Plant Foods Hum Nutr (2009) 64:1824
DOI 10.1007/s11130-008-0106-8
ORIGINAL PAPER
Blue Maize: Morphology and Starch Synthase
Characterization of Starch Granule
Rubi G. Utrilla-Coello & Edith Agama-Acevedo &
Ana Paulina Barba de la Rosa &
Jose L. Martinez-Salgado &
Sandra L. Rodriguez-Ambriz & Luis A. Bello-Perez
Published online: 20 January 2009
# Springer Science + Business Media, LLC 2009
Abstract The use of pigmented maize varieties has increased
due to their high anthocyanins content, but very few studies
are reported about the starch properties of these grains. The
aim of this work was to isolate the starch granules from
pigmented blue maize and carry out the morphological,
physicochemical, and biochemical characterization studies.
The proximate composition of starch granules showed high
protein contents, after purification, the blue maize starch
presented lower protein amount than starch from white maize
(control). Although the purity of starch granules was
increased, the damaged starch (determined for the Maltase
cross absence) was also increased. Scanning electron micros-
copy showed the presence of some pores and channels in the
blue maize starch. The electrophoretic protein profiles showed
differences in the bands that correspond to the enzymes
involved in the starch biosynthesis; these differences could
explain the variation in morphological characteristics of blue
maize starches against starch from white maize.
Keywords Blue maize . Starch . Morphology .
Starch synthases
R. G. Utrilla-Coello : E. Agama-Acevedo (*) :
S. L. Rodriguez-Ambriz : L. A. Bello-Perez
Centro de Desarrollo de Productos Biticos del IPN.
Km 8.5 carr Yautepec-Jojutla, colonia San Isidro,
P.O. Box 24, 62731 Yautepec, Morelos, Mxico
e-mail: eagama@ipn.mx
A. P. B. de la Rosa : J. L. Martinez-Salgado
Instituto Potosino de Investigacin Cientfica y Tecnolgica,
Camino de la Presa San Jos, Lomas 4a Seccin,
78216 San Luis Potos, Mexico
Introduction
Maize is one of the cereals of worldwide importance; this grain
is used for human consumption and also has important
industrial applications. This cereal shows a genetic diversity,
giving origin a great number of varieties, hybrids and
genotypes, and recently the pigmented materials have received
increased interest due to the anthocyanin contents. The
antioxidant properties of flavonoids, including anthocyanins,
were revised and the beneficial effects on health have been
discussed [1]. Most of the pigmented maize studies are
focused in the extraction, characterization and use of its
colorants in the food industry [2]. Although the starch is the
main component of pigmented maize, there are few studies on
this component. The characterization of starch granules, and
even more the knowledge of enzymes involved in their
biosynthesis have generated information that might explain
the physicochemical and functional properties of maize-based
products such as tortilla. Starch is a versatile and useful
polymer, in recent years, the characterization of the enzymes
that are involved in starch biosynthesis has increased, these
enzymes are responsible of the granule starch formation
which influences in its morphologic, molecular and structural
characteristics, and consequently the physicochemical and
functional properties of starchy products [3]. Although have
been characterized approximately thirteen enzymes in starch
granules, only three of them are considered key points in the
starch synthesis: starch synthase (SS), starch branching
enzyme (SBE), and starch debranching enzyme (SDBE).
Two isoforms of the SS are responsible of the production of
linear chains of 14 glucosyl units in the amylose
(GBSS, granule bound starch synthase) and amylopectin
synthesis (SSS, soluble starch synthase). A granule-bound
isoform, GBSSI, which is encoded by waxy (wx) locus in
cereals, functions specifically to elongate amylose [4, 5].
Plant Foods Hum Nutr (2009) 64:1824
Although GBSSI activity and wx gene dosage are linearly
proportional, wx dosage is not proportional to amylose
content, as shown in early studies of maize [6]. Cereal
endosperms contain at least four SSS isoforms that are
categorized according to conserved sequence relationships.
In maize, SSSI elongates lineal chains until a degree of
polymerization (DP) between 615 glucose units, SSSII
synthesize larger chains (DP>24), and SSSIII intermediate
chains (DP 1624) [7]. Loss of SSII activity reduces starch
content, amylopectin chain-length distribution, and reduced
