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Food Hydrocolloids 45 (2015) 218e226

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Physicochemical and structural properties of debranched waxy rice,


waxy corn and waxy potato starches
Nuengmaysa Klaochanpong a, Chureerat Puttanlek b, Vilai Rungsardthong c,
Santhanee Puncha-arnon a, Dudsadee Uttapap a, *
a
Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, 49 Soi Thianthale 25,
Bangkhunthian-Chaithale Road, Tha-kham, Bangkhunthian, Bangkok 10150, Thailand
b
Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakhon Pathom 73000, Thailand
c
Department of Agro-Industrial Technology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, 1518 Pibulsongkram Road,
Bangsue, Bangkok 10800, Thailand

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 27 May 2014
Accepted 12 November 2014
Available online 26 November 2014

Starch gels of waxy rice (WR), waxy corn (WC) and waxy potato (WP) were hydrolyzed with pullulanase,
and the products obtained were analyzed for unit chain length distribution and certain physicochemical
properties. Average chain lengths of debranched WR, WC and WP starches were 18.2, 19.2 and 25.6,
respectively. The debranched starches had a greater ability to form complexes with iodine and possessed
higher solubility but lower viscosity compared with their corresponding dispersed native starches. Their
complexing abilities with fatty acids (palmitic and butyric acids) were found to be dependent on the unit
chain length. Among the three debranched starches, debranched waxy potato starch exhibited the
greatest ability to form complexes with iodine and fatty acids, while debranched waxy rice starch had the
highest solubility and lowest viscosity. The debranched starches formed stable gels at high concentrations (10e20%) and formed precipitates at concentrations up to 5%.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Debranched waxy starch
Molecular structure
Solubility
Complexing ability
Viscosity

1. Introduction
Native starches are used in food and non-food applications, in
which starch properties such as viscosity, retrogradation, solubility,
gelation, gel appearance and texture are the main criteria for
choosing an appropriate starch for a certain end-use. In most applications, starch is used in a gelatinized native form, either in
cooked or uncooked products. It is well-known that starch is biosynthesized as granules in higher plants and consists of two types
of a-D-glucose polymers: amylose and amylopectin. Amylose is a
mixture of lightly branched and linear molecules, whereas amylopectin is a much larger molecule with a highly branched structure
consisting of about 95% a-1,4 linkages and 4e5% a-1,6linkages
(Hizukuri, Abe, & Hanashiro, 2006; Tester, Karkalas, & Qi, 2004).
The ratio of amylose to amylopectin in starch varies depending on
the botanical source. Generally, normal starches consist of 20e30%

* Corresponding author. Tel.: 66 2 470 7754; fax: 66 2 452 3479.


E-mail address: dudsadee.utt@kmutt.ac.th (D. Uttapap).
http://dx.doi.org/10.1016/j.foodhyd.2014.11.010
0268-005X/ 2014 Elsevier Ltd. All rights reserved.

amylose and 70e80% amylopectin, whereas waxy starches contain


essentially 100% amylopectin.
Starch can be debranched at a-1, 6 linkages by debranching
enzymes (e.g. isoamylase and pullulanase) under specific conditions. The debranched products are a mixture of short linear chain
glucans with different degrees of polymerization (DP) (Hizukuri
et al., 2006; Manners, 1989). For amylose-containing starch
(normal starch), both amylose and amylopectin are debranched,
producing a mixture of long linear chains derived from amylose
with average chain lengths between 350 and 550 (Charoenkul,
Uttapap, Pathipanawat, & Takeda, 2006; Morrison & Karkalas,
1990;
Tester et al.,
2004;
Thitipraphunkul,
Uttapap,
Piyachomkwan, & Takeda, 2003), and short linear chains derived
from amylopectin with average chain lengths between 20 and 30
(Charoenkul et al., 2006; Hizukuri et al., 2006; Tester et al., 2004;
Thitipraphunkul et al., 2003). On the other hand, only short linear
chains are released when waxy starch is debranched, and therefore
these chains have a relatively narrower MW distribution (Shi, Cui,
Birkett, & Thatcher, 2006). The chain length (CL) distribution of
debranched amylopectin is highly related to its crystalline polymorphs (Hizukuri, 1985). In general, amylopectins of A-type

