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International Journal of Biological Macromolecules 66 (2014) 325–331

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

asting and rheological properties of rice starch as affected by pullulan

ong Chena,b, Qunyi Tonga,b,∗, Fei Renb,c, Guilan Zhub

The State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, ChinaSchool of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, ChinaCollege of Life Science and Technology, Southwest University of Science and Technology, Mianyang 621010, China

r t i c l e i n f o

rticle history:eceived 23 January 2014eceived in revised form 17 February 2014ccepted 23 February 2014vailable online 6 March 2014

eywords:

a b s t r a c t

Effect of pullulan (PUL) on the pasting, rheological properties of rice starch (RS) was investigated. Theswelling power, amylose leaching, and confocal laser scanning microscopy (CLSM) observation of thesamples were also conducted to explore the possible interaction between starch and pullulan. Rapidvisco-analysis (RVA) showed that PUL significantly changed viscosity parameters of rice starch–pullulan(RS–PUL) mixtures. Dynamic rheological measurements revealed that the modulus (G′, G′′) of the mixturesincreased with the increase of pullulan concentration from 0.01% to 0.07%, but then decreased with

ice starchullulanheological propertiesacromolecular interaction

the increase of pullulan concentration from 0.07% to 0.50%. The pasting and rheological properties ofsamples indicated that pullulan could blend well with rice starch and promote the gelatinization ofstarch granules at low concentration of pullulan, but suppress the gelatinization of starch granules athigh concentration of pullulan. The results of swelling power, leached amylose and CLSM observation ofsamples further suggest that the interaction between starch and pullulan occurred in the RS–PUL systemand the interaction was hypothesized to be responsible for these results.

. Introduction

Starch is one of the most important and abundant materials inature, and possesses extensive applications, such as thickening,elling, stabilizing, and binding agent in food industry [1]. How-ver, native starch sometimes does not satisfy the requirements ofroduction due to its retrogradation tendency [2–4] and instabilitynder shear and acidic conditions [5]. These changes would bringndesirable effects on the quality of starch–based products.

Physical blending starch with non-starch polysaccharides, as aafe and effective method to modify the quality of native starch,as extensively reported by many researchers. The specific mod-

fication of the properties of starch is significant. Wang et al. [6]nvestigated the effect of flaxseed gum on the rheological proper-ies of maize starch and found that the addition of flaxseed gumas in favor of forming stronger gels and could be used togetherith starch to get a high apparent viscosity in industrial produc-

ion. Kim and Yoo [7] indicated that xanthan gum could promotehe recrystallization at the beginning of aging, and so increased the

odulus of the rice starch–xanthan gum mixtures. Also, Sasaki andohyama [8] reported the effects of different non-starch polysac-harides on the characteristics of mixture systems and Tischer et al.

∗ Corresponding author. Tel.: +86 510 85919170; fax: +86 510 85919170.E-mail addresses: 973611919@qq.com (L. Chen), qytong@263.net (Q. Tong).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.02.052141-8130/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

[9] reported on the influence of iota-carrageenan on the rheologi-cal properties of different starches. It was widely accepted that theaddition of non-starch polysaccharides in starch system evidentlychanged the characteristics of starch and the interaction betweenstarch and non-starch polysaccharides was considered to play animportant role in determining the properties of these combinationsystems [10,11].

Pullulan is a typical linear exocellular polysaccharide producedby Aureobasidium pullulans. It has a starch-like structure of link-age ˛-d-glucan primarily consisting of maltotriose repeating unitsinterconnected by ˛-(1→6) linkages, resulting in a stair-step struc-ture [12]. The regular structure conferred pullulan some distinctiveproperties compared to other polysaccharides, such as enhancedsolubility and preferable film forming ability [13,14]. Pullulan, asan important non-starch polysaccharide, has been widely used infood industry, especially the production of edible film [15]. Physi-cochemical properties of pullulan have been extensively studied inmodel aqueous system [16,17].

