Research Paper. Paste and Gel Properties and In Vitro Digestibility of Tef [Eragrostis tef (Zucc.) Trotter] Starch. 1 Introduction

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1 20 DOI /star Starch/Stärke 56 (2004) Geremew Bultosa a, John R. N. Taylor b Paste and Gel Properties and In Vitro Digestibility of Tef [Eragrostis tef (Zucc.) Trotter] Starch a Department of Chemistry, Alemaya University, Dire Dawa, Ethiopia b Department of Food Science, University of Pretoria, Pretoria, South Africa Properties of tef starch from five varieties were compared with commercial maize starch. In most tef varieties the paste clarity (measured as % T) was similar to that of maize starch, but the paste was visually less white in colour. Tef starch gel texture was short and in most varieties was slightly firmer than that of maize starch. Tef starch adhesiveness was less than maize starch. Retrogradation extent of tef starch evaluated, as % gel syneresis under storage at 4 C and -18 C at 3, 7, 10 and 21 storage test days, was lower than that of maize starch. Storage with three freeze-thaw cycles (-18 C 24 h; 23 C 6 h) gave a similar trend. In tef starch initial digestion by α-amylase and hydrolysis by mild HCl treatment was slightly higher than in maize starch, probably in part because of the smaller granule size and higher amorphous portion of tef starch. Alpha-amylase degradation of tef starch granules was by surface erosion, probably due to the absence of surface pores in the granules. Keywords: Eragrostis tef; Paste clarity; Gel texture; Retrogradation; α-amylase digestion; Acid hydrolysis Research Paper 1 Introduction Grain tef has compound type starch granules comprising many simple polygonal (2 6 µm in diam.) shaped individual granules [1, 2]. Studies of starch composition and physico-chemical properties of five grain tef varieties indicate that tef starch has lower paste consistency (peak, breakdown and setback) compared to maize starch [2]. The research further showed that tef starch granule composition (amylose/amylopectin ratio, protein, ash and crude fat) is similar to normal native cereal starches [2]. Tef starch gelatinisation temperature measured by Kofler hot stage microscopy (68 80 C) [2] and differential scanning calorimetry (64 82 C) [3] is high, like with other tropical cereal starches. Lipid and phosphorus contents are similar in level to normal native rice starch [3]. The swelling factor and crystallinity level of tef starch is lower than that of maize starch [3], but the extent of amylose leaching is higher, probably in part due to lower crystallinity level and lower lipid level in tef starch than in maize starch [3]. In Ethiopia, grain tef is traditionally used in various baked foods, particularly injera (fermented pancake-like spongy flat bread) [4]. It is also used in traditional opaque beer, local spirits, porridge and atmit (thin porridge) [5]. In all these food and beverage products, tef starch granules are structurally transformed. During the baking of injera, starch is completely gelatinised to form a steam-leavened, spongy textured matrix, in which fragments of bran, embryo, micro-organisms and organelles are embedded [4]. Gelatinised starches have a tendency to retrograde, which can affect the texture and shelf-life acceptability of foods [6]. The nutritional value of foods can be also negatively affected because of starch staling and the possible formation of resistant starch type III on starch retrogradation [6]. The smaller setback and low cold paste viscosity of tef starch compared to maize starch is an indicator of slow retrogradation tendency [2], which might have a positive role in respect of storage stability of food products made with tef starch. Additionally, depending on the degree of enzyme [6] or acid [6, 7] treatment, starch can be depolymerised to different types of oligosaccharides (maltodextrins and glucose). The mode of starch granule attack by enzymes can vary with starch and enzyme type [6]. For tef starch, this type of information is limited to the fact that during fermentation for injera preparation, a few starch granules are eroded by amylase [4, 5]. In this paper, some functional properties of tef starch are described: paste clarity, gel texture, retrogradation, and hydrolysis with porcine pancreatic α-amylase and hydrochloric acid. 2 Materials and Methods 2.1 Grain tef samples and starch granule extraction Correspondence: John R. N. Taylor, Department of Food Science, University of Pretoria, Pretoria 0002, South Africa. Fax: ; jtaylor@postino.up.ac.za. Starch granules from four Ethiopian grain tef varieties (DZ , DZ-01-99, DZ and DZ-Cr-37) and one South African grain tef variety (South African Brown)

