Food Chemistry 143 (2014) 156–162

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Food Chemistry

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Structure and digestibility of endosperm water-soluble a-glucans fromdifferent sugary maize mutants

0308-8146/$ - see front matter Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.07.109

⇑ Corresponding author. Tel.: +86 (0) 510 853 27859; fax: +86 (0) 510 859 19161.E-mail address: miaoming@jiangnan.edu.cn (M. Miao).

Ming Miao a,⇑, Rong Li a, Bo Jiang a, Steve W. Cui a,b, Keyu Lu a, Tao Zhang a

a State Key Laboratory of Food Science & Technology, Ministry of Education Key Laboratory of Carbohydrate Chemistry & Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi,Jiangsu 214122, PR Chinab Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ont. N1G 5C9, Canada

a r t i c l e i n f o

Article history:Received 2 May 2013Received in revised form 13 July 2013Accepted 20 July 2013Available online 29 July 2013

Keywords:Water-soluble polysaccharideSugary maizeNanoparticleFine structureDigestibility

a b s t r a c t

The structure and digestibility of endosperm water-soluble a-glucans from different sugary-1 maizemutants (Zhongtian 8#, Zhongtian 2# and Pintian 8#) were investigated. The yield of pure glucan wasin the range of 25.91–34.38%. The a-glucan belonged to a typical native nano-scale particle and the aver-age particle size was in the following order: Zhongtian 8# >Pintian 8# >Zhongtian 2#. The weight-aver-age molar mass of glucans ranged from 1.69 to 2.08 � 107 g/mol. The branch densities and a-1,6 linkagesof Zhongtian 8#, Zhongtian 2# and Pintian 8#, were 8.60%, 8.77% and 9.51%, 7.71%, 6.58% and 6.81%,respectively. The resistant starch (10.06%) of Pintian 8# was lower than other two cultivars. The studyshowed that water-soluble glucan exhibited a-1,4-linked backbone with a-1,6 branch sites and digest-ibility was influenced by granule size, ratio of a-1,4 to a-1,6 linkages, molecular fine structure in thisset of sugary maize mutants.

Crown Copyright � 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction sists of tip cap, pericarp, germ and endosperm as prominent botan-

Naturally occurring polysaccharides are by far the most abun-dant renewable resource on the earth. Plant polysaccharides in-clude cellulose, starch, pectin, and hemicelluloses, which aresynthesised by the sun’s energy. They played a critical role in thedevelopment of new products ranging from foods, nutraceuticals,pharmaceuticals, textiles, paper, and biodegradable packagingmaterials (Cui, 2005). Generally, most food polysaccharides areclassified as water-soluble polysaccharides and the functionalproperties are water binding capacity, enhancing viscosity and pro-viding desired textures for many foods (Charalampopoulos, Wang,Pandiella, & Webb, 2002; Cui, 2005). Recently, water-soluble poly-saccharides have received considerable attention as an importantclass of bioactive substances due to their potential biological activ-ities, especially immunological, antitumor and prebiotic effects(Kosaraju, 2005; Wu, Cui, Tang, Wang, & Gu, 2007; Zhang, Cui, Che-ung, & Wang, 2007). Discovery and evaluation of new bioactivepolysaccharides for functional foods has emerged as one of thehot research fields in chemistry and biology.

Maize (Zea mays L.) is one of the most important food crops ofthe world, exceeded only by rice and wheat in terms of quantityproduced. Besides being consumed as such, maize kernel is usedfor various industrial applications, such as production of feed, fueland sweetener (Creech, 1965; Hallauer, 2001). Maize kernel con-

ical tissues. Mature endosperm, which is the most important majorconstituents, is rich sources of starch, protein and fat. It is valuableto exploit them. Sugary maize, a variety of maize mutants withhigh sugar content, is a kind of favourable and profitable crop,and is much admired for its high nutritional value. In addition toits use in production of flour, bread and pasta, it has been com-monly used as vegetable (Hallauer, 2001). Recent studies haveshown that sugary mutation leads to a deficiency of starch debran-ching enzyme in amyloplasts, which caused the biosynthesis ofmore highly branched a-glucan rather than amylopectin (Fujitaet al., 2012; Huang & Yao, 2011; Wong et al., 2003). However, thereis relatively little information pertaining to water-soluble polysac-charide from endosperm of sugary maize, especially detailed stud-ies of the structural characterisation. Since structure and functionsare intimately related, an in-depth study of the structure of thepolysaccharides would be of interest. Therefore the aim of this re-search was to elucidate its structural characterisation and in vitrodigestion by a combination of chemical and instrumental analysis,which could be useful for utilisation of water-soluble polysaccha-ride in specialty foods.

