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The contribution of flour components to the structure of injera, an Ethiopian fermented bread made from tef (Eragrostis tef)
Journal of Cereal Science 10 (1989) 93-104
The Contribution of Flour Components to the Structure of Injera, an Ethiopian Fermented Bread Made from Tef (Eragrostis tel) MARY L. PARKER*, MELAKU UMETAt and RICHARD M. FAULKS AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA, Norfolk, u.K. Received 23 January 1989
Injera, a pancake-like fermented bread prepared from white or red tef (Eragrostis tel) flour, is the traditional staple food of Ethiopia. The fate of the major components of the bran and endosperm during the two-stage fermentation and baking has been examined by light and electron microscopy. Angular starch granules released from compound grains during milling showed a range of erosion effects typical of enzymic degradation during fermentation. The appearances of bran and embryo fragments, cell walls and protein bodies were unaffected by fermentation or baking. Microorganisms, the natural contaminants of tef grains, produced strands of fibrillar material during fermentation that bound the flour particles together. Apart from the presence of polyphenolic material in the testa cells of red tef, no structural differences were observed between red and white grain during the preparation of injera. The portion of dough that was thinned, boiled and returned to the mixture for the second fermentation period contained swollen gelatinised starch. During cooking, the starch within the injera was totally gelatinised to form a steam-leavened, spongy starch matrix, in which fragments of bran and embryo, micro-organisms and organelles were embedded. The protein bodies played no role in the formation of the matrix-gas bubble interface.
Introduction Tef [Eragrostis tef(Zucc.) Trotter] is Ethiopia's most widely cultivated cereal food crop. The red or white grain is used for making the several types of ~at breads that form the basic traditional diet of Ethiopia. Tef is also used, to a lesser extent, in porridge and native beer. Tefis not suitable for making leavened bread because the flour lacks gluten, but when available, wheat and other cereal flours may be mixed with tef flour for varietyl. The most popular type of flat bread is injera (Fig. 1), a flexible, spongy, pancake-like product perforated with 'eyes'2. Before it is cooked, the soft dough is fermented twice over a period of 2-3 days, acquiring its characteristic sourness and flavour. To cook injera, the dough is thinned to a thick batter and poured onto a lightly-oiled pan, which is then covered with a tightly-fitting lid to retain the steam. Traditionally, sufficient injera are cooked to last until the next batch is fermented, so the process is continuous with the remains of the previous batch being used as a 'starter' (Fig. 2). This type of
* To whom correspondence should be addressed. t Current address: Ethiopian Nutrition Institute, P.O. Box 5654, Addis Ababa, Ethiopia. 0733-5210/89/050093 + 12 $03.00/0
© 1989 Academic Press Limited
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FIGURE 1. Freshly prepared Ethiopian injera, showing 'eyes' perforating the surface
fermentation and steam-leavening is similar to that used in Sudan for the preparation of Kisra bread from sorghum or millet flours. Several investigations of the microbial flora involved in the fermentation process have been undertaken l ,4,5. It was thought that the yeast Candida guillermondii (Cast.) Langeron and Guerra was the principal fermenting agentl, with the less numerous bacteria contributing flavour and variation, and occasionally spoiling the batter with offflavours. However, recent investigations have shown that fermentation is initiated by gas-producing members of the Enterobacteriaceae 5 present in the flour and starter. As the pH falls to 5,8, these bacteria are succeeded by various other species, including Lactobacillus spp., that contribute the characteristic lactic-acid sour taste and odour to the injera. At pH 4 the yeast population predominates 5 . Very little is known of the effects of the microflora on the nutritional components of fermenting tef dough. Soluble components such as amino acids, glucose, maltose and minerals4, and vitamins l are found in the discarded acidic supernatant from the primary fermentation, and levels of reducing sugars are known to vary widely during fermentation'l.
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TEF FLOUR + WATER + 'IRSHO' STARTER (from 0 previous batch)
Primary fermentation 30-72h
Thin paste fermented in a previously-used container
Secondary fermentation 30 min-2h
FERMENTED PASTE thinned and cooked to form 'INJERA
FIGURE 2. Traditional method of preparing tef injera.
In order to investigate changes in protein, carbohydrate, vitamin, mineral and fatty acid levels during fermentation and baking, injera were prepared under laboratory conditions, taking care to exclude micro-organisms not present originally in the flour, and samples of the fermenting dough and the cooked injera were analysed 6 • 7, and examined by electron microscopy. This present paper describes the ultrastructural aspects of the interaction between the microflora and the major cell components of the flour, and examines the contribution of protein, starch and bran to the structure of cooked injera. Experimental Preparation of injera Two samples of tef grain, red and white, were obtained from the Agricultural Marketing Corporation, Addis Ababa, Ethiopia. Grain was ground in an ultracentrifugal mill to a particle size of less than 0·5 mm. Flour was mixed 2: 3 (w jw) with sterile distilled water, and blended at high speed for 2 min in a domestic mixer. The resultant soft dough was allowed to ferment at 22°C in a covered sterilised beaker. After 72 h, at the end of the first stage of fermentation, the liquid
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. irsho' which covered the dough was discarded. About 10 % of the dough was mixed I: 3 (v Iv) with boiling water, and heated for a further 10--15 min with continuous stirring. It was then mixed back, without cooling, into the fermenting dough, and sufficient sterile water was added to make a thin batter. The batter was left 2 h for a period of secondary fermentation, during which it rose and subsided. More sterile water was added to form a pouring mixture. At both stages of fermentation, sterile water and glassware were used to minimise the contamination of the dough, so that any micro-organisms present were those naturally associated with the tef grain. To cook injera, a frying pan with a non-stick surface was wiped with vegetable oil and heated until very hot on an electric hot plate. A thin stream of batter was poured into the pan in a circular motion, starting from the outside. The pan was covered tightly with aluminium foil to retain the steam, and the heat turned down. After 4-5 min the injera, which resembled a thin flexible pancake with the upper surface covered with perforations, was removed from the pan.
