Food Chemistry 131 (2012) 500–507

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Food Chemistry

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Effect of gelatinisation on slowly digestible starch and resistant starch ofheat-moisture treated and chemically modified canna starches

Juraluck Juansang a, Chureerat Puttanlek b, Vilai Rungsardthong c, Santhanee Puncha-arnon a,Dudsadee Uttapap a,⇑a Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 49 Soi Tientalay 25, Bangkhuntien-Chaitalay Road,Takham, Bangkhuntien, Bangkok 10150, Thailandb Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakhon Pathom 73000, Thailandc Department of Agro-Industrial Technology, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, 1518 Pibulsongkram Road, Bangsue, Bangkok10800, Thailand

a r t i c l e i n f o

Article history:Received 31 May 2011Received in revised form 20 July 2011Accepted 6 September 2011Available online 16 September 2011

Keywords:Canna starchGelatinisationHeat-moisture treatmentModified starchOctenyl succinylationResistant starchSlowly digestible starch

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.09.013

⇑ Corresponding author. Tel.: +66 2 470 7754; fax:E-mail address: dudsadee.utt@kmutt.ac.th (D. Utta

a b s t r a c t

The effects of gelatinisation on slowly digestible (SDS) and resistant starch (RS) of native and modifiedcanna starches were investigated. Starch slurries (10% w/w) were gelatinised at 100 �C for 5, 10, 20and 40 min using a rapid visco analyzer (RVA). Significant change in the degree of gelatinisation (DG) val-ues of all starch samples was observed during the initial 10 min of gelatinisation; after that the DG valuesincreased gradually with gelatinisation time. The RS contents in all gelatinised starches decreased withincreasing gelatinisation time, while the SDS values fluctuated. Chemical modification affected DG valuesas well as RS/SDS contents. The RS contents in 10% (w/w) acetylated, hydroxypropylated, octenyl succiny-lated and cross-linked canna starches gelatinised at 100 �C for 40 min were 26.6%, 32.0%, 45.3% and 19.8%,respectively, which were higher than that of the native starch (12.4%). Canna starch modified by cross-linking had the highest SDS content when gelatinised for 20–40 min. Modification of canna starch byheat-moisture treatment resulted in a lower content of RS for all treated samples. However, the Vt-HMT25 (canna starch containing moisture content of 25% during heat treatment) when gelatinised for5–20 min contained a higher amount of SDS, compared to unmodified starch. The most effective modifi-cation method for RS and SDS formation was octenyl succinylation, where the sum of RS and SDSapproached that of Novelose260.

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1. Introduction

Starch is the main carbohydrate in human nutrition, and is oneof the most important sources of biological fuel for humans. Fornutritional purposes, starch is generally classified into rapidlydigestible starch (RDS), slowly digestible starch (SDS) and resistantstarch (RS), depending on the rate and extent of its digestion(Englyst, Kingman, & Cummings, 1992). RDS induces a rapid in-crease of blood glucose and insulin levels after ingestion. SDSprolongs the release of glucose, thus preventing hyperglycaemia-related diseases. RS reduces starch availability for digestion andproduces short-chain fatty acids in the large bowel through fer-mentation, which is beneficial for colon health and protectionagainst colorectal cancer (Lehmann & Robin, 2007). Consequently,starch ingredients with high levels of SDS and RS can improve thenutritional function of foods. RS has been categorised into four

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+66 2 452 3479.pap).

types, of which chemically modified starches are classified as RStype 4 (Eerlingen & Delcour, 1995).

A decrease in the susceptibility of chemically modified starches,either in native granular form or in a gelatinised state, to enzymehydrolysis was shown in a recent report (Lehmann & Robin,2007). Substitution of starch hydroxyl groups with citryl (Wolf,Bauer, & Fahey, 1999; Xie & Liu, 2004), acetyl (Chung, Shin, &Lim, 2008; Hoover & Sosulski, 1985), octenylsuccinyl (Han & BeM-iller, 2007; He, Liu, & Zhang, 2008; Heacock, Hertzler, & Wolf,2004) and hydroxypropyl (Conway & Hood, 1976; Han & BeMiller,2007) substituents has been found to decrease starch digestibilityby a-amylase. However, studies examining the in vitro digestion ofcross-linked starches have yielded contradictory observations. Asignificant reduction in digestibility of starch cross-linked by aphosphate bridge was reported by Sang and Seib (2006) and Wooand Seib (2003); whereas no (or only slight) changes were foundby Chung et al. (2008), Östergaard, Björck, and Gunnarsson(1988) and Wolf et al. (1999). The discrepancy in the digestibilityof cross-linked starches might be caused by the inherent propertiesof each type of starch, as well as the conditions used for starch

J. Juansang et al. / Food Chemistry 131 (2012) 500–507 501

modification. Although the digestibility of chemically modifiedstarch has been studied by many researchers, analyses of the nutri-tional fractions of starch (RDS, SDS and RS) have only recentlydrawn more attention.

