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Food Hydrocolloids 24 (2010) 60–65

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Food Hydrocolloids

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The effect of enzymatic pretreatments on subsequent octenyl succinicanhydride modifications of cornstarch

Qiang Huang, Xiong Fu*, Xiao-wei He, Fa-xing Luo, Shu-juan Yu, Lin LiCollege of Food Science, South China University of Technology, 381 Wushan Road, Guangzhou 510640, China

a r t i c l e i n f o

Article history:Received 25 August 2008Accepted 7 August 2009

Keywords:Starch granulea-AmylaseOS-StarchPasting propertiesThe group distribution

* Corresponding author. Tel.: þ8620 87113845; faxE-mail address: lfxfu@scut.edu.cn (X. Fu).

0268-005X/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.foodhyd.2009.08.005

a b s t r a c t

Cornstarch granules were allowed to react with a-amylase and then esterified with octenyl succinicanhydride (OSA). The paste properties and the distribution of octenyl succinate (OS) groups of OS-starchgranule were studied. Results show that the a-amylase pretreatments significantly decreased the degreeof substitution (DS) of OS-starch compare with a non-pretreated control group, while the retrogradationproperties, freeze-thaw stability and shearing resistant stability of a-amylase pretreated OS-starch didn’tchange markedly. Enzymatic pretreatment appears to influence the distribution of the OS groups frombeing mostly on the surface and amorphous part of the starch granules to being in the inner amorphousand crystalline regions of the granules, as shown by X-ray photoelectron spectroscopy (XPS) andlintnerisation studies.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

When starch is modified by the treatment with octenyl succi-nate anhydride (OSA), the normally hydrophilic starch moleculesacquire useful surface-active properties by the incorporation ofhydrophobic and hydrophilic alkenyl groups. OS-starch is appli-cable in a variety of oil-in-water emulsions for food, pharmaceuticaland industrial products such as beverages and salad dressings,flavor-encapsulating agents, clouding agents, processing aids, bodypowders, and lotions (Jeon, Viswanathan, & Gross, 1999; Park,Chung, & Yoo, 2004).

In recent years, the preparation and physicochemical propertiesof OS-starch have been reported (Angellier, Molina-Boisseau,Belgacem, & Dufresne, 2005; Bao, Xing, Phillips, & Corke, 2003;Song, He, Ruan, & Chen, 2006). It has been suggested that theoctenyl succinate (OS) groups are located mainly on the surface ofstarch granules (Shogren, Viswanathan, Felker, & Gross, 2000).Normally, the reaction of OSA and starch is retarded due to poorpenetration of the big oily droplets of OSA into the starch granulesin an aqueous suspension, and the site of reaction is limited to thegranule surface. As a consequence, the OS groups are not evenlydistributed throughout the starch granule.

Amylase treatments have been used to produce porous starchand for structural analysis of starch granules (Franco & Ciacco, 1992;Jone, Richard, & Bemiller, 1992; Sreenath, 1992), relatively little

: þ8620 87113848.

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work has been done on starch modification after pretreatmentswith amylase. Theoretically, pore formation in the starch granuleswill increase the surface area of the starch particles and make iteasier for the chemical reagent to infiltrate into the inner parts ofthe starch, especially for the water insoluble OSA. The amylasepretreatment would be expected to change the distribution ofsubstitution groups and consequently influence the paste stabilityof starch. Huber and BeMiller (2000) have reported on the archi-tecture and permeability of cornstarch granules. They have shownthat pores on the surface of starch granules, internal cavities at thegranule hilum, and channels connecting the two could significantlyinfluence granule reactions to chemicals. Accordingly, the fact thatthere exist holes or ravine on starch granules after treatments withamylase is interesting for mechanistic studies.

