Abstract: The mature grains of two Egyptian and six imported Triticum species were analyzed for protein patterns, total protein and amino acids content to characterize the variations between them. The data showed twenty two bands of total proteins with an obvious variation in the number and position of bands from one species to another. The highest numbers of proteins bands were recorded in T. paleocolchicum (18 bands) whereas, the lowest ones in T. durum (12 bands). The eight examined Triticum species share nine bands (6, 13, 16, 19, 20 and 21), while there were also some bands which characterize each species. The band (10) is characteristic for T. paleocolchicum and the band (12) for T. dicoccoides. Cladistic analysis support the delimitation of studied species in the two subgenera Triticum and Boeoticum. A high degree of similarity (94%) was observed between T. aestivum and T. spelta. Total protein of the grains varied from a minimum of 9.3% in T. aestivum to a maximum of 14.8% in T. dicoccoides. Most amino acids showed a significant variation between the examined Triticum species and all essential amino acids compared well with FAO/WHO reference pattern. The nutritional quality of proteins as measured by their essential amino acids chemical scores ranged from 14.29% for tyrosine in T. spelta grain protein to 239.29% for isoleucine in T. durum grain protein. Most essential amino acid of grain protein in T. aestivum and T. dicoccoides record higher values than the FAO/WHO (1990) recommended pattern. All the studied Triticum species had high content from the non-essential amino acids, particularly glutamic acid that had the greatest proportion ranging from 24.86 g/100 g protein in T. dicoccoides to 36.13 g/100 g protein in T. spelta. The six studied Triticum species imported from outside Egypt had pronounced total protein and amino acids content and consistent the most suitable condition for growing under Ismailia conditions.
INTRODUCTION
Wheat is most important temperate cereals (17000 cultivars) of complex ancestry involving closely allied Aegilops species (Mabberley, 1997). Five Triticum species were distinguished by Boulos (2005) from Egypt (T. aestivum, T. durum, T. dicoccum, T. pyramidale and T. turgidum). He also mentioned that the wheat is one of the most important cereal crops grown successfully in Egypt for the grains using principally as flours.
Galili and Feldman (1983 and 1985) observed twenty one discrete bands in T. aestivum and T. durum and considered that Triticum species were rich source of glutenins and gliadins which have proved to be important for technological properties. Ciaffi et al. (1993) mentioned that the endosperm proteins of T. dicoccoides of two classes proteins; gliadin and glutenin, which have a good deal in determining the nutritional and technological properties. El-Akkad (1998) studied the protein electrophoresis of two cultivars of T. aestivum and noticed distinct variation between them. In the same time, Hassan and Eid (1998) examined the protein patterns of T. aestivum and T. durum and Triticale genotypes and found that glutenins, gliadins and subunits of these proteins bands were varied among wheat and Triticale genotypes. Tarekegne et al. (2000) identified Ethiopian wheat cultivars by grain protein electrophoresis on the basis of gliadins and glutenin subunit banding patterns, which were unique for all cultivars and distinguish between them. El-Akkad and El-Abd El-Kariem (2002) studied the protein patterns of 12 cultivars of T. aestivum and T. durum and found obvious variation in the number and position of the protein bands.
Concerning the protein content, Ciaffi et al. (1992) found that the variation in T. dicoccoides protein content was large, ranging from 16-27%, compared to diploid wheat (20-28%), confirming the finding that wild wheat relatives have a wider range of variation for grain protein content than cultivated ones. Borghi et al. (1996) studied twenty-five T. monococcum lines and found that the protein content of these lines ranged from 13.2 to 22.8% higher than those found for bread wheat which ranged from 10.8 to 13.3%. Mesfin et al. (2000) studied the suitability of Triticum aestivum for many food products depends on its grain protein content. Oliveira (2001) studied T. dicoccum and T. spelta to obtain information about their agronomical and grain quality characteristics and found that their protein content was higher in the cultivars. In addition, Bramble et al. (2002) studied the protein variance structure in Triticum aestivum to quantify the variation of protein structure.
