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Proximate Analysis¸ Minerals, Tannin and Phytate Content of Some

Local and Imported Legume Seeds

By Nahla Elgaili Ali Ebrahim

B.Sc. (1999) Gezira University

A dissertation submitted in partial fulfillment of the requirements of the degree of Master of science in tropical animal production

Supervisor

Prof. Khadiga Abbas Abdel Atti

Department of Animal Nutrition Faculty of Animal Nutrition

University of Khartoum

April 2011

i

DEDICATION

To my parents To my dear husband

To dear Friends and Colleagues

WITH LOVE

ii

AKNWOLEDGEMENTS

Most of all, great thanks to Allah (Subhanahu Wa Taala) for helping

me complete this study.

It is my great pleasure to express my deepest appreciation to my

supervisor Prof. Khadiga Abbas Abdel atti for her great efforts and advice to

make my study possible and worthwhile. My thanks extended to Prof. Amir

Mohamed Salih and Prof. Mohamed Khair for their amicable personalities

and great help in making this research possible. My deepest gratitude and

appreciation to my husband Dr.Hassan Mohamed Doka who furnished me

with all kinds of help and guidance regarding my work and life.

My extended thanks to Mr.Mahjoub Sayed for his willing and guidance

in laboratory data analysis and treatments.

Thanks to the Ministry of Animal Resources and Fisheries in Sudan,

my colleagues and administrators of KuKu Artificial Insemination Centre for

their great efforts and help to make this study possible by allowing me to

carry out this research.

The deepest and special gratitude and thanks to the spirit of my aunt

the late Haja Fatima Ali Saadallah “in Heaven Insha allah”, my Mother

Ustazah Salha Alsayed, and my great Father Elgaili Ali Saadallah for their

continues encouragements and help through out my life.

My special thanks to Mr. Hamza Ibrahim for his great help in

arranging and editing the thesis.

iii

LIST OF CONTNETS

DEDICATION ............................................................................................................... i

AKNWOLEDGEMENTS............................................................................................. ii

LIST OF CONTNETS ................................................................................................. iii

LIST OF TABLES ........................................................................................................ v

LIST OF FIGURES ...................................................................................................... v

ABSTRACT IN ENGLISH ........................................................................................ vi

ABSTRACT IN ARABIC .......................................................................................... vii

CHAPTER ONE: INTRODUCTION ........................................................................... 1

CHAPTER TWO: LITERATURE REVIEW ............................................................... 3

2.1 Nutritive value of legume seeds .............................................................................. 3

2.1.1 Chemical composition ..................................................................................... 3

2.1.2 Mineral content ................................................................................................ 3

2.1.3 Anti-nutritional factors..................................................................................... 4

2.1.4 Uses in animal diets ......................................................................................... 4

2.2 Faba bean (Vicia faba) ............................................................................................ 4


2.2.2 Mineral content ................................................................................................ 5


2.2.3.1 Tannin ....................................................................................................... 6

2.2.3.2 Phytates ..................................................................................................... 6

2.3 Pea (Pisum sativum) ................................................................................................ 6


2.3.2 Mineral content ................................................................................................ 7


2.3.3.1 Tannin ....................................................................................................... 8

2.3.3.2 Phytates ..................................................................................................... 8

2.4 Chick pea (Cicer areitnum) .................................................................................... 8


2.4.2 Mineral content ................................................................................................ 9

iv


2.4.3.1 Tannin ....................................................................................................... 9

2.4.3.2 Phytates ..................................................................................................... 9

2.5 Cow Peas (Vigna unguiculata) ............................................................................. 10

2.5.1 Chemical composition ................................................................................... 10

2.5.2 Mineral content .............................................................................................. 10

2.5.3 Anti-nutritional Factors .................................................................................. 11

2.5.3.1 Tannin ..................................................................................................... 11

2.5.3.2 Phytates ................................................................................................... 11

2.6 Lupins (Lupinus spp.) ........................................................................................... 11


2.6.2 Mineral content .............................................................................................. 12

2.6.3 Anti-nutritional factors................................................................................... 13

2.6.3.1 Tannins .................................................................................................... 13

2.6.3.2 Phytates ................................................................................................... 13

2.7 Common beans (Phasolus vulgaris) ..................................................................... 13


2.7.2 Mineral content .............................................................................................. 14

2.7.3 Anti-nutritional factors................................................................................... 14

2.7.3.1 Tannins .................................................................................................... 14

2.7.3.2 Phytates ................................................................................................... 14

CHAPTER THREE: MATERIALS AND METHODS ............................................. 15

CHAPTER FOUR: RESULTS AND DISCUSSION ................................................. 18

4.1 Chemical Composition results .............................................................................. 18

4.2 Mineral content results .......................................................................................... 22

4.3 Anti-nutritional factors results .............................................................................. 27

CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS ......................... 31

5.2 Conclusion ............................................................................................................ 31

5.3 Recommendations ................................................................................................. 32

REFRENCES .............................................................................................................. 33

APPENDICES ............................................................................................................ 42

v

LIST OF TABLES

Table Page

1 Scientific, English and local names of legume seeds 16

2 Chemical composition results of some local and imported legume seeds 19

3 Macro elements results% of some local and imported legume seeds 23

4 Micro elements results (%) of some local and imported legume seeds 25

5 Anti-nutritional factors (phytate and tannin %) results of some local and imported legume seeds 27

LIST OF FIGURES

Figure Page

1 Chemical composition results of some local and imported legume seeds 20

2 Macro elements results% of some local and imported legume seeds 24

3 Micro elements results (%) of some local and imported legume seeds 26

4 Anti-nutritional factors (phytate and tannin %) results of some local and imported legume seeds 28

vi

Proximate Analysis, Minerals Tannin and Phytate Content of Some Local and Imported Legume Seeds

M.Sc. in Tropical Animal Production

Nahla Elgaili Ali Ebrahim

ABSTRACT

The present study was designed to determine the nutrient composition

of some Local and Imported Legume seeds.

Seeds of ten legume crops, namely local and imported faba bean

(Vicia faba), field pea (Pisum sativum), chickpea (Cicer arietnum),lupin

(Lupinus termis.) and local seeds of cowpea (Vigna unguiculata L.),and

common haricot bean (Phaseolus vulgaris) were investigated for

determination of proximate composition, minerals, tannin and phytate.

