Food Chemistry 114 (2009) 821–828

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Food Chemistry

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Effects of Alcalase/Protease N treatments on rice starch isolation and their effectson its properties

Yue Li a, Charles F. Shoemaker b, Jianguo Ma a, Changrong Luo c, Fang Zhong a,*

a State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR Chinab Department of Food Science and Technology, University of California, Davis, CA 95616, USAc Huabao Edible Essence and Spice (Shanghai) Co., Ltd., Shanghai 201821, PR China

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 April 2008Received in revised form 13 September2008Accepted 13 October 2008

Keywords:Rice starchStarch isolationAlcalase/Protease NPastingRheology

0308-8146/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.foodchem.2008.10.023

* Corresponding author. Tel.: +86 13812536912; faE-mail address: yue_li1@yahoo.com.cn (F. Zhong).

The effects of different protease treatments on rice starches and their properties were studied. The ricestarches produced from protease N exhibited higher pasting viscosities than those produced from alca-lase. The hot pastes of the starches produced from protease N also showed higher elastic moduli, zero-order Newtonian viscosities and yield stresses than those produced from alcalase. No differences werefound in the crystalline pattern, thermal properties, granules appearance, and average molecular weight(Mw) of the rice starches between the two protease treatments. But the Mw of the pasted starch producedfrom protease N was higher than that produced from alcalase. When additional protease was added tothe isolated starches and the mixture pasted, the Mw of the starches pasted with added alcalase was sig-nificant lower than that of the starches pasted with added protease N. The reduction in molecular weightsuggested that alcalase had modified the starch molecules during pasting.

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1. Introduction starch separation process, are preferred for rice starch isolation.

Starch is the major component of rice grains and an importantfunctional ingredient in prepared foods. High purity rice starch isdesired to provide minimum rancidity during storage, a high de-gree of whiteness, a bland flavor, and small granules (5 lm). It alsoserves as a starting material for chemical modification, fermenta-tion, and industrial applications (Lumdubwong & Seib, 2000; Puc-hongkavarin, Varavinit, & Bergthaller, 2005).

Alkaline steeping (using 0.2–0.5% NaOH, w/w) has been conven-tionally used for isolation of rice starch with good recovery and lowresidual protein content (Landers & Hamaker, 1994; Resurreccion,Li, Okita, & Juliano, 1993; Tanaka, Sugimoto, Ogawa, & Kasai, 1980;Yang, Lai, & Lii, 1984), but this method causes effluent problems.Chiou, Martin, and Fitzgerald (2002) have reported that the struc-ture of the starch granule can be damaged when the rice grainswere steeped in 0.2 M aqueous ammonia solution. A physical pro-cess which employed high-pressure homogenisation was studiedfor recovering rice starch and protein fractions by partial mechan-ical breaking of the starch–protein matrix (Guraya & James, 2002).The residual protein in the starch yield was 2.7%, which was great-er than that of an alkaline process.

Gains in efficiency, while preserving the native structure of therice starch granules and limiting the waste products from the

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The removal of proteins in starch granules using protease appearedto provide an effective method for producing rice starch and mayserve as an alternative to an industrial alkaline process. Most foodscientists believed that protease treatments without additionalchemicals result in starch granules retaining most of their nativecharacteristics. Chiou et al. (2002) used a shorter period for prote-ase starch isolation and compared the isolated rice starch with astarch recovered from an ammonia treatment and a starch ex-tracted by means of a laundry detergent. They concluded that thefine structure of the amylopectin could be preserved upon isolationof starch by either protease or detergent but not with alkalinemethods. Wang and Wang (2004) applied high-intensity ultra-sound together with a neutral protease and produced rice starchwith 0.50–0.96% residual protein content, and no damage was ob-served with scanning electron microscopy on the starch granulesurface. However, peak viscosities were increased by sonicationtreatment. Recently, effect of protease purification on the function-ality of maize starches was studied (Tester, Yousuf, Karkalas, Kett-litz, & Röper, 2008). The authors reported that commercialproteases, such as Promod 25P, which was used for maize starchextraction, could cause modification of starch by solubilisation ofa-glucans from swollen granules.

Rice starch is often used to provide various textural propertiesin foods, but few reports have been found to study the applicationproperties of the rice starch produced from protease treatment.In our previous study (Li, Shoemaker & Shen et al., 2008), two

822 Y. Li et al. / Food Chemistry 114 (2009) 821–828

food-grade enzyme (an alkaline protease, Alcalase and a neutralprotease, protease N) preparations were found to be more effectivethan other proteases for protein removal in the isolation of ricestarch from wet-milled flour. With regard to the properties of theisolated starches, it was found that Protease N treatments pro-duced rice starch with similar pasting viscosities to the native riceflour, while the starch produced from Alcalase treatments had low-er pasting final viscosities than the flour (Li, Shoemaker & Shenet al., 2008). Since the functional or physiochemical properties ofrice starch are closely related to its structure, the difference inpasting properties may have resulted from granular or molecularstructure alterations.

In this paper, pasting and rheological properties of starches thatwere isolated from four rice varieties with Alcalase or Protease Nwere investigated. Additional studies of the effect of enzyme treat-ment on the granular and molecular structure of the starches werealso studied.

