Food Chemistry 127 (2011) 1555–1561 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Proximate composition, histochemical analysis and microstructural localisation of nutrients in immature and mature seeds of marama bean (Tylosema esculentum) – An underutilised food legume Minah Mmoni Mosele a,b, Åse Solvej Hansen a,⇑, Michael Hansen c, Alexander Schulz c, Helle Juel Martens c a b c Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark Department of Extension and Training, National Food Technology Research Centre, Mpuutsane Industrial Area, Private Bag 008, Kanye, Botswana Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark a r t i c l e i n f o Article history: Received 31 May 2010 Received in revised form 12 January 2011 Accepted 3 February 2011 Available online 26 February 2011 Keywords: Marama bean Morama bean Tylosema esculentum Proximate composition Histochemistry CLSM Seed microstructure a b s t r a c t Marama bean (Tylosema esculentum) is a wild-growing legume adapted to semi-arid conditions in southern Africa. Both immature and mature seeds are used as food by locals and marama bean has potential as a crop plant. Physicochemical and histochemical methods were used to study the accumulation of nutrients and their localisation in immature and mature seeds. The immature seeds had a high content of moisture (67%) and protein (21%), and a low content of lipid (1.5%). At maturity, proteins formed spherical bodies that were embedded in a droplet lipid matrix. The mature seeds are exceptional as they have a high content of protein (32%) and lipid (40%) and no starch. Staining of polysaccharides indicated increases of pectin and cellulose during maturation, parallel with the general increase of cell wall thickness; however, lignin was absent. The content and distribution of protein, lipid and carbohydrates in immature and mature marama beans make this underutilised nutritive legume a prospective crop plant and interesting for food processing applications. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Marama bean (Tylosema esculentum Burchell A. Schreiber) is a wild perennial legume species and a prospective new crop in Southern Africa because of its exceptionally high nutritional value (Hartley, Tshamekeng, & Thomas, 2002). The species is native to the Kalahari Desert and neighbouring sandy semi-arid regions of Southern Africa, in particular Botswana, Namibia and the northern part of South Africa (Castro, Silveira, Coutinho, & Figueiredo, 2005; Hartley et al., 2002). Botanically, Tylosema esculentum belongs to the tribe Cercideae in the subfamily Caesalpinioideae within Fabaceae, and is thus related to Cercis and Bauhinia (Wunderlin, Larsen, & Larsen, 1981). Species of the genus Tylosema were previously included in Bauhinia, but were later established as a separate genus (Castro et al., 2005). Apart from T. esculentum, there are four other known species within the genus, namely, T. fassoglense, T. argenteum, T. humifusum and T. angolense (Castro et al., 2005); among these, T. fassoglense is also edible (Brink, 2006). The species of Tylosema are unique within the family Fabaceae in the fact that their flowers exhibit heterostyly, meaning that they are self incompatible because of spatial ⇑ Corresponding author. Tel.: +45 35333241; fax: +45 35333245. E-mail address: aah@life.ku.dk (Å.S. Hansen). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.02.017 separation of the stigma and anthers (Hartley et al., 2002). This may reduce propagation potential, and obviously reduces seed production rates for cropping. In a field experiment carried out in Texas (USA) the marama bean plant took 4.5 years to produce edible seeds, with flowering at 2 years, development of fruits or seeds at 3.5 years, and final harvesting at full maturation one year later (Powell, 1987). The mature seeds of marama bean are encapsulated in hard, woody seed coats, reddish to brownish-black in colour (Van der Maesen, 2006). The seeds, commonly called marama or morama bean, tsin bean or gemsbok bean, are an important component of the diet among