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Food Hydrocolloids 25 (2011) 1702e1709

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Food Hydrocolloids

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Effect of heat-moisture treatment on the formation and physicochemicalproperties of resistant starch from mung bean (Phaseolus radiatus) starch

Suling Li a, Rachelle Ward b, Qunyu Gao a,*

aCarbohydrate Laboratory, College of Light Industry and Food Sciences, South China University of Technology, WuShan, Guangzhou 510640, PR Chinab Industry and Investment NSW, Yanco Agricultural Institute, Yanco, NSW 2703 Australia

a r t i c l e i n f o

Article history:Received 12 September 2010Accepted 10 March 2011

Keywords:Mung bean starchHeat-moisture treatmentResistant starchPhysicochemical properties

* Corresponding author. Tel.: þ86 020 87113845.E-mail addresses: suling2011@gmail.com (S. Li),

gov.au (R. Ward), qygao@scut.edu.cn (Q. Gao).

0268-005X/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.foodhyd.2011.03.009

a b s t r a c t

Mung bean starch was subjected to a range of heat-moisture treatments (HMT) based on differentmoisture contents (15%, 20%, 25%, 30%, and 35%) all heated at 120 �C for 12 h. The impact on the yields ofresistant starch (RS), and the microstructure, physicochemical and functional properties of RS wasinvestigated. Compared to raw starch, the RS content of HMT starch increased significantly, with thestarch treated at 20% moisture having the highest RS content. After HMT, birefringence remained at theperiphery of the granules and was absent at the center of some granules. The shape and integrity of HMTstarch granules did not change but concavity was observed under scanning electronic microscopy.Apparent amylose contents of HMT starch increased and the HMT starch was dominated by highmolecular weight fraction. Both the native and HMT starches showed A-type X-ray diffraction pattern.Relative crystallinity increased after HMT. The gelatinization temperatures (To, Tp, and Tc), gelatinizationtemperature range (TceTo) and enthalpies of gelatinization (DH) increased significantly in HMT starchcompared to native starch. The solubility increased but swelling power decreased in HMT starches. Thisstudy clearly shows that the HMT exhibited thermal stability and resistance to enzymatic hydrolysisowing to stronger interactions of starch chains in granule.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The advent of novel and innovative products is a strategic fieldin the food industry. With an increasing trend toward health andnutrition, consumers are not merely interested in traditionalnutritional aspects of the food, but are also concerned withsupplementary health merits derived from its regular ingestion(Aparicio-Saguilán et al., 2007). In response, food manufacturers,researchers and producers have aimed to improve the digestionand health benefits of starch as it is the main carbohydrate in thehuman diet and serves as an energy source. Resistant starch (RS) isdefined as the sum of starch and the fraction of starch degradationthat escapes digestion in the small intestine of healthy people(EURESTA, 1993), but may be fermented by the colonic microor-ganism. Potential physiological value of the resistant starcheswas reported to have some close relationship with colonic healthfor the effects on fecal bulk and short chain fatty acid metabolism

rachelle.ward@industry.nsw.

All rights reserved.

(Jenkins et al., 1998; Sajilata, Singhal, & Kulkarni, 2006). Due to itsphysiological effects, RS is considered to be a promising andinnovative food ingredient, which could be parallel with dietaryfibers.

Heat-moisture treatment (HMT) commonly occurs at a low-moisture content (<35% w/w), and at elevated temperatures abovethe glass transition temperature (Tg) but below gelatinizationtemperature for a certain period of time (15 mine16 h) (Gunaratne& Hoover, 2002; Jacobs & Delcour, 1998). Such physical modifica-tions are more receptive to the consumer as it avoids traditionalchemical means to modify starch. Great effort has been made todemonstrate that HMT could enhance the yields of RS. Sievert andPomeranz (1989) prepared RS from normal and waxy starches byHMT at 18% moisture and showed that HMT reduced enzymesusceptibility. Chung, Liu, and Hoover (2009) reported that the RSyields of corn, pea, and lentil starches had increased by 7.7%, 2.3%and 5.6% respectively after HMT. Luo, Gao, and Yang (2003) sug-gested boil-stable resistant starch was prepared from high-amylosecorn starch by HMT.

Heat-moisture treatment is an important physical method ofimproving poor functional properties of native starch and partic-ularly favorable for food applications (Hoover & Manuel, 1996a;Maache-Rezzoug, Zarguili, Loisel, Queveau, & Buléon, 2008).