The results indicated that the imported lupin seeds contained the

highest protein (39%) content, while the lowest was recorded for the local

cowpea seeds (17%) content. The ten legumes had high crude fiber content

ranging between 5.0% and 19.8%. Ether extract ranged between 0.07% and

9%,a local faba bean have the highest phosphorous content(1.3%),imported

lupin was the highest in calcium (0.7%),and local chickpea was the highest

in magnesium (0.3%) and sodium range between 0.04% and

0.1%.Regarding micro elements, selenium range was between (0.1and 0.6

ppm) ,zinc range between (0.04 and 0.1 ppm),cooper range between (0.01

and 0.03 ppm). High manganese was found in local chickpea (7.7ppm), and

the highest cobalt was found in imported faba bean(1.6ppm) .

High phytate was recorded in imported faba bean seeds 1.7% content, and

in imported lupin seeds 1.0 % content. The highest tannin was found in

local field pea seeds 0.8% and local faba bean seeds 0. 4%.

vii

تيتالتانين والفايالمعادن التحليل التقريبي ومحتوى

البقوليات المحلية والمستوردة بذور لبعض

طق الحارة ماجستير اإلنتاج الحيواني فى المنا

براهيمنهلة الجيلي علي إ

المستخلص

المحليـة (البقوليـات محاصيل لبذور عشرة منتركيب المغذيات الدراسة لتحديد هذه أجريت

والتي تـم البيضاء والفاصولياواللوبياء،والترمس،الحمص،،والبسلةي،والفول المصر: وهي ،)توردةوالمس

.إجراء التحليل التقريبي عليها،والمعادن، ومحتوى مادتي التانين والفايتيت

بينمـا أدناهـا ، % 39وتين سجلت للترمس المستورد أشارت نتائج الدراسة إلى أن أعلى نسبة للبر

محليـة (العشرة البقوليات المختَبرة كما أشارت النتائج إلى أن . %17) الحنيطير( المحليةاء للوبيسجلت

تراوحـت نسـبة و ، %19.8 و% 5.0 خام والتي تفاوتت نسبها بيناأللياف المحتويات غنيةٌ ب) ومستوردة

.% 9.2 و% 0.07المستخلص اإلثيري بين

المحلـي ر سـجلت للفـول المصـري ى نسبة للفسـفو أعلإلى أن كما أشارت نتائج الدراسة

المحلي أعالها فى الحمصو ،%0.7يات المختبرة فى الكالسيوم الترمس المستورد أعلى البقولو،1.3%

.% 0.1و % 0.04بين الصوديوم فتراوحت قيم ،أما % 0.3نيسيوم جالم

أما العناصر الصغرى فقد س0.6و 0.1بين تتراوحي لتاوفيها للسيلينيوم أعلى قيمةلت ج

تراوحت قيم عنصر ,جزء من المليون 0.1و 0.04تراوحت بين قيم عنصر الزنك ، جزء من المليون

، أعلى نسبة للكوبالت ُسجلت في الفول المصري المستورد جزء من المليون 0.03و 0.01النحاس بين

. جزء من المليون 7.7محلي الالحمص فى سجلت لمنغنيزل أعلى قيمة جزء من المليون،و 1.6

1.0، وفي الترمس المستورد %1.7المستورد فول المصريأعلى قيمة للفايتيت سجلت فى ال

%. 0.7 )الحنيطير(ة المحلي اللوبياءفي و % 0.8لبسلة المحلية في اأعلى قيمة للتانين سجلت ، و%

1

CHAPTER ONE

INTRODUCTION

The recent increasing demand for alternative protein sources used

in animal nutrition renews the interest in legume seeds. The cheapness of

most legume seeds as plant sources compared to animal ones can

encourage their utilization in animals and poultry feeds especially in

under developing tropical countries, where climatic conditions are

suitable for their growth and occurrence.

Nutritionally, legume seeds are appreciated especially for the

agronomic advantage as nitrogen fixing and the high crude protein

content. Legume seeds show lower lipid contents and are free of

cholesterol as well as holding different minerals and vitamins: they as

well are good sources of complex carbohydrates to all monogastrics

(Sebastia et al., 2001; Deka and Sarker, 1990; Kandelwal et al., 2009;

Elhardallou and Walker, 1994).

The total world production of seeds was estimated to exceed 176

million tones in 2003, 40 million of which were produced in Africa and

more than 63 million in India (Reddy et al., 2003). About 70% of the

total world legume production is used for human consumption (Monti

and Grillo, 1983). Among legumes cultivated for human nutrition, some

are today broadly cultivated in many countries, while others’ cultivation

is limited in local societies of some poor countries. Chickpeas (Cicer

arietinum L.) is an important food source in southern Europe, Middle

East, North Africa and India, but they are also consumed worldwide.

2

Common beans (Phaseolus vulgaris L.), are an important part of diets in

Latin America, East Africa and India and also in some Mediterranean

and Middle Eastern countries (Graham and Ranalli, 1997). Other

worldwide-cultivated legumes for human food are faba beans (Vicia

faba), which are mostly consumed in developing countries of Asia and

Africa, with China being the largest producer (Duc, 1997). Cowpea

(Vigna unguiculata L.) is one of the most widely adapted grain legumes.

However, two thirds of its production and more than three-fourths of the

production area spreads over the vast Sudan Savanna and Sahelian zones

of sub-Saharan Africa, while substantial quantities are produced in South

America (mostly Brazil), Asia and southwestern regions of North

America (Ehlers and Hall, 1997). Lupins (Lupinus spp.) consumption by

humans is generally limited, despite their high protein content and

nutritive value, due to their high content of alkaloids that make the seeds

bitter and potentially toxic (Friedman, 1996).

The aim of this study is to determine the nutrient potential of some

local and imported legume seeds to encourage their utilization in animal

feeding.

3

CHAPTER TWO

LITERATURE REVIEW

2.1 Nutritive value of legume seeds

2.1.1 Chemical composition

Proximate composition varies considerably not only among the

various legumes, but also within the same genus. Furthermore,

significant compositional differences have been observed even within

varieties, cultivars, or genotypes of the same species.

The protein in legumes varies considerably, but legumes have

generally higher protein contents when compared to all other plant foods,

thus they are characterized as 'the poor man's meat' (Duranti and Gius,

1997). In general the legumes are rich in crude fiber, which usually

exceeds 20% of total dry weight, while in lupins they can reach up to

40% (Farrell and Mannion, 1997). Some legumes significantly contain

lower oil contents, less than 10% (Duranti, 2007).

2.1.2 Mineral content

In general legumes are rich in potassium and magnesium, and

consumption of even small quantities of less than a portion per day is

sufficient to cover the adult recommended daily allowance for Mg, P and

Fe (Wang and Daun, 2006). The micronutrients contents vary

considerably even within each legume species and are strongly affected

by their environmental contents (Johnson et al., 2005).