N. Klaochanpong et al. / Food Hydrocolloids 45 (2015) 218e226

starches contain a large proportion of short branch chains, whereas


those of B-type starches have a larger proportion of long chains
(Hanashiro, Abe, & Hizukuri, 1996). The distinct branch CL distributions of amylopectin from A- and B-type starches can affect the
functionalities of the debranched products.
Apart from using starch in the native form, debranched starch
(DBS) derived from enzyme hydrolysis of gelatinized starch is now
recognized as a potential source of natural linear polymers. DBS has
a totally different structure from native starch; therefore, their
functionalities are supposed to be different. To date, research work
on applications of DBS has mainly focused on resistant starch formation (Miao, Jiang, & Zhang, 2009). DBS is also being used as a fat/
protein replacer in food products (Chiu & Mason, 1998), as well as a
tableting excipient in drug formulation (Arends-Scholte et al.,
2000; Shi et al., 2006).
To determine suitable utilizations of these DBSs for industrial
purposes, information on their basic properties (structural and
physicochemical properties) should be elucidated. So far, there
have been many investigations on the molecular structure of DBSs
(Cai & Shi, 2010; Cai, Shi, Rong, & Hsiao, 2010; Kurimoto &
Sugimoto, 1975; Yotsawimonwat et al., 2008). This study aimed to
investigate both the structural and physicochemical properties of
DBSs. Waxy-type starches (waxy rice, corn and potato starches)
were chosen for the reason that the debranched products obtained
would have more uniform unit chain length. Fatty acid-complexing
ability, solubility, viscosity and gelation of the DBSs were determined and discussed in relation to unit chain length distribution.
Information on these characteristics was also discussed in view of
potential industrial applications of DBSs.
2. Materials and methods
2.1. Materials
Waxy rice (RD 8) and waxy corn (Kwpsx 7253) were provided
by the Sakon Nakhon Rice Research Center (Sakon Nakhon,
Thailand) and the National Corn and Sorghum Research Center
(Pak Chong, Thailand), respectively. Waxy potato starch (3.92%
amylose content) was acquired from National Starch Food Innovation (Bangkok, Thailand). Bacillus acidopullulyticus pullulanase
(EC 232-983-9P, 400 U/ml), maltoheptaose, and two pullulan
standards (P6000 and P12000) were purchased from SigmaeAldrich (St. Louis, MO). All chemicals used in this experiment
were analytical grade.
2.2. Starch isolation
Waxy rice and waxy corn with amylose contents of 0.24% and
3.80%, respectively, were isolated according to the procedures
described by Jiranuntakul, Puttanlek, Rungsardthong, PunchaArnon, and Uttapap (2011). De-hulled waxy rice grains were
steeped in distilled water at 4  C for 24 h, while corn kernels were
steeped in water containing 0.16% sodium hydrogen sulte at
50  C for 24 h. The supernatant was discarded, and the steeped
waxy rice grains/corn kernels were ground with a blender and
then passed through a 63 mm screen. The slurry was kept at 4  C
for 48 h. The supernatant was removed, and the starch cake was
re-suspended in 0.35% sodium hydroxide solution and kept at
4  C for 48 h. The supernatant was decanted and the starch layer
was re-slurried with water. The starch slurry was passed through
a 63 mm sieve and kept at 4  C for 48 h. The steps of washing with
water were repeated four times until the pH of the starch slurry
reached 7, and then it was stored at 4  C for 48 h. Finally, the
supernatant was removed and the starch cake was dried in an
oven at 40  C for 24 h.

219

2.3. Waxy starch debranching


Starch debranching was carried out according to the method of
Yotsawimonwat et al. (2008) with a slight modication. Waxy
starch was suspended in 0.05 M acetate buffer, pH 5.0 (10%, w/w).
The suspension was cooked in a boiling water bath with stirring for
60 min, followed by autoclaving at 121  C for 60 min to complete
gelatinization. The gel was cooled to 55  C and hydrolyzed with
pullulanase (45 U/g of starch) at 55  C in a shaking water bath for
20 h. The enzyme was then deactivated with threefold volume of
99% ethanol. The precipitated debranched starch was recovered by
ltration with Whatman No.4 lter paper, washed with 95%
ethanol three times, dried in an oven at 50  C for 18 h, and sifted
through a 106 mm sieve. Yields of DBSs, based on dry weight basis of
initial starch, ranged between 89.9 and 92.3%. Debranched waxy
rice, corn and potato starches were denoted as DWRS, DWCS and
DWPS, respectively.
2.4. Molecular structure
Average chain length and unit chain distribution of DBSs were
determined by high-performance size-exclusion chromatography
(HPSEC), according to the procedures described by Jiranuntakul
et al. (2011). The HPSEC system consisted of a pump (LC-20AD;
Shimadzu, Tokyo, Japan), an injector, and two 30 cm columns connected in series and packed with 5 mm porous silica microspheres
(Zorbax PSM 60S; Agilent Technologies, Santa Clara, CA). The temperature of the columns was maintained at 50  C. The mobile phase
was 90% (v/v) dimethyl sulfoxide (DMSO) in water with a 0.5 ml/min
ow rate. Debranched samples (4 mg) were individually dispersed
in 1 ml of 90% DMSO and heated for 5 min in a boiling water bath.
After dispersion, the solution was ltered using 0.45 mm lter paper.
A 40 ml aliquot was then injected into the HPSEC system. A calibration curve for the chromatograms was constructed using maltoheptaose and two pullulan standards (P6000 and P12000). The yaxis of the chromatogram was converted from the refractive index
(RI) value to a molar response using the calibration curve (Yuan,
Thompson, & Boyer, 1993). Chain-length distributions were categorized into DP of 6e12, 13e24, 25e36 and 37, according to the
classication proposed by Hanashiro et al. (1996).
2.5. Fatty acid-complexing ability
2.5.1. Iodine staining index (ISI)
Iodine staining index was used as a tool to quantify and evaluate
the iodine binding and fatty acid-complexing abilities of the native
starches and DBSs (Kaur & Singh, 2000; Yotsawimonwat et al.,
2008). Measurement of ISI was modied from the procedure
described for iodine-binding capacity determination by Knutson
(1986). Each debranched starch sample (0.25 g dwb) was
dispersed in 2.5 ml of 90% DMSO and cooked in a boiling water bath
for 30 min, and then 22.5 ml of deionized water was added. For
non-debranched starch, the native starch sample (0.25 g dwb) in
25 ml of deionized water was directly cooked in a boiling water
bath for 30 min. Then 50 ml of this solution was transferred into a
test tube, which was wrapped with aluminum foil for light protection and then incubated with 5 ml of 0.6 mM iodine solution in
10% DMSO for 30 min. The absorbance of the DBSeiodine complex
was scanned at wavelengths from 400 to 700 nm at 1 nm intervals
to obtain a spectrum. The iodine staining indexes of the DBSeiodine complex was expressed as the absorbances at 460 and 570 nm.
2.5.2. Fatty acid-complexing ability
Fatty acid-binding capacity was determined using the method of
Yotsawimonwat et al. (2008) with slight modications. Twenty-ve

