Comparative Study on the Starch Noodle Structure of Sweet Potato and Mung Bean

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JFS C: Food Chemistry and Toxicology

Comparative Study on the Starch NoodleStructure of Sweet Potato and Mung BeanH-Z TAN, W-Y GU, J-P ZHOU, W-G WU, AND Y-L XIE

ABSTRACT: Fine structure of sweet potato starch (SPS) and mung bean starch (MBS) by gel-permeation chromatog-raphy (GPC) showed that the amylose in SPS and MBS had 9.0 and 1.8 chains, respectively. The long chains of amy-lopectin in MBS (Ap-MB) were longer than those of amylopectin in SPS (Ap-SP), but the short chains of Ap-SP wereshorter than those of Ap-MB. The structures of starch noodles of sweet potato (SPSN) and mung bean (MBSN) wereanalyzed by GPC, scanning electron micrograph (SEM), differential scanning calorimetry (DSC), and X-ray diffrac-tion after hydrolysis by acid and enzymes. The results showed that the residues obtained with acid and enzymes inMBSN contained large amounts of high molecular weight fractions, and a relatively small amount of low molecularweight fractions, whereas those in SPSN contained some high molecular weight fractions and large amounts of lowmolecular weight fractions. SPSN exhibited higher digestibility by HCl, α-amylase, β-amylase, and pullulanase thanMBSN. The surface of MBSN was more smooth than that of SPSN and the inside of MBSN contained long, thick, andorderly filaments, while there were many pore spaces inside SPSN from SEM. The DSC thermogram of the resistantresidues from both starch noodles after acid/enzyme hydrolysis showed a broad endotherm peak near 100 ◦C (96to 115 ◦C) due to the presence of the complexes of amylose-lipid and lipid-(long chains in amylopectin). Becauseof a lower content of branched amylose and a higher content of amylopectin in SPS, the structure of SPSN had aless distinct crystalline pattern and higher adhesiveness, whereas there was a higher content of amylose with a littlebranch and moderate amylopectin in MBS. Thus, the structure of MBSN had a stronger distinct crystalline patternand good cohesiveness.

Keywords: hydrolysis, mung bean, starch, starch noodle, structure, sweet potato

Introduction

Sweet potato starch noodle (SPSN), produced from purified sweetpotato starch, is one of the principal starch products in China.

However, sweet potato starch noodle is dull, opaque, and moder-ately elastic, and has high cooking loss and swelling when cooking.Numerous studies have dealt with the properties of sweet potatostarch (SPS) and the quality of SPSN (Collado and Corke 1997; Col-lado and Corke 1999; Zhang and Oates 1999; Chen and others 2003).Some attempts have been also made to improve the quality of SPSN(Baek and others 2001; Collado and others 2001). Why does SPSNhave poor cooking quality compared to transparent, glossy, and elas-tic mung bean starch noodle (MBSN)? Traditionally, these differ-ences in the quality of starch noodles have been attributed to thecontent of amylose (Cheng and Shuh 1981), the ratio of amyloseand amylopectin (Kim and others 1996), fat and protein in starch(Kim and others 1996), and starch granule size (Chen and others2003). However, chemical structures of both starches, such as amy-lose molecular size, chain length, and branched property of amyloseand amylopectin, also differ. Mestres and others (1988) and Xu andSeib (1993) investigated the structure of MBSN by hydrolyzing MBSNwith acid and enzymes, and then described MBSN as a ramified 3-dimensional network held together by short segments of stronglyretrograded amylose that melts at temperatures above the boilingpoint of water.

MS 20060184 Submitted 3/30/2006, Accepted 7/24/2006. Authors Tan, Gu,and Xie are with School of Food Science and Technology, Southern YangtzeUniv., Wuxi, Jiangsu, P. R. China, 214036. Authors Tan, Zhou, and Wu arewith School of Food Science and Technology, Hunan Agricultural Univ., Hu-nan Changsha, P. R. China, 410128. Direct inquiries to author Gu (E-mail:[email protected]).

The structure of mung bean starch noodles has been partly un-derstood; however, the structure of sweet potato starch noodles hasnot received enough scientific attention. An understanding of thestructure of SPSN is a prerequisite to undertaking additional effortsto improve its quality. Thus, the aim of this study was to investi-gate elaborately the structure of SPSN by utilizing the methods ofMestres and others (1988) and Xu and Seib (1993), to use MBSN forreference, and to combine the analysis of the properties of SPS andmung bean starch (MBS). The systematic study on the structure ofboth starch noodles would be useful and will serve as a foundationfor improving the quality of Chinese sweet potato starch noodles.

Materials and Methods

MaterialsSweet potato, XuShu18 variety, used in this research is the most

popular variety in China, cultivated in many provinces, and was ob-tained from a local benefactor in Xuzhou, Jiangsu, P. R. China. Themung bean, QingDou2 variety, used in this research was purchasedfrom a local supermarket in Wuxi, Jiangsu, China. Alpha-amylase(TypeII-A, 9000 units per milligram of solids), β-amylase (E.C. nr3.2.1.2, 10 units/mg solid), and pullulanase (E.C. n 3.2.1.41; 12200units/mL) were purchased from Sigma Chemical Co. (St. Louis,Mo., U.S.A). Sepharose CL-2B, Sephadex G-50, G-100 and standarddextrans (T-series) were from Pharmacia Fine Chemicals, Uppsala,Sweden.

Starch isolationStarches from sweet potato and mung bean were isolated accord-

ing to the method of Gonzalez-Reyes and others (2003).

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Comparative study on the starch noodle structure . . .