the nomadic ‘‘hunter-gatherers’’ in remote settlements, where few conventional crops can survive (National Research Council, 1979). Raw mature seeds of marama beans store well and remain edible for years under dry storage conditions (Van der Maesen, 2006). The immature seeds of marama bean, inclusive of seed coat, are used as vegetables and the mature seeds are normally eaten boiled or roasted, and have a sweet flavour, with some bitterness in some varieties (Van der Maesen, 2006). The roasted mature seeds can be used to make butter, similar to peanut butter. Flour prepared from mature marama seeds can be used to prepare beverages and can also be added to cereals to increase the nutritive value (Van der Maesen, 2006). 1556 M.M. Mosele et al. / Food Chemistry 127 (2011) 1555–1561 It is known that mature marama seeds store large quantities of lipids and proteins in the storage cotyledons, both well above 30% (Amarteifio & Moholo, 1998; Bower, Hertel, Oh, & Storey, 1988; Holse, Husted, & Hansen, 2010), making them comparable to soybeans (Glycine max) and peanuts (Arachis hypogaea). The marama beans are a source of edible oil used for cooking, especially in Botswana. Both, the composition of fatty acids in marama oil and the content of phytosterols and vitamin E have been investigated (Ketshajwang, Holmback, & Yeboah 1998; Mitei, Ngila, Yeboah, Wessjohann, & Schmidt, 2009). The essential amino acids present are tyrosine, being the highest, followed by arginine, leucine and lysine (Bower et al., 1988). Recently it has been shown that marama bean has a very high content of dietary fibre with variation between 19% and 27%, a high content of lignans, and no content of cyanogenic glycosides, as well as the potent allergens found in peanut and lupin (Holse, Husted, & Hansen, 2010). The effects of food processing have only been studied on the functional properties of marama flour (Jideani, Van Wyk, & Cruywagen, 2009; Maruatona, Duodu, and Minnaar (2010). The domestication of marama bean should be encouraged because of its nutritional value and also due to its potential use as a food crop in arid areas (Bower et al., 1988) and regions with erratic rainfall (National Research Council, 1979). Despite the potential of marama bean as a healthy nutritive crop for developing countries, not much is known about in situ localisation of nutrients. The nutritional value of immature seeds has not been investigated at all. Any developmental studies have been limited to the flowering parts of marama bean plant (Castro et al., 2005; De Frey, Coetzer, & Robbertse, 1992; Hartley et al., 2002). Legumes store lipids, proteins and various types of polysaccharides as major reserves in their seeds. Although the basic microstructure of mature legume seeds is well-known, especially from commercial crops such as soybean (Webster & Leopold 1977) and peanut (Lott & Buttrose 1977), less is known about the early deposition of seed reserves. To our knowledge, there is one study on mature marama bean protein bodies (Amonsou, Taylor, & Minnaar, 2011), and that study did not present the complete microstructure of the seeds. Marama bean has commercial potential for agriculture in the areas where it grows, because of its high oil and protein contents and it is therefore important to study the nutritional value of the immature pods and the mature marama seed since the bean is consumed at both stages. The distributions of the various food components are best understood by way of histochemistry and this is important information in its processing and industrial applications. The aim of our study is to characterise the development and localisation of chemical components in marama bean seeds at two developmental stages, termed ‘‘immature’’ and ‘‘mature’’ stages of consumption, by means of physicochemical and histochemical analyses and electron microscopy. 