Blue Maize: Morphology and Starch Synthase Characterization of Starch Granule
- First Online:
- 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
- 265 Views
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.
KeywordsBlue maizeStarchMorphologyStarch synthases
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 . Most of the pigmented maize studies are focused in the extraction, characterization and use of its colorants in the food industry . 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 . 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 . 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) . 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 . The amylopectin chains are radially ordered when starch granule grows and the granule size depends of the DP of branching . 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 . 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
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.
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 . Total starch content was analyzed according to Goñi et al. , 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 .
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. . 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).
An analysis of Student’s t-test was performed (SPSS, V.6.0., 1996).
Results and Discussion
Chemical composition of dry-milled endosperms and isolated starches from blue and white maizes (dry basis)
8.3 ± 0.2a
7.5 ± 0.0b
0.2 ± 0.1a
0.5 ± 0.1a
6.3 ± 0.3a
7.4 ± 0.8b
0.3 ± 0.0a
0.6 ± 0.0b
23.10 ± 0.5a
26.3 ± 0.2b
84.1 ± 0.3a
78.7 ± 1.8b
4.5 ± 0.3a
4.2 ± 0.2a
1.9 ± 0.2a
2.5 ± 0.0b
93.0 ± 0.7a
93.8 ± 1.0a
12.6 ± 0.3a
5.1 ± 0.2b
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
Starch Biosynthetic Enzymes
2D-PAGE Separation of Tightly Associated Starch Granule Proteins
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.
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.
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.