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N. Klaochanpong et al. / Food Hydrocolloids 45 (2015) 218e226

ml of each starch solution was prepared according to the procedure


described in Section 2.5.1. Palmitic acid (0.25 g) in 0.5 ml of 99%
ethanol or 0.25 ml of butyric acid was added to the starch solution.
The mixture was incubated in a boiling water bath for 15 min with
vigorous stirring, then cooled down to 25  C and placed at this
temperature for 30 min to facilitate complex formation. After that,
50 ml of the solution was taken for ISI measurement, as described in
Section 2.5.1.
2.6. Solubility
Native and debranched starch samples were suspended in water
and in 0.2 and 2.0 M NaCl solutions at concentrations of 1, 5 and 10%
w/w in a centrifuge tube. After heating in a shaking water bath at
30, 70 or 95  C for 30 min, each solution was well-mixed by a vortex
mixer for 2 min and then centrifuged at 3,130 g for 10 min. The
collected supernatant as well as the initial starch suspension was
analyzed for total starch by the phenolesulfuric acid method. The
solubility was determined as follows:

Solubility%

Starch concentration in supernatant%w=v


Initial starch concentration%w=v
 100

of variance (ANOVA) and expressed as mean values standard


deviations. A Duncan test was conducted to examine signicant
differences among experimental mean values (a  0.05).
3. Results and discussion
3.1. Average chain length and unit chain length distribution
Chain length (CL) distributions of the DBSs are shown in Fig. 1,
and the corresponding data is presented in Table 1. Chromatograms
of DWRS and DWCS were similar to each other, while that of DWPS
was different. Average chain lengths, in terms of degrees of polymerization (DP), of DWRS, DWCS and DWPS were 18.2, 19.2 and
25.6, respectively. DWRS contained the highest amount of short
chains (DP 6e12, 47.8%) and the lowest amount of long chains
(DP  37, 9.1%), while DWPS showed the opposite (31.0% for short
chains and 17.7% for long chains). Unit chain elements of DWCS lay
between DWRS and DWPS. These results are in agreement with
several previous reports, i.e. that amylopectin of A-type starches
(such as waxy maize, maize, rice, wheat, taro, tapioca, and sweet
potato) has relatively shorter chain lengths, as compared to B-type
starches (such as potato, canna, high-amylose maize, and tulip)
(Hanashiro et al., 1996; Hizukuri, 1985; Jane et al., 1999; Jane, Wong,
& McPherson, 1997).

2.7. Viscosity

3.2. Fatty acid-complexing abilities

Individual starch samples were suspended in water at concentrations of 1, 5, 10 and 20% (dwb) in a 50 ml centrifuge tube and
then cooked in a boiling water bath for 1 h. The starch solutions
were cooled down by leaving the tubes at room temperature until
the temperature was reduced to 90, 70, 50 and 30  C. At these
temperatures, the viscosity of the starch solution was determined
by a viscometer (model LVDV-IP; Brookeld Engineering Laboratories, Middleborough, MA) equipped with a suitable spindle (nos.
61e64) that rotated at a xed speed of 60 rpm.

Absorption spectra of native starcheiodine and debranched


starcheiodine complexes are presented in Fig. 2. Two observable
peaks were found at wavelengths between 400 nm and 530 nm and
between 530 nm and 650 nm. It is known that polyiodide ions align
inside the cavities of starch chains and give off faint red, red, redpurple, purple, blue-purple, or blue color, (in the web version)
depending on starch chain length (DP). According to the report of
Bailey and Whelan (1961), the peak wavelength (lmax) increased
linearly with starch chain length between DP 12 and 50; thereafter,
the rate of increase dropped and attained a steady value at 645 nm
(DP 350e400). The starch chain length at which the complex
started giving off a blue color was about DP 45, where lmax was at
570 nm. The transition wavelength (530 nm) in this study corresponded to the absorption wavelength of the complex of iodine
with amylodextrin having a chain length of 28 (Bailey & Whelan,
1961). When the absorption spectra (Fig. 2) were considered in

