Plant Foods for Human Nutrition

, Volume 64, Issue 1, pp 18–24

Blue Maize: Morphology and Starch Synthase Characterization of Starch Granule

Authors

  • Rubi G. Utrilla-Coello
    • Centro de Desarrollo de Productos Bióticos del IPN.
    • Centro de Desarrollo de Productos Bióticos del IPN.
  • Ana Paulina Barba de la Rosa
    • Instituto Potosino de Investigación Científica y Tecnológica
  • Jose L. Martinez-Salgado
    • Instituto Potosino de Investigación Científica y Tecnológica
  • Sandra L. Rodriguez-Ambriz
    • Centro de Desarrollo de Productos Bióticos del IPN.
  • Luis A. Bello-Perez
    • Centro de Desarrollo de Productos Bióticos del IPN.
Original Paper

DOI: 10.1007/s11130-008-0106-8

Cite this article as:
Utrilla-Coello, R.G., Agama-Acevedo, E., de la Rosa, A.P.B. et al. Plant Foods Hum Nutr (2009) 64: 18. doi:10.1007/s11130-008-0106-8

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 microscopy 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 maizeStarchMorphologyStarch synthases

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 α–1–4 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]. 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 6–15 glucose units, SSSII synthesize larger chains (DP > 24), and SSSIII intermediate chains (DP 16–24) [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 H–511 (vitreous endosperm) with white grains and blue variety Chalqueño cónico (floury endosperm), were obtained from INIFAP-Texcoco, México. 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., México, D.F.), sieved through 50 U.S. mesh (300 μm), and stored at room temperature in sealed plastic containers.

Chemical Composition

Moisture content was determined by gravimetric heating (130 ± 2 °C for 2 h) using a 2–3 g sample. Ash, protein, fat and damage starch were analyzed according to AACC methods 08–01, 46–13, 30–25, and 76–31, respectively [11]. Total starch content was analyzed according to Goñi 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 amyloglucosidase (14 units/mg protein) (Roche, Indianapolis, IN). Released of glucose was assessed using a glucose oxidase/peroxidase assay (SERA-PAK® Plus, Bayer de México, S.A. de C.V., Edo. de México). These analyses were carried out in 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 6000 × g 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 13,000 × g for 30 min. The supernatant was mixed with an equal volume of cold (−20 °C) 30% TCA in acetone to precipitate proteins during 2 h at −20 °C, the mixture was centrifuged at 13, 000 × g for 10 min. The pellet was washed twice with cold acetone and dried under a gentle stream of air. SDS-PAGE was performed with 12.5% self-cast gels with the Laemmli buffer system at 10 mA constant current per gel during 12 h, in a Protein Electrophoresis-Standard Vertical Systems SE 600 (Hoefer, San Francisco, CA). The protein concentration loaded was 9.8 µg/µL (20 µL were used). The gels were stained with Coomassie blue.

Two-dimensional Gel Electrophoresis (2D-PAGE)

The procedure was performed according to GE Healthcare, Uppsala, Sweden (Berkelman and Stenstedt, 2002). Thio-urea buffer (350 mL) was added to the dried protein pellet, vortexed and centrifuged. The protein concentration in the supernatant was measured with the Bio Rad Protein Assay (Bio Rad Laboratories, Hercules, CA), with a volume of supernatant containing a desired amount of protein. 114 μg were loaded onto 11 cm IPG strips with a linear pI gradient of 4–7 (GE Healthcare, Bioscience Uppsala, Sweden) in gel-rehydration at 50 V for 12 h. Isoelectric focusing (IEF) was performed at 20 °C for 0.54 kVh on an IPGphore (GE Healthcare, Bioscience Uppsala, Sweden). Prior to second dimension SDS-PAGE the IPG strips were first equilibrated for 15 min in 6 M urea; 30% glycerol; 2% SDS; 50 mM Tris.HCl, pH 8.8; 15 DTT, and secondly for 15 min in 6 M urea; 30%; 2% SDS; 50 mM Tris.HCl, pH 8.8; 2.5% iodoacetamide. SDS-PAGE was performed with 12.5% self-cast gels with the Laemmli buffer system at 10 mA constant current per gel during 12 h, in a Protein Electrophoresis-Standard Vertical Systems SE 600 (Hoefer, San Francisco, CA). The gels were stained with Coomassie blue. Spots were cut off and analyzed by MALDI-TOF according to the Proteomics Labs services (CINVESTAV- Campus Guanajuato).

