC-PAD profiles of WTNPS-9 and WXTPS-10 lintners were matchable (Fig. 5f and g) despite slightly longer chains in WTNPS-9 lintner than in WXTPS-10 lintner (Table 6). The crystallites were pure B-type and both potato lintners displayed the same crystallinity. Among the lintners obtained from normal and high-amylose starches, WTNPS-9 lintner was the only one which did not exhibit chains with a DP higher than 43 (Table 6, Fig. 5). Consequently, in the case of potato, amylose is not involved in crystallites as was already suggested by Bertoft (2004) and in opposition to the putative amylose involvement in B crystallites reported by Saibene and Seetharaman (2010). WTMS-12 lintner showed three minor groups at DP 24, DP 30 and DP 38 and a maximum DP detected of 57 (Table 6, Fig. 5d). Similar data had been reported for normal maize by Bogracheva et al. (1999). WTCS-2 lintner showed two minor groups at DP 26 and DP 39 and a maximum DP detected of 56 (Table 6, Fig. 5a). These long chains are probably linked to the presence of amylose in these lintner residues. Moreover, these two lintners were less crystalline than their waxy homolog counterparts, and exhibited 10e15% of B-type crystallinity. In the case of WTMS-12, an enhancement of the proportion of B-type crystallites was observed during lintnerization. These acid resistant long chains could then correspond to B-type crystallites formed by a reordering of initial amylose segments, as already mentioned above. In the case of WTCS-2, the proportion of B-type crystallites is still the same before and after lintnerization. Amylose may be involved in crystallites, as already proposed by Gérard et al. (2002), although amylopectin alone is assumed to be responsible for starch crystallinity. The involvement of amylose chains in normal cassava B-type crystallites seemed to contribute to more resistant structures against acid hydrolysis, comparing to WXCS-1 B-type crystallites. AMYCS-3 lintner displayed the longest chains as its HPAEC-PAD profile showed three minor groups at DP24, DP33

and DP46 and a maximum DP detected of 75 (Table 6, Fig. 5c). Even if the AMYCS-3 lintner exhibited mainly B-type crystallinity (90%), it did not show a HPAEC-PAD pattern similar to potato lintners, suggesting that the AMYCS-3 ultrastructure was different from typical B-type starches. AMYCS-3 lintner exhibited more chains of DP > 30 and less chains of DP 9e17 and DP 23e30 than waxy and normal cassava lintners, i.e. fewer single-chains (DP around 14) and short double helices. The long chains were not attacked by the acid suggesting that they were not in the amorphous phase, but probably involved in “long” double helices. Alternatively, or concomitantly, the abundance of these acid resistant long chains could be the result of a reordering phenomenon of residual linear chains in B-type crystallites. In the native starch, these long chains could then either belong to amylose molecules involved in crystals; this involvement existing preferentially in high-amylose starch, by analogy to what Gérard et al. (2002) suggested for amylose rich maize mutants. Alternatively these long chains could belong to extra long chains of an intermediate material. 4. General discussion 4.1. PCA analysis The principal component analysis of the data obtained by CLI, A4FeMALLS, X-ray diffraction, DSC and granulometry gave an overview of the starches samples. The two significant principal components (PCs) generated by PCA explained 55 and 27% of the variation, respectively. The loading plot (Fig. 6) showed that amylose content (amylose %), lmax values and dGappw were closed to each other. It is logical to observe a strong correlation between amylose content and lmax values (Sánchez, Dufour, Moreno, & Ceballos, 2010), but the amylose content and dGappw represent independent features, linked here in PC1. It is interesting to notice that both are representative of molecular features, emphasizing the importance of this level of organization on the global diversity of starches. A positive correlation was also expected between crystallinity and DSC data (To, Tp and DH), in line with previous results (Zobel et al., 1988). This is in line with the cooperative nature of gelatinization phenomena, with the swapping of the crystalline/ double helical for a coiled conformation.

