Kwame Nkrumah University of Science and Technology

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Legumes are one of the main groups of food commodities which are globally consumed and cultivated but are often utilized in their whole form. With increasing proof of nutritional benefits, legumes are potentially good sources of nutrients including starches. Accordingly, the objective of this study is to investigate the starch-pasting properties of Soyabean (Glycine max), Pigeon pea (Cajanus cajan) and Bambara groundnut (Vigna subterranea) in relation to Cassava (Manihot esculenta) to obtain information on their suitability as food thickeners in the food industry.

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The Rapid Visco Analyzer was used to determine the pasting properties of the various starches.

Legumes are crops that belong to one of the main agricultural commodities. They are seeds of plants associated with the family Leguminosae. Legumes are relatively inexpensive agricultural commodities but they are very important in human diets. In many countries, legumes are stapled diets and provide extremely important nutrients for many consumers. Legumes are important in human diets as they are very good sources of protein and starch (energy).

According to Hedley (2000), legumes contain relatively high amounts of carbohydrate of about 50-65% out of which starch constitutes about 25-50%, 20-30% of protein and oils not greater than 2% for starch-storing legumes. In recent times, the consumption of legumes for starch (energy) and protein has been replaced with cereals and root and tuber crops such as cassava (Erbersdobler, 2017). However, with increasing evidence on their nutritional benefits, legumes are poorly utilized in Ghana. Plant sources including legumes have the potential to provide starches with useful characteristics for the development of new food products.

Lee (2007) reported that starches obtained from different plant sources differ in their physical and chemical characteristics as well as their pasting properties. As a result, starches from particular plants find specific applications during food processing. Generally, the potential of legumes in many industries especially the food industry has not been exploited to its maximum.

Starch is an important component of food providing a large amount (60-70%) of calorie intake for both humans and livestock (Lawton, 2004). Aside it being nutritionally beneficial, starch is a raw material which is versatile with a wide scope of applications in industries namely: food, pharmaceutical, feed, textile, cosmetic and paper industries. In the food industry, starch is used as a thickener, filler contributing to the solid content of stews and soups, a binder to compact the mass of food and prevent it from drying out during cooking and also as a stabilizer. Some non-food applications of starch include; adhesives in the packaging industry, printing and fabric finishing in the textile industry, dispersing and pill coating agents in pharmaceuticals, and also employed in the processing of dry cell batteries and biodegradable plastics (Burrell, 2003; Lawton, 2004; FAO, 1998; Moorthy, 2002).

The major constituent of carbohydrate in legumes is starch; which varies greatly depending on the type of legumes. The starch component of legumes consists mainly of amylose and amylopectin. In legume starches, the amylose content is higher than the amylopectin (Ashogbon and Akintayo, 2014). Despite being rich in starch, legumes have remained underutilized, though starch from these crops could be used in different industrial applications (Lee, 2007). The current industrial demand for starch is being met by a restricted number of crops mainly cassava, corn, potato and wheat (Ellis et al., 1998). In order to increase the competitiveness of starches from legumes on the world markets, the unveiling of the characteristic properties of starches from legume crops is required.

The pasting properties of starch determine its suitability in the food product as it involves the changes that occur when the starch granules are heated and cooled. During the gelatinization process, starch granules absorb water and swell resulting in the leaching of amylose out of swollen granules, which causes an increase in viscosity of the medium (Hermansson and Svegmark, 1996).

In recent times, the consumption of legumes for starch (energy) has been replaced with cereals and root and tuber crops such as cassava (Erbersdobler, 2017). The utilization of legumes in the form of starches remains unpopular in food formulations and novel food products and so a way of improving the interest of legumes industrially is to isolate and study the functional properties of their starches and apply them in both food and non-food systems. Legumes contain starches which can be incorporated into food systems as thickeners. However, there is limited information on the starch-pasting properties of Glycine max, Cajanus cajan and Vigna subterranea in relation to Manihot esculenta which is commonly used in the food industry. Thus, this work seeks to provide information on the food application of legumes (Glycine max, Cajanus cajan and Vigna subterranea) starches as thickeners in the food industry.