4

2.1.3 Anti-nutritional factors

Legume seeds contain several anti-nutritional protein and non-

protein compounds. The presence of these anti-nutritional factors is often

the result of an evolutionary adaptation enabling survival and completion

of plant life cycle (Duranti and Gius, 1997). Anti-nutritional factors of

legumes can be broadly divided into four groups: (a) factors affecting

protein utilization and digestion, such as protease inhibitors, lectins and

tannins, (b) factors affecting mineral utilization, such as phytates and

glucosinolates, (c) anti-vitamin factors and (d) miscellaneous substances

including cyanogens, alkaloids, phytoestrogens and saponins (Francis

et al., 2001).

2.1.4 Uses in animal diets

Beans are used in the diets of all the major classes of farm animals.

Levels of the seeds in the diets of calves up to 3 months of age are

usually about 150 kg/t, whereas mixtures containing 250 kg/t have been

used for intensively fed steers. Beans are usually cracked, or coarsely

ground for feeding but it appears that whole beans are quite satisfactory

for older ruminants, which rapidly adapt for chewing them. The inclusion

of peas in ruminant diets may be at 400 kg/t, peas are particularly useful

in replacing soya bean in poultry diets, whereas beans are largely

confined to ruminant diets (Mc Donald, et al., 2002).

2.2 Faba bean (Vicia faba)


The crude protein of faba bean was found to be about 25% as

reported by (Saharan et al., 2002). Other findings by (Brand et al., 2004)

5

showed that the crude protein was 26%, while Hussein and Mortuza

(2006) reported a value of 27.67%.

Fiber content of faba bean is 15.4% as reported by Saharan et al.

(2002). Brand et al. (2004) found that the percentage of crude fiber is

13.3, while Hussein and Mortuza (2006) reported a value of 5.67%.

The percentage of ether extract was estimated to be 2.7 (Saharan et

al., 2002). In another study carried out by Brand et al. (2004) the ether

extract was 1.38%, while Hussein and Mortuza (2006) in their study

found that the percentage of ether extract was 3.12.

The ash content was found to be about 5.1% as reported by

Saharan et al. (2002). Brand et al. (2004) reported 2.79% for ash content.

A study by Hussein and Mortuza (2006) showed that ash percentage was

found to be 5.67 calculated from dry matter.

Generally beans make significant contribution to the energy of the

animal; the metabolizable energy content is 13.5MJ/kg DM for

ruminants, 12.0 MJ/kg DM for poultry (McDonald et al., 2002). The

carbohydrates were 53.2% (Saharan et al., 2002). Another study by

Brand et al. (2004) showed 54.2% carbohydrates while 45% was

reported by (Hussein and Mortuza, 2006).


McDonald (2002) reported that beans contain about (1.00, 5.5, 2.0

and 0.1%) g/kg DM for macro elements (Ca, P, Mg and Na) respectively.

Also McDonald reported values of (14.0, 16.0, 46.0 and 0.20%) g/kg DM

for micro elements (Cu, Mn, Zn and Co) respectively.

6


2.2.3.1 Tannin

Polyphenols or condensed tannins are products of secondary plant

metabolism. They cause reduction in protein and amino acid digestibility

by forming indigestible linkages with protein and in particular by

reacting with lysine and methionine (Duranti and Gius, 1997; Hickling,

2003). Also tannins decrease carbohydrate digestibility by forming

enzyme-resistant complexes with starch. Tannin in faba bean about

1.60% as reported by Farrell and Mannion (1997) and Saharan et al.

(2002).

2.2.3.2 Phytates

Phytates are mainly met in two forms; as phytic acid (IP6) and as

phytin (phytate salts). Phytates are chelating agents; their anti-nutritional

activity is mainly due to their ability to bind metals (Ca+2, Mg+2, Fe+2,

Zn+2 and Cu+2), thus leading to their deficient absorption (Morris, 1986).

Additionally the formation of protein-phytate complexes causes negative

effects on protein utilization (Selle et al., 2000). Also, phytates impact

amylase, pepsin and trypsin activity (Urbano et al., 2000).

Phytate content in faba bean was estimated by 1.012% as

demonstrated by (Farrell and Mannion, 1997; Saharan et al., 2002).

2.3 Pea (Pisum sativum)

Also regarded as sources of protein, having a better balance of

amino acids content, the metabolizable energy content is about 13 MJ/kg

DM for ruminants and 12.7 MJ/kg (McDonald et al., 2002).

7


Peas also regarded as sources of protein, having a better balance of

amino acids content. The crude protein ranged between 15.5 and

39.7percent (Monti and Grillo, 1983). Mesquita and Giada (2005)

reported a value of 14.7%. Studies have been carried out on the protein

composition by (Nikolopoulou et al., 2007) revealed that the crude

protein range was 24.3 and 32.5%. Crude fiber of pea seeds found to be

about 4.25 (El-Adawy et al., 2003). Brand et al. (2004) reported that, the

dietary fiber was 8.30%, while 10.4% was reported by Mesquita and

Giada (2005).

The ether extract in raw mature seed of pea seed was found to be

0.84% (Alonso et al., 2000). Another study by Brand et al. (2004)

reported 1.23%. A range between 1.68 and 2.34% was reported in a study

conducted in Brazil by Mesquita and Giada (2005). Ash percentage in

pea seeds was found to be 2.7 as reported by Alonso et al. (2000).

Mesquita and Giada (2005) reported a range of 2.47 and 3.0%, while

other experiments by Nikolopoulou et al. (2007) reported a range of 3.05

and 4.06%.Carbohydrates in pea seeds as reported by El-Adawy et al.

(2003), was 53.9%. Also Brand et al. (2004) found that (CHO) % was

45.4. A range between 33.4-47.5% was reported by Nikolopoulou et al.

(2007).


McDonald (2002) stated that peas contain about (1.5, 4.4, 1.4 and

0.5%) g/kg DM for macro elements (Ca, P, Mg and Na) respectively. He

reported 33% g/kg DM for Zn.

8


2.3.3.1 Tannin

Tannin content in pea seeds was found to range between 0.05 and

2.0% (Alonso et al., 2000, Urbano et al., 2003, Nikolopoulou et al.,

2007).

2.3.3.2 Phytates

Phytates content was found to be 0.27-1.2% as reported by Alonso

et al. (2000); Habiba (2002) and El-Adawy et al. (2003).

2.4 Chick pea (Cicer areitnum)

Chick Pea ranks as one of the most valuable legume crops from

nutritional view point. Information from the Centre of Legumes in

Mediterranean Agriculture (CLIMA) shows that chick pea help to reduce

cholesterol and regulate blood sugar.


The lowest protein content, have been observed for the common

chickpea, being less than 15% on dry matter basis ((Monti and Grillo,

1983; Mesquita and Giada, 2005). A range of 13.27-18.5 was reported by

Mesquita and Giada, (2005), while 24.0%was reported by Iqbal et al.