2. Materials and methods

2.1. Materials

Four rice varieties were studied. Waxy (CM 101), short (Koshi),medium (M202), and long (Cocodrie) grain rice samples weregrown and milled by the California Cooperative Rice ResearchFoundation, Biggs, CA. The commercial enzyme, Alcalase 2.4L(activity 2.4 AU/g) was provided by Novo Nordisk (Beijing, China)and Protease N ‘‘Amano” (activity 150,000 units/g) was purchasedfrom Amano Pharmaceutical Co., Ltd (Nagoya, Japan). They wereboth food-grade and used without further purification. Ricestarches (protein content < 0.5%, total starch content > 96%, dry ba-sis) isolated by conventional alkaline steeping were provided byJiangsu Baobao group Co., Ltd in Jiangsu, China.

2.2. Isolation of rice starch

Milled rice (50 g) was soaked in 150 ml deionised water for 18 hat 4 �C in a refrigerator and then blended in a Waring blender for3 min. The rice flour slurry was transferred to a jacketed beaker,and the temperature was maintained at the optimal condition(50 �C) of each protease with a circulator bath. The initial pH ofthe dispersion was adjusted to 8.5 for Protease N and 10.5 for Alca-lase treatments, using 1.0 M NaOH. Two hundred units per ml Pro-tease N or Alcalase were added, and the protease hydrolysis wasconducted for 4 h with constant stirring using a magnetic stirrer.After the hydrolysis the dispersion was centrifuged at 10,000 gfor 10 min. The supernatant and the surface brown layer of thestarch were removed and the lower white starch layer was washedwith deionised water and centrifuged 3 times. The washed starchwas freeze-dried, passed through a 200-mesh sieve and stored un-til analysed.

2.3. Measurement of protein content

Nitrogen was determined in duplicate by the Kjeldahl proce-dure, according to AACC Approved Method 46-13 (2000), and pro-tein content of isolated starches was calculated by multiplyingnitrogen contents by 5.95.

2.4. Measurement of amylose content

Amylose contents of isolated rice starches were analysed intriplicate with an amylose/amylopectin assay kit (Megazyme Inter-national Ireland Ltd., Bray, Co., Wicklow, Ireland) based on the con-canavalin A method (Yun & Matheson, 1990).

2.5. Measurement of moisture content

The moisture content of isolated starches was analysed in trip-licate using AACC method 44–40 (2000).

2.6. Pasting and rheological properties

The pasting properties of rice flour and the starches were mea-sured on a rotational rheometer (Rheolyst AR 1000N, TA Instru-ment, New Castle, DE) as described by Zhong et al. (in press). Acone and plate geometry was used. The cone made from a polysulf-one plastic, had a 4� angle and was 40 mm in diameter. Starch dis-persions (8%), (w/w, dry weight basis) were prepared and acalibrated pipette was used to deliver 1.1 ml of a dispersion be-tween the cone and plate, which was the exact volume to fill thegap. Three replicates were analysed of each dispersion. A thin layerof silicon oil was dispensed around the perimeter to minimizeevaporation during the measurements.

A program was written with the TA Advantage Software Version4.0 (TA Instrument) and used for the pasting and rheological mea-surements of starch slurries with the rheometer. After loading, thestarch dispersion was held at 50 �C for 1 min at 200 s�1. The tem-perature was raised to 95 �C at a rate of 12 �C/min, and held at95 �C for 2 min 30 s. The temperature was then decreased to50 �C at the same rate, and finally held at 50 �C for 1 min. The shearrate during the pasting was maintained at 200 s�1. Next, the sam-ple temperature was raised to 65 �C and the sample was held for5 min at rest before continuing.

In the next step, the viscoelastic properties of the paste weremeasured at 65 �C. An oscillatory stress sweep was made at a con-stant frequency of 1 Hz over an oscillatory stress range of 0.1 to10 Pa at 5 points per log cycle. The storage (G’) and loss moduli(G00) were determined in triplicate from measurements withinthe identified linear viscoelastic range of each paste.

The measurement of viscosity under a controlled shear stressrange was made in triplicate following the oscillatory measure-ments at 65 �C. Different shear stress ranges were used for eachsample, based on an observed apparent yield value of each paste,which had been observed in initial measurements. For the mea-surement of a flow curve, measurements were made at a seriesof shear stresses, starting with the lowest value. At each shearstress the paste viscosity was observed until either an equilibriumvalue was obtained or at the end of a 2 min cycle. The final value ofviscosity was recorded and the next shear stress was applied.

2.7. X-ray diffractometry of isolated starches

The X-ray diffraction patterns were made with the rice starchpowders (10% moisture), using a diffractometer (BrukerD8 AXS,Bruker BioSpin, Rheinstetten, Germany) in duplicate at target volt-age 40 kV and target current 100 mA with 0.154 nm CuK radiation(Ni filter). The typical widths of the divergence, scattering andreceiving slits are 1.0, 1.0 and 0.2 mm, respectively. Diffractogramswere recorded from 3� to 55� with a step of 0.5�. Relative crystal-linity of the starches was calculated using the method of Nara, Sak-akura, and Komiya (1983), using peak-fitting software (Origin 7.0,Microcal Inc., Northampton, MA).