2. Materials and methods 2.1. Plant material Seeds of marama bean (T. esculentum) were collected in 2008, from multiple plants growing in their natural habitat in the southern region of Botswana. Whole pods with immature seeds, at the stage where they are normally consumed, were stored at 20 °C. Mature seeds were kept at 4 °C. 2.2. Linear dimensions Linear measurements were taken according to the method of Mpotokwane, Gaditlhatlhelwe, Sebaka, and Jideani (2008), with slight modifications, as reported for marama bean by Jideani et al. (2009). Twenty seeds were randomly selected by collecting a handful from a random location in a bag for containing the seeds. The length (L), width (W) and thickness (T) were measured to an accuracy of 0.001 mm, using a vernier calliper. 2.3. Proximate composition The samples were decorticated with a hammer and knife, and the cotyledons were milled into flour in a laboratory mill (IKA A10, Labortechnik, Staufen, Germany) for 15 s. Marama flour was passed through a 1 mm diameter mesh sieve, except for the flour from immature seeds because it formed a paste and was used in that state. All samples were analysed in triplicate. Proximate composition was determined by approved standard methods of analysis with a few modifications, as reported by Holse, Husted, and Hansen (2010). The methods used were AOAC (2000) for protein, moisture, lipid and ash. Results were expressed as a percentage in wet basis (as is). 2.4. Transmission electron microscopy (TEM) Storage cotyledons were isolated from thawed immature seeds and dry mature seeds. Small 3  3  5 mm pieces of seed coat (in immature seeds only), and outer and central parts of cotyledons were fixed for 4 h in Karnovsky’s fixative (5% glutaraldehyde, 4% paraformaldehyde, 0.1 M sodium cacodylate buffer), including a vacuum treatment, washed in cacodylate buffer at pH 7.3 and post-fixed in 1% osmium tetroxide (with 0.1 M cacodylate buffer) for 8 h at 4 °C. After washing in buffer and water, the samples were dehydrated in a graded acetone series, infiltrated with three different ratios of Spurr resin to acetone and embedded in Spurr resin within flat moulds. The resin was polymerised in an oven at 60 °C for 8 h. Ultra-thin sections were cut with a diamond knife, using a Reichert-Jung/LKB Supernova ultramicrotome and collected on pioloform-coated copper grids. Sections were contrasted with 1% uranyl acetate and lead citrate (2.7% in 3.5% sodium citrate) and examined in a Philips CM 100 TEM at 60 kV. 2.5. Light microscopy (LM) Samples for LM were collected from the pool of samples prepared for TEM, as described above, except samples of endosperm which were collected in fresh tissue of immature and mature seeds. Semi-thin sections of 2 lm were cut with glass knives on the ultramicrotome and stained with 1% Aniline Blue Black in 7% acetic acid for proteins, periodic acid Schiffs (PAS) for insoluble carbohydrates containing 1,2-glycol groups, Sudan Black for lipids (saturated solution in 70% ethanol), 2% iodine solution (I2 KI) for starch, phloroglucinol (mixture of 0.1 g phloroglucinol, 16 ml concentrated HCl and 84 ml 95% ethanol) for lignin and 0.01% Calcofluor White M2R for b-1, 4 linked glucans (cellulose), following the methods of O’Brien and McCully (1981). Coriphosphine O (0.03%) was used for staining pectin (Ueda & Yoshioka, 1976). Sections were viewed in immersion oil in a Nikon Eclipse 80i light and fluorescence microscope. 2.6. Scanning electron microscopy (SEM) For SEM, specimens were cut with a razor blade into small wedges (3  5  5 mm) and fixed overnight in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7, at 4 °C. After washing overnight in 0.1 M cacodylate buffer with 5% sucrose, pH 7, samples were washed for 5 min in the same solution and post-fixed for 1 h in 2% osmium tetroxide, in 0.1 M cacodylate buffer with 5% sucrose, pH 7, at room temperature. Following washing steps in buffer and water, samples were dehydrated in a graded ethanol series. M.M. Mosele et al. / Food Chemistry 127 (2011) 1555–1561 Further dehydration was done in hexamethyldisilazane (HDMS) for 30 min. Samples were mounted onto metal stubs with doublesided carbon tape and sputter-coated with a thin layer of gold/palladium under vacuum, using an automated sputter coater SC7640 (Quorum Technologies, Newhaven, UK). The samples were viewed in a Quanta 200 Microscope (FEI Company™). 