Microscopy of the fermenting dough and injera Dough was sampled during the first fermentation at 0, 24, 48, 72 h (after decanting the supernatant), during the secondary fermentation, and after cooking, and then freeze dried. Samples for scanning electron microscopy were sputter-coated with a layer of gold, approximately 20 nm thick, and viewed and photographed at 30 kVin a Philips SEM 501B. Freeze-dried samples for transmission electron microscopy were fixed in 3 % glutaraldehyde in 0'05 M cacodylate buffer, pH 7,2, post-fixed in I % aqueous osmium tetroxide and dehydrated in a graded series of ethanol. Samples were transferred to acetone, and infiltrated and embedded in Spurr resin. Sections, I 11m thick, were stained with I % toluidine blue in 1 % borax, pH II, for light microscopy. Sections, approximately 70 nm thick, were collected on copper grids, stained sequentially with uranyl acetate and lead citrate, and viewed and photographed at 60 kV in an AEI 801 transmission electron microscope.
Results
Tel grains and flour
The grains of tef were extremely small, less than 1·5 mm in length. As in other smallseeded cereals, the embryo, which is rich in protein and lipid, occupied a relatively large proportion of the grain [Fig. 3(a)]. When milled in the traditional way, the whole of the grain is utilised, producing an extremely nutritious flour. However, the outer pericarp (pc), which is thin and membranous (Fig. 3(a)] and equivalent to the beeswing bran of wheat, may be separated readily from the grain by soaking before milling. The cells of the outer endosperm were horny or vitreous [Fig. 3(a)], and contained most of the protein reserves of the endosperm. On milling, pieces of these cells tended to remain attached to the bran layers so that the compound starch grains survived intact, surrounded by separate, but tightly-packed, spherical protein bodies [Fig. 3(b)]. Very little cytoplasmic matrix protein was visible between these protein bodies. In some seeds, the central endosperm cells were mealy [Fig. 3(a)], and tended to break open during milling, releasing into the flour the individual angular starch granules from the compound starch grains, and also small groups of protein bodies. The aleurone layer was one cell thick, and contained aleurone grains and lipid bodies. The seed coat layers, which were closely adherent to the aleurone layer, varied in thickness depending on the colour of the grain. The testa of red tef was thicker than that of white tef, due to the electron-opaque material deposited in the lumen of the testa cells during grain development.
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FIGURE 3. (a) Scanning electron micrograph of a halved tef grain showing the thin pcricarp (pc), the starchy endosperm (en) with outer horny region and mealy centre (arrowed), and relatively large embryo (em). (b) Fragment of flour of red tcf showing intact and broken endosperm cells attached to the bran. Starch granules are either within compound grains (cs) surrounded by protein bodies (p), or released singly (arrowed). The lipid-rich aleurone cell contents (a) are disrupted by milling. (c) Scanning electron micrograph of dough fermented for 48 h showing angular starch granules (s) with bacteria attached (arrows). (d) Dough fermented for 72 h showing numerous bacteria (b), protein bodies (p), starch granules (s), lipid (1) and cell wall material (cw). The bacteria and cell components me interconnected by a network of fine fibrils (arrows). Scale bar markers are in j..lffi. CER 10
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FIGURE 4. Dough after 72 h fermentation. (a) Protein bodies (p) within particles of flour show no evidence of digestion, but have concentric rings of electron-dense material. Some starch granules are internally eroded (arrows). (b) Severely-eroded starch granules (s) with bacteria (b) attached. (c) Starch granule with concentricallylayered substructure. (d) Starch granule with surface pits (arrowheads) and internal erosion. The erosion of the concentric layers (arrows) has produced a saw-tooth effect. Scale bar markers are in J-lm.