2.8. Determination of thermal properties of isolated starches

Thermal properties of starches were analysed in triplicate byusing a Perkin–Elmer Pyris 1 DSC (Norwalk, CT). Starch (3 mg,dry weight basis) was weighed precisely into aluminum samplepans and mixed with water to obtain a ratio of 1:2 (w/w). The panswere hermetically sealed and stored at room temperature for 15 h.After loading on the DSC, the pans were then held at 25 �C for

Y. Li et al. / Food Chemistry 114 (2009) 821–828 823

1 min, heated from 25 to 95 �C at 10 �C/min and cooled to 25 �C atthe same rate.

2.9. Scanning electron microscopy of isolated starches

Scanning electron micrographs of starches isolated with Prote-ase N or Alcalase were recorded with Quanta-200 scanning elec-tron microscopy (FEI Company, Eindhoven, Holland) at anaccelerating voltage of 5 kV. Starch granules were sprinkled ontodouble-backed, cellophane tape attached to an aluminium stub be-fore coating with gold–palladium in an argon atmosphere.

2.10. Determination of molecular weight distributions of isolated ricestarches

2.10.1. HPSEC analysis of isolated rice starchesThe isolated starch samples for the HPSEC analysis were pre-

pared following the method used by Yokoyama, Renner-Nantz,and Shoemaker (1998), with minor modification. Starch was addedto DMSO (HPLC grade, Sigma Chemical Co., St Louis, MO) contain-ing 50 mM LiBr (Fisher Scientific, Fair Lawn, NJ) at 0.4% (w/v, dryweight basis) and then heated at 95 �C for 15 min with constantstirring. After heating, samples were cooled and continuously stir-red overnight and then centrifuged for 10 min at 10,000 g, filteredthrough a 1.2 lm nylon syringe membrane filter and analysed intriplicate with a high performance size-exclusion chromatography(HPSEC) system.

2.10.2. HPSEC analysis of pasted rice starchesStarches isolated with Alcalase or Protease N were dispersed in

deionised water at a concentration of 8.0% (w/v, dry weight basis).The dispersions were preheated at 50 �C for 3 min, and then thetemperature was raised from 50 �C to 95 �C at the rate of 12 �C/min with constant stirring. When the temperature reached 95 �C,the dispersions were immediately transferred to a Value ULT-1386-3V Ultra-low temperature freezer (Thermo Fisher ScientificInc., Waltham, MA.) at �80 �C and then freeze-dried. The freeze-dried samples were dispersed in DMSO (containing 50 Mm LiBr)at a concentration of 0.4% (w/v, dry weight basis) and heated at95 �C for 15 min with constant stirring. Then the samples werecooled to 40 �C and continuously stirred overnight. After centrifu-gation at 10,000g for 10 min, the samples were filtered through a1.2 lm nylon syringe membrane filter for HPSEC-MALLS-RI systemanalysis. Three replicates were analysed for each sample.

2.10.3. HPSEC analysis of rice starches pasted with added proteaseStarches isolated with Alcalase or Protease N were dispersed in

0.1 M NaNO3 (Reagent grade, Fisher Scientific, NJ) at a concentra-tion of 0.4% (w/v, dry weight basis) together with 100 U/ml Alca-lase or Protease N. The dispersions were preheated at 50 �C for3 min, and then the temperature was raised from 50 �C to 95 �Cat the rate of 12 �C/min with constant stirring. When the temper-ature reached 95 �C, the dispersions were immediately transferredinto centrifugal tubes and centrifuged at 10,000g for 10 min at45 �C. The supernatants after centrifugation were filtered through1.2 lm nylon syringe filters and analysed in triplicate by HPSEC-MALLS-RI.

2.10.4. HPSEC-MALLS-RI systemThe HPSEC system consisted of an HP 1050 series pump and

auto-injector (Hewlett Packard, Valley Forge, PA) fitted with a100 ll injection loop. The system also employed a multi-angle la-ser light scattering detector (MALLS) (Dawn DSP-F, Wyatt Tech.,Santa Barbara, CA) with He–Ne laser source (k = 632.8 nm), a K-5flow cell, and a differential refractometer detector (RI) (modelERC-7512, ERMA Inc., Tokyo, Japan).

Two different mobile phases and column banks were used forthe determination of the molecular weight distributions. For thedetermination of the molecular weight distributions of starchesbefore or after pasting, a bank of Waters Stragel columns (HMW6E, 6E and 2) were used with 50 mM LiBr in DMSO as a mobilephase with a flow rate of 0.4 ml/min. The refractive index of1.479 and the dn/dc value of 0.066 for starch in DMSO/50 mM LiBrwere used for the molecular weight calculations. For the determi-nation of the molecular weight distributions of starches pastedwith added protease, four aqueous Waters SEC columns (Ultrahy-drogel 120, 250, 500 and 1000; Millipore Co., Milford, MA) wereused with 0.1 M NaNO3 as a mobile phase with a flow rate of0.6 ml/min. A refractive index of 1.334 and dn/dc value of 0.160was used for the analysis of starch in H2O/NaNO3. The columnswere maintained at 60 or 40 �C, respectively for the organic andaqueous SEC experiments.