2. Materials and methods

2.1. Materials

Three different mutants of mature allelic sugary-1 maize inbredlines (Zhongtian 8#, Zhongtian 2# and Pintian 8#) were purchased

M. Miao et al. / Food Chemistry 143 (2014) 156–162 157

from Chinese Academy of Agricultural Sciences (Beijing, China).Waxy maize starch was a generous gift from Changchun DachengIndustrial Group Co., Ltd., (Changchun, Jilin, China). Pancreatinfrom porcine pancreas (Cat. No. P-1625, activity 3 � USP/g) andpanose (P2407) were purchased from Sigma–Aldrich ChemicalCo., (St. Louis, MO, USA). Amyloglucosidase (EC 3.2.1.3., 3300 U/ml) and glucose oxidase–peroxidase assay kits (Cat. No. K-GLUC)were from Megazyme International Ireland Ltd., (Wicklow, Ire-land). All chemicals were reagent grade and were obtained fromSinopharm Chemical Reagent Co., Ltd., (Shanghai, China).

2.2. Isolation of water-soluble polysaccharides

The isolation of water-soluble a-glucan was performed accord-ing to the method of Wong et al. (2003) with a slight modification.Mature sugary-1 maize kernels were dehulled, endosperm was sep-arated from germ and soaked in five times their weight of deionizedwater at 20 �C overnight. The softened grains were ground in a lab-oratory blender. The mixture was filtered through 270-mesh sievesand then centrifuged at 10,000g for 10 min. The supernatant wascollected whereas the sediment was further extracted twice withdeionised water. The resulting decant was heating in a boiling waterbath for 30 min to denature protein. After centrifugation, the vol-ume of liquid was measured and 3 volumes of ethanol were addedto precipitate the polysaccharide. The precipitate was then col-lected and placed in a fume hood to remove residual ethanol. Thedried solids were ground to form a powder, which was the materialused for characterisation studies within the next few months.

2.3. Proximate analysis

The following standard Association of Official Analytical Chem-ists (AOAC) (2000) procedures were used for proximate analysis:moisture (convection oven, 105 �C, AOAC 930.15), crude protein(leco, total combustion method, AOAC 968.06), and ash (furnace,AOAC 942.05). The total carbohydrate content was determinedby phenol–sulphuric acid colorimetric method (Dubois, Gilles,Hamilton, Rebers, & Smith, 1956). Yields of soluble polysaccharidewere expressed as percentage of the total carbohydrate content ofendosperm and expressed on a dry matter basis.

The monosaccharide composition was determined using high-performance anion-exchange chromatography coupled withpulsed amperometric detection (HPAEC-PAD) as suggested by Cui(2005). Polysaccharide (4 mg) was hydrolysed with 2 M H2SO4

(2 ml) at 105 �C for 8 h to release monosaccharide completely.After neutralised with BaCO3 and centrifuged at 10,000 rpm for10 min, supernatants were analysed by ICS-5000 HPAEC-PAD (Dio-nex, Sunnyvale, CA) equipped with an electrochemical detectorusing a gold working electrode and Ag/AgCl as reference electrode.A CarboPac PA20 column (3 � 150 mm, 6 lm, Dionex, Sunnyvale,CA) connected to a CarboPac PA-1 guard column (4 � 50 mm, Dio-nex, Sunnyvale, CA) was used. Gradient elution system was usedwith a mixture eluent: 250 mM NaOH solution (A), 1 M sodiumacetate solution (B) and ultrapure water. The initial mobile phasewas only 1.8% elute solution A for 21 min. A gradient from 5% to20% elute solution B was performed in next 9 min and 1.8% elutesolution A was maintained in the period. Finally, single 80% elutesolution A was used as mobile phase from 30.1 to 50 min. It wascarried out with a flow rate of 0.5 ml/min at 30 �C. A set of mono-saccharides were used as standard samples, including glucose,fructose, galactose, sorbose, mannose and rhamnose.