1557 supplementation or fortification of diets of the local communities. From the chemical results we can conclude that lipid, protein and carbohydrate accumulate as the seeds mature and, like soybean and peanut, these major intracellular food reserves are stored in the thick cotyledons during seed development. 3.3. Histochemistry of seed coat and endosperm 2.7. Confocal laser scanning microscopy (CLSM) Mature marama bean seeds, on average, were 19.9 (±1.6) mm in lengths in a range of 17.0–23.0 mm, 17.6 (±2.5) mm in widths in a range of 11.5–20.5 mm and 13.1 (±0.9) mm in thicknesses in a range of 12.0–15.0 mm. Our results are similar to those reported by Jideani et al. (2009) for the same species. Immature and mature marama seeds are seen in Fig. 1A. The colour of the seed coat is cream in the immature seeds and brown in the mature seeds. The seed coat consists of a double layer of lignified macrosclereids with a callose-rich light line, underlying spongy parenchyma cells (Fig. 1B) and an inner epidermis. In contrast to e.g. soybean, a subepidermal layer of so-called hourglass cells storing proteins (Moïse, Han, Gudynaite-Savitch, Johnson, & Miki, 2005) does not exist in marama bean. The seed coat could potentially be a source of phenolic compounds for application as natural antioxidants (Oomah, Cardador-Martínez, & Loarca-Piña, 2005; Zadernowski, Borowska, Naczk, & NowakPolakowska, 2001). Direct microscopic localisation of phenolics is, however, restricted to anthocyanin-containing compartments, while other phenolics are auto-fluorescent and can be detected by fluorescence microscopy (Hutzler et al., 1998), as also noted in our study (not shown). At the immature stage, also, a nuclear endosperm is present (Fig.1D) which provides a temporal source of nutrition for the growing embryo. Later, cell walls develop in the endosperm (Fig. 1E), consisting of peripheral cells with dense proteins and lipids, but no starch (not shown). As in other Fabaceae species, e.g. Trifolium (Jakobsen, Martens, & Lyshede, 1994), the endosperm is used up during seed development and the nutrients are translocated into the thick cotyledons (Fig. 1F). This contrasts with the closely related species Cercis siliquastrum, where the endosperm does persist in the mature seeds (Baldan et al., 1995). 3.2. Proximate composition 3.4. Histochemistry of cell walls in storage cotyledons The protein content of mature seeds was above 30%, but it was already ca. 20% at the immature stage (Table 1) which means that the protein is stored in the seed from the early stage of development. The content of lipids was as high as 40% in the mature seeds but was negligible at the immature stage. Accordingly, lipid storage occurs in late stages of seed maturation. The carbohydrate content of mature seeds was twice that of immature seeds. The results of major chemical components in mature seeds are within the amounts reported by Bower et al. (1988), Amarteifio and Moholo (1998) and Holse, Husted, and Hansen (2010). The content of lipids in the immature seeds was similar to that reported by Redondo-Cuenca, Villanueva-Suárez, Rodríguez-Sevilla, and Mateos-Asparicio (2006) for green (immature) soybeans at 0.93 g/100 g. The ash and moisture contents for mature seeds were below ca. 5%. Immature seeds had a moisture content of ca. 67%. The results confirm that mature marama beans have high contents of protein and lipid, comparable to those of other oil seeds such as soybean and peanut (Adsule, Kadam, & Salunkhe, 1989; Nkama & Filli, 2006; Redondo-Cuenca et al., 2006; Vaidehi & Kadam, 1989). However, marama bean protein content was slightly above that of peanut, and the content of lipids was almost twice that of soybean. Thus, marama bean would be a good crop for Coriphosphine O, Calcofluor White, and PAS were used as histochemical tests, specific for pectin, cellulose and 1–4 bound polyglucans (e.g. starch, cellulose and hemicelluloses), respectively (Luza, van Gorsel, Polito, & Kader, 1992; Marcus et