As regard to the amino acids, Molino et al. (1988 and 1989) isolated the amino acids from the grains of three Triticum aestivum varieties and found that they had the greatest proportion of glutamic acid. Nevo and Beiles (1992) showed the highest values of lysine and isoleucine contents in Triticum dicoccoides compared to other studied species of wild wheat and the lowest one of proline was observed in T. aestivum. Acquistucci et al. (1995) found positive correlations between grain protein content and amino acid values of T. monococcum strains for glutamine and proline and a negative correlation for threonine, cystine, valine, isoleucine, leucine, asparagine, serine, glycine and alanine. Moreover, Cervantes et al. (2002) studied the amino acid composition of commercial wheat and found high variations with high content from the essential amino acids.
The present study carry out for the comparison of protein electrophoretic patterns, total protein and amino acid composition of the most common two Egyptian Triticum species with six imported Triticum species to distinguish the variation between them. In addition, assessment was made for the quality of total proteins and amino acids for the six imported Triticum species and their suitability for the cultivation in Ismailia Governorate, Egypt.
MATERIALS AND METHODS
The grains of investigated eight Triticum species were obtained from three different sources; Agricultural Research Institute, Dokki, Giza; Center International de Mejoraminento de Maiz Y Trigo, int., Mexico (CIMMYT) and National Small Collection, USDA-ARS Aberdeen, USA (NSGC) as in Table 1. These grains were cultivated in the Experimental Farm of the Faculty of Agriculture, Suez Canal University, Ismailia Governorate, Egypt on fifteen November of 2004/2005. The grains were collected to study the proteins and amino acids.
Protein electrophoresis: Total endosperm storage protein were extracted from grain after embryo remove by treatment with a solution containing 2% (w/v) sodium dodecyl-sulphate (SDS), 6M urea and 1.5% (w/v) 2-mercaptoethanol (2-ME). The extraction solvent was prepared fresh for each electrophoretic run. The solution used for extraction included 0.002% (w/v) of tracking dye (bromophenol blue). Distal half grains were crushed individually and placed in a 1.5 mL plastic microcentrifuge tube to which was added 0.4 mL of the extracting solution. The samples were left overnight at room temperature. Then 10 μL of the samples were used directly for electrophoresis (Mahgoub, 1988).
Endosperm storage proteins are separated and classified into their subunits by sodium dodectyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The method fractionated proteins mainly according to their molecular weight. The discontinuous buffer system of the high resolution one-dimensional SDS-PAGE used to fractionate the proteins was adapted from that described by Laemmli (1970) with some modifications proposed by Payne et al. (1981) for wheat storage proteins. The separating gel contained 0.36 M Tris-HCL, pH 9.1 and 0.1% (w/v) SDS. Gels were made from either 10% (w/v) acrylamide and 0.125% (w/v) Bis (N, N methylenbis-acrylamide) or 5% acrylamide and 0.26% Bis.