Preparation of starch noodlesStarch (1.2 g) was cooked in 10 mL of distilled water in a stainless

steel bowl using a double boiler to prepare starch paste and used asdough binder. The cooked gelatinized starch (starch paste) was thenmixed with 25 g of starch and water calculated to give 50% moisturecontent in the dough and then mixed and stirred at the rate of 100 r/min in a blender (SM-25, Sinmag B.M. Co. China) for about 10 min todistribute water evenly and to obtain a smooth ball (starch dough)that does not stick to the hands. The starch dough was extrudedusing a 50-mL syringe into boiling water for 30 s, transferred to coldwater, and drained. Strands were separated and hung to partiallydry, kept at 4 ◦C for 2 h and −10 ◦C for overnight, dried at 40 ◦C inconvection dryer, and cooled to room temperature. Triplicates perbatch were analyzed.

Hydrolysis of starch noodlesAnalysis of starch noodles was made according to the method of

Xu and Seib (1993) with modifications. Starch noodles from sweetpotato and mung bean (1 cm, 18 g) were soaked separately overnightin 0.8% aqueous sodium azide (250 mL) at 25 ◦C. Both mixtures wereheated in a boiling water bath for 1 h, cooled to 25 ◦C, and thenmilled (Model SHG262 Shunhui ELC CO. China) to pass through a0.5-mm sieve. After an equal volume of 2 M aqueous HCl was added,each sample was maintained at 35 ◦C in a water bath and gentlyshaken once a day for up to 20 d. An aliquot (0.1 mL) of a digest wastaken at various times and made to 10 mL volume with water. Aftercentrifuging, triplicate aliquots of the supernatant were assayed fordegree of hydrolysis. Degree of hydrolysis (hydrolysis percent) wasdefined as (Zhang and Oates 1999):

Hydrolysis percent =Reduced sugar provided by enzyme/acid hydrolysis × 100%

Reduced sugar produced by acid hydrolysis∗

Reducing sugar was determined as outlined by Somogyi (1952)and using D-glucose as a standard. Acid hydrolysis∗ was carried outby treating starch noodle with HCl (1 g starch noodle mixed with 20mL 1N HCl) at 100 ◦C for 2 h.

In a similar manner, boiled and milled samples were mixed with0.2 M phosphate buffer (pH 6) containing α-amylase (9000 units).Each mixture was maintained at 25 ◦C for up to 20 d. In anotherdigestion, a mixture of β-amylase (E.C. nr 3.2.1.2, 10 units/mg solid)and pullulanase (E.C. nr 3.2.1.41; 12200 units/mL) was used to digestthe dispersions in 0.01 M acetate buffer (pH 3.8) at 25 ◦C for 60 h.

At the end of both the acid and enzyme digestion, the mixtureswere independently centrifuged, the supernatant discarded, and theresidue rinsed with water. Those steps were repeated 4 times, andthe residue was dried under vacuum. The dried residues were ana-lyzed by gel-permeation chromatography (GPC), differential scan-ning calorimetry (DSC), and X-ray diffractometry.

Starches analysisAmylose content measurement. Absolute amylose contents

were calculated following the Kasemsuwan and others (1995)method. Analysis was replicated at least 3 times.

Differential scanning calorimetry (DSC). DSC analysis wasperformed with a DSC instrument (DSC-7, Perkin-Elmer Co., Nor-walk, Conn., U.S.A.). SPS, MBS, and their amylose and amylopectinwere each weighed into aluminum pans (Perkin Elmer Co.). Deion-ized water was added by micropipette to achieve a mixture with arequired water content of 70% (w/w) in starch; that is, a water-to-starch ratio of 2:1 was used in all DSC runs. The sample pans were

sealed and equilibrated at room temperature for 24 h before analy-sis. The samples were heated at 10 ◦C/min over a temperature rangeof 10 to 180 ◦C using an empty pan as a reference.

Gel-permeation column chromatography. Sweet potatostarch and mung bean starch (20 mg) were solubilized in 90% (v/v)dimethyl sulfoxide (DMSO, 4.0 mL) by heating the dispersion ina small beaker at 90 ◦C with continuous stirring for 16 h using aheating block with magnetic stirrer (SH23-2, Meyinpu InstrumentCo., Shanghai, China). Samples were then diluted to 25 mL. Thesolubilized starch was centrifuged (3000 × g) and filtered througha 0.45-µm filter (Carptoon Filter Co. Zhejiang, China) beforechromatographic analysis. Size-exclusion chromatography wasdone on a Sepharose CL-2B gel (22 i.d. × 900 mm) (PharmaciaLKB, Uppsala, Sweden). The columns were run in the ascendingmode. A sample solution (5 mL) was injected into the column.The eluant was a NaCl aqueous solution (0.02%) with a flowrate of 16 mL/h. Fractions of 4 mL each were collected andsubjected to total carbohydrate analysis with a phenol-sulfuricacid reaction measured at 490 nm (Dubois and others 1956). Theexcluded and total volumes of the column were calibrated usingDextran T-2000 and D-glucose, respectively. All chromatogramswere done in triplicates. The log molecular weight comparedwith elution-volume plot for the dextran standards was linear(r2 = 0.9977).

Fractionation of amylose and amylopectin from bothstarches. Amylose and amylopectin of sweet potato starch (Am-SP, Ap-SP) and mung bean starch (Am-MB, Ap-MB) were fractionedaccording to Jideani and others (1996). Starches were defatted by3 replicates of dissolution in hot dimethyl sulfoxide solution andprecipitation with ethanol to remove trace amounts of lipids, whichinterfere with complete dispersion. Fractionation of defatted starch(10 g) into amylose and amylopectin was performed by the methodof Lansky and others (1949) with modifications (Jane and Chen1992). Amyloses were purified by ultracentrifugation, followed by re-peated recrystallization from 10% aqueous 1-butanol. Ultracentrifu-gation was required to remove microgel-like amylopectin (MGA)from amylose. Significant quantities of MCA were obtained fromboth starches.

