Journal of Cereal Science 52 (2010) 321e326
Contents lists available at ScienceDirect
Journal of Cereal Science
journal homepage: www.elsevier.com/locate/jcs
Identification of botanical origin of starches by SDS-PAGE analysis of starch
granule-associated proteins
Jae-Wook Yoon a, Jae-Yeon Jung a, Hyun-Jung Chung a, Mi-Ryung Kim b,
Chan-Wha Kim b, Seung-Taik Lim a, *
a
b
Graduate School of Life Sciences and Biotechnology, Korea University, 5-1 Anam-dong, Sungbuk-ku, Seoul 136-701, South Korea
Department of Bio-Food Materials, Silla University, San 1-1 Gwaebop-dong, Sasang-gu, Busan 617-736, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 January 2010
Received in revised form
20 June 2010
Accepted 22 June 2010
Starch granule-associated proteins (SGAPs) were extracted from various starches and analyzed using
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. The SDSPAGE results showed that granule-bound starch synthase (GBSS) of approximately 60 kDa was found in
most starches, except waxy-type starches. Starches exhibited the presence of proteins specific to the
botanical origin, including those of 22 kDa for maize; 160 and 98 kDa for potato; 140, 115, 90, and 80 kDa
for wheat. These proteins could be detected from noodles prepared with the corresponding starches. The
detection of the specific proteins by SDS-PAGE may be used to identify the origin of starch incorporated
in various foods and industrial products.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Starch granule-associated protein (SGAP)
SDS-PAGE
Starch noodle
Botanical origin
1. Introduction
The identification of botanical origin is often required in quality
control for commercial starches or various starch-based products.
The analysis typically includes indirect techniques measuring the
differences in physical and chemical properties of starch (Chatel
et al., 1996). The tools often used to identify starch origin include
optical and electronic microscopy, enzymology, rheology, chromatography, NMR, X-ray diffraction pattern, viscometer profiles, and
FTIR spectroscopy (Bernetti et al., 1990). The identification of
botanical origin is relatively simple when one starch is present.
However, it becomes more difficult for identification when more
than two starches are mixed. Moreover, once the starch has been
already processed under heat or other mechanical forces, it may be
almost impossible to determine the identity of starch by using the
indirect techniques.
Starch noodles, produced from purified starches of various
origins, are popular staple foods in many Asian countries (Tan et al.,
2009). Traditionally, clear noodles of mung bean starch are regarded as one of the best starch noodles since the starch provides
texture and appearance favorable to Asian people. Due to its high
amylose content, the mung bean starch exhibits restricted swelling
* Corresponding author. Tel.: þ82 2 3290 3435; fax: þ82 2 921 0557.
E-mail address: [email protected] (S.-T. Lim).
0733-5210/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2010.06.015
for gelatinization and high shear resistance during pasting (Lii and
Chang, 1981). However, researchers have attempted to explore
other starches that have an equivalent quality as noodle starch to
mung bean starch in order to replace the expensive mung bean
starch (Tan et al., 2009). Starch of some legumes such as broad
bean, pea, cowpea, and tuber, or root and tuber starches such as
potato, sweet potato, and cassava were reported to be competitive
with the mung bean starch (Tan et al., 2009). Among the potential
mung bean starch substitutes, sweet potato starch is widely
consumed on a commercial scale as a noodle starch in Korea, China,
Vietnam and Taiwan because it has a similar gelling ability and
eating quality (Lee et al., 2005). The sweet potato starch noodles
which are popular in Korea are referred to as “dangmyun”.
However, sweet potato starch is substantially more expensive
(more than twice as expensive) than common cereal starches such
as corn, wheat, and rice starches. Recently, starch noodles have
been manufactured from mixtures of corn and sweet potato
starches to reduce the material cost (Yook and Lee, 2001). However,
adulteration of sweet potato starch by adding other less expensive
starches not only for the commercial starch products but also for
starch noodles is often practiced. Therefore, the analysis of starch
used in noodles to identify its origin is highly needed for quality
control of commercial starches and starch-based noodles.