Inorganic acid treatment has been widely used to demonstratestarch granule arrangement. During acid hydrolysis, the amorphousparts of starch are preferentially hydrolyzed and dissolved, but themain crystalline structures (acid-resistant residues) remain intact(Robin, Mercier, Duprat, & Guilbot, 1975; Srichuwong, Isono,Mishima, & Hisamatsu, 2005). Hydrolysis in mild conditionsconsists of two stages: an initial fast step at the amorphous regionsof the granule followed by a second, slower step at the crystallineregions (Robin et al., 1975). Franco, Cabral, and Tavares (2002) alsofound that the acid hydrolysis on all starches occurred in two steps.Thus, the distribution of OS groups in amorphous or crystallineregion can be presumed by determining the DS of residualOS-starch during specific hydrolysis time.

In this study, the granular structure of cornstarch was firstaltered by the pretreatment with a-amylase and then the OS-starch

Q. Huang et al. / Food Hydrocolloids 24 (2010) 60–65 61

was prepared to determine the change of the paste properties andthe distribution of the OS groups in the modified starch. We furthercompared the OS-group distribution in both amorphous/crystallineand surface/inner region by acid hydrolysis and X-ray photoelec-tron spectroscopy (XPS) analyses.

2. Materials and methods

2.1. Materials

Commercially available cornstarch (13.5% moisture) wasobtained from DACHENG Company (Chang Chun, China). Highpurity 2-Octen-1-ylsuccinic anhydride (OSA) was obtained fromSigma–Aldrich Chemical Co. (Milwaukee, WI). Food grade bacteriala-Amylase (EC 3.2.1.1) was donated by Novozymes called BacterialAmylase Novo (BAN, 800 KU/g). One unit of BAN was defined as5.6 mg of starch was liquefied in 1 h at pH 6.0 and 60 �C. The otherchemicals used in the study were analytical grade.

2.2. Enzymatic hydrolysis

Cornstarch (800 g, dry starch base) suspended in acetate buffer(pH 6.0) at a concentration of 30% (w/v), were incubated withdifferent units (0.7 m/g, 3.5 m/g, 6.6 m/g, 19.8 m/g) of BAN at 60 �C for7 h (Franco & Ciacco, 1987). After incubation, the enzymes in thedispersion were inactivated by adding HCl (0.1 M) to reach pH 3.0,followed by agitation for 15 min, and neutralization with NaOH(0.1 M). The starch was recovered by vacuum filtration, lightlywashed on a filter with distilled water, and air-dried. The degree ofhydrolysis (DH) was determined by the phenol-sulphuric acidmethod (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). It was

Shearing resistant stability ¼ The beginning viscosity of the held stage at 50+CTerminal viscosity after holding 2h

� 100

defined as soluble saccharides per initial total saccharides in thestarch suspension. The preparation of enzymatic treated cornstarchwas performed in duplicate.

2.3. Preparation of OS-starch

OS-starch was prepared by treating starch with octenyl succinicanhydride (OSA) according to the procedure of Jeon et al. (1999).Cornstarch and BAN pretreated cornstarch (500 g respectively)were suspended in distilled water (750 mL) and then the pH of thesuspension was adjusted to 8.5 with 3% (w/v) NaOH. OSA (20 mL)was added slowly over 2 h. The reaction was allowed to continue for5 h and at the same pH. After the reaction was completed, thesuspension was neutralized to pH 7.0 with 3% (w/v) HCl. Theresultant starch suspension was vacuum-filtered through filterpaper and washed three times with distilled water and once with95% ethanol to remove the residual reagents. The recovered starchwas then dried in an oven at 37 �C over night. The dried sample wasground gently in a mortar to pass through a 100-mesh standardsieve.