Primary fermentation - 72 h duration During the primary fermentation period, the soft dough prepared from tef flour became progressively more acidic and rose considerably, due to gas evolution from microorganisms that were associated naturally with the grain. Scanning electron micrographs
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of dough fermented for 48 h then freeze-dried [Fig. 3(c)), showed angular starch granules interconnected by strands of material, and short chains of coccobacilli (arrowed). In sectioned dough [Fig. 3(d)), numerous bacteria were found attached to starch granules, protein bodies and cell wall material, and all these organelles were linked by fine strands of fibrillar material, which were particularly associated with the bacteria. There was considerable variation in the effect offermentation on the individual starch granules. In Fig. 4(a), some of the starch granules in the protein-rich sub-aleurone cells have eroded cores (arrowed) whilst others are apparently intact. Erosion or digestion in some starch granules was so far advanced that the starch granules were disintegrating [Fig. 4(b)). Other granules exhibited concentric rings and hollow cores [Fig. 4(c)), and as these grains were eroded, a sawtooth effect was produced [Fig. 4(d), arrows]. Surface erosion, initiated as small pits [Fig. 4(d) arrowheads] was also observed. In contrast, protein bodies appeared not to be eroded during fermentation, although the concentric lamellae within their matrix were more pronounced [Fig. 4(a)]. Second fermentation - 2 h duration
At the end of the primary fermentation period, the yellow liquid 'irsho' that had separated from the solids in the dough was discarded, and some of the dough was diluted, boiled and returned uncooled to the fermentation vessel. The addition of this 'absit' initiated a second period of fermentation. Within this fermentation mixture, therefore, was material that had undergone the primary fermentation only, and also material that had been fermented and boiled. This is illustrated in Fig. 5(a), where partly gelatinised starch granules that have become swollen and misshapen during boiling are attached to a large fragment of flour containing angular ungelatinised starch granules still within the cells. Considerable gas evolution took place in the batter during secondary fermentation, and bacteria were found in close association with both gelatinised and un-gelatinised starch [Fig. 5(b)]. Starch that had been boiled had a characteristic appearance in sectioned material. It is clear from Fig. 5(c) that the fragment of flour had been boiled, because it contained swollen, partly gelatinised starch granules in compound grains, whereas the starch granule adhering to the surface retained its original angular form. The appearance of the boiled protein bodies was similar to that in fermented tef. Partly gelatinised starch had a characteristic amorphous outer layer with a central core of loose fibrils [Fig. 5(d)]. Preparation and structure of injera
After secondary fermentation, the batter was thinned further with water, and poured onto a hot, oiled frying pan, which was then covered tightly to create a steamy atmosphere. Crumbs of cooked injera had an open spongy structure in which were embedded fragments of bran [Fig. 6[(a)] and embryo. Sections of the crumb viewed by light microscopy [Fig. 6(b)], showed a continuous network of completely gelatinised starch (sg) incorporating air cavities, bran layers with protein bodies from the outer endosperm, and micro-organisms. Staining with toluidine blue differentiated between bacteria and protein bodies, which are both approximately 1 !-Lm in diameter; bacteria
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FIGURE 5. Dough at start of secondary fermentation, with boiled' absit ' returned. (a) Scanning electron micrograph of a flour particle with starch-filled endosperm cells attached to the aleurone layer (a) and seed coat (sc). Adhering to the surface are bacteria (arrow) and partly gelatinised starch (sg) from the' absit'. (b) Bacteria (arrows) attacheq to angular starch (s). Partly gelatinised starch (sg) is distorted. (c) Sectioned flour particle from' absit' showing partly gelatinised starch (sg) and intact protein bodies (p). Ungelatinised starch (s) from the batter is also present. (d) Partly gelatinised starch with fibrillar centre, and attached bacterium (b). Scale bar markers are in ~lm.
STRUCTURE OF ETHIOPIAN FERMENTED INJERA
FIGURE 6. Cooked injera. (a) Scanning electron micrograph of part of a crumb showing a large bran fragment (b) embedded in a network of gelatinised starch (sg). (b) Light micrograph of sectioned injcra crumb showing bran and endosperm fragments embedded in the gelatinised starch network (sg). Protein bodies (p), bacteria (arrows), air cavities (ac) and fragments of embryos (em) are also present. (c) Gelatinised starch (sg) within a fragment of flour showing vestigial concentric layering. Protein bodies (p) and cell walls (cw) are recognisable in cooked injera. (d) Part of the gelatinised starch matrix at its interface with an air cavity (ac), showing trapped bacteria (arrows), protein bodies (p), lipid (I) and cell wall material (cw). Scale bar markers arc in ~m.
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stained a purplish-blue, and protein bodies appeared blue. Completely gelatinised starch stained pale pink, in contrast to ungelatinised starch, which remained colourless. Fragments of embryo and oil drops within the starch matrix of the injera had a yellowish tinge as a result of their reaction with osmium tetroxide during fixation. In the steamy conditions essential for cooking injera, most of the starch within the body of the injera gelatinised completely, the granules fusing into a continuous amorphous matrix (sg) in which bubbles of gas were trapped [Fig. 6(b)]. However, some of the starch granules deep within fragments of the outer endosperm were only partly gelatinised [Fig. 6(c)], possibly due to the insulating effect of the surrounding protein bodies and cell wall. Typically, each partly ge1atinised starch granule had a swollen irregular shape, and showed vestiges of its concentric sub-structure in the form of alternating layers of loosely- and tightly-packed fibrils. The effect of cooking on other components ofinjera is illustrated in Fig. 6(d). Protein bodies, although distorted, appeared similar to those in ungerminated tef, and played no role in the retention of gas bubbles. The structure of the cell wall material also appeared relatively unchanged. Bacteria and yeast (not illustrated) embedded in the starch matrix retained their characteristic appearance. The interface between the starch matrix and the gas bubbles was smooth, but may be interrupted by fragments of bran. Two types of lipid-rich inclusions were presented in injera; fragments of embryo or aleurone containing small oil droplets and larger oil drops, possibly originating from the oil used to prevent the injera sticking to the pan during cooking. Discussion