Fine structure of amyloses and amylopectins. Purity of amy-loses and amylopectins was determined using columns (22 i.d. ×900 mm) packed with Sepharose CL-2B gel (Pharmacia LKB) ac-cording to the method mentioned above for gel-permeation columnchromatography of both starches. Fine structure of amylose andamylopectin from SPS and MBS was determined according to themethod of Yuan and others (1993) and Takeda and Hizukuri (1987).Starch samples (10 mg) were added to 10 mL 0.05 M sodium acetatebuffer (PH 3.5) containing 0.2 mL pullulanase solution (81 units/mLof 0.05 M sodium acetate buffer, PH 3.5) and then incubated at 37◦C in a water bath shaker for 1 d. Under these conditions, the amy-loses and amylopectins were debranched completely. The samplesolutions (0.5 mL) were diluted with water (9.5 mL), centrifuged,and filtered through a 0.45-µm syringe filter (Carptoon Filter Co.).An aliquot (5 mL) of the mixture was immediately injected into aSephadex G-50 (17 × 700 mm). Components were eluted with 0.1M aqueous potassium hydroxide at a flow rate of 0.5 mL/min inthe ascending direction. Fractions (3.5 mL per tube) were collectedand analyzed for total carbohydrate (Dubois and others 1956) andreducing power (Somogyi 1952), from which degrees of polymer-ization (DP) were calculated. The excluded and total volumes ofthe column were calibrated using Dextran T-2000 and D-glucose,respectively. All chromatograms were done in triplicates.

Mean chain length (CL) and mean degree of polymerization (DP)of both starches were determined according to the methods of

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Madhusudhan and Tharanathan (1996). The number of chains permolecule is DP/CL.

The β-amylolysis limit was determined by hydrolyzing the speci-mens at 37 ◦C for 4 h in 20 mM acetate buffer (pH 4.8) with 25 U (µmolof matose released per min, at 37 ◦C and pH 4.8) of β-amylase permg of substrate (Takeda and others 1984). The addition of the pul-lulanase to a β-amylase hydrolysate (25 U/mg, 35 ◦C, 3 h), withoutinactivation of β-amylase, resulted in the complete degradation ofthe amyloses and amylopectins (Takeda and Hizukuri 1987). Percentβ-amylolysis (or percent β-amylolysis with pullulanase) = [Reduc-ing capacity (as maltose)] ×100/[Total carbohydrate (as maltose)].

Starch noodle analysesScanning electron microscopy (SEM). After both dry starch

noodles were cross split with a sharp blade, the surfaces and the crosssections were mounted on specimen holders with colloidal graphiteand then sputter-coated with gold, respectively. Both starch noodleswere examined by scanning electron micrograph (Sem Quanta-200,FEI Co., Eindhoven, The Netherlands) at an accelerating voltage of15 kV. Photomicrographs were taken on Agfapan-Apx 100 films.

Gel-permeation chromatography. Gel-permeation chro-matography was done on a Sephadex G-100 (17 × 700 mm).The samples were pretreated according to the method of Xu andSeib (1993). The residues (10 mg) isolated after acid and enzymedigestion of cooked starch noodles were dissolved in 0.5 mL of 2Maqueous potassium hydroxide at 25 ◦C. After each solution wasdiluted with water (9.5 mL), the latter steps were done according tothe method mentioned above for amylose and amylopectin.

Figure 1 --- Sepharose CL-2B elution profiles of defattedand deproteined starches from sweet potato (SPS) andmung bean (MBS)

Table 1 --- Properties of starch fractions from sweet potato and mung bean

Sweet potato starch Mung bean starch

Fractions Ap IM Am Ap IM Am

Absolute amylose content (%)a --- --- 28.9 ± 0.35 --- --- 33.7 ± 0.41Yield (%) 67.0 ± 0.33 11.0 ± 0.13 22.0 ± 0.29 56.6 ± 0.38 4 ± 0.09 39.4 ± 0.26Average molecular weight (Da) 2.23 × 107 8.96 × 104 3.28 × 105 1.82 × 107 1.54 × 104 7.25 × 105

± 508 ±114 ± 93 ±606 ±235 ±89DP ∼137600 553 2030 ∼112300 95 4500β-amylolysis limit (%) 60 ± 3.1 --- 93 ± 5.3 76 ± 5.7 --- 98 ± 5.0β-amylolysis with pullulanase (%) 99 ± 3.5 --- 99 ± 2.7 100 ± 4.0 --- 99.5 ± 2.2CL 60 --- 226 130 --- 2500Number of chains per molecule --- --- 9.0 --- --- 1.8

a w/w, on a starch basis.Data were reported in means ± standard deviation.The number of chains per molecule, DP/CL.Ap = amylopectin; Ax = intermediate fraction; Am = amylose; --- = not determined; CL = average unit chain length value; DP = average degree of polymerization.

Differential scanning calorimetry. Both original starch noo-dles and their hydrolysis residues were dried and milled. DSC anal-yses were performed in accordance with the method mentionedabove for starches.

X-ray diffractometry. The dried starch noodles (8% moisture)and the vacuum-dried residues (4% moisture) produced by acid orenzyme hydrolysis were milled into powder in a mortar prior toanalysis. Their X-ray diffraction patterns were determined with anAustralia X-ray diffractometer (Bruker D8S AXS). The operating con-ditions for the diffractometer were copper Ka radiation; high tensionvoltage, 35 KV; current, 20 mA; chart speed, 5 mm/min; scanning an-gular velocity, 2 ◦θ/min; 2θ = 2 to 40 ◦.

Results and Analysis

Fine structure of SPS and MBSGel-permeation chromatography of both starches. SPS and

MBS separated into 2 major peak fractions (amylopectin and amy-lose) and a minor peak of intermediate materials (IM) as shown inFigure 1. The large molecular size amylopectin molecules of bothstarches apparently eluted as a single symmetrical peak at or nearthe void volume of the column, indicating that the fractions were freefrom other low molecular weight contaminants, whereas the amy-lose molecules, which were smaller, eluted at 144 mL and 120 mLfor SPS and MBS, respectively. Table 1 shows the properties of starchfractions from sweet potato and mung bean. The Ap-SP possessed amolecular weight of 2.23 × 107 Da, corresponding to approximately137600 (DP) which was characteristic of hydroglucose residues. Incomparison, the Ap-MB possessed a molecular weight of 1.82 × 107

Da, which corresponded to a chain length mass of approximately112300 (DP) characteristic of hydroglucose residues, and impliedsmaller amounts of branches for the Ap-MB fraction than the Ap-SPfraction.

