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COMPARATIVE STUDIES ON THE EFFECTS OF DIFFERENT MODIFICATIONS ON THE COLD WATER SOLUBILITY OF STARCH FROM SELECTED UNDERUTILIZED LEGUMES

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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

PropertiesAmyloseAmylopectin
Chain length   ArrangementLong, straight   Densely packedBranched   More open, lower density
SizeSmallLarge
  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).


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