| Table 1: | Classification, chromosome number (Dorofeev et al., 1979) and source of the studied Triticum species |
| Beni Swif3 and Sakha 69: From Agricultural Research Institute, Dokki, Giza. CIMMYT: Center International de Mejoraminento de Maiz Y Trigo, int., Mexico. NSGC: National Small Collection, USDA-ARS Aberdeen, USA | |
The stacking gel consisted of 3% (w/v) acrylamide, 0.25% (w/v) Bis, 0.1% (w/v) SDS and 0.006 M Tris-phosphate (pH 6.7). Both gels were polymerized by adding ammonium per sulphate and TEMED (N, N, N, N-tetramethylethlenediamine) immediately before pouring the gels. The cathode buffer was 0.04 M Tris-Glycine (pH 8.9), containing 0.1% (w/v) SDS. The anode buffer was 0.12 M Tris-HCL (pH 8.1). The proteins were electrophoresis at a constant current of 50 mA and 150 volts until the bromophenol blue front has entered the separating gel and then at 90 mA until the tracking dye front migrated to about 0.5 cm from the end of the gel. The gels were stained with a solution of 0.01% (w/v) coomassie brilliant blue R and 0.003% (w/v), coomassie brilliant blue G dissolved in 18% (w/v) methanol, 5% (w/v) trichloroacetic acid (TCA) and 6% (w/v) acetic acid (HAC) (the dye was dissolved first in methanol, filtered and then added to a TCA-HAC solution). Gels were stained overnight, then distained for at least two days in distilled water and photographed. In order to compare protein bands and obtain an index of similarity between the species, the percent coefficient of similarity was calculated as described by Todaria et al. (1983) as follow:
| W | = | No. of similar bands between the two compared species a and b |
| A | = | Total No. of bands in species a |
| B | = | Total No. of bands in species b. |
Protein content: Protein was determined according to improved Kjeldahl methods of AOAC (1970) modified by distilling the ammonia into boric solution and titrated with standard acid according to Page et al. (1982) at control Laboratory of Food Science, Faculty of Agriculture, Cairo University.
Amino acids: Samples of 1 g taken from different studied Triticum species dried grains were defatted and weighed in the screw-capped tubes; 5 mL of HCl 6.0 N were added to each tube. The hydrolysis was attached to a system, which allows the connection of nitrogen and vacuum lines without disturbing the sample. The tubes were capped 7 mol placed in an oven at 110°C for 24 h. (AOAC, 1995). The tubes were then opened and the content of each tube was filtered and then evaporated for dryness in a rotary evaporator. A suitable volume of sodium citrate buffer (pH 2.2) was added to each dried film of the hydrolyzed sample. After all soluble materials completely dissolved the samples were then filtered using a 0.2 μm membrane filter, (Winder and Eggum, 1966). The samples were analyzed using High Performance Amino Acid Analyzer, Biochrom 20 (Auto sample version) Pharmcia Biotch constructed at Nuclear Centre for Radiation Researches Technology, Cairo (NCRRT). Data analysis of chromatogram apparatus, which was done by EZChromTM Chromatography Data System Tutorial and Users Guide-Version 6.7.
The contents of the different amino acids recovered were expressed as g per 100 g protein and were compared with the FAO/WHO (1990) reference pattern (Vijayakumari et al., 1997; Shaheen and Hamed, 2003).
The essential amino acid chemical score was calculated as follows:
Statistical analysis
For protein patterns: The data matrix was built based on the presence/absence
of bands obtained from the protein analysis of the grains in the electrophoresis
unit. These results used to construct a neighbor-joining tree of individuals
using Euclidean distances computed between all pairs of individuals. A neighbor-joining
tree of the relationships among individuals was constructed using the UPGMA
method on the matrix of Euclidean distances.
RESULTS AND DISCUSSION
Protein electrophoretic patterns: The total proteins of the grains of the eight studied Triticum species were analyzed and the electrophoretic pattern is shown in Table 2-4 and Fig. (1) with twenty two bands. These bands were distributed along the gel with molecular weights ranging from 143 to 6 KD. There is an obvious variation in the number and position of bands from one species to another. The highest number of bands were 18 for total proteins and 4 for high molecular weight glutenin in T. paleocolchicum; 5 for high molecular weight gliadin in T. aestivum, T. spelta, T. dicoccoides
| Fig. 1: | Polyacrylmide gel electrophoresis SDS-PAGE for total proteins
of the eight studied Triticum species. 0. Molecular weight of marker
bands (97, 66, 45, 29 and 14 kD), 1. T. aestivum, 2. T. spelta,
3. T. monococcum, 4. T. timopheevii, 5. T. dicoccoides,
6. T. durum, 7. T. paleocolchicum and 8. T. polonicum.
( |
| Table 2: | Distribution of total protein, glutenin and gliadin bands in the different examined Triticum species. (HMW = High Molecular Weight; LMW = Low Molecular Weight) |
| Table 3: | Distribution of protein bands in the different examined Triticum species (1 = Present; 0 = Absent) |
| Table 4: | Percent coefficient of similarity between the different studied Triticum species on the basis of the number of protein bands |
and T. paleocolchicum and 10 for low molecular weight subunits in T. spelta. On the other hand, the lowest number of bands (12) were recorded in T. durum; one band of high molecular weight glutenin in T. dicoccoides; two bands of high molecular weight gliadin in T. monococcum and 7 bands of low molecular weight subunits in T. durum.