crystallinity with the result of altered granule morphology,
suggesting that the SSII has important influence on starch
structure [8]. The amylopectin chains are radially ordered
when starch granule grows and the granule size depends of
the DP of branching [9]. The granule size is correlated with
some physicochemical and functional properties of starch
such as temperature, enthalpy of gelatinization, pasting
characteristics, enzymatic susceptibility, granule crystallinity,
swelling, and solubility [10]. In this sense, the aim of this
work was to isolate and purify starch granules from blue corn
endosperm using a refined dry-mill method, and carry out the
morphological and physicochemical characterization, as well
the identification of proteins attached to these granules.
Materials and Methods
Starch Isolation
Maize hybrid H511 (vitreous endosperm) with white grains
and blue variety Chalqueo cnico (floury endosperm), were
obtained from INIFAP-Texcoco, Mxico. The pericarp and tip
of the grains were removed manually using a blade. The
starchy endosperms were ground in a commercial hammer
grinder (Mapisa Internacional S.A. de C.V., Mxico, D.F.),
sieved through 50 U.S. mesh (300 m), and stored at room
temperature in sealed plastic containers.
Chemical Composition
Moisture content was determined by gravimetric heating
(1302 C for 2 h) using a 23 g sample. Ash, protein, fat and
damage starch were analyzed according to AACC methods
0801, 4613, 3025, and 7631, respectively [11]. Total
starch content was analyzed according to Goi et al. [12],
briefly 50 mg of sample were dispersed in 3 mL of distilled
water and mixed with 3 mL of 4 M KOH, the mixture was
intensively stirred with a magnetic bar during 30 min at room
temperature; the dispersed samples were treated with amylo-
glucosidase (14 units/mg protein) (Roche, Indianapolis, IN).
Released of glucose was assessed using a glucose oxidase/
peroxidase assay (SERA-PAK Plus, Bayer de Mxico, S.A.
de C.V., Edo. de Mxico). These analyses were carried out in
19
triplicate. The apparent amylose content was determined as
described by the test of Hoover and Ratnayake [13].
Protein Extraction
Maize starch (1 g) was stepped in 5 ml of 1% sodium
metabisulfite solution at 45 C for 48 h. Then, the suspension
was transferred at 4 C, allowed to settle for 2 h and drained.
The starch was resuspended in 10 ml of 0.5 M NaCl solution
and stored at 4 C for 1 h, centrifuged at 6000g at 4 C for
10 min. The supernatant was drained and 10 mL of ethanol
was added, and allowed to settle for 20 min at room
temperature. The suspension was centrifuged as mentioned
above. The supernatant was drained and 20 mL of distilled
water was added, the suspension was centrifuged and the
wash with water was repeated. Finally, the supernatant was
discarded and the starch dried at 40 C for 3 h.
Polarized Light Microscopy
Birefringence of individual starch granules was evaluated
using a polarized microscope (LEICA, DMLB and Nikon,
model Alpha-Phot II, Japan) with an objective of 10 X,
equipped with a digital camera (Cannon, PowerShot S40,
Japan and Dage, model MTI DC-330, Japan). Starch was
sprinkled in a glass microscope slide with a drop of distilled
water. Starch granules were randomly selected and the
presence of Maltese cross was observed.
Scanning Electron Microscopy
For SEM tests, the samples were fixed to a conductive tape
of copper of double glue, which was covered with 20 nm
thick layer of coal and deposited in vacuum with an
evaporator in a JEOL JSMP 100 (Tokyo, Japan) electron
microscope. Later on, samples were covered in the ionizer
metals JEOL with 50 nm thick gold layer. All samples were
examined using an accelerating voltage of 5 kV.
Extraction of Starch Biosynthetic Enzymes
Starch biosynthetic enzymes were extracted using the method
of Peng et al. [14]. The isolate starch (1 g) was washed four
times with 10 mL extraction buffer (50 mM Tris HCl, pH
7.5; 1 mM EDTA; 1 mM DTT) and two times with SDS-
wash buffer (62.5 mM Tris-HCl, pH 6.8; 10 mM DTT; 2%
SDS). After each wash, the sample was mixed for 15 min at
4 C. Thereafter, the sample was centrifuged at 13,000 rpm
for 10 min at 4 C. SDS-wash buffer (30 mL) was added to
the pellet and boiled for 15 min under constant agitation. The
paste was frozen at 20 C for 1 h and thawed in a water
bath for 20 min before centrifugation at 13,000g for
30 min. The supernatant was mixed with an equal volume of
20 Plant Foods Hum Nutr (2009) 64:1824