2.4. Transmission electron microscopy (TEM)

The images of polysaccharide specimens were captured using atransmission electron microscopy (JEM-2100 LaB6, JEOL Ltd.,

Tokyo, Japan) operated at 200 kV. Droplets of around 0.01% (w/v)glucan suspension in water were deposited on glow-dischargedcarbon-coated 400 mesh grids. The liquid in excess was blottedaway with filter paper prior to drying. All the samples wereobserved at a magnification of 60,000�, using a high-resolutiondigital CCD camera (US 1000, Gatan, Inc., Pleasanton, USA),150 lm condenser aperture, and 60 lm objective aperture.

2.5. Particle size analysis

Particle size analysis of a-glucan from different sugary maizemutants was done using a laser light scattering particle size ana-lyser (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK).The focal length was 100 nm.

2.6. Molecular weight distribution profiles

Glucan samples (10 mg) were added to 5 ml of deionized waterand boiled with stirring for 15 min to completely dissolve the sam-ples. The dissolved samples, filtered through 5 lm cellulose acetatefilters (Whatman, Maidstone, UK), were injected into a high perfor-mance size exclusion column chromatography system with multi-angle laser light scattering detector and a refractive index detector(HPSEC-MALLS-RI) (Wyatt Technology, Santa Barbara, CA). Twoseries tandem columns (300 � 8 mm, Shodex OH-pak SB-806 and804, Showa Denko K.K., Tokyo, Japan) with a OH-pak SB-G guardcolumn, a DAWN HELEOS laser photometer fitted with He-Ne laser(k = 632.8 nm) with a K-5 flow cell, and a OPTILAB� T-rEX Interfer-ometric refractometer were used. The flow rate was set at 0.5 ml/min with a mobile phase of distilled-deionized water (pH 6.8,18.2 MX cm) containing 0.02% NaN3. A dn/dc value of 0.138 wasused in molecular weight calculation, and data processing was per-formed using ASTRA software (Version 5.3.4.14, Wyatt Technol-ogy). Weight-average molar mass (Mw, g/mol) and z-root meansquare radius of gyration (Rz, nm) were obtained using the sec-ond-order Berry method. The molecular density (q, g/mol nm3)was calculated as Mw=R3

z .

2.7. Branch chain length distribution profiles

The chain length distribution of a-glucan was determined byhigh pressure anion exchange chromatography with pulsedamperometric detection (HPAEC-PAD). The samples (10 mg) weredissolved with 2 ml of NaNO3 solution (pH 4.0, 0.1 M) and heatedin a boiling water bath for 10 min. Isoamylase (0.5 U) was addedto each dispersion, and the mixtures were incubated at 40 �C withshaking for 24 h. Then, the solution was heated in a boiling waterbath for 10 min to deactivate the enzyme. The debranched samplesolutions were filtered through a 0.45-lm membrane filter andthen injected into the HPAEC-PAD system (50 ll sample loop).The HPAEC-PAD system consisted of a Dionex DX 600 equippedwith an ED 50 electrochemical detector with a gold working elec-trode, GP 50 gradient pump, LC 30 chromatography oven, and AS40 automated sampler (Dionex Corporation, Sunnyvale, CA, USA).The standard triple potential waveform was employed, with thefollowing periods and pulse potentials: T1 = 0.40 s, with 0.20 ssampling time, E1 = 0.05 V; T2 = 0.20 s, E2 = 0.75 V; T3 = 0.40 s,E3 = �0.15 V. Data were collected using Chromeleon software, ver-sion 6.50 (Dionex Corporation, Sunnyvale, CA, USA). Eluents wereprepared in distilled deionized water with helium sparging; eluentA was 150 mM NaOH, and eluent B was 50 mM sodium acetate in150 mM NaOH. Linear components were separated on a DionexCarboPac™ PA1 column with gradient elution (40% of eluent B at0 min, 50% at 2 min, 60% at 10 min, and 80% at 40 min) at 30 �Cand a flow rate of 1 ml/min. The number average chain lengthwas determined by the following equation: CL =

P(AN)/

PA. A

Tabl

e1

Chem

ical

com

posi

tion

s,st

ruct

ural

para

met

ers

and

Invi

tro

dige

stio

nof

thre

edi

ffer

ent

endo

sper

mw

ater

-sol

uble

a-g

luco

poly

sacc

hari

des.

