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RESEARCH ARTICLE Physicochemical properties and in vitro digestibility of resistant starch from mung bean (Phaseolus radiatus) starch Suling Li 1 , Qunyu Gao 1 and Rachelle Ward 2 1 Carbohydrate Laboratory, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, P. R. China 2 NSW Department of Primary Industries Yanco Agricultural Institute, Yanco NSW, Australia RS from mung bean starch was prepared by autoclaving, pullulanase debranching, and retrogradation. Physicochemical properties, crystalline structure, and in vitro digestibility of selected RS samples with different RS content were investigated. Compared to native starch, AAM content of RS increased but MW decreased greatly. SEM clearly showed RS samples exhibited irregular shaped fragments with compact structure. XRD pattern indicated that RS samples had typical B-type pattern with sharp peaks at 17.08, 22.28, and and 23.98 2u. The relative crystallinity, gelatinization temperatures, and enthalpy increased with increasing RS content. The a-amylase digestibility of RS was lower than that of native starch. The results suggested that the decrease in enzymatic digestion of RS might due to compact and ordered crystalline structures after debranching and recrystallization. Received: August 15, 2010 Revised: November 28, 2010 Accepted: November 29, 2010 Keywords: Digestibility / Mung bean starch / Properties / Pullulanase / Resistant starch 1 Introduction In response to consumer awareness and demands for healthy food options, great efforts have been made to explore new sources to prepare nutraceuticals and func- tional foods with beneficial physiological effects [1–4]. One example of new nutrition-valued food is to increase the RS within food products [5]. RS are the starch portions that escape digestion in the small intestine, but might be fermented and absorbed in the large intestine [6]. RS imparts desirable physiological benefits to human health, which mainly includes the increase in faecal bulk, and the production of some SCFAs from fermentation in the large intestine, improvement in cholesterol problem, the reduction in glycemic and insulinemic response to food, and the decrease in the risk of ulcerative colitis and colon cancer [5, 7, 8]. RS features a unique combination of physiological and functional properties, while traditional fiber has a coarse texture [9]. These properties enable RS to receive increasing attention as a beneficial food component. RS can be classified into five categories, as follows; RS1: physically inaccessible starch; RS2: native granule or ungelatinized starch; RS3: retrograded starch [10]; RS4, chemically modified starch [4]; RS5, V-form, inclusion complexes formed by AM and polar lipids [11]. RS3 remains thermally stable in normal cooking and it is the most important type of starches in processed foods [1]. RS3 closely relates to highly retrograded AM fractions. Different processing methods and conditions can influence the formation and properties of RS. For example, pullulanase debranching is an important technology for gelatinized starch to produce linear glucans and therefore helps starch recrystallization [5]. Correspondence: Professor Qunyu Gao, Carbohydrate Laboratory, College of Light Industry and Food Sciences, South China University of Technology, 381 WuShan Road, Guangzhou 510640, P. R. China. E-mail: [email protected] Fax: þ86-020-87113848 Abbreviations: HPGPC, high performance gel permeation chromatography; RDS, rapidly digestible starch; SDS, slow digestible starch. DOI 10.1002/star.201000102 Starch/Sta ¨ rke 2011, 63, 171–178 171 ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com

Physicochemical properties and in vitro digestibility of resistant starch from mung bean (Phaseolus radiatus) starch

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Page 1: Physicochemical properties and in vitro digestibility of resistant starch from mung bean (Phaseolus radiatus) starch

RESEARCH ARTICLE

Physicochemical properties and in vitro digestibility ofresistant starch from mung bean (Phaseolus radiatus)starch

Suling Li1, Qunyu Gao1 and Rachelle Ward2

1 Carbohydrate Laboratory, College of Light Industry and Food Sciences, South China University of Technology,Guangzhou, P. R. China

2 NSW Department of Primary Industries Yanco Agricultural Institute, Yanco NSW, Australia

RS from mung bean starch was prepared by autoclaving, pullulanase debranching, and

retrogradation. Physicochemical properties, crystalline structure, and in vitro digestibility of

selected RS samples with different RS content were investigated. Compared to native starch,

AAM content of RS increased but MW decreased greatly. SEM clearly showed RS samples

exhibited irregular shaped fragments with compact structure. XRD pattern indicated that RS

samples had typical B-type pattern with sharp peaks at 17.08, 22.28, and and 23.98 2u. Therelative crystallinity, gelatinization temperatures, and enthalpy increased with increasing RS

content. The a-amylase digestibility of RS was lower than that of native starch. The results

suggested that the decrease in enzymatic digestion of RS might due to compact and ordered

crystalline structures after debranching and recrystallization.