2 Starch/Stärke 56 (2004) Paste and Gel Properties and In Vitro Digestibility of Tef 21 were extracted by dry/wet milling, sieving and centrifugation as described [2]. The varieties were chosen because they are intensively cultivated. Maize starch (Merck Uni- LAB, Code: , Halfway House, Gauteng, South Africa) was analysed for comparison. 2.2 Starch paste clarity Starch paste clarity was studied by suspending ca. 50 mg ± 0.1 mg starch (dry basis, db) in 5 ml distilled water and heating the suspension to 95 C in a boiling water bath by shaking thoroughly every 5 min for 30 min [8]. While cooling to room temperature (ca. 5 min), 1 ml aliquots of the solutions were diluted to 10 ml (0.1%) with distilled water. Percentage transmittance (% T) of the 0.1 % starch solution was determined against a water blank at 650 nm in glass cuvettes using a spectrophotometer (Lambda EZ 150, Perkin Elmer, Norwalk, USA). With the Lambda EZ 150 spectrophotometer, the 1% starch solution gave % T < 2, because of this a 0.1% starch solution was used. 2.3 Starch gel texture Starch (10%, db) was cooked in a Rapid Visco Analyser (RVA model 3D, Newport Scientific, Warriewood, Australia) using the RVA program described in [2]. The starch paste from the RVA canister was immediately hot filled into shallow circular plastic dishes (ca. 16 mm height 37 mm diam.) and stored for 24 h at ca. 23 C [9]. Before hot filling the starch paste, the height of each dish had been increased by approximately 5 mm (i.e. from 11 mm to ca. 16 mm height) with adhesive tape around the rim. After 24 h of storage, the adhesive tape was removed and a smooth surface was created by cutting the excess gel above the rim with a thread. Gel texture was analysed using a TA-XT2 texture analyser (SMS Stable Micro systems, Godalming, England). The starch gel ca. 11 mm in height was compressed to a distance of 5 mm into the gel with a test speed of 0.5 mm/s using a cylindrical plunger P 20 (20 mm diam.) of pre test speed 2.0 mm/s and post test speed of 10.0 mm/s. The peak height force (g-force) at 5 mm compression was termed firmness, and the negative force area of the curve during retraction of the probe was termed adhesiveness (gforce s). 2.4 Starch gel syneresis and freeze-thaw stability A starch slurry (8%, w/w, db) was cooked at 90 C in a water bath for 35 min with constant agitation using a glass rod. The paste was cooled at room temperature (23 C) over 3 h. Gel syneresis and freeze-thaw stability were evaluated according to [10] using ca g starch paste. Syneresis (% gel weight) was determined gravimetrically by centrifuging the sample at 3030 g for 10 min and then decanting off the water separated. Samples were stored at 4 C and 18 C and at the intervals of 3, 7, 10 and 21 days of storage syneresis was measured. For freeze-thaw stability evaluation, a sample was treated three times with a freeze-thaw cycle (freeze storage at 18 C for 24 h, thawed at ca. 23 C for 6 h and refrozen at 18 C) and at the end of each thaw cycle syneresis was measured. 2.5 In vitro digestibility with porcine pancreatic α-amylase (PPA) Starch (40 mg ± 0.1 mg, db) was suspended in 20 ml distilled water. A 5 ml (10 mg) starch suspension was treated with 2 µl PPA (A 6255) (Sigma, St. Louis, USA) in phosphate buffer (ph 6.9) with gentle mixing and incubated in a constant temperature (37 C) water bath for 5 h [11]. The concentration of α- amylase was 33.3 mg/ml in 2.9 M NaCl containing 3 mm CaCl 2. The PPA had an activity of 1020 units/mg. One unit is defined as the activity of α-amylase which liberates 1 mg maltose in 3 min at ph 6.9 and 20 C [11]. At timed intervals of: 0.5, 1, 2, 3, 4 and 5 h, 1 ml was removed into 5 ml 99.8% (v/v) ethanol and the remaining solution was gently mixed. To the sample removed, 1 ml 0.1 M hydrochloric acid was added immediately to stop enzyme activity. The sample solution was centrifuged at 2500 g for 5 min and 1 ml of the supernatant was analysed for soluble carbohydrate by the phenol-sulphuric acid method [12]. A blank was treated the same way and its absorption was subtracted from the sample absorption. The amount of solubilised carbohydrate was determined using a D-maltose standard (5 120 µg/ml) calibration curve [12]. For scanning electron microscopy (SEM), enzyme attacked starch after 2 h hydrolytic reaction was washed with ethanol (96%) (2 3 ml) and dried in a forced air draught oven at 50 C for about 8 h. The samples were prepared for SEM as described [2]. Samples were viewed using a JEOL JSM 840 SEM (JEOL, Tokyo, Japan) operated at an accelerating voltage of 5 kv. 2.6 Acid hydrolysis Starch samples (ca. 0.4 g ( 0.1 mg (db)) were suspended in 16 ml 2.2 M aqueous HCl (1 g/40 ml) and incubated at 35 C for up to 24 days [7]. The samples were re-suspended daily by shaking and at the intervals of 1, 2, 3, 4, 8, 12, 16, 20 and 24 days, 1 ml was withdrawn, neutralised (ca. 1 ml, 2.2 M NaOH) and centrifuged at 2500 g for 5 min. From the supernatant 125 µl was analysed for soluble carbohydrate by the phenol-sulphuric acid method and the amount of solubilised carbo-