2.8. Gelling property


Gelling property was investigated in terms of appearance/
morphology, turbidity, gel consistency, and sediment volume.
Starch solutions obtained from the viscosity experiment were
transferred into glass tubes and stored at 4 and 30  C for 24 h.
Appearance of starch gels/solutions was then recorded and photographs were taken. Turbidity of well-mixed starch solutions/gels
was determined by measuring light transmittance (% T) at a
wavelength of 600 nm using a spectrophotometer (Chiu, 1989).
Turbidity (%) was calculated as 100 e %T. To analyze the gel consistency, the tubes containing starch solutions/gels were laid
horizontally over ruled paper graduated in millimeters; 30 min
later the length of the gel was measured along the length of the
test tube. Types of gel consistency were categorized as hard gel
consistency (27e35 mm), medium gel consistency (36e49 mm)
and low gel consistency (50 mm and higher) (Cagampang, Perez, &
Juliano, 1973). For the determination of gel/precipitate volume,
each starch sample was centrifuged at 8,013 g for 10 min and the
volume of sediment was measured. The sediment volume was
calculated as a percentage ratio of volume of sediment to total
volume of starch gel/solution.
2.9. Statistical analysis
All analyses of sample characteristics were carried out in triplicate, except that ve replicates were performed for the solubility
determination. The experimental data were analyzed using analysis

Fig. 1. HPSEC chromatograms of waxy rice, waxy corn and waxy potato starches after
debranching with pullulanase (molar basis).

N. Klaochanpong et al. / Food Hydrocolloids 45 (2015) 218e226


Table 1
Molar-based average chain length and unit chain distributions of debranched
starches determined from HPSEC chromatograms.
Starch
sample
DWRS
DWCS
DWPS

Average chain
length
c

18.2 0.1
19.2 0.4b
25.6 0.2a

Distribution (%)
DP 6e12
a

47.8 0.1
42.4 0.4b
31.0 0.6c

Table 2
Iodine staining indexes of native (N) and debranched (D) starches, with and without
fatty acid (FA) complexation.
Samples

DP 13e24
c

36.4 0.1
40.3 0.2b
41.5 0.5a

DP 25e36
c

6.8 0.1
7.7 0.1b
9.8 0.1a

DP  37

Waxy rice
NWRS

9.1 0.1
9.6 0.3b
17.7 0.2a

Means with different letters in the same column are signicantly different
(p  0.05).

221

Waxy corn
DWRS

Without FA complexation
ISI460
0.196aB
0.282aA
ISI570
0.089aB
0.490aA
With butyric acid complexation
ISI460
0.116bB
0.267aA
D460
0.080
0.015
ISI570
0.058bB
0.447bA
D570
0.031
0.043
With palmitic acid complexation
0.068cB
0.218bA
ISI460
D460
0.128
0.064
0.028cB
0.290cA
ISI570
D570
0.061
0.200

Waxy potato

NWCS

DWCS

NWPS

DWPS

0.237aB
0.173aB

0.315aA
0.527aA

0.403aB
0.322aB

0.451aA
0.846aA

0.142bB
0.095
0.089bB
0.084

0.283bA
0.032
0.490bA
0.037

0.364bB
0.039
0.298bB
0.024

0.440aA
0.011
0.795bA
0.051

0.103cB
0.134
0.067bB
0.106

0.252cA
0.063
0.307cA
0.220

0.298cB
0.105
0.195cB
0.127

0.377bA
0.074
0.542cA
0.304

Capital letters (A, B) indicate a signicant difference (p  0.05) between native and
debranched starches of the same starch.
Small letters (a, b) indicate a signicant difference (p  0.05) among samples at the
same wavelength in the same column.
D difference in ISI values of free starch and starcheFA complex.

Fig. 2. Absorption spectra of dispersed native (N) starcheiodine complexes and


debranched (D) starcheiodine complexes.

association with the unit chain length distributions (Fig. 1), it could
be estimated that wavelengths of less than approximately 530 nm
reected the light absorption of iodine and short starch chains (<DP
30), while those between 530 and 645 nm reected the light absorption of iodine and longer starch chains (around DP 35e300).
The iodine staining indexes (ISIs) herein were thus expressed as the
absorbance at 460 nm (representative of short chains) and at
570 nm (representative of longer chains); the values are summarized in Table 2. DBSs exhibited higher ISI values than their corresponding intact starch molecules (NWSs). The higher ISI of the DBSs
as compared with the intact amylopectin molecules is thought to be
associated with inner and outer branch chains in the cluster
structure of amylopectins. Embedding of the inner branch chains in
organized clusters makes it difcult for them to form complexes
with iodine. Hydrolysis of the branch points with pullulanase
released these chains from the ordered structure, enabling them to
have a higher possibility to form complexes. Among the three waxy
starches, DWPS had the highest ISI, followed by DWCS and DWRS.
These ISI values had a positive correlation with the average chain
length of the DBS, suggesting that longer branch chains were more
effective in forming complexes.
The complex-forming ability of DBS with fatty acids (FA) was
determined by measuring the free DBSs remaining in the solution
using iodine staining because the procedure is fast and simple. The
method was also able to reveal the chain lengths of DBS that
preferentially form inclusion complexes with FA according to the
decrease in the absorbance at different wavelengths
(Yotsawimonwat et al., 2008). Palmitic acid (PA, 16:0) and butyric
acid (BA, 4:0) were used as representatives of long-chain and shortchain fatty acids, respectively. It should be noted that the starcheFA
complexes measured by this procedure represented those that
were more stable than the complex between iodine and starch. The