2.10.5. Data analysisData analysis methods were the same as used by Yokoyama et al.

(1998). Astra software (Version 4.7.07, Wyatt Technology, SantaBarbara, CA) was used for data analysis. Starch samples were ana-lysed for Mw using the second order Berry method (Berry, 1966).

2.11. Measurement of the a-amylase activity in protease

An aliquot of the protease (Alcalase or Protease N) was dilutedwith phosphate buffer at pH 5.2, to adjust the pH and enzyme con-centration just before the experiments. The protease preparations(0.5 ml) were mixed with 0.5 ml rice starch solutions (1%, w/v)and incubated at 40 �C or 80 �C for 5 min. The amount of reducingfragments liberated was measured by adding 1 ml 3,5-dinitrosali-cylic (DNS) acid reagent (1%, w/v), heating in boiling water for10 min and measuring the absorbance at 540 nm (Kondo, Urabe,& Yoshinaga, 1996). A maltose standard series was prepared forcalibration. The units of activity were calculated, where 1 unit willliberate 1 ll maltose (reducing disaccharide) from starch underanalysis conditions. Three replicates were analysed of each sample.

2.12. Statistical analysis

The averages and Duncan t-test were performed by SPSS 13.0for Windows software (SPSS Institute Inc., Cary, NC).

3. Results and discussion

3.1. Chemical composition of isolated starches

The starches isolated with Alcalase or Protease N were analysedfor their residual protein and amylose contents (Table 1). In allcases the remaining protein contents were less than 0.7%. Thisindicated a successful removal of protein using the Alcalase or Pro-tease N treatments. No significant differences in the protein con-tents were found between the starches isolated from the twodifferent proteases (p < 0.05). The amylose contents showed theexpected trends among varieties, and there were no significant dif-ferences between the starches of the same variety isolated with thetwo different proteases.

3.2. Pasting properties of isolated starches

In our previous study (Li et al., 2008), it was found that ProteaseN treatments produced rice starch with similar pasting viscosities(trough and final viscosity) to the native rice flour, while the starchproduced from Alcalase treatments had lower final viscosities thanthat isolated with protease N. In order to study the effect of

Table 1Protein and amylose contents of rice starches isolated with Alcalase (Al) or Protease N(N)a.

Rice starch Protein content (%) Amylose content (%)

Cocodrie-Al 0.59 ± 0.02ab 21.3 ± 0.4a

Cocodrie-N 0.62 ± 0.03a 20.4 ± 0.4a

Koshi-Al 0.56 ± 0.02ab 14.7 ± 0.3b

Koshi-N 0.60 ± 0.03a 14.2 ± 0.2b

M202-Al 0.51 ± 0.02c 13.4 ± 0.5c

M202-N 0.59 ± 0.03ab 12.9 ± 0.3c

CM101-Al 0.58 ± 0.03ab 0.9 ± 0.1d

CM101-N 0.62 ± 0.02a 0.8 ± 0.1d

Each of the protein and amylose contents is expressed on a dry weight basis.Mean values with different superscript letters are significantly different (p < 0.05).

824 Y. Li et al. / Food Chemistry 114 (2009) 821–828

different protease treatments on the pasting properties of the ricestarch, pasting curves measured on a rotational rheometer withcone and plate fixtures were recorded for rice starches isolatedfrom four varieties using Alcalase or Protease (Fig. 1). No observeddifferences of pasting onset and peak temperatures were found be-tween the rice starches isolated with Alcalase and Protease N, butthe pasting viscosities (the peak, trough and final viscosities) weredifferent. Overall, the starches isolated with Protease N exhibitedhigher pasting viscosities than those isolated with Alcalase. Thepasting curves were also different among different rice varieties.The pasting viscosity of the waxy starch (CM101), increased morerapidly than the non-waxy starches, which exhibited a shoulder atthe beginning of pasting, and the pasting onset temperature of

Fig. 1. Pasting curves of (a) Cocodrie, (b) Koshi, (c) M202, and (d) CM

CM101 was lower than those of non-waxy starch. With smallamounts of amylose present in the waxy starch, the granules areeasily swollen (Varavinit, Shobsngob, Varanyanond, Chinachoti, &Naivikul, 2003). It was found out that viscosities of starch disper-sions during heating were principally dependent on granular vol-ume fraction (Li, Shoemaker, Ma, Kim, & Zhong, 2008). The rapidincreasing in pasting viscosities of waxy starch was due to thequick swelling of the starch granules during pasting.

3.3. Rheological properties

Immediately following the pasting program on the rotationalrheometer, the temperature on the paste was changed from 50 to65 �C and the elastic (G’) and loss (G00 moduli were recorded withinthe linear viscoelastic range of each paste. For the same variety, therice starch isolated with Protease N had higher G’ values than thatisolated with Alcalase. The Protease N starches also had smaller tand (tan d = G00/G’), which indicated higher degrees of elasticity rela-tive to the viscous nature of the pastes, in comparison with theAlcalase starches.