There were some bands in common between the eight studied species (6, 13, 16, 19, 20 and 21), while there were also some bands which characterize each species. The band (10) is characteristic for T. paleocolchicum; the band (12) for T. dicoccoides; the band (4) for both T. paleocolchicum and T. polonicum and the band (22) for both T. aestivum and T. spelta. These results are in harmony with the data obtained by Galili and Feldman (1983 and 1985), Pogna et al. (1990) and Hassan and Eid (1998) who observed that 17 to 20 total protein bands were resolved in the common wheat cultivar and durum wheat with three main groups; 1-4 bands of high molecular weight glutenin; 5-9 bands of high molecular weight gliadin and 10-21 bands of low molecular weight subunits.
The neighbor-joining tree illustrated the relationships between the eight studied Triticum species based on the UPGMA clustering is presented in Fig. 2 and the values of similarity percentage in Table 4. This tree consists of two major groups, one comprising the two species of subgenus Boeoticum moderately supported with 85% similarity percentage and the other the six species of subgenus Triticum, which are separated into three subgroups. The first subgroup comprises the two hexaploids; T. aestivum and T. spelta of section Triticum which is strongly supported with 94% similarity percentage. The two remaining subgroups have the four tetraploids species of section Dicoccoidea with the same taxonomic distance; The two species of Emmer wheats group; T. dicoccoides and T. paleocolchicum form moderately supported subgroup with 85% similarity percentage. The other two species of Naked tetraploids group; T. durum and T. polonicum form the other moderately supported subgroup with 85% similarity percentage. These results confirm the classification of Dorofeev et al. (1979) and in agreement with the morphological, anatomical and pollen grains data obtained by Gowed (2003).
Total protein percentage: Data in Table 5 show that there is notable variation in total protein values determined in the grains of the eight examined of Triticum species. The maximum value of total protein percentage (14.8%) was recorded in T. dicoccoides, while, their minimal values (9.3%) were observed in T. aestivum. Ciaffi et al. (1992) found also the protein content of T. dicoccoides ranged from 16-27%. The variation in the amount of grain proteins was the main factor responsible for the differences in bread (Ciaffi et al., 1993). These results are in agreement with those obtained by Borghi et al. (1996) who observed that the protein content of twenty-five T. monococcum from 13.2 to 22.8% higher than those found for bread wheat which ranged from 10.8 to 13.3%.
Amino acids: Table 5 and 6 show obvious differences in the amounts of different essential and non-essential amino acids existing in the grains of the eight studied Triticum species.