(2006). The crude fiber in chick pea as reported by Mesquita and Giada

(2005) was found to be 9.88%. Another study in Egypt by El-Adawy

(2002) showed that crude fiber was 3.82%.while 13.2% reported by

Nikolopoulou et al. (2006).The ether extract percentage was 6.48 (El-

Adawy, 2002). A range between 5.96 and 6.69% was reported by

9

(Mesquita and Giada, 2005), while Nikolopoulou et al. (2006) reported

4.40-7.33%.

The ash content in chick pea seeds was found to be 3.72% as

reported by El-Adawy (2002). A range between 2.94 and 3.15% was

reported by Mesquite and Giada (2005), while Nikolopoulou et al. (2006)

reported a range between 2.7 and 3.3%. Carbohydrates in chick pea were

found to be 68.43%as reported by Mesquita and Giada (2005). Another

study by Nikolopoulou et al. (2006), showed that carbohydrates ranged

between 42.7 and 50.8%.


In a recent study carried out by Daur et al. (2008),it was found

that, mineral content of raw seed of chick pea was 1.003% for Na,

1.171% for K, 2.527% for P, 0.194 for Ca, 1.137% for Cu, 0.687% for

Zn, 0.193% for Mn and 0.047% for Mg.


2.4.3.1 Tannin

Tannin content in chick pea ranged between 0.49 and 0.86% as

reported by El-Adawy (2002) Ryan et al. (2007), while Daur et al.

(2008), in their study reported that tannins content in chick pea was

found to be 3.16%.

2.4.3.2 Phytates

Phytic acid content in chick pea seeds was found to range between

1.2 and 1.8% as reported by El-Adawy (2002); Ryan et al. (2007).

10

2.5 Cow Peas (Vigna unguiculata)

The crop is grown for its mature seeds, and/or for its immature

fruits and leaves (which are used as vegetables); haulms are also fed to

livestock.


Crude protein of cow pea seeds was found to range between 20.1

and 25.8 (Giami et al., 2001). Another study by Arinathan et al. (2003)

showed 15.9%., while 24.7% was reported by Iqbal et al. (2006).Crude

Fiber of cow pea seeds was found to be 1.02-2.6% as reported by Giami

et al. (2001) and Ojimelukwe (2002), while 4.56% was reported by

Arinathan et al. (2003) and Iqbal et al. (2006). Ether extract of cow pea

seeds was ranged between 1.21 and 3.71% as reported by Giami et al.

(2001). Another study by Arinathan et al. (2003) showed that ether

extract percentage was 4.55, while 1.16% was reported by Ghavidel and

Prakash (2007).Ash content of cow pea seeds as reported in the study by

Giami et al. (2001) ranged between 1.48 and 3.6%, 4.2% was reported

by Iqbal et al. (2006), and 3.62% was reported in a study by Ghavidel

and Prakash (2007).Carbohydrates of cow pea seeds ranged between 20.2

and 64.6% (Giami et al., 2001). Ghavidel and Prakash (2007) reported

38.1% for Carbohydrates for cow pea seeds.


In a study carried out by Thangadurai (2003), it was reported that

mineral content of cow pea seeds was, Na 0.41%, K 4.79%, Ca 1.5%,

Mg 0.24%, P 3.5, Zn 0.057%, Mn 0.029% and Cu 0.011%.

11

2.5.3 Anti-nutritional Factors

2.5.3.1 Tannin

Tannin content in cow pea seeds as reported by Giami et al.

(2001); Arinathan et al. (2003) was found to range between 0.29 and 0.38

percent.

2.5.3.2 Phytates

Phytic acid content in cow pea seeds ranged between 0.12 and

0.29% as reported by Giami et al. (2001); Arinathan et al. (2003).

2.6 Lupins (Lupinus spp.)

The name 'Lupin' derives from the Latin word 'lupinus' (meaning

wolf). Like most members of legume family, lupins can fix nitrogen from

the atmosphere into ammonia via a rhizobium-root nodule symbiosis,

fertilizing the soil for other plants; this adaptation allows lupins to be

tolerant of infertile soils and capable of pioneering change in barren and

poor quality soils. The yellow legume seeds of lupins, commonly called

lupin beans. Lupin beans are commonly sold in a salty solution in jars

(like olives) and can be eaten with or without the skin. Lupins are also

cultivated as forage and grain legumes.

The Mediterranean L. albus (white lupin), L. angustifolius (blue

lupin) and Lupinus hirsutus are also edible after soaking the seeds for

some days in salted water. They are known as altramuz in Spain and

Argentina .The Mediterranean species of lupin, blue lupin, white lupin

and yellow lupin (L. luteus) are widely cultivated for livestock and

12

poultry feed. Both sweet and bitter lupins in feed can cause livestock

poisoning.


Monti and Grillo (1983) reported protein range between 17.0 and

38.7% for lupin seed. Lupins (Lupinus spp.), also have a very high

protein content, similar to soybean (Ródriguez-Ambriz et al., 2005),

reaching up to 45.4% (Ruiz and Sotelo, 2001), 39.3% was reported by

Brand et al. (2004).Crude Fiber was found to be 16.8% (Ruiz-Lopez et

al., 2000), while (Ruiz and Sotelo, 2001) reported 15.4%, Brand et al.

(2004) report 20.2%.Higher oil contents have been observed for lupins.

Ródriguez-Ambriz et al. (2005) have mentioned ranges of 20%, although

most of the literature shows lower oil contents in lupins, not exceeding

15% (Porres et al., 2005). Ruiz-López et al. (2000) reported 7.9 %, while

in another study in Mexico wild yellow lupin (Ruiz and Sotelo, 2001)

8.89% was observed.

Ash content in lupins ranged between 3.59 and 3.8% (Ruiz-López

et al., 2000; Ruiz and Sotelo, 2001). Brand et al. (2004) report 3.3%.

Carbohydrates as reported by Ruiz-López et al. (2000) were 34.6%,

Brand et al. (2004) report 31.65%.


Porres et al. (2005) in their study report that, mineral content of

lupins seed was K 0.955%, Na 0.112%, P 1.420%, Ca 0.139%, Mg

0.145%, Zn 0.004%, Cu 0.072% and Mn 0.09%.

13


2.6.3.1 Tannins

Tannins content in lupin seeds 0.22% (Ruiz-Lopez et al., 2000).

0.16% was reported by (Ruiz and Sotelo, 2001).

2.6.3.2 Phytates

Phytic acid content of lupins 1.169%(Ruiz-López et al., 2000)

1.109% (Ruiz and Sotelo, 2001).