cold (20 C) 30% TCA in acetone to precipitate proteins higher lipid and ash contents were determined in white
during 2 h at 20 C, the mixture was centrifuged at 13, starch compared with the blue starch (Table 1). When the
000g for 10 min. The pellet was washed twice with cold starch purity (tested as total starch) was quantified, the blue
acetone and dried under a gentle stream of air. SDS-PAGE maize starch had higher value than white starch; this is
was performed with 12.5% self-cast gels with the Laemmli mainly because the total starch method eliminates proteins
buffer system at 10 mA constant current per gel during 12 h, but not lipid and ash content. Lower amylose content was
in a Protein Electrophoresis-Standard Vertical Systems SE determined in the blue maize starch, although the values of
600 (Hoefer, San Francisco, CA). The protein concentration both samples (blue and white) are considered as normal
loaded was 9.8 g/L (20 L were used). The gels were starches (Table 1). No differences were found in the level of
stained with Coomassie blue. damaged starch (Table 1), the extraction method used in
this work avoided mechanic stress in the sample and no
Two-dimensional Gel Electrophoresis (2D-PAGE) damage of the starch granule ocurred; the damage observed
in the microscopical study could be due to the damage that
The procedure was performed according to GE Healthcare, take place during the endosperm filling [16, 17]. One
Uppsala, Sweden (Berkelman and Stenstedt, 2002). Thio-urea purification step was carried out for removing the proteins
buffer (350 mL) was added to the dried protein pellet, and lipids contamination in the starch samples.
vortexed and centrifuged. The protein concentration in the
supernatant was measured with the Bio Rad Protein Assay Protein Extraction
(Bio Rad Laboratories, Hercules, CA), with a volume of
supernatant containing a desired amount of protein. 114 g The protein content, although at minor level in the blue
were loaded onto 11 cm IPG strips with a linear pI gradient of maize than white maize starch, decreased significantly after
47 (GE Healthcare, Bioscience Uppsala, Sweden) in gel- the extraction process (Table 1). This result might indicate
rehydration at 50 V for 12 h. Isoelectric focusing (IEF) was that there are differences in the protein type associated or
performed at 20 C for 0.54 kVh on an IPGphore (GE bound to the starch granules. Due to the elimination of
Healthcare, Bioscience Uppsala, Sweden). Prior to second proteins, the purity of the starch (tested as total starch) was
dimension SDS-PAGE the IPG strips were first equilibrated increased (Table 1), but the treatment with diverse solvents
for 15 min in 6 M urea; 30% glycerol; 2% SDS; 50 mM Tris. produced higher level of damaged starch in the blue starch
HCl, pH 8.8; 15 DTT, and secondly for 15 min in 6 M urea; (from 4.52 to 12.62%), only a slight increase of starch
30%; 2% SDS; 50 mM Tris.HCl, pH 8.8; 2.5% iodoaceta- purity was found in the white starch (Table 1).
mide. SDS-PAGE was performed with 12.5% self-cast gels
with the Laemmli buffer system at 10 mA constant current Polarized and Scanning Electron Microscopy
per gel during 12 h, in a Protein Electrophoresis-Standard
Vertical Systems SE 600 (Hoefer, San Francisco, CA). The The polarized photographs are show in Fig. 1. The Blue
gels were stained with Coomassie blue. Spots were cut off maize starch (Fig. 1a) had some granules that did not show
and analyzed by MALDI-TOF according to the Proteomics
Labs services (CINVESTAV- Campus Guanajuato). Table 1 Chemical composition of dry-milled endosperms and
isolated starches from blue and white maizes (dry basis)
Statistical Analysis
Content (%) Blue Maize White Maize
An analysis of Students t-test was performed (SPSS, Proteina 8.30.2a 7.50.0b
V.6.0., 1996). Lipid 0.20.1a 0.50.1a
Moisture 6.30.3a 7.40.8b
Ash 0.30.0a 0.60.0b
Results and Discussion Apparent amylose 23.100.5a 26.30.2b
Total starch 84.10.3a 78.71.8b
Damaged starch 4.50.3a 4.20.2a
Chemical Composition
Proteinb 1.90.2a 2.50.0b
Total starchb 93.00.7a 93.81.0a
A higher amount of total protein was obtained in the blue Damage starchb 12.60.3a 5.10.2b
maize starch than in the white maize; these results are in
agreement with previous reports indicating that blue maize Values with different letters in the same column are statistically
different (p0.05)
had higher protein and lysine content than white maize Mean of three replications; dry basis
[15]. The high level of protein is due mainly to some parts a
N6.25
b
of germ that are co-extracted with the starch; however, After protein extraction
Plant Foods Hum Nutr (2009) 64:1824 21