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d

Relative peak area (%)

9

WTCS-2

8 7 6 5 4 3 2

10 9

Relative peak area (%)

a 10

7 6 5 4 3 2

0

0

5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75

5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75

DP

e

7 6 5 4 3 2

DP 10 9

WXCS-1

8

Relative peak area (%)

Relative peak area (%)

9

7 6 5 4 3 2

0

0

5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75

5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75

DP

DP

AMYCS-3

9 8

f

7 6 5 4 3 2 1

10 9

Relative peak area (%)

Relative peak area (%)

WXMS-11

8

1

1

c 10

WTMS-12

8

1

1

b 10

171

WTPS-9

8 7 6 5 4 3 2 1

0 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75

0 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75

DP

DP

g

10

Relative peak area (%)

9

WXTPS-10

8 7 6 5 4 3 2 1 0

5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75

DP

Fig. 5. Chain length distribution of 10 days lintners obtained using HPAEC-PAD. WTCS-2 (parental waxy cassava) (a), WXCS-1 (waxy cassava) (b), AMYCS-3 (5G160-13 cassava) (c), WTMS-12 (normal (wild type) maize) (d), WXMS-11 (waxy maize) (e), WTNPS-9 (normal (wild type) potato) (f) and WXTPS-10 (waxy potato transgenic) (g). DP: degree of polymerization.

According to the loading plot, it was possible to point out that the PC1 emphasized essentially the variation due to dGappw and the amylose content on one hand (positive values) and, on the other hand the crystallinity variation (negative values). The PC2 emphasized mainly the M w variations, and then the abundance of B-type crystallites, the granule size and the DP/B values. The loading plot confirmed that gM and M w were negatively correlated. The score plot (Fig. 7) showed a distribution of starches in four groups: potato starches, cassava mutants starches with small granules, waxy and

normal starches (different from potato). Potato starches showed high negative scores in PC1 and PC2 owing to their high crystallinity, low M w, high proportion of B-type crystallites and large granule diameter. Small granule mutants showed high positive score in PC1 due to their high-amylose content, their high density and their low crystallinity and thermal properties. Their medium position according to PC2 could be the result of a low Mw weighted by a small granule size. Anyway, this behavior differs from maize (Utrilla-Coello, Agama-Acevedo, de la Rosa, Rodriguez-Ambriz, &

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different. Waxy starches and normal ones were discriminated by PC1, so by their amylose content and their crystallinity. Potato starches were discriminated by PC2, i.e. mainly by their granule size and the abundance of their B-type crystallites. Finally, the score plot showed that the small granule mutants starches were well different from the three other starches groups, according to PC1 and PC2. 4.2. Deposition inside the starch granule

Fig. 6. Loading plot of PC1 and PC2. Amylose content, amylose %; weight average molar mass, Mw; z-average radius of gyration, RG; the slope of the logelog plot of the relation linking molar mass and radius of gyration, nG; average shrinking factor, gM; average number of glucosyl units in a linear chain per branching point, DP/B; apparent particle density, dGappw; crystallinity, cryst %; proportion of B-type crystallites, B-type %; average granule diameter, B; To, Tp and DH obtained by DSC.

Bello-Perez, 2010) and wheat (Salman et al., 2009) starch’s granules, where A and B granules are less different. Waxy cassava and waxy maize starches presented a high negative score in PC1, due to their high crystallinity and their low amylose content and a high positive score in PC2 because of their high Mw ; they were strongly discriminated from waxy potato starch by PC2. Within the fourth group WTCS-6 and WTCS-7 were slightly shifted due to their high Mw . According to this PCA analysis, normal maize and normal cassava properties were not that

Fig. 7. Score plot of PC1 and PC2. Waxy cassava, WXCS-1; parental waxy cassava, WTCS-2; 5G160-13 cassava, AMYCS-3; 5G160-16 cassava, AMYCS-4; parental (wild type) 5G160 cassava, WTCS-5; normal (wild type) cassava MCOL1505, WTCS-6; normal (wild type) cassava MTAI8, WTCS-7; normal (wild type) cassava HMC-1, WTCS-8; normal (wild type) potato, WTNPS-9; waxy potato transgenic, WXTPS-10; waxy maize, WXMS-11; normal (wild type) maize, WTMS-12.