3 22 Bultosa et al. Starch/Stärke 56 (2004) hydrate was determined with a D-maltose calibration curve [12]. For SEM, samples at the intervals of 1, 4, 8, 16 and 24 days of reaction were filtered and sequentially washed with ethanol (2 10 ml) (70 %, v/v, followed by 96 %, v/v) and centrifuged at 2000 g for 5 min. The washed samples were dried at 50 C in a vacuum oven for more than 12 h. Samples for SEM were prepared and viewed as described in section Statistical analysis At least three replicate experiments were analysed by one-way analysis of variance (ANOVA) and compared at p < 0.05 using Fisher s least significant difference (LSD) test, using Statistica for Windows (Statsoft, Tulsa, USA, 1995). 3 Results and Discussion 3.1 Paste clarity The paste clarity (% T) of most of the tef starches (DZ , DZ and DZ ) was similar to that of the maize starch (Tab. 1). This is probably due to the difference in amylose and lipid contents of the starches not being substantial (Tab. 1), because the paste clarity is influenced, in part, by amylose/amylopectin ratio and lipid contents of the starches [13]. The starch pastes from the red grain tef varieties (DZ-01-99, DZ and South African Brown) appeared less white than the pastes from white tef varieties (DZ and DZ-Cr-37) and maize starch (visual observation). The presence of polyphenols reported in the red tef varieties [14] might have influenced the starch colour, as occurs with sorghum starch [15]. In addition, sulphite, a powerful inhibitor of polyphenol oxidase [16], was not used for tef starch extraction [2], whereas it is used in commercial maize starch extraction [17]. In the absence of sulphite, polyphenol oxidase might have catalysed phenolic compounds [16] to contribute colour to the starches of some of the tef varieties. The paste clarity of starch is reported to be influenced by the interplay of: the degree of swelling, granular remnants in the paste, junction zone formation of starch molecules in the paste, existence of amylose-lipid complexes, cross-linking, trace non-starch components (protein, lipids, phosphorus and polyphenols) and ph [8]. Previously, the order of starch paste clarity was reported to be: maize < waxy maize < wheat < cassava < potato [8]. On the basis of this and the work reported here the paste clarity of tef starch can be categorised as the same or slightly less than that of maize starch. 3.2 Gel texture At 10% starch, the pastes of all tef starches were short textured like maize starch, and on cooling the pastes became gels. The starch gel of DZ was softer than that of maize starch, whereas DZ and South African Brown formed firmer gels than maize starch (p < 0.05) (Tab. 1). The highest gel firmness was observed for South African Brown and the lowest was for DZ The gel firmness of DZ and DZ- Cr-37 tef starches were similar to maize starch gels (p < 0.05). The gel of normal cereal starches of sufficient concentration (ca. 6 20%) is a composite of leached continuous amylose matrix with swollen granular remnants (deformable particles) acting as fillers in the continuous phase [18]. The gel firmness of normal cereal starch has been positively correlated with leached amylose [18] and starch granule associated proteins [19], negatively with starch lipids complexed with amylose [9], and positively with phosphorus contents [20] and free starch lipids, because free lipids will not restrain the leaching of amylose [21]. In normal rice and maize starches, the interaction of leached amylose with remnants of swollen granular structures was considered a major factor for the reinforcement of the starch gel matrix rather than re-associ- Tab. 1. Paste clarity and gel texture of starch from different tef varieties and maize a. Variety Transmittance Amylose Protein Ash Lipids Phosphorus Firmness Adhesiveness [%] [%] b [%] b [%] b [mg/g] c [mg/g] c [g force] [g force s] DZ b ± 0.8 a a ± b ± 1.8 DZ b ± c ± d ± 3.0 DZ c ± b ± c ± 2.0 DZ-Cr a ± b ± c ± 2.5 S. A. Brown 24.8 a ± d ± a ± 1.8 Tef (mean) 27.5 ± ± ± 8.4 Maize 29.3 bc ± b ± e ± 0.7 a Values within the same column with different letters are significantly different (p < 0.05). b Mean values from Bultosa et al. [2] and c Bultosa and Taylor [3].