ISI values of DBS/PA and DBS/BA solutions after adding an iodine


reagent and the difference in ISI values (D) of free starch and
starcheFA complex, are shown in Table 2. The ISI values of the FAcomplex solutions (for either non-debranched or debranched
starches) were lower than those of the solutions without FA
complexation, indicating that starch chains either in intact or
debranched forms could form complexes with long-chain (PA) and
short-chain (BA) fatty acids. However, the complexing ability of PA
was greatly superior to that of BA, as evidenced by the magnitude of
the difference in ISI values (Table 2), i.e. D in case of PA were
signicantly higher than those of BA. This result was consistent
with the ndings of several previous reports, i.e. that fatty acids
with chain lengths of 8e10 or fewer carbon atoms had either no
ability or poor ability to form complexes with amylose (Godet, Tran,
Colonna, & Buleon, 1995; Karkalas & Raphaelides, 1986; Lebail,
Buleon, Shiftan, & Marchessault, 2000; Tufvesson, Wahlgren, &
Eliasson, 2003). Tufvesson et al. (2003) studied the complex formation of potato amylose and fatty acids with chain length between 3 and 22 carbons and found no DSC peak corresponding to
the transition of amyloseelipid complex when butyric acid was
used as a complexing agent. Karkalas and Raphaelides (1986)
proposed that poor complex formation of short-chain fatty acids
is due to electrostatic repulsion and possibly to the absence of
adequate hydrophobicity. Longer lipid chain lengths are more hydrophobic and less soluble in water; therefore, they have a stronger
preference for residing within the hydrophobic cavity of the
amylose single helix instead of remaining in the surrounding hydrophilic aqueous environment (Fanta, Shogren, & Salch, 1999;
Putseys, Derde, Lamberts, Goesaert, & Delcour, 2009). Based on Xray diffraction data, however, Takeo, Tokumura, and Kuge (1973)
suggested that butyric acid can form a complex with amylose (7
glucosyl residues-helical conformation).
As shown in Table 2, the complexing abilities of debranched
starch with fatty acids were also different from those of the corresponding branch chains in intact molecules. These differences
were found to be related to the length of amylopectin unit chains as
indicated by the differences in D values of each pair of debranched
waxy starch (DWS) and native waxy starch at wavelengths of 460
and 570 nm. That is, long chains (D570) in debranched form had
greater abilities to form complexes with fatty acids than those in
intact molecules (except for the complex of waxy corn with butyric
acid), while short chains (D460) displayed the reverse. The explanation for long-chain behavior is similar to that described for the

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N. Klaochanpong et al. / Food Hydrocolloids 45 (2015) 218e226

Fig. 3. Solubility values of native (N) and debranched (D) waxy starches in water at different starch concentrations and temperatures.

complexation of long chains with iodine. Long chains located inside


made it difcult for them to complex with the fatty acids. However,
the superior complex-forming abilities of the short chains
anchored to the molecules were unexpected and is still not well
understood. It is possible that there are some conformation differences between the free short chains (the debranched starch) and
the short chains anchored to the intact molecules, and this would
affect the ability of complexation. A comparison among all starch
samples revealed that debranched waxy potato starch showed the
greatest abilities to form complexes with both PA and BA.
3.3. Solubility
Solubility values of native and DBSs in water and NaCl solutions
are shown in Figs. 3 and 4. Native starches had very limited solubility in water (<0.6%) at low temperature (30  C). Their solubility
values increased signicantly at temperatures higher than the
gelatinization temperature (~70  C); however, they were still less
than 50%. On the other hand, all DBSs displayed much higher solubility. In most cases, solubility values were more than 95% at 95  C.
The solubility of 1% DWRS at 30  C was as high as 63.2%. Among the
three waxy starches, DWRS displayed the highest solubility value,
followed by DWCS and DWPS, respectively. The differences in these
values were more obvious when the DBSs were solubilized at low
temperatures (30 and 70  C). The solubility value results were
consistent with the unit chain lengths of the DBSs, i.e. the longer
the chain length, the lower the solubility. The solubility of all DBSs
increased with increasing temperature, but decreased with
increasing starch and salt concentrations. Although the solubility
decreased in the presence of salt due to the salting-out effect, the
magnitude of change was not signicant. Temperature appeared to
be a more important factor, especially at high starch
concentrations.
When the 1% and 5% DBS solutions dissolved at 95  C were
further kept at room temperature for 2 h, it was found that the
solubility values of DWRS, DWCS and DWPS decreased by 2.5% and
3.3%, 8.0% and 8.8%, and 15.9% and 22.2%, respectively (Table 3).

These results suggested that some soluble DBSs were associated


into insoluble aggregates, presumably by interchain bonding with
H-bond (retrogradation). The aggregation of amylose in dilute
(<0.5% w/v) aqueous solutions has been studied by a number of
researchers. A major nding of such studies is that the rate of aggregation of amylose is strongly dependent on the chain length.
With increasing chain lengths, amylose aggregation is at rst slow
(for DP < 50), increases rapidly to a maximum rate (at DP 80), and
then becomes steadily slower until, for DP > 2,000, only slow and
limited aggregation is observed in dilute solutions (Husemann,
Burchard, & Pfannemller, 1964; Husemann, Pfannemller, &
Burchard, 1963). Gidley and Bulpin (1989) found that short-chain
amyloses (DP < 110) precipitated at all concentrations up to 5.0%
upon cooling hot aqueous solutions. Amyloses of DP 40 and 65 gave
ne, dense precipitates, whereas precipitates of DP 90 and 110 were
less dense, occupying a greater volume fraction of the solution.
Accordingly, the greater aggregation of DWPS compared with
DWCS and DWRS is due to the chain length characteristics of the
DBSs. DWPS possesses a noticeably higher proportion of chains
between DP 30e100 (Fig. 1), and therefore has a higher tendency to
form insoluble aggregates.
3.4. Viscosity
Viscosities of the native and debranched starches at various
starch concentrations and temperatures are shown in Table 4.
Native starches exhibited much higher viscosity than the DBSs,
especially at 5% concentration. Native waxy potato starch had the
highest viscosities, followed by waxy corn and waxy rice starches,
respectively. On the other hand, viscosity values of all DBSs at 1%
concentration were very low (1.6e2.4 cP) and approached the
viscosity of deionized water (1.0 cP at 20  C). Viscosity increased
continuously as the concentration increased from 1% to 20% and as
the temperature dropped from 90  C to 30  C.
Among the three waxy starches, DWPS displayed higher viscosity values as compared with DWCS and DWRS. These viscosity
values were seemingly correlated with the chain length of the DBSs,