Received: August 15, 2010

Revised: November 28, 2010

Accepted: November 29, 2010

Keywords:

Digestibility / Mung bean starch / Properties / Pullulanase / Resistant starch

1 Introduction

In response to consumer awareness and demands for

healthy food options, great efforts have been made to

explore new sources to prepare nutraceuticals and func-

tional foods with beneficial physiological effects [1–4]. One

example of new nutrition-valued food is to increase the RS

within food products [5]. RS are the starch portions that

escape digestion in the small intestine, but might be

fermented and absorbed in the large intestine [6]. RS

imparts desirable physiological benefits to human health,

which mainly includes the increase in faecal bulk, and the

production of some SCFAs from fermentation in the

large intestine, improvement in cholesterol problem, the

reduction in glycemic and insulinemic response to food,

and the decrease in the risk of ulcerative colitis and colon

cancer [5, 7, 8]. RS features a unique combination of

physiological and functional properties, while traditional

fiber has a coarse texture [9]. These properties enable

RS to receive increasing attention as a beneficial food

component.

RS can be classified into five categories, as follows;

RS1: physically inaccessible starch; RS2: native granule or

ungelatinized starch; RS3: retrograded starch [10]; RS4,

chemically modified starch [4]; RS5, V-form, inclusion

complexes formed by AM and polar lipids [11]. RS3

remains thermally stable in normal cooking and it is the

most important type of starches in processed foods [1].

RS3 closely relates to highly retrograded AM fractions.

Different processing methods and conditions can

influence the formation and properties of RS. For example,

pullulanase debranching is an important technology for

gelatinized starch to produce linear glucans and therefore

helps starch recrystallization [5].

Correspondence: Professor Qunyu Gao, CarbohydrateLaboratory, College of Light Industry and Food Sciences, SouthChina University of Technology, 381 WuShan Road, Guangzhou510640, P. R. China.E-mail: [email protected]: þ86-020-87113848

Abbreviations: HPGPC, high performance gel permeationchromatography; RDS, rapidly digestible starch; SDS, slowdigestible starch.

DOI 10.1002/star.201000102Starch/Starke 2011, 63, 171–178 171

� 2011WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.starch-journal.com

Page 2: Physicochemical properties and in vitro digestibility of resistant starch from mung bean (Phaseolus radiatus) starch

Many studies about RS have been conducted on

starches from maize, wheat, potato, pea, and wx maize

[12]. It is necessary to look for alternative sources of starch

that have the potential to exhibit better physicochemical

and functional characteristics [13]. Mung bean (Phaseolus

radiatus) or green bean is native to the northeastern India–

Burma (Myanmar) region of Asia. It can be grown in Asia,

Africa, South America and Australia [14]. Traditionally,

mung bean starch is the best raw material for making

starch noodle due to the properties of high AM content,

restricted swelling and high shear resistance of its paste

[15].

Mung bean is mainly cultivated in China and a good

source of starch (�50%). The information regarding the

production and properties of RS from mung bean starch

is extremely scarce. Therefore, it is worthwhile to explore the

potential to derive value-added products from mung bean

starch. The purpose of this study was to evaluate physico-

chemical properties and structural characteristics of mung

bean starch with different RS content. The in vitro enzymatic

digestibility of RS was also studied. Understanding of the

properties and enzymatic digestibility of mung bean RS will

establish a foundation for further application.