S. Li et al. / Food Hydrocolloids 25 (2011) 1702e1709 1703

Reported changes to the granular surface, swelling factor, amyloseleaching, gelatinization temperatures, X-ray diffraction patternand crystallinity, gelatinization parameters on starch from anextensive range of botanical sources and a range of HMT conditionsare numerous (Adebowale, Afolabi, & Olu-Owolabi, 2005;Adebowale, Olu-Owolabi, Olawumi, & Lawal, 2005; Franco,Ciacco, & Tavares, 1995; Gunaratne & Hoover, 2002; Hoover,Swamidas, & Vasanthan, 1993; Hoover & Vasanthan, 1994;Watcharatewinkul, Puttanlek, Rungsardthong, & Uttapap, 2009).The changes of properties vary with moisture content duringtreatment and the starch source (Hoover & Manuel, 1996a).

A starch that has not been investigated as a potential RS ingre-dient is mung bean starch. Mung bean (Phaseolus radiatus) isa member of the legume family with 63% carbohydrate and 16%dietary fiber (US Department of Agriculture, 2001, p. 14). Mungbeans are great source of starch and yield 50e60% starch based onvarious processing methods (Fan & Ai, 1996). Mung bean starchexhibits high-amylose content, restricted swelling, high shearresistance of its paste and high granular stability (Oates, 1991).Mung bean starch is potential for yielding a higher resistant starchby HMT with higher amylose content. Since there is a generalperception that mung bean commodity is of poor economic value,the need to provide background research about the possibleexploitation of value-adding products from mung bean starchthrough production of RS by HMT is of significant economical andnutritional benefit.

The objective of this study was to evaluate the effect of heat-moisture treatment on resistant starch, apparent amylose content,morphology, swelling power, solubility, size distribution, crystal-line structure, gelatinization characteristics of mung bean starch.This study will present a comprehensive understanding of thechanges in physicochemical and functional properties followingheat-moisture treatment. Outcomes of this research will form theplatform for future investigations and applications of the func-tionality of mung bean resistant starch by HMT.

2. Materials and methods

2.1. Materials

Mung bean starch was purchased from Hada Starch Factory(Harbin, China). Moisture, fat, protein and ash of the native mungbean starch (P. radiatus) were 12.93%, 0.13%, 1.34%, and 0.15%,respectively. Resistant starch assay kit was purchased from Mega-zyme International Ireland Ltd. (Wicklow, Ireland). Standardsamylose and amylopectin were obtained from Sigma ChemicalCompany (St. Louis, MO, USA). Chemicals and solvents used in thiswork were of analytical grade.

2.2. Heat-moisture treatment

Themoisture levels of starch samples were adjusted to 15%, 20%,25%, 30% and 35% (the moisture level of native starch was pre-determined) by dispersing in appropriate amount of distilled water.All samples were then held in sealed containers (500 mL) and keptfor 24 h at ambient temperature. The sealed containers wereheated in a thermostatically controlled convection oven (DHG-9140A, Shanghai Shengxian Instrument Manufacturing Company,China) at 120 �C for 12 h. All containers were cooled to ambienttemperature. All samples were removed from the containers anddried at 45 �C for 12 h to achieve uniform moisture content (w8%).Based on the treatment moisture content, the HMT starch sampleswill be referred to as HMT-15, HMT-20, HMT-25, HMT-30 andHMT-35.

2.3. Resistant starch determination

Resistant starch content was assessed using Resistant StarchAssay Kit based on the AOAC (2002.02). In brief, starch (100 mg)and 4 mL of enzyme mixture (pancreatic a-amylase, 10 mg/mL, andamyloglucosidase, 3 U/mL) was added to each test tube, and thenincubated in a shaking water bath (Wisebath@, Feedback ControlDigital Timer Function, Sweden) for 16 h (37 �C, 200 strokes/min)to hydrolyze digestible starch. The resistant portion was precipi-tated with 95% ethanol and the residue obtained was washed with50% ethanol twice, and treated with potassium hydroxide solution(4 M, 2 mL) to solubilize the RS. The RS solution obtained wasadjusted to pH 4.75 with 8 mL of 1.2 M sodium acetate buffer (pH3.8). After incubation with amyloglucosidase (0.1 mL, 3300 U/mL)at 50 �C for 30 min, the samples were centrifuged at 3000g for10 min. 3 mL of Glucose-oxidase-peroxidase-aminoantipyrine(GOPOD)was added to aliquots (0.1 mL) of the supernatant, and themixture was incubated at 50 �C for 20 min. Absorbance wasmeasured using a spectrophotometer (Model 722, ShanghaiAnalytical Instrument Company, China) at 510 nm.