Although refined starch is devoid of most proteins, it still
contains small amounts of proteins that are strongly associated
with the surface or reside inside the starch granules (Goldner and
322
J.-W. Yoon et al. / Journal of Cereal Science 52 (2010) 321e326
Boyer, 1989). These proteins are called starch granule-associated
proteins (SGAPs) most of which are related to starch synthesis. The
SGAPs from different starches have been identified and known to
be specific to starch origin (Mu-Forster et al., 1996). However, no
study has been made in the utilization of SGAPs as biomarkers for
the identification of starch origins. The objective of the present
study was to develop reliable and accurate methods to identify the
botanical origin of starches, even after thermal processing for
noodle manufacture, by analyzing the SGAPs.
2. Materials and methods
2.1. Materials
Maize (Zea mays), waxy(wx) maize, tapioca (Manihot utilissima
Pohl.), sorghum (Sorghum bicolor L. Moench) and sago (Metroxylon
sagu Rottb.) starches were obtained from Samyang Genex Corp.
(Seoul, Korea), and high-amylose maize starch (HYLONÒVII) was
obtained from National Starch (Bridgewater, NJ, USA). Cowpea
(Vigna unguiculata L. Walp.) and wheat Triticum aestivum starches
were provided by Pulmuone Co., Ltd. (Seoul, Korea) and CJ Corp.
(Seoul, Korea), respectively. Potato (Solanum tuberosum L.) and
sweet potato (Ipomoea batatas Lam) starches were purchased
from Avebe Corp. (Veendam, Netherlands) and Jiangsu Jinwu
General Foods, Ltd. (Lianyungang, China), respectively. Buckwheat (Fagopyrum esculentum) starch was kindly provided by
Kangnung National University (Kangnung, Korea). Rice (Oryza
sativa L. cv. Ilpum), chestnut (Castanea crenata Sieb. Et Zucc.),
barley (Hordeum vulgare L.), acorn (Lithocarpus glabra) and mung
bean (Vigna radiata L.) were purchased from a local market
(Seoul, Korea) and each starch was isolated using 0.2% sodium
hydroxide as described by Chung et al. (1998). The crude protein
content of the starches was measured using an automatic
Kjeldahl system (K-350, BÜCHI Labortechnik AG, Postfach,
Switzerland).
2.2. Analysis of starch granule-associated proteins from various
starches
Protein extracts from isolated starches were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
using a Hoefer (Hollison, MA) electrophoresis system. Starches
(25 mg) were dissolved in 500 ml extraction buffer containing 2%
2-mercaptoethanol, 2% SDS, 10% glycerol, and 66 mM Tris (pH 6.8).
Suspensions were boiled for 10 min with intermittent stirring. After
immediate centrifugation at 10,000 rpm for 20 min, the supernatant was filtered through a 0.2 mm Nanosep MF microfilter (Pall
Corporation, East Hills, NY). An aliquot (40 ml) of the filtrate was
loaded into each well of a 6% and 10% polyacrylamide gel (acrylamide: bisacrylamide ¼ 29:1, w/w) to examine proteins of both
high and low molecular size ranges. The gel was run at a constant
voltage (30 V) for 3 h and stained with silver nitrate (Celis, 2006).
The molecular size of the proteins was estimated by comparison
with marker proteins.
Fig. 1. (a) 6% and (b) 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of total starch granule-associated proteins from cereal starches: maize
(lane 1), high-amylose maize (lane 2), waxy maize (lane 3), rice (lane 4), wheat (lane 5), barley (lane 6), buckwheat (lane 7), and sorghum (lane 8) starches.
J.-W. Yoon et al. / Journal of Cereal Science 52 (2010) 321e326
2.3. Preparation of starch noodle and analysis of its SGPs
Starch noodles were prepared according to the method of Lee
et al. (2005). Equivalent amounts of other starches (maize, potato,
or wheat) were mixed with sweet potato starch and the mixture
was dispersed in water (40%, db). The slurry was heated at 65 C for
6 min with mechanical stirring, and then carefully poured onto
a glass plate rimmed with metal strips (1.5 mm high). The plate was
placed in a steam bath, cooked for 2 min and cooled to room
temperature. The starch paste was placed at 4 C for 48 h and cut
into noodle strands (2 mm wide) with a roller cutter. The noodle
strands were dried at room temperature for 24 h.