[bookmark: _Toc534388642]Legumes are plants that belong to the family Leguminosae and are principally cultivated for their seeds. They contain nutrients like proteins, carbohydrates, fiber, vitamin (niacin and thiamine) and minerals (calcium and iron) with starch being the major constituent of legume carbohydrate (Salwa et al., 2010). Cereals are the main source of human and animal nutrition with legumes being the second. The blend of varied nutrient composition in legumes makes it undeniably a preferred choice over cereals especially in Africa where malnutrition is on the rise. Aside from providing a lot of nutrients to humans when consumed, they also enhance the soil fertility by the fixation of atmospheric nitrogen. Non-starch storing legumes such as soybeans have relatively increased level of edible oil of up to 20% since they do not store starch as their main energy source whereas the starch-storing ones such as Bambara groundnut and Pigeon pea usually have an oil content of 2% or less (Hedley, 2000). Some varieties of legumes mostly grown for human consumption include peanuts, bambara groundnut, kidney beans, pigeon pea, cowpea, green beans, chickpeas, lentils and soyabeans.

Plants of legumes belong to the family generally known as Leguminosae or Fabaceae within the order Fabeles. Approximately, thirteen to eighteen thousands of legume species have been discovered (Encyclopedia Britannica, 2006). A mature legume seed has the embryo, two cotyledons and a seed coat (testa). The seed coat protects the embryo and reduces the rate at which microbial contamination and moisture loss takes place (Lee, 2007). Although certain legumes possess an endosperm structure, in most legumes the cotyledon represents the major constituent of the dry legume seeds and typically contains a reserve supply of starch and protein for growth (Verbersax and Occena, 1993). They are classified into starch-storing and non-starch storing legumes. Legume seeds which contain components other than starch as a principal energy reserve, a typical example being soybean in which oil is present in considerable quantities. In some important legumes, the dimensions and arrangement of the cotyledon cells influence the texture of legumes and the nutrient availability after processing (Fujimora and Kugimiya, 1994).

[bookmark: _Toc534388644]Legumes are cultivated because they provide a wide range of usages, mainly for human consumption and stock feed. In developing countries, legumes are generally used for human consumption, whereas in developed countries and industrialized economies they are principally used as stock feed. In agriculture, they aid in increasing the soil fertility as they have the ability to fix atmospheric nitrogen through their root nodules, which helps to gradually minimize the use of chemical fertilizers. Legumes also have numerous applications as they can be employed not only in food applications but also in textile, adhesive and pharmaceutical applications.

Legumes serve as a good source of nutrients such as carbohydrates, proteins, fat, minerals (calcium, copper and iron) and other vitamins (thiamin and folic acid). Hoover and Zhou (2003) reported that legumes are high in complex carbohydrate content ranging from 24-68% on a dry weight basis. They supply the body with a significant amount of energy when consumed. Legume starch comprises 24-56% of the total carbohydrate content. Legumes can be a good source of protein and therefore are primarily cultivated for their protein source. According to Verbesax and Occena (1993), the protein content of legumes usually ranges from 20-40%. Legumes are known to be higher in protein than any other food and have twice the protein content of most cereals. They are also referred to as the “poor man’s meat” (Aykroyd and Doughty, 1964), as they regularly replace meat, cheese, eggs and fish in the diet and are relatively a cheaper alternative source of protein as compared to animal products. Although most legumes contain little fat, there are few exceptions like soyabean which has a relatively low carbohydrate content (30-40%), high protein (35-45%) and fat (20%).

Although legumes are classified as highly nutritional, they contain antinutrients which can be detrimental to the health when consumed. Antinutrients are substances that reduce the intake, absorption and utilization of nutrients and may produce other adverse effects. Legume seeds in their raw state contain a wide variety of antinutrients which are potentially harmful. Some examples of antinutrients include tannins, phytic acid, saponins, lectins, cyanogenic glycosides and gossypol oxalates amongst others. For instance, lectins tend to agglutinate erythrocyte (red blood cells). Also, the antinutrient phytic acid reduces the absorption of minerals such as zinc, magnesium, copper and iron by strongly binding to the nutrients which ultimately results in the precipitation making it difficult for adsorption. To effectively reduce the high levels of antinutrients in legume seeds, methods such as soaking in water, increased cooking time and fermentation can be employed. Most of the antinutrients present in foods are water-soluble thus they dissolve during soaking. Soaking pigeon pea for 6 to 18 hours reduces the amount of lectin by 38-50% while soaking kidney beans for 12 hours decreases the lectin content by 49% (Ballantyne, 2017). Deactivation methods can however be used to decrease a lot of the antinutrients present. The method of deactivation depends on the legume type and mode of preparation.