The intermediate materials, IM, has been described as a mixtureof very short linear amylose chains and branched, either normalor long-chained amylopectin (Eliasson 2004). In our research thepresence of IM in SPS was discernible in GPC analysis by its elutionat 128 mL along with a 2nd peak of Am eluting at 144 mL, whilethe presence of IM in MBS was also discernible in GPC analysis byits elution at 104 mL along with a 2nd peak of Am. The IM-SP was,however, present in higher yields (approximately 11.0%) than IM-MB (approximately 4.0%). Wang and others (1993) also found IMwas fractionated by GPC on Sepharose CL-2B and found to be abranched component smaller than the amylopectin but in differentproportions depending on the type of mutation.

Amylose in SPS and MBS. The absolute amylose content in MBSwas 33.7%, greater than that of SPS (28.9%) (Table 1). This could be

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attributed to Ap-MB having longer peak chain length of long branchchains than Ap-SP (DP 40 compared with DP35 at peak (Figure 2).Amylose is the most important factor affecting the starch gel strengthbecause of its prompt association and retrogradation and its inter-action with lipids to form helical complex and with amylopectin togive strong gel networks (Jane and Chen 1992).

The maximumβ-amylolysis limits for Am-SP and Am-MB were ap-proximately 93% and approximately 98%, respectively (Table 1). Theincomplete β-amylolysis of both amyloses was most likely due tothe presence of a relatively small number of α-(1→6) linkages in themolecule. Both amylose molecules possessed 9.0 and 1.8 chains withvarious chain lengths 226 and 2250 for Am-SP and Am-MB, respec-tively, indicating that the Am-MB contained low molar fractions ofbranched molecules whereas the Am-SP contained a high molar frac-tion. Most amylose comprised 3 to 5 chains per molecule on average,whereas Hizukuri and others (1981) reported that potato, tapioca,and kuzu amylose were composed of approximately 9 to 20 chains.Our results agree with those of Takeda and Hizukuri (1987), who alsoreported that sweet potato amylose was composed of 9.8 chains. Itwas improbable that the branched structure was due to the smallproportion of accompanying amylopectin, as the amylose sampleswere recrystallized 6 times using 1-butanol in our experiment. Theβ-amylolysis with pullulanase for Am-SP and Am-MB were approxi-mately 99% and 99.5%, respectively (Table 1), suggesting that thesechains were joined by α-(1→6) linkages, since the amyloses werecompletely degraded into maltose with β-amylase and pullulanase(Takeda and Hizukuri 1987).

Amylopectin in SPS and MBS. Debranching of both Ap-SP andAp-MB gives elution profiles as shown in Figure 2. Upon pullulanasedebranching, amylopectins from SPS and MBS were separated bythe Sephadex G-50 system into distinct peak fractions consisting ofslightly high molecular weight linear amylose (119 mL of elution vol-ume) and a range of lower molecular weight linear oligosaccharides.Although the boundaries were somewhat arbitrary due to overlap-ping of the peaks, the fractions were designated as A, B1, B2, B3, andB4 in reverse order of elution. The distribution profiles indicated that

Figure 2 --- Sephadex G-50 elution profiles of debranchedamylopectin from SPS and MBS using pullulanase. Thenumber in parenthesis indicates DP, average degree ofpolymerization.

Table 2 --- The corresponding DP and proportion of these fractions of Ap-SP and Ap-MB

F1 F2

B4 B3 B2 B1 A

Fractions DP ratio (%) DP ratio (%) DP ratio (%) DP ratio (%) DP ratio (%)

Ap-SP 35 27 28 38 21 20 15 10 8 5Ap-MB 40 10 35 27 28 38 21 22 15 3

Ap-SP = amylopectin in sweet potato starch; Ap-MB = amylopectin in mung bean starch; DP = average degree of polymerization.

the fraction F1 was composed of fractions B2, B3, and B4, and thatfraction F2 contained fractions A and B1. The corresponding DP andproportion of the whole amylopectin of these fractions are listed inTable 2. The resolution of the linear oligosaccharide peak fractionsrevealed 5 populations for Ap-MB and Ap-SP of chain length dis-tributions from the amylopectin molecules. Ap-SP contained moreshort chains than long chains. The chemical analysis indicated thatthe long chains of Ap-MB (DP40) were longer than Ap-SP (DP35), butthe short chains of Ap-SP (DP8) were shorter than those of Ap-MB(DP15).

The distribution characteristics of the chain lengths (Figure 2 andTable 2) are consistent with the cluster models of French (1972). Thegeneral features of these models are that amylopectin is composedof compact parts of oriented chains (clusters) which are randomlyor somewhat regularly branched, and that the clusters are linkedby long chains that extend into 2 or more clusters. Consequently,the chain lengths are distributed in a polymodal manner with pe-riodic peaks at multiple lengths between 2 adjacent clusters. Theprofiles shown in Figure 2 reveal such periodical distributions andcan be interpreted according to the cluster structure models with thefollowing assumptions: (1) fractions A and B1-B4 are the A- and B-chains, respectively, which bind at C-6 of the other chains throughtheir reducing residues (the A-chains carry no chains and the B-chains carry the A- or other B-chains); (2) the chains in fractions Aand B1 make a single cluster; (3) the chains in fractions B2 and B3extend into 2 and 3 clusters, respectively, and the chains in fractionB4 stretch across more than 4 clusters (Hizukuri 1986). It is acceptedthat the crystalline domains of starch granules appear to be com-posed of A-chains and the exterior parts of B-chains. Some 22 to25 chains may be included in a single cluster, assuming that the pe-riods (99 to 110 A) seen on X-ray small-angle diffraction correspondto the distance between clusters and are based on the packing of thedouble helical chains in crystalline cells (French 1972).