Both red and white tef are used to make injera, that made from the red grain has a rustyred speckled appearance, whilst that made from the costlier white grain is considered more desirable. The red coloration in the bran was due to the presence of pigmented material, possibly tannins as in some sorghum grains 8 , in the vacuoles of a layer of cells in the testa. The deposition of this osmiophilic material in developing red tef grain has been examined (Parker, Umeta and Faulks, in preparation). Apart from the presence of this layer, no other structural differences between red and white grain were noted in either fermenting or cooked material. The presence of tannins may affect the nutritional value of red tef injera, however, as tannins are known to have an inhibiting effect on alpha-amylase 9 • 1o and enzymes in the brewing of sorghum beer l l , and may be responsible for the lower levels of maltose and maltotriose found in the fermenting dough of red tef. The thin bran layers and endosperm cell walls in tef probably account for the low levels of dietary fibre 6 of less than 5 % compared with 14 % in wheat, and these components appear to be unaffected by fermentation 7 • There have been several investigations, with conflicting results, of the succession of micro-organisms involved in the fermentation oftef dough 1 ,4,5, but it is agreed that both bacteria and yeasts contribute to the characteristic odour and flavour of injera. In the present study, the microflora involved in the fermentation process were natural contaminants of the grain probably originating from the oxen of the threshing floor 4 • In Ethiopia, a 'starter', or a deliberately unwashed fermentation vessel from a previous successful fermentation might also be used (Fig. 2), and airborne contaminants, which include wild yeasts, are not specifically excluded. During fermentation, fibrillar material
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produced by the microflora bound together bacteria and flour debris, such as protein bodies, starch, bran and wall material, as well as lipid released from the aleurone layer and embryo during milling. It is suggested that the aggregation of these previously suspended particles may initiate the separation of fermented dough into a sediment and supernatant 'irsho'. Loss of up to 9 % of starch in the primary fermentation period? was evidenced by observations of starch granules with eroded cores, and surface pitting typical of enzyme attack 12 • Whilst some starch hydrolysis must result from the action of endogenous amylases present in the grain, starch was probably the main energy source for the microorganisms? Even after 72 h fermentation, however, the appearance of the starch granules was variable, some being severely eroded, whilst others embedded in bran fragments were apparently unaffected. It is not known why the layered appearance of some starch granules became more pronounced during fermentation, but the increasing acidity of the dough (c. pH 4?) may have modified the starch, loosening the layers so that they became more susceptible to enzyme attack. Gelatinised starch is known to be more readily attacked by enzymes 13 , so that by returning hot 'absit' to the dough, ideal conditions for a second period of fermentation were achieved, and resulted in a rapid loss of starch 7 • The storage protein bodies of tef, with their characteristic substructure, were similar to those found in sorghum and millet and other small grains 14 • During milling, some protein bodies were released individually into the flour, but a proportion were retained in fragments of protein-rich outer endosperm cells attached to bran layers. Although total protein levels decreased slightly during the preparation of injera 6 , most likely as a result of discarding soluble components in the 'irsho', the endosperm protein bodies showed little evidence of proteolytic digestion, either during fermentation or baking. In bread made from wheat flour, the endosperm storage proteins form a continuous gluten network, in which starch granules and gas bubbles are held 15 • It is clear from this investigation, however, that tef storage proteins played no part in the structural integrity of cooked injera, although they may add to the texture, and that the major contributor to the injera matrix was gelatinised starch. This type of steam-leavened starch matrix is also found in commercially-produced wafers made from wheat flour 16 • In wafer production, as in the cooking of injera, it is important that the batter-like dough should have a high water content, that a steamy atmosphere should be maintained throughout the cooking period, and that heat should be efficiently transferred from the cooking surface 16 • These are conditions that favour rapid gelatinisation of starch and entrapment of gas bubbles. In addition, the proportion of flour particles below 40 Ilm is of importance 1 ? In tef, this fraction included the readily-gelatinised individual starch granules that were released from the compound grains during milling, particularly those from the less tightly packed mealy endosperm in the centre of some grains. In wafers, the continuous phase of gelatinised starch surrounding the gas bubbles contains no visible organised structure ll\ whereas in injera, there were recognisable fragments of bran and embryo, protein bodies and micro-organisms. The financial support of the World Health Organisation is gratefully acknowledged by Melaku
Vmeta.
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References 1. Stewart, R. B. and Asnake, G. Econ. Bot. 16 (1962) 127-130. 2. Ebba, T. T'ef (Eragrostis tef). The cultivation, usage and some of the known diseases and insect pests. Experimental Station Bulletin. No. 60. Haile Sellassie I University, College of Agriculture: Dire Dawa, Ethiopia (1969). 3. El Tinay, A. H., Abdel Gadir, A. M. and EI Hidai, M. J. Sci. Food Agric. 30 (1979) 859-863. 4. Berhanu, A. G., Meaza, G. and Abraham, B. SINET: Ethiop. J. Sci. 5 (1982) 69-76. 5. Berhanu, A. G. J. Food Sci. 50 (1985) 800-801. 6. Umeta, M. Studies on the fem1entation ofTef (Eragrostis ref) and its nutritional significance. MSc thesis, University of East Anglia (1986). 7. Umeta, M. and Faulks, R. M. Food Chem. 27 (1988) 181-189. 8. Morrall, P., Liebenberg, N. V. D. W. and Glennie, C. W. Scanning Electron Microsc. III (1981) 571-576. 9. Tamil', M. and Alumot, E. J. Sci. Food Agric. 20 (1969) 199-203. 10. Strumeyer, D. H. and Malin, M. 1. Biochim. Biophys. Acta 184 (1969) 643-648. II. Daiber, K. H. J. Sci. Food Agric. 26 (1975) 1399-1411. 12. Gallant, D. J., Derden, A., Aumaitre, A. and Guilbot, A. Stiirke 25 (197;3) 56-64. 13. Shetty, R. M., Lineback, D. R. and Seib, P. A. Cereal Chem. 51 (1974) 364-375. 14. Adams, C. A., Novellie, 1. and Liebenberg, N. V. D. W. Cereal CiJem. 53 (1976) 1-12. 15. Bechtel, D. B., Pomeranz, Y. and Francisco, A. Cereal Chem. 55 (1978) 392-401. . 16. Stevens, D. J. Starke 28 (1976) 25-29. 17. Pritchard, P. E. and Stevens, D. 1. Food Trade Review 44 (6) (1974) 12.