Heat-moisture treatment (HMT) refers to the exposure of starchgranules to a temperature above the glass transition temperature,but below the gelatinisation temperature for a certain time periodand at restricted moisture content (below 35%) (Jacobs & Delcour,1998). It is a physical modification technique that is considered tobe natural and safe, when compared to chemical modifications(Lawal, 2005). Watcharatewinkul, Puttanlek, Rungsardthong, andUttapap (2009) and Watcharatewinkul, Uttapap, Puttanlek, andRungsardthong (2010) have shown that HMT canna starch dis-played equivalent pasting properties to starch cross-linked by so-dium trimetaphosphate. When gelatinised, the granules of HMTcanna starches exhibited fewer breakages compared to the nativestarch, and the extent of disintegration decreased with increasingmoisture content of starch during HMT. Therefore, it was supposedthat the cooked HMT starches would have less susceptibility to en-zyme hydrolysis due to their limited gelatinisation.

Most starch consumed by humans has undergone some form ofprocessing, which usually involves heating in the presence of mois-ture, with or without shear, and then cooling. During heat process-ing, the starch granules swell and gelatinise. As a result, themolecular order of the starch granule is destroyed and the starchis easily digested due to increasing accessibility of enzymes tothe starch substrate. Thus, the digestibility of starch dependsstrongly on the degree of gelatinisation, which is governed bythe intrinsic properties of starch, as well as, by gelatinisation con-ditions, such as starch concentration, gelatinisation temperatureand time and heating process. The appearance of gelatinised starchgranules varies from swelled granules, to a combination of starchfragments and dispersed starch molecules, to completely dispersedstarch molecules, depending on the degree of gelatinisation. Underidentical gelatinisation conditions, starch from the same botanicalsource but treated with different modifications might display dif-ferent starch gel morphologies. As a consequence, the key factorsthat determine the RDS, SDS and RS contents in cooked starchare the extent of granule disintegration, which is determined bythe degree of gelatinisation, and the structural change of starchby each chemical/physical treatment.

In this study, canna starch extracted from rhizomes of the edi-ble canna plant (Canna edulis Ker.) was chosen as a starch base formodification, since its paste viscosity is quite stable at high tem-perature and shear force, it has high tendency towards retrograda-tion (Thitipraphunkul, Uttapap, Piyachomkwan, & Takeda, 2003;Wandee, Puttanlek, Rungsardthong, & Uttapap, 2011) and also highresistance to enzyme hydrolysis (Hung & Morita, 2005). Therefore,it would contain some initial amounts of RS and SDS in the nativeform. These fractions were expected to be increased by chemical/physical modifications (acetylation, hydroxypropylation, octenylsuccinylation, crosslinking and HMT). The modified canna starcheswere subjected to heat gelatinisation at various conditions, andRDS, SDS and RS content were analysed. The amounts of these frac-tions were compared and discussed in relation to the degree of gel-atinisation and structural changes of the modified starches. Acommercial RS product (Novelose260) was used as a referencefor comparison.

2. Materials and methods

2.1. Materials

Two varieties of edible canna (Thai-green [TG] and Vietnam[Vt]) were grown on experimental plots at the Rayong Field Crops