4 Starch/Stärke 56 (2004) Paste and Gel Properties and In Vitro Digestibility of Tef 23 ation of leached amylose alone [18]. The higher amylose leaching [3] at 10% concentration and protein content [2] in tef starch than in maize starch presumably contribute positively to the greater gel firmness of some of the tef starches, whereas the higher phosphorus [3] content would negate this. Comparison of starch gel texture parameters determined by different workers is difficult due to differences in experimental conditions (sample size and shape, ratio of compressing probe size versus sample, extent of deformation, cross-head speed, number of bites and replicates per mean value) [22]. However, under uniform experimental condition, gel firmness (7 25% starch) of normal native maize starch is smaller than that of normal native wheat starch [9] but higher than normal native oats [23] and rice starches [18]. On the basis of this, the gel firmness of tef starches at 10% concentration can be placed somewhere between maize and wheat starches in gel firmness. The adhesiveness of tef starch gels (mean 16.9 g-force s) was in all cases significantly (p < 0.05) less than that of maize starch (31.8 g-force s) (Tab. 1). Adhesiveness is the ability of starch gel to stick to other objects [22]. When the attractive force within the starch gel matrix is strong, the stickiness of the gel surface toward the probe is reduced [22]. The data suggest that the intermolecular forces in tef starches gel are stronger than in maize. 3.3 Retrogradation The amount of water separated from fresh tef starches pastes and the fresh maize starch paste was very similar in all three tests (p < 0.05) (Tab. 2). In research using essentially the same method [10] the amount of water separated from fresh normal native cereal starch pastes was ordered: maize > barley > oat > rice. After three days storage at 4 C and 18 C, syneresis was substantially lower in tef starches than maize starch in all cases, due to the slow retrogradation tendency of tef starches. A similar trend was observed at 7, 10 and 21 storage days, and with the freeze-thaw cycle treatments. Starch retrogradation for various botanical sources was ordered as folows: wheat, maize > rice, cassava, potato >> waxy maize [24]. The data in our study suggest that the retrogradation tendency of tef starches is less than that of maize and wheat starches. In tef and maize starches stored at 4 C and 18 C, syneresis increase was higher between 3 to 10 days than between 10 to 21 days, probably in part because of available mobile phase in the first period. For all tef starches and maize starch, syneresis was greater at 4 C than 18 C. Three freeze thaw cycles, which exposed starch gels to 18 C for 3 days in total, resulted in as much retrogradation as storage at 18 C for 10 days (Tab. 2). The data suggest that retrogradation in both tef starches and maize starch was enhanced more by freeze-thaw treatments than freeze storage. This is because on freezing, ice crystals are formed leaving a non-frozen starch-rich phase and on thawing water can be easily expressed leading to more syneresis than freeze storage alone [25]. Starch gel retrogradation is reported to be affected by the interplay of storage temperature and water proportion in the gel [26], starch granule composition [26], amount of amylose solubilised [21], degree of polymerisation (dp) of amylopectin A chains and dp of amylose [26, 27]. Water influences the glass transition temperature (T g ) of the starch gels [26]. For a starch gel of 45 50% water, the T g is ca. 5 C and retrogradation is high at ca C and very low at freezer storage temperatures < -5 C [26]. In this work retrogradation was higher at 4 C storage than at 18 C, in agreement with theory. Nucleation for recrystallisation is known to in- Tab. 2. Syneresis of starch [%] from different tef varieties and maize after storage at 4 C and 18 C, and after freeze thaw treatments. Storage at 4 C Fresh paste 3 days 7 days 10 days 21 Days Variety Tef (mean) 2.5 ± ± ± ± ± 0.7 Maize 2.9 ± ± ± ± ± 1.9 Storage at 18 C Tef (mean) 2.5 ± ± ± ± ± 0.4 Maize 3.0 ± ± ± ± ± 1.7 Freeze thaw cycle* treatment Fresh paste 1st cycle 2nd cycle 3rd cycle Variety Tef (mean) 2.5 ± ± ± ± 0.5 Maize 3.0 ± ± ± ± 0.6 Where: * is one freeze thaw cycle = freeze storage at 18 C for 24 h., thaw to ca. 23 C for 6 h and refreeze at 18 C.

