MICROSTRUCTURE OF PROTEIN BODIES IN MARAMA BEAN SPECIES

Get Complete Project Material File(s) Now! »

Protein types

Legume proteins have been classified as albumins (water soluble), globulins (salt soluble), prolamins (alchohol soluble) and glutelins (alkali/acid soluble) (Osborne, 1924). Although, globulins constitute the major storage protein of most legumes (Table 2.4) (Mohan & Janaharnan, 1995; Lqari et al., 2002; Chavan et al., 2001), some storage proteins especially among the dry beans have been found to contain albumins as their major protein fraction (Adebowale et al., 2007; Idouraine et al., 1994). Water-soluble albumin has been reported as the major storage protein fraction in mucuna bean species, followed by the glutelin (Adebowale et al., 2007). The albumin was found to constitute 83% of the storage protein in terapy bean (Idouraine et al., 1994), 73% in adzuki bean (Vigna angularis var Takara) and 53% in faba bean (Rahman, 1988). The navy bean showed similar proportions of albumin and globulin (approx. 35%). Albumin and globulin were found to be the predominant protein fractions in two species of Bauhinia including B. purpera and B. vahlii, whereas glutelin was predominant in B. race (Rajaram & Janardhanan, 1991). The high proportion of water-soluble albumin mainly in dry bean implies that a less complex procedure would be required for their protein extraction.

Globulins

Storage globulins are often composed of two major fractions, namely 7S (vicilin) and 11S (legumin). Variations in contents between the vicilin-like (7S) and legumin-like (11S) fractions of legume globulins have been reported (Meng & Ma, 2001; Adebowale et al., 2007; Horax et al., 2004). Both 7S and 11S have been found to be major storage protein fractions of lima bean (Chel-Guerrero et al., 2007), faba bean (Kimura et al., 2008) and lupin (Duranti et al., 1981), similar to soya bean (Kinsella, 1979). On the other hand, the 7S globulins have been found to be abundant in mucuna bean (Adebowale et al., 2007), terapy bean (Idouraine et al., 1994), and red bean (Phaseolus angularis) (Meng & Ma, 2001), similar to cowpea (Horax et al., 2004). Using Size Exclusion High Performance Liquid Chromatography (SE-HPLC) and analytical ultracentrifugation, Rahman et al. (2000) demonstrated that 7S globulin was the major protein component in mung bean. Chavan et al. (2001) reported 7S as major and 11S as minor in beach pea legume protein. Protein extraction using the traditional alkaline extraction/isoelectric precipitation and micellisation, did not affect the 7S content of mung bean (Rahman et al., 2000). Vicilin-like proteins have been also found to be the major storage protein fraction in bambara groundnuts and pigeon pea (Benjakul et al., 2000).
In addition to the two protein fractions associated with globulins, another 7S globulin designated as basic 7S has been reported in soya bean (Yamauchi, Sato & Yamagishi, 1984; Omi et al., 1996). This protein is called basic 7S because of its basic isoelectric point in the pH range 9.05–9.26 (Yamauchi et al., 1984) and has been found to account between 0.5-3% of the total seed protein (Omi et al., 1996; Yamauchi, et al., 1984). The basic 7S globulin protein fraction has been found to be immunologically distinguished from 11S and 7S globulin (Sato et al., 1987; Omi et al., 1996). The basic 7S globulin (Bg) of soya bean, which was released by pressurisation has been found to be localized in the dermal tissue of seed cotyledons and to consist of 16 and 27 kDa subunits (Omi et al., 1996). Although the basic 7S globulin constitutes a minor component of seed protein, it was found to be a sulphur-rich protein and could be heat coagulated to form soluble and insoluble aggregates (Sathe et al., 1989; Yamuachi et al., 1994).
The variation in contents between 7S and 11S of legume globulins may be attributed to differences in genotypes. The relative amount of 7S and 11S protein fractions may influence the functionality of proteins. For instance, Arrese et al. (1991) reported that the gelation characteristic of soya protein was related to the amount of 7S and 11S. High ratios of 11S to 7S in proteins led to increased textural properties of hardness, cohesiveness, and gumminess of gels (Tay & Perera, 2004). The globulin of some legumes has further been characterised to gain understanding into its functionality in food systems.