To extract the starch granule-associated proteins in the
prepared starch noodle, the dried starch noodle was ground with
a homogenizer. From the finely ground starch noodle, starch
granule-associated proteins were extracted and analyzed using the
above procedure.
3. Results and discussion
3.1. Total protein content of various starches
The amount of total proteins present in the starch samples was
measured. The protein content of those starches tested was in
a range of 0.114e0.675% and was similar to previous reports
(BeMiller and Whistler, 2009). Therefore, it was confirmed that
323
most proteins not associated with the starch granules were properly removed.
3.2. SGAPs of cereal starches
Fig. 1 shows the 6% (a) and 10% (b) SDS-PAGE results for the
SGAPs extracted from various cereal starches. All cereal starches
tested, except the waxy maize starch, showed an intense band at
a size range between 61 and 55 kDa, which was from a protein
known as ‘GBSSI’ (granule-bound starch synthase I) (Goldner and
Boyer, 1989). This protein is the primary enzyme responsible for
the synthesis of amylose molecules, which is the reason that waxy
maize starch showed no GBSS bands on a gel. GBSSI exists as several
isoforms and its molecular size slightly varies among starches from
different botanical sources (Nakamura et al., 1993; Shure et al.,
1983).
For normal maize starch (lane 1), two distinct bands were
observed at a larger molecular size range than the 60 kDa GBSSI
(Fig. 1a), which we assumed to be 85 kDa SBEIIb and 76 kDa SSI
(Mu-Forster and Wasserman, 1998). Mu-Forster and Wasserman
(1998) suggested that the starch granule-associated proteins
included starch-biosynthetic enzymes such as GBSSI, SBEIIb, and
SSI, which could not be removed from intact starch granules by
protease treatment or detergent washing. It was suggested that
those proteins were tightly associated or entrapped within starch
granules. In addition, many protein bands were detected below the
Fig. 2. (a) 6% and (b) 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of starch granule-associated total proteins from different starches:
chestnut (lane 1), acorn (lane 2), cowpea (lane 3), mung bean (lane 4), potato (lane 5), sweet potato (lane 6), tapioca (lane 7), and sago (lane 8) starches.
324
J.-W. Yoon et al. / Journal of Cereal Science 52 (2010) 321e326
60 kDa GBSSI band (Fig. 1b, lane 1). Among them, 22 and 19 kDa
protein subunits have been reported to represent a-zein, a storage
protein that exists mainly in the protein bodies of the maize
endosperm and also on the surface of starch granules (Mu-Forster
and Wasserman, 1998). The protein band at 32 kDa and the
multiple bands between 45 and 60 kDa were believed to be GBSSI
fragments that formed due to the heating procedure used during
protein extraction (Mu et al., 1998).
High-amylose maize starch exhibited a similar banding pattern
(lane 2) to normal maize starch (lane 1). However, there was no
band corresponding to the 85 kDa SBEIIb (Fig. 1a, lane 2). This result
was in agreement with a previous report which showed that SBEIIb
played a specific role in the short-chain transfer of amylopectin,
and a lack of SBEIIb generated to produce high-amylose starch
(Nishi et al., 2001). In contrast, waxy maize starch did not show any
protein bands except for faint bands at 22 and 19 kDa, which corresponded to a-zein.
For rice starch, two protein bands were observed at 82 and
72 kDa (Fig. 1a, lane 4), corresponding to SBEIIb and SSI, respectively. A similar result was reported by Umemoto and Aoki (2005).
No distinct protein bands were observed in the smaller size range
(Fig. 1b, lane 4), and obscure bands between 32 and 60 kDa seemed
to be heat-induced GBSSI fragments, as was observed for normal
maize starch.