2 Materials and methods

2.1 Materials

Native mung bean starch was purchased from Hada

Starch Factory (Harbin, China) to prepare a series of

products with different RS content. The RS products were

labeled as RS 11.7%, RS 29.9%, RS 36.8%, RS 43.0%,

and RS 51.0%. The samples with RS 29.9–51.0% were

subjected to autoclaving, pullulanase debranching, and

crystallization processes. An aqueous slurry of starch

(8% w/w, dry basis) was added with an acetate-acetate

buffer solution and autoclaved at 1218C for 20 min. After

cooling to 558C, pH was adjusted to 4.6 with 0.5 N acetic

acid. The gelatinized starch was treated with different

concentrations of pullulanase (20, 40, 60, and 50 ASPU/

g dry starch, Diazyme1 P10, Danisco) and incubated at

558C for 24 h to prepare RS 29.9%, RS 36.8%, RS 43.0%,

and RS 51.0%, separately. The enzyme was inactivated at

1008C for 15 min and then the debranched starch was

stored at 48C for 24 h. The retrograded starch waswashed

with 90% ethanol to pH 7 and dried at 458C to �10%

moisture content. Sample RS 11.7% was not debranched

by pullulanase, but underwent autoclaving and crystalliza-

tion. The undebranched starch (RS 11.7%) was the control

sample. The RS samples were ground and packed in

plastic bags for further analysis.

RS assay kit was brought from Megazyme International

Ireland Ltd. (Wicklow, Ireland). Porcine a-amylase (260 U/g),

AM and AP were obtained from Sigma Chemical Company

(St. Louis, MO, USA). Standard dextrans were purchased

from Pharmacia Ltd. (Germany). All other chemicals and

solvents in this work were of analytical grade.

2.2 Resistant starch determination

The RS content was determined using a Megazyme

Resistant Starch Assay Kit with the description of

Association of Official Analytical Chemists (AOAC)

2002.02 [16]. In short, starch (100 mg) and 4 mL of

enzyme mixture (pancreatic a-amylase, 10 mg/mL, and

amyloglucosidase, 3 U/mL) was added to each test tube,

and then incubated in a shaking water bath (Wisebath@,

Feedback Control Digital Timer Function, Sweden) for

16 h (378C, 200 strokes/min) to hydrolyze digestible

starch. The resistant portion was precipitated with 95%

ethanol and the residue obtained was washed with 50%

ethanol twice, and treated with potassium hydroxide

solution (4 M, 2 mL) to solubilize the RS. The RS solution

obtained was adjusted to pH 4.75 with 8 mL of 1.2 M

sodium acetate buffer (pH 3.8). After incubation with amy-

loglucosidase (0.1 mL, 3300 U/mL) at 508C for 30 min,

the samples were centrifuged at 3000 � g for 10 min.

Threemilliliter of glucose-oxidase-peroxidase-aminoantipyrine

(GOPOD) was added to aliquots (0.1 mL) of the super-

natant, and the mixture was incubated at 508C for 20 min.

Absorbance was measured using a spectrophotometer

(Model 722, Shanghai Analytical Instrument Company,

China) at 510 nm. The analyses were determined in

triplicate and the average value was recorded.

2.3 Apparent amylose content

Apparent AM content in the sampleswas determined using

themethod of Juliano et al. [17]. Starch (100 mg, dry basis)

was wetted with 1 mL ethanol (95%) and gelatinized by

treatment with 9.2 mL of 1 N NaOH and storage for 24 h at

ambient temperature. After adjusting to 100 mL of distilled

water, an aliquot of 5 mL of the solution was transferred to

100 volumetric flasks and 1 mL of 1 N acetic acid was

added. Then 2 mL of 0.2% w/v I2 and 2% w/v KI were

added to the volumetric flask and the volume was adjusted

to 100 mL with distilled water. The solution was allowed to

stand for 20 min at ambient temperature prior to absorb-

ance measurements at 620 nm. A standard curve was

plotted for mixtures of pure AM and AP from potato starch.

AAM content was calculated from the standard curve.

2.4 High-performance gel permeationchromatography (HPGPC)

Sample (50 mg) was dissolved in 10 mL of 90% DMSO

and the solution was centrifuged at 3000 � g for 20 min.

172 S. Li et al. Starch/Starke 2011, 63, 171–178

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Sample solution (100 mL) was filtered through cellulose–

acetate membrane filters with 0.45 mm pore size. The

solution was fractionated through Waters 600 HPLC sys-

tem (Waters Corp., Milford, MA, USA) with analytical col-

umns (UltrahydrogelTM Linear 300 mm � 7.8 mm). The

column temperature was maintained at 458C. The mobile

phase of 100 mM NaNO3 containing 0.02% NaN3 at a rate

of 0.9 mL/min and monitored by differential RI detectors

(Model 2410, Waters Corps., Milford, MA, USA). Standard

dextrans with different MWs were used for MW calibration.