The flow curves of the hot pastes (65 �C) were measured imme-diately after the oscillatory test. This was a measurement of viscos-ity as a function of shear stress. All flow curves of the starch pastesshowed an apparent yield stress and shear-thinning behaviour(Fig. 2). For each variety, the rice starch isolated with Protease Nhad higher yield stresses (go than those isolated with Alcalase. ANewtonian region (go) at low shear stresses for each paste was ob-served, which was followed by a shear-thinning region. The transi-tion between the Newtonian and shear-thinning region wasseparated by a rapid fall of viscosity of several orders of magnitude

101 rice starches isolated with Alcalase (Al) or Protease N (N).

Fig. 2. The viscosity dependence on shear stress of (a) Cocodrie, (b) Koshi, (c) M202, and (d) CM101 rice starch pastes isolated with Alcalase (Al) or Protease N (N).

Y. Li et al. / Food Chemistry 114 (2009) 821–828 825

over a narrow range of shear stress. The shear stress at this transi-tion has been interpreted as the ‘‘yield stress” (Barnes, 2000).

The go and ro for the starch pastes produced from the two pro-tease treatments are shown in Table 2. Both go and go for the Pro-tease N treatment were higher than those from the Alcalasetreatment. All the rice varieties exhibited the same trend. Thiswould indicate that the Protease N treatment produced hot starchpastes which were more viscous than the pastes from the Alcalasetreatment.

For the same rice variety, big differences were found betweenthe pasting or rheological properties of the rice starches isolatedwith Alcalase or Protease N. It was hypothesised that the enzy-matic treatment, especially the Alcalase treatment, may damagethe molecular structure of the rice starch during the isolation pro-cess, because the functional or physiochemical properties of starchare closely related to its molecular structure. In order to test thishypothesis, several tests were carried out.

Table 2The rheological parameters of rice starch pastes isolated with Alcalase (Al) or Protease N

Rice starch Storage modulus G’ (Pa) Loss modulus G00 (Pa) Tan d (

Cocodrie-Al 27.2 ± 5.0a 8.72 ± 0.80a 0.32 ±Cocodrie-N 45.6 ± 6.3b 10.5 ± 0.50b 0.23 ±

Koshi-Al 9.36 ± 1.2c 5.77 ± 0.30c 0.62 ±Koshi-N 38.8 ± 3.0d 11.4 ± 0.45d 0.29 ±

M202-Al 7.16 ± 0.40e 5.41 ± 0.21e 0.76 ±M202-N 45.6 ± 3.2f 10.5 ± 0.65f 0.23 ±

CM101-Al 32.2 ± 5.3g 8.40 ± 0.50g 0.26 ±CM101-N 49.8 ± 3.6h 9.51 ± 0.47h 0.19 ±

Mean values within the same rice variety and variable column with different superscrip

3.4. Test of physiochemical properties for the isolated rice starches

Wide angle X-ray diffraction (XRD) measurements of the ricestarches produced from these two proteases were performed. Allstarches displayed typical A-type X-ray diffraction patterns. Therelative crystallinity (RC) of the waxy starch (CM101) was slightlyhigher than that of the normal starch (Table 3). It was recognisedthat crystallinity in native starches occurs in the amylopectin con-tent, for a major amount of helical order exists within these crys-talline amylopection regions (Noosuk, Hill, Pradipasena, &Mitchell, 2003). For the same variety, no difference of RC was foundbetween the starches isolated with Alcalase and Protease N, andthe RC of the starch extracted from conventional alkaline steepingwas similar to that of the starches isolated with Alcalse or ProteaseN (Table 3). Scanning electron micrography of the rice starchesshows no apparent damage to the granule surfaces, and therewas also no obvious difference between the scanning electron

(N).

G00/G’) Zero-order Newtonian viscosity go (kPa s) Yield stress ro (Pa)

0.2a 23.3 ± 2.0a 5.0 ± 0.2a

0.1a 105.4 ± 9.8b 34.2 ± 2.2b

0.2b 2.85 ± 0.19c 1.58 ± 0.10c

0.1c 66.0 ± 5.0d 21.5 ± 3.1d

0.2d 1.12 ± 0.09e 1.00 ± 0.08e

0.1e 98.3 ± 8.8f 21.5 ± 1.9f

0.1f 35.9 ± 4.0g 35.2 ± 2.2g

0.1f 119.0 ± 11.0h 46.6 ± 3.8h

t letters are significantly different (p < 0.05).

Table 3Moisture content and relative crystallinity of rice starches isolated with Alcalase (Al)or Protease N (N).

Rice starch Moisture content (%) Relative crystallinity (%)

Conventional method 10.1 ± 0.2a 40.2 ± 0.4a

Cocodrie-Al 10.8 ± 0.3ab 39.7 ± 0.5a

Cocodrie-N 10.5 ± 0.4a 40.3 ± 0.4a

Koshi-Al 11.2 ± 0.4b 40.8 ± 0.4ab

Koshi-N 10.9 ± 0.5ab 41.6 ± 0.5b

M202-Al 12.2 ± 0.3bc 39.9 ± 0.5a

M202-N 11.9 ± 0.5b 40.8 ± 0.6ab

CM101-Al 13.2 ± 0.4d 42.3 ± 0.6b

CM101-N 12.8 ± 0.3c 42.8 ± 0.5b

Mean values within the same rice variety and variable column with differentsuperscript letters are significantly different (p < 0.05).

Fig. 3. Scanning electron micrograph of M202 rice starch isolated with Alcalase (A)or Protease N (B). Magnification �5000.