Statistical Analysis

An analysis of Student’s t-test was performed (SPSS, V.6.0., 1996).

Results and Discussion

Chemical Composition

A higher amount of total protein was obtained in the blue maize starch than in the white maize; these results are in agreement with previous reports indicating that blue maize had higher protein and lysine content than white maize [15]. The high level of protein is due mainly to some parts of germ that are co-extracted with the starch; however, higher lipid and ash contents were determined in white starch compared with the blue starch (Table 1). When the starch purity (tested as total starch) was quantified, the blue maize starch had higher value than white starch; this is mainly because the total starch method eliminates proteins but not lipid and ash content. Lower amylose content was determined in the blue maize starch, although the values of both samples (blue and white) are considered as normal starches (Table 1). No differences were found in the level of damaged starch (Table 1), the extraction method used in this work avoided mechanic stress in the sample and no damage of the starch granule ocurred; the damage observed in the microscopical study could be due to the damage that take place during the endosperm filling [16, 17]. One purification step was carried out for removing the proteins and lipids contamination in the starch samples.
Table 1

Chemical composition of dry-milled endosperms and isolated starches from blue and white maizes (dry basis)

Content (%)

Blue Maize

White Maize

Proteina

8.3 ± 0.2a

7.5 ± 0.0b

Lipid

0.2 ± 0.1a

0.5 ± 0.1a

Moisture

6.3 ± 0.3a

7.4 ± 0.8b

Ash

0.3 ± 0.0a

0.6 ± 0.0b

Apparent amylose

23.10 ± 0.5a

26.3 ± 0.2b

Total starch

84.1 ± 0.3a

78.7 ± 1.8b

Damaged starch

4.5 ± 0.3a

4.2 ± 0.2a

Proteinb

1.9 ± 0.2a

2.5 ± 0.0b

Total starchb

93.0 ± 0.7a

93.8 ± 1.0a

Damage starchb

12.6 ± 0.3a

5.1 ± 0.2b

Values with different letters in the same column are statistically different (p ≤ 0.05)

Mean of three replications; dry basis

aN × 6.25

b After protein extraction

Protein Extraction

The protein content, although at minor level in the blue maize than white maize starch, decreased significantly after the extraction process (Table 1). This result might indicate that there are differences in the protein type associated or bound to the starch granules. Due to the elimination of proteins, the purity of the starch (tested as total starch) was increased (Table 1), but the treatment with diverse solvents produced higher level of damaged starch in the blue starch (from 4.52 to 12.62%), only a slight increase of starch purity was found in the white starch (Table 1).

Polarized and Scanning Electron Microscopy

The polarized photographs are show in Fig. 1. The Blue maize starch (Fig. 1a) had some granules that did not show 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 properties 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].
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Fig. 1

Polarized light microscopy of starch granules from: a blue maize and b white maize

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Fig. 2

Scanning electron microscopy (SEM) of starch granules from: a blue maize and b white maize

Starch Biosynthetic Enzymes

SDS-PAGE profiles of the protein extracted from starch granules are shown in Fig. 3, the lanes 2 to 4 correspond to the supernatant from the extraction process, lanes 5 and 6 are soluble proteins in the washing step and lane 7 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 biosynthesis conduce to different starch structure and consequently modifications in its physicochemical and functional properties [26]. In the extraction step (Fig. 3a and b, lanes 2–4) the band of 90 kDa (SSSII) was present, but its intensity decreased after the washing step (lanes 5–6) 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].
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Fig. 3

SDS-PAGE of the protein extraction of starch granules. a blue maize and b white maize. The lane 1 correspond to molecular weight standards, lanes 2–4 to supernatant of extraction process, lanes 5–6 to washing step and lane 7 to concentrated extract

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 samples in a pI range between 5 and 6, where approximately 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.
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Fig. 4

2D-PAGE of blue maize starch granule bound proteins isolated after SDS washing of the granules. A total of 114 µg of proteins isolated from blue a and white b maize starch granules were separated using first dimension 11 cm, pI 4–7 NL IPG strips, followed by second dimension 12.5% linear SDS-PAGE. Proteins were visualized with Coomassie brillant blue staining

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Fig. 5

Mascot results from MS data of one 60 kDa spot from Fig. 4

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 properties, 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 morphological 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-México.

Copyright information

© Springer Science+Business Media, LLC 2008