2.7 Common beans (Phasolus vulgaris)


Crude Protein in common beans seed ranged between 17.0 and

39.4% (Monti and Grillo, 1983), while 15.9 and 20.9% was reported by

Mesquita and Giada (2005). Another study by Yoshida et al.(2005)

reported that crude protein was 21.4 and 23.1%. Crude fiber was 8.55% as

reported by (Mesquita and Giada, 2005), 16.4% as reported by Carmona-

Garcia et al. (2007), while 22.2% was reported by Kahlon et al. (2005).

Ether extract was found to range between 1.26 and 2.49% (Mesquita and

Giada, 2005), a range between 1.26 and 2.49% was reported by De

Almeida Costa et al. (2006). A range between 1.98-2.12% was reported

by Carmona-Garcia et al. (2007).Mesquita and Giada (2005) reported a

range between 3.7 and 3.8% for ash content in common beans seed. Also

3.79 and 4.54% was reported in Mexico by (Carmona-Garcia et al.,

2007). Carbohydrates in common beans was 67.9% (Mesquita and Giada,

2005), while 43.5% was reported by Carmona-Garcia et al. (2007).

14


According to Prolla et al.(2008). Common beans mineral content

was found as Zn 0.32%, Mn 0.14%, Cu 0.11%, Ca 0.26%, Mg 0.22% and

P 0.36%.


2.7.3.1 Tannins

Tannin content in common beans was 1.7-2.62% as reported by

De Mejia et al. (2005); Ryan et al. (2007).

2.7.3.2 Phytates

Phytic acid content of common bean seed was 0.007-0.009% as

reported by De Mejia et al. (2005); Ryan et al. (2007).

15

CHAPTER THREE

MATERIALS AND METHOD

The study held on February 2010 at the Faculty of Animal

Production, University of Khartoum, Sudan.

This study was confined to six local and imported legume seeds of

faba bean (Vicia faba), Pea (Pisum sativum), chick pea (Cicer areitnum),

lupins (Lupinus spp.) local cow peas (Vigna unguiculata L.), and

common beans (Phaseolus vulgaris L.). Table (1) shows English and

local names of the investigated seeds. The seeds were purchased from the

local market in Khartoum North – Sudan, cleaned and then ground in the

laboratory mill.

Chemical analysis

Dry matter content, Ash content, crude fibre content where

determined according to AOAC (1984) method (appendix 1).

Ether extract content was determined according to the method

AOAC (1984) using soxhelt apparatus. Crude protein was determined by

using Kjeldahal method according to AOAC (1984).

The nitrogen free extract (NFE) was obtained by difference

(appendix 1).

Tannin determination

Quantitative estimation of tannin was carried out using the

modified vanillin – HCL method of price et al. (1978) (appendix 1).

16

Table (1): Scientific, English and local names of legume seeds

English name Botanical name Local name

1. Faba bean Vicia faba L. Fool masri

2. Pea Pisum sativum L. Bessela

3. Chick pea Cicer arietinum L. Homos

4. Common bean Phaseolus vulgaris Fasolia bida

5. Cow pea Vigna unguiculata Lubia (Henateer)

6. Lupins Lupinus termis Tormos

17

Absorbance was read on spectrophotometer (Jenway 6305 UV/Vis.

Spectrophotometer), and the concentration of the condensed tannin was

determined from the standard curve.

Phytic Acid determination

The phytic acid content was determined by the method described

by wheeler and Ferrel (1971). The concentration was read using a

Spectrophotometer within one minute (appendix 1).

Mineral content analysis

Macro elements Sodium and Potassium were determined by PEP7

flame Photometer, phosphorus by 6505 UV/Vis. Spectrophotometer,

calcium and magnesium by titration.

Micro elements copper, zinc, manganese, cobalt and selenium

were determined by atomic absorption spectrophotometer version AA-

6800 SHIMADZU.

Data analysis

The data obtained of the investigated seeds was analyzed by

working out the means and standard errors for the various groups.

18

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Chemical Composition results

The results of chemical analysis of the current study were presented

in Table (2) and Fig. (1), showed that the crude protein content of faba

bean (Vicia faba L.) locally produced was higher (32.4%) than the

imported one, this disagreed with previous results that reported by

Saharan et al. (2002) 25%; Brand et al. (2004) 26% and Hussein and

Mortuza (2006) 27.7%, and this may be due to several reasons such as

soil characteristics and genotype. The crude protein of the local pea seeds

(Pisum sativum) was higher (26.6%), than the imported pea seed

(21.8%), this was similar to the findings that reported by Nikolopoulou et

al. (2007), who reported 24.3-32.5%. The protein of imported chick pea

(Cicer areitnum) was higher (24.5%) than the local chick pea (21.3%),

this agreed with the results reported by Iqbal et al. (2006)who reported

(24.0%), but disagreed with that reported by Mesquita and Giada (2005)

who reported (13.3-18.5%). The higher protein content (39.2%) was

observed for imported lupins, similar results reported (39.3%) for lupins

(Lupinus spp.) by Brand et al. (2004). The protein of local common

beans (Phasolus vulgaris)was (25.6%) this result agreed with the range

reported by Yoshida et al. (2005) 21.4-23.1%, while the lowest protein

content was reported for local cow pea seeds (Vigna unguiculata)

(16.9%), this result similar to that showed by Arinathan et al. (2003)

who reported 15.9%., while this disagreed with that reported by Iqbal et

al. (2006) (24.7%).

19

Table (2): Chemical composition (%) of some local and imported legume seeds

Seed type Dry Matter DM (%)

Crude Protein (%)

Ether Extract (%)

Crude Fiber CF (%) Ash (%)

Nitrogen Free Extract

(NFE) %

Faba beans ** (L) 93.74±0.03 32.38±0.43 0.12±0.00 10.55±0.21 3.22±0.00 57.29±0.41 *** (I) 93.46±0.05 28.48±0.56 0.09±0.00 7.47±0.005 3.3±0.00 53.66±0.18

Pea seeds ** (L) 93.88 ±0.02 26.57±0.43 0.07±0.00 4.97±0.00 1.09±0.00 58.25±0.02

***(I) 93.24 ±0.04 21.75±1.20 0.85±0.02 8.60±0.06 2.73±0.03 57.58±0.35

Chick peas ** (L) 95.06 ±0.03 21.30±1.26 6.35±0.005 9.93±0.05 3.5±0.005 52.85±0.03 ***(I) 93.13 ±0.01 24.50±0.20 6.13±0.005 12.82±0.005 2.83±0.00 45.09±0.2