Fig. 4. Solubility values of 5% (w/w) debranched waxy starches in 0, 0.2 and 2 M NaCl solutions at different temperatures.

N. Klaochanpong et al. / Food Hydrocolloids 45 (2015) 218e226


Table 3
Solubility values of 1% and 5% (w/w) debranched waxy starches in water at 95  C and
after leaving at room temperature for 2 h.
Starch
sample

Starch
concentration (%)

DWRS

1
5
1
5
1
5

DWCS
DWPS

Solubility (%)
0h
100.8
99.9
99.5
95.6
100.2
99.3

D (%)

2h

98.3
96.6
91.5
86.8
84.3
77.1

0.8
2.3A
1.8A
4.4A
1.9A
1.2A

AB

5.8
1.1AB
7.5B
1.3B
7.4B
2.8B

2.5
3.3
8.0
8.8
15.9
22.2

Capital letters (A, B) indicate a signicant difference (p  0.05) between DBS solutions at 95  C and those after leaving at room temperature for 2 h for the same starch
and starch concentration.
D percentage difference of solubility values at initial time and after leaving at
room temperature for 2 h.

in
accordance
with
the
empirical
equation
of
MarkeHouwinkeSakurada, in which the viscosity of polymers ()
is a function of the molecular weight (M) [ kMa, where k and a
are constants related to the degree of molecular expansion]. It was
also observed that the difference was greater at high DBS concentration (10e20%) and low temperature (50 and 30  C). Therefore,
not only the chain length of the DBSs, but their ability to retrograde
into a network structure might also affect the viscosity of the DBS
solutions. The superior re-association ability of DWPS compared
with DWCS and DWRS resulted in a signicant difference in their
viscosity values, especially at high DBS concentration and low
temperature.
3.5. Gelling properties
It is known that when starch is subjected to gelatinization,
either by heating in excess water or suspending in some chemical
solutions, the starch granules will be destroyed to some extent,
depending on gelatinizing conditions. The dissolved starch molecules, especially amylose, can re-associate by H-bonding (between
hydroxyl groups or via a water bridge), resulting in a precipitate or
gel (Biliaderis, 2009). The ability of starch to retrograde depends on
many factors, such as starch composition (amylose content, protein
content, lipid content), structure of starch molecules (amylose/
amylopectin structure), storage conditions (e.g. temperature, time),
starch concentration, etc. Generally, retrogradation of highTable 4
Viscosities of native (N) and debranched (D) waxy starches at different starch concentrations and temperatures.
Starch Starch Viscosity (centipoise, cP)
sample conc.
90  C
70  C
(% w/v)
NWRS
DWRS

NWCS
DWCS

NWPS
DWPS

1
5
1
5
10
20
1
5
1
5
10
20
1
5
1
5
10
20

11.0
2696.7
1.7
2.0
4.1
5.5
13.9
3433.3
1.8
1.9
3.0
7.8
325.7
4266.7
1.6
1.8
3.9
26.8

50  C

30  C

0.4
14.6 0.6
16.8 1.7
20.1 1.6
66.6 2843.3 41.6 3163.3 80.8 3216.7 40.4
0.1
2.0 0.1
2.1 0.1
2.1 0.1
0.1
2.2 0.1
2.5 0.1
3.0 0.1
0.2
5.5 0.4
6.5 0.2
7.6 0.2
0.1
13.1 0.1
28.4 0.3
45.5 0.7
0.4
21.1 1.2
21.4 0.7
22.9 1.2
72.3 3623.3 76.4 4256.7 65.1 4450.0 75.5
0.1
1.8 0.1
2.0 0.1
2.4 0.1
0.1
2.1 0.1
2.3 0.1
2.8 0.1
0.1
3.8 0.2
6.5 0.3
8.3 0.3
0.1
17.7 0.1
30.5 0.9
42.1 1.8
4.3
337.0 4.4
353.5 3.8
380.7 4.5
66.6 4553.3 60.3 4676.7 95.0 4936.7 76.4
0.1
1.7 0.1
1.9 0.1
2.1 0.1
0.1
2.5 0.1
4.8 0.6
12.2 0.8
0.1
10.7 0.4
25.9 0.8
39.3 1.1
0.4
39.0 0.7
85.8 1.8
135.8 2.5