The average of duplicate of MW was recorded.

2.5 Scanning electron microscopy (SEM)

RS and native samples were mounted on an aluminum

specimen holder by double-sided tape. The samples were

coated with gold palladium, with a thickness of about 30 nm.

The specimen holder was then transferred to an environ-

mental scanning electron microscope (Quanta 200, FEI,

Holland) and examined at an accelerating potential of 20 kV.

2.6 X-ray diffraction (XRD)

XRD analysis of native and RSs was examined by a D/

Max-2200 X-ray diffractometer (Rigaku Denki Co., Tokyo,

Japan). The samples were scanned with a CuKa target at

40 kV and 30 mA. XRD patterns were recorded using a

scintillation detector scanning from 48 to 358 (2u) with the

speed rate of 48/min. The degree of relative crystallinity of

the samples was quantitatively estimated following the

method of Nara and Komiya (1983) [18].

2.7 Differential scanning calorimeter (DSC)

Thermal properties of starches were measured by a differ-

ential scanning calorimeter (DSC) (Perkin-Elmer DSC 8000,

Norwalk, CT, USA). Starch (7.5 mg) was weighed into high-

pressure screw type DSC pans and distilled water (15.0 mg)

was added tomake suspensionswith 70%moisture content.

Pans were hermetically sealed and equilibrated for 2 h at

ambient temperature before heating in the DSC. The scan-

ning temperature range and the heating rate were 30–1808Cand 108C/min, respectively. Indium and zinc were used for

the calibration and an empty pan was used as a reference.

Onset temperature (To), peak temperature (Tp), conclusion

temperature (Tc), and enthalpy (DH, J/g) for gelatinization

were determined. Results were presented as an average of

four repeats.

2.8 In vitro a-amylase hydrolysis rate ofdigestion

The determination of in vitro enzymatic hydrolysis index

was conducted according to the modified method of

Jenkins et al. (1987) and Fredriksson et al. (2000) [19,

20]. Briefly, 4 mL of a-amylase solution (100 U/mL) was

added to RS samples held in 15 cm dialysis bag. The

dialysis bag was placed into a beaker containing

400 mL distilled water at 378Cwith agitation. The dialysate

was obtained every 30 min from 0.5 to 5 h. The enzyme

was inactivated at boiling water for 5 min to ensure further

hydrolysis did not occur after the prescribed time. Maltose

concentration was determined by phenol–sulfuric acid

reagent method. The rate of starch digestion was calcu-

lated as the percentage of starch digestion products to

maltose (maltose equivalents) at the different incubation

times. The samples were run in triplicate.

2.9 Statistical analysis

The test data were statistically analyzed using one-way

analysis of variance (ANOVA) on SPSS version 13.0 soft-

ware for Windows (USA). Least significant difference

(LSD) test was used to determine differences between

means. p < 0.05 was considered to be significant. The

results reported were average and SD values.

3 Results and discussion

3.1 Apparent AM content

Native starch of AM content in this study was 30.1%

(Table 1), which is comparative to previous mung bean

AM contents of 30.9–34.3% [21] and defatted mung bean

starch 32.7–34.3% [14]. The AM content of unbranched

RS 11.7% was 6.0% greater than native starch, and the

debranched RS samples were 22.7–28.7% greater than

native starch. Results show that AM content increased with

the enhancement of RS, that AM content was somewhat

related to RS and that the RS could be further enhanced

by debranching with pullulanase. Upon retrogradation,

rearrangement or recrystallization of starch fractions

would generate RS3 resulting from highly retrograded

AM chains [22].

Table 1. AAM content of native and RSs

Samples AAM content (%)

Native starch 30.1 � 0.2a

RS 11.7% – undebranched 36.1 � 0.6b

RS 29.9% – debranched 52.8 � 0.8c

RS 36.8% – debranched 58.9 � 0.9d

RS 43.0% – debranched 57.6 � 0.6d

RS 51.0% – debranched 58.8 � 1.1d

Mean values with different letters within each column aresignificantly different (p < 0.05).