Research Center, Rayong, Thailand. Eight-month-old rhizomeswere harvested, and starch was isolated according to a proceduredescribed by Thitipraphunkul et al. (2003). Chemically modifiedcanna starch samples were prepared according to the methods de-scribed in previous reports, as follows: acetylated canna starch(TG-AC; DS = 0.06) (Saartrat, Puttanlek, Rungsardthong, & Uttapap,2005); hydroxypropylated canna starch (TG-HP; DS = 0.02)(Chuenkamol, Puttanlek, Rungsardthong, & Uttapap, 2007); octenylsuccinylated canna starch (TG-OSA; DS = 0.025) (Kweon, Choi, Kim,& Lim, 2001); and cross-linked canna starch (TG-CL; 0.2% w/w so-dium trimetaphosphate) (Emrat, Uttapap, Puttanlek, & Rungsard-thong, 2007). Heat-moisture treated canna starches, namedVt-HMT18, Vt-HMT22 and Vt-HMT25, were treated at 100 �C for16 h, and had moisture contents of 18%, 22% and 25%, respectively(Watcharatewinkul et al., 2009). Novelose260 (N260) was pro-vided by the National Starch and Chemical Co. (Bangkok). Alpha-amylase (type VI-B from porcine pancreas, A-3173, 28 U/mg) andamyloglucosidase (EC 3.2.1.3, from Aspergillus niger, 300 U/ml)were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aglucose assay kit (liquicolor; Ref. No. 10121) was purchased fromHuman GmbH (Wiesbaden, Germany).

2.2. Gelatinisation of starch and light microscopy of starch gel

Starch slurries (10% w/w; 3 g of starch in 27 g of distilled water)were gelatinised at 100 �C for 5, 10, 20 and 40 min using a RVA-3Drapid visco analyzer (Newport Scientific, Warriewood, Australia)with a paddle rotated at a fixed speed of 160 rpm. The starch slurrywas heated from room temperature to 100 �C, at a rate of 3 �C/min,maintained at 100 �C for 5 min (or 10, 20, 40 min), and then cooledto 40 �C at the same rate. One set of freshly prepared starch sam-ples was analysed for SDS and RS. Another set of the gelatinisedsamples was dried at 40 �C for 15 h, and then ground with a mortarand pestle and passed through a 140-mesh sieve. The fine powderobtained was analysed for degree of gelatinisation. The gelatinisedstarch gels were stained with 0.2% I2/KI and observed under a lightmicroscope (E200 Eclipse, Nikon Corp., Tokyo, Japan).

2.3. Determination of degree of gelatinisation (DG) by amylose–iodinecomplex formation

The degree of gelatinisation (DG) was determined according tothe method of Baks, Ngene, van Soest, Janssen, and Boom (2007),with modifications. Each gelatinised starch sample (0.04 g) wasdissolved in 50 ml of 0.15 M KOH, and then centrifuged at5000 rpm for 10 min. One ml of the supernatant was taken andneutralised with 9 ml of 0.17 M HCl. Subsequently, 0.1 ml iodinereagent (1 g iodine and 4 g potassium iodide in 100 ml water)was added, to form a blue complex with the dissolved amylosepresent in the sample. The absorbance was measured at 25 �Cand 600 nm (A1). The same amount of gelatinised starch samplewas dissolved in 50 ml of 0.40 M KOH, and boiled in a water bathat 95 �C for 10 min. The supernatant was neutralised with 0.45 MHCl. After adding 0.1 ml iodine reagent, the absorbance at 25 �Cand 600 nm was measured (A2). The DG was equal to a percentageratio of A1 (0.15 M KOH) to A2 (0.40 M KOH).

2.4. Determination of RDS, SDS and RS

RDS, SDS and RS were determined according to the method ofEnglyst et al. (1992), with modifications. Porcine pancreatic a-amylase (0.357 g) was dispersed in 50 ml sodium acetate buffer(0.1 M, 4 mM CaCl2, pH 5.2), and centrifuged at 5000 rpm for10 min. The supernatant (4.5 ml) was transferred to a beaker,and 0.5 ml of amyloglucosidase (15 U/ml) was added to the solu-tion. This enzyme solution was freshly prepared for each digestion.

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Gelatinised starch samples (100 mg, db), 10 glass beads (0.5 mmdiameter), 5 ml of pH 5.2 sodium acetate buffer and 5 ml of the en-zyme solution were added to an Erlenmeyer flask, mixed for 1 minand then incubated in a shaking water bath (37 �C, 130 strokes/min). After 20 and 120 min of incubation, 0.2 ml aliquots wereadded to 4 ml of absolute ethanol, mixed well and centrifuged at5000 rpm for 10 min. The liberated glucose in the supernatantwas determined using a glucose assay kit. Starch fractions that di-gested within 20 min, between 20 and 120 min, and that remainedundigested within 120 min, were classified as RDS, SDS and RS,respectively.