Sam

ple

Moi

stu

re(%

)C

rude

prot

ein

(%)

Ash

(%)

Yie

ld(%

)M

w(1

07g/

mol

)R z

(nm

)q

(g/m

ol�n

m3)

aC

L(D

P)B

ran

chde

nsi

ty(%

)ba

-1,6

lin

kage

s(%

)R

DS

(%)

SDS

(%)

RS

(%)

Zhon

gtia

n8#

10.0

1±

0.15

1.37

±0.

141.

51±

0.28

34.3

8±

0.12

2.08

±0.

0236

.0±

0.1

445.

8±

1.7

11.6

3±

1.15

8.60

±0.

197.

71±

0.13

69.1

3±

2.17

16.8

2±

1.35

14.0

5±

1.82

Zhon

gtia

n2#

9.73

±0.

241.

53±

0.30

1.10

±0.

1725

.91

±0.

151.

69±

0.01

30.7

±0.

258

4.1

±3.

611

.40

±0.

938.

77±

0.17

6.58

±0.

2067

.87

±1.

3619

.67

±0.

9212

.46

±2.

34

Pin

tian

8#9.

69±

0.18

1.26

±0.

231.

09±

0.22

30.5

3±

0.20

1.81

±0.

0030

.5±

0.2

637.

9±

2.5

10.5

2±

1.26

9.51

±0.

226.

81±

0.23

72.5

5±

3.09

17.3

9±

1.60

10.0

6±

2.15

Mw

,wei

ght-

aver

age

mol

arm

ass;

Rz,

z-ro

otm

ean

squ

are

radi

us

ofgy

rati

on;q

,dis

pers

edm

olec

ula

rde

nsi

ty;

CL,

nu

mbe

r-av

erag

ech

ain

len

gth

;R

AG

,rap

idly

dige

stib

lest

arch

;SD

S,sl

owly

dige

stib

lest

arch

;R

S,re

sist

ant

star

ch.

aM

olec

ula

rde

nsi

ty(q

)w

asca

lcu

late

das

Mw=R

3 z.

bB

ran

chde

nsi

tyw

asca

lcu

late

das

the

inve

rse

ofn

um

ber-

aver

age

chai

nle

ngt

h.

158 M. Miao et al. / Food Chemistry 143 (2014) 156–162

was the peak area at molecular weight of N with units of DP. Thebranch density was determined as the inverse of CL.

2.8. UV/visible spectrophotometer

The structural analysis of the a-glucan was measured usingKrismann’s solution with UV/visible Spectrophotometer (UV-2102PC, Unico Instrument Co., Ltd., Shanghai, China) as suggestedby Curá and Krisman (1990). A Krismann’s reagent was prepared byadding 0.5 ml of iodine/potassium iodide solution (0.13 g I2 and1.3 g KI in 5 ml of deionized water) to 120 ml of a saturated CaCl2

solution. Dried glucan (50 mg) was dissolved in deionized solution(10 ml) in 30 ml screw-cap vials. 0.05 ml of the diluted solutionwas mixed with Krismann’s solution (1.5 ml) and then adjustedto a final volume 2 ml of with water. The absorbance spectra andwavelength of maximum absorption (kmax) were analysed over awavelength scan of 360–600 nm.

2.9. Fourier transform infrared spectroscopy (FT-IR)

The IR spectrum of a-glucan was recorded on a Nicolet Nexus470 FT-IR spectrometer (Thermo Electron Corporation, Waltham,MA, USA) at room temperature. The a-glucan powder was blendedwith KBr powder in the ratio 1:100, and pressed into tablets beforemeasurement. A region from 400 to 4000 cm�1 was used for scan-ning at 4 cm�1 resolution by 32 scans.

2.10. Proton nuclear magnetic resonance spectroscopy (1H NMR)

1H NMR analyses of a-glucan samples were performed using anAVANCE III 400 MHz Digital NMR Spectrometer (Bruker BiospinInternational AG, Switzerland) according to the method of Gidley(1985). Samples were first dissolved in 1 ml of deuterium oxide(D2O), incubated for 2 h at room temperature, and then lyophi-lized. The D2O-treated samples were dissolved in 0.6 ml of D2Oagain, and 1H NMR spectra were obtained at 80 �C. The ratio ofa-1, 4- to a-1, 6-glucosidic linkages was determined from integra-tion of the anomeric resonance at 5.4 and 5.0 ppm, respectively.