5 24 Bultosa et al. Starch/Stärke 56 (2004) crease exponentially with decreasing temperature down to T g, whereas crystal growth is favoured at a temperature below the melting temperature (T m ) (ca. 50 C) of amylopectin [13, 26, 27]. In waxy maize starches, retrogradation was positively correlated to dp amylopectin unit chains and negatively to dp 6-9 unit chains [25]. The slower retrogradation tendency of tef starches than maize starch suggests that it may be due a higher proportion of 6-9 dp amylopectin A chains in tef starch than in maize starch, whereas the proportion of dp amylopectin A chains may be less than in maize starch. Monoacyl lipids are also known to retard starch retrogradation by directly forming complexes with amylose, slightly interacting with amylopectin outer chains, interfering with the possible slight co-recrystallisation of amylose and amylopectin, and changing water distribution in the gel [26]. In the lipid fractions, monoacyl lipids including free fatty acids are known to form complexes with amylose [6]. Tef starches had total lipids slightly lower than maize starch [3] but the extent of amylose-lipid complex formation may probably not follow the same trend. In fact, in the X-ray diffraction of native tef and maize starch granules, the intensity (I) for an indicator of V-type diffraction of the amylose-lipid complex (d = 4.4 Å) is stronger in tef starches (I = 36%) than in maize starch (I = 24%) [3], which suggests the extent of amylose-lipid complex formation is slightly larger in tef starches than maize starch. Thus, the slower retrogradation of tef starch, in part could probably be related to the extent of amylose-lipid complex formation. Fig. 1. SEM of porcine pancreatic α-amylase treated starches from different tef varieties and maize. A and B are untreated and C F are treated. A = DZ , B = Maize, C = DZ , D = DZ , E = South African Brown and F = Maize, sa = smooth surface and sharp edged, nsp = no surface pores, sp = surface pores, se = surface erosion, sh = surface eroded hole, la = less attacked and pt = pit.

6 Starch/Stärke 56 (2004) Paste and Gel Properties and In Vitro Digestibility of Tef 25 Tab. 3. In vitro porcine pancreatic α amylase digestion and mild hydrochloric acid hydrolysis of tef and maize starches (db) at C. Variety α Amylase digestion [%] over time [h] Time [h] Tef (mean) 4.7 ± ± ± ± ± ± 2.2 Maize 1.4 ± ± ± ± ± ± 0.7 Variety Acid hydrolysis [%] over time [days] Day Tef (mean) 2.0 ± ± ± ± ± ± ± ± ± 1.1 Maize 1.3 ± ± ± ± ± ± ± ± ± Starch in vitro digestibility with PPA For the tef starches (mean) at 1, 3 and 5 h hydrolysis, the digestibilities were: 10.1, 22.6 and 32.6%, respectively, and for maize starch 3.8, 20.8 and 31.0%, respectively (Tab. 3). Digestibility up to 2 h was relatively higher in the tef starches than in maize starch and thereafter the difference was minimal. This is probably, in part, because of a higher portion of amorphous regions in tef starches (63%) than in maize starch (60%) [3], because the amorphous portion is known to be readily degraded during the early digestion period [28, 29]. The native tef starch granule is smooth with no surface pores (Fig. 1 A), whereas in maize starch surface pores are evident (Fig. 1 B). The mode of PPA attack on all tef starch varieties appeared to be the same. Because of this SEM (Fig. 1) is only given for DZ (C), DZ (D) and South African Brown (E) starches. The mode of attack was surface erosion that enlarged at each shell level into the granule (Fig. 1 C, D and E). In some granules, surface eroded large holes were observed (Fig. 1, C, E), whereas some granules appeared less attacked (Fig. 1, C, D, E). This is because enzymic starch degradation within the same starch preparation is known not to be the same and will differ granule by granule, with an already attacked granule being hydrolysed more rapidly [28]. Wheat, barley and rye starch granules (A type) as well as cassava and sweet potato starch granules have susceptibility zones and they are attacked by endocorrosion (channels from selected points on the surface towards the centre of the granule) creating pits that sink into the interior [28]. In normal maize and rice starches the mode of attack is also endocorrosion with pitted channels enlarged at each shell level through the granules [28]. In tef hydrolysis is also by endocorrosion in nature (Fig. 1 C, D and E). However, in potato and some B type starches the granules are slightly eroded by exocorrosion on the entire granule surface or portion of it [28]. 