Starch noodle analysesGel-permeation chromatography. Gel-permeation chro-

matography of the acid- or enzyme-resistant molecules in bothcooked starch noodles is shown in Figure 3. The gel-permeationchromatography of the acid-resistant molecules (Figure 3a) in SPSNshowed 5 peaks with DP68, 55, 49, 41, and 22, respectively, whereasthose in MBSN showed 2 peaks with DP68 and 49. This impliesthat retrograded amylopectin in SPSN was degraded partly toshorter chain segments during acid treatment, whereas retrogradedamylopectin in MBSN was difficult to degrade to shorter chains andretained a large number of long chains. The α-amylase resistantresidues (Figure 3b) in SPSN showed 6 peaks at DP57, 50, 43, 31, 14,and 6, respectively, whereas those in MBSN showed 4 peaks withDP57, 50, 43, and 35. It implies that the population of long chainsin SPSN decreased and the fraction with short chains increasedduring α-amylase treatment. The oligosaccharides with very shortchains may be represented by segments of α-amylase-degradedlong chains. In MBSN, the long chains were still dominant, whichmay be due to differences in the arrangement of the long chains in


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the amylopectin clusters of the mung bean starch noodle comparedto that of SPSN. The β-amylase and pullulanase resistant residues(Figure 3c) in SPSN showed 7 peaks at DP70, 61, 45, 36, 25, 16, and 8,whereas those in MBSN showed 4 peaks with DP70, 57, 40, and 30.This implies that SPSN was hydrolyzed more rapidly than MBSNbecause of more A chains (external chain) in Ap-SP than in Ap-MBand the greater ratio of long chains to short chains in MBS thanin SPS (Figure 2). The residues from acid and enzymes in MBSNcontained mainly high molecular weight fractions that appeared atthe void volume, and some low molecular weight fractions such aslimit dextrins, indicating the difficulty to hydrolyze MBSN. Thosehigh molecular weight fractions may be the short amylose chains,generated by the degradation of amylose, which can form doublehelices again to resist hydrolysis. This phenomenon was analogouswith enzyme-resistant retrograded starch, and based on restrictedenzyme access to potential substrates arranged in double helicalaggregates (Gidley and others 1995). Gidley and others (1995) found

Figure 3 --- Gel-permeation chromatography of residues ofcooked starch noodles from SP and MB resistant to (a)1M HCl at 35 ◦C for 20 d; (b) α-amylase for 20 d; (c) a mix-ture of β-amylase and pullulanase for 60 h. The numberindicates DP, average degree of polymerization.

that X-ray diffraction and C CP/MAS NMR spectroscopy indicatedlevels of crystalline and double helical order to be 25% to 30% and 60to 70%, respectively, in enzyme-resistant retrograded starches. Theresidues from acid and enzyme treatment of SPSN contained somehigh molecular weight fractions and large amounts of low molecularweight fractions such as limit dextrin, including maltotriose andmaltose (Inouchi and others 1987; Eliasson 2004), indicating thefacility to hydrolyze SPSN. Both starch noodles hydrolyzed byacid contained fewer small molecular weight materials than thosehydrolyzed by enzymes, indicating the possibility to attack starchnoodles by enzymes.

Hydrolysis properties. The hydrolysis profiles for both cookedstarch noodles are presented in Figure 4. The 2-stage hydrolysis

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Figure 4 --- Hydrolysis of both cooked starch noodles using1M HCl at 35 ◦C for 20 d (a) α-amylase at 35 ◦C for 20 d; (b)a mixture of β-amylase and pullulanase at 35 ◦C for 60 h;(c) SPSN: sweet potato starch noodle; MBSN: mung beanstarch noodle




pattern was quite obvious in all cases. A fast hydrolysis rate dur-ing the 1st 6 d followed by a slower rate between 7 to 20 d forboth starch noodles hydrolyzed with 1M HCl at 35 ◦C was ob-served. When both starch noodles were hydrolyzed with α-amylaseat 35 ◦C they displayed a pattern that was a considerably fast hy-drolysis rate during the 1st 3 d followed by a slower rate between4 to 20 d. Another fast hydrolysis rate during the 1st 12 h followedby a slower rate between 13 to 60 h for both starch noodles hy-drolyzed with a mixture of β-amylase and pullulanase at 35 ◦C wasalso observed. Comparatively, the SPSN had a higher digestibilitywith 1 M HCl, α-amylase, β-amylase, and pullulanase than thoseof the MBSN. The lower digestibility of the latter can be attributedto its high amylose content (approximately 40%), which is of a rel-atively high molecular weight, as well as due to comparatively lessbranching. On the other hand, the high digestibility of SPSN couldbe due to its low amylose content and the presence of very highlybranched amylopectin and low molecular weight of the constituentfractions.

Microscopic structure. Figure 5 shows SEM of uncooked starchnoodles from sweet potato and mung bean. The surfaces of bothstarches were crimpy to a different extent due to shrinkage duringdrying. The smoother surface of MBSN than that of SPSN (Figure 5aand 5c) might be due to a stronger gel strength and elasticity ofMBSN, which can withstand shrinkage better during drying, thanSPSN. The inside of MBSN (Figure 5d) contained long, thick, and or-derly filaments that may be cellulose-like crystalline areas becausea higher amylose content and longer chain length of amylopectin inMBS lead to ease of retrogradation. The leakage of water during cool-ing generated a compact structure inside MBSN, while there were

Figure 5 --- Scanning electronmicrographs (150 to 300×) ofboth uncooked starch noodles:(a) the surface of SPSN; (b) thecross section of SPSN; (c) thesurface of MBSN; (d) the crosssection of MBSN

many pore spaces on the inside of SPSN (Figure 5b) because a higheramylopectin content and shorter chain length of amylopectin leadto less retrogradation and loose inside structure; and the leakage ofwater after freezing and drying generated many pores on the insideof SPSN.