Subunit composition

One-dimensional SDS-PAGE has been employed to determine the protein profile of legume globulins. Differences have been reported in terms of number and molecular weight of constituent polypeptide subunits of legume proteins such as bambara groundnuts (Odeigah & Osanyinpeju, 1998), faba bean (Ghandorah & El-shawaf, 1993) and terapy bean (Idouraine et al., 1994). Terapy bean protein extracts showed major polypeptide bands at 29, 45 and 49 kDa (Idouraine et al., 1994). The globulin fraction of moth bean (Vigna aconfitifolia L.) protein was composed of three major polypeptides with an estimated mass of 45-55 kDa and several additional polypeptides in the range 14-32 kDa (Sathe & Venkatakchalam, 2007). Lupin (Lupinus albus) globulins analysed by ion exchange chromatography, gel filtration, and cellulose acetate electrophoresis revealed eight fractions, all with acidic character (Cerletti et al., 1997). The seed globulin of alupin specie has been found to contain four subunits corresponding to the vicilin and two corresponding to the legumin (Duranti et al., 1981).
Differences in structures and subunit composition of the two protein fractions (7S and 11S) of legume globulin have been reported. The 11S legume proteins are oligomeric, consisting of six acidic (α) and six basic (β) subunits disulphide-bonded as αβ pairs (Derbyshire, Wright & Boulter, 1976). The nomenclature of the storage soya bean globulin is shown in Figure 2.6. Many studies have confirmed the oligomeric structure of 11S protein in soya bean (Kinsella, 1979; Mujoo et al., 2008; Liu et al., 2007).
The protein band distribution of some legumes compared with those of soya is shown in Figure 2.7 (A-C). The vicilin (7S) of most legumes contains a variable number of subunits and these often lack disulphide bonds (Rahma et al., 2000; Mujoo et al., 2003; Tang, 2008). The vicilin fraction of soya protein has been found to be composed of three major subunits (Mujoo et al., 2003; Liu et al., 2007). Adebowale et al. (2007) reported two subunits with apparent molecular weight of 36 kDa and 17 kDa for the vicilin-like storage protein fraction of mucuna bean. Four subunits were separated by SDS-PAGE when the vicilin structure of broad bean (Vicia faba) has been analysed (Bailey & Boulter, 1972). The variation in the number of vicilin-like (7S) storage protein was attributed to post-translational proteolytic processing of the pre-proprotein and/or the differential extent of glycosylation (Muntz, 1998; Sathe, 2002).
Similarity in protein band distribution under reducing and non-reducing conditions was reported among Bambara groundnut var. HT, bambara groundnuts var. T, cowpea and pigeon pea (Fig. 2.7) (Benjakul et al., 2000), suggesting an absence of disulphide bonds. This similarity may be due to fact that these legumes have vicilin as major storage protein (Benjakul et al., 2000). Rahman et al. (1988) analysed the subunit pattern of mung bean (Phaseolus aureus) by means of SE-HPLC and SDS-PAGE. These authors reported a major protein fraction of molecular weight: 54 ± 2kDa (Rt:
84.1 min), which was similar under reducing and non-reducing conditions, suggesting again that this was a vicilin-like storage protein. Based on the results of both one and two dimensional gel electrophoresis, Rahman et al. (2000) elucidated the absence of disulphide bonds in polypeptides of 7S vicilin of mung bean protein fraction. Horax et al. (2004) reported on electrophoretic characteristics of three cowpea varieties in comparison with soya. These authors found that cowpea protein isolates were concentrated at 60 and 40 kDa, whereas soya bean protein bands were at 95, 65, 60, 40, and 35 kDa. Similarity in electrophoresis bands between cowpea and soya have been found in the range 40-62 kDa (Horax et al., 2004). This study confirms that vicilin (7S) is the major storage fraction in cowpea compared with soya protein, which has both vicilin (β-conglycinin) and legumin (glycinin) (Kinsella, 1979; Mujoo et al., 2003) as major storage protein fractions. The molecular size distribution reported for cowpea (Horax et al., 2004) was similar to that reported for navy bean and kidney bean (Kohnhorst et al., 1990). The latter had a major band ranging from 43-47 kDa and a minor band with size 26-28 kDa (Kohnhorst et al., 1990).
The patterns of proteome maps of globulin protein have been found to vary in their constituent polypeptides depending on legume type. Natarajan et al. (2007) reported significant variations in the five glycinin subunits (G1-G5) among sixteen soya bean genotypes analysed by proteomic techniques and genetic analysis. Major variation was observed among wild and cultivated genotypes rather than within the same group. The glycinin to β-conglycinin ratios were also found to vary significantly among 14 commercial soya bean cultivars (Zarkadas et al., 2007). Similar variations in the ratio of glycinin to β-conglycinin have been also reported by Yagasaki et al. (1997); although low ratio values compared with those of Zarkadas et al. (2007) were found. Protein quality of soybeans varieties can be determined from their ratio of the major seed storage protein components of glycinin and β-conglycinin.
The proteome map of lupin (Lupine albus) revealed an intrinsically complex pattern of lupin storage proteins with several spots of same molecular weight but with different pIs. The digital image processing of the map detected 357 spots, seed protein families (Magni et al., 2007). The profiles of total seed proteins isolated from mature seeds of four peanut cultivars were studied using two-dimensional gel electrophoresis coupled with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC–MS/MS) (Kottapalli et al., 2008 ). The four peanut cultivars revealed distinct differential expression of storage proteins, with the number of polypeptides varying from 457-556. Abundant polypeptides identified belong to the globulin fraction consisting of Arachin (glycinin and Arah3/4) (Kottapalli et al., 2008).
Chemical characteristics of storage proteins from legume seeds in terms of subunit composition and structure have been found to significantly influence the functional properties of proteins (Kinsella, 1979; Nakai, 1983). The remarkable variability in protein expression among legume storage proteins will bring about differences in their functionality. The protein expressions of many existing indigenous legumes such as the marama bean are not known and this information would be required to gain understanding of their function in foods.