2.4. Determination of the degree of substitution

The degree of substitution (DS) is the average number ofhydroxyl groups substituted per glucose unit. The DS of OS-starchwas determined by titration as previously reported (Song et al.,

2006).OS-starch sample (5 g, dry weight) was accurately weighedand suspended by stirring for 30 min in 25 mL 2.5 M HCl-isopropylalcohol solution. 100 mL 90% (v/v) aqueous isopropyl alcoholsolution was added and the slurry stirred for an additional 10 min.The suspension was filtered through a glass filter and the residuewas washed with 90% isopropyl alcohol solution until no Cl� couldbe detected any longer (using 0.1 M AgNO3 solution). The starchwas re-dispersed in 300 mL distilled water, and then the dispersionwas heated in a boiling water-bath for 20 min with stirring. Thestarch solution was titrated with 0.1 M standard NaOH solution,using phenolphthalein as an indicator. A blank was simultaneouslytitrated with native starch as a control. The DS was calculated by thefollowing equation:

DS ¼ 0:162� ðA�MÞ=W1� ½0:210� ðA�MÞ=W�

where: A is the titration volume of NaOH solution (mL), M is themolarity of NaOH solution, and W is the dry weight (g) of theOS-starch.

2.5. Pasting properties and shearing resistant stability

Pasting properties and shearing-resistant stability of modifiedstarch was analyzed on a Brabender viscograph E (Brabender OHG,Germany). A 28 g dry sample was suspended in 432 g distilled water(6%). A 700 cm/g cartridge was fitted with the rotation speed set at75 rpm, The suspension was heated at 1.5 �C/min to 95 �C from30 �C, then held for 30 min at 95 �C, cooled to 50 �C at 1.5 �C/min,and held for 30 min for pasting properties and 2 h for shearingresistant stability at 50 �C. The shearing resistant stability wascalculated as follow:

2.6. Freeze-thaw stability

The starch pastes (6%, w/v) were kept in 100 mL plastic bottlessealed with screw caps and stored at �18 �C (freezer) for 12 h. Andthen the paste was thaw at 25 �C for 6 h (One freeze and thaw cycledefined 1 time.) The process was repeated till water expelled or thepaste turned into sponginess. And the number of cycles for freeze-thaw represents the property of freeze-thaw stability.

2.7. Water release ratio of the starch paste

Starch pastes (1%, w/v) were prepared and put into 25 mLmeasuring cylinder in 25 �C for 24 h. The water separated from thepaste reflects the retrogradation properties (Huang, Gao, Zhou, &Zhang, 2001), which was calculated as follow:

Water release ratio ¼ The volume of supernatant layerTotal volume of the paste

� 100

2.8. Acid hydrolysis

OS-starch was hydrolyzed with 2.2 M HCl at 35 �C (1.0 g starch/10 mL acid) for periods ranging from 1 to 4 days. The starch slurrieswere shaken daily by hand to re-suspend the deposited granules.Aliquots taken at specific time intervals were neutralized andcentrifuged (10 min at 2000 � g). The granular residues obtained

Q. Huang et al. / Food Hydrocolloids 24 (2010) 60–6562

were washed three times with de-ionized water and acetone, thedegree of substitution of residual OS-starch was determined. Andthe total carbohydrate content in the supernatant was measureddaily using the phenol-H2SO4 reagent (Dubois et al., 1956).

2.9. X-ray photoelectron spectroscopy (XPS) analyses

The surface morphology of the samples was analyzed usinga KRATOS AXIS Ultra (DLD) X-ray photoelectron spectrometer (UK).The X-ray gun was operated at 15 kV voltages and 10 mA. Surveyand high-resolution spectra were collected using 160 eV and 40pass energy, respectively. The analyzer chamber was degasified andpressure in the analyzer chamber was kept as low as w10�8 torr.A thin film of thickness 0.3 mm was used for XPS evaluation.Gaussian/Lorentzian in 7:3 ratio peak deconvolution and presen-tation output is produced by integrated VISION control and infor-mation system software attached with the unit. All spectra arepresented charge balanced and energy referenced to C 1s at284.8 eV.

2.10. Statistical analysis

Analyses of variance and the mean separations were performedby Duncan’s least significant test (r < 0.05) using SPSS 12.0statistical software for Windows (SPSS, Inc., Chicago, IL, USA).

3. Results

3.1. Paste properties of OS-starch

The Brabender viscograms of native cornstarch, OS-starch andBAN pretreated OS-starch are presented in Table 1. The data showthat the OS-starch exhibits lower pasting temperature, higherviscosity, higher breakdown and setback value compare with nativestarch, which is consistent with previous studies (Bao et al., 2003;Song et al., 2006).