2.4. Apparent amylose contents

Apparent amylose contents in the samples were determinedaccording to the procedure of Juliano et al. (1981).

2.5. Scanning electron microscopy (SEM)

Starch samples were prepared by sprinkling the starch ondouble-sided adhesive tape attached to a circular aluminum stub,and then coated with 20 nm gold under vacuum. The samples wereviewed and photographed with a scanning electron microscope(model S-3700 N, Hitachi, Japan) at an acceleration potential of20 kV and magnification of �1000.

2.6. Polarized light microscopy

Birefringence of native and modified mung bean starch granuleswere observed under an optical microscope (model BH-2, Olympus,Japan). All samples were dispersed in solution (glycerine/deionisedwater; 1:1 v/v) and the imageswere recorded at 400�magnification.

2.7. High-performance gel permeation chromatography (HPGPC)

Samples were prepared by dissolving 50 mg of sample in 90%DMSO and the solution was centrifuged at 3000 rotate/min for20 min. Sample solution (100 mL) was filtered through celluloseeacetate membrane filters with 0.45 mm pore size. The solutionswere fractioned through a Waters 600 high-performance liquidchromatography system (Waters Corp., Milford, MA) with two in-line, identical analytical columns (Ultrahydrogel� Linear300 mm� 7.8 mm). The column temperatures were maintained at45 �C. The mobile phase of NaNO3 (100 mM) containing NaN3(0.02%) was circulated at a rate of 0.9 mL/min and the eluted starchdetected by with a differential refractive index (DRI) detector(Model 2410, Waters Corps., Milford, MA). The percentage of peakarea was calculated using the Origin 6.0 software (Microcal Inc.,Northampton, MA).

2.8. X-ray diffraction (XRD)

X-ray patterns were obtained with a D/Max-2200 X-raydiffractometer (Rigaku Denki Co., Tokyo, Japan). Starch sampleswere equilibrated in a saturated relative humidity chamber for 24 hat ambient temperature. The samples were scanned in the range of

Table 1Resistant starch content and apparent amylose content of native and heat-moisturetreated (HMT) starches.a

Samples Resistant starch (%) Apparent amylosecontent (%)

Native starch 11.2� 0.1a 29.7� 0.2a

HMT-15 30.9� 0.5b 31.9� 0.8b

HMT-20 45.2� 0.1c 35.0� 0.6c

HMT-25 36.6� 0.2d 32.3� 0.6b

HMT-30 30.8� 0.8b 32.0� 0.5b

HMT-35 17.1� 0.2ab 31.5� 0.1b

a Experimental data are the means of triplicate determinations� standard devi-ation. Data for resistant starch and apparent amylose content are denoted assignificantly different (P< 0.05) according to the superscripts for each HMTtreatment.

S. Li et al. / Food Hydrocolloids 25 (2011) 1702e17091704

4e35� (2q), with target voltage 40 kV, target current, 30 mA, and ata scanning rate of 4 �/min. Relative crystallinity was calculated asthe ratio of the areas of crystalline and amorphous regions of X-raydiffractograms (Nara & Komiya, 1983).

2.9. Fourier-transform infrared spectroscopy (FT-IR)

All infrared spectra were obtained on Vector 33 spectrometer(Bruker, Germany) equipped with an attenuated total reflectance(ATR) attachment with a resolution of 4 cm by 64 scans. Spectrawere baseline-corrected by drawing a straight line between1200 cm�1 and 800 cm�1. A half-band width of 26 cm�1 anda resolution enhancement factor of 2.4 were employed. Absorbanceheights at 1047 cm�1 and at 1022 cm�1 obtained on the spectrawasdenoted as ordered starch and amorphous starch, respectively. Allstarch samples were saturated in relative humidity chamber over-night at ambient temperature before FT-IR measurement.

2.10. Differential scanning calorimetry (DSC)

Gelatinization characteristics were measured and recorded ona PerkineElmer DSC8000 (Norwalk, CT, USA) differential scanningcalorimeter, equipped with a thermal analysis software, Pyriswindow (PerkineElmer). Water (15.0 mL) was added with a micro-syringe to starch (7 mg dry basis) in the DSC pans, which were thensealed, reweighed and kept at room temperature for 24 h to ensureequilibration of the starch samples and water. The samples werescanned from 30 �C to 150 �C at 10 �C/min. An empty pan was usedas a reference.

