STRUCTURE AND CHEMICAL COMPOSITION OF COWPEA SEEDS

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LITERATURE REVIEW

INTRODUCTION

This review discusses the science and mechanisms involved in the development of the hard-to cook (HTC) defect in legumes. It also reviews the current knowledge on the effects of high temperature and high humidity storage conditions (HTHH) on legume seed in respect of the HTC defect. It also focuses on the principles involved in γ-irradiation and reviews the effects of γ irradiation on the major components of legume seeds, which have been cited as being involved in the HTC defect.

STRUCTURE AND CHEMICAL COMPOSITION OF COWPEA SEEDS

Cowpeas are small dicotyledonous legume seeds which vary in shape (kidney or globular) (Taiwo, 1998) and have a 100 seed weight of 2-28 g (Langyintuo, Ntoukam, Murdock, Lowenberg-Deboer and Miller, 2004). The two major structural components of cowpea seeds are the seed coat and the cotyledon (Figure 2.1). The cowpea seed coat like other typical legumes contains numerous specified tissues, which include the micropyle, hilium and raphe (Ma, Cholewa, Mohammed, Reterson and Gijzen, 2004). The seed coat (testa) serves as a protective barrier of the cotyledons from the external environment. The seed coat is rich in minerals such as magnesium, iron, calcium, zinc, potassium, and iron as well as phytate, tannins, and phenolics (Adebooye and Singh, 2007). The colour of the seed coat may vary depending on the phenolic compounds present. The range of colours is wide, and includes red, brown, purple, white and black, with the most common being white, brown and a combination of both (Taiwo, 1998; Affrifah, Chinnan and Phillips, 2005). The cowpea seed coat can be tightly bound to or loosely bound to cotyledons. The surface of the seed coat can be rough, smooth or wrinkled (Olapade, Okafor, Ozumba and Olatunji, 2002).
The cowpea seed like all legumes contains two cotyledons protected by the seed coat. The cotyledons form the major part of the seed with respect to both weight and volume (Sefa-Dedeh, Stanley and Voisey, 1979). The cotyledons consist of large parenchyma cells, which range in size from 70 – 100 μm (Tiwari and Singh, 2012). The parenchyma cells are bound together by the cell walls and middle lamellae. The middle lamella contains pectic substances and protein which help bind adjacent cells together. The parenchyma cells contain starch granules and storage protein. The starch granules are embedded in a protein matrix and the protein bodies are surrounded by a membrane of lipoprotein (Figure 2.2) (Liu, McWatters, Phillips, 1992). The protein bodies contain crystalline inclusions known as globoids that are rich in phytin. The structure and composition of the cotyledons are of importance as they influence the cooking quality of the seeds.
The proximate composition of cowpea seeds varies according to variety, climate, growth location and agriculture practices (Hsieh, Pomeranz and Swanson, 1992). Cowpeas are not only an important source of proteins and carbohydrates but are also a good source of several B-complex vitamins, minerals and dietary fibre (Table 2.1). In addition, they are a rich source of minerals, such as calcium, magnesium, potassium, and phosphorus.

LEGUME COTYLEDON CELL WALL STRUCTURE AND COMPOSITION

Plant cell walls are dynamic structures, composed of complex polysaccharides (cellulose, hemicellulose, and pectin), small amounts of phenolic compounds (lignin and hydroxycinnamic acids esterified to the cell wall polysaccharides) and proteins (extensin and enzymes) with ionic and covalent linkages stabilising its components (Rodionova and Bezborodov, 1997). An understanding of the cell wall polysaccharide structure and composition is essential in order to understand the changes in the structural organisation of the polysaccharides that occurs during cooking. The mature cotyledon cell walls of pulses typically contain about 25-30% cellulose, 15-24% hemicelluloses, 0.4-0.6% lignin and 28-41% pectin (Tiwari and Singh, 2012). Structural proteins constitute about 1-5% on dry basis (Ochoa-Villarreal, Vargas-Arispuro, Aispuro-Hernández and Martínez-Téllez, 2012). The rigidity of the pulse cell wall is brought about by cellulose and hemicellulose polymers, with pectin providing fluidity through the gelatinous polysaccharides matrix, ensuring a strong yet dynamic and flexible properties of the cell wall (Ochoa-Villarreal et al., 2012). Cellulose and hemicelluloses are embedded in the amorphous pectin polymers with stability provided by the proteins and phenolic compounds (Sorieul, Dickson, Hill and Pearson, 2016). Hemicelluloses are bound to the surface of the cellulose network preventing direct contact among microfibrils, and pectin is linked to hemicelluloses forming a gel phase (Ochoa-Villarreal et al., 2012).

CELLULOSE

Cellulose is the major biopolymer which provides structural support in the plant cell wall (Figure 2.3). Cellulose comprises a third of the total mass of the plant cell wall (Ochoa-Villarreal et al., 2012). It is a linear unbranched polymer composed of β-1,4-linked glucan chains organised in more or less crystalline microfibrils. The cellulose polymers are associated with each other via hydrogen bonding and van der Waals forces.

HEMICELLULOSES

Hemicelluloses can be defined as cell wall non-starch polysaccharides that are not solubilised by hot water or chelating agents but are solubilised by aqueous alkali (Rose, 2003). In dicotyledonous plants, these include several polymers, mainly xylans, xyloglucans, and glucomannans which are characterised by having a backbone of β-1,4-linked sugars with an equatorial linkage configuration (Scheller and Ulvskov, 2010). The structural similarity of hemicelluloses and cellulose allows for the formation of strong non-covalent associations between the two types of polysaccharides.

Xyloglucans

Xyloglucans are heteropolysaccharides made up of a repeated structure of different monosaccharides. These are the predominant hemicellulosic polysaccharides in the primary cell walls of dicotyledons. Xyloglucans have a ‘cellulosic backbone’ consisting of 1,4-linked β-D-glucose, with several regularly spaced xylose side chains with D-xylose at C6 (α) for most glucose residues (Hedley, 2001). Disaccharides (α-L-fucose-1,2-β-D-galactose) and sometimes β (1→2)-linked L-arabinose may substitute some of the xylose residues (Gibeaut and Carpita, 1994). Cellulose and xyloglucans are found in equal proportions in Type I plant cell walls, which is predominant in dicotyledonous plants. Some xyloglucan chains play a role in supporting rigidity and cell maintenance, being crosslinked to cellulose microfibrils and pectin polymers (Ochoa-Villarreal et al., 2012).

Xylans

Xylans are a heterogeneous group of polysaccharides with a backbone of β-(1→4)-linked xylose residues (Heinze, Koschella and Ebringerova, 2004). The substitutions which may occur vary widely in composition and distribution according to plant species. They may have substitutions with α-(1→2)-linked glucuronosyl and 4-O-methyl glucuronosyl residues (Ebringerova and Heinze, 2000). Xylans generally contain many arabinose residues attached to the backbone, and these are known as arabinoxylans and glucuronoarabinoxylans (Wertz and Bédué, 2013). Primary cell walls of dicotyledonous plants have small amounts of glucuronoarabinoxylans, while the endosperm of cereals has high amounts of arabinoxylans (Brett and Waldron, 1996). Xylan polymers in the cell wall can crosslink themselves through ferulic acid residues (De O Buanafina, 2009) and also be involved in crosslinking cellulose microfibrils and lignin (Imamura, Watanabe, Kuwahara and Koshijima, 1994; Balakshin, Capanema, Gracz, Chang and Jameel, 2011). Xylan polymers have a significant influence on the integrity of cell wall due to this immense cross-linking (Faik, 2013). Xylans have been documented as a constituent of legume seed cell walls (Stolle-Smit, Beekhuizen, van Dijk, Voragen and Recourt, 1995; Shiga and Lajolo, 2006).

Mannans and Glucomannans

The β-(1,4)-linked polysaccharides rich in mannose or with a backbone consisting of mannose are referred to as mannans and galactomannans (Boraston, Lammerts van Bueren, Ficko-Blean and Abbott, 2007). However, if the polysaccharide consists of mannose and glucose in a nonrepeating pattern, they are the glucomannans and galactoglucomannans. Mannans and glucomannans are often acetylated (Zhang, Rogowski, Zhao, Hahn, Avci, Knox and Gilbert, 2014). The mannans are present in low concentration in the plant primary cell wall of dicotyledons (Liepman, Nairn, Willats, Sørensen, Roberts and Keegstra, 2007).

PECTIN

Pectins are heterogenous cell wall polysaccharides characterised by a high content of α-(1,4)-D-galacturonic acid residues (Broxterman and Schols, 2018). In the primary plant cell wall, the pectic polysaccharides which can be detected in the cell wall include homogalacturonan (HG), xylogalacturonan (XYA), apiogalacturonan, rhamnogalacturonan I (RGI) and rhamnogalacturonan II (RGII) (Figure 2.4) (Harholt, Suttangkakul, and Scheller, 2010). The ratio between HG, XGA, RG-I, and RG-II varies in the cell wall, but typically the most abundant polysaccharide is HG, which constitutes about 65% of the pectin, with RGI representing 20% to 35% (Mohnen, 2008). The primary role of pectin in the plant cell wall in concert with other polymers is to provide physical strength and provide a barrier to the outside environment (Harholt et al., 2010). Pectin polysaccharides constitute a major part of the middle lamella, providing the principal adhesion between adjacent cells in legume seeds (Hoondal, Tiwari and Tewari, 2002). An understanding of pectin structure and composition is thus important to be able to understand and explain changes which occur during cooking and in textural defects that affect legume seeds. The methods used to study pectin involve sequential extraction to acquire the different fractions. Pectins can thus be classified according to their method of extraction, with chelator soluble pectins (CSPs)/ cyclohexane-trans-l,2-diamine tetraacetate (CDTA) soluble pectin fractions representing the pectin which is ionically bound to Ca2+ (Brummell, 2006). The CSPs are enriched in homogalacturonan in the middle lamella (Brummell, 2006). Alkali can also be used in pectin extraction to isolate sodium carbonate-soluble pectins (SSPs) (Brummell, 2006). These pectins show characteristically high ratios of neutral sugars to uronic acid, suggesting enrichment in rhamnogalactorunan I (RG-I) from the primary wall (Brummell, 2006). Water soluble pectins (WSP) represent the freely soluble pectin in the apoplast which can be extracted using water (Redgwell, Melton and Brasch, 1992).

Homogalacturonan (HG)

HG is the major pectin polymer in the primary cell walls of dicotyledonous plant seeds (Figure 2.4). It is a linear polymer made up of 1,4-linked α-D-galacturonic acid residues, with some of the carboxyl groups partially methyl-esterified at C-6 and acetyl-esterified at positions O-2 and/or O-3 (Ochoa-Villarreal et al., 2012). HG units with more than 50% of galacturonic acid residues esterified with methyl (or methoxy) are referred to as high methyl-esterified HGs and those with less as low methyl esterified HGs. Methyl esterification of HGs usually gets more attention as it influences the physical properties of pectin (Wolf, Mouille, and Pelloux, 2009). HG that is unmethylated has a negative charge and may ionically interact with Ca2+ to form a stable gel, which is associated with an increase in cell wall strength (Willats, McCartney, Mackie and Knox, 2001). This occurs when greater than 10 consecutive unmethylated esterified galacturonic acid residues are coordinated and is sometimes referred to as the egg-box model (Caffall and Mohnen, 2009). The egg-box model explains the gelation mechanism by which low methoxyl pectin forms a gel in the presence of calcium. Approximately 70% of pectin in plant cell wall is bound to Ca2+ (Jarvis and Apperley, 1995).

Rhamnogalacturonan-I (RGI)

RG-I is a group of pectin polysaccharides characterised by a backbone of alternating galacturonic acid and rhamnose residues [α-(1,2)-D-GalA-α-(1,4)-L-Rha]n, consisting of large linear or branched arabinose and galactose as the main side chains (Harholt et al., 2010). The side chains are diverse depending on plant source and may include α-L-fucose; β-D-glucuronic acid and 4-O -methyl, as ferulic and coumaric acid. They represent the second most abundant pectin in the plant cell wall. RG-I has been suggested to function as linkage support to other pectic polysaccharides HG and RG-II, which are covalently attached as side chains (Ochoa-Villarreal et al., 2012). According to Zykwinska, Ralet, Garnier and Thibault (2005), RG-I can hydrogen bond to cellulose through some high molecular weight side chains, especially those rich in arabinose and/or galactose.

Rhamnogalacturonan-II (RG-II)

RG-II is the most branched and complex group of pectic polysaccharides. In dicotyledonous plant cells, they are a minor pectic component and represent only 0.5 to 0.8% (Matsunaga, Ishii, Matsunamoto, Higuchi, Darvill, Albersheim and O’Neill, 2004). RG-II comprises a backbone consisting of at least eight α-GalA residues to which five different types of side chains are attached (Ulvskov, 2010). The cluster of side chains is linked to positions O-2 and O-3 on galacturonan backbone (Harholt et al., 2010). The side chains contain rare and unique glycosyl residues and glycosidic linkages (e.g 2-O-methyl-1-Fuc, L-aceric acid and α-1, 3-xylofuranose) (Harholt et al., 2010). RG-II exists in primary walls mainly as a dimer cross-linked by 1:2 borate-diol ester (Ishii, Matsunaga, Pellrin, O’Neill, Darvill and Albershiem, 1999). Almost 95% of RG-II polymers exist as dimmer complex form (dRG-II). The dimerization helps to ensure the integrity of the cell wall (O’Neill, Ishii, Albersheim and Darvill, 2004).

Xylogalacturonan (xyg) and apiogalacturonan (apa)

XYG is a group of pectic polysaccharides consisting of a HG backbone substituted by xylose residues at carbon 3 (O’Neill and York, 2003). In addition, xylose residues can connect to the first substituted xylose with β-1-4 linkage (Zandeleven, Beldman, Bosveld, Schols and Voragen, 2006). It is also a minor component of the cell wall, constituting less than 10% (Zandeleven et al., 2006; Mohnen, 2008). However, XYG was suggested to be in a higher proportion than HG in legumes such as common beans (Phaseolus vulgaris) (Shiga and Lajolo, 2006). XYG is present in peas (Le Goff et al., 2001) and soybeans seeds (Huisman, Brüll, Thomas-Oates, Haverkamp and Schols, 2001). APA is a group of pectin polysaccharides which are actually HG substituted by D-apiose. On the HG chain, apiose residues can be β-2-linked, 3-linked as well as 2- and 3-linked to single galacturonan residues (Wertz and Bédué, 2013).

DECLARATION 
ABSTRACT 
DEDICATION 
ACKNOWLEDGEMENTS
TABLE OF CONTENTS 
LIST OF TABLES
LIST OF FIGURES 
1. INTRODUCTION
2. LITERATURE REVIEW
2.1 INTRODUCTION
2.2 STRUCTURE AND CHEMICAL COMPOSITION OF COWPEA SEEDS
2.2.1 LEGUME COTYLEDON CELL WALL STRUCTURE AND COMPOSITION
2.2.1.1 CELLULOSE
2.2.1.2 HEMICELLULOSES
2.2.1.3 PECTIN
2.3 THEORIES ON MECHANISMS RESPONSIBLE FOR THE HARD-TO-COOK DEFECT
2.3.1 PHYTASE-PHYTATE-PECTIN HYPOTHESIS
2.3.2 LIGNIFICATION HYPOTHESIS
2.3.3 CHANGES IN STORAGE PROTEIN HYPOTHESIS
2.3.4 MEMBRANE DEGRADATION HYPOTHESIS
2.3.5 PROTEIN-STARCH INTERACTIONS HYPOTHESIS
2.3.6 MULTIPLE MECHANISMS HYPOTHESIS
2.4 IRRADIATION
2.4.1 PRIMARY CHEMICAL EFFECTS
2.4.2 SECONDARY EFFECTS
2.4.3 EFFECTS OF γ-IRRADIATION ON MAJOR COMPONENTS OF FOOD
2.5 CONCLUSIONS
3 HYPOTHESES AND OBJECTIVES
3.1 HYPOTHESIS 1
3.2 HYPOTHESIS 2
4 RESEARCH
4.1 EFFECTS OF γ-IRRADIATION ON COTYLEDON CELL SEPARATION AND PECTIN SOLUBILISATION IN HARD-TO-COOK COWPEAS
4.2 EFFECTS OF γ-IRRADIATION ON THE FUNCTIONAL AND THERMAL PROPERTIES OF INDUCED HARD-TO-COOK COWPEA (VIGNA UNGUICULATA L. WALP.) SEEDS AND COOKED PREPARED PASTES
5 GENERAL DISCUSSION 
5.1 A CRITICAL REVIEW OF EXPERIMENTAL DESIGN AND METHODOLOGIES
5.2 SUMMARY OF THE MAIN RESEARCH FINDINGS
5.3 PROPOSED MECHANISMS FOR HTC DEVELOPMENT BASED ON FINDINGS ON THE EFFECTS OF γ-IRRADIATION
5.4 FUTURE RESEARCH
6 CONCLUSIONS AND RECOMMENDATIONS
7 REFERENCES 
8 PUBLICATIONS, PRESENTATIONS AND POSTERS BASED ON THIS RESEARCH
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