2.5. Statistical analysis

Unless otherwise stated, analyses of starch characteristics andproperties were carried out in duplicate. Experimental data wasanalysed using analysis of variance (ANOVA), and expressed asmean values. A Duncan test was conducted to examine significantdifferences among experimental mean values (a 6 0.05).

3. Results and discussion

3.1. Starch gel morphology

Gel morphologies of chemically and HMT modified cannastarches gelatinised at 100 �C for 5 and 20 min are shown in Figs.1 and 2, respectively. Gels of native canna starch from differentvarieties (Thai-green variety in Fig. 1, and Vietnam variety inFig. 2) displayed a similar feature. After 5 min gelatinisation, thenative starch granules were broken into small pieces, and the spacesurrounding the granules became blue, due to the complexes ofleached amylose and iodine. The extent of disintegration increased,with an increase in gelatinisation time.

Canna starch modified by hydroxypropylation with a degree ofsubstitution (DS) of 0.02 was the most fragile, compared to theother starch samples (Fig. 1, TG-HP). The granules of hydroxypro-pyl starch were almost completely broken after 20 min gelatinisa-tion, i.e. the granule fragments were hardly observed. Granuleappearances of octenyl succinyl starch (DS = 0.025), after 5 and20 min, were similar to those of the native starch. However, theremaining fragments seemed to be larger than those of the nativestarch (Fig. 1, TG-OSA). Acetylated starch with DS of 0.06 exhibiteda different feature from the other two substituted starches. Itsgranules were obviously less broken, compared to the native andthe other two modified starches (Fig. 1, TG-AC). These morpholo-gies were in accordance with a higher viscosity of hydroxypropylcanna starch and a lower viscosity of acetylated canna starch, com-pared to the native starch, as reported by Chuenkamol et al. (2007)and Saartrat et al. (2005), respectively. On the other hand, starchgranules of cross-linked canna starch were highly swollen, but stillremained in granular form (Fig. 1, TG-CL). This indicated the highresistance of cross-linked canna starch to heat and shear, whichis a typical characteristic of cross-linked starches.

As shown in Fig. 2, gel morphologies of HMT starches were to-tally different from that of the native starch. The HMT starches dis-played more extensive resistance to heat and shear. The HMT18%granules were less broken, compared to the native starch, whilethe HMT22% and HMT25% granules were swollen but unbroken.Gel appearance of HMT starch was similar to that of the chemicallycross-linked starch. A decrease in the extent of disintegration withincreasing moisture content of starch samples, during heat treat-ment, has been reported by Watcharatewinkul et al. (2009). Forthe commercial Novelose260 (HMT high amylomaize), very limitedswelling was observed even after 40 min gelatinisation, indicatingthat the Novelose260 granules were very resistant to gelatinisation.

3.2. Effect of gelatinisation time on DG values

The effect of gelatinisation time on the DG values of 10% (w/w)native and modified starches is shown in Fig. 3. The extent ofgranule disintegration can be indicated by the DG value, i.e. ahigher DG value indicates a greater extent of granule dispersion.As shown in the figure, all starch samples displayed a similar pat-tern: a significant change in the DG value was observed duringthe initial 10 min of gelatinisation, after which the DG value in-creased gradually with gelatinisation time. However, the DG val-ues and magnitude of changes were different among the starchsamples. The native starches from the two varieties – Thai-green(TG) and Vietnam (Vt) – exhibited a very similar profile. The DGvalues of native canna starch from Thai-green and Vietnam vari-eties, gelatinised for 5–40 min were 68.7–84.3% and 67.0–84.6%,respectively.

Acetylated starch (TG-AC), hydroxypropylated starch (TG-HP)and octenyl succinylated starch (TG-OSA) had an increase in DGvalues as compared to the native starch. The DG values of substi-tuted starches conformed to the following order: TG-HP > TG-AC > TG-OSA. After gelatinisation for 40 min, the DG values ofTG-AC, TG-HP and TG-OSA were 92.0%, 94.4% and 87.8%, respec-tively (Fig. 3A). Except for TG-AC, the DG values obtained wereconsistent with the gel morphologies, mentioned in the previoussection. Being less broken after gelatinisation, as revealed inFig. 1, it was expected that TG-AC would have a lower DG valuethan the native starch. However, it was found that TG-AC had aslightly higher DG value, implying that the amylose of TG-ACcould be dispersed and reacted with I2 better than the nativestarch. The substitution groups might facilitate the penetrationand absorption of water into the starch granules by H-bond dis-ruption, by enlarging the inter-space between starch chains,and/or because of the hydrophilic nature of the substituent. Therapid increase in water content inside the granules, as well asan increase in the plasticising effect of water, rendered the mod-ified starch easy to gelatinise. The diverse effects of differenttypes of substitution on gelatinisation, as found in this study,were most probably due to the nature of the substituted groups(molecular size, hydrophobicity/hydrophilicity, functional groups)as well as the degree of substitution.