In the case of wheat starch, a number of bands were observed in
the larger molecular size range (Fig. 1a, lane 5). A unique band at
145 kDa for wheat starch might be related to SBEI according to an
immunoblotting study conducted by Peng et al. (2000). Three
closely related polypeptides having molecular sizes of 100, 108 and
115 kDa were also observed. Denyer et al. (1995) demonstrated that
Fig. 3. (a) 6% and (b) 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of starch granule-associated total proteins extracted from noodles made
from different starch mixtures. Sweet potato:maize ¼ 1:1 (lane 1), sweet potato:potato ¼ 1:1 (lane 2), sweet potato:wheat ¼ 1:1 (lane 3), and sweet
potato:maize:potato:wheat ¼ 1:1:1:1 (lane 4).
J.-W. Yoon et al. / Journal of Cereal Science 52 (2010) 321e326
proteins with these sizes displayed starch synthase activity. The
protein at 92 kDa in wheat starch was reported to have a similar
amino acid sequence as SBEIIb from maize and rice (Takaoka et al.,
1997). The protein at 77 kDa might be from a starch synthase,
which was present in both soluble and granule-bound forms
(Takaoka et al., 1997).
In the case of barley starches, two protein bands were
observed in the large molecular size region (Fig. 1a, lane 6). Based
on their molecular sizes (90 and 77 kDa), these protein subunits
might correspond to SSII and SSI, respectively (Boren et al., 2004;
Hylton et al., 1996). For buckwheat starch, a doublet band of
approximately 80 kDa and multiple bands between 30 and
60 kDa were observed in addition to the 60 kDa GBSSI (Fig. 1, lane
7), whereas only a faint GBSSI band was shown in sorghum
starch (Fig. 1, lane 8).
Based on the SGAPs from cereal starches, it could be suggested that the presence of protein bands for zeins is a characteristic feature observed only in maize varieties, and due to
their distinct location apart from other protein bands, the maize
starch can be easily identified. In addition to the zeins, other
indicators, such as the 60 kDa GBSSI and 85 kDa SBEIIb, could be
used to distinguish waxy and high-amylose maize, respectively.
Similar banding without zeins may correspond to rice starch
and bands at 145 and 100e115 kDa are an indication of wheat
starch.
3.3. SGAPs of nut, legume, and tuber starches
SGAPs were also extracted from other starches: chestnut and
acorn for nuts; cowpea and mung bean for legumes; potato, sweet
potato, tapioca and sago for tubers and roots. In all the starches
tested, a distinct band appeared around 60 kDa, which was in the
molecular size range of GBSSI proteins. However, the shape and
breadth of the bands slightly varied depending on the starch
source.
SDS-PAGE results of chestnut (lane 1) and acorn (lane 2)
starches showed only a major band around 60 kDa GBSSI, with
minor faint bands (Fig. 2). Legume starches showed a relatively
complex SGAPs profile. Cowpea starch showed protein bands at
105, 72, 60, 56, 54, 32, 30 and 20 kDa with additional minor bands
(Fig. 2, lane 3). It was reported that cowpea seed proteins consisted
of globulins with molecular weights of 65, 60, 59, and 50 kDa and
albumins with molecular weights of 99, 91, 32 and 30 kDa,
depending on their solubility (Chan and Phillips, 1994). Mung bean
SGAPs were identified as 105 kDa starch phosphorylase, 96 kDa
branching enzyme, 85 kDa methionine synthase, 58 kDa GBSSI, and
53 and 32 kDa starch synthases (Ko et al., 2005).
Potato starch exhibited a characteristic SGAPs banding pattern
with a unique band at a very large molecular size (160 kDa) (Fig. 2a,
lane 5). This protein has been called the R1 protein, which is bound
to starch granules in potato leaves and tubers and is responsible for
the incorporation of phosphate into starch glucans (Lorberth et al.,
1998). In addition, potato starch exhibited a dark band around
98 kDa, which was reported to be the two SBEII isoforms (Larsson
et al., 1998). There were also multiple bands below the 60 kDa
GBSSI, which probably corresponded to the starch phosphorylase
precursors involved in starch synthesis (Brisson et al., 1989). In
contrast to potato starch, sweet potato and tapioca starches
exhibited only a GBSSI band at 59 kDa (Wang et al., 1999) and
58.6 kDa (Salehuzzaman et al., 1993), respectively.