The Contribution of Flour Components to the Structure of Injera, an Ethiopian Fermented Bread Made from Tef (Eragrostis tel) MARY L. PARKER*, MELAKU UMETAt and RICHARD M. FAULKS AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA, Norfolk, u.K. Received 23 January 1989
Injera, a pancake-like fermented bread prepared from white or red tef (Eragrostis tel) flour, is the traditional staple food of Ethiopia. The fate of the major components of the bran and endosperm during the two-stage fermentation and baking has been examined by light and electron microscopy. Angular starch granules released from compound grains during milling showed a range of erosion effects typical of enzymic degradation during fermentation. The appearances of bran and embryo fragments, cell walls and protein bodies were unaffected by fermentation or baking. Microorganisms, the natural contaminants of tef grains, produced strands of fibrillar material during fermentation that bound the flour particles together. Apart from the presence of polyphenolic material in the testa cells of red tef, no structural differences were observed between red and white grain during the preparation of injera. The portion of dough that was thinned, boiled and returned to the mixture for the second fermentation period contained swollen gelatinised starch. During cooking, the starch within the injera was totally gelatinised to form a steam-leavened, spongy starch matrix, in which fragments of bran and embryo, micro-organisms and organelles were embedded. The protein bodies played no role in the formation of the matrix-gas bubble interface.
Introduction Tef [Eragrostis tef(Zucc.) Trotter] is Ethiopia's most widely cultivated cereal food crop. The red or white grain is used for making the several types of ~at breads that form the basic traditional diet of Ethiopia. Tef is also used, to a lesser extent, in porridge and native beer. Tefis not suitable for making leavened bread because the flour lacks gluten, but when available, wheat and other cereal flours may be mixed with tef flour for varietyl. The most popular type of flat bread is injera (Fig. 1), a flexible, spongy, pancake-like product perforated with 'eyes'2. Before it is cooked, the soft dough is fermented twice over a period of 2-3 days, acquiring its characteristic sourness and flavour. To cook injera, the dough is thinned to a thick batter and poured onto a lightly-oiled pan, which is then covered with a tightly-fitting lid to retain the steam. Traditionally, sufficient injera are cooked to last until the next batch is fermented, so the process is continuous with the remains of the previous batch being used as a 'starter' (Fig. 2). This type of
* To whom correspondence should be addressed. t Current address: Ethiopian Nutrition Institute, P.O. Box 5654, Addis Ababa, Ethiopia. 0733-5210/89/050093 + 12 $03.00/0
© 1989 Academic Press Limited
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FIGURE 1. Freshly prepared Ethiopian injera, showing 'eyes' perforating the surface
fermentation and steam-leavening is similar to that used in Sudan for the preparation of Kisra bread from sorghum or millet flours. Several investigations of the microbial flora involved in the fermentation process have been undertaken l ,4,5. It was thought that the yeast Candida guillermondii (Cast.) Langeron and Guerra was the principal fermenting agentl, with the less numerous bacteria contributing flavour and variation, and occasionally spoiling the batter with offflavours. However, recent investigations have shown that fermentation is initiated by gas-producing members of the Enterobacteriaceae 5 present in the flour and starter. As the pH falls to 5,8, these bacteria are succeeded by various other species, including Lactobacillus spp., that contribute the characteristic lactic-acid sour taste and odour to the injera. At pH 4 the yeast population predominates 5 . Very little is known of the effects of the microflora on the nutritional components of fermenting tef dough. Soluble components such as amino acids, glucose, maltose and minerals4, and vitamins l are found in the discarded acidic supernatant from the primary fermentation, and levels of reducing sugars are known to vary widely during fermentation'l.
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TEF FLOUR + WATER + 'IRSHO' STARTER (from 0 previous batch)
Primary fermentation 30-72h
Thin paste fermented in a previously-used container
Secondary fermentation 30 min-2h
FERMENTED PASTE thinned and cooked to form 'INJERA
FIGURE 2. Traditional method of preparing tef injera.
In order to investigate changes in protein, carbohydrate, vitamin, mineral and fatty acid levels during fermentation and baking, injera were prepared under laboratory conditions, taking care to exclude micro-organisms not present originally in the flour, and samples of the fermenting dough and the cooked injera were analysed 6 • 7, and examined by electron microscopy. This present paper describes the ultrastructural aspects of the interaction between the microflora and the major cell components of the flour, and examines the contribution of protein, starch and bran to the structure of cooked injera. Experimental Preparation of injera Two samples of tef grain, red and white, were obtained from the Agricultural Marketing Corporation, Addis Ababa, Ethiopia. Grain was ground in an ultracentrifugal mill to a particle size of less than 0·5 mm. Flour was mixed 2: 3 (w jw) with sterile distilled water, and blended at high speed for 2 min in a domestic mixer. The resultant soft dough was allowed to ferment at 22°C in a covered sterilised beaker. After 72 h, at the end of the first stage of fermentation, the liquid
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. irsho' which covered the dough was discarded. About 10 % of the dough was mixed I: 3 (v Iv) with boiling water, and heated for a further 10--15 min with continuous stirring. It was then mixed back, without cooling, into the fermenting dough, and sufficient sterile water was added to make a thin batter. The batter was left 2 h for a period of secondary fermentation, during which it rose and subsided. More sterile water was added to form a pouring mixture. At both stages of fermentation, sterile water and glassware were used to minimise the contamination of the dough, so that any micro-organisms present were those naturally associated with the tef grain. To cook injera, a frying pan with a non-stick surface was wiped with vegetable oil and heated until very hot on an electric hot plate. A thin stream of batter was poured into the pan in a circular motion, starting from the outside. The pan was covered tightly with aluminium foil to retain the steam, and the heat turned down. After 4-5 min the injera, which resembled a thin flexible pancake with the upper surface covered with perforations, was removed from the pan.