al., 2008). Parenchyma cell walls in the storage cotyledons are rich in pectin, especially within the middle lamella, as seen by Coriphosphine O fluorescent dye (Fig.1G–I). In the immature tissue the new cell walls are pectin-positive, visible as a thin straight line clearly depicting newly divided cells (Fig.1G). In mature seeds, pectin is a prominent constituent of epidermis cell walls and around intercellular spaces of the storage parenchyma cells (Fig.1I). These cells have primary cell walls which increase in thickness during maturation, consisting mainly of cellulose as indicated by Calcofluor White staining under UV excitation (Fig.1J,K). Provascular strands were noted (Fig. 3B), but lignification of prospective xylem elements had not started yet, as tested by phloroglucinol staining (not shown). PAS-staining (Fig. 1L–N), as well as iodine-staining (not shown), revealed that marama bean, in contrast to other legumes, e.g. peanut (Schadel, Walter, & Young, 1983; Young, Pattee, Schadel, & Sanders, 2004), does not store detectable amounts of starch. Insoluble polysaccharides, strongly positive with PAS, were deposited in the cell walls in both immature and mature seeds (Fig.1L–N). In these images, post fixation contrasting is seen as grey and black areas labelling lipophilic and proteinaceous substances. Compatible with the changes in proximate composition, it can be concluded that the thickening of cell walls during maturation is accompanied by an increase of insoluble carbohydrates, namely cellulose and pectin. It should further be noted that, within the outermost cells in immature cotyledons, the cytoplasm is somewhat shrunken, possibly due to some extent Samples were cut from fresh samples with a razor blade into thick hand sections. They were specifically stained for neutral lipids with Nile Red (0.1 lg/ml) according to the method by Greenspan, Mayer, and Fowler (1985). After rinsing in water, images from intact cells were recorded in a Leica SPII confocal laser scanning microscope, using the 543 nm excitation line and 570– 595 nm emission. 2.8. Statistical analysis The quantitative data was presented as means of three replicates, using Microsoft Excel. Images were processed with Adobe Photoshop CS2. 3. Results and discussion 3.1. Linear dimensions Table 1 Proximate composition (%) of immature and mature marama bean cotyledon (as is). Sample Moisture Ash Protein Lipid Carbohydratea Immature Mature 66.8 (0.4) 5.3 (0.2) 2.2 (0.1) 3.0 (0.0) 20.8 (0.4) 32.3 (0.8) 1.5 (0.1) 40.0 (0.7) 8.6 19.4 Values in parentheses indicate standard deviations. a Carbohydrate by difference. 1558 M.M. Mosele et al. / Food Chemistry 127 (2011) 1555–1561 Fig. 1. Histochemistry of the cell wall of immature (i) and mature (m) marama seeds. Presence of pectin is indicated by Coriphosphine O, cellulose by Calcofluor White and 1,4 bound polyglucans by the PAS reaction. Opened pod of immature (i) and mature (m) seeds (A). Bar = 20 mm. Cross section of the seed coat showing the palisade layer of macrosclereids (s) with the light line of callose (l) and parenchyma tissue (p) (B). Bar = 25 lm. Cross section through a group of macrosclereids in the seed coat (C). Bar = 25 lm. Cellular (c) and nuclear (n) endosperm (D). Bar = 100 lm. Cells from the cellular endosperm (E). Bar = 100 lm. Block of storage cotyledon tissue, as trimmed for SEM (F). Bar = 1 mm. Pectin in the cell walls of developing storage cotyledon is seen as yellow fluorescence after Coriphosphine O staining (G). Newly formed cell walls (arrow). Bar = 100 lm. Pectin in central part of developing storage cotyledon (H). Note appearance of a green fluorescence from proteinaceous substances (arrow). Bar = 100 lm. Pectin and protein bodies (arrow) in mature tissue (I). Bar = 100 lm. Cellulose (blue) in immature (J) and mature cotyledons (K). Bar = 100 lm. Insoluble carbohydrates (red) detected by PAS reaction in immature cotyledons (L, M) and mature seed (N). Some cytoplasmic compounds are osmiophilic and stained grey to black due to OsO4 fixation (arrows). Bar = 20 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) of dehydration during the collection period or non-optimal freezing conditions during transport. 