The actual paradigm of starch organization is that amylopectin is the major structuring factor, amylose acting more as filler within the internal starch granule (Zeeman, Kossmann, & Smith, 2010). Secondly, the primary structure of amylopectin and the amylose level are directly related to the expression of the different enzymes involved in the biosynthesis inside the amyloplast (Zeeman et al., 2010). For waxy varieties the branching degrees and densities of amylopectin depend on the botanical origin: maize and cassava have similar branching degrees and densities whereas potato exhibits a less dense structure with longer chains. After their synthesis, clustered short chains of amylopectin crystallize. The crystallinities of the corresponding starches are similar but waxy potato has shown to have longer chains involved in crystallites. Normal starches amylopectins exhibit generally lower M w and RG than corresponding waxy types, potato being the exception. The branching degrees and densities depend on the botanical origin, as well as debranched chain length distributions. The amylose content seems therefore to have no influence on the amylopectin branching structure which remains stable for each botanical origin. The crystalline allomorphs proportions are the same as waxy varieties, however crystallinity is lower for the normal starches, except for potato. The chain length distributions of lintnerized granules exhibit longer chains for normal cassava and maize than for their waxy varieties, meaning that a portion of amylose chains have not been hydrolyzed by the acid. Then, in cassava and maize starches, amylose could be either involved in crystallites or in an organized form within amorphous regions of the granules. Increasing the amylose content using the 5G160 mutation has been shown to induce important modifications in cassava amylopectin, decreasing the M w and RG , increasing the branching degree and changing the distribution of linear chain length, then modifying the branching pattern. These modifications were influencing starch granule ultrastructure such as the decrease of starch crystallinity. This primary structure of starch is expected to play a central role in the pattern of higher order structures of starch, and therefore in starch properties. In preliminary experiments (Ceballos et al., 2008), 5G160 mutant cassava have consistent differences in the pattern of isoamylase (ISA) band in electrophoresis. The structure of the 5G160 mutant amylopectin with more DP 6e12 chains and less DP 13e24 chains comparatively to the normal is in agreement with observations reported on ISA mutants from Arabidopsis by Wattebled et al. (2005). However the high-amylose content is not explained by ISA mutation and would rather be due to a mutation affecting SS. The phenotype observed for AMYCS-3 and AMYCS-4 could then be the result of multiple mutations that occurred in different sites due to gamma ray exposition and leading to the alteration of different synthesis enzymes activities. The primary structure of amylopectin (M w and branching pattern) and the amylose content are directly linked to the extent of crystallinity and the allomorph type. The starch granule is also changed with these enzymatic modifications. Using a simplified model, it is possible to evaluate the number of amylopectin molecules in a spherical starch granule of a density of 1.5 using the