Fig. 1 Polarized light microscopy of starch granules from: a blue

maize and b white maize
Fig. 2 Scanning electron microscopy (SEM) of starch granules from:
a blue maize and b white maize
the Maltase cross (see circles), indicating the lost of
molecular order. This behavior was presented in minor
level in the white maize starch (Fig. 1b). These results are
in agreement with the amount of damaged starch (Table
1). Due to the softer conditions used in this study for
starch isolation [18], the presence of some pores in the
blue maize starch are observed (Fig. 2a). It has been
reported that the granule growth is carried out by starch
synthases that goes inside the granule through the pores or
channels [16, 17, 19]; additionally, these structures affect
the physicochemical, functional and digestibility proper-
ties of starch. A smoother surface was showed in the white
maize starch (Fig. 2b), which might be related to the lower
damaged starch level in this sample. The morphological
characteristics of maize starches analyzed might be related
to functional properties (retrogradation) and digestibility
of maize-based products such as that showed in tortillas
[20].

Starch Biosynthetic Enzymes

SDS-PAGE profiles of the protein extracted from starch

Fig. 3 SDS-PAGE of the protein extraction of starch granules. a blue
granules are shown in Fig. 3, the lanes 2 to 4 correspond to maize and b white maize. The lane 1 correspond to molecular weight
the supernatant from the extraction process, lanes 5 and 6 standards, lanes 24 to supernatant of extraction process, lanes 56 to
are soluble proteins in the washing step and lane 7 washing step and lane 7 to concentrated extract
22 Plant Foods Hum Nutr (2009) 64:1824

Fig. 4 2D-PAGE of blue maize

starch granule bound proteins
isolated after SDS washing of
the granules. A total of 114 g
of proteins isolated from blue
a and white b maize starch
granules were separated using
first dimension 11 cm, pI 47
NL IPG strips, followed by
second dimension 12.5% linear
SDS-PAGE. Proteins were
visualized with Coomassie
brillant blue staining