As described above, cowpea, mung bean, and potato starches
showed characteristic banding patterns with distinctive indicator
proteins. However, the other starches exhibited only a GBSSI band,
which would make them difficult to identify solely by SDS-PAGE
profiles of SGAPs.
325
3.4. SGAPs from starch noodle
Starch noodles that were made from the equivalent mixtures of
sweet potato starch with maize (Fig. 3, lane 1), potato (lane 2) and
wheat (lane 3) starches showed the same SGAPs banding pattern as
the corresponding raw starches (Figs. 1 and 2). Although the sweet
potato starch, which exhibited only a GBSSI band, was indistinguishable from the other starches, the proteins from each starch in
the SGAPs profile of the noodles made from the mixture of all four
starches were clearly distinguished (Fig. 3, lane 4). The R1
(160 kDa), SBEI (145 kDa), and zein (22 kDa) proteins were key
indicators of potato, wheat and maize starches, respectively. In
addition, the other SGAPs could also be used to verify the starch
identity.
4. Conclusions
It is often difficult to determine the botanical origin of starches
included in processed products because conventional methods
measuring the physical and chemical properties of native starches
are useless due to the changes of starch during processing.
However, the residual proteins that tightly associate to starch may
be used for the identification of starches in processed starch-based
foods such as noodles. SDS-PAGE analysis is a routine method
currently used to assess the presence of proteins which can provide
reliable results from the analysis of starch granule-associated
proteins with ease.
References
BeMiller, J., Whistler, R., 2009. Starch: Chemistry and Technology, third ed.
Academic Press, New York, NY.
Bernetti, R., Kochan, D.A., Trost, V.W., Young, S.N., 1990. Modern methods of analysis
of food starches. Cereal Foods World 35, 1100e1105.
Boren, M., Larsson, H., Falk, A., Jansson, C., 2004. The barley starch granule proteome
e internalized granule polypeptides of the mature endosperm. Plant Science
166, 617e626.
Brisson, N., Giroux, H., Zollinger, M., Camirand, A., Simard, C., 1989. Maturation and
subcellular compartmentation of potato starch phosphorylase. Plant Cell 1,
559e566.
Celis, J.E., 2006. Cell Biology: A Laboratory Handbook. Elsevier Academic,
Amsterdam, Boston.
Chan, C.W., Phillips, R.D., 1994. Amino-acid-composition and subunit constitution of
protein-fractions from cowpea (Vigna Unguiculata L. Walp) seeds. Journal of
Agricultural and Food Chemistry 42, 1857e1860.
Chatel, S., Voirin, A., Luciani, A., Artaud, J., 1996. Starch identification and determination in sweetened fruit preparations. Journal of Agricultural and Food
Chemistry 44, 502e506.
Chung, H., Cho, S., Chung, J., Shin, T., Shon, H., Lim, S.-T., 1998. Physical and
molecular characteristics of cowpea and acorn starches in comparison with
corn and potato starches. Food Science and Biotechnology 7, 269e275.
Denyer, K., Hylton, C.M., Jenner, C.F., Smith, A.M., 1995. Identification of multiple
isoforms of soluble and granule-bound starch synthase in developing wheat
endosperm. Planta 196, 256e265.
Goldner, W.R., Boyer, C.D., 1989. Starch granule-bound proteins and polypeptides e
the influence of the waxy mutations. Starch-Starke 41, 250e254.
Hylton, C.M., Denyer, K., Keeling, P.L., Chang, M.T., Smith, A.M., 1996. The effect of
waxy mutations on the granule-bound starch synthases of barley and maize
endosperms. Planta 198, 230e237.
Ko, Y.T., Chang, J.Y., Lee, Y.T., Wu, Y.H., 2005. The identification of starch phosphorylase in the developing mungbean (Vigna radiata L.). Journal of Agricultural
and Food Chemistry 53, 5708e5715.