Microscopy of the fermenting dough and injera Dough was sampled during the first fermentation at 0, 24, 48, 72 h (after decanting the supernatant), during the secondary fermentation, and after cooking, and then freeze dried. Samples for scanning electron microscopy were sputter-coated with a layer of gold, approximately 20 nm thick, and viewed and photographed at 30 kVin a Philips SEM 501B. Freeze-dried samples for transmission electron microscopy were fixed in 3 % glutaraldehyde in 0'05 M cacodylate buffer, pH 7,2, post-fixed in I % aqueous osmium tetroxide and dehydrated in a graded series of ethanol. Samples were transferred to acetone, and infiltrated and embedded in Spurr resin. Sections, I 11m thick, were stained with I % toluidine blue in 1 % borax, pH II, for light microscopy. Sections, approximately 70 nm thick, were collected on copper grids, stained sequentially with uranyl acetate and lead citrate, and viewed and photographed at 60 kV in an AEI 801 transmission electron microscope.
Results
Tel grains and flour
The grains of tef were extremely small, less than 1·5 mm in length. As in other smallseeded cereals, the embryo, which is rich in protein and lipid, occupied a relatively large proportion of the grain [Fig. 3(a)]. When milled in the traditional way, the whole of the grain is utilised, producing an extremely nutritious flour. However, the outer pericarp (pc), which is thin and membranous (Fig. 3(a)] and equivalent to the beeswing bran of wheat, may be separated readily from the grain by soaking before milling. The cells of the outer endosperm were horny or vitreous [Fig. 3(a)], and contained most of the protein reserves of the endosperm. On milling, pieces of these cells tended to remain attached to the bran layers so that the compound starch grains survived intact, surrounded by separate, but tightly-packed, spherical protein bodies [Fig. 3(b)]. Very little cytoplasmic matrix protein was visible between these protein bodies. In some seeds, the central endosperm cells were mealy [Fig. 3(a)], and tended to break open during milling, releasing into the flour the individual angular starch granules from the compound starch grains, and also small groups of protein bodies. The aleurone layer was one cell thick, and contained aleurone grains and lipid bodies. The seed coat layers, which were closely adherent to the aleurone layer, varied in thickness depending on the colour of the grain. The testa of red tef was thicker than that of white tef, due to the electron-opaque material deposited in the lumen of the testa cells during grain development.
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FIGURE 3. (a) Scanning electron micrograph of a halved tef grain showing the thin pcricarp (pc), the starchy endosperm (en) with outer horny region and mealy centre (arrowed), and relatively large embryo (em). (b) Fragment of flour of red tcf showing intact and broken endosperm cells attached to the bran. Starch granules are either within compound grains (cs) surrounded by protein bodies (p), or released singly (arrowed). The lipid-rich aleurone cell contents (a) are disrupted by milling. (c) Scanning electron micrograph of dough fermented for 48 h showing angular starch granules (s) with bacteria attached (arrows). (d) Dough fermented for 72 h showing numerous bacteria (b), protein bodies (p), starch granules (s), lipid (1) and cell wall material (cw). The bacteria and cell components me interconnected by a network of fine fibrils (arrows). Scale bar markers are in j..lffi. CER 10
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FIGURE 4. Dough after 72 h fermentation. (a) Protein bodies (p) within particles of flour show no evidence of digestion, but have concentric rings of electron-dense material. Some starch granules are internally eroded (arrows). (b) Severely-eroded starch granules (s) with bacteria (b) attached. (c) Starch granule with concentricallylayered substructure. (d) Starch granule with surface pits (arrowheads) and internal erosion. The erosion of the concentric layers (arrows) has produced a saw-tooth effect. Scale bar markers are in J-lm.