2.11. Swelling power and starch solubility

The swelling power and starch solubility were measuredaccording to the method of Adebowale, Afolabi, et al. (2005),Adebowale, Olu-Owolabi, et al. (2005). Starch suspension (1% w/w)was prepared and mixed well using a vortex mixer. Aliquots ofsolution (10 mL) was transferred to a glass tube and heated byshaking at 55, 65, 75, 85, and 95 �C for 30 min, separately. Afterincubation, the samples were cooled to room temperature andcentrifuged at 4000g for 15 min. The supernatant was decantedcarefully and the residual sediment was determined. The residueobtained after drying the supernatant represented the amount ofstarch dissolved in water. Solubility was calculated per 100 g ofstarch on dry weight basis. The solid fractionwas dried at 105 �C for3.5 h. Swelling powerwas reported as the content of sediment in thedried solids sample.

2.12. Statistical analysis

The test data were statistically analyzed using one-way analysisof variance (ANOVA) on SPSS version 13.0 software for Windows(USA). Triplicate determinations were performed for each test forcalculation of average and standard derivation. LSD (p< 0.05) testwas applied to determine differences between means for thetreatments at the 5% significance level.

3. Results and discussion

3.1. Resistant starch contents and apparent amylose contents

The contents of resistant starch (RS) and apparent amylose ofnative and HMT starches are presented in Table 1. The RS wasgreatest in the sample with 20% moisture content (HMT-20), andthe RS in HMT starch 2e4 times greater than the control. Marutaet al. (1998) claimed that an increase in enzyme-resistant

portions was attributed to strong associations between molecularchains and a tight structure within starch granules after HMT. Thecompact structure of HMT starch would be more resistant toenzymatic hydrolysis and, as expected a greater RS was observed(Table 1). Moisture content is the key factor in RS formation duringheat-moisture treatment. It has been shown that water createshydrogen bonds between molecular chains within the starchgranule (Kurakake, Noguchi, Fujioka, & Komaki, 1997), so it isfeasible to suggest that the moisture content of the starch prior toHMT can be optimized to maximize the resistant starch contentduring HMT.

The apparent amylose content of un-treated mung bean starchwas 29.7% (Table 1). Compared to that of native starch, theapparent amylose content of HMT starch was raised between 1.8%and 5.3%. The difference in apparent amylose content betweennative and HMT starch was statistically significant. The apparentamylose content of HMT-20 was highest (35.0%) of all samples.The increase in amylose content may be ascribed to interactionbetween starch chains within the amorphous area of the granule.Gunaratne and Hoover (2002) proposed that HMT might causeadditional interactions occurred between amyloseeamylose and/or amyloseeamylopectin chains. An increased amount of amylosechains could better interact with the chains within the starchgranules. Thus, the result of greater amylose after HMT can reflectthe association of amyloseeamylose and/or amyloseeamylopectinto some extent.

3.2. Scanning electron microscopy

The shape and surface characteristics of native and treatedstarches are shown in Fig. 1. Mung bean starch granules werekidney-shaped, spherical or oval varied with size. The smallerstarch granules were spherical, while the larger starch granulesappeared oval and the kidney shape was common to all granulesizes. HMT did not alter granule shape and the integrity of starchgranules. This concurred with observations of HMT on finger milletstarch (Adebowale, Afolabi, et al., 2005), canna starch(Watcharatewinkul et al., 2009), newcocoyam starch (Lawal, 2005),wheat starch (Kulp & Lorenz, 1981), potato starch (Lorenz & Kulp,1982), and a series of legume starches including green arrow peastarch, black bean, othello pinto beans starch, express field pea andeston lentil starch (Hoover & Manuel, 1996a).

The surface of native starch granules was smooth and noevidence of fissure was observed under the SEM. Cavitiesappeared on the samples of HMT-15, HMT-30 and HMT-35(Fig. 1B, E, and F), and pitting and indentation occurred on thesamples of HMT-20, HMT-25, HMT-30, and HMT-35 (Fig. 1CeF).These observations could be attributed to the re-association ofthe starch chains within the granule where the tissue structure

Fig. 1. Scanning Electron Micrographs (SEM) of native and heat-moisture treated (HMT) starches: (A) Native starch, (B) HMT-15, (C) HMT-20, (D) HMT-25, (E) HMT-30, (F) HMT-35.Scanning electron micrographs of all starches were recorded at 1000� magnification.