However, researches focused on the effect of pullulan on thephysicochemical properties of starch were limited, and the knowl-edge about the interaction between pullulan and starch was stillinsufficient. Rice and rice starch products are staple foods in ori-ental countries, therefore, in the present study, the rice starch was

chosen as a model to assess the interaction between starch andpullulan. The main objectives of this work were: (1) to investigatethe effect of pullulan on the pasting, and rheological properties of

3 iological Macromolecules 66 (2014) 325–331

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26 L. Chen et al. / International Journal of B

ice starch; (2) to explore the interaction between rice starch andullulan; (3) to offer the possibility of modifying the quality of ricetarch by means of blending starch with pullulan. Such studies areeaningful in exploring the potential utilization of microbiological

olysaccharides to improve the processing characteristics, storagetability of rice starch-based foods.

. Materials and methods

.1. Materials

Rice starch was obtained from Jiangsu Baby CorporationSuqian, China). It contained 0.23% proteins, 0.68% free lipids,3.30% amylose, and 11.82% moisture. Pullulan was purchasedrom Hayashibara Biochemical Inc. (Shanghai, China). The mois-ure content and molecular weight of pullulan were 4.50%, and00,000 MW, respectively.

.2. Sample preparation

The rice starch (5%, w/v)–pullulan (0.01, 0.04, 0.07, 0.1, 0.3, and.5%, w/v) mixtures (RS–PUL) were prepared as follows. Briefly, var-

ous quantities of pullulan were dissolved thoroughly in 100 mLf distilled water in closed tubes, and then 5 g of rice starch (dryeight) was added. The mixtures were dispersed at room temper-

ture by magnetic stirring for 30 min. Then, the RS–PUL mixturesere heated at 95 ◦C in a water bath for 20 min with continuous

tirring to obtain a complete gelatinization of the rice starch. Lastly,he hot mixtures were allowed to cool down slowly to 25 ◦C. Ricetarch without pullulan was used as a control.

.3. Pasting properties

Pasting properties of RS and RS––PUL mixtures were deter-ined by using a rapid visco-analyzer (RVA-RECHMASTER,ewport Scientific Pty. Ltd., Sidney, Australia). The pasting param-ters were measured following the general pasting method (STD 2).ample was equilibrated at 50 ◦C for 1 min, heated to 95 ◦C within.5 min, and then held at 95 ◦C for 5 min. The hot sample was sub-equently cooled to 50 ◦C within 7.5 min, and maintained at 50 ◦Cor 4 min. Paddle speed was 960 rpm for the beginning 10 s to dis-erse the sample, and then the speed of paddle was set at 160 rpmuring the measurement. Pasting parameters included peak viscos-

ty, trough viscosity, final viscosity, breakdown value, and setbackalue and the viscosity parameters were expressed in cP units inresent study.

.4. Rheological measurements

Rheological measurements were carried out using an AR G2heometer (TA instrument Inc., USA). Parallel plate geometry60 mm) at gap 500 �m was used for both steady shear and smallmplitude oscillatory measurements. Samples were transferred tohe rheometer plate at 25 ◦C. The excess material was wiped offith a spatula. Silicon oil was applied to the exposed surfaces of

amples to prevent evaporation during experiments. The sampleas allowed to rest for 5 min in order to equilibrate stresses before

tarting the test.Steady shear experiments were performed over the shear rate

ange of 0.1–10 s−1. According to Barnes et al. [18], the shear rateange of present study included three parts and could simulate

he shear effect which the food materials subjected in producingrocedures.

The frequency sweep tests were performed at 25 ◦C overhe frequency range of 0.1–10 Hz, and the strain value for

Fig. 1. Effect of pullulan on the apparent viscosity of rice starch (RS) and ricestarch–pullulan (RS–PUL) mixtures at 25 ◦C.

measurements was selected as 3%. The 3% strain was in the lin-ear viscoelastic region according to the strain sweep results (datanot shown).

For the rheological properties of samples, one representativedatum was drawn in Figs. 1 and 2, because there were no differ-ences in data from triplicate measurements. For the power lawparameters in Table 2, however, data were presented as means oftriplicate.