826 Y. Li et al. / Food Chemistry 114 (2009) 821–828

micrograph of the starches isolated with the two proteases (Fig. 3).Thermal properties were measured using differential scanning cal-orimetry (DSC) (Table 4). For the same variety, no significant differ-ences among onset (To), peak (Tp), completion (Tc) temperaturesand enthalpy (DH) were found between the two protease treat-ments. The results of the physicochemical studies suggested thatthe rice starches produced from the two different proteases gener-ally had the same crystalline pattern, thermal properties and starchgranule appearance.

3.5. Test of molecular weight distributions for the isolated rice starches

3.5.1. HPSEC analysis of rice starches before and after pastingIn order to confirm that the rice starches remained their native

characteristics during the isolation process, detailed molecularweight distributions of rice starches isolated with Alcalase or Pro-tease N were analysed by the HPSEC-MALLS-RI system. In our pre-vious study, it was found out that 50 mM LiBr in DMSO was abetter solvent than aqueous solvent and 10% H2O in DMSO, basedon its ability to dissolve starch and to reduce the size of starchaggregates (Zhong, Yokoyama, Wang, & Shoemaker, 2006). LiBr(50 mM) in DMSO was therefore used as the mobile phase. Whenthe isolated rice starch was directly dispersed into the mobilephase (unpasted) and analysed by HPSEC, no significant differencesin average molecular weight (Mw) were observed between the ricestarches produced from Alcalase and Protease N (Table 5). It indi-cated that neither Alcalase nor Protease N would damage the struc-ture of the rice starch during the isolation process.

Since the rheological differences between the two protease-iso-lated starches appeared during the latter part of pasting, it is sug-gested that there would be an effect of the protease on the starchmolecular structure, which occurred during the pasting process.The molar masses of the pasted starches were then measured.There were varying degrees of reductions in the Mw of all thestarches after pasting (Table 5). The reduction in Mw of the pastedstarch was probably due to amylopectin degradation. It has beenreported that rice starch heated at 80 �C for 1 h (Mizukami, Takeda,& Hizukuri, 1999) or steamed 10 min (Lu, Chen, & Lii, 1996) dis-played amylopectin degradation. For the same rice variety, theMw of the pasted starch produced from Alcalase treatment showedgreater reductions than the pasted starch produced by Protease Ntreatment. When comparing the elastic and flow parameters of thepastes to the Mw of the pasted starches (Table 2 and 5), there is acorresponding ranking between the Mw values and either the elas-tic moduli (G’), zero-order Newtonian viscosity (g) or yield stress(ro) over the rice varieties for the same enzyme used for theisolation.

After the starch isolation, remaining protease was removed bywashing the starch with deionised water several times after hydro-lysis. It has been reported that the surface of many rice starch gran-ules are covered with pores of varying diameter (Jayakody &Hoover, 2002). Thus, the micro-pore structure of the starch granulecould have retained some enzyme. If this enzyme or other enzymeimpurities had sufficient heat stability, then there may have beenmodifications during the pasting. Hydrolysis of intergranular pro-teins during pasting by residual protease may have been possible,or enzyme impurities with amylase activity may have caused lim-ited starch hydrolysis during pasting as well.

3.5.2. HPSEC analysis of rice starches pasted with added proteaseIn order to test the hypothesis, that residual enzyme activity

could have damaged the starch structure during pasting, additionalprotease (Alcalase or Protease N) was added to the isolatedstarches, and the mixture was pasted. Then the mixture was di-rectly analysed by HPSEC with aqueous columns and 0.1 M NaNO3

as a mobile phase. The HPSEC analysis showed dramatically differ-ences in the molar mass between the rice starch pasted with addedAlcalase and Protease N. The Mw of rice starches pasted with addedAlcalase was significant lower than that of the starches pasted withadded Protease N (Table 5). The reduction in molecular weight

Table 4Thermal properties of rice starches isolated with conventional alkaline steeping method and protease treatment.

Rice starch To (�C) Tp (�C) Tc (�C) Tc–To (�C) DH (J/g)

Conventional method 62.3 ± 0.5c 67.5 ± 0.4c 72.6 ± 0.5a 10.3 ± 0.5b 10.5 ± 0.4a

Cocodrie-Al 63.4 ± 0.4d 67.2 ± 0.5c 72.1 ± 0.4a 8.7 ± 0.6a 11.7 ± 0.3b

Cocodrie-N 64.5±0.5d 66.6 ± 0.4c 71.9 ± 0.6a 8.2 ± 0.5a 10.9 ± 0.6ab

Koshi-Al 59.4 ± 0.5b 64.8 ± 0.3b 78.1 ± 0.6b 18.7 ± 0.7c 12.3 ± 0.5c

Koshi-N 59.2±0.6b 64.4 ± 0.5b 79.1 ± 0.4b 19.9 ± 0.6c 12.8±0.4c

M202-Al 61.3 ± 0.5c 66.2 ± 0.6c 71.3 ± 0.4a 10 ± 0.5b 10.6 ± 0.3a

M202-N 62.2 ± 0.5c 66.9 ± 0.6c 71.3 ± 0.5 a 9.1 ± 0.6b 10.1 ± 0.4a

CM101-Al 56.2 ± 0.4a 63.5±0.3a 80.2 ± 0.5bc 24 ± 0.4d 13.2 ± 0.4d

CM101-N 56.8 ± 0.3a 64.3 ± 0.5a 81.1 ± 0.6c 24.3 ± 0.5d 12.9 ± 0.5d

Mean values within the same rice variety and variable column with different superscript letters are significantly different (p < 0.05).