3.5. Lipids and proteins in immature storage cotyledons After biosynthesis at the endoplasmic reticulum (ER), seed intracellular storage proteins bud off as separate organelles (protein bodies, PBs), which either accumulate in the cytoplasm or are sequestered into vacuoles (protein storage vacuole, PSV) by autophagy (Herman and Larkins 1999). The terminology is, however, not clear, and we will (as many authors) refer to these storage vacuoles as protein bodies (PBs). In the SEM, many tiny globules, presumably PBs, are present in the developing storage tissue (Fig. 2A). Intracellular protein depositions, as well as M.M. Mosele et al. / Food Chemistry 127 (2011) 1555–1561 1559 Fig. 2. The early deposition of protein and lipid in seeds of immature marama bean. SEM of developing storage parenchyma showing small spherical protein bodies (PB) (arrow) (A). Light microscopy labelling of cytoplasmic and cell wall proteins with Aniline Blue Black (blue). Also note black labelling of osmiophilic substances (B). TEM of the amorphous appearance of a PB (C). Aggregation of PBs (D). Spherical PBs in close association with ER (straight arrows) and ribosomes (bent arrow) (E). Bars = 10 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) proteins within the cell walls, are seen in the light microscope after staining with Aniline Blue Black (Fig. 2B). The pale blue substances within the parenchyma cell indicate the early onset of protein deposition, also recognised in TEM (Fig. 2C–E). The amorphous PBs show cytosolic inclusions (Fig. 2C) and seem to fuse with each other (Fig. 2D). At this developmental stage the PBs were closely associated with long strands of rough ER and sometimes small protein deposits were encircled by ER (Fig. 2E). From our electron microscopy data we hypothesise that the marama bean PBs are actually of the cytosolic type, forming large aggregates and not the PSV type, which we would expect to be smooth-surfaced vesicles. In consistency with the chemical data, lipid bodies (LBs) are not present at the early stage of maturity. 3.6. Lipids and proteins in mature storage cotyledons At the mature stage of seed development, the voluminous cotyledons almost completely occupy the embryo sac cavity. Cells of the central part of the cotyledon are 30 ± 5 lm in length and 22 ± 5 lm in width. Protein bodies (13 ± 3 lm diameter) and lipid bodies (0.87 ± 0.17 lm diameter) are the dominant features of the cytoplasm (Figs. 3 and 4). PBs decorated with LBs can be seen in mature storage parenchyma, using SEM (Fig. 3A). Large PBs (up to 40 lm diameter) are located within the subepidermal cell layers (Fig. 3G and H). We observed a trend of small PBs in the epidermal layer, which increase in size but decrease in number in the sub-epidermis, and finally increase in number but decrease in size at the centre of the cotyledon (Fig. 3G–I). Interestingly, protein bodies are labelled green with Coriphosphine O (Fig. 3B, see Fig. 1H–I, for comparison). The finding that this pectinspecific dye (yellow emission) also specifically labels cytosolic proteins (green emission) was confirmed by control-staining of other protein-rich materials, e.g. sunflower seeds and Phaseolus beans (not shown). The PBs stained blue with Aniline Blue Black (Fig. 3C). In post-fixed samples PBs are surrounded by a greyish substance indicative of osmiophilic LBs (Fig. 3D). This was proved by Sudan Black staining (Fig. 3E) in which PBs and cell walls are clearly unlabelled. PBs consist of an electron-dense proteinaceous matrix, often containing spherical cytosolic inclusions (Fig. 3F and H) called globoids (Lott & Buttrose, 1977; Lott, Ockenden, 1560 M.M. Mosele et al. / Food Chemistry 127 (2011) 1555–1561 Fig. 3. Storage protein and lipid in seeds of mature marama bean. SEM of parenchyma cells with large proteins bodies (PBs) covered with lipid bodies (LBs) (A). Bar = 10 lm. Coriphosphine O-stained section, showing green fluorescence from PBs and yellow