Most legumes cultivated are utilized without further processing. Legumes have a wide range of applications in food products, pharmaceutical, dyes and in beverages yet to date most of them have been studied to its maximum. Some underutilized legumes with much industrial potential include bambara groundnut and pigeon pea. More importantly, there is the need to utilize these legumes as they have the potential of curbing food security problems such as shortage and unreliable supply as well as the elevated cost of highly nutritious food. These underutilized legumes are an alternative and reliable means of providing an inexpensive source of rich protein diet.

Starch is a naturally occurring polymer of α-D glucose. It is the main energy reservoir of plants, and also a major source of dietary energy for humans and animals. Starch is found in leaves of all green plants, in seeds, fruits, stems, roots and tubers of most plants. Starch granules are formed in amyloplasts of plants. They are also formed in chloroplasts where they serve as a temporary store of energy and carbon (Lawton, 2004). Aside from its nutritive value, starch is a very useful raw material with a wide range of applications in both the food and non-food industries. Some uses of starch include; food additives to control consistency and texture of sauces and soups, to resist the breakdown of the gel during processing and to increase the shelf-life of an end product in the food industry. With increasing industrial demand for starches, there is the need to explore new and alternative sources of starch. However, for long their role has mostly been that of staple food and food security crops in the developing countries (Scott et al., 2000).

Starch consists of two major components; amylose and amylopectin. The amylose molecule is a linear structure which comprises of α-D-(1,4) glycosidic bonds whereas that of the amylopectin comprises of a highly branched polymer and are linked by α-D-(1,4) glycosidic linkages and α-D-(1,6) glycosidic linkages at branch points. Normal starches contain 20-30% amylose, the difference being made up by amylopectin. However, the relative proportion of amylose to amylopectin may vary from crop to crop and with variety (Shujun et al., 2006). The amylose content values ranging from 13.6-23.8% for cassava, 20-25% whereas the amylose content for legume seeds is 10-66% depending on the type and variety. Legume starches have a high content of amylose than amylopectin so it tends to form a gel more easily (Ashogbon and Akintayo, 2014). Gelatinization temperature is much higher in legumes due to its high amylose content because generally, amylose requires more energy to break its bonds and gelatinize the starch as a result of its more extensive linear hydrogen bonds. The amylopectin structure imparts viscosity while the amylose structure is associated with the gel strength. Amylose linear structure can easily re-associate themselves to form gels through hydrogen bonding whereas amylopectin structure does not form good gels as a result of the larger molecule size which cannot realign that easily due to its branched chain. Amylose is a function of gelation whiles amylopectin indicates viscosity.

Starches in their native form have limited applications in the industries such as the food industry due to extreme conditions like temperature, pH and shear during processing. Native starches are directly obtained from starch-storing plants and have low shear stress resistance, thermal decomposition, high retrogradation and syneresis while the modified starches are native starches which have undergone chemical, physical and enzymatic processing methods (Fleche, 1985). Modification of starch changes starch properties and extends the scope of starch applications. In the cause of widening the application of starches and promoting their utilization, there is a need to enhance their functional properties such as freeze-thaw stability, retrogradation rate, and acid or alkali resistance. Starch modification can be attained by altering the structure and affecting the hydrogen bonding without damaging its granular structure. Some physical starch modification techniques are heat-moisture treatment and pre-gelatinization. Some chemical starch modification techniques involve cross-linking, conversion and stabilization. Usually, modified starches have functional properties that native starches do not provide (Jobling, 2004).

Size distribution and size of starch granules are important properties that influence the functionality of the starches such as swelling, pasting, gelatinization and solubility. For example, due to the small granule size of cocoyam starches, they are employed as fillers in biodegradable plastics and in aerosols as well (FAO, 2000). Granule size functional studies are important in different food and non-food applications (Li et al., 2015). The size of starch granules may range from 1 to 110µm (Hoover, 2001). The morphology of starch varies from a sphere, ellipsoid, polygon, lenticular and irregular tubules (Nepolo et al., 2015; Hoover, 2001). Legume starch granules are bigger and are generally oval in shape with size ranging from 4-80μm (Ashogbon et al., 2011). Morphological studies have reported cassava starch to be round with a flat surface on one side with granule size ranging from 4-43μm (Wickramasinghe et al., 2009). Larger granules are reported to have higher amylose content and generally imply high viscosity but tend to gel easily since the molecules easily re-associate hence they have higher setback values than smaller ones (Tiwari and Singh, 2012). Large legume starch granules can therefore be used in the production of noodles since it gelatinizes faster. The factors affecting granular morphology and size distribution includes plant genotype, cultivar, amyloplast biochemistry, biological origin, seasonal changes, cultural practices and plant physiology (Lu et al, 2008; Singh and Kaur, 2009). According to Cardoso et al. (2006), the morphology of starch granules is also affected by the method of extraction, physical operations such as drying, grinding/milling, and seed coat removal or purification process.