2.11. Enzymatic starch hydrolysis

The digestibility was analysed according to the procedure ofEnglyst, Kingman, and Cummings (1992) with a slight modifica-tion. To prepare Enzyme Solution I, amyloglucosidase solution(0.14 ml) was diluted to 6.0 ml with deionized water. EnzymeSolution II was prepared by suspending porcine pancreatic a-amy-lase (12.0 g) in water (80.0 ml) with magnetic stirring for 10 minthe centrifuging the mixture for 10 min at 1500g. Finally, a portion(54.0 ml) of the supernatant was transferred into a beaker. EnzymeSolution III was prepared immediately before use by mixing water(4.0 ml), Enzyme Solution I (6.0 ml), and Enzyme Solution II(54.0 ml).

The glucan sample (200 mg) was dissolved in 15 ml of phos-phate buffer (0.2 M, pH 5.2) by vortexing. After the solution wasequilibrated at 37 �C for 5 min, seven glass balls (10 mm diameter)and Enzyme Solution III (5.0 ml) were added. Then, the sampleswere shaken in a 37 �C water bath at 150 rpm. Aliquots of hydroly-sed solution (0.5 ml) were taken at different time intervals andmixed with 4 ml of absolute ethanol to deactivate the enzymes.The glucose content of the hydrolysates was determined usingglucose oxidase/peroxidase assay kits. The percentage of hydroly-sed glucan was calculated by multiplying a factor of 0.9 with theglucose content. Each sample was analysed in triplicate.

The values of different carbohydrate nutritional fractions(rapidly digestible starch, RDS, slowly digestible starch, SDS, andresistant starch, RS) were obtained by combining the values of

1 10 100 1000

0

6

12

18

24

30

Pintian 8#

Zhongtian 8#

Num

ber (

%)

Size (nm)

Zhongtian 2#

Fig. 2. Particle size analysis of three different endosperm water-soluble a-glucans.

M. Miao et al. / Food Chemistry 143 (2014) 156–162 159

G20 (glucose released after 20 min), G120 (glucose released after120 min), FG (free glucose), and TG (total glucose) using the fol-lowing formulas:

% RDS = (G120 � FG) � 0.9 � 100% SDS = (G120 � G20) � 0.9 � 100% RS = (TG � FG) � 0.9 � 100 � %RDS � %SDS.

2.12. Statistical analysis

All results were analysed by the Duncan test using the statisti-cal analysis system (SAS Institute, Cary, NC). A level of 0.05 was setto determine statistical significance.

3. Results and discussion

3.1. Chemical composition analysis

The results of the chemical analyses performed on three differ-ent endosperm water-soluble a-glucans are summarised in Table 1.The purity of the water-soluble polysaccharides was judged on thebasis of composition (low ash and low endosperm protein content).The yields (on a total seed basis) of pure water-soluble polysaccha-rides (25.91–34.38%, Table 1) was in the range (7.8–34.8%) re-ported for sweet corn cultivars (Creech, 1965; Hallauer, 2001).The differences in the chemical composition and yield of thewater-soluble glucan from sugary maize mutants may be attrib-uted to the variation in genotypic, soil characteristics, kernel ageand isolation method (Cui, 2005; Hallauer, 2001; Miao, Zhang, &Jiang, 2009). The neutral sugar compositions of three differentendosperm water-soluble polysaccharides were homopolymerscontaining only one constituent monosaccharide (namely glucose)through a-1,4 linked and a-1,6 branched glycosidic bonds fromHPAEC and NMR analysis. This was similar with the homogeneouscomposition of starch or glycogen reviewed by Ball and Morell(2003) and Manners (1991). They have reported that a-1,4 linkedand a-1,6 branched glucose polymers define the most widespreadform of storage polysaccharides in living cells. Especially, glycogenis an essentially homogeneous water-soluble polymer with 8–12%branches whilst starch is a complex semi-crystal structure made oftwo distinct polysaccharide fractions: amylose and amylopectin.According to the studies of Cuevas, Gilbert, and Fitzgerald (2010)and Shin, Simsek, Reuhs, and Yao (2008), phytoglycogen as awater-soluble starch-related a-glucans from cereal grains contain-ing the su1 mutation, which is the primary starch analogue in theendosperms of rice, corn, sorghum or barley.