Lupins ** (L) 95.25 ±0.00 27.35±0.20 9.24±0.15 19.77±0.005 3.59±0.03 38.04±0.23 *** (I) 93.39 ±0.08 39.22±0.98 8.87±0.09 15.60±0.005 3.28±0.03 41.92±0.13

Common beans ** (L) 94.98 ±0.01 25.64±0.92 1.77±0.09 9.13±0.005 4.76±0.005 55.79±0.28

Cow pea ** (L) 93.52 ±0.09 16.85±0.90 0.85±0.90 10.40±0.005 3.91±0 005 52.54±0.48

All values are the means expressed on a dry matter basis ± standard error L: Local legume seeds I: Imported legume seeds

20

0

10

20

30

40

50

60

70

80

90

100

(%)

DM CP Fat CF Ash NFE DM CP Fat CF Ash NFELocal Imported

Faba beans Pea seeds Chick peas Lupins Common beans Cow pea

Fig. (1): Chemical composition (%) of some local and imported legume seeds

21

Fiber content of local (Vicia faba L.) was higher (10.6%) than the imported

faba bean, the results obtained disagreed with that reported by Saharan et al.

(2002) (15.4%) and Brand et al. (2004) (13.3,), also disagreed with that results

reported (5.7%).by Hussein and Mortuza (2006),and this may be due to several

environmental factors including rain fall during seed fill, water stress, soil type and

cultivation.

In the current study the crude fiber of the local pea seeds (Pisum sativum)

obtained (4.9%), lower than the imported pea seeds (8.6%) the results similar to

that reported by El-Adawy et al. (2003) (4.3%), and Brand et al. (2004) who

reported (8.3%).In the current study fiber content of imported chick pea (Cicer

areitnum) was higher (12.8%) than the local chick peas (9.9%),the results observed

for imported chick pea disagreed with previous studies(3.9% and 13.2%) reported

by (El-Adawy, 2002; Nikolopoulou et al., 2006),similar findings in the present

study of local chick peas fiber content agreed with that reported (9.9%) by

Mesquita and Giada (2005). In the current study the highest crude fiber was

observed for the local lupins (Lupinus spp.) (19.8%), these results were similar to

that reported by Brand et al. (2004) 20.2%, also the results observed for imported

lupins seed fiber content agreed 15.4% with that reported (Ruiz and Sotelo,

2001).Fiber content result of local common beans (Phasolus vulgaris) in the

present study was ((19.3%) disagreed with previous results 8.55% as reported by

(Mesquita and Giada, 2005), 16.4% as reported by Carmona-Garcia et al. (2007),

and 22.2% as reported by Kahlon et al. (2005) Results of local cow pea seeds

(Vigna unguiculata) disagreed (10.4%)with previous results (1.02-2.6% as reported

by Giami et al. (2001), while 4.56% was reported by Arinathan et al. (2003) and

Iqbal et al. (2006).

22

In the current study, all investigated legume seeds have low lipid

content (Duranti, 2007) as shown in Table (2).The highest lipid content

observed for local and imported lupins seeds (9.24 and

8.87%)respectively

The ash content of the investigated seeds (Table 2) would be

important to the extent that it contains the nutritionally important mineral

elements, which were presented in Table (3) and Fig. (2). all the analyzed

seeds have a range (1.09 and 4.76%), these results agreed with all

previous results reported.

In the present study all legume seeds showed a range of Nitrogen

Free extract between (38 and 58.3%), this agreed with that reported in

previous studies, as presented in (Table 2).

4.2 Mineral content results

Table (4) and Figs

(3) presents the macro elements content of the investigated seeds. High

potassium level was observed for all legumes followed by phosphorous.

Micro elements presented in Table (4), levels of (Mn) of the local chick

pea (7.7%) and imported faba beans (2.08%) were the highest among all

legumes. All currently investigated seeds have high contents of (Co) the

highest was reported for local lupins seeds (4.55%). Similar findings

have been observed for both elements (Cu) and (Zn). For (Se) content the

highest have been reported for local lupins seeds (0.614%) while in some

legumes are not detected.

These micronutrients contents vary considerably even within each

legume species and are strongly affected by the environmental factors

(Johnson et al., 2005).

23

Table (3): Macro elements (%) of some local and imported legume seeds

Seed type Calcium % (Ca)

Phosphorus % (P)

Potassium % (K)

Magnesium % (Mg)

Sodium % (Na)

Faba beans **(L) 0.490±0.005 1.280 ±0.006 1.283 ±0.004 0.17±0.005 0.09±0.003

***(I) 0.320±0.000 0.970 ±0.004 1.137 ±0.04 0.14±0.01 0.085±0.002

Pea seeds **(L) 0.280±0.005 0.810 ±0.004 1.08±0.005 0.1±0.00 0.056±0.001 ***(I) 0.330±0.010 0.690±0.004 0.90±0.003 0.14±0.005 0.072±0.005

Chick peas ** (L) 0.320±0.005 0.810±0.003 0.670±0.500 0.250±0.020 0.069±0.001 ***(I) 0.490±0.010 0.490±0.004 0.670±0.500 0.170±0.010 0.055±0.002

Lupins ** (L) 0.33±0.02 0.72±0.001 1.05±0.003 0.21±0.06 0.099±0.001 *** (I) 0.660±0.020 0.610±0.005 0.690±0.010 0.190±0.010 0.062±0.002

Common beans (L) **(L) 0.550±0.020 1.030±0.004 1.420±0.001 0.130±0.005 0.049±0.001

Cow peas (L) ***(L) 0.380±0.005 0.890±0.001 1.220±0.020 0.180±0.020 0.054±0.001

*All values are the means expressed on a dry matter basis ± standard error * * L: Local legume seeds *** I: Imported legume seeds

24

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

(%)

Ca P K Mg Na Ca P K Mg NaLocal Imported

Faba beans Pea seeds Chick peas Lupins Common beans Cow peas

Fig. (2): Macro elements (%) of some local and imported legume seeds

25

Table (4): Micro elements (ppm) of some local and imported legume seeds

Seed type Se Zn Cu Mn Co

Faba beans **(L) 0.298±0.001 0.043±0.010 0.022±0.010 0.023±0.010 1.16±0.5

***(I) 0.300±0.241 0.108±0.005 0.031±0.005 2.075±0.050 1.59±0.1

Pea seeds **(L) 0.237±0.020 0.055±0.005 0.014±0.010 0.022±0.000 0.43±0.01

***(I) 0.455±0.027 0.068±0.010 0.014±0.010 0.030±0.005 0.46±0.10

Chick peas **(L) ****ND 0.098±0.010 0.025±0.005 7.67±0.500 4.55±0.40

***(I) ****ND 0.085±0.010 0.023±0.005 0.085±0.005 0.46±0.10

Lupins **(L) 0.614±0.290 0.096±0.010 0.023±0.000 0.0139±0.050 1.43±0.40

***(I) ****ND 0.045±0.005 0.020±0.005 0.027±0.005 1.28±0.30

Common beans **(L) 0.141±0.150 0.084±0.005 0.029±0.010 0.0132±0.050 0.19±0.40

Cow peas **(L) 0.371±0.190 0.047±0.020 0.020±0.005 0.035±0.005 0.78±0.02

*All values are the means expressed on a dry matter basis ± standard error ** L: Local legume seeds *** I: Imported legume seeds **** ND: Non Detected