corresponds to precipitated protein. As it was observed on

Fig. 3, several proteins of different molecular weights were
eliminated during the extraction and washing steps in the
process to obtain the starch granule associated proteins
(SGAPs), where the main proteins in this fraction are SS
and GBSS. The cleaning procedure has been used to
eliminate the storage proteins that do not correspond to
the enzymes involved in the starch biosynthesis or SGAPs
[21]; however, as was observed in blue maize starch profile
(Fig. 3a, lane 7), some starch biosynthetic enzymes (SSSII,
90 kDa; AGPase, 50 kDa) were eliminated in the extraction
process, but the GBSS isoform I of 60 kDa [22] was
concentrated; this could be due to the fact that this enzyme
is found inside the starch granule and it can be extracted
only after heating (gelatinization) as was carried out in the
concentration step. White maize starch (Fig. 3b, lane 7)
showed less recuperation of the GBSSI but bands at 120, 90
and 50 kDa were not found. These differences in the
electrophoretic pattern of both maize starches, blue and
white, suggest that starch biosynthesis differs among them
[23, 24, 25]. Alterations or changes in the starch biosyn-
thesis conduce to different starch structure and consequent-
ly modifications in its physicochemical and functional
properties [26]. In the extraction step (Fig. 3a and b, lanes
24) the band of 90 kDa (SSSII) was present, but its
intensity decreased after the washing step (lanes 56) and
when the extract was concentrated (lane 7), indicating that
this protein was solubilized in the aqueous media; this
corresponds with the literature. This enzyme is found in the
outer layer of starch granule and it is easily removed after
washing steps [27].

2D-PAGE Separation of Tightly Associated Starch

Granule Proteins

In Fig. 4 is shown the 2D-PAGE profile of purified SGAPs.

The presence of a 60 kDa protein is observed in both Fig. 5 Mascot results from MS data of one 60 kDa spot from Fig. 4
Plant Foods Hum Nutr (2009) 64:1824
samples in a pI range between 5 and 6, where approxi-
mately 7 spots were resolved. Those spots were cut and
tryptic digested for MALDI-TOF analysis, as shown in
Fig. 5, all the spots correspond to the GBSS1, this indicates
that GBSSI is a multi-enzymatic complex, and has been
reported that depending of the number of GBSSI isoforms,
influences the amylose biosynthesis [28, 29]. In maize
starch GBSS has been reported in a range of molecular
weight between 58 and 60 kDa and pI between 6 and 6.5
[30, 31, 23]. Nakamura et al. [23, 30] found that GBSS is
observed in SDS-PAGE in a single band at 60 kDa, but in
2D-PAGE can be detected from 3 to 5 spots of different pI.
An explanation for GBSSI (in wheat cv Chinese Spring)
having several isoforms has been given by Nakamura et al.
[23], who attributed these isoforms to correspond to
different genes located on chromosomes 7A, 4A and 7D
(which correspond to the known positions of the WAXY
loci [32, 28]. The functional significance (if any) of
multiple forms of GBSSI still has to be discovered [33].
In this sense, is not possible to associate the difference in
amylose content and the expression level of GBSSI
between samples, in maize seeds Tsai [6] showed that the
amylose content was not linearly related to the activity of
GBSSI.
The differences showed between the white maize and
blue maize starches are not related with the color of the
seed, the maize variety might be responsible of this pattern
independently of the pigments. However, the biosynthesis
of the pigments can alter starch biosynthesis, producing
starch with different physicochemical and functional prop-
erties, but further studies should be carried out.
Conclusions
Differences in the starch granules between blue and white
maize were observed, granule starch from blue maize has
more propensity to present damage during the purification
process. The protein profiles showed differences in the bands
that correspond to the enzymes involved in the starch
biosynthesis that could explain the differences on morpho-
logical characteristics of differences of both starches. The
main enzyme found was the GBSS and this enzyme has
different isoforms as determined by 2D-PAGE. These results
indicate structural differences in the starch of the maize
varieties analyzed and consequently different characteristics
of the maize products like tortillas.
Acknowledgements We appreciate the financial support from SIP-
IPN, COFAA-IPN and EDI-IPN. One of the authors (RGUC) also
acknowledges the scholarship from CONACYT-Mxico.
23
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