Starch/Starke 2011, 63, 171–178 Properties and digestibility of mung bean resistant starch 173

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Pullulanase can not only catalyze hydrolysis of a-D-

glucosidic linkages of pullulan, but also cleave

a � (1 ! 6) branch linkages of AP and glycogen [23,

24]. The resulted short linear chains had similar molecular

characteristics to AM and mainly linked by a � (1 ! 4)

linkage. Guraya et al. [25] (2001) suggested that amount of

debranching would affect the retrogradation process

during storage because shorter chain length was favorable

for precipitation, rather than longer chain length. This was

due to strong gel networks between AM interaction of AM

or AP during retrogradation [26]. Thus, treatment of mung

bean starch with pullulanase can produce RS3 starch

by chain elongation and folding, which will promote

recrystallization.

Addition of excess pullulanase does not increase the

RS of the mung bean starch; e.g., 50 and 60 ASPU/g (dry

starch) of pullulanase resulted in the samples with RS

content of 51.0 and 43.0%, respectively. Eerlingen et al.

[27] (1993) indicated that too many number of short chains

were unfavorable for nucleating and propagating into

stable crystallites whereas appropriate starch chain length

was good for starch recrystallization. In other words,

pullulanase treatment could generate appropriate chain

lengths of AM that helps starch recrystallization.

3.2 Molecular weight distribution by HPGPC

The elution profiles of MW of RSs were examined by

HPGPC (Table 2). Native mung bean starch was a complex

molecule with high Mw and it was difficult to fraction. The

Mw of debranched products showed a dramatic decrease

in Mw with increasing RS content. These results suggested

that themolecule of starch decreased greatly after debranch-

ing. Pullulanase can rapidly hydrolyze a�(1!6) glucosidic

bonds from the parent AP molecule. The technology of

pullulanase debranching is promising for producing linear

and low Mw chains. With the degradation of polymer,

debranching treatment might provide sufficient AM chains

for the crystallization process. When the debranched

starch was recrystallized, the short linear chains would

aggregate and realign into double helical strands and be

stabilized by hydrogen bonds [28]. This result is consistent

with the study of Leong et al. [24] (2007), which showed

that a 20–25 glucose residue was favorable for starch

retrograded or recrystallized and resulted in RS formation.

The aggregated starch chains with suitable Mw could lead

to precipitation and formation of denser structures during

the periods of retrogradation. The potential substrates in

RS3 with double helical aggregations might increase its

resistance to enzyme hydrolysis [29].

3.3 Morphology (SEM)

Morphology of native and RS was obtained from SEM

(Fig. 1). Native mung bean starch granules appeared small

and either elliptical or spherical shape. Large granules

displayed kidney-shaped or oval, whereas small granules

exhibited spherical. The granular size of mung bean starch

ranged from 7.9 to 31.6 mm.However, granular structure of

starch was destroyed after modifications. RS3 samples

showed bigger, irregular shaped fragments with compact

structure (Figs. 1B, C, and D). The size of large fragments

ranged from 30 to 80 mm, and that of the smaller ones

varied from 10 to 30 mm. For RS samples, the difference in

microscopic appearance was influenced by autoclaving

and retrogradation relative to crystalline structure in RS

samples [30].

When starch was heated in excessive water at gelati-

nization temperature, Ratnayake and Jackson [31]

observed that molten starch granules or beams would

connect with one another to form a sponge-like structure

observed from different starch. Previous studies

suggested that starch molecules re-associated as double

helices, and formed tightly packed structures stabilized by

hydrogen bonding upon retrogradation. RS was charac-

terized as an ordered double helix structure and appeared

to be located in inner regions [31–33]. Thus, the newly

formed structure with irregular shape of RS would

decrease enzymatic hydrolysis.

3.4 X-ray diffraction (XRD)

Figure 2 shows the XRD patterns and relative crystallinity

(%) of native starch and the selected RS3 samples. Native

mung bean starch showed strong diffraction peaks at

15.08, 17.18, 19.78, 23.08 2u (Fig. 2, curve A). Native mung

bean starch exhibited A-type crystalline pattern. Some

confusion in literature about the crystalline pattern of mung

bean starch is reports of C-type and A-type [34, 35].