Functional properties of legume proteins

Functional properties are physicochemical properties which affect the behaviour of proteins in food systems during preparation, processing, storage and consumption (Kinsella, 1979). These properties include solubility, water and oil absorption, emulsifying and foaming properties as well as rheological properties (Kinsella, 1979). The thermal and rheological properties of legume protein are discussed in the following sections.

Thermal properties

The temperature of denaturation (T_d) and enthalpy (∆H) are the two parameters commonly used to describe the thermal characteristics of legume proteins (Hermanson, 1986; Renkema et al., 2001; Horax et al., 2004). T_d and ∆H provide information on the type of protein structure (simple or complex). The enthalpy is the measure of heat flow into the protein. The greater the heat flow the greater the state of nativity and the more complex the protein structure (Hermanson, 1986; Sorgentini et al., 1995).
A study on thermal denaturation of cowpea storage protein by Differential Scanning Calorimetry (DSC) revealed only 1 major peak corresponding to the vicilin fraction as compared to soya protein which showed two peaks (Horax et al., 2004) corresponding to the β–conglycinin (7S) and glycinin (11S) protein fractions (Hermanson, 1986; Wagner & Annor, 1990). The temperatures of denaturation of 7S cowpea protein (85- 88.4^oC) have been found to be similar to that of 7S soya protein. But, variable denaturation enthalpies ranging from 8.4-10.33 J/g protein for 7S cowpea protein and 0.6-1.01 J/g protein for 7S soya protein have been found (Horax et al., 2004). The difference in enthalpy (heat flow) may be attributed to differences in 7S contents between soya and cowpea proteins
Differences in thermal stability between vicilin (7S) and legumin (11S) fractions of legume protein have been reported (Sorgentini et al., 1995; Hermanson, 1986). The denaturation temperature (96.3^oC) and enthalpy (4 J/g) of glycinin (11S) were high compared to those of β-conglycinin (7S) (T_d: 82.5 and ∆H: 0.7-1.0 J/g protein) for soya bean protein. Similarly, Sorgentini et al. (1995) reported high thermal transition point for glycinin (92^oC) compared with β–conglycinin (72^oC), suggesting thatglycinin is more thermally stable than β-conglycinin. Glycinin showed a denaturation temperature of 90^oC at neutral pH and an ionic strength of 0.25, whereas ß- conglycinin already unfolded at 74^oC under the same condition (Hermanson, 1986). Differences in network structure and interaction have been found to be responsible for the differences in thermal stability between 7S and 11S proteins (Barac et al., 2001; Renkema et al., 2001). β-conglycinin has been reported to be a trimer consisting of α’, α, and β subunits (Thanh & Shabadaki, 1979; Mujoo et al., 2003), while the glycinin has been found to be a hexamer consisting of pairs of acidic and basic polypeptide subunits; each pair linked by disulphide bonds (Wagner & Annor, 1990; Mujoo et al., 2003).
Variation in thermal stability among dry bean legumes including kidney bean, red bean and mung bean has been reported (Tang, 2008). For instance, the T_d of kidney bean vicilin protein (90.2ºC) was high compared with those of red bean (87.1ºC) and mung bean (84.6ºC). Since T_d is the measure of thermal stability, these results suggest that kidney bean is more thermal stable than red bean and mung bean. Values of thermal properties reported for red bean by Tang (2008) were similar to those of Meng and Ma (2001).
The influence of heating temperature on thermal denaturation of legume proteins has been investigated (Sorgentini et al., 1995, Remondetto et al., 2001). Sorgentini et al. (1995) reported that heating protein at 100^oC for 30 min led to the denaturation of both 7S and 11S proteins. At 80^oC the 7S was totally denatured but the 11S still retained its conformational structure.