3.2. Particle size analysis

TEM depictions of endosperm water-soluble a-glucans fromdifferent sugary maize cultivars are presented in Fig. 1. The glucans

Fig. 1. TEM images of three different endosperm

were shown as clustered spherical particles from the TEM image,indicating that native individual particle tended to aggregate insolution. The particle size, measured by scale bar of microscopy,was in the range of 30–80 nm, which belongs to a typical nativenano-scale particle. Putaux, Buléon, Borsali, and Chanzy (1999)reported the phytoglycogen particle from maize mutant su1 exhib-ited a spheroidal shape with a diameter ranging from 30 to 100 nmusing cryo-TEM, which was consistent with our observation.Zhongtian 8# or Pintian 8# had more large-sized particles thandid Zhongtian 2#. The differences in particle morphology may beattributed to the biological origin, biochemistry of the amyloplastand physiology of the plant (Ball & Morell, 2003; Manners, 1991;Miao et al., 2009).

The particle size distributions of the isolated endosperm a-glu-cans are shown in Fig. 2. The granule size distributions of glucansfrom Pintian 8#, Zhongtian 2# and Zhongtian 8# have beenobserved to exhibit ranges of 43–110, 28–95 and 36–105 nm,respectively. The average particle size was in the following order:Zhongtian 8# (73.02 nm) >Pintian 8# (72.24 nm) >Zhongtian 2#(69.14 nm). The results of endosperm water-soluble a-glucan fromdifferent sugary maize cultivars with particle size analyser con-firmed the morphological results obtained with TEM. The particlegranule size distribution of polysaccharide has been reported to af-fect the various physicochemical properties, including digestibilityand emulsification, to a considerable extent (Cui, 2005; Fujita et al.,2012; Shin et al., 2008).

3.3. Molecular weight distribution analysis

The molecular weight and radius of gyration of isolated water-soluble polysaccharides determined by HPSEC-MALLS-RI are sum-marised in Table 1. The weight average molecular weight (Mw) of

water-soluble a-glucans. Scale bar: 50 nm.

Fig. 3. 1H NMR of three different endosperm water-soluble a-glucans. (A) Zhongtian 8#, (B) Zhongtian 2#, (C) Pintian 8#, (D) panose.

160 M. Miao et al. / Food Chemistry 143 (2014) 156–162

endosperm a-glucans from different sugary maize cultivars rangesfrom 1.69 to 2.08 � 107 g/mol, and z-average radius of gyration (Rz)ranges from 30.5 to 36.0 nm, respectively. The Mw was in the fol-lowing order: Zhongtian 8# >Pintian 8# >Zhongtian 2#. The molec-ular weight increased as the particle size content increased fordifferent maize cultivars, which meant a positive relationship be-tween molecular weight and particle size. Zhang, Ao, and Hamaker(2006) reported that Mw and Rg of amylopectin and amylose inmaize starch were 1.08 � 108 g/mol, 220.7 nm, and 5.62 � 106 g/mol, 129.3 nm, respectively. The Mw of water-soluble a-glucanwas lower than that of amylopectin, but higher than that of amy-lose, which may be attributed to the differences in biosyntheticpathway in cereal endosperm (Ball & Morell, 2003). In the biosyn-thesis of starch, starch synthase, starch branching enzyme andstarch debranching enzyme work coordinately to produce starchcrystals and granules. In the absence of starch debranching en-zyme, the highly branched glycogen is formed to replace starch.The primary role of starch debranching enzyme is to form branchesby cleaving a section of linear glucan and attaching it to anotherchain by an a-1,6-glucosidic linkage, but also selectively cleavethe branches to allow for the formation of ordered structureamongst linear chains, which inhibit the formation of starch gran-ules with semi-crystal ring structure. Dispersed molecular densityof polysaccharides from Zhongtian 8#, Zhongtian 2# and Pintian8# were 445.8, 584.1 and 637.9 g/mol nm3, respectively. Thewater-soluble polysaccharides from Zhongtian 8# was much den-ser than Zhongtian 2# or Pintian 8# in aqueous dispersion. Wonget al. (2003) observed that phytoglycogen from sugary-1 mutantswas not only composed of multiple components of smaller molec-

ular weight and gyration radii, but also more tightly packed mole-cule exhibiting greater dispersed density than amylopectin. Similarobservations for phytoglycogens have been reported by Huang andYao (2011), the dispersed molecular density of phytoglycogen frommaize is over 1000 g/mol � nm3 compared to approximately 40 g/mol � nm3 for amylopectin.