The use of starch in various industries is determined by its functional properties such as gelatinization, pasting, retrogradation, viscosity, swelling and solubility which vary considerably depending on the source of starch. The functional properties of starches are dependent on the composition and molecular structures of the starches which include amylose/amylopectin ratio, phosphorus content, granular size, the molecular weight of the starches and chain length distribution of amylopectin (Lu et al., 2005; Fredriksson et al., 1998). Therefore characterization of starches for their physicochemical, functional and structural properties is essential in order to unravel their potential for use in the food and non-food industries.

In determining the application of starches in food very important characteristics like viscosity, stability and clarity of starch pastes need to be identified. For starch-based food products where starches are employed as thickeners, clarity of gel becomes a desirable function as it influences the opacity and brightness of a food product. For instance, starches used to thicken salad dressings are preferred to be opaque while those in pies are meant to be transparent. In regulating the texture and consistency of foods such as soups and sources, the paste stability of the starch is an important feature of starch as it increases the shelf-life of products (FAO, 1998).

Pasting is an important property of starch that governs its application. The Rapid visco-analyzer (RVA) has been generally used in evaluating the pasting properties of starch and starch-containing products. It is an important tool for measuring viscosity and other quality parameters of starch necessary for its wide applications. Other instruments used include the Ottawa starch viscometer (OSV) and Micro Visco-amylograph (Zhou et al., 1998). The Rapid visco-analyzer is preferred because of its versatility, faster and stronger mixing and the fact that it can analyze multiple properties at a time.

Pasting involves a sequence of events which begins with gelatinization and ends with retrogradation. Gelatinization occurs when starch is dissolved in excess water and heated at room temperature, the temperature gradually increases until it reaches 95ºC with gelling or swelling beginning at 50ºC. During gelatinization, granules hydrate progressively by imbibing in water, hydrogen bonds are distorted resulting in crystalline regions being converted into amorphous regions, starch granules begin to imbibe in water and ultimately the granule swells so much that the short chains of amylose leach out of the starch granules resulting in the formation of a viscous medium (Testler and Karkalas, 2004). Retrogradation occurs when the starch is cooled or stored at low temperatures. It involves the re-association of molecules resulting in crystalline aggregates and a gelled structure. Retrogradation is an important property needed in the formulation of food products as it influences the shelf-life quality and acceptability of starch-containing foods. For starch to ascertain its suitability in food and non-food systems, important parameters like Peak viscosity, Pasting time and temperature, Hot paste viscosity, Setback value, Breakdown value and the Final viscosity needs to be determined. For example, the higher the breakdown viscosity the lower the ability of the starch to withstand shear and heat during cooking (Adebowale et al., 2005) Factors that influence the pasting properties of starch are the granule size, the amylose, amylopectin and lipid component, chemical and enzymatic modifications as well as ingredients employed during food processing.

The temperature at which the starch granules swell or gel when dissolved in boiling/ hot water usually at 50ºC. It is a useful parameter as it indicates the minimum temperature and time needed for cooking and other applications. Maskan and Altan (2016) reported that pasting temperature is dependent on the starch type, starch concentration processing method and ingredients. The onset of paste formation begins with gelatinization which involves the breakdown of the intermolecular bonds of starch molecules when it is subjected to water and heat causing starch granules to swell to form a gel, by incorporating in water. Higher gelatinization temperature signifies the resistance of the starch granules to swelling and may be attributed to bonding forces of the granules interior components (Opata et al., 2007). According to Chen et al. (1999), the gelatinization temperature of starch increases with high amylose content and high starch concentration.

It is the viscosity at the end of the heating period (95ºC) usually followed by holding at this temperature for about 15-20 minutes. It is an important property in the handling of food systems during processing and indicates the stability of the starch granules towards shear (Campbell et al., 2013). In pasting analysis viscosity decreases during the holding period at 95ºC. Granule disintegration due to shearing of the stirrer and the vessel results in the reduction of viscosity.

During the heating process of starch, the maximum viscosity obtained by the slurry before breakdown is referred to as the peak viscosity (Maskan and Altan, 2016). It gives an indication of the elasticity, swelling ability, and gel strength of the starch and hence informs decision making on their food application. Peak time is the time needed for the peak viscosity to be attained during heating.