S. Li et al. / Food Hydrocolloids 25 (2011) 1702e1709 1705

was weak. The concavities and indentation were the evidence ofthe weak tissue of granules that was affected by thermal forcegiven by HMT. Furthermore, the pressure and heating outside thestarch would lead comparatively loose granules into compactones during HMT. The granules of starch were reported to becometight and compact after HMT (Watcharatewinkul et al., 2009). It islikely that the stronger interaction of starch chains would formtight structure within amorphous regions and this might reducethe susceptibility to enzymatic digestion to occur on HMT. Thechange in HMT starch might decrease the enzymatic hydrolysisbecause the compact granule of HMT starch reduces the acces-sibility between enzyme and substrate (Svihus, Uhlen, & Harstad,2005). The granule stability of mung bean starch is favorable forRS granules formation during HMT and the RS products by HMTare preferable for adding in low-moisture products. Thus, thechange in morphology of starch could reflect the interactions ofmolecular chains within starch granules and the influence of HMTon starch.

3.3. Birefringence

The birefringence patterns of native and heat-moisture treatedmung bean starch are shown in Fig. 2. Birefringence is a symbol ofthe average radial orientation of helical structures. A pronouncedbirefringence was displayed at the center of native starch (Fig. 2A).For HMT starch, some starch granules remained highly birefringentwhereas other granules exhibited a decrease in birefringence onlyin the center of the granule (Fig. 2CeF). For the very high moisturecontent treatments of HMT-30 and HMT-35 (Fig. 2E and F) loss ofbirefringence occurred at the center but not at the periphery of thegranule.

The observed loss of birefringence at the center of the granulewas in agreement with the investigation of Chung et al. (2009), whoreported the decrease in birefringence was more pronounced at thegranule center of corn and pea starches. The granule center of HMTstarch appeared hollow in both corn and pea starches. They sug-gested that HMTwith high temperature offer thermal energy to the

Fig. 2. Polarized Light Microscopy of native and heat-moisture treated (HMT) starches. (A) Native starch, (B) HMT-15, (C) HMT-20, (D) HMT-25, (E) HMT-30, (F) HMT-35. Photo-micrographs of all starches were recorded are at 400� magnification.

S. Li et al. / Food Hydrocolloids 25 (2011) 1702e17091706

double helices (forming the crystallites) and thismay promote theirmobility, which leads to a loss of radial orientation. The formationof tight helical starch in the periphery has been confirmed bystrong birefringence (Chung, Liu, & Hoover, 2010). A similar resultwas observed for HMT potato starchwhereby a loss in birefringencewas found at the center of the HMT potato granules with thedevelopment of the voids (Vermeylen, Goderis, & Delcour, 2006).Watcharatewinkul et al. (2009) reported that thin channels (mightbe equivalent to the voids/hollows) were found on HMT cannastarch inside the granules with blue staining. Consequently, thevariation in birefringence can be the reflection to change of internalgranule by HMT.

The observed loss of birefringence at the periphery of thegranule (Fig. 2E and F) might due to disorientation of chains withincreased moisture. It has been noted that the increased tempera-ture during HMTcould decrease birefringence at center and even atthe periphery of starch granules (Chung, Hoover, & Liu, 2009). The

loss of birefringence at the periphery is consistent with theconcavity or cracks shown by SEM (Fig. 1E and F).

3.4. Size distribution elution profile

The size distribution of native and heat-moisture treatedstarches were determined by high-performance gel permeationchromatography (HPGPC). HPGPC separates on the basis of mole-cule size so larger molecules elute before smaller molecules. TheHPGPC distribution was generally bimodal. In this study, Peak I isdefined as the larger molecules that were eluted first and Peak II asthose smaller molecules that eluted latter. The percentage of peakarea occupied by each fraction is presented in Table 2. The nativemung bean starch had a single Peak I, whereas the HMT starchfeatured both Peak I and II. Relative to native mung bean starch(100%), the area of Peak I decreased to a range from 64.8% to 73.0%,and so Peak II increased from 27.6% to 35.2%. These results indicate

Table 2Peak area of native and heat-moisture treated starches by HPGPC.