Cross-linked canna starch (TG-CL) showed a decreased DG valuecompared to the native starch. Extending the gelatinisation timefrom 5 to 40 min resulted in a slight increase of DG value. This indi-cated that the strength of the additional covalent bonds of thecross-linked starch enabled the granules to withstand the gelatin-isation temperature, and that the cross-linking agents helpedmaintain the granular integrity. The DG value of TG-CL gelatinisedfor 40 min was 55.9%.

DG values of Vt-HMT18 were much closer to those of the nativestarch (Fig. 3B), although their gel appearances were significantlydifferent. An increase in moisture content of starch samples duringheat treatment resulted in a lower DG value, i.e. DG values of Vt-HMT25 < Vt-HMT22 < Vt-HMT18. The DG values of Vt-HMT18,Vt-HMT22 and Vt-HMT25 gelatinised for 40 min were 86.8%,74.6% and 69.4%, respectively. The results obtained could be ex-plained in relation to the free amylose dispersed in starch gel, asreported by Watcharatewinkul et al. (2009). They found that amy-lose leaching of HMT canna starch tended to be lower when themoisture content of the starch increased. Therefore, a lesseramount of amylose dispersed in starch gel resulted in poorer inten-sity of the blue colour produced by the amylose–iodine complex.As a consequence, the DG value of heat-treated high-moisturestarch became lower. Novelose260, the commercial HMT high-amylose maize starch, had the lowest DG value (13.6%) amongthe starches tested.

Fig. 1. Gel morphologies of chemically modified canna starches gelatinised, at 100 �C, for 5 min (left) and 20 min (right).

J. Juansang et al. / Food Chemistry 131 (2012) 500–507 503

3.3. Effect of gelatinisation time on SDS and RS contents

Changes in the SDS and RS contents of 10% (w/w) native andmodified starches, with gelatinisation time, are shown in Table 1.In general, RS decreased with an increase in gelatinisation time,while SDS values fluctuated. Native starches from the two varietiesof canna had comparable SDS and RS contents. The initial content

of RS in canna starch was relatively high (88%), but was reducedextensively upon gelatinisation: to 10.1–12.4%, after 40 min gela-tinisation. Gelatinisation at 100 �C, for only 5 min, resulted in amarked decrease of RS in native canna starch (from 88% to 23–26%), signifying the unstable nature of RS in raw canna starch.The RS fraction of Novelose260 changed only slightly withgelatinisation time (from 70.7% in the raw sample to 54.4% after

Fig. 2. Gel morphologies of heat-moisture treated canna starches and Novelose 260 gelatinised, at 100 �C, for 5 min (left) and 20 min (right).

504 J. Juansang et al. / Food Chemistry 131 (2012) 500–507

gelatinisation for 40 min), whereas its SDS contents were quite low(3.8–7.4%) throughout the gelatinisation time.

3.4. RS contents

Regardless of the modification methods used, the RS contents ofthe modified starches were lower than that of the Novelose260.Among the modified starches, OSA starch displayed the highest

RS content at all gelatinisation times, and its RS content was notmuch different from the Novelose260. All substituted cannastarches had a higher resistance to enzyme hydrolysis, comparedto the native starch. The resistance conformed to the following or-der: OSA starch > HP starch > AC starch. The results suggested thatthe nature of the substituted groups had more influence on resis-tance to enzyme hydrolysis than the degree of gelatinisation ofstarch granules. The most obvious case was the HP starch: its gran-

0

20

40

60

80

100

0 10 20 30 40

% D

egre

e of

gel

atin

izat

ion

Gelatinization time (min)

TG-HP

TG-AC

TG-OSA

Native TG

TG-CL

0

20

40

60

80

100

0 10 20 30 40

% D

egre

e of

gel

atin

izat

ion

Gelatinization time (min)

Vt-HMT18

Native Vt

Vt-HMT22

Vt-HMT25

N 260

(A)

(B)

Fig. 3. Effect of gelatinisation time on the DG values of 10% (w/w) native andmodified canna starches ((A) chemically modified starches, (B) heat-moisturetreated starches).