26

0

1

2

3

4

5

6

7

8

(%)

Se Zn Cu Mn Co Se Zn Cu Mn CoLocal Imported

Faba beans Pea seeds Chick peas Lupins Common beans Cow peas

Fig. (3): Micro elements (ppm) of some local and imported legume seeds

27

4.3 Anti-nutritional factors results

Table (5) and Fig. (4) Showed the results of certain anti-

nutritional factors such as tannins and phytic acid. In the current study

tannin content in both local and imported faba bean seeds disagreed with

that reported(1.60%) in Farrell and Mannion (1997);Saharan et al.

(2002). Tannin content in pea seeds agreed with the range (0.05-2.0%)

reported by Alonso et al. (2000); Urbano et al. (2003); Nikolopoulou et

al. (2007).In this study similar findings of tannins content in chick pea

(0.49-0.86%) was reported by El-Adawy (2002); Ryan et al. (2007),

whereas higher value of tannin (3.16%) reported recently by Daur et al.

(2008) as showed in Table (5).

Tannins in both local and imported investigated lupins seeds disagreed

with the results reported by Ruiz and Sotelo (2001). Common beans

contains the smallest amount of tannins among all investigated legumes,

this result obtained disagreed with that (1.7-2.62%) reported in De Mejia

et al. (2005); Ryan et al. (2007).

Tannins in cow pea seeds found to be higher than in reported studies

(Giami et al., 2001; Arinathan et al., 2003) (0.29-0.38%).

In the present study the highest phytic acid content (Table 4) was

observed for imported faba bean seeds, this result disagreed (1.012%)

with that mentioned by Farrell and Mannion (1997); Saharan et al. (2002).

Phytic acid content of both local and imported pea seeds disagreed (0.27-

1.2%) with that reported by Alonso et al. (2000); Urbano et al. (2003).

The content of phytic acid in local chick pea was higher (0.099%) than the

28

imported (0.027%) chick pea seeds, this result disagreed with that (1.2-

1.8%) reported in El-Adawy (2002); Ryan et al. (2007).

Phytic acid content of imported lupins seed was higher than the local

lupins, the results obtained disagreed (1.109%) with that reported in Ruiz-

López et al. (2000); Ruiz and Sotelo (2001).

Phytic acid results of local common bean seeds agreed (0.007-0.009%)

with that reported in De Mejia et al. (2005); Ryan et al. (2007).

The results obtained (0.15) for phytic acid of local cow pea seeds agreed

with that (0.12-0.29%) reported in Giami et al. (2001); Arinathan et al.

(2003).

29

Table (5): Anti-nutritional factors (phytate and tannin %) of some local and imported

legume seeds

Seed types Phytate (%) Tannin (%)

Faba beans **(L) 0.021 ± 0.05 0.44 ± 0.30

***(I) 1.67 ± 0.0005 0.05 ± 0.20

Pea seeds **(L) 0.088 ± 0.0005 0.83 ± 0.08

***(I) 0.074 ± 0.0005 0.084 ± 0.08

Chick pea **(L) 0.099 ± 0.000 0.33 ± 0.02

***(I) 0.027 ± 0.001 0.084 ± 0.05

Lupins **(L) 0.025 ± 0.000 0.075 ± 0.05

***(I) 0.98 ± 0.0005 0.054 ± 0.03

Common beans **(L) 0.035 ± 0.000 0.03 ± 0.01

Cow pea **(L) 0.15 ± 0.0007 0.73 ± 0.05

*All values are the means expressed on a dry matter basis ± standard error (L): Local legume seeds : (I) Imported legume seeds

30

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Phytate Tannin Phytate TanninLocal Imported

Faba beans Pea seeds Chick pea Lupins Common beans Cow pea

Fig. (4): Anti-nutritional factors (phytate and tannin %) of some local and imported legume seeds

31

CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

Conclusion

1. In the current study imported Lupins (Lupinus spp.) have the highest

protein content up to 40%, also high either extract content 9.24% and

crude fiber 19.7%.

2. In this study, generally legumes have high mineral content, local faba

bean have the highest Phosphorus contentn1.6%, imported lupins was

the highest in Calcium 0.7%, and local chick pea was the highest in

Magnesium 0.3%.

3. For micro elements high Manganese was found in local chick pean7.7

ppm,highest Cobalt was in faba bean 1.6 ppm.

4. High phytate content was reported for imported faba bean seeds 1.67%,

and imported lupin seed phytate content was 0.98%.Highest tannin

found in local pea seeds 0.8 and local faba beanlevel 0.4%.

32

Recommendations

1) Several environmental factors should be considered. However, further

and more systematic research is required to outline these environmental

implications.

2) The presence of certain anti nutritional factors such as tannins and

physic acid decrease protein digestibility of the seed, further research

for other anti nutritional factor should be done.

33

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42

APPENDICES Appendix (1)

Methods of analysis

Proximate analysis:

1. Moisture content:

Moisture content was determined according to the AOAC (1984) as

follows:

Two gram of sample was weighed using a sensitive balance in clean dry

and pre-weighed crucible and then placed in an oven at 105oC and left

overnight. The crucible was transferred to a desicator and allowed to cool and

then weighed. Further placements in the oven were carried out until

approximately constant weight was obtained. Moisture content was calculated

using the following formula:

MC % = (W2 – W1) – (W3 – W1) X 100 (W2 – W1)Where:

MC = Moisture contentW1 = Weight of empty crucible W2 = Weight of crucible with the sample W3 = Weight after drying

2. Ash content:

Ash content of the sample was determined according to the method of

AOAC (1984) as follows:

Two gram of sample was placed in a clean dry pre-weighed crucible.