3.4. Fine structure analysis

Branch chain length distribution resulting from the debran-ching of three different water-soluble a-glucans are summarisedin Table 1. The number-average chain length (CL) of polysaccha-rides ranges from DP 10.52 to DP 11.63 and the CL was in the fol-lowing order: Pintian 8# <Zhongtian 2# <Zhongtian 8#. These datawere comparable with those reported by Boyer and Liu (1983). Inthat study, the CL values of maize phytoglycogen, sorghum phyto-glycogen and rabbit liver glycogen were reported to be DP 10–16,DP 12–16 and DP 12–14, respectively. The branch densities calcu-lated were 8.60, 8.77 and 9.51% for three different water-solublepolysaccharides from sugary maize mutants including Zhongtian8#, Zhongtian 2# and Pintian 8#, respectively. Shin et al. (2008) re-ported that the branch density of phytoglycogens ranges from 8.8%to 9.5%, nearly twice that of starch from normal corn (4.6%) andwaxy corn (5.7%), which was in accordance with the branch densi-ties obtained with HPAEC-PAD. According to Inouchi, Glover, andFuwa (1987), fine structures of maize phytoglycogen and amylo-pectin were modelled, in which the chain length and degree ofbranching were 10.3%, 9.7% and 18.5%, 5.4%, respectively. There

401

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9192

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1152

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1383

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1640

.82

2928

.70

3384

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-0

5

10

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20

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70

%T

500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

531.

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65849.

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75

80

%T

500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

529.

5257

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86765.

31854.

9892

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1077

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1153

.87

1383

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1632

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2359

.41

2920

.66

3382

.29

5

10

15

20

25

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35

40

45

50

55

60

65

70

75

%T

500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

407

16

527.

2157

6.81

709.

8376

4.70

860.

6092

8.51

1018

.00

1081

.64

1157

.05

1383

.03

1642

.04

2931

.99

3420

.55

-10

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

%T

500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

Zhongtian 8# Zhongtian 2#

Pintian 8# Waxy maize starch

Fig. 4. FT-IR spectra of three different endosperm water-soluble a-glucans.

M. Miao et al. / Food Chemistry 143 (2014) 156–162 161

were no long B chains in phytoglycogen with highest degree ofbranching.

The absorption spectra in the presence of Krisman’s solution ofthree different endosperm water-soluble a-glucans showed a kmaximum of 460–474 nm followed the order: Zhongtian 2#(460 nm) <Pintian 8# (463 nm) <Zhongtian 8# (474 nm). The amy-lose is made up of a (1 ? 4) bound linear glucose polymer with thelowest degree of branching (1.0–2.0%) and a kmax 580–640 nm;amylopectin is an intermediate type branched compound with2.1–5.9% of glucoses in the branching points and a kmax 390–570 nm; and phytoglycogen is the highest branched molecule witha branching degree of 6.0–7.5% and a kmax 460–480 nm as sug-gested by Curá and Krisman (1990). They also found that an in-crease in the percentage of the branching points was correlatedwith a decrease in the kmax, or conversely a higher kmax with a low-er degree of branching in the presence of Krisnian’s iodine reagent,which were similar to the results obtained in this study.

To further characterise the structural properties of water-solu-ble glucans, the ratios of a-1,4 and a-1,6 linkages were analysedusing 1H NMR spectroscopy and the results are present in Table 1.As shown in Fig. 3, panose had 50% a-1,4 and 50% a-1,6 linkages,which was used as a standard to verify the positions for H-1 ofa-1,4- and a-1,6-linked units. The a-1,6 linkages of Zhongtian8#, Zhongtian 2# and Pintian 8# were 7.71, 6.58 and 6.81%, respec-tively. Yun and Matheson (1993) reported that glycogen has A:Bchain ratio (where A chains contain only (1 ? 4) linkages and Bchains also contain (1 ? 6) linkages) of <1 and amylopectin ratiosof >1, which led to average frequencies of substitution of B chainsover the whole molecule of <2 for the glycogens and >2 for theamylopectins. Our results indicated that the endosperm water-sol-uble glucans from three sugary maize mutants may be a polysac-charide backbone consisted of 1,4-linked a-D-glucopyranosylresidues, which were, to some extent, branched through 1,6 glyco-sidic bonds as evidenced by HPAEC and 1H NMR.