Thermal properties. Table 3 shows the thermal properties forboth starches, both starch noodles, and their resistant residues hy-drolyzed with acid and enzymes. The DSC thermogram of originalSPSN at 70% moisture level between 10 to 180 ◦C showed a singleand faint endotherm at 47.7 to 54.7 to 61.2 ◦C (To to Tp to Tc) with�H 0.97 J/g, and was much smaller than SPS at 64.6 to 72.1 to 80.7◦C (To to Tp to Tc) with �H 1.5 J/g. Sweet potato starch noodle,which is composed mainly of retrograded amylopectin, gelatinizedeasier than its original starch. An endotherm at approximately 50 ◦C,characteristic of crystalline retrograded amylopectin (Ring and oth-ers 1987), was also observed in our thermogram of uncooked SPSN.Many researchers had also reported that the endothermic transitionfor retrograded starch began at a temperature about 20 ◦C lower thanthat for gelatinization of starch granules in waxy maize starch withhigh amylopectin content (White and others 1989; Shi and Seib 1992;Yuan and others 1993). During storage at 4 ◦C, gelatinized starchmolecules reassociate in the SPSN, but in less ordered and henceless stable forms than in the native starch granular state. The re-sistant residues after HCl hydrolysis showed the largest endotherm(99.0 to 106.7 to 112.6 ◦C with �H 24.5 J/g) among these resistantresidues with acid and enzymes. This is indicative of the fact thatthe retrograded sweet potato starch was more resistant to acid thanto α-amylase, β-amylase, and pullulanase, especially in the initialstages up to 5 d (Figure 4).


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The DSC thermogram of uncooked mung bean starch noodles at70% moisture level between 10 to 180 ◦C also showed a single andbroad peak at 68.3 to 72.5 to 83.5 ◦C(To to Tp to Tc) with �H 5.4 J/g,which was higher than those of original mung bean starch (at 57.6to 64.8 to 75.9 ◦C with �H 2.6 J/g). This shows that it is difficult togelatinize MBSN, which is composed of mainly retrograded amy-lose. These findings are in agreement with previous findings, whichreported that the mung bean starch noodles have crystals that meltat 67 to 72 to 78 ◦C (Xu and Seib 1993). The resistant residues afterHCl hydrolysis showed the highest endotherm at 104.3 to 111.2 to115.5 ◦C with �H 44.8 J/g. This also indicates that the retrogradedmung bean starch is more resistant to acid than to enzymes, which isconsistent with those of SPSN. A possible explanation might be thatmore long B-chains from amylopectin are released by acid hydroly-sis (Figure 3a) than by enzyme (Figure 3b and 3c). Those chains couldbehave like short amylose chains, capable of forming lipid complexand double helices (Chung and others 2003), both of which requireda higher enthalpy to melt. Mestres and others (1988) reported �H7.9 J/g at Tp119 ◦C for the acid-resistance residue from uncookedmung bean starch noodles while Xu and Seib (1993) reported �H18 J/g at Tp128 ◦C for the same sample but cooked.

The α-amylase-resistant residues showed only 1 faint peak at 96.7to 99.4 to 104.2 ◦C with �H 0.07 J/g for SPSN, and at 98.5 to 108.3to 110.4 ◦C with �H 0.72 J/g for MBSN. The findings of Xu and Seib(1993) show that α-amylase-resistant residues of MBSN do not showa peak in the temperature range tested (7 to 147 ◦C). The residues

Table 3 --- Thermal property of both starches, both starch noodles, and their resistant residues hydrolyzed with acidand enzymes

Samples To (◦C) Tp (◦C) Tc (◦C) ∆H (J/g)

Original SPS 64.6 ± 0.3 72.1 ± 0.5 80.7 ± 0.4 1.5 ± 0.1Original SPSN 47.7 ± 0.3 54.7 ± 0.4 61.2 ± 0.3 0.97 ± 0.1SPSN hydrolyzed with HCl for 20 da 99.0 ± 0.4 106.7 ± 0.3 112.6 ± 0.3 24.5 ± 0.2SPSN hydrolyzed with α-amylase for 20 da 96.7 ± 0.4 99.4 ± 0.4 104.2 ± 0.3 0.07 ± 0.0SPSN hydrolyzed with β-amylase/pullulanase for 60 ha 96.4 ± 0.5 103.0 ± 0.6 107.7 ± 0.8 2.0 ± 0.1Original MBS 57.6 ± 0.8 64.8 ± 0.7 75.9 ± 0.3 2.6 ± 0.2Original MBSN 68.3 ± 0.3 72.5 ± 0.6 83.5 ± 0.9 5.4 ± 0.3MBSN hydrolyzed with HCl for 20 da 104.3 ± 1.0 111.2 ± 0.9 115.5 ± 0.7 44.8 ± 0.2MBSN hydrolyzed with α-amylase for 20 da 98.5 ± 0.7 108.3 ± 1.2 110.4 ± 0.6 0.72 ± 0.1MBSN hydrolyzed with β-amylase/pullulanase for 60 ha 101.2 ± 0.9 105.9 ± 1.1 109.3 ± 0.8 6.3 ± 0.2

aThe resistant-residues of cooked starch noodles.Data were reported in means ± standard deviation.SPS = sweet potato starch; SPSN = sweet potato starch noodle; MBS = mung bean starch; MBSN = mung bean starch noodle; To = onset temperature; Tp = peaktemperature; Tc = temperature at the end of gelatinization; �H = enthalpy of gelatinization.