Compare with OS-starch, a rise in pasting temperature anda drop in peak viscosity and cold paste viscosity were observed asa result of BAN pretreatment (r< 0.05, Table 1). Pasting properties ofstarch are affected by amylose, lipid contents and by branch chain-length distribution of amylopectin. Amylopectin contributes toswelling of starch granules and pasting, whereas amylose and lipidsinhibit the swelling and increase the pasting temperature (Tester &Morrison, 1990). Furthermore, the amylopectin chain-length and

Table 1Pasting characteristics* of native cornstarch, OS-starch and BAN pretreatedOS-starch in different DH.a

DH (%) DS Tp(�C) Pv(BU) Cv(BU) BD(BU) SB(BU)

0 0 78.0b

(0.57)290f

(2.83)414f

(5.66)88g

(2.12)211f

(4.95)0 0.022b

(0.0017)62.2f

(0.28)1167b

(4.95)959b

(7.07)536b

(1.41)328c

(4.24)4.3e

(0.14)0.010e

(0.0014)69.5e

(0.42)725c

(2.83)714d

(5.66)387c

(1.41)376d

(4.24)10.9d

(0.28)0.016d

(0.0011)70.3de

(0.28)587d

(1.41)625e

(7.07)253d

(1.41)291e

(4.24)18.4c

(0.14)0.018c

(0.0013)70.8cd

(0.14)585d

(2.83)751c

(5.66)143f

(1.41)309b

(4.24)31.6b

(0.42)0.015d

(0.0011)71.7c

(0.42)478e

(4.95)427f

(2.83)243e

(4.24)192g

(1.41)

a DH, Degree of hydrolysis. DS, Degree of substitution, Tp, pasting temperature,Pv, peak viscosity, Cv, cold paste viscosity, BD, breakdown, SB, setback, BU, Bra-bender units. The data are averages of two measurements with standard deviation.Means within a column with different letters are significantly different (r < 0.05).*Starch concentration 6% (w/w).

amylose molecular size produce synergistic effects on the viscosityof starch pastes (Jane & Chen, 1992). Starch with short averageamylopectin branch chain lengths (e.g., waxy rice and sweet ricestarch), and with large proportions of short branch chains (dp11–16) relative to the shoulder of dp 18–21 (e.g., wheat and barleystarch) displayed low gelatinization temperatures (Jane et al., 1999).Therefore, the increases in pasting temperature of BAN pretreatedOS-starch may be due to the degradation of short branch chain ofamylopectin in crystalline region and lipid-free amylose in amor-phous region by a-amylase, which made the residual starch granulepossess more longer branch chain of amylopectin and moreproportion of amylose lipids complex.

The breakdown viscosity is the difference between the peakviscosity and the viscosity after holding for 30 min at 95 �C (Huang,Li, & Fu, 2007; Muhammad, Hussin, Man, Ghazali, & Kennedy,2000). The higher breakdown viscosity indicates granule disrup-tion or the less tendency of starch to resist shear force duringheating. The breakdown value of OS-starch paste was higher thanthose of BAN pretreated OS-starch (r< 0.05, Table 1). The OS-starchshows higher peak viscosity than both native counterpart and BANpretreated OS-starch (r < 0.05, Table 1). Therefore, the lowerbreakdown viscosity of BAN pretreatment OS-starch does notnecessarily indicate the higher resistance to shear force comparedwith OS-starch. The setback value (SB), an index of retrogradationtendency in the starch paste, also decreased after pretreatment(r< 0.05, Table 1). However, the setback value is more likely relatedto the retrogradation tendency of amylose, for the initial gelnetwork development is dominated by amylose gelation (Miles,Morris, Orford, & Ring, 1985). Therefore, the setback value is a moresuitable parameter to evaluate the retrogradation properties ofnative starch than modified starch.