3.5 Starch acid hydrolysis lintnerisation At all incubation times tef starch was slightly more etched by mild hydrochloric acid (HCl) treatment than maize starch (Tab. 3). In both tef starch and maize starch, the increase in hydrolysis was slightly faster before 8 days than thereafter. This is because in part during the earlier stages, acid rapidly attacks the amorphous regions, thereafter the crystalline regions are attacked slowly [30]. Acid treated tef starch varieties observed by SEM appeared to be the same. Because of this only SEM of DZ at intervals of 1, 4, 8, 16 and 24 days are shown (Fig. 2 A). At 1 day, the surface of tef starch granule appeared without spots, but with slight etching (Fig. 2, A1), whereas small spots were seen on the surface of maize starch granule (Fig.2, B1). At 4 days, the tef starch granules were slightly surface etched and denuded (Fig.2, A4), whereas in maize starch granules, the surface appeared without spots but on one side, in some granules, a crater started to appear (Fig. 2, B4). At 8 and 16 days, tef and maize starch granules underwent shrinkage due to loss of the granule solids (Fig. 2, A8, B8, A16 and B 16). After 24 days most features of the tef starch granules were either deformed or lost and the granules appeared to fuse together (Fig. 2, A24), whereas in the maize starch granules more shrinkage and craters appeared (Fig. 2, B24). At 24 days of hydrolysis the yield of lintnerised tef starch was 5 % (Tab. 3) below than that of lintnerised maize starch. Comparing the data of [7] and [31], the rate of hydrolysis with mild HCl can be ordered: waxy maize > rice > maize oats > wheat > finger millet > cassava > potato. On the basis of this, the hydrolysis of tef starch is similar to to that of normal cereal starches (e.g. rice) that are hydrolysed slightly faster than normal maize starch.

7 26 Bultosa et al. Starch/Stärke 56 (2004) Fig. 2. SEM of mild HCl treated (lintnerised) starch granules from tef variety DZ and maize at the intervals of 1, 4, 8, 16 and 24 days. A = Tef, B = Maize, cl = clean, sp = spots, set = surface etching, fr = fragmented, df = deformed, sb = solubilised and cr = crater.

8 Starch/Stärke 56 (2004) Paste and Gel Properties and In Vitro Digestibility of Tef 27 4 Conclusions The paste clarity of tef starch is similar to maize starch. The gel texture of tef starch is short and the gel is generally firmer than maize starch. The retrogradation tendency is lower than that of maize starch, which is beneficial for applications where starch staling is preferred to be reduced. Alpha-amylase degradation of tef starch appears to be by surface erosion, endocorrosion in nature, enhanced by channels that enlarge through granules at each shell level. Mild HCl treatment of granules denudes the granule surface and removes starch throughout the granules which deform into collapsed granules. Acknowledgements This research was supported by the Ethiopian Government and partly by the co-funding of International Foundation for Science, Stockholm, Sweden, and the Organisation for the Prohibition of Chemical Weapons, The Hague, The Netherlands, through a grant to Mr. G. Bultosa (No. E/3173 1). Tef improvement program of the DZARC (Ethiopia) and the ARC of South Africa are acknowledged for providing the Ethiopian tef varieties and South African Brown tef variety, respectively. Mr. Alan N. Hall is acknowledged for his technical assistance on SEM. Mr. Lemma Wogi is indebted for his technical assistance to the first author on some of the works conducted at Alemaya University. References [1] M. Umeta, M. L. Parker: Microscopic studies of the major macro components of seeds, dough and injera from Tef (Eragrostis tef). SINET: Ethiop. J. Sci. 1996, 19, [2] G. Bultosa, A. N. Hall, J. R. N. Taylor: Physico chemical characterisation of grain Tef [Eragrostis tef (Zucc.) Trotter] starch. Starch/Stärke 2002, 54, [3] G. Bultosa, J. R. N. Taylor: Chemical and physical characterisation of grain Tef [Eragrostis tef (Zucc.) Trotter] starch granule composition. Starch/Stärke 2003, 55, [4] M. L. Parker, M. Umeta, R. M. 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