The difference in diffraction pattern of native mung bean

starch might due to the starch origin, isolation method,

sample preparation method, and X-ray analysis operation

conditions.

Compared to native starch, the sample of RS 11.7%

(non-debranched) had additional diffraction peak at 5.88 2u(Fig. 2, curve B). The debranched starch products dis-

played similar XRD patterns with sharper peaks (Fig. 2,

curve C, D, E, and F). The RS samples with pullulanase

Table 2. Summary of MW distributions of RS

Samples Mwa)

RS 29.9% 3.50 � 103

RS 36.8% 3.70 � 103

RS 43.0% 3.90 � 103

RS 51.0% 3.97 � 103

a) Mean values of average MW.

174 S. Li et al. Starch/Starke 2011, 63, 171–178

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debranching had sharp peaks at 17.08, 22.28, 23.98 2u, andnew peaks occurred at 5.78, 14.08, and 25.98 2u (Fig. 2,

curve C, D, E, and F). The result showed that RS3 dis-

played a typical B-type diffraction pattern. The typical B-

type X-ray pattern was the result of the retrogradation of

both AM and AP.

Miao et al. [36] reported that the differences in starch

crystallinity could mainly due to crystal size, amount of

crystalline regions affected by AP content and AP chain

length, orientation of the double helices within the crystal-

line domains, and degree of interaction between double

helices. The hydrolysis of a�(1!6) glycosidic bondswould

produce more free linear chains in the hydrolysate and RS

crystallites exhibited more tightly packed structure and/or

more arranged to XRD than did native starches. Wu and

Sarko [32] suggested that the extent of diffraction intensity

could reflect the change in retrograded AM. During

heating and cooling, AM chains were recrystallized and

even debranched AP chains were re-crystallized [5].

Therefore, the change in starch crystalline structure will

cause difference in diffraction pattern.

The relative crystallinity of nativemung bean starch was

the lowest value (21.6%) of all samples. With increasing

RS content, the relative crystallinity of samples increased.

The value of relative crystallinity (21.9%) of un-debranched

sample was similar to native starch (Fig. 2, curve B).

However, the debranched RS samples showed much

higher value in relative crystallinity and the relative crys-

tallinity ranged from 38.8 to 42.8%. (Fig. 2, curve C, D, E,

Figure 1. Micrographs of native and RSs. (A) Native (T3000), (B) RS 11.7% (T600), (C) RS 36.8% (T600), (D) RS 51.0%(T600).

5 10 15 20 25 30 35

FEDCBA

Inte

nsity

Diffraction angle

Figure 2. XRD patterns of native and RSs. (A) Native,(B) RS 11.7%, (C) RS 29.9%, (D) RS 36.8%, (E) RS43.0%, (F) RS 51.0%.

Starch/Starke 2011, 63, 171–178 Properties and digestibility of mung bean resistant starch 175

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Page 6: Physicochemical properties and in vitro digestibility of resistant starch from mung bean (Phaseolus radiatus) starch

and F). The increase in relative crystallinity was found on

the debranched and recrystallized cassava starch [37].

The increased relative crystallinity further illustrated the

ordering arrangement of starch chains in RS.

3.5 Thermal analysis by DSC

Thermal properties of native and the resistant products

were determined by DSC analysis. The native mung bean

starch showed a clear gelatinization peak at 76.58Cwith an

endothermic enthalpy of 2.3 J/g (Table 3). This gelatiniza-

tion enthalpy reported here were lower than previous

studies with the difference in results, attributed to different

starch source, isolation methods and/or DSC pan. After

gelatinization and retrogradation treatment, the RS prod-

ucts exhibited broader peaks and the gelatinization tem-

peratures shifted to higher temperatures. The enthalpy of

RS product increased from 4.1 to 14.1 J/g with increasing

RS content. The RS product showed obvious endothermic

transition temperatures ranging from 65.3 to 103.38Cwith the peak temperatures of 66.0–109.98C. The result

can be mainly associated to the dissociation of AM double

helices and formation of the re-association of leached

AM upon retrogradation. Hence, the gelatinization

temperature of RS was increased and RS products exhib-

ited more thermal stability than that of native starch.