2.5. Determination of swelling power and amylose leaching

Swelling power of samples during heating was determined fol-lowing the method reported by Chaisawang and Suphantharika[19] with a slight modification. Firstly, suspensions of 5% rice starchalone and rice starch (5%)–pullulan (0.01, 0.04, 0.07, 0.10, 0.30, and0.50%) blends were prepared in centrifuge tubes with closed screwcaps. Then the samples were heated in a shaking water bath atdesignated temperatures in the range of 50–90 ◦C for 30 min. Afterheating, samples were cooled to room temperature and then cen-trifuged at 10,000 × g for 30 min. The supernatant was separated todetermine the leached amylose by the iodine colorimetric reactionaccording to the method described by Chrastil [20], and the resultsshould be regarded as “apparent amylose”, which included not onlyamylose but also segmental amylopectin leached from starch gran-ules during gelatinization [21]. The leached amylose was calculatedby dividing the amylose content in the supernatant by the originalweight of the rice starch. The precipitate was weighed and thendried to constant weight in a hot air oven at 105 ◦C. The swellingpower was expressed as the ratio of the wet weight of the residueto its dry weight.

2.6. Confocal laser scanning microscopy (CLSM)

The ultrastructure observation of samples was carried out usingconfocal laser scanning microscopy (Carl Zeiss Inc., Braunschweig,Germany) to visualize the distribution pattern of pullulan aroundthe surface of starch granules according to the method proposed byFunami et al. [21] with a slight modification. The rice starch (5%),rice starch (5%)–pullulan (0.01%, 0.5%) and pullulan (0.5%) were dis-

persed and heated in boiling water bath for 20 min to gelatinize therice starch. Two types of fluorescent dyes, fluorescein isothiocya-nate (FITC) (20 �L at 2 mg/mL) and rhodamine B (20 �L at 2 mg/mL)were used to identify pullulan and starch. It had been proved that

L. Chen et al. / International Journal of Biological Macromolecules 66 (2014) 325–331 327

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he multiple staining could lead to an intuitionistic understandingf interactions between components [22] and a better comprehen-ion of macroscopic rheological behavior of polymer system [21].hen the samples and dyes mixtures were kept in dark place atoom temperature for 4 h. Dyed sample was deposited on a micro-cope slide, dried in the air, and washed with distilled water toemove the excess dyes. The sample was observed within 10 minfter sample preparation. Emission was within 500–525 nm for FITCnd 600–635 nm for rhodamine B, respectively.

.7. Statistical analysis

Statistical significance was assessed by one-way analysis ofariance (ANOVA) using SPSS 20.0 (SPSS Inc., Chicago, USA) forindows program. The level of significance was set at p < 0.05.

. Results and discussion

.1. Pasting properties

The pasting properties of RS and RS–PUL mixtures at differ-nt concentration of pullulan are summarized in Table 1. Pullulanffected the pasting properties of rice starch effectively and thenfluence was highly dependent on the concentration of pullulan.enerally, pullulan caused a slight increase in peak viscosity atoncentration of pullulan ≤ 0.07%. This could be attributed to theossible interactions between starch molecules and hydrocolloiduring the pasting procedure [23]. Though the interaction between

mylose and pullulan are probably mainly responsible for the vis-osity increase, the interaction between pullulan and amylopectin,specially the fraction with long exterior chains, should be also con-idered as a factor to raise the viscosity of samples, for the reason

arch (RS) and rice starch–pullulan (RS–PUL) mixtures.

that amylopectin with long exterior chains might behave in a simi-lar way to amylose and leach out from starch granules upon heating.In addition, at low addition concentration, pullulan could not coverthe starch granules, but could promote an increase of the capac-ity of the starch granules to swell freely, therefore the viscosity ofrice starch increased. Based on the pronounced increase of leachedamylose and swelling power (Section 3.4) at low concentration ofpullulan, we would also expect an increase in peak viscosity. How-ever, a sharp decrease in peak viscosity occurred when the additionlevels of pullulan were higher than 0.07%. This might be ascribed tothe inhibition effect of pullulan on the swelling of starch granule.