1. INTRODUCTION 
2. LITERATURE REVIEW 
2.1. Distribution, habitat and morphological characteristics of marama seed
2.2. Legume seed microstructure
2.3. Protein composition of legumes
2.3.1. Amino acid compositions
2.3.2. Protein types
2.3.3.1. Globulins
2.3.3.2. Subunit composition
2.4. Functional properties of legume proteins
2.4.1. Thermal properties
2.4.2. Rheological properties
2.5. Conclusions
2.6. Hypotheses
2.7. Objectives
3. RESEARCH 
3.1. MICROSTRUCTURE OF PROTEIN BODIES IN MARAMA BEAN SPECIES
3.1.1. Introduction
3.1.2. Materials and methods
3.1.2.1. Materials
3.1.2.2. Sample preparation
3.1.2.3. Proximate composition
3.1.2.4 Mineral composition
3.1.2.4. Light Microscopy (LM), Transmission Electron Microscopy (TEM) and Confocal Laser Scanning Microscopy (CLSM)
3.1.2.5. Proteinase K digestion of marama bean and soya bean cotyledons
3.1.2.6. Statistical analyses
3.1.3. Results and discussion
3.1.3.1. Chemical composition of marama bean
3.1.3.2. Microscopy of protein bodies in marama bean
3.1.4. Conclusions
3.2. COMPOSITION OF MARAMA BEAN PROTEIN
3.2.1. Introduction
3.2.2. Materials and methods
3.2.2.1. Materials
3.2.2.2. Chemicals
3.2.2.3. Protein preparations
3.2.2.4. Amino acid analysis
3.2.2.5. SDS-PAGE
3.2.2.6. Two-dimensional gel electrophoresis
3.2.2.7. Statistical analysis
3.2.3. Results and discussion
3.2.3.1. Protein contents and yields of marama protein extract
3.2.3.2. Amino acid profile
3.2.3.3. Protein subunit composition
3.2.3.4. Two-dimensional gel electrophoresis
3.2.4. Conclusions
3.3. THERMAL AND RHEOLOGICAL PROPERTIES OF MARAMA BEAN PROTEIN
3.3.1. Introduction
3.3.2. Materials and methods
3.3.2.1. Chemical composition of protein preparations
3.3.2.2. Thermal properties
3.3.2.3. Rheological properties
3.3.2.4. Rheological properties of peroxidase treatred protein dough
3.3.2.5. Statistical analysis
3.3.3. Results and discussion
3.3.3.1. Chemical composition of protein preparations
3.3.3.2. Thermal properties of marama protein
3.3.3.3. Rheological properties of marama protein
3.3.3.4. Rheological properties of peroxidase treated marama protein dough
3.3.4. Conclusions
4. GENERAL DISCUSSION 
4.1. Critical review of methodology
4.2. Relationship between marama protein composition and functionality
4.3. Potential applications
5. CONCLUSIONS AND RECOMMENDATIONS 
6. REFERENCES 
7. PUBLICATION, PRESENTATIONS AND CONFERENCES ATTENDED BASED ON THIS WORK
GET THE COMPLETE PROJECT