Samples Peak area (%)

Peak I Peak II

Native starch 100.0 0.00HMT-15 72.4 27.6HMT-20 67.8 32.2HMT-25 70.5 29.5HMT-30 73.0 27.0HMT-35 64.8 35.2

S. Li et al. / Food Hydrocolloids 25 (2011) 1702e1709 1707

that HMT degraded the native starch into smaller molecules andHMT starch was dominated by large molecules.

3.5. X-ray diffraction pattern and relative crystallinity (RC)

The relative crystallinity (% e RC) and X-ray diffraction patternof native and HMT starches are shown in Fig. 3. The RC of HMTstarches ranged from 29.1% to 34.5%. The relative crystallinity (RC)of HMT starch was more than the native starch whereby theextent of the increased RC on HMT was 7.5e12.9% (Fig. 2, data inparenthesis). The better arrays could originate from suggestion thatheat-moisture treatment may cause associations with new crys-tallization and perfection of the small existing crystalline regionsof the starch granule (Donovan, Lorenz, & Kulp, 1983; Hoover &Vasanthan, 1994) and/or new crystallites in amorphous region(Adebowale & Lawal, 2003; Hoover & Manuel, 1996a). Conse-quently, the ordered and/or new crystallites impart an increase indiffraction intensity and RC compared to that of the native granule.

Native mung bean starch exhibited the ‘A’ type crystallinepattern and showed a weak diffraction peak at 5.7� 2q and twoboard peaks at 16.9� and 19.7� 2q. Compared with native starch, theheat-moisture treated starch had more intense diffraction peaks at16.9� and 22.9� 2q. Moreover, the HMT starch showed newdiffraction peaks occur at 15.0� 2q, plus an unresolved doubletbetween 16.9� and 17.5� 2q.

After heat-moisture treatment, the ‘A’ type crystalline patternremained unchanged with increased diffraction intensity (Fig. 3).The increased X-ray intensity is in agreement with previous studyHMT starch from wheat, oat (Hoover & Vasanthan, 1994), fingermillet (Adebowale, Afolabi, et al., 2005), cocoyam (Lawal, 2005),some legumes such as green arrow pea (Hoover & Manuel, 1996a)and lentil (Hoover & Vasanthan, 1994), maize starches originatedfrom waxy, normal maize, dull waxy maize and amylomaize(Hoover &Manuel, 1996b). In contrast, a decrease in X-ray intensitywas reported for HMT starches derived from some other legumes

Fig. 3. X-ray diffraction pattern and relative crystallinity (in parenthesis) of native andheat-moisture (HMT) treated starches.

including black bean, express field, pinto bean, eston lentil (Hoover& Manuel, 1996a), potato and yam (Hoover & Vasanthan, 1994),normal corn (Chung et al., 2009) andwaxy corn (Franco et al., 1995).As results in this study confirm, Hoover and Manuel (1996a)suggested that the change in X-ray diffraction intensity variedgreatly from starch source and heat-moisture conditions. Thereforeliterature comparisons between HMT on starch should only becompared to those with similar botanical source and HMTtreatments.

HMT starch had similar X-ray diffraction patterns with differentmoisture treatments. However, the diffraction intensity and RC ofHMT starch did not increase with increasing heat-moisture levels.Therewas a slight decrease in X-ray intensity fromHMT-15 to HMT-20, whereas an increase in X-ray intensity from HMT-20 to HMT-25was observed. The change in X-ray diffraction intensity is based onboth the starch source and heat-moisture treated conditions.Adebowale, Afolabi, et al. (2005) reported a slight decrease inintensity at the moisture contents of 20%, 25% and 30%. Franco et al.(1995) observed a decrease in intensity for waxy corn starch treatedfrom 18% to 27% moisture. They also indicated that a decrease inwater-binding capacity and enzymatic hydrolysis was found at 18%moisture level and higher moisture level would disrupt theprevious crystalline order. It has been shown that differences inX-ray intensity were linked to the re-alignment or arrangement ofdouble helices within the crystalline domains of the granules(Hoover & Vasanthan, 1994). Adequate moisture content is favor-able for structural rearrangement with re-alignment of bondingforces in starch granules and formation of ordered double helicalamylopectin side chain clusters (Lawal, 2005). Results obtainedfrom X-ray diffraction pattern revealed that the magnitude of X-rayintensity changed with the moisture content of the starch sampleand supported the previous assumptions. In this work, appropriatemoisture level was preferable for the crystalline formation andsubsequent increase in moisture would decrease the crystallinityafter HMT. The X-ray pattern of mung bean starch showed thatmoisture level had impact on diffraction intensity and relativecrystallinity. So it is important to select appropriate moisture tocontrol the change in X-ray intensity and prevent disrupting theoriginal and newly developed crystallites. In this study, mung beanstarch with 20% moisture treated with HMT at 120 �C for 12 h isfavorable for RS formation.