Pullulan induced an evident reduction of final viscosity, break-down value, and setback value of the rice starch at high pullulanconcentration. The decrease in breakdown values suggested thatthe disruption of starch granules integrity were inhibited by addi-tion of pullulan. It might be interpreted by assuming that thepullulan could cover around the starch granules step by stepwith the increasing concentration of pullulan. The decrease insetback value of rice starch revealed that retrogradation (espe-cially the short-term retrogradation of amylose) of starch mightalso be retarded by pullulan. The higher the pullulan concen-tration used, the higher the setback reduction of the RS–PULmixtures. The inhibition of retrogradation might be attributedto the interaction between leached amylose and pullulan. Highconcentration of pullulan might interact with amylose moleculesto form pullulan–amylose intermolecular connection, and thendecrease the quantity of amylose–amylose interactions, whichwere very important to retrogradation of starch. From the applied

point of view, the inhibition effect of pullulan on the recrystal-lization process of starch molecule is worth investigating furtherto develop starch-based products with longer shelf lives and goodmouthfeel.

328 L. Chen et al. / International Journal of Biological Macromolecules 66 (2014) 325–331

Table 1Pasting properties of rice starch with and without the addition of pullulana.

Sample Viscosity (cP)

Peak Trough Breakdown Final Setback

5%RS 728.7 ± 4.5d 645.0 ± 2.6d 83.7 ± 2.1d 1155.3 ± 5.0f 510.3 ± 2.5g5%RS + 0.01%PUL 731.7 ± 2.5d 651.0 ± 3.6d 80.7 ± 1.5d 1135.7 ± 2.5d 484.7 ± 1.5f5%RS + 0.04%PUL 740.3 ± 4.5d 661.3 ± 5.1e 79.0 ± 1.0d 1127.3 ± 4.0d 466.0 ± 2.0e5%RS + 0.07%PUL 763.3 ± 6.0e 684.0 ± 5.3f 79.3 ± 2.5d 1145.3 ± 4.0e 461.3 ± 2.5d5%RS + 0.10%PUL 705.7 ± 6.5c 631.7 ± 4.2c 74.0 ± 2.6c 1072.3 ± 5.5c 440.6 ± 2.1c5%RS + 0.30%PUL 626.3 ± 8.0b 568.0 ± 4.6b 58.3 ± 3.5b 972.7 ± 5.1b 404.7 ± 0.6b

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as observed by means of confocal laser scanning microscopy (Sec-tion 3.5). As a result, amylose molecules were restricted to diffusedinto the starch paste, and therefore the formation of elastic gel wasinhibited. Furthermore, the reduction of G′ also might be resulted

Table 2Influence of pullulan concentration on power law parameters (K, n) of the ricestarch–pullulan mixturesa.

PUL conc. (%) K (Pa sn) n (-) R2

0.00 3.153a 0.737a 0.9950.01 4.032b 0.800ab 0.9970.04 9.183e 0.775ab 0.9970.07 11.694f 0.881b 0.9990.10 7.502d 0.849ab 0.9930.30 7.081d 0.796ab 0.997

5%RS + 0.50%PUL 567.0 ± 7.0a 517.7 ± 4.5a

a Tests were performed in triplicate. Mean ± standard deviation values in the sam

It could be concluded that the pullulan could effectively affectasting properties of rice starch, and different interaction betweentarch molecule and pullulan occurred at different pullulan con-entration.

.2. Steady shear rheological properties

The flow steady curves of RS–PUL mixtures are shown in Fig. 1.t could be seen that all samples exhibited non-Newtonian shear-hinning flow behavior due to the fact that the apparent viscosityf the samples decreased as shear rate increased. Similar results fortarch–polysaccharide system were reported [6,7].

Pullulan could effectively influence the steady rheological prop-rties of rice starch. The effect of pullulan on apparent viscosityf RS–PUL mixtures was dependent on the concentration. Thepparent viscosity of RS–PUL mixtures increased initially with thencrease of pullulan from 0.01% to 0.07%, but then decreased withhe further increase of pullulan from 0.07% to 0.5%. This trend wasn good agreement with the results of pasting characteristics.