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relation: namp ¼ 4=3pðB=2Þ3  ð1:5=M w Þ. For waxy maize, waxy cassava and waxy potato starches, 3.25  106, 5.59  106, and 1.55  108 were obtained respectively. A potato starch granule would be therefore constituted by about fifty times more amylopectin molecules than a maize starch granule. As the M w values of amylopectin is stable for all the species, the granule size, which depends on botanical origin, cannot be related to the amylopectin size and structure. It would be rather governed by the stop of the amylopectin synthesis (Zeeman et al., 2010). Acknowledgment The authors thank S. Guilois, B. Pontoire, E. Perrin and M. De Carvalho (Research Unit on Biopolymers, Interactions and Assemblies, INRA, Nantes, France) for excellent technical support. The authors thank N. Stephant (Service commun de Microscopie à Balayage et Microanalyse (SMEBM), University of Nantes, France) for the SEM experiments. The authors acknowledge the Conseil Régional des Pays de la Loire for its financial support. The authors are grateful to Higher Education and Training Service (DESI-CIRAD) for the financial support in training Teresa Sánchez, from CIAT, Cali, Colombia in CIRAD (Montpellier, France) and INRA (Nantes, France) laboratories. References Ball, S., Guan, H. P., James, M., Myers, A., Keeling, P., Mouille, G., et al. (1996). From glycogen to amylopectin: a model for the biogenesis of the plant starch granule. Cell, 86(3), 349e352. Bertoft, E. (2004). Lintnerization of two amylose-free starches of A- and B-crystalline types, respectively. Starch/Stärke, 56(5), 167e180. Biliaderis, C. G., Grant, D. R., & Vose, J. R. (1981). Structural characterization of legume starches. 2. Studies on acid-treated starches. Cereal Chemistry, 58(6), 502e507. Bogracheva, T. Y., Cairns, P., Noel, T. R., Hulleman, S., Wang, T. L., Morris, V. J., et al. (1999). The effect of mutant genes at the r, rb, rug3, rug4, rug5 and lam loci on the granular structure and physico-chemical properties of pea seed starch. Carbohydrate Polymers, 39(4), 303e314. Burchard, W. (1983). Static and dynamic light scattering from branched polymers and biopolymers. Advances in Polymer Science, 48, 1e124. Buléon, A., Colonna, P., Planchot, V., & Ball, S. (1998). Starch granules: structure and biosynthesis. International Journal of Biological Macromolecules, 23(2), 85e112. Ceballos, H., Fregene, M., Lentini, Z., Sánchez, T., Puentes, Y. I., Pérez, J. C., et al. (2006). Development and identification of high-value cassava clones. Acta Horticulturae, 703, 63e70. Ceballos, H., Sánchez, T., Morante, N., Fregene, M., Dufour, D., Smith, A. M., et al. (2007). Discovery of an amylose-free starch mutant in cassava (Manihot esculenta Crantz). Journal of Agricultural and Food Chemistry, 55(18), 7469e7476. Ceballos, H., Sánchez, T., Tofiño, A. P., Rosero, E. A., Denyer, K., Smith, A. M., et al. (2008). Induction and identification of a small-granule, high-amylose mutant in cassava (Manihot esculenta Crantz). Journal of Agricultural and Food Chemistry, 56(16), 7215e7222. Charles, A. L., Chang, Y. H., Ko, W. C., Sriroth, K., & Huang, T. C. (2004). Some physical and chemical properties of starch isolates of cassava genotypes. Starch/Stärke, 56(9), 413e418. Colonna, P., & Buléon, A. (2010). Thermal transitions of starches. In A. C. Bertolini (Ed.), Starches, characterization, properties and applications (pp. 59e102). Boca Raton, London, New York: CRC Press. Davis, J. P., Supatcharee, N., Khandelwal, R. L., & Chibbar, R. N. (2003). Synthesis of novel starches in planta: opportunities and challenges. Starch/Stärke, 55(3e4), 107e120. French, D. (1984). Organization of starch granules. In R. L. Whistler, J. N. BeMiller, & E. F. Paschall (Eds.), Starch: Chemistry and technology (2nd ed.). (pp. 183e247) Orlando: Academic Press. Gérard, C., Barron, C., Colonna, P., & Planchot, V. (2001). Amylose determination in genetically modified starches. Carbohydrate Polymers, 44(1), 19e27. Gérard, C., Colonna, P., Buléon, A., & Planchot, V. (2002). Order in maize mutant starches revealed by mild acid hydrolysis. Carbohydrate Polymers, 48(2), 131e141. Gidley, M. J., Hanashiro, I., Hani, N. M., Hill, S. E., Huber, A., Jane, J.-L., et al. (2010). Reliable measurements of the size distributions of starch molecules in solution: current dilemmas and recommendations. Carbohydrate Polymers, 79(2), 255e261. Gomand, S. V., Lamberts, L., Derde, L. J., Goesaert, H., Vandeputte, G. E., Goderis, B., et al. (2010). Structural properties and gelatinization

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List of symbols B: average number of branching points in the ABC three-functional polycondensation model. BmH : average number of branching points obtained using theoretical amylopectin structures as proposed by Hizukuri (1986). BD: branching degree. BDmH: branching degree obtained using theoretical amylopectin structures as proposed by Hizukuri (1986). CL: average chain length. dGappw: apparent particle density calculated on the basis of a smeared uniform 3 density in the particle using the following equation: dGappw ¼ Mw =ð4p=3ÞRGw . B: average granule diameter. DP: degree of polymerization

DP w : weight average degree of polymerization. DP w =B: average number of glucosyl units in a linear chain per branching point. DP w =BmH : average number of glucosyl units in a linear chain per branching point obtained using theoretical amylopectin structures as proposed by Hizukuri (1986). DH: gelatinization enthalpy. DSC: differential scanning calorimetry. gM: average shrinking factor or average branching parameter. IBC: iodine binding capacity. KG: constant. lmax: wavelength at maximum absorption of the iodine complexes with starch polymers. M: molar mass of a fraction. Mn : number average molar mass. Mw : weight average molar mass. Mw =M n : dispersity index. nG: hydrodynamic coefficient. RG: radius of gyration of a fraction. RGw : weight average radius of gyration. RGwðbrÞ : weight average radius of gyration of the branched polymer. RGwðlinÞ : weight average radius of gyration for the corresponding linear polymer. RGz : z-average radius of gyration. RH: hydrodynamic radius. Tc: conclusion temperature. To: onset temperature gelatinization. Tp: peak temperature gelatinization.