3.6. Fourier-transform infrared spectroscopy

Van Soest, Tournois, De Wit, and Vliegenthart (1995) proposeda convenient way to quantitatively determine the starch short-range structure with infrared spectroscopy, as distinct from thelong-range order with packing of double helices from X-ray

Native HMT-15 HMT-20 HMT-300.82

0.84

0.86

0.88

0.90

0.92

Abs

orba

nce

Rat

io o

f 10

47/1

022c

m-1

Fig. 4. Ratio between 1047 cm�1 and 1022 cm�1 of FT-IR spectra of native and heat-moisture treated starches.

Table 3Thermal characteristics of native and heat-moisture treated starches.

Samples Toa (�C) Tpa (�C) Tca (�C) TceTob (�C) DHc (J/g)

Native starch 67.0 71.9 76.2 9.2 5.1HMT-20 75.9 91.4 99.5 23.7 22.1HMT-30 75.6 81.9 91.2 15.6 16.2

a To, Tp, Tc indicate the temperature of the onset, peak, conclusion of gelatini-zation, respectively.

b ToeTo indicates the gelatinization temperature range.c DH indicates enthalpy of gelatinization.

S. Li et al. / Food Hydrocolloids 25 (2011) 1702e17091708

diffraction. It was suggested that the IR absorbance band at1047 cm�1 is associated with the amount of ordered or crystallinestarch and the band at 1022 cm�1 is sensitive to amorphous starch.The ratios of the heights of the bands at 1047e1022 cm�1

demonstrated the ratio of ordered/crystalline regions to amor-phous regions of starch granules (Capron, Robert, Colonna, Brogly,& Planchot, 2007; Van Soest et al., 1995).

The ratio of 1047e1022 cm�1 on HMT starch was greater thanthat of native starch (Fig. 4). The result was probably attributed tothe alignment and associations of amyloseeamylose and/or amy-loseeamylopectin in the amorphous regions of starch during HMT.HMT-30 had the highest ratio between 1047 cm�1 and 1022 cm�1;this was consistent with the increase in RC from X-ray diffractionpattern (Fig. 3). It is presumed that the decrease of the ratiobetween 1047 cm�1 and 1022 cm�1 of HMT-15 and HMT-20happens due to a double helical reorientation within crystallinedomains and/or to disruption of very few of the hydrogen bondslinking adjacent double helices.

3.7. Thermal properties by DSC

The thermal parameters of native, HMT-20 and HMT-30 starchesdetermined by DSC are summarized in Table 3. Native mung beanstarch had an endothermic transition ranging from 67.0 �C to76.2 �C with enthalpy of 5.1 J/g (dry sample). Results clearly showthat HMT starch had an endothermic transition over a range ofapproximately 75.6 �Ce99.5 �C, with an increase of onset meltingtemperature of HMT starch about 9 �C. The increase in meltingenthalpy for HMT starches were about 11.1e17.0 J/g than in nativestarch. These results indicate that HMT starch showed more heatstability than that of native starch.

The difference in gelatinization temperature varied according toamylose content, the size, form and distribution of starch granules,and to the internal interaction and/or re-alignment of starch chainswithin the granule. Transition temperatures were impacted by the

Table 4Effect of temperature on swelling power and solubility of native and heat-moisture trea

Assay temperature (�C) Starch samples

Native starch HMT-15

Swelling power (g/g)a

55 3.8� 0.1 3.4� 0.165 4.2� 0.2 3.8� 0.375 13.8� 0.4 4.2� 0.185 17.9� 0.6 8.1� 0.195 22.1� 0.2 13.8� 0.9

Solubility (g/100 g)b

55 0.8� 0.0 2.5� 0.365 1.4� 0.7 3.5� 0.775 6.2� 0.1 7.6� 0.185 20.3� 1.1 48.5� 1.095 28.3� 0.7 63.2� 0.6

a Swelling power calculated as g/g starch on dry weight basis.b Solubility calculated as g/100 g starch on dry weight basis.

molecular architecture of the crystalline region (Miao, Zhang, &Jiang, 2009). The extent of starch chain associations within theamorphous regions and the degree of crystalline order increasedduring HMT (Gunaratne & Hoover, 2002; Watcharatewinkul et al.,2009). In addition, difference in TceTo reflected the extent ofheterogeneity of crystallites within the granules of native starch,HMT-20 and HMT-30. Cooke and Gidley (1992) suggest that the DHvalues represent the number of double helices that unravel andmelt during gelatinization. The increase in DH values in HMT starchcould be a result of greater amounts of double helices or strongerinteraction between starch chains within the crystalline domains ofstarch.