Primary fermentation - 72 h duration During the primary fermentation period, the soft dough prepared from tef flour became progressively more acidic and rose considerably, due to gas evolution from microorganisms that were associated naturally with the grain. Scanning electron micrographs
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of dough fermented for 48 h then freeze-dried [Fig. 3(c)), showed angular starch granules interconnected by strands of material, and short chains of coccobacilli (arrowed). In sectioned dough [Fig. 3(d)), numerous bacteria were found attached to starch granules, protein bodies and cell wall material, and all these organelles were linked by fine strands of fibrillar material, which were particularly associated with the bacteria. There was considerable variation in the effect offermentation on the individual starch granules. In Fig. 4(a), some of the starch granules in the protein-rich sub-aleurone cells have eroded cores (arrowed) whilst others are apparently intact. Erosion or digestion in some starch granules was so far advanced that the starch granules were disintegrating [Fig. 4(b)). Other granules exhibited concentric rings and hollow cores [Fig. 4(c)), and as these grains were eroded, a sawtooth effect was produced [Fig. 4(d), arrows]. Surface erosion, initiated as small pits [Fig. 4(d) arrowheads] was also observed. In contrast, protein bodies appeared not to be eroded during fermentation, although the concentric lamellae within their matrix were more pronounced [Fig. 4(a)]. Second fermentation - 2 h duration
At the end of the primary fermentation period, the yellow liquid 'irsho' that had separated from the solids in the dough was discarded, and some of the dough was diluted, boiled and returned uncooled to the fermentation vessel. The addition of this 'absit' initiated a second period of fermentation. Within this fermentation mixture, therefore, was material that had undergone the primary fermentation only, and also material that had been fermented and boiled. This is illustrated in Fig. 5(a), where partly gelatinised starch granules that have become swollen and misshapen during boiling are attached to a large fragment of flour containing angular ungelatinised starch granules still within the cells. Considerable gas evolution took place in the batter during secondary fermentation, and bacteria were found in close association with both gelatinised and un-gelatinised starch [Fig. 5(b)]. Starch that had been boiled had a characteristic appearance in sectioned material. It is clear from Fig. 5(c) that the fragment of flour had been boiled, because it contained swollen, partly gelatinised starch granules in compound grains, whereas the starch granule adhering to the surface retained its original angular form. The appearance of the boiled protein bodies was similar to that in fermented tef. Partly gelatinised starch had a characteristic amorphous outer layer with a central core of loose fibrils [Fig. 5(d)]. Preparation and structure of injera
After secondary fermentation, the batter was thinned further with water, and poured onto a hot, oiled frying pan, which was then covered tightly to create a steamy atmosphere. Crumbs of cooked injera had an open spongy structure in which were embedded fragments of bran [Fig. 6[(a)] and embryo. Sections of the crumb viewed by light microscopy [Fig. 6(b)], showed a continuous network of completely gelatinised starch (sg) incorporating air cavities, bran layers with protein bodies from the outer endosperm, and micro-organisms. Staining with toluidine blue differentiated between bacteria and protein bodies, which are both approximately 1 !-Lm in diameter; bacteria
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FIGURE 5. Dough at start of secondary fermentation, with boiled' absit ' returned. (a) Scanning electron micrograph of a flour particle with starch-filled endosperm cells attached to the aleurone layer (a) and seed coat (sc). Adhering to the surface are bacteria (arrow) and partly gelatinised starch (sg) from the' absit'. (b) Bacteria (arrows) attacheq to angular starch (s). Partly gelatinised starch (sg) is distorted. (c) Sectioned flour particle from' absit' showing partly gelatinised starch (sg) and intact protein bodies (p). Ungelatinised starch (s) from the batter is also present. (d) Partly gelatinised starch with fibrillar centre, and attached bacterium (b). Scale bar markers are in ~lm.
STRUCTURE OF ETHIOPIAN FERMENTED INJERA
FIGURE 6. Cooked injera. (a) Scanning electron micrograph of part of a crumb showing a large bran fragment (b) embedded in a network of gelatinised starch (sg). (b) Light micrograph of sectioned injcra crumb showing bran and endosperm fragments embedded in the gelatinised starch network (sg). Protein bodies (p), bacteria (arrows), air cavities (ac) and fragments of embryos (em) are also present. (c) Gelatinised starch (sg) within a fragment of flour showing vestigial concentric layering. Protein bodies (p) and cell walls (cw) are recognisable in cooked injera. (d) Part of the gelatinised starch matrix at its interface with an air cavity (ac), showing trapped bacteria (arrows), protein bodies (p), lipid (I) and cell wall material (cw). Scale bar markers arc in ~m.
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stained a purplish-blue, and protein bodies appeared blue. Completely gelatinised starch stained pale pink, in contrast to ungelatinised starch, which remained colourless. Fragments of embryo and oil drops within the starch matrix of the injera had a yellowish tinge as a result of their reaction with osmium tetroxide during fixation. In the steamy conditions essential for cooking injera, most of the starch within the body of the injera gelatinised completely, the granules fusing into a continuous amorphous matrix (sg) in which bubbles of gas were trapped [Fig. 6(b)]. However, some of the starch granules deep within fragments of the outer endosperm were only partly gelatinised [Fig. 6(c)], possibly due to the insulating effect of the surrounding protein bodies and cell wall. Typically, each partly ge1atinised starch granule had a swollen irregular shape, and showed vestiges of its concentric sub-structure in the form of alternating layers of loosely- and tightly-packed fibrils. The effect of cooking on other components ofinjera is illustrated in Fig. 6(d). Protein bodies, although distorted, appeared similar to those in ungerminated tef, and played no role in the retention of gas bubbles. The structure of the cell wall material also appeared relatively unchanged. Bacteria and yeast (not illustrated) embedded in the starch matrix retained their characteristic appearance. The interface between the starch matrix and the gas bubbles was smooth, but may be interrupted by fragments of bran. Two types of lipid-rich inclusions were presented in injera; fragments of embryo or aleurone containing small oil droplets and larger oil drops, possibly originating from the oil used to prevent the injera sticking to the pan during cooking. Discussion