The paste stability of OS-starch was presented in Table 2.Compare with the OS-starch without pretreatment, the BANpretreatment did not have a significant effect on retrogradation,freeze-thaw and shearing resistant stability of counterpartOS-starch, although the degree of substitution (DS) is significantlyreduced (r < 0.05, Table 1), which may be the result of removal ofthe amorphous areas. This should be attributed to the change ofdistribution of OS groups in starch granule after BAN pretreatment.

3.2. Distribution of OS groups in the starch granules

3.2.1. Amorphous and crystalline regionThe relationship between lintnerized time and DS of residual

OS-starch were shown in Fig. 1 A. The DS of OS-starch without BANpretreatment decreased drastically in 1 days of hydrolysis, andremained low for the first 3 days, while in 3–4 days of hydrolysis,the DS of residual OS-starch increased a little. As for BAN pretreatedOS-starch, the DS also decreased in 1–3 days of hydrolysis, anddidn’t increase any more in 3–4 days of hydrolysis. Fig. 1 B showsthe solubility of OS-starch during lintnerisation. Generally, thelintnerisation rate of BAN pretreated OS-starch is higher than

Table 2Paste stability of OS-starch and BAN pretreated OS-starch.a

DH (%) DS water releaseratio/%

freeze-thawstability/times

Shearing resistantstability/%

0 0.022 3.3b(0.2) 2 62.7c(2.2)4.3 0.010 3.6b(0.3) 2 61.6c(1.7)10.9 0.016 3.2b(0.4) 2 69.3b(1.9)18.4 0.018 3.4b(0.3) 2 63.2c(2.1)31.6 0.015 3.5b(0.2) 2 63.6c(2.0)

a DH, Degree of hydrolysis; DS, Degree of substitution; The data are averages oftwo measurements with standard deviation. Means within a column with differentletters are significantly different (r < 0.05).

0.000

0.005

0.010

0.015

0.020

0.025A

B

Hydrolysis time (d)

Deg

ree

of s

ubst

itut

ion

(DS)

DH: 0

DH: 4.3

DH: 10.9

DH: 18.4

DH: 31.6

0

10

20

30

40

50

0 1 2 3 4

0 1 2 3 4

Hydrolysis time (d)

Solu

bilis

atio

n (%

)

DH:0

DH:4.3

DH:10.9

DH:18.4

DH:31.6

Fig. 1. The relationship between hydrolysis time and DS (A), solubility (B) of OS-starchand BAN pretreated OS-starch.

Q. Huang et al. / Food Hydrocolloids 24 (2010) 60–65 63

OS-starch. And the higher degree of hydrolysis by BAN, the higher isthe lintnerisation rate by hydrochloric acid. The result should berelated to the pits or holes of starch granules by BAN pretreatment.The increased surface area of BAN pretreated starch granules wouldincrease the accessibility of substrate to the acid and allow the acidto penetrate to the internal structures more readily.

Normal cereal starch consists of three distinct components: 1)highly crystalline regions formed from double-helical amylopectinchains, 2) solid-like regions formed from lipid-complexed amylose,and 3) completely amorphous regions associated with amylopectinbranches and possibly the lipid-free amylase (Lauro, Forssell,Suortti, Hulleman, & Poutanen, 1999; Morgan, Furneaux, & Larsen,1995). And former studies have shown that the substitute groupsare mostly located in the amorphous region near the branch pointof amylopectin (Hood & Mercier, 1978; Shogren et al., 2000). In theinitial time of lintnerisation (3 days), the amorphous region ofstarch granules with OS groups was released and DS of the residualgranules decreased rapidly, while in 3–4 days of hydrolysis, thesemi-crystalline and crystalline region should have been hydro-lysised, for the maltese-cross of native OS-starch began to vanish asobserved by polarizing microscope, and the residual starch becomedissolved in water and turned into a sticky state. The result impliesthat the crystalline regions are less substituted than the amorphousregion. For BAN pretreated OS-starch granules, the hydrolysis rateare faster than OS-starch, which means the crystalline regions have

more chance to be hydrolysis in 3–4 days. The DS keeps decreasingslowly instead of increasing during linterisation of BAN pretreatedOS-starch. The results suggest that the OS groups also mainlydistributed in amorphous, but more in crystalline region than non-pretreated OS-starch.