| Fig. 2: | A neighbor-joining tree illustrating the relationships among the studied species of Triticum |
| Table 5: | Mean values of total protein and essential amino acids content existing in mature grains of the studied eight Triticum species (expressed as g per 100 g protein). (Practice values = essential amino acid chemical score) |
| Table 6: | Mean values of non essential amino acids existing in mature grains of the studied eight Triticum species (expressed as g per 100g protein) |
Essential Amino Acids (EAA): The nutritional quality of proteins as measured by their essential amino acids chemical scores range from 14.29% for tyrosine in T. spelta grain protein to 239.29% for isoleucine in T. durum grain protein (Table 5). Most essential amino acid of grain protein in T. aestivum and T. dicoccoides record higher values than the FAO/WHO (1990) recommended pattern (threonine, 3.67 and 3.62; valine, 5.14 and 4.61; isoleucine, 2.94 and 3.29 and leucine 7.34 and 7.90 g/100 g protein, respectively). Methionine (sulphar containing amino acid) is presenting in a very low levels with scores of 0.45, 0.47 and 0.50 g/100 g protein in the grain protein of T. timopheevii, T. monococcum and T. paleocolchicum, respectively, while lacking in the other studied species. However, valine (aliphatic amino acid) is present at high levels in all the studied species ranging from 4.29 in T. durum to 5.14 g/100 g protein in T. aestivum compared with the FAO/WHO (1990) recommended pattern (3.50 g/100 g protein). As well as, the other aliphatic amino acid (leucine) is found in high levels in T. polonicum, T. monococcum, T. aestivum, T. spelta and T. dicoccoides (6.70, 7.16, 7.34, 6.71 and 7.90 g/100 g protein, respectively) comparing with the FAO/WHO (1990) reference pattern (6.60 g/100 g protein). In addition, leucine was accumulated in higher levels compared with the other essential amino acids ranging from 6.31 to 7.90 g/100 g protein in T. timopheevii and T. dicoccoides. In contrast, the levels of three essential amino acids; lysine (basic amino acid), tyrosine (aromatic amino acid) and methionine (sulphar containing amino acid) show lower values than those of FAO/WHO (1990) recommended pattern in all studies species and a great differences were found between tyrosine and methionine values comparing with the other essential amino acids values. These results are in harmony with the data obtained by Nevo and Beiles (1992) who showed that T. dicoccoides contains high values of lysine and isoleucine compared to the other studied wild wheat species. All the studied Triticum species had high content from the essential amino acids as recorded by Cervantes et al. (2002) in commercial wheat, or higher than that found by Shaheen and Hammed (2003) in Eragrostis aegyptiaca (Poaceae) and Fimbristylis bisumbellata (Cyperaceae).
Non-essential Amino Acids (EAA): The content of glutamic (acidic amino acid) is very high in all studied Triticum species ranging from 24.86 g/100 g protein in T. dicoccoides to 36.13 g/100 g protein in T. spelta (Table 6). As well as, proline (heterocyclic amino acid) show the same result, being present with a high values in all the examined species varying from 10.72 in T. durum to 13.17 g/100 g protein in T. dicoccoides. However, cystine (sulphar containing amino acid) is the limiting amino acid in all protein of the different species with the lowest amounts in T. polonicum (1.79), T. timopheevii (1.80) and T. paleocolchicum (1.98 g/100 g protein). T. aestivum contains the highest content of aspartic acids, glycine, alanine and histidin (6.61, 5.14, 4.40 and 2.75 g/100 g protein, respectively), however T. dicoccoides records high values of proline and serine (13.17 and 6.26 g/100 g protein, respectively). As well as, T. monococcum protein contains the highest value of phenylalanine (aromatic amino acids, 7,16); T. durum with greatest content of cystine (3.22) and T. polonicum with highest content of arginine (basic amino acid, 4.46 g/100 g protein). In contrast, T. spelta has the smallest amounts of most non-essential amino acids content; aspartic acid (5.16), arginine (3.61), glycine (4.13), alanine (3.61) and histidin (2.07 g/100 g protein). However, T. durum has the lowest contents of three non-essential amino acids (proline, 10.72; serine, 4.83 and phenylalanine, 4.83 g/100 g protein). All the studied species had the greatest proportion of glutamic acid in agreement with the finding of Molino et al. (1988 and 1989) in three Triticum aestivum varieties. These results agree with Nevo and Beiles (1992) who reported that T. aestivum recorded low value of proline. Most non-essential amino acids values in this study seems to be higher than the previous findings in Eragrostis aegyptiaca and Fimbristylis bisumbellata (Shaheen and Hammed, 2003).
It is concluded that the six studied Triticum species imported from outside Egypt had pronounced total protein and amino acids content and consistent the most suitable condition for growing under Ismailia conditions. The same conclusion obtained by Gowed (2003) and Hassan et al. (2005) on the basis of the morphological data.