Breakdown viscosity measures the difference between peak viscosity and the hot paste viscosity whereas the setback value is the difference between the final viscosity and the hot paste viscosity. The breakdown viscosity is an indication of paste stability. Adebowale et al. (2005) reported that the higher the breakdown in viscosity, the lower the ability of the starch sample to withstand heating and shear stress during cooking. Setback value is a measure of the tendency of the starch to undergo retrogradation. Low setback values show greater resistance to retrogradation (cooling and re-association of molecular components) and so it is a quality attribute parameter especially in the food industry as it affects the final quality and shelf life of the product.

It is the viscosity at the end of the cooling period (50ºC) and indicates the end of retrogradation. The final viscosity is important in food processing for textural and sensory properties of a product (Campbell et al., 2013). It is by far the most important attribute since that’s what a consumer sees in a food product.

The food industry has massively relied on cassava, maize, potato and wheat starches for use in various applications. With the increasing demand for starch, there is the need to explore other indigenous crops locally grown by farmers for subsistence purposes as alternative sources of starch. Attempts to do so far have focused on cassava. Legumes, just like cassava have great potential for this purpose, however, their applications in most industries have been very limited due to lack of information on its functional properties. Therefore, accessing the structural, physicochemical and functional properties of the starches from legumes is a laudable step in a positive direction. Detailed information on the characteristics of these starches would enhance their use in industries. The pasting properties provide varied applications in both food and non-food systems. For instance, higher peak, final and setback viscosity of starch granules makes it suitable for products requiring high gel strength such as noodles and pasta whereas lower peak, final and setback viscosities make them appropriate in applications requiring low viscosity, especially in infant food formulation.

Legume seeds (Glycine max, Cajanus cajan and Vigna subterrenea) and cassava were obtained from local markets in Kumasi, Ghana. Sorting was done first to remove impurities and damaged seeds. Cassava peels were removed after which it was washed in excess water to remove dirt and reduced into smaller sizes. After, the legume seeds and cassava were dried separately in a solar tent for a maximum of three days and fourteen days respectively. The seed coat of the legumes was manually removed. After which the different legumes seeds and cassava were separately pulverized into legume flour and cassava flour respectively using the Preethi Trio heavy duty grinder (MG 182/00).

Starch was extracted from 500g of each legume flour and cassava flour using the alkaline deproteination method of Gomez-Brenes et al. (1983). The flours were defatted using petroleum ether at a 1:3 (w/v) ratio and then agitated on a Gallenhamp orbital shaker at 200 rpm at room temperature for 3 hrs. It was made to sediment and the supernatant was discarded. Proteins were removed by adding 0.01M NaOH at a 1:5 (w/v) ratio. The slurry was then agitated at 200 rpm for 3 hours after which it was allowed to settle and the supernatant was discarded off. The resulting precipitate was again agitated with distilled water at 1:3 (w/v) ratio after which it was strained with cheese cloth to obtain the starch slurry.

Purification of the various starches was obtained using the method as described by Segura-Campos et al. (2015). Each starch sample was suspended in distilled water at 1:4 (w/v) ratio containing sodium hypochlorite at 25 g/L, agitated at 200 rpm for 10 min and centrifuged (Kubota, KS-5000P) at 2500 rpm for 12 min. This step was repeated. The precipitate obtained was then suspended in distilled water at a 1:4 (w/v) ratio containing 1M HCl, was agitated at 200 rpm for 10 min and was centrifuged at 2500 rpm for 12 min. Lastly, the starch was suspended in distilled water at a 1:4 ratio (w/v), agitated at 200 rpm for 10 min and centrifuged at 2500 rpm for 12 min. The purified starch obtained was solar dried for 48 hours, weighed and packed in Ziploc bags.

Pasting properties were determined using a Rapid Visco Analyzer (RVA) Model 4500 (Newport Scientific, Warriewood, Australia). The Profile method for pasting of flour was used for the samples. The moisture content of all starch samples was determined based on which the ratio of starch to distilled water (w/v) will also be determined for the pasting. On the average, 3g of starch was suspended in 25 mL of distilled water. Based on the profile method, the equipment was set to rotate at an initial speed of 960 rpm and to start pasting at 50°C. After 10 sec, the speed was reduced and kept constant at 160 rpm and after 1 min, the temperature was elevated to 95°C for a holding time of 4.42 min after which it was cooled to 50°C for a further holding time of 2 minutes. The data was plotted as time (min) versus viscosity in centipoise (cP).