The phenomena above might be attributed to the differentnteraction between rice starch and pullulan. When the pullulanddition level was low (0.01–0.07%), the pullulan could thoroughlyisperse in blend system and promote the swelling of starch gran-les duo to the excellent hydrophilic ability of pullulan [24]. Inuch a case, several pullulan molecules might be adhered on theurface of rice starch through hydrogen bond during pasting orurface absorption of starch granules, these pullulan moleculescted like hydrophilic radical to improve the hydration and swellingharacteristics of starch granules, therefore, increased the viscos-ty of mixture samples. In addition, the interaction between leachedmylose and pullulan might be occurred at low pullulan concentra-ion, and the amylose-pullulan molecular composite might haveigher viscous characteristic than amylose–amylose system. As aesult, the viscosity of RS–PUL samples increased at low concen-ration of pullulan. However, the pullulan might wrap around theurface of the starch granules at the relative high concentration. Thewelling levels of starch granules were therefore decreased, and sohe viscosity decreased.

The power law model (Eq. (1)) was applied to describe the varia-ion in rheological properties as a function of shear rate for RS–PUL

ixtures. The model was used extensively to describe the flowroperties of non-Newtonian liquids in both theoretical researchnd practical applications [18]. The power law parameters (n, Knd R2) were summarized in Table 2.

= K.�

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where � is the shear stress (Pa), � the shear rate (s−1), K theonsistency index (Pa sn), and n is the flow behavior index.

The experimental data of flow steady tests for RS and RS–PULixtures were fitted well with the power law equation with high

etermination coefficient (0.993–0.999), as shown in Table 2. Thereere little differences between n values, whereas K values were

49.3 ± 3.1a 866.3 ± 6.5a 348.6 ± 2.1a

n for each sample followed by different letters are significantly different (p < 0.05).

dependent on the concentration of pullulan (p < 0.05). When addi-tion levels were less than 0.07%, the K values increased with theincrease of pullulan concentration. On the contrary, the K valuesdecreased as the concentration of pullulan increased from 0.07% to0.5%. Compared with starch system, the RS–PUL system had higherK values. These results indicated that the addition of pullulan couldsignificantly influence the steady rheological properties of ricestarch, suggesting possible interactions between starch moleculesand pullulan.

3.3. Dynamic rheological properties

The variation of storage modulus (G′), loss modulus (G′′) andloss tangent (tan ı) as a function of frequency at 25 ◦C were shownin Fig. 2. The G′, G′′ of all samples increased with the frequencyincreased, and the addition of pullulan apparently changed thedynamic modulus (G′, G′′). The result indicated that the additionof pullulan influenced the structure of rice starch gel. When theaddition level of pullulan was relative low (0.01, 0.04, 0.07, and0.10%), the G′ values of samples were greater than rice starch, whichmight be attributed to the interaction between pullulan and starch,especially the interaction between leached amylose and pullulan[23].

In order to evaluate the effect of pullulan concentration on thedynamic rheological properties for RS and RS–PUL, the modulus(G′, G′′) of samples at defined frequency (0.1 Hz, 1.0 Hz and 10 Hz)were compared with those of starch, as could be seen from Table 3.G′, of the RS–PUL mixtures increased gradually with the increaseof pullulan concentration over the range of 0–0.07%, suggestingthat pullulan could pronouncedly influence the elastic characteris-tic of rice starch at quite a low concentration of pullulan. However,the G′ decreased with the increase of pullulan concentration overthe range of 0.07–0.5%. This decrease might be ascribed to thefact that pullulan wrapped around most of the starch granules,

0.50 5.709c 0.815ab 0.994

K, consistency index; n, flow behavior index; R2: determination coefficient.a All data represent the mean of triplicates. Means within a column with different

letters are significantly different (p < 0.05).

L. Chen et al. / International Journal of Biological Macromolecules 66 (2014) 325–331 329

Table 3Storage (G′) and loss (G′′) modulus at different frequencies for RS–PUL system withdifferent pullulan concentrationsa.