Figure 6 --- X-ray diffraction patterns of both starch noodles and their resistant residues hydrolyzed with a mixture ofβ-amylase and pullulanase at 35 ◦C for 60 h: (a) SPSN; (b) MBSN; A: original starch noodles; B: hydrolyzed residuesfrom starch noodles

resistant to the combination of β-amylase and pullulanase fromcooked MBSN gave a higher peak temperature (Tp 105.9 ◦C) and ahigher enthalpy of gelatinization(�H 6.3 J/g) than those of SPSN (Tp

103.0 ◦C and �H 2.0 J/g), resulting from the higher amylose contentand lower amylopectin content in MBS than those in SPS. After thesurface density of the amylopectin has been reduced by β-amylase,the task of pullulanase in penetrating the interior must become pro-gressively easier, because the relative density of the branch point inspace decreases. With cooking, the melting of amylopectin crystal-lites in starch noodles accelerated a successive attack by β-amylaseand pullulanase, while the difficulty of melting amylose crystallitesin starch noodles when cooking prohibited β-amylase and pullu-lanase from attacking the crystalline zones.

X-ray analysis. The X-ray diffraction patterns of both starchnoodles and their dried resistant residues are shown in Figure 6.For original SPSN 3 peaks were observed at 2θ values of 16.3, 22.0,and 27.2 A, corresponding to d-spacing (inter planar distances) of5.4, 4.1, and 2.6 A, respectively. For resistant residues hydrolyzed us-ing a mixture of β-amylase and pullulanase, 1 peak disappeared and2 peaks remained at 2θ values of 17.1 and 21.9 A, corresponding tod-spacing of 5.2 and 4.1 A, respectively. It can be inferred that crys-tallites within enzyme-resistant residues from SPSN were smallerand/or less perfectly packed than in original SPSN because of theirweaker retrograded amylopectin state of crystallinity.

The X-ray diffraction pattern of the MBSN gave strong peaks at2θ =17.0, 23.0, and 22.1 A, corresponding to d-spacing of 5.2, 4.0, and




3.9 A, respectively, which can be attributed to different crystallinestructures that are typical patterns of B-type peak (Mestres and oth-ers 1988) and should be distinguished from that of SPSN. Upon cook-ing and then hydrolysis with β-amylase and pullulanase, the X-raydiffraction pattern changed and was indicated by 3 smaller peaks at2θ of 16.8, 19.4, and 22.0 A, corresponding to d-spacing of 5.3, 4.6, and4.0 A, respectively. This can be attributed to the fact that retrogradedamyloses are still partly hydrolyzed by enzymes. Such a descriptionis in line with model studies on amylose gels and enzyme-resistantmaterial from amylose gels that show weak X-ray diffraction (Cairnsand others 1990). Cairns and others (1990) suggested that networkdisruption by enzyme hydrolysis did not allow increased crystallinepacking to occur. Similar observations were made on both acid-and α-amylase-treated starch noodles (not shown in figures). Theacid/enzyme-resistant residues exhibited weaker diffraction peaksthan original starch noodles, which showed the presence of poorB-patterns, especially MBSN in our research. This was in agreementwith findings of Sievert and others (1991). The appearance of broaddiffraction lines strongly suggested that smaller and/or less perfectcrystallites were present in acid/enzyme-resistant residues than inMBSN, where the sharp, well-resolved pattern reflected a higher de-gree of crystallite perfection. However, generally, crystallinity is aproperty of the amylopectin fraction. Combined with the results offine structure of amyloses and amylopectins (Table 1 and Figure2), X-ray diffraction patterns showed weaker crystallinity of SPSNthan that of MBSN, resulting from insufficient amylose crystallinityand more short chain crystallinity in Ap-SP, and more amylose crys-tallinity and long chain crystallinity in Ap-MB, respectively.

DiscussionThe differences between DP and CL of the residues indicate some

branch points in the resistant residues. The residues resistant tothe pullulanase and β-amylase combination contain mainly highmolecular weight fractions that appear at the void volume and de-crease only slightly, indicating the presence of long linear chains.Because the size-exclusion limit (Vo) of the gel is approximately100000 Daltons, the molecules resistant to the pullulanase and β-amylase combination must have been amylose. Double helical (an-tiparallel helix or “hairpin” model; French 1972) conformation ofthese long chains in the crystallites would be compatible with thecrystallite dimension and explain their resistance to acid hydroly-sis. Amylopectin and short-chain linear material are the main crys-talline entity of the granule of legume starches and the acid-resistantresidues. The overall crystalline structure in this starch would be veryresistant to acid hydrolysis because of strong intermolecular hydro-gen bonding among the aggregated amylose molecules (Biliaderisand others 1981).

The faster hydrolysis pattern corresponds to the hydrolysis of themore amorphous parts of all starch noodles. During the 2nd stage,the crystalline starch is slowly degraded. This is analogous to thephenomenon observed with cellulose and a number of semicrys-talline synthetic polymers. Hydrolytic action in these materials oc-curs most rapidly in the disordered regions, whereas the crystallineareas are more resistant (Banks and Greenwood 1975). The slowerhydrolysis rate of the crystalline parts of the starch noodles may bedue to 2 reasons: (1) the dense packing of starch chains within thecrystallites of starch noodles does not readily allow the penetrationof HCl and enzymes into these regions; (2) acid hydrolysis of a glu-cosidic bond may require a change in conformation for the glucoseunit, from chair to half-chair. Obviously, if the hydrolyzed bond ex-ists within a crystallite, this change in conformation would requirea high energy of activation. All glucosidic oxygens are buried in theinterior of the double helix in starch crystallites and are, therefore,

far less accessible to acid or enzyme attack (Biliaderis and others1981).