J. Juansang et al. / Food Chemistry 131 (2012) 500–507 505

ules exhibited much more complete breakage than the native andacetylated starches, but its RS content was higher. There was also atendency that, as the molecular sizes of the substituted group be-came larger, the modified canna starch became more difficult tohydrolyse. The results obtained in this study were similar to those

Table 1SDS and RS contents of 10% (w/w) native, chemically modified and physically modified ca

%SDS

Gelatinisation time (min) 0 5 10 20

Chemical modificationTG y8.8a z11.5e x5.6f w2.0f

TG-AC x2.9h yz15.7b y14.4b y13.7b

TG-HP w5.1d y14.0c x13.0c y14.2b

TG-OSA w7.2c z23.4a y17.6a x12.7c

TG-CL w3.9f x8.6g x9.5e y17.9a

Physical modificationN260 z7.4b x5.2h w3.8g y6.1e

Vt y4.4e w2.4i x3.7g w2.2f

Vt-HMT18 y2.9g z10.5f x5.6f w0.5g

Vt-HMT22 x2.2i z5.5h z5.6f xy2.8f

Vt-HMT25 v0.6j z13.0c y11.3d x9.9d

Means with different letters (a, b, . . .) in the same column are significantly different (p 6Means with different letters (x, y, . . .) in the same row are significantly different (p 6 0.

reported in previous literature. Chung et al. (2008) found that theamounts of RS in gelatinised corn starches were 19.5%, 14.3%,13.0% and 7.0% in hydroxypropylated, acetylated, oxidised andcross-linked starches, respectively, whereas the unmodified starchcontained 7.3%. Hoover, Hannouz, and Sosulski (1998) and Woot-ton and Chaudhry (1981) observed that hydroxypropylation de-creased the enzyme susceptibility of pea and wheat starches,even after the starches were pasted. Östergaard et al. (1988) alsofound that substitution with acetyl or hydroxypropyl groups re-duced the enzyme susceptibility in gelatinised starches. They alsosuggested that the hydroxypropyl groups had a greater effect thanthe acetyl groups on enzyme resistance, because the bulkierhydroxypropyl groups provide greater hindrance to attack by en-zymes (Hoover & Sosulski, 1986; Wootton & Chaudhry, 1981). Ithas also been suggested that the presence of bulky substituentson C2 of the glucose unit would sterically hinder the proper posi-tioning of the substrate into the active site of a-amylase, and alsoeffectively restrict enzyme attack on the adjacent glycosidic bondsof unsubstituted glucose residues (Hoover & Sosulski, 1986; Hoo-ver & Zhou, 2003; Östergaard et al., 1988).

Cross-linked canna starch had the highest RS content in its rawstate (94.1%), but when cooked, the RS content was reduced rapidlywith extended gelatinisation time. The remaining RS after gelatin-isation for 40 min was 19.8%. Except for few cases, the RS contentsin cross-linked starch were lower than in the substituted starches,although its starch granules were obviously less broken. Again,these data confirmed that the susceptibility of starch to enzymehydrolysis was influenced more by the substituted groups in starchmolecules, than by the degree of disintegration of starch granules.

Raw HMT canna starches contained considerably high amountsof RS (92.2–94.8%); however when cooked, their RS was reducedmarkedly. At the same period of gelatinisation, the RS contents ofall HMT starches were lower than that of the native starch. Themost obvious case was Vt-HMT18; even after only 5 min of gelatin-isation, the RS content in Vt-HMT18 was decreased to 3.1%, and italmost disappeared (0.2%) after 40 min gelatinisation. The resultobtained was contrary to our presupposition that the HMT starchwould have a significantly higher resistance to enzyme hydrolysis,due to its limited gelatinisation (in other words, would contain ahigher amount of RS), compared to the native starch. Being keptin a granular form, with less dispersion of HMT starch granulesafter gelatinisation (as shown in Fig. 2), would seemingly obstructthe accessibility of enzymes to amylose/amylopectin inside thestarch granules. However, the results showed that HMT converselyfacilitated the susceptibility of the treated granules to enzymehydrolysis. This was possibly due to the degradation of starch

nna starches.