Both red and white tef are used to make injera, that made from the red grain has a rustyred speckled appearance, whilst that made from the costlier white grain is considered more desirable. The red coloration in the bran was due to the presence of pigmented material, possibly tannins as in some sorghum grains 8 , in the vacuoles of a layer of cells in the testa. The deposition of this osmiophilic material in developing red tef grain has been examined (Parker, Umeta and Faulks, in preparation). Apart from the presence of this layer, no other structural differences between red and white grain were noted in either fermenting or cooked material. The presence of tannins may affect the nutritional value of red tef injera, however, as tannins are known to have an inhibiting effect on alpha-amylase 9 • 1o and enzymes in the brewing of sorghum beer l l , and may be responsible for the lower levels of maltose and maltotriose found in the fermenting dough of red tef. The thin bran layers and endosperm cell walls in tef probably account for the low levels of dietary fibre 6 of less than 5 % compared with 14 % in wheat, and these components appear to be unaffected by fermentation 7 • There have been several investigations, with conflicting results, of the succession of micro-organisms involved in the fermentation oftef dough 1 ,4,5, but it is agreed that both bacteria and yeasts contribute to the characteristic odour and flavour of injera. In the present study, the microflora involved in the fermentation process were natural contaminants of the grain probably originating from the oxen of the threshing floor 4 • In Ethiopia, a 'starter', or a deliberately unwashed fermentation vessel from a previous successful fermentation might also be used (Fig. 2), and airborne contaminants, which include wild yeasts, are not specifically excluded. During fermentation, fibrillar material
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produced by the microflora bound together bacteria and flour debris, such as protein bodies, starch, bran and wall material, as well as lipid released from the aleurone layer and embryo during milling. It is suggested that the aggregation of these previously suspended particles may initiate the separation of fermented dough into a sediment and supernatant 'irsho'. Loss of up to 9 % of starch in the primary fermentation period? was evidenced by observations of starch granules with eroded cores, and surface pitting typical of enzyme attack 12 • Whilst some starch hydrolysis must result from the action of endogenous amylases present in the grain, starch was probably the main energy source for the microorganisms? Even after 72 h fermentation, however, the appearance of the starch granules was variable, some being severely eroded, whilst others embedded in bran fragments were apparently unaffected. It is not known why the layered appearance of some starch granules became more pronounced during fermentation, but the increasing acidity of the dough (c. pH 4?) may have modified the starch, loosening the layers so that they became more susceptible to enzyme attack. Gelatinised starch is known to be more readily attacked by enzymes 13 , so that by returning hot 'absit' to the dough, ideal conditions for a second period of fermentation were achieved, and resulted in a rapid loss of starch 7 • The storage protein bodies of tef, with their characteristic substructure, were similar to those found in sorghum and millet and other small grains 14 • During milling, some protein bodies were released individually into the flour, but a proportion were retained in fragments of protein-rich outer endosperm cells attached to bran layers. Although total protein levels decreased slightly during the preparation of injera 6 , most likely as a result of discarding soluble components in the 'irsho', the endosperm protein bodies showed little evidence of proteolytic digestion, either during fermentation or baking. In bread made from wheat flour, the endosperm storage proteins form a continuous gluten network, in which starch granules and gas bubbles are held 15 • It is clear from this investigation, however, that tef storage proteins played no part in the structural integrity of cooked injera, although they may add to the texture, and that the major contributor to the injera matrix was gelatinised starch. This type of steam-leavened starch matrix is also found in commercially-produced wafers made from wheat flour 16 • In wafer production, as in the cooking of injera, it is important that the batter-like dough should have a high water content, that a steamy atmosphere should be maintained throughout the cooking period, and that heat should be efficiently transferred from the cooking surface 16 • These are conditions that favour rapid gelatinisation of starch and entrapment of gas bubbles. In addition, the proportion of flour particles below 40 Ilm is of importance 1 ? In tef, this fraction included the readily-gelatinised individual starch granules that were released from the compound grains during milling, particularly those from the less tightly packed mealy endosperm in the centre of some grains. In wafers, the continuous phase of gelatinised starch surrounding the gas bubbles contains no visible organised structure ll\ whereas in injera, there were recognisable fragments of bran and embryo, protein bodies and micro-organisms. The financial support of the World Health Organisation is gratefully acknowledged by Melaku
Vmeta.
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References 1. Stewart, R. B. and Asnake, G. Econ. Bot. 16 (1962) 127-130. 2. Ebba, T. T'ef (Eragrostis tef). The cultivation, usage and some of the known diseases and insect pests. Experimental Station Bulletin. No. 60. Haile Sellassie I University, College of Agriculture: Dire Dawa, Ethiopia (1969). 3. El Tinay, A. H., Abdel Gadir, A. M. and EI Hidai, M. J. Sci. Food Agric. 30 (1979) 859-863. 4. Berhanu, A. G., Meaza, G. and Abraham, B. SINET: Ethiop. J. Sci. 5 (1982) 69-76. 5. Berhanu, A. G. J. Food Sci. 50 (1985) 800-801. 6. Umeta, M. Studies on the fem1entation ofTef (Eragrostis ref) and its nutritional significance. MSc thesis, University of East Anglia (1986). 7. Umeta, M. and Faulks, R. M. Food Chem. 27 (1988) 181-189. 8. Morrall, P., Liebenberg, N. V. D. W. and Glennie, C. W. Scanning Electron Microsc. III (1981) 571-576. 9. Tamil', M. and Alumot, E. J. Sci. Food Agric. 20 (1969) 199-203. 10. Strumeyer, D. H. and Malin, M. 1. Biochim. Biophys. Acta 184 (1969) 643-648. II. Daiber, K. H. J. Sci. Food Agric. 26 (1975) 1399-1411. 12. Gallant, D. J., Derden, A., Aumaitre, A. and Guilbot, A. Stiirke 25 (197;3) 56-64. 13. Shetty, R. M., Lineback, D. R. and Seib, P. A. Cereal Chem. 51 (1974) 364-375. 14. Adams, C. A., Novellie, 1. and Liebenberg, N. V. D. W. Cereal CiJem. 53 (1976) 1-12. 15. Bechtel, D. B., Pomeranz, Y. and Francisco, A. Cereal Chem. 55 (1978) 392-401. . 16. Stevens, D. J. Starke 28 (1976) 25-29. 17. Pritchard, P. E. and Stevens, D. 1. Food Trade Review 44 (6) (1974) 12.