3.8. Swelling power and starch solubility

The effect of temperature on swelling power and solubility ofnative and HMT starch are presented in Table 4. HMT starchsamples that were treated with different moisture contentsexhibited different swelling power. The swelling power of HMTstarch was less than that of un-treated starch. The decreasedswelling power of HMT starch is consistent with previous on fingermillet starch (Adebowale, Afolabi, et al., 2005), maize starch(Kurakake et al., 1997), and mucuna starches (Adebowale & Lawal,2003). These studies suggested that a reduction in swellingcapacity of HMT starch accounts for the ordering rearrangement ofstarch molecule and restriction of starch hydration. It is known thatstarch granules start to swell in relatively mobile amorphousfraction (Donovan, 1979). During HMT, with the rearrangement ofmolecular chains and formation of ordered double helical amylo-pectin side chain clusters, a rigid structure within HMT starchgranule would limit starch swelling (Franco et al., 1995; Kurakakeet al., 1997; Lawal, 2005). Thus the decrease in swelling capacityis ascribed to the structural rearrangement and/or re-associationsof starch chains caused by the heat-moisture treatment.

Compared to the native starch, solubility of all the HMT starchsamples was greater (Table 4). A similar increase in solubility afterHMT was observed for wheat (Kulp & Lorenz, 1981), maize(Kurakake et al., 1997), and finger millet starches (Adebowale,Afolabi, et al., 2005). Changes to the solubility were independentof the moisture content of the starch from which the HMT wereprepared, and this result is consistent with to a previous study ofthe HMT potato starch (Eerlingen, Jacobs, VanWin, & Delcour,1996). Here, it is presumed that the increase in solubility isdirectly linked to the occurrence of weak structure on the surface ofstarch with enlarged voids inside starch granule during HMT(Vermeylen et al., 2006), which was supported by the results in

ted starches.

HMT-20 HMT-25 HMT-30 HMT-35

3.2� 0.2 3.2� 0.3 3.4� 0.6 3.9� 0.33.3� 0.3 3.4� 0.4 3.8� 0.5 4.0� 0.23.9� 0.1 4.1� 0.1 4.2� 0.1 5.5� 0.16.5� 0.3 7.5� 0.0 8.2� 0.0 9.1� 0.4

13.1� 0.5 13.4� 1.1 13.9� 0.9 14.7� 0.3

2.2� 0.2 3.1� 0.4 4.0� 0.8 3.6� 0.22.9� 0.3 3.2� 0.1 5.1� 0.1 4.1� 0.46.2� 0.1 6.5� 0.8 11.6� 0.3 10.4� 0.3

35.9� 1.4 37.4� 0.3 39.3� 0.1 30.0� 0.140.1� 1.7 52.9� 0.5 55.4� 0.5 41.0� 0.2

S. Li et al. / Food Hydrocolloids 25 (2011) 1702e1709 1709

Figs. 1 and 2. It is suggested that HMT starch may be easy for waterto access to starch amorphous regions and the remaining un-associated starch chains could solubilize into water, and thereforeincrease the solubility of starch.

4. Conclusions

Mung bean starch can be modified by heat-moisture treatmentto yield a product with increased resistant starch, which wascharacterized by granules with a pitted surface, decreased bire-fringence at the center of granules, increased apparent amylosecontents, greater in relative crystallinity, increased thermalstability, reduced the swelling capacity. Not only do these resultsclearly show opportunity to increase resistant starch for beneficialnutritional properties, but also the associated moisture dependentcharacteristics are ready to be exploited for alternate applicationsfor the HMT starch. These results are important for exploring theuse of mung bean with special advantages, and are good forunderstanding the potential characteristics of HMT starch fromtraditional Chinese crop.

Acknowledgments

The authors gratefully acknowledge the financial assistanceprovided by the National High-tech R&D Program of China(2007AA10Z309) and Guangdong Province Program of China(2009B090300274).

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