%RS

40 0 5 10 20 40

w2.1f z88.2g y22.9f x20.4g w18.1g v12.4f

z16.7b z91.4f y35.9d x33.8e w32.2d v26.6d

z16.2b z93.4d y45.5c x43.5c w37.6c v32.0c

x12.5c z92.2e y54.2b x51.3b w49.4b v45.3b

z23.6a z94.1c y36.6d x35.3d w25.4e v19.8e

z7.2e z70.7i y62.5a x60.1a w56.9a v54.4a

z10.3d z88.1h y26.1e x24.7f w21.6f v10.1g

w0.4g z94.4b x3.1h w1.6j y3.8j v0.2i

x1.5f z94.8a y22.6f x12.5i w5.5i v1.9h

w6.9e z92.2e y19.1g x17.3h w12.8h v10.5g

0.05).05).

Fig. 4. Relationships between DG value and RS content of 10% native and modifiedcanna starches ((A) chemically modified starches, (B) heat-moisture treatedstarches).

506 J. Juansang et al. / Food Chemistry 131 (2012) 500–507

molecules by HMT, or because the swelling state of HMT starchgranules might accelerate the sequential catalytic action of the en-zyme by increasing the probability of substrate–enzyme binding.As the moisture in starch samples during HMT increased, in mostcases (but not all) the RS content in HMT starch was found to in-crease (Vt-HMT25 > Vt-HMT22 > Vt-HMT18). This indicated thatthe extent of hydrolysis is co-influenced by many factors, such asthe physical appearance of starch granules, their molecular struc-ture and/or the capability of starch–enzyme binding. The effectof HMT on susceptibility of canna starch to enzyme hydrolysiswas different from those reported previously on other starches.Brumovsky and Thompson (2001) found that HMT high-amylosecorn starch contained 43.9% resistant starch, compared to 18.4%in native starch. Güzel and Sayar (2010) reported that RS andSDS contents in cooked (in a boiling-water bath for 20 min) HMTbean starches were significantly higher than those in correspond-ing native starches. According to Chung, Liu, and Hoover (2009),when the starch was heat-moisture treated at 120 �C for 2 h, theRS content of corn, pea, and lentil starches increased from 4.6%,5.2% and 5.3% to 12.3%, 16.4% and 15.7%, respectively, as comparedto gelatinised unmodified starches.

3.5. SDS contents

All starch samples displayed fluctuations of SDS content withgelatinisation time, because it could be transformed from RS orchange into RDS. Native canna starch and Novelose260 containedless than 10% SDS, whereas acetylated and hydroxypropylatedstarches had considerably higher amounts of SDS (13–17%), at allgelatinisation times. The SDS contents of cooked octenyl succiny-lated and cross-linked starches varied between 12.5–23.4% and8.6–23.6%, respectively. The SDS content of octenyl succinylatedstarch was high at the initial period of cooking, but became loweras the cooking time was prolonged. The opposite was found withthe cross-linked starch, i.e. the SDS content increased with longercooking time. When the sum of RS and SDS was taken into consid-eration, it was found that the RS + SDS of octenyl succinylatedstarch was a bit less than, or similar to, that of the Novelose260,and was somewhat higher when the starch was cooked for a shortperiod of time (5–10 min). The RS + SDS of octenyl succinylatedstarch after cooking for 5 and 10 min were 77.6% and 68.9%,whereas those of Novelose260 were 67.7% and 63.9%, respectively.

The HMT starches (Vt-HMT18, Vt-HMT22 and Vt-HMT25) con-tained much lower contents of SDS (0.6–13.0%) than the chemi-cally modified starches (2.9–23.6%). Their SDS decreased withincreasing gelatinisation time (5–40 min). The SDS of HMT starcheswas higher than the native starch after cooking for a short period(5–10 min), but became lower when the cooking time was ex-tended to 20 and 40 min – except in the case of Vt-HMT25 gelatin-ised for 20 min, which still contained a higher amount of SDScompared to the native starch.