The resistant residues from both starch noodles after HCl and en-zyme hydrolysis in our research all show a broad endotherm peaknear 100 ◦C (96 to 115 ◦C; Table 3). Apparently, this is difficult to rec-oncile with the results from Mestres and others (1988), who reported�H 7.9 J/g at Tp119 ◦C for the acid-resistance residue from uncookedmung bean starch noodles, and Xu and Seib (1993), who reported�H 18 J/g at Tp 128 ◦C for the same sample but cooked. We deducethat it may be due to the presence of the complexes of amylose-lipid and lipid-(long chains in amylopectin). It agrees with the find-ings by Morrison and others (1993), Jacobson and BeMiller (1998),and Chung and others (2003), in which the acid/enzyme-resistantresidues had a greater tendency to form amylose-lipid complex. Asimilar result was also found by Godet and others (1995), who re-ported the melting temperature of the different amylose-lipid com-plexes was in the range 78 to 115 ◦C. The acid/enzyme hydrolysismight produce amylose chains of reduced chain lengths, which haveincreased mobility and thus complex more readily with lipids.

Morrison and others (1993) reported that the residual amount ofamylose-lipid complexes (Single V6-amylose helices) increased byacid hydrolysis. It was because the amylose-lipid complex was re-sistant to the acid/enzyme hydrolysis. In accordance with their re-sult, the resistant-acid/enzyme residues contain the single helicesof amylose-lipid complexes. This phenomenon is also supportedby Sievert and others (1991) and Chung and others (2003), who re-ported the reflection of amylose-lipid complexes appeared at 0.449nm (about 22 A) and about 20 A, respectively. If we accept that the 2θ

value of about 22 A reflection arose from amylose-lipid complexes,then the reduced intensities of this peak could be interpreted asamylose-lipid crystallites being melted out near 100 ◦C. During theDSC scanning of 10 to 180 ◦C, we observed a transition of meltingenthalpy at about 96.4 to 112.6 ◦C for the acid/enzyme-resistantresidues in SPSN and about 98.5 to 115.5 ◦C for those residues inMBSN. This is in agreement with results obtained from Sievert andothers (1991), who reported that about 105 ◦C corresponds to dis-sociation of amylose-lipid complexes.

Mestres and others (1988) and Xu and Seib (1993), based ontheir findings of acid and enzyme hydrolysis of uncooked andcooked MBSN at 35 ◦C, proposed that junction zones anchor the3-dimensional structure. In our results, the cause of SPSN loosestructure compared to MBSN allows further speculation based onthe 3-phase theory (micelle, paracrystalline fringe, and filler mass)proposed by Xu and Seib (1993).

We conjecture that SPSN has loose structure due to its crystallineinferiority to MBSN from Figure 5b. In MBSN, the micelle con-tains retrograded segments of amylose molecules and is resistantto acid and enzymes (Xu and Seib 1993). This can be explained us-ing our investigation that the most highly organized zone contain-ing crystallites is caused by moderate chain length in Am-MB, inorder in close juxtaposition due to fewer amylose branches com-prised 1.8 branch chains per molecule, facilitating chains juxtaposeclosely. However, much shorter chains in Am-SP and more amy-lose branches comprising 9.0 chains per molecule were adverse toordered and juxtaposed chains. Thus SPSN did not have a morecompact micelle than MBSN. The hydrolysis-resistant crystallinezone is considered to be the structural center, a composite of in-tensity features from ordered (double helical), which is produced byamyloses and long chains in amylopectin, and a small amount ofnonordered (amorphous single chain) materials, which consists ofamylose-lipid and lipid (long chains in amylopectin). Attached tothe micelle is the paracrystalline finger composed of less organizedmaterial. Xu and Seib (1993) argued that the molecules in this zone


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are all linear; that zone does not swell sufficiently. But our findingsprovide additional information that the 2nd zone is composed ofbranched amylopectin, which can form a network-like frameworkdue to its cohesiveness. Both amylopectins possess 5 fractions anddifferent length branched chains (Figure 2), but large amounts ofshort branched chains in SPS form network-like frameworks anddecrease the ability for crystallization in SPSN, while large amountsof long branched chains in MBS can crystallize so that this zone isstill organized in MBSN. The 3rd and most prominent zone in thestarch noodle is the filler mass or amorphous zone. The filler massis composed of cracked gelatinized starch granules and their frag-ments, which exhibit good viscosity and cling tightly to the other 2zones. Besides occupying a large volume in a starch noodle, the fillermass would be hydrolyzed by acid and enzymes in SPSN and MBSN.The structure of starch noodles is thus composed of 3 phases accord-ing to our conjecture: hydrolysis-resistant crystalline zone (doublehelical and amorphous single chain), network-like framework (amy-lopectin), and filler mass (cracked gelatinized starch granules andtheir fragments). Because of a low content of branched amyloseand much more amylopectin in SPS, SPSN has lower crystallinityand higher adhesiveness, whereas there is a high content amy-lose with little branching and moderate amylopectin in MBS; thus,MBSN has higher crystallinity, good cohesiveness, and excellentquality.

Conclusion

In this study, the structure of SPSN and mung bean was analyzedby GPC, SEM, DSC, and X-ray diffraction after hydrolysis by acid

and enzymes. Meanwhile the structure of their starches was also in-vestigated by GPC and used to explain complementarily the struc-ture of both starch noodles. The surface of MBSN was more smooththan that of SPSN and the inside of MBSN contained long, thick,and orderly filaments, while there were many pore spaces inside ofSPSN from SEM. The structure of both starch noodles was composedof 3 phases according to our conjecture: hydrolysis-resistant crys-talline zone, network-like framework, and filler mass. Because of alower content of branched amylose and a higher content of amy-lopectin with many short chains SPS, SPSN has lower crystalline,higher adhesiveness, and a high sensitivity to hydrolysis with acidand enzymes, whereas there is a high content amylose with a littlebranch and moderate amylopectin content with many long chainsin MBS; therefore, MBSN has higher crystallinity, good cohesiveness,excellent quality, and a low sensitivity to hydrolysis with acid andenzymes. Owing to the difference in structure of SPS and MBS, thestructure and quality of their starch noodles were different markedly.Further research is needed to increase our insight into the efforts ofthe improvement of SPSN.

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Comparative Study on the Starch Noodle Structure of Sweet Potato and Mung Bean