Table 5Average molecular weights (Mw) of the pasted or unpasted rice starches isolated with Alcalase (Al) or Protease N (N).

Rice starch Mw (�107)

Unpasted rice starch Pasted rice starch Pasted rice starch (with added N) Pasted rice starch (with added Al)

Cocodrie-Al 7.40 ± 0.23a 5.41 ± 0.19c 4.12 ± 0.31b 0.989 ± 0.13ab

Cocodrie-N 7.00 ± 0.18b 6.49 ± 0.22d 6.07 ± 0.28d 1.59 ± 0.14d

Koshi-Al 11.7 ± 0.78c 4.30 ± 0.20b 4.03 ± 0.33b 1.32 ± 0.15c

Koshi-N 11.8 ± 0.74c 5.42 ± 0.18c 4.88 ± 0.29c 2.86 ± 0.22e

M202-Al 9.84 ± 0.65d 3.23 ± 0.35a 3.02 ± 0.38a 0.896 ± 0.12a

M202-N 10.7 ± 0.62d 6.47 ± 0.32d 6.02 ± 0.36d 1.38 ± 0.18cd

CM101-Al 12.7 ± 0.72e 9.50 ± 0.78e 9.01 ± 0.58e 1.22 ± 0.09c

CM101-N 14.3 ± 0.68e 13.5 ± 0.62f 12.5 ± 0.52f 1.07 ± 0.11b

Mean values within the same rice variety and variable column with different superscript letters are significantly different (p < 0.05).

Y. Li et al. / Food Chemistry 114 (2009) 821–828 827

suggested that Alcalase had modified the starch molecules duringpasting.

Since food-grade proteases are not highly purified, there may beother activities present such as amylase activity. No amylase activ-ity was found in either Alcalase or Protease N, by a reducing sugarassay at 40 �C. As the pasting period occurs from 50 to 95 �C, and a-amylase is temperature stable, it is necessary to determine the a-amylase activity at a high temperature. Amylase activities of 0.5and 2.4 units/g were found for Protease N and Alcalase, respec-tively. Trace amounts of endo-amylase activity could have causedsignificant reductions in viscosity, because the interchain hydroxylbonds of the starches were broken and part of the granules weredisrupted (Atwell, Hood, Lineback, Varrianomarston, & Zobel,1988; Olkku & Rha, 1978) during pasting, which would make thepasted rice starch more sensitive to the enzyme and much moreeasily damaged.

Although protease treatments provide an effective method forthe purification of native rice starches, commercial protease shouldbe selected carefully in order to avoid damage to the starch struc-ture by contamination with the protease during or after isolationprocess. On the other hand, the presence of small amounts of otherenzyme activities may provide a novel method to modified starchproperties.

4. Conclusion

The rice starches produced from Protease N treatment exhibitedhigher pasting viscosities than those produced from Alcalase treat-ment. The hot starch pastes of the starches produced from ProteaseN treatment also showed higher elastic moduli, zero-order Newto-nian viscosity and yield stress than those produced from Alcalasetreatment. The results of the physicochemical studies suggested

that protease can produce rice starch without damaging its naturalcharacteristics during isolation. The Mw of the pasted starch pro-duced from Protease N was higher than that produced from Alca-lase. When additional protease was added to the isolatedstarches and the mixture pasted, the Mw of the starches pastedwith added Alcalase was significantly lower than that of thestarches pasted with added Protease N. It is suggested that Alcalasehad modified the starch molecules during pasting. The differencesin pasting viscosities and the rheological properties between therice starches isolated with Alcalase and Protease N were due tothe protease remaining in the rice starches. Protease N was a pref-erable enzyme, it could effectively separate rice protein and starchwithout damaging the molecular structure of the rice starch duringisolation process and pasting, as compared with Alcalase. More de-tails of the cost and energy use in industrial production need fur-ther investigation.

Acknowledgements

This work was financially supported by National 863 Program2006AA10Z327, Nature Science Foundation of Jiangsu Province,China (BK 2007502), NSFC 30871744, 111 project-B07029 andPCSIRT0627. The authors also thank the Rice Experiment Station,Biggs, CA, USA for providing the rice samples.

References

AACC (2000). Approved methods of the AACC. St Paul, MN: American Association ofCereal Chemists.

Atwell, W. A., Hood, L. F., Lineback, D. R., Varrianomarston, E., & Zobel, H. F. (1988).The terminology and methodology associated with basic starch phenomena.Cereal Food World, 33, 306–311.

828 Y. Li et al. / Food Chemistry 114 (2009) 821–828

Barnes, H. A. (2000). A handbook of elementary rheology. Aberystwryth, England:University of Wales Institute of Non-Fluid Mechanics (pp. 71–76).