3.6. Relationship between DG value and RS content

Relationships between DG value and RS content of 10% nativeand modified starches are shown in Fig. 4. RS contents in all samplesdecreased with an increase in the degree of gelatinisation. The de-crease of RS content in Thai-green canna starches displayed a fairlylinear function of the DG values (R2 = 0.992). This indicated that theRS content in native starches depended mainly on the level of starchgranule disintegration. In spite of substitution by an acetyl group,acetylated canna starch also showed a linear relationship of RS con-tent with DG value (R2 = 0.997); however, the slope was much lowerthan that of the native starch. This result suggested that the acetylgroup could impede the susceptibility of starch to enzyme hydroly-sis. The effect of the substitution group was more clearly observed in

hydroxypropyl starch, which displayed a lower slope than nativeand acetylated starches. However, the RS content dropped rapidlywhen the DG value was higher than 90%. Some reports have indi-cated that the amorphous regions (amylose and branch points ofamylopectin) are more accessible to the hydroxypropylating re-agent, and thus are modified to a greater extent than the crystallineregions (amylopectin) of starch granules. It is generally acceptedthat amylose molecules are leached out first at the early stage of gel-atinisation. Therefore, at a lower degree of gelatinisation (<90%), theamylose molecules (which had a higher tendency than the amylo-pectin to carry the hydroxypropyl groups) would be the majorityin the starch dispersion. Consequently, at this stage the hydroxypro-pyl starch exhibited a higher resistance to enzyme hydrolysis, com-pared to the corresponding native starch. Above 90% DG, a greaterextent of starch dispersion, as well as the increased populations ofunmodified starch chains in the dispersion, rendered the starch mol-ecules more easily accessed by the enzyme, thus the RS contentdropped rapidly. Octenyl succinylated starch displayed a trend sim-ilar to the hydroxypropyl starch, but its DG values were lower.

J. Juansang et al. / Food Chemistry 131 (2012) 500–507 507

The change in RS content of cross-linked starch with the DG val-ues was slightly higher than that of the native starch at the earlystage of gelatinisation (<50%), but a significant difference was ob-served after that. A significant reduction in the digestibility of starchcross-linked by a phosphate bridge was also reported by Sang andSeib (2006) and Woo and Seib (2003). It is still uncertain why thecross-linked starch displayed higher susceptibility to enzymehydrolysis. Typically, cross-linked starch can resist pasting and willmaintain its granular integrity better than native starch upon gela-tinisation. It might be possible that the enzyme is able to accessstarch molecules more readily when starch granules are in a swollenstate. Additionally, at higher degrees of gelatinisation the phosphatediester bonds could be destroyed, and the resulting phosphatemonoester might enhance the binding/catalysing capabilities ofthe enzyme to the substrate. The decrease in RS content of cross-linked starch was accompanied by an increase of SDS. Within thegelatinisation period of 5–40 min, the summation of RS and SDScontents of cross-linked starch was nearly constant (Table 1), indi-cating that most of the RS reduced was transformed into SDS.Cross-linked starch contained the highest amount of SDS (16.5–23.1%) among the starches gelatinised for 20–40 min. Also, it had atendency to have a higher SDS content with extended gelatinisationtime.

Native Vietnam cv. canna starch showed characteristics similarto the native Thai-green cultivar. The RS content of all HMT starchesdisplayed a significantly higher change with the DG value, com-pared to the native starch. For Novelose260, the RS content declinedlinearly with the DG value, although its DG values were very limited(less than 20%). Changes in SDS contents of starch samples with theDG values were in an irregular pattern (Figure not shown). It is dif-ficult to estimate the SDS content, at a particular degree of gelatin-isation, because SDS can be increased by transformation of RS intoSDS, and can also be decreased by transformation of SDS into RDS.

4. Conclusions

Octenyl succinylation has shown to be a promising method forpreparation of a high-RS and SDS product from a potential sourceof raw material, canna starch. The sum of its RS and SDS was a bitless than, or similar to, that of the Novelose260, and was somewhathigher after a short period of cooking. The RS content of octenylsuccinylated canna starch was reduced only slightly by gelatinisa-tion, even at 40 min gelatinisation. The results from this studyshowed that heat-moisture treatment was not an efficient meansof producing a high-RS product from canna starch. However,starches from other sources, or starches treated by using otherHMT conditions, might be examined to confirm this conclusion.

Acknowledgements

We gratefully acknowledge the Thailand Research Fund, Thai-land (TRF-MAG Project MRG-WII525S057) and the Higher Educa-tion Research Promotion and National Research UniversityProject of Thailand, Office of the Higher Education Commission,for their financial supports.

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