fluorescence from pectin in middle lamella (B). Bar = 100 lm. Aniline Blue Black staining of PBs and cell wall proteins without postfixation (C), and with lipids visible after postfixation (D). Bar = 25 lm. Sudan Black labels lipid material surrounding unstained PBs (E). Bar = 25 lm. Mineral globoids (arrow) in PBs (F). Bar = 25 lm. TEM of epidermis and subepidermal layers. Note that small size PBs in epidermis (black arrow) and large PBs in subepidermal cells (white arrow) (G). Bar = 10 lm. Subepidermal cells with LBs, proplastids (black arrow) and large PBs with numerous globoids (white arrow) (H). Bar = 5 lm. LBs encircling PBs. Plasmodesmata through cell walls (arrow) (I). Bar = 5 lm. Section showing large-lobed nucleus (n) (J). Bar = 5 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Raboy, & Batten, 2000). Studies of the elemental composition and distribution of mineral reserves within globoids in legumes revealed P, K, Mg and Ca salts of phytic acid (Lott & Buttrose, 1977). Cereal grains and oil seeds are particularly rich sources of phytate. Lipids are abundant at the mature stage of the seeds. Numerous electron-transparent lipid bodies (LBs) encircle the PBs and line the cytoplasmic side of the plasma membrane (Fig. 3I). LBs do not fuse and appear to be bound by a thin membrane, presumably a half unit membrane. LBs, like PBs, probably originate from the ER. The distribution of LBs within individual parenchyma cells was judged by the application of Nile Red and confocal imaging in the xyz-plane. The fat is mostly neutral lipids, in droplet form, as Nile Red staining is specific for neutral lipids (Fig. 4A–C), usually triaglycerols or cholesteryl esters (Fowler & Greenspan, 1985; Greenspan et al., 1985). Fig. 4. Three-dimensional distribution of proteins and lipids in a parenchyma cell from seed of mature marama bean. Confocal recordings of Nile Red stained lipid bodies in top view (A) and sections obtained at two other focal planes (B and C) revealing the unlabelled PBs. Bars = 10 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) M.M. Mosele et al. / Food Chemistry 127 (2011) 1555–1561 4. Conclusions and perspectives Despite its exceptional nutritional quality, marama bean has not been domesticated and is thus not listed with other legume oilseeds, such as peanut and soybean. The present study employed physicochemical and histochemical methods and electron microscopy to elucidate the changes from the edible immature stage of seed development to the mature stage. It revealed a high protein content (21%) and moderate content of insoluble carbohydrates (9%) at the immature stage of the storage cotyledons, and a high protein (32%) and lipid (40%) content accompanied by increased carbohydrate content (19%) at the mature stage. The major carbohydrates in marama bean are insoluble polysaccharides (pectin and cellulose) stored as cell wall components. In the present study, it turned out that variation in seed development was considerably high between seeds collected at the same stage. Therefore seed variation should be kept in mind if selecting individual seeds for plant breeding work. Marama bean, being a wild legume, is a promising crop, worth domestication and cultivation for diverse food applications in the diets of communities and the food industry. Marama bean may, furthermore, have potential as a substitute for genetically modified legumes due to its naturally occurring high protein and lipid contents. Acknowledgements We are grateful to the National Food Technology Research Centre (NFTRC) for providing marama beans, to Piotr Binczycki for skilled preparation of the material for LM and TEM and to Professor Søren Balling Engelsen, Faculty of Life Sciences, University of Copenhagen for valuable comments on the manuscript. This work is part of a PhD project (M.M. Mosele) with the financial support of The Government of Botswana, Ministry of Infrastructure, Science and Technology. References Adsule, R. N., Kadam, S. S., & Salunkhe, D. K. (1989). Peanut. In D. K. 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