Then the crucible with its content ignited in a muffle furnace at about 550oC

for 3 hr or more until light gray ash was obtained. The crucible was removed

from the furnace to a desiccator to cool and then weighed. The crucible was

43

reignited in the furnace and allowed to cool until constant weight was

obtained. Ash content was calculated using the following equation:

AC % = (W2 – W1) X 100 Ws Where:

AC = Ash contentW1 = Weight of empty crucible W2 = Weight of crucible with ash Ws = Weight of sample

3. Fat content

Fat was determined according to the method of AOAC (1984) using

Soxhelt apparatus as follows:

An empty clean and dry exhaustion flask was weighed. About 2 g of

sample was weighed and placed in a clean extraction thimble and covered

with cotton wool. The thimble was placed in an extractor. Extraction was

carried out for 6-8 hr with petroleum ether. The heat was regulated to obtain at

least 15 siphoning per hour. The residual ether was dried by evaporation. The

flak was placed in an oven at 105oC till it dried completely and then cooled in

a desiccator and weighed. The fat content was calculated using the following

equation:

Fat % = W2 – W1 X 100 WsWhere:

W1 = Weight of extraction flask W2 = Weight of extraction flask with fatWs = Weight of sample

44

4. Crude fiber

Crude fibre was determined according to AOAC (1990). Two gram of

deflated sample was treated successively with boiling solution of H2SO4 and

KOH (0.26 N and 0.28 N, respectively). The residue was then separated by

filtration, washed and transferred into a crucible then placed into an oven

adjusted to 105oC for 18-24 hr. the crucible with the sample was weighed and

ashed a muffle furnace at 500oC and weighed. The crude fibre was calculated

using the following equation:

CF % = W1 – W2 X 100 WsWhere:

CF = Crude fibre W1 = Weight of crucible before ashingW2 = Weight of crucible after ashing Ws = Weight of crucible sample

5. Crude protein

The crude protein was determined by using the micro-kjeldahal method

according to AOAC (1984) as follows:

a. Digestion:

2g of the sample was weighed and placed in small digestion flask (50ml).

About 0.4g catalyst mixture (96% anhydrous sodium sulphate and 3.5%

copper sulphate) was added. 3.5ml of approximately 98% v/w of H2SO4 was

added. The contents of the flask were then heated on an electrical heater for 2

hr or till the colour changed to blue-green. The tubes were then removed from

the digester and allowed to cool.

45

b. Distillation:

The digested sample was transferred to the distillation unit and 20ml of

40% sodium hydroxide were added. The ammonia was received in 100ml

conical flask containing 10ml of 2% boric acid plus 3-4 drops of methyl-red

indicator. The distillation was continued until the volume reached 50ml.

c. Titration:

The content of the flask were titrated against 0.02 N HCl. The titration

reading was recorded.

The crude protein was calculated using the following equation:

CP % = (T-B) x N x 14 x 100 x 6.25Ws x 1000

Where: CF = Crude protein T = Titration reading B = Blank titration reading N = HCl normality Ws = Sample weight

1000= To convert to mg

6. Carbohydrates:

Carbohydrates were determined by difference according to the

following equation:

Carbohydrate= 100 – (DM + Ash + Fat + FC + PC)

7. Phytic acid

The phytic acid content was determined by the method described by

Wheeler and Ferrel (1971). Two g of milled sample were weighed in125ml

conical flask. The sample was extracted with 50ml of 3% trichloroacetic acid

(TCA) for 3 hr with mechanical shaker. The suspension was centrifuged for 5

minutes and 10ml aliquot of the supernatant was transferred to 40ml tube.

46

Four ml of FeCl3 solution (made to contain 2mg ferric ion per ml in 3% TCA)

were added to the aliquot. The tube was heated in boiling water bath for 45

min. one or two drops of 3% sodium sulphate in 3% TCA were added. The

tube was cooled and centrifuged for 10-15 min. the clear supernatant was

decanted, the precipitate was washed twice by dispersing well in 25ml 3%

TCA, heated for 10-15 min in a boiling water bath, then centrifuged again.

Washing was repeated once more with water, the washed precipitate was

dispersed in a few ml of water and 3 ml of 1.5 N NaOH were added and the

volume completed to approximately 30ml with water. Then the tube was

heated in boiling water both for 30 min and hot filtered using Whatsman No.

2: the precipitate was washed with 60-70ml hot water and the washings were

decanted. The precipitate was dissolved from the filter paper with 40ml hot

3.2 N HNO3 into 100ml volumetric flask.

The paper was washed with hot water; the washing was collected in the

same flask, then completed to volume. 0.5ml aliquot was taken from the

above solution and transferred into 10ml volumetric flask, then 2ml 1.5N

KSCN was added and completed to volume by water, then immediately read

at 480nm using a spectro-photometer within one min.

Calculation:

A standard curve of different Fe (NO3)3 concentrations was plotted to

calculate the ferric ion concentration. The phytate phosphorous was calculated

from the ferric ion concentration assuming 4.6 iron: phosphorous molar ratio.

Phytate = A x C x 20 x 10 x 50 x 100 Mg/100g 1000 x 2Where:

A = Optical density

47

C = Concentration corresponding to the optical density

Tannin content

Quantitative estimation of tannin was carried out using the modified

vanillin – HCl method of Price et al. (1978).

The vanillin – HCl reagent was prepared by mixing equal volumes of

8% concentrated HCl in methanol and 1% vanillin methanol. The two

solutions of the reagents were mixed just prior to use. It was discarded if a

trace of colour appeared.

Catechin was used to prepare the standard curve. This was done by

adding 600mg of catechin to 100ml of 1% HCl in methanol. From this stock

solution various dilutions were prepared. Five ml of vanillin-HCl reagent were

added to 1ml of each dilution. The absorbance was read using a

spectrophotometer at 500nm after 20 min. from addition of the reagent at

30oC. The absorbance was plotted against catechin concentration. 0.5g of

ground sample was placed in a test tube. Then 10ml of 1% concentrated HCl

in methanol were added. The test tube was capped and continuously shaken

for 20 min. one ml of supernatant after centrifugation was pipettes into each of

the tubes and then proceeding as described in the standard curve above.

For zero setting before absorbance was read, 1 ml of blank solution (1%

HCl in methanol) was mixed with 5ml 4% concentrated HCl and 5ml of

vanillin-HCl reagent in a test tube and incubated for 20 min at 30oC.

Absorbance at 500nm was read on a spectrophotometer and the concentration

of the condensed tannin was determined from the standard curve. Tannin

concentration was expressed as catechin equivalent as follows:

48

Tannin % = C x 50 x 100 500

Where: C = Concentration corresponding to the optical density 50 = Volume of extract (ml)

500= Sample weight (mg)

49

Appendix (2)

Local and imported legume seeds Pictures a) Local and Imported lupins seed

b) Local and Imported pea seed

Local

LocalImported

Imported

50

c) Local and Imported faba bean seed

d) Local caw pea seed

Local

LocalImported

51

e) Local common bean seed

Local

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