223

concentration gelatinized starch results in a gel structure, while


that of low-concentration gelatinized starch results in a precipitate
structure (Eliasson & Gudmundsson, 2006). Therefore, the
appearance of intact or debranched starch solution after leaving for
a certain period could possibly be: 1) a turbid solution resulting
from very small aggregates of starch molecules; 2) a solution with
precipitates resulting from agglomeration of small aggregates of
starch molecules; 3) a gel resulting from a network structure of
starch molecules; or 4) a combination of these appearances,
depending on the factors mentioned above. Thus, phase change
behavior would involve aqueous phase (clear solution), gel phase,
and solid phase (precipitates).
Appearances of cooked native and debranched starches at
different starch concentrations after storing at 4 and 30  C for 24 h
are shown in Fig. 5. Turbidity, gel consistency (which indicated the
stability of the gel), and sediment volume (which implied the phase
change behavior) are summarized in Table 5. Gels of all native waxy
starches were viscous and homogenous; however, waxy corn starch
gel displayed the most turbidity, followed by waxy rice and waxy
potato gels, respectively. Starch gel turbidity has been reported to
be affected by several factors, such as granule swelling, granule
remnants, leached amylose and amylopectin, amylose and amylopectin chain length, intra- or interbonding, lipid content and crosslinking substitution (Jacobson, Obanni, & BeMiller, 1997). For waxy
starches, amylose is a major factor affecting gel turbidity. Waxy
corn starch used in this study contained 3.80% amylose, while
amylose content in waxy rice starch was only 0.24%. Although waxy
potato starch had a comparable amount of amylose to waxy corn
starch (3.92%), the exceptionally high level of phosphate monoesters in waxy potato starch caused its gel to be transparent. Data
on gel consistency (consistency  35 mm) as well as sediment
volume (100%) indicated that all native starch gels were stable at
the conditions used in this study.
For the DBSs, various appearances e such as turbid solution,
precipitate, gel lump, semi-rm gel and rm gel, as well as a
mixture of these forms e were observed, depending on the type
and concentration of starch and the storage temperature. Regardless of the type of waxy starch, the following characteristics were
observed: precipitates/gel aggregates at 1% concentration; rm gel
that did not ow when the tube was turned upside down at 20%
concentration; and either precipitate, gel aggregate, semi-rm gel
or rm gel at 5% and 10% concentrations. Changes in the appearance
of starch samples were more prominent when starch solutions
were stored at 4  C, likely due to the higher rate of retrogradation at
low temperature. Starch gels/solutions at concentrations 5% were
very turbid, i.e. turbidities of all samples approached 100%. However, differences in turbidities could be observed at 1% concentration. The DWPS solution was the most turbid, followed by DWCS
and DWRS solutions, respectively. The debranched starch samples
had a higher proportion of gel phase when the concentration was
increased to 5% and 10%. These appearances were supported by gel
consistency as well as sediment volume values, as shown in Table 5.
Gel consistency of debranched starch samples became harder
(shorter ow distance) and sediment volume was higher when the
concentration of starch samples was increased. DWRS displayed
the best gel-forming ability, i.e. rm gel (gel consistency around
20 mm) could be formed even at 5% concentration. However, the
other two types of starches (DWCS and DWPS) could also produce
rm gel at a concentration of 20%. These gels were not sticky and
exhibited a smooth, glossy surface.
For dispersed native starch, precipitation or gelation can occur
during storage, depending on concentration. Solutions of low
concentration (approximately 2% or less) become increasingly
cloudy due to the progressive association of starch molecules
(mainly amylose) into larger particles (precipitates), while pastes of

224

N. Klaochanpong et al. / Food Hydrocolloids 45 (2015) 218e226

Fig. 5. Appearances of cooked native (N) and debranched (D) starches at different starch concentrations after storing at 30  C (left column) and 4  C (right column) for 24 h.

higher concentration set into a gel, which on aging becomes


steadily rmer (Collison, 1968). Similarly, phase-change behavior of
the DBS solution was also considered to be a result of re-association
of the debranched starch chains. Gidley and Bulpin (1989) examined the inuence of amylose chain length (DP 40e2,800) on aggregation behavior in aqueous solutions of 0.5e5.0% w/v
concentrations. They found that amyloses having chain lengths of
<110 residues precipitated from aqueous solutions, while both
precipitation and gelation occurred for chain lengths of 250e660
residues, and gelation was predominant over precipitation for
longer chains (>1,100 residues). It has also been reported that DP 10

is the minimum chain length required for recrystallization during


retrogradation (Robin, Mercier, Charbonniere, & Guilbot, 1974). If a
considerable amount of longer chains (DP > 10) is present, the short
chains can behave as co-crystals (Gidley & Bulpin, 1987). In our
experiment, the average DP of the DBSs was approximately 18e26,
and more than 80% of the DBSs had chain lengths of less than 37
residues. These starch chains could form a rm gel at high concentrations (20% for corn and potato starches and 5e10% for rice
starch). Therefore, in these cases the gel structure might be formed
by junction formation in the overlapping portions of several starch
chains. The superior gelling ability of DWRS compared with DWCS

N. Klaochanpong et al. / Food Hydrocolloids 45 (2015) 218e226

225

Table 5
Turbidity, gel consistency and sediment volume of cooked native (N) and debranched (D) starches at different starch concentrations after storing at 30  C and 4  C for 24 h.
Starch sample

Starch conc. (% w/v)