3.6 In vitro a-amylase hydrolysis rate ofdigestion

In vitro a-amylase hydrolysis (0–5 h) of RS is presented in

Fig. 3. With increasing RS content, the a-amylase hydroly-

sis rate of RS decreased. The control sample (RS 11.7%)

had the highest a-amylase hydrolysis rate among RS

samples. Moreover, a rapid hydrolysis rate of all samples

was observed within the first 2 h of incubation. This result

might mainly ascribe to hydrolysis of rapidly digestible

starch (RDS) and slow digestible starch (SDS). After

2.5 h, the enzymatic digestion rate slowed down to a

plateau. The a-amylase hydrolysis rate of RS 29.9%,

RS 36.8%, RS 43.0%, and RS 51.0% was lower than

that of RS 11.7%. The enzymatic digestibility decreased

as RS content increased and the sample of RS 51.0%

showed lowest enzymatic digestion of all starch samples.

Pongjanta et al. [5] reported similar results that the incom-

pletely debranched RS3 sample from high amlyose rice

starch had stronger resistance to a-amylase digestion

than native starch. The enzyme hydrolysis rate of banana

RS products was less than in native starch [30]. The

resistance to enzymatic hydrolysis is due to starch recrys-

tallization with tight and packed structures [1]. Shin et al.

[38] suggested that RDS, SDS, and RS fractions formed

into a single crystallite structure. RDS and SDS mainly

consisted of amorphous regions, while RS considered as

an arrangement and ordered double helix structure in

inner regions. Thick and dense crystallites would make

it difficult for a-amylase to access the double helices in a

crystalline structure [32], which decreased the hydrolysis

rate of starch digestion and even enhanced the resistance

to a-amylase hydrolysis.

Table 3. Thermal properties of native and RSs

Samples T0a) (8C) Tp

a) (8C) Tca) (8C) DHb) (J/g)

Native starch 76.5 � 0.3a 81.0 � 0.2a 86.1 � 0.2a 2.3 � 0.1a

RS11.7% 65.3 � 0.3b 66.0 � 0.3b 68.3 � 0.2b 4.1 � 0.1b

RS29.9% 90.2 � 0.2c 104.4 � 0.5c 109.8 � 0.2c 9.2 � 0.1c

RS36.8% 93.8 � 0.4d 106.3 � 0.1d 111.8 � 0.2d 11.0 � 0.1d

RS43.0% 94.0 � 0.3d 109.9 � 0.3ab 114.4 � 0.3ab 12.0 � 0.2ab

RS51.0% 103.3 � 0.2ab 108.7 � 0.1bc 115.2 � 0.1bc 14.1 � 0.2bc

a) To, Tp, and Tc indicate the temperature of the onset, peak, and conclusion of gelatinization, respectively.b) DH indicates enthalpy of gelatinization.Average values in the column with different superscripts are significantly different (p < 0.05).

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6Time (h)

Hyd

roly

sis r

ate

(%)

Native11.6%29.9%36.8%43.0%51.0%

Figure 3. In vitro �-amylase hydrolysis rate of native andRS samples.

176 S. Li et al. Starch/Starke 2011, 63, 171–178

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4 Conclusions

Mung bean RS products with various RS contents pos-

sessed differences in physicochemical properties, crystal-

line structure, and enzyme hydrolysis profiles. The

differences in properties amongst debranched RS

starches indicated that the debranching technique com-

bined with crystallization could be used to design different

RS products.With respect to AAM content andMWresults,

pullulanase was an effective method to yield short chain

AM, which was helpful for RS formation during retrogra-

daton. After recrystallization, compact and ordering crystal

structure of starch might be developed and it led to a

decrease in a-amylase hydrolysis rate of starch. In

addition, RS products exhibited thermal stability with

increasing RS contents, which was favorable for food

processing. RS products with different properties and in

vitro digestion profiles are desired for satisfying different

marketing demand.

The study was carried out with financial support

of the National High-tech R&D Program of China

(2007AA10Z309), and Guangdong Province Program

of China (2009B090300274).

The authors declared no conflict of interest.

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