Larsson, C.T., Khoshnoodi, J., Ek, B., Rask, L., Larsson, H., 1998. Molecular cloning and
characterization of starch-branching enzyme II from potato. Plant Molecular
Biology 37, 505e511.
Lee, S.-Y., Woo, K.-S., Lim, J.-K., Kim, H.I., Lim, S.-T., 2005. Effect of processing
variables on texture of sweet potato starch noodles prepared in a nonfreezing
process. Cereal Chemistry 82, 475e478.
Lii, C.Y., Chang, S.M., 1981. Characterization of red bean (Phaseolus radiatus var.
Aurea) starch and its noodle quality. Journal of Food Science 46, 78e91.
Lorberth, R., Ritte, G., Willmitzer, L., Kossmann, J., 1998. Inhibition of a starchgranule-bound protein leads to modified starch and repression of cold sweetening. Nature Biotechnology 16, 473e477.
Mu-Forster, C., Huang, R.M., Powers, J.R., Harriman, R.W., Knight, M.,
Singletary, G.W., Keeling, P.L., Wasserman, B.P., 1996. Physical association of
326
J.-W. Yoon et al. / Journal of Cereal Science 52 (2010) 321e326
starch biosynthetic enzymes with starch granules of maize endosperm e
Granule-associated forms of starch synthase I and starch branching enzyme II.
Plant Physiology 111, 821e829.
Mu-Forster, C., Wasserman, B.P., 1998. Surface localization of zein storage proteins
in starch granules from maize endosperm e Proteolytic removal by thermolysin
and in vitro cross-linking of granule-associated polypeptides. Plant Physiology
116, 1563e1571.
Mu, H.H., Mu-Forster, C., Bohonko, M., Wasserman, B.P., 1998. Heat-induced fragmentation of the maize waxy protein during protein extraction from starch
granules. Cereal Chemistry 75, 480e483.
Nakamura, T., Yamamori, M., Hirano, H., Hidaka, S., 1993. The waxy (Wx) proteins of
maize, rice and barley. Phytochemistry 33, 749e753.
Nishi, A., Nakamura, Y., Tanaka, N., Satoh, H., 2001. Biochemical and genetic analysis
of the effects of amylose-extender mutation in rice endosperm. Plant Physiology 127, 459e472.
Peng, M.S., Gao, M., Baga, M., Hucl, P., Chibbar, R.N., 2000. Starch-branching
enzymes preferentially associated with A-type starch granules in wheat
endosperm. Plant Physiology 124, 265e272.
Salehuzzaman, S.N.I.M., Jacobsen, E., Visser, R.G.F., 1993. Isolation and characterization of a cdna-encoding granule-bound starch synthase in cassava (Manihot-
Esculenta Crantz) and its antisense expression in potato. Plant Molecular Biology
23, 947e962.
Shure, M., Wessler, S., Fedoroff, N., 1983. Molecular-identification and isolation of
the waxy locus in maize. Cell 35, 225e233.
Takaoka, M., Watanabe, S., Sassa, H., Yamamori, M., Nakamura, T., Sasakuma, T.,
Hirano, H., 1997. Structural characterization of high molecular weight starch
granule-bound proteins in wheat (Triticum aestivum L.). Journal of Agricultural
and Food Chemistry 45, 2929e2934.
Tan, H., Li, Z., Tan, B., 2009. Starch noodles: history, classification, materials, processing, structure, nutrition, quality evaluation and improving. Food Research
International 42, 551e576.
Umemoto, T., Aoki, N., 2005. Single-nucleotide polymorphisms in rice starch synthase IIa that alter starch gelatinisation and starch association of the enzyme.
Functional Plant Biology 32, 763e768.
Wang, S.J., Yeh, K.W., Tsai, C.Y., 1999. Molecular characterization and expression of
starch granule-bound starch synthase in the sink and source tissues of sweet
potato. Physiologia Plantarum 106, 253e261.
Yook, C., Lee, W.K., 2001. Production of starch vermicelli (Dangmyun) by using
modified corn starches. Korean Journal of Food Science and Technology 33,
60e65.