Berry, G. C. (1966). Thermodynamic and conformational properties of polystyrene. I.Light-scattering studies on dilute solutions of linear polystyrenes. Journal ofChemical Physics, 44, 4550–4564.

Chiou, H., Martin, M., & Fitzgerald, M. (2002). Effect of purification methods on ricestarch structure. Starch/Stärke, 54, 415–420.

Guraya, H. S., & James, C. (2002). Deagglomeration of rice starch–protein aggregatesby high-pressure homogenization. Starch/Stärke, 54, 108–116.

Jayakody, L., & Hoover, R. (2002). The effect of lintnerization on cereal starchgranules. Food Research International, 35, 665–680.

Kondo, A., Urabe, T., & Yoshinaga, K. (1996). Adsorption activity and conformation ofa-amylase on various ultrafine silica particles modified with polymer silanecoupling agents. Colloids Surfaces A, 109, 129–136.

Landers, P. S., & Hamaker, B. R. (1994). Antigenic properties of albumin, globulin andprotein concentrate fractions from rice bran. Cereal Chemistry, 71, 409–411.

Li, Y., Shoemaker, C. F., Ma, J. G., Kim, J. M., & Zhong, F. (2008). Structure–viscosityrelationships for starches from different rice varieties during heating. FoodChemistry, 106, 1105–1112.

Li, Y., Shoemaker, C. F., Shen, X., Ma, J., Ibáñez-Carranza, A. M., & Zhong, F. (2008).The isolation of rice starch with food grade proteases combined with othertreatments. Food Science and Technology International, 14, 215–224.

Lu, S., Chen, C.-Y., & Lii, C.-Y. (1996). Gel-chromatography fractionation and thermalcharacterization of rice starch affected by hydrothermal treatment. CerealChemistry, 73, 5–11.

Lumdubwong, N., & Seib, P. A. (2000). Rice starch isolation by alkaline proteasedigestion of wet-milled rice flour. Journal of Cereal Science, 31, 63–74.

Mizukami, H., Takeda, Y., & Hizukuri, S. (1999). The structure of the hot-watersoluble components in the starch granules of new Japanese rice cultivars.Carbohydrate Polymers, 38, 329–336.

Nara, S., Sakakura, M., & Komiya, T. (1983). On the acid resistance of starch granules.Starch/Stärke, 35, 266–270.

Noosuk, P., Hill, S. E., Pradipasena, P., & Mitchell, J. R. (2003). Structure–viscosityrelationships for Thai rice starches. Starch/Stärke, 55, 337–344.

Olkku, J., & Rha, C. K. (1978). Gelatinization of starch and wheat-flour starch –review. Food Chemistry, 3, 293–317.

Puchongkavarin, H., Varavinit, S., & Bergthaller, W. (2005). Comparative study ofpilot scale rice starch production by an alkaline and an enzymatic process.Starch/Stärke, 57, 134–144.

Resurreccion, A. P., Li, X., Okita, T. W., & Juliano, B. O. (1993). Characterization ofpoorly digested protein of cooked rice protein bodies. Journal of CerealChemistry, 70, 101–104.

Steeneken, P. A. M. (1989). Rheological properties of aqueous suspensions ofswollen starch granules. Carbohydrate Polymers, 11, 23–42.

Tanaka, Y., Sugimoto, T., Ogawa, M., & Kasai, Z. (1980). Isolation andcharacterization of two types of protein bodies in the rice endosperm.Agricultural and Biological Chemistry, 44, 1633–1639.

Tester, R. F., Yousuf, R., Karkalas, J., Kettlitz, B., & Röper, H. (2008). Properties ofprotease-treated maize starches. Food Chemistry, 109, 257–263.

Varavinit, S., Shobsngob, S., Varanyanond, W., Chinachoti, P., & Naivikul, O. (2003).Effect of amylose content on gelatinization, retrogradation and pastingproperties of flours from different cultivars of Thai rice. Starch/Stärke, 55,410–415.

Wang, L., & Wang, Y. J. (2004). Rice starch isolation by neutral protease and high-intensity ultrasound. Journal of Cereal Science, 39, 291–296.

Yang, C., Lai, H. M., & Lii, C. Y. (1984). The modified alkaline steeping method for theisolation of rice starch. Food Science (Chinese), 11, 158–162.

Yokoyama, W., Renner-Nantz, J. J., & Shoemaker, C. F. (1998). Starch molecular massand size by size-exclusion chromatography in DMSO–LiBr coupled withmultiple angle laser light scattering. Cereal Chemistry, 75, 530–535.

Yun, S. H., & Matheson, N. K. (1990). Estimation of amylose content of starchesafter precipitation of amylopectin by concanavalin-A. Starch/Stärke, 42,302–305.

Zhong, F., Li, Y., Ibanz, A. M., Oh, M. H., McKenzie K. S., & Shoemaker, C. F. (in press).The effect of rice variety and starch isolation method on the pasting andrheological properties of rice starch pastes. Food Hydrocolloids, doi:10.1016/j.foodhyd.2008.02.003.

Zhong, F., Yokoyama, W., Wang, Q., & Shoemaker, C. F. (2006). Rice starch,amylopectin, and amylose: molecular weight and solubility in dimethylsulfoxide-based solvents. Journal of Agricultural and Food Chemistry, 54,2320–2326.