Robin, Alain, Kari, and Eric (2000) have found that the initial rate ofacid hydrolysis (lintnerisation) was increased in cationised andoxidized potato starch compared with native granules. And thedegree of substitution decreased not significantly in wet-cationisedgranules after high level of lintnerisation. They conclude that the wet-cationised products were modified throughout the granules. In ourstudy, the insoluble OSA is suspended in starch slurry for the esteri-fying reaction, which is different fromwater-soluble etherifying agent(2,3-epoxypropyltrimethylammonium chloride). The different solu-bility and reaction mechanism of esterify and etherify can explain theinconsistency between this study and Robin et al.’s report.

3.2.2. Surface and inner regionXPS is a powerful tool to investigate chemical changes resulting

from surface modification since the investigated thickness is of theorder of about 4–10 nm (Angellier et al., 2005; Labet, Thielemans, &Dufresne, 2007). Surface compositions of native cornstarch and OS-starch as determined by XPS are displayed in Fig. 2. The signalsobserved around binding energies of 531, 399, and 285 eV corre-spond to the 1s orbital electrons of oxygen, nitrogen, and carbon,respectively. After modification with OSA, there appear a new Naabsorption peak at about 1069 eV (Fig. 2), and the peak corre-sponding to carbon atoms of OS-starch observed around bindingenergies of 285.09, 283.53 and 286.23 eV, for the new bond of–C]O and –C]C. Also, the peak corresponding to oxygen atoms ofOS-starch observed around binding energies of 531.57, 530.04 and532.11 eV, for the bond of –C–O–, –C]O and –O–C–O– respectively(Fig. 3), indicating clearly that carbon atoms were esterified andthereby that chemical modification occurred.

Surface compositions of native cornstarch, OS-starch and BANpretreatment OS-starch as determined by XPS are given in Table 3. Inorder to determine the molecular composition from data in Table 3,it can be assumed that the elements observed arise from the glucoseunits of starch, OSA, etc. A series of simultaneous equations can bewritten as follows (Shogren et al., 2000):

hD6gD4:8pD12a [ nc (1)

5gD1:9pD3a [ no (2)

a [ nNa (3)

1:3p [ nN (4)

where n represents the number fraction� 100 of an experimentallyobserved element, g is the number of glucose residues, a is thenumber of OS groups, p is the number of amino acid residues and his the number of hydrocarbon (CH2) groups. Proteins are found insmall amounts on starch granules and were approximated by theformula, C4.8O1.9N1.3H7.7.

The solutions to equations (1)–(4) for OS-starch and BAN pre-treated OS-starch are given in Table 4. Taking the ratio of a/g, valuesfor the surface D.S. of 0.048 were calculated for the (bulk) D.S. 0.022of OS-starch samples. This suggests that the surface of OS-starch isenriched with OS groups by a factor of 2 over that of the bulkgranule. The result was consistent with Shogren et al.’s (2000)report.

Chemical elements of BAN pretreated OS-starch are presented inTable 3 and Table 4. Relatively low level Na was detected in all BAN

Fig. 2. Survey XPS spectra for native cornstarch (A) and OS-starch (B).

Q. Huang et al. / Food Hydrocolloids 24 (2010) 60–6564

pretreated sample with similar DS. The ratio of a/g, values for thesurface DS is lower than the DS of whole granule, which impliesthat the OS groups is distributed primarily in the interior of the BANpretreated cornstarch granule.

Although XPS is a powerful tool to investigate chemical changesresulting from surface modification, it should be noted that theinvestigated thickness is limited (w4–10 nm), which is about thelength of one cluster (w9 nm) of branch chain double helixes inamylopectin (Jane, 2006).