3.5. FT-IR spectra analysis

The FT-IR spectra in the mid-infrared region of water-soluble a-glucans are presented in Fig. 4. In the fingerprint region of the poly-saccharide spectrum, five characteristic peaks appear between 900and 1300 cm�1, which have been attributed to C–O bond stretching(Cui, 2005; Santha, Sudha, Vijayakumari, Nayar, & Moorthy, 1990).In the curves in Fig. 4, the peak around 1020 cm�1 was attributedto the C–O stretch of the C–O–C in polysaccharide, peaks near1080 and 1155 cm�1 were characteristic of the anhydroglucosering C–O stretch, peak near 928 was assigned to the skeletal modevibrations of a-1,4 glycosidic linkage, and peak near 854 cm�1 wascorresponded to C–H and CH2 deformation (Santha et al., 1990). Abroad peak appeared around 3400 cm�1, indicating the presence ofhydroxyl groups. The spectra also showed C–H stretching at2930 cm�1, adsorbed water bending vibration at 1640 cm�1, andC–H deformation at 1380 cm�1. Shingel (2002) reported that thesharp peaks at 1020 cm�1 in the spectra indicated the presenceof the a-1,6 linkage, suggesting that both water-soluble polysac-charide and waxy maize starch exhibited an a-1,4 backbone andan a-1,6 branched structure. The main difference between water-soluble polysaccharides and starch was the sharp of characteristicpeaks, which may be due to biological origin, biochemistry of theamyloplast and physiology of the plant (Ball & Morell, 2003; Halla-uer, 2001; Miao et al., 2009).

3.6. In vitro digestion

Enzymatic digestibility of three different endosperm water-sol-uble a-glucans is shown in Table 1. According to the review of Eng-lyst et al. (1992), starch is divided into RDS, SDS and RS fractions,which are the three consecutive digestion fractions divided byreaction time and represent three starch materials found instarches and processed foods. The results showed that RDS, SDS

162 M. Miao et al. / Food Chemistry 143 (2014) 156–162

and RS were 69.13%, 16.82%, and 14.05%, 67.87%, 19.67%, and12.46%, 72.55%, 17.39%, and 10.06% for Zhongtian 8#, Zhongtian2#, and Pintian 8#, respectively. Starch digestibility has beenshown to be influenced by source, granule size, ratio of a-1,4 toa-1,6 linkages, crystallinity, molecular structure, surface poresand interior channels (Miao et al., 2009; Zhang et al., 2006). Thissuggests that the value of in vitro digestibility (RDS + SDS) of Pin-tian 8# was higher than that of other two a-glucans, which mightbe due to its lower a-1,6 linkages (Table 1) and larger proportion ofsmall particle size (Fig. 2). Pazur and Ando (1960) reported that thea-1,4 linkage in maltose is hydrolysed at about 30 times the ratefor an a-1,6 linkage in isomaltose. Thus, it is likely that the hydro-lysis of a-1,6 linkage is the rate-limiting step of glucose release,resulting in water-soluble a-glucan with a lower digestibility thanstarch. Similar observations for soluble a-glucans have been re-ported by Lin et al. (2012). The structure of a-limit dextrin influ-encing the glucose generation was a crucial factor for in vitroenzyme digestibility. Clearly, highly branched a-glucan extractedfrom sugary maize showed a reduced digestibility as comparedwith starch and this study shows that digestibility was influencedby granule size, ratio of a-1,4 to a-1,6 linkages, molecular finestructure in this set of sugary maize mutants.

4. Conclusions

This study showed that the water-soluble a-glucans, from threesugary maize mutants (Zhongtian 2#, Zhongtian 8#, Pintian 8#)studied, belonged to a typical native nano-scale particle and exhib-ited a-1,4-linked backbone with a-1,6 branch sites. The a-glucanfrom Pintian 8# differed significantly from the other two cultivars,with higher molecular density, branch density, rapidly digestiblestarch, and lower number-average chain length and resistantstarch. Further work is underway to obtain structure–propertyrelationships for these a-glucans. These experimental results willprovide useful information for consumers and food industriesusing sugary maize.

Acknowledgements

The research was financially supported by the National NaturalScience Foundation of China (31000764, 31230057), National HighTechnology Research and Development Program of China (2013AA102102) and Science & Technology Pillar Program of JiangsuProvince (BE2012613, BY2012049).

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