Turbidity (%)
30  C

NWRS
DWRS

NWCS
DWCS

NWPS
DWPS

5
1
5
10
20
5
1
5
10
20
5
1
5
10
20

69.4
17.8
99.4
100.0
100.0
89.1
66.4
99.9
100.0
100.0
22.5
99.5
99.9
100.0
100.0

4 C

0.3
1.8
0.2
0.1
0.1
0.3
1.9
0.1
0.1
0.1
1.3
0.1
0.1
0.1
0.1

70.4
99.2
100.0
100.0
100.0
88.7
99.7
100.0
100.0
100.0
36.3
99.8
100.0
100.0
100.0

and DWPS was unexpected, since it had the shortest average chain
length. Therefore, besides chain length, other factors e such as the
remaining a-1,6 linkages, phosphate ester in glucose residues, and
conformation of the debranched chains e might also be involved in
the efciency of gel formation. It is worth noting that DWS gels
appeared as particle gels (gure not shown) rather than as a
continuous network in the case of native starch gels. Therefore, the
gel of DWS would have signicantly different characteristics from
the gel of native starch.
3.6. Potential applications of DBSs
In view of the potential industrial applications of DBSs, information on each attribute derived from this study indicated the
following potential uses.
3.6.1. Complexing ability
The results indicated a signicant increase in complex formation
with iodine and fatty acids when starches were debranched.
Debranched potato starch displayed the highest ability to form
complexes. Therefore, DBSs could be an alternative source of carriers for drug delivery and encapsulating agents for active compounds such as avonoids, vitamins and antioxidants. Similar to
cyclodextrins, the complex-forming mechanism of DBSs with other
guest molecules is molecular inclusion; thus, the guest molecules
must have the appropriate size and conformation to t into the
cavities of the DBSs. DBSs can increase the dispersion of highly
insoluble compounds, control the release of compounds, and protect sensitive compounds against the environment.
3.6.2. Solubility and viscosity
The DBSs displayed much higher solubility and extremely low
viscosity compared with native starches; therefore, they might be
suitable for use as an ingredient in products that need a highly
dispersed bulking agent ller/carrier. The low viscosity of DBSs also
facilitates product processing, such as pumping through a pipeline,
mixing by agitation, or atomizing in a spray dryer. The DBSs from
rice starch would be the rst choice if a DBS that can be solubilized
at low temperature is required.
3.6.3. Gelling properties
The DBSs formed gels at high concentration and formed precipitates at low concentration. DBS gels are non-sticky, smooth, and
have a glossy appearance; therefore, they can be used as a gelling
agent in food, cosmetics and pharmaceutical products. Due to the
textural appearance of these gels, they have also received attention

0.2
0.4
0.1
0.1
0.1
0.4
0.1
0.1
0.1
0.1
1.1
0.1
0.1
0.1
0.1

Gel consistency (mm)

Sediment volume (%)

30  C

30  C

29.7
144.7
141.7
20.0
20.7
17.3
149.3
90.0
24.0
20.3
19.0
156.3
60.0
26.3
20.3

4 C

1.5
2.1
4.5
1.0
2.1
0.6
7.6
8.0
1.0
1.2
1.0
3.8
2.0
1.5
0.6

26.3
150.7
18.0
19.7
19.7
20.0
150.7
27.3
23.7
20.7
18.3
114.8
29.0
25.0
19.7

2.5
2.5
1.0
0.6
1.2
1.0
4.0
2.1
0.6
0.6
0.6
4.0
1.0
1.0
0.6

100.0
0.4
3.3
42.1
52.9
100.0
0.1
15.4
26.7
45.4
100.0
4.2
20.3
36.7
62.9

4 C

0.1
0.7
1.2
2.6
0.7
0.1
0.1
1.2
0.7
0.7
0.1
1.4
0.6
1.9
1.9

100.0
3.8
34.3
74.2
76.2
100.0
5.2
22.4
31.2
52.8
100.0
5.0
21.3
39.6
66.7

0.1
2.1
1.5
1.4
1.3
0.1
2.5
1.4
1.3
1.9
0.1
2.5
1.2
2.6
1.4

for use as a fat replacer. In addition to serving as a substitute for fat,


they would also function as a thickener, bulking agent and/or
binder. At low concentrations, DBSs can be used as a clouding agent
in juices and beverages, as they can form a particle gel; however,
additional experimentation with DBSs is needed to improve their
suspension stability. For use as a gelling agent, DBS from waxy rice
starch would be the rst choice, while for use as a clouding agent;
DBS from waxy potato starch is preferable.
4. Conclusions
In this study, four important attributes e complexing ability,
solubility, viscosity and gelling property e of debranched waxy rice,
corn and potato starches were investigated and compared with those
of non-debranched starches. The results obtained indicated signicant differences in such properties between the DBSs and their corresponding non-debranched starches. Also, DBSs from different
sources of starch exhibited certain differences in the magnitude of
each attribute, mostly related to average chain lengths of the DBSs.
Potential applications of these DBSs have been suggested; however,
further investigations should be performed to verify their suitable
utilizations. Other attributes, such as adhesiveness, compressibility,
interaction with other substances, etc., that are specic for any
particular end use should also be investigated.
Acknowledgments
The authors gratefully acknowledge nancial support from: the
Thailand Research Fund, via the Royal Golden Jubilee Ph.D. Program, for Miss Nuengmaysa Klaochanpong; and the Higher Education Research Promotion and National Research University
Project of Thailand, Ofce of the Higher Education Commission.
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