4. Discussion

It has been suggested that a-amylases, unlike acid, are able tosimultaneously solubilize the amorphous and crystalline areas of

Fig. 3. High resolution XPS spectrum of native corn

starch granules (Colonna, Buleon, & Lemarie, 1988; Lauro et al.,1999; Leach & Schoch, 1961). And crystalline polymorphs, accessi-bility of substrate to enzyme, fraction of crystalline structures,amylopectin chain-length distribution and granule size influencethe enzyme susceptibility for granular starches. As A-type starch(such as cornstarch), the first alteration in structure is seen to bepitting at the surface, and then small pits become holes, after whichendocorrosion (i.e. from the inside out) appears to occur. In thiscase the internal preferential loss of material from the amorphousgrowth rings becomes clear (Donald, 2004). Alfa-amylase such asBAN is an endo-specific enzyme that randomly catalyzes thehydrolysis of a-(1 / 4) glycosidic linkages in amylose and amylo-pectin chains. Although a-amylase can hydrolyze both the amor-phous and crystalline regions in starch granules, the enzyme has

starch (A) and OS-starch (B) for C1s and O1s.

Table 3Surface elemental composition of native cornstarch, OS-starch and BAN pretreatedOS-starch Determined from XPS (number fraction � 100).

DH (%) DS C O Na N

0 0 68.061 31.067 0 0.6080 0.022 66.044 33.192 0.302 0.4624.3 0.010 64.430 35.524 0.046 010.9 0.016 62.986 36.975 0.039 018.4 0.018 63.577 36.305 0.118 031.6 0.015 64.839 35.094 0.066 0

DH, Degree of hydrolysis, DS, Degree of substitution.

Table 4Calculated molecular compositions of OS-starch and BAN pretreated OS-starch.

DH (%) DS g A h p a/g

0 0.022 6.365 0.302 22.526 0.355 0.0484.3 0.010 7.077 0.046 21.415 0 0.00610.9 0.016 7.372 0.039 18.286 0 0.00518.4 0.018 7.190 0.118 19.021 0 0.01631.6 0.015 6.979 0.066 22.172 0 0.009

g, glucose residue (C6O5H10); a, OSA(C12O3H17Na); h, hydrocarbon(CH2); p,protein(C4.8O1.9N1.3H7.7); DH, Degree of hydrolysis; DS, Degree of substitution.

Q. Huang et al. / Food Hydrocolloids 24 (2010) 60–65 65

more ready access to the amorphous region than the crystallineregion. The radial channels in starch granules are believed to makethe amorphous region inside granule more accessible to theenzymes (Gallant, Bouche, & Baldwin, 1997). Removal of theamorphous region could induce the residual of BAN pretreatedstarch granules are not easy reacting with OSA reagent, in agree-ment with the DS data (Table 1). In addition, the OSA droplets couldreact with the granular surface or, depending on the droplets sizeand the size of pore in the starch granules which made by BANpretreatment, travel into the channels and interior cavity. By thistoken, the exiting pinholes will promote the reaction of OSA andinner part of starch granules (XPS results).

Since functional properties of starch derivatives such as pastingtemperature, gelatinisation and cooking characteristics, solidsviscosity relationships and retrogradation tendencies are deter-mined by the type of substituent, the level of substitution and topo-chemical aspects (Van der Burgt et al., 1998), the methods whichcan produce evenly distribution and characterization of substitutedgroups in OS-starch granules by other methods needed furtherstudy.

5. Conclusions

Amylase of BAN attack preferentially occurred in the amorphousparts of starch granule, which make the DS of amylase pretreatedOS-starch in low level compare with the normal OS-starch. Theretrogradation properties, freeze-thaw stability and shearing-resistant stability have little change although in lower DS of BANpretreatment OS-starch. OS groups were distributed mostly on thesurface and amorphous region of the untreated starch granules,while the substitution groups were found more in the inner, crys-talline region of the BAN pretreated OS-starch granule.

Acknowledgements

The authors gratefully thank Dr. Andrew Tang for his help in thepreparation of the manuscript, and would like to acknowledge thegrant (20436020) by NFSC and the Key Project (2008B021100005)of agriculture of Guangdong Province in China.

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