PUL conc. (%) G′ (Pa) G′′ (Pa)

0.1 Hz 1 Hz 10 Hz 0.1 Hz 1 Hz 10 Hz

0 20.63c 22.54c 29.86a 1.101a 2.928a 9.608a0.01 21.75d 23.79c 28.95a 1.050a 2.980a 9.922a0.04 24.71e 28.53d 40.02bc 2.018d 4.482c 11.740c0.07 26.03f 30.67e 40.71c 2.528f 5.310d 12.150c0.10 24.15e 28.59d 38.99b 2.194e 5.015d 12.230c0.30 17.09b 20.55b 30.24a 1.721c 4.165c 10.960b0.50 16.20a 19.02a 29.06a 1.335b 3.638b 10.130a

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a All data represent the mean of triplicates. Means within a column with differentetters are significantly different (p < 0.05).

rom the competition of pullulan and amylose molecules to estab-ish an intermolecular connection through hydrogen bond, thusecreasing the quantity of amylose–amylose interactions, whichere fundamental to elastic character of starch gelatin. Similarly,

he G′′ increased with the increase of pullulan at low addition levels≤0.07%), but were inversely proportional to the concentration ofullulan at high addition levels (>0.07%).

In comparison with Fig. 2A and B, the G′ values were higherhan the G′′ values in the experimental range for all samples, both

odulus were dependant on frequency, and they did not crossoverach other throughout the frequency range varied from 0.1 to 10 Hz,hese results suggested that both RS and RS–PUL mixtures exhibitedypical biopolymer gel system [25]. The changes in G′ values afterdding pullulan to rice starch were more evident in comparisonhose of G′′. It indicated that there was a more pronounced effectf pullulan on the elastic properties of rice starch gelatin than theiscous properties.

As could be seen in Fig. 2C, the loss tangent (tan ı) of the RS–PULlends increased initially with the increase of pullulan concentra-ion, but then decreased when pullulan concentration was higherhan 0.3%, indicating that the total structure of starch gel becameeaker when the addition levels were lower than 0.3%. On the

ontrary, the tan ı of mixtures decreased with the higher pullu-an concentration (>0.3%). So, pullulan had a double-faces effectn rice starch, which would change the gel network of rice starchrom viscoelastic to more viscous-like at low pullulan concentra-ion or more solid-like at high pullulan concentration. This could bettributed to the different interaction between starch and pullulan,s discussed above.

.4. Swelling power and leached amylose

The variation of leached amylose during heating was shownn Fig. 3. As could be seen, the leached amylose of samples wasery low when the temperature was lower than 60 ◦C, but thenncreased quickly with the temperature increased. This variationendency could be interpreted by the fact that starch could notelatinize when the temperature was lower than pasting tempera-ure. However, starch granules could swell and gelatinize graduallyhen the temperature was higher than starting point of gelatiniza-

ion temperature [26,27]. The amylose leaching curves of starchlone and RS–PUL samples were nearly overlapped with each othern the temperature range of 50–80 ◦C. However, when the tem-erature increased up to 90 ◦C, leached amylose of samples wasignificantly different (p < 0.05). The leached amylose of RS–PULixtures increased with the increase of pullulan concentration over

he range of 0.01–0.07%, which could be attributed to the positive

ffect of pullulan on gelatinization of rice starch at low additionaloncentration. However, an inhibition effect was observed with theelatively low leached amylose content when the concentration ofullulan was high. It could be ascribed to the restricted influence on

Fig. 3. Variation of leached amylose with temperature for rice starch (RS) and ricestarch–pullulan (RS–PUL) mixtures. Data are shown as mean ± SD (n = 3).

starch gelatinization when the concentration of pullulan was high.This explanation could be further supported by the swelling powerof samples measured at 90 ◦C (Fig. 4).

As could be seen from Fig. 4, the RS–PUL mixtures exhib-ited greater swelling power (SP) (g/g) at low concentration thanthe control, however SP decreased as the concentration of pullu-lan increased from 0.07% to 0.5%. Low concentration of pullulanincreased the swelling power of starch because of the promotingeffect on water absorption and granule gelatinization of starch,as discussed in the results of pasting and rheological propertiesabove. On the other hand, pullulan decreased the swelling powerat high addition level. Pullulan might compete with starch gran-ules to absorb water and lead to incomplete swelling of the starchgranules.

3.5. Confocal laser scanning microscopy (CLSM)

Fig. 4. Swelling power of rice starch (RS) and starch–pullulan (RS–PUL) mixtures at90 ◦C. Data are shown as mean ± SD (n = 3).

330 L. Chen et al. / International Journal of Biological Macromolecules 66 (2014) 325–331

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y FITC, but stained less intensively by rhodamine B. This could beonfirmed by our result (Fig. 5A). At the same time, pullulan coulde dyed well with rhodamine B (Fig. 5D). Double fluorescent dyesould be used to confirm the distribution of pullulan in the RS–PULixtures system (Fig. 5B and C). In the micrographs, green areas

orresponded to the fluorescence of FITC, revealing the presence ofelatinized starch. Red areas corresponded to the fluorescence ofhodamine B, allowing the localization of pullulan in the mixture.

The starch ghosts [29] were observed in micrographs exceptor Fig. 5D. In Fig. 5B, the pullulan was dispersed in rich starchel system to form relative homogeneous gels, suggested that thetarch granules were free to swell and gelatinize throughly becauseo restrictive network was formed at low pullulan concentration0.01%). However, agglomerated particles were observed and thetarch components were prevented from leaching out of the starchranules when the addition concentration of pullulan was high0.5%), as could be seen in Fig. 5C. The agglomerated particles wereomposed of rice starch and pullulan, in which the pullulan estab-ished the network-like structure and starch granules were partlymbedded in the network. Interestingly, the color of the network-ike structure was glassy yellow rather than red. This might bexplained by the occurrence of colocalization between pullulannd amylose, where the pullulan and the leached amylose jointly

ormed the network-like structure. This phenomenon further indi-ated that the interactions between rice starch (especially amylose)nd pullulan indeed occurred in the RS–PUL system. Therefore, theellow of the network-like area was the superposition of green

tarch; (B) 5% rice starch–0.01% pullulan; (C) 5% rice starch–0.5% pullulan; and (D)

(dyed starch) and red (dyed pullulan). The restricting effect ofpullulan promoted association effect among starch granules andrestricted swelling of granules. The results were in good agreementwith those of rheological and pasting experiments, which could beseen in Table 1 and Figs. 2–4.

4. Conclusions

Rice starch–pullulan (RS–PUL) mixtures were prepared, andthe pasting and rheological properties of mixtures were studied.RVA results showed that pullulan might promote the gelatiniza-tion of rice starch at low pullulan concentration (0.01–0.07%),whereas inhibited the gelatinization at high pullulan concentra-tion (0.07–0.5%). Steady flow tests showed that both RS and RS–PULmixtures were shear-thinning flow. Dynamic viscoelastic measure-ments on the RS–PUL mixtures indicated that modulus (G′, G′′)increased with the increasing of pullulan concentration from 0.01%to 0.07%, and then decreased with the increasing of pullulan con-centration from 0.07% to 0.5%. The results of swelling power andleached amylose of samples also suggested that the gelatinizationof starch could be promoted at low pullulan concentration andinhibited at high pullulan concentration. The phenomenon mightbe ascribed to a different distribution of pullulan around the sur-

face of starch, as could be confirmed in the CLSM observation ofsamples. The present work indicated that the pasting and rheo-logical characteristics of the rice starch were evidently affected bypullulan, and the extent of influence was apparently dependent

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n the concentration of pullulan. The interactions between starchranules, amylose molecule and pullulan were hypothesized to beesponsible for these results.

cknowledgements

The authors thank Dr. Zipei Zhang, Dr. Marwan, and Dr. Xiaoyuhang for helpful discussion and reviewing the manuscript, Mr.uojun Liu for the use of the rheological instrument, Mrs. Weijunang for technical assistance with CLSM experiment.

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