X

SD9800010

Biochemical and NutrfMonal Studies Of . Lupin .'•*.-

Seed Cultivated in The Sudan

Malik EL Mubarak Mohamed ELamin B.Sc .(Food Science) • Faculty Of Agriculture. University Of Zagazig (1987)

M.Sc .(Food Biochemistry ) Faculty Of Agriculture. U. of K. (1993).

Thesis:

Submitted In Partial Fulfilment Of Requirment For The Degree Of Doctor Of Philosophy In Biochemistry and Food Science .

Department Of Biochemsitry and Soil Science Faculty Of Agriculture.

U. of K. Dec.

2 9-T CONTENTS

NO TITLE PAGE LIST OF TABLES 1 LIST OF FIGURES II ACKNOWLEDGMENT^ ITI ABSTRACT IN ENGLISH IV - V ABSTRACT IN ARABIC VI - VII

CHAPTER ONE

INTRODUCTION 2

CHAPTER TWO

LITERATURE REVIEW 2-1 NUTRITIONAL VALUE OF LUPIN SEEDS 5 2-2 PHYSICAL PROPERTIES OF LUPIN SEEDS 8 2-3 CHARACTERISTICS OF SEED 9 2-4 CHEMICAL COMPOSITION OF LUPIN SEED 9 2-4-1 CONTENT.. 10 2- 4-2 OIL AND FATTY ACID 12 2-4-3 14 2-4-4 MINERALS CONTENT , 15 2-5 PROTEIN FRACTIONS OF LUPIN SEEDS 15 2-6 CLASSIFICATION OF LUPIN PROTEIN FRACTIONS 18 2-6-1 ALBUMINS AND GLOBULINS 19 2-6-2 ALCOHOL EXTRACTABLE PROTEINS (AEP) 21 2-6-3 GLUTELINS < 21 2-6-3-1 Gi-GLUTELIN 22 2-6-3-2 Cb-GLUTELIN 22 2-6-3-3 G> GLUTEUN 23 2-6-4 INSOLUBLE PROTEIN (RESIDUE) 24 2-7 IN VITRO PROTEIN DIGESTIBILITY OF LUPIN SEED. ./?#?. Ifjn • 25 2-8 ANTINUTRrnONAL FACTORS 26 2-8-1 PHYTIC ACID 26 2-8-2 TANNINS 27 CHAPTER THREE \ . ' MATERIALS AND METHODS . 3-1 MATERIALS 31 3-1-1 SAMPLES 31 3-1-2 DEBITTERING OF LUPIN SEEDS 31 3-2 METHODS 31 3-2-1 PHYSICAL PROPERTIES - 31 3-2-1-1 SEED DIMENSION 32 3-2-1-2 DENSITY 32 3-2-1-3 BULKDENSUY 32 3-2-1-4 POROSITY 32 3-2-1-5 SPECIFIC VOLUME AND BULK DENSITY 32 3-2-2 DEFATTING LUPIN SEED 34 3-2-3 PROXIMATE ANALYSIS 34 3-2-4 34 3-2-5 DETERMINATION OF FATTY ACIDS PROFILE 34 3-2-6 DETERMINATION OF ANTINUTRITIONAL FACTORS 35 3-2-6-1 TANNINS 35 3-2-6-2 PHYTIC ACID 37 3-2-7 DETERMINATION OF MINERALS 41 3-2-7-1 SODIUM AND POTASSIUM '. 41 3-2-7-2 PHOSPHOROUS 42 3-2-8 FRACnONATION OF LUPIN PROTEIN 45 3-2-8-1 MENDEL - OSBORNE (1914) 45 3-2-8-2 LANDRYANDMOUREAUX ,. ' 46 3-2-9 IN VITRQ PROTEIN DIGESTIBILITY WITH PEPSIN 49

CHAPTER FOUR

• • - - 4 RESULTS AND DISSECTION 4-1 PHYSICAL PROPERTIES OF LUPIN SEEDS 50 4-1-1 SEED WEIGHT .DENSITY AND VOLUME 50 4-1-2 SEED DIMENSION 50 4-2 CHEMICAL COMPOSITION OF LUPIN SEEDS 51 4-2-1 MOISTURE CONTENT 52 4-2-2 FIBRE CONTENT .". 52 4-2-3 ASH CONTENT 56

4-2-4 CONTENT y 57 4-2-5 FATTY ACIDS 58 4-2-6 ANTINUTRITIONAL FACTORS 64 4-2-6-1 PHYTIC ACID CONTENT 64 4-2-6-2 TANNIN CONTENT 64 4-2-7 CONTENT 64 4-2-7-1 CALCIUM CONTENT 65 4-2-7-2 POTASSIUM CONTENT 69 4-2-7-3 MAGNESIUM CONTENT 70 4-2-7-4 IRON CONTENT 70 4-2-7-5 PHOSPHOROUS CONTENT 70 4-2-7-6 CONTENT 71 4-2-7-7 SODIUM CONTENT 72 4-2-7-8 MANGANESE CONTENT 72 4-2-7-9 LEAD CONTENT 73 4-2-8 TOTAL PROTEIN 73 4-3 PROTEIN FRACTIONS 75 4-3-1 MENDEL-OSBORNE METHOD 75 4-3-1-1 ALBUMIN 75 4-3-1-2 GLOBULIN 76 4-3-1-3 ALCOHOL EXTRACTABLE PROTEINS 77 4-3-1-4 GLUTELIN 78 4-3-1-5 INSOLUBLE PROTEIN 81 4-3-2 LANDRY AND MOUREAUX METHOD 82 4-3-2-1 GLOBULIN 82 4-3-2-2 ALBUMIN 83 4-3-2-3 ALCOHOL EXTRACTABLE PROTEINS 84 4-3-2-4 GLUTEL1NS '. 85 4-3-2-4-1 Gi- GLUTELIN 85 4-3-2-4-2 Gi- GLUTELIN 85 4-3-2^-3 G3- GLUTELIN 88 4-3-2-5 ' INSOLUBLE PROTEIN , 89 4 - 4 EFFECT OF DEBITTERING ON JN VITRO PROTREIN DIGESTIBILITY OF TWO LUPIN CULTIVARS 89 4^-1 IVPD OF PROTEIN 89 4-4-2 IVPD OF PROTEIN FRACTIONS 93 4-4-2-1 GLOBULIN 93 4-4-2-2 ALBUMIN ' 94 4-4.2-3 ALCOHOL EXTRACTABLE PROTEINS 95 4-4-2-4 Gi- GLUTELIN 95 4-4-2-5 , G3- GLUTELIN 96 4-4_2-6 Gs- GLUTELIN 96 SUMMARY AND CONCLUSIONS 98 REFERENCES 99 LIST OF TABLES

NO TITLE PAGE

1 PROTEIN EXTRACTION PROCEDURE FOR SEQUENCE Ao AND Do :.. 47 2 PHYSICAL PROPERTIES OF TERMIS CULTIVARS 51 3 CHEMICAL ANALYSIS OF RAW AND DEBITTERED CULTTVARS OF LUPINUS TERMIS 1... 53 4 FATTY ACID COMPOSITION OF LUPINUS TERMIS

SEEDOIL_..7 60 5 PHYTATEfTANNIN, OF LUPINUS TERMIS * CULTIVARS 66 6 MINERALS IN LUPINUS TERMIS SEED 67 7 PROTEIN FRACTIONS OF RAW AND DEBITTERED CULTIVARS OF LUPINUS TERMIS ACCORDING TO MENDEL - OSBORNE METHOD (1914) 79 8 PROTEIN FRACTIONS OF RAW AND DEBITTERED CULTIVARS OF LUPINUS TERMIS ACCORDING TO LANDRY AND MOUREAUX METHOD (1970) 86 9 EFFECT OF DEBITTERING ON IVPD OF LUPIN CRUDE PROTEIN AND PROTEIN FRACTIONS 90

(I) LIST OF FIGURES

NO TITLE PAGE

1 SHAPES OF TWO CULTIVARS 33

2 STANDARD CURVE OF TANNIN 36

3 STANDARD CURVE OF PHYTIC ACID 40

4 STANDARD CURVE OF PHOSPHOROUS 44

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(III) Abstract

Two lupin varieties ( Rubatab and Dongola) purchased from the local market were used hi this study . Physical properties showed that the weight and volume of cv.DongoIa were higher than those of cv.Rubatab while thickness and density of cv.Rubatab were higher than those of cv.DongoIa . Lupin varieties were debittered by cooking in water for 30 and 60 mins then steeped for three days in tap water for debittering . Proximate analysis showed that the moisture content ranged from 4.4 to 5.7%. Fiber content ranged from 8.8 to 10.9 % of whole seed and for kernels and testa it ranged from 0.9 to 1.8% and from 45 to47% respectively . The ash content ranged from 1.3 to 1.9% . Oil content ranged from 9.9 to 10.9% in whole seed (raw, debittered) and in kernels ranged from 11.5 to 12.0% while the testa had very low oil content ( 0.6- 1.1%). Protein content ranged from 49.4 to 50.4% for raw whole seed and was 47.1 to 50.4% for debittered whole seeds . For kernels it ranged from 56 to 59%. The protein content of testa ranged from 5.6 to 9.3%. carbohydrates content was about 23% for raw whole seed and ranged from 25 to 26% for debittered whole seed . It ranged from 21 to 24 % for kernels and increased to 39% in testa. Calorific value for 100 g of whole seed ranged from 381 to 433tcal. It decreased in testa ranging from 187 to 193/caI. In this study both varieties did not contain antinutritional factors v trypsin inhibitors . The content lannins and phytate was very low presumably due to debittring. Fatty acid composition of lupin seed oil showed that it is composed of ten fatty acids . Oleic acid was the major fatty acid . It ranged from 52-60% * Linoieic acid was about 16-19% . Palmatic acid ranged from 10-11% . Other fatty acid of lupin oil were palmitoleic, lauric , myristeic , stearic , largeric , linolenic , and arashidic . The mineral content of lupin seed showed that Ca was 0.1%, k" ranged from 0.4-0.5% and was 0.7% in kernels . Mg content ranged from 0.15-0.22% . Fe content was 0.03% . P content ranged from 0.2 to 0.3% . Zn content ranged from 82 to 127 ug/g for debittered whole seed , for kernels it ranged from 107 to 135 ug/g. Na content ranged from 56 to 72 ug/g. Mn content ranged from 4 to 6 ug/g . Pb content ranged from 9 to 10 ug/g. In this study lupin proteins were fractionated by two methods : Mendel-Osborne method and Landry and Moureaux . Protein fractions obtained by the first method were albumin which ranged from 3. Ho 24.8% , globulin ranged from 1.9 to 3 % , glutelin ranged from 13.2 to 62% . The Landry and Moureaux method gave globulins in the range of 24 to 66.4% , albumin ranged from 1.1 to 3.2% „ glutelin (G?) ranged

(IV) from l.Oto 14.6 % , glutelin ( G?) ranged from 5.3to 63.1% . Debittiing . increased glutelin and decreased albumin and globulin fractions . In vitro protein digestibility of total lupin proteins ranged from 73.9 to 81.0% for cv.Rubatab and from 73to 80 % for cv. Dongola. IVPD of the globulin fractions for cv.Rubatab ranged from 97 to 77% for whole seed and 96.5/- for kernels showing asignificant decrease as a result of debittring . For cv.Dongola it increased from 45 to 87.7% in whole seed ( raw and debittered) and from 61.2 to 78.9% for kernels . IVPD of albumin decreased significantly/for cv.Rubatab ranged from 78 to 33% for whole seed ( raw and debittered ) and 29% for kernels . For cv.Dongola it ranged from 96 to 76.6 % for whole seed (raw and debittered ) , from 92.4 to 69.7% for kernels . IVPD of glutelin £ Gi) for whole seed ( raw and debittered ) decreased significantly! for cv.Rubatab it ranged from 64.4to 53.5% an^l from 55 to 48.9% for kernels . For cv.Dongola it decreased significantly from 94.2 to 48.5% forwhole seed ( raw and debittered) but for kernels it increased significantryffrom 58.9 to 74.2 % . IVPD of glutelin ( G2) of cv.Rubatab ranged from 74.6 to 79.7% for whole seed ( raw and debittered ) and for kernels it ranged from 61 to 57% . For cv.Dongola mcreased significantly ( P <0.05 ) from 63.7 to 91% . IVPD of glutelin ( GJ) for cv.Rubatab increased significantly from 45.1 to 88.5% for whole seed . For kernels it decreased significantly ( P < 0.05) from 94.0 to 78.7% . For cv.Dongola for whole seed (raw and debittered ) it was significantly increased from 35.5 to 76.7% and for kernels was decreased insignificantly (P < 0.05) from 78.7to75% . