ABSTRACT
The industrial utilization of native starch is limited by its imperfect nature, hence the need for modifications. The continued use of conventional crops such as cassava, maize, rice and potato as sources of starch for industrial uses adds to the demand pressure on these crops. On the other hand, the isolation of starch from underutilized legumes will not only reduce this pressure, but will also add value to boost the economic potential of these legumes. This work was aimed at comparing the effects of different modifications on the cold water solubility and functional properties of starch isolated from Vigna subterranea, Sphenostylis stenocarpa, Cajanus cajan, and Mucuna pruriens var pruriens. The recovered starch yield ranged between 70 99 %. Most of the modifications enhanced the desirable properties of the starches. The cold water solubility of the modified starches were in the range of 35 81 %, with acid-alcohol, alcohol- alkaline and acid hydrolysis modifications giving the best solubility, while heat moisture treatment and carboxymethylation modifications gave the least solubility. Most of the modified starches had high swelling power, with the exception of the acid treated starches. The pH of the modified starches were around neutrality (pH 7) excluding acid-treated which was neutralised with a 1 M NaOH solution. The water absorbing capacity of the starches increased with increasing solubility for most modified starches excluding the acid-treated starches. There were reductions in the amylose content of most of the modified starches with the exception of the acid hydrolysed, pyrodextrinized and osmotic pressure treated starches. The gelatinisation temperature of the modified starches reduced with increasing solubility. The gelatinisation temperature analyses of the starches
ranged between 68 and 72 0C for the native starches and 34 and 60 0C for the  modified starches. The moisture contents of the modified starches were relatively low. The clarity of the starches varied, with the oxidized and acid-alcohol  treated starches giving the  highest clarity.  The  modified  starch  yield  ranged  between  70  %  and  99  %.  Based  on  visual assessments, the samples were relatively clean and white with a minor contribution of colour from the seed coat of the grains. There were some variations in the transmittance properties of the pastes of the native and modified  starches.  These  properties  of modified  starches suggest that starches from underutilized  legumes could  be used in the production  of cold water soluble starch thereby increasing its value addition
CHAPTER ONE
INTRODUCTION
Starch is an abundant organic polysaccharide molecule in nature with two main components- amylose and amylopectin. The industrial utilisation of native starch is limited because of its imperfect nature therefore the importance of modifications for both industrial and food applications (Kavlani et al., 2012). Legumes are fruits or seeds of prime importance in human and animal nutrition and contain moderate amounts of carbohydrate and protein. The legumes in the study (Cajanus cajan, Mucuna pruriens var pruriens, Sphenostylis stenocarpa, and Vigna subterranea) is usually cultivated by the rural community. Such subsistence cultivation makes this legume, which show promise despite its shortcomings, underutilized and neglected. In order to increase legume production and utilisation, one of the approaches is to exploit its major components, starch through value- added product design and development strategy (Shimelis et al., 2006). Harnessing the potentials of underutilized legumes as invaluable sources of starch could be a way out (Adebowale et al., 2002; Adebowale and Lawal 2003; Shimelis et al., 2006). Producing starch and starch derivatives from conventional plants like cassava, maize, rice and potato places too much demand on them, particularly as they have to meet the needs of both domestic and industrial uses; thus, the need to include legumes more so, underutilized ones (Lawal, 2004). The industrial utilization of native starch is limited because of its imperfect nature such as water insolubility, low thermal resistance, tendency to easily retrograde, unstable pastes, low thickening power, and viscosity (Akpa and Dagde, 2012; and Ashogbon and Akintayo, 2014). Modification of starch is carried out to enhance the positive attributes, and to eliminate the shortcomings of native starch (Vaclavik and Christian, 2008). Starch modification could be by physical, chemical, enzymatic and genetic methods (Kavlani et al., 2012). Subsequently, studies have been performed by preparing cold water soluble starch by various modifications (Sair, 1964; Trubiano, 1987; Khalil et al., 1990; Chen and Jane, 1994; Bello-Perez et al., 2000; Atichokudomchai and Varavinit, 2003; Sanchez-rivera et al., 2005; Sang et al., 2007; Chatakanonda et al., 2011; Xin et al., 2012, Yu et al., 2015).
This work was aimed at comparing different physical and chemical modifications on the cold water solubility of some underutilised legume starches alongside evaluating the functional properties of the starches to improve the method for industrial application.
1.1 Legumes
Legumes are second only to the Graminiae in their importance to humans. The seeds are of prime importance in human and animal nutrition due to their high protein content (20 50
%) which is significantly more than the level found in their root crop counterparts; yam and cassava (Ustimenko-Bakumovsky, 1983). Legumes are important ingredients of diet in many parts of the world and have been considered as the most significant food sources for people of low incomes (Bressani and Elias, 1979) Legumes contain about 60 % carbohydrates in which starch constitutes the major portion (Sathe and Salunkhe, 1981). Refined starches from several cereals, roots and tubers are used widely in industrial and food applications but legume starches have few commercial uses (Hoover and Sosulski
1989; 1991). Legume plants belong to the family variously referred to as Fabaceae or Leguminosae within the order Fabales. To date, approximately thirteen to eighteen thousand species of legume have been discovered (Aykroyd and Doughty 1964).
1.1.1 Underutilized Legumes
The concentration on a few major staple crops has resulted in an alarming reduction not only on crop diversity but also the variability within them, especially the neglected and underutilized species (NUS). NUS are indigenous, relatively common, available, accessible, well-adapted, easy and cheap-to- produce crops. Moreover, they are culturally linked to the people who use them traditionally (Jaenicke and Pasiecznik, 2009). Underutilized species are usually ignored by policy makers probably because their economic value is not apparent and hence are excluded from research and development agenda of research and academic institutions (Stifel, 1990).
Across the world, many of the plant species that are cultivated for food are neglected and underutilized while they play a crucial role in food security, nutrition, and income generation of the rural poor (CGIAR, 1999). The term neglected and underutilized species refers to a category of non-commodity cultivated and wild species, which are part of a large agro-biodiversity portfolio today falling into disuse for a variety of agronomic, genetic, economic, social and cultural factors (CGIAR, 1999). Neglected and underutilized species are traditionally grown by farmers in their centres of diversity. While these crops continue to be maintained by cultural preferences and traditional practices, they remain inadequately characterised and neglected by research and conservation. Lack of attention indicates that their potential value is underestimated and underexploited. It also places
them in danger of continued genetic erosion and disappearance which would further restrict development options for the poor. Many neglected and underutilized crop species (NUCS) are nutritionally rich (Dansi et al., 2012); therefore their erosion can have immediate consequences on the nutritional status and food security of the poor while their enhanced use can bring about better nutrition and reduce hunger. A lot of NUCS are recorded to be adapted to difficult environments unfit for other crops where they can provide sustainable productions (Dansi et al., 2012). In this way, they contribute significantly to maintain diversity rich and hence more stable agro-ecosystems. Only about
30 crop species provide 95 % of the worlds’ food energy whereas over 7,000 species have been known to be used for food and are either partly or fully domesticated. This large array of plant species includes those recognized to be underutilized as well as those that are recognised as important minor crops. Uses also vary from place to place for instance, the legume Lathyrus is largely used for fodder in Turkey but in South Asia is mostly used as human food (Dansi et al., 2012).
In developing strategic approaches there has been the tendency to build on successful experiences with underutilized crops; Africa hosts thousands of edible plants, but only a small number dominate agriculture. However, these crops decline rapidly and their potential is often overlooked. Worldwide, farmers are abandoning them as globalisation, population growth and urbanisation change agricultural and food systems (Dansi et al.,
2012). There is growing international recognition that nutritious NUS crops are important in improving the livelihoods of smallholder farmers in Africa and because many NUS are well adapted to marginal environments, they also offer opportunities for climate change adaptation (Dansi et al., 2012).
Bambara groundnut, for instance, is drought tolerant, which helps farmers manage risks. It has high nutritional value yet its constrained by weak value chains that largely involve local markets. The knowledge acquired in developing value chains of this crop can be applied also to many other NUS. Commercialising NUS requires a holistic value chain approach, supportive policies and receptive markets. The need to harness the potentials of underutilized legumes as invaluable sources of starch and protein concentrates have been emphasised (Adebowale et al., 2002, Adebowale and Lawal, 2003). Producing starch and starch derivatives from conventional food crops like cassava, maize, rice and potato, places too much demand on them, particularly as they have to meet the nutritional needs of a high percentage of the population (Adebowale and Lawal, 2003).
1.1.1.1 Cajanus cajan (Pigeon Pea)
Though largely considered an orphan crop, pigeon pea has a huge untapped potential for improvement both in quantity and quality of production in Africa. Typically, the average nutritional composition of pigeon pea is 19.2 % protein, 57.3 % carbohydrates, 1.2- 1.5 % fat, 8.1 % fibre and 3.8 % ash (Smartt, 1976). In addition to the benefit of serving as a starch source, such efforts could lead to a reduction in the over-dependence on cassava starch for food and industrial purposes, reduce post- harvest losses and increase the utilization and potential of these largely underutilized crops in Nigeria and most parts of Sub-Saharan Africa (Smartt, 1976). In a study comparing the properties of thermal alkaline treated pigeon pea, Roskhrua et al. (2013) reported an increase in the granule morphology and gelatinisation temperature alongside a reduction in the swelling power and viscosity of the modified pigeon pea starch. Srijunthongsiri et al. (2014) also reported a decrease in viscosity and an increase in gelatinisation temperature of Cajanus cajan starch.
Plate 1- Cajanus cajan Fig. 1: Image showing Cajanus cajan plant
(Smartt, 1976)
1.1.1.2 Mucuna pruriens var pruriens
Mucuna pruriens var pruriens also known as velvet bean is a trailing vine yielding seeds which vary in colour and shape. The seed has a starch content of about 51.5 % (Sridhar and Seena, 2006).
Plate 2- Mucuna pruriens var pruriens Fig. 2: Image showing Mucuna pruriens plant
(Burkill, 1995)
M. pruriens had a great taxonomic confusion about its varietal difference but now it is accepted that there are two varieties namely, M.pruriens var. utilis and M. pruriens var. pruriens (Carsky et al., 1998; Sridhar and Seena, 2006). The main differences are the pubescent hairs on the pods, the seed coat colour and duration of harvesting. M. pruriens (itching beans) have long stinging hairs on their pods and contact on them results in itching dermatitis, whereas M. utilis possess silky hairs on their pods; Interestingly, unconventional legumes as these are promising in terms of nutrition, provision of food security, agricultural development, crop rotation and industrial purposes in developing countries (Sridhar and Seena, 2006). Mucuna pruriens var pruriens is an annual perennial, herbaceous, vigorous climbing vine that grows to 3-18 cm in height. The M. pruriens is traditionally used as food by certain ethnic groups in a number of countries including India, Philippines, Nigeria, Ghana, Malawi and Brazil (Carsky et al., 1998). Although the M. pruriens contains high levels of protein and carbohydrate, its utilization is limited due to the presence of a number of anti-nutritional/ anti-physiological compounds such as phenolics, tannins, L-Dopa, lectins and protease inhibitors, which reduce the nutrient utilization potential (Pugalenthi et al., 2005). Adebowale and Lawal (2003) reported an increase in moisture content and a decrease in solubility for M. pruriens starch.
1.1.1.3 Sphenostylis sternocarpa (African Yam Bean)
The plant, Sphenostylis stenocarpa, is a tuberous underutilized legume of tropical Africa used in human and animal nutrition (Adebowale et al., 2002; Eke, 2002). Like most grain legumes cultivated in Africa, African Yam bean is rich in protein (19.5 %), carbohydrates (62.6 %), fat (2.5 %), vitamins and minerals (Iwuoha and Eke, 1996). The protein is made up of over 32 % essential amino acids, with lysine and leucine being predominant (Onyenekwe et al., 2000). In spite of its composition, it has a low consumption rate. This is mainly due to its long cooking time of about 145 minutes (Nwokolo, 1996).
Plate 3 Sphenostylis stenocarpa Fig. 3: Image showing Sphenostylis stenocarpa plant
(Iwuoha and Eke, 1996)
African yam bean is a vigorous herbaceous climbing vine reaching 1.5 – 2 metres in height producing pods as well as small spindle shaped tubers about 5 – 8 cm long, just like sweet potato. It is usually cultivated as a secondary crop with yam in Ghana and Nigeria. A few farmers who still hold some seed stocks, especially the white with black-eye pattern, plant it at the base of yam mounds in June or July. The crop flourishes and takes over the stakes from senescing yam. It flowers and begins to set fruits from late September and October. The large bright purple flowers result in long linear pods that could house about 20 seeds (Potter, 1992). Adebowale et al. (2009) reported a decrease in moisture content making it a good source of starch with a capacity for prolonged shelf life; also, they reported spherical granule morphology.
1.1.1.4 Vigna subterranea (Bambara Groundnut)
Though considerably not popular throughout the world, cultivation of Vigna subterranea occur in some African countries like Zimbabwe, Ghana, South Africa and Nigeria alongside some countries like India, Sri Lanka, and Malaysia (Goli, 1997).
Plate 4- Vigna subterranea Fig. 4: Image showing Vigna subterranea plant (De Kock,
2004).
Bambara groundnut is highly nutritive and contains high quantities of carbohydrate, protein and fat. The starch exhibits higher swelling power, breakdown and set back but lower gelatinization temperature, pasting temperature, water and oil absorption capacity (Gidley, 1987). Bambara groundnut contains 60 % carbohydrate, 20 % protein, 6 % oil and rich in micronutrients, they are reported to provide more methionine than other grain legumes (De Kock, 2004). A few studies have reported on the structure and functional properties for bambarra groundnut starch and flour. Adebowale et al. (2002) reported that the swelling capacity increased with increase in temperature for both starch and flour of bambarra groundnut. This bean flour had higher water absorption capacity than that of Great Northern bean, reported by Sathe and Salunkhe (1981). Piyarat (2007) reported an increase in swelling power and reductions in gelatinisation temperature and absorption capacity.
1.2 Starch
Starch is a natural glucose-based polymer; it is the major carbohydrate storage material which exists in form of granules in many higher plants, and is composed essentially of
-D-glucopyranosyl units and small amounts of non-carbohydrate components , particularly lipids, proteins, phosphorus (Liu, 2005; Xie, 2008).
1.2.1 Sources of Starch
Starch occurs mainly in the seeds, roots and tubers of higher plants (Ashogbon and Akintayo, 2014). Plants synthesize starch during photosynthesis. It is synthesized in plastids as a storage compound for respiration at dark periods, also, synthesized in amyloplasts found in tubers, seeds and roots as a long-term storage compound in which large amounts of starch accumulate as water-insoluble granules (El-Fallal et al., 2012). The shape and diameter of these granules depend on the botanical origin. Regarding to commercial starch sources, the granule sizes range from 2
synthesis of starch. Sucrose is the starting point of starch synthesis. It is converted into the nucleotide sugar ADP-glucose that forms the actual starter molecule for starch formation. Subsequently, enzymes such as soluble starch synthase and branching enzyme synthesize the amylopectin and amylose molecules (Smith, 1999; El- Fallal et al., 2012).
Starch-containing crops form an important constituent of the human diet. Besides the direct use of starch-containing plant parts as a food source, starch is harvested and chemically or enzymatically processed into a variety of different products such as starch hydrolysates, glucose syrups, fructose, starch or maltodextrin derivatives, or cyclodextrins. In spite of the large number of plants able to produce starch, only a few plants are important for industrial starch processing. The major industrial sources of starch are maize, cassava, potato, and wheat (El-Fallal et al., 2012).
1.2.2 Structure and Properties of Starch
Starch, as extracted from various plant tissues, is obtained in the form of granules with typical particle sizes between 1-100 microns. However, the shape and size of the granules depend on the source (French, 1984; Chen et al., 2010). These granules are present in the chloroplasts of green leaves and in the amyloplasts of storage organs such as seeds and tubers. Starch granule differences amongst various plant species are accounted for, not only
by the ratio of constituent molecules, but also by their location and interaction (Zobel,
1988a, 1988b). The crystalline composition consists of around 15 to 45 % of the starch granules. The diameter of starch granules ranges from 2 to 100 (Whistler and Daniel, 1985). The crystallinity of native starch varies between 15 and 45 % depending on the origin and pre-treatment (French, 1984). According to the currently accepted concept, amylopectin forms the crystalline component whereas amylose exists mainly in the amorphous form (Zobel, 1992; Hanashiro et al., 1996; Marc et al., 2002; El- Fallal et al., 2012). Granules are (in fact) insoluble in cold water but swell and form a gel if the outer membrane has been removed by grinding. On the other hand, if a granule is treated in warm water, a soluble portion of the starch diffuses through the granule wall and the remainder of the granules swells to such an extent that they burst (Ashogbon and Akintayo, 2013; 2014).
Starch is made up mostly of amylose and amylopectin chains (Table 1). The structural and molecular arrangement is peculiar for amylose and amylopectin fractions and their ratios in starch vary depending on the source (Ashogbon and Akintayo, 2014). The activity of the enzymes involved in starch biosynthesis may be responsible for the variation in amylose content of the various starches (Krossmann and Lloyd, 2000; Marc et al., 2002). Starch in its native form is insoluble in cold water but can be solubilised by heating with excess water. The change in the conformation during application of moist heat results in the loss of the crystallisation of amylopectin followed by swelling, hydration & solubilisation (Krossmann and Lloyd, 2000). The process is called gelatinisation.
Table 1: Properties of amylose and amylopectin fractions of starch
Properties | Amylose | Amylopectin |
Chain length Arrangement | Long, straight Densely packed | Branched More open, lower density |
Size | Small | Large |
Rate of digestion | Slow | Faster |

Source: (McWilliams, 2001)
1.2.3 Components of Starch
1.2.3.1 Major Components of Isolated Starch
1.2.3.1.1 Amylose
Amylose is a linear molecule consisting of -(1-4) linked glucose residues with a small
-(1-6) linkages (Fig. 1). It makes up a minor fraction of the starch granule where it generally accounts for 20 – 30 % of the total starch content (Chen and Jane, 1994). Each macromolecule of the linked glucose residues bears a reducing and non-reducing end. Amylose is located in the granule as bundles between amylopectin clusters and randomly dispersed. They could be located therefore between the amorphous and crystalline regions of the amylopectin clusters (Robin et al., 1974). Though amylose is the minor component in most granules, it has a large influence on the properties of starch (Takeda et al., 1992). The length of the amylose chain is not the same for every source; it varies among different plant species but usually ranges between 102 – 104 glucose units. The amylose is essentially linear but not purely and its properties when dissolved are generally regarded as typical of a linear polymer (Biliaderis, 1990). In a study on the properties of thermal alkaline treated pigeon pea, Roskhrua et al. (2013) reported a reduction in amylose content after modification.
Fig. 5: Structure of amylose (Zhong et al., 2006).
1.2.3.1.2 Amylopectin
Amylopectin is the major constituent of starch which consists of large highly branched
– (1-4) linked gluco- (1-6)
linkages (Manners, 1989). The average frequency of branching points in amylopectin is 5
% but varies with the botanical origin (Thompson, 2000). The complete amylopectin molecule contains about 2,000,000 glucose units; making it one of the largest molecules in
nature (Marc et al., 2002).The multiplicity in branching is a common feature of both amylopectin and glycogen. The outer chains are linked by glycosidic bonds at their potential reducing group through C6 of a glucose residue to an inner chain (Fig. 2). Such chains are in turn defined as chains bearing other chains as branches. The single other chain per molecule likewise carries other chains as branches but contains the sole reducing terminal residue. The ratio of chain to chain is an important parameter which is also referred to as the degree of multiple branching (Marc et al., 2002). Roskhrua et al. (2013) reported an increase in the amylopectin content of Cajanus cajan starch after modification.
1.2.3.2 Minor Components of Isolated Starch
Besides proteins, other minor constituents including lipids, phosphorus, and trace elements, are commonly found in isolated starch (Champagne, 1996). The minor components are categorized into three groups: particulate material, surface components and internal components (Galliard and Bowler, 1987). Particulate materials are fragments of non-starch material that separate with starch, surface components are associated with the surface of granules and can be removed by extraction, and internal components are materials that are buried within the granule and are inaccessible to extraction unless the granule has been subjected to disruption (Galliard and Bowler, 1987). Starch proteins can be classified as surface proteins which can be extracted in aqueous solutions, or as integral proteins, which are extractable only when a starch solution is heated to temperatures near the gelatinisation temperature (Morrison and Karkalas, 1990; Majoobi et al., 2011). Starch contains several different minerals in small amounts, but the most important mineral is phosphorus (Buleón et al., 1998). Phosphorus plays an extremely important role in starch functional properties, such as, paste clarity, viscosity consistency, and paste stability. Phosphorus in starch is mainly present in two forms; phosphate-monoesters and phospholipids. In tuber starches, lipids are only found on the granule surface, while starches from cereal endosperm have
surface and integral lipids (Morrison et al., 1984 and Davis et al., 2003). It is well established that polar lipids, e.g. monoglycerides and fatty acids, form a helical inclusion complex with the amylose molecule (Zobel et al., 1988a; Biliaderis, 1990; Rutschman and Solms, 1990).
This material content is developed to serve as a GUIDE for students to conduct academic research
COMPARATIVE STUDIES ON THE EFFECTS OF DIFFERENT MODIFICATIONS ON THE COLD WATER SOLUBILITY OF STARCH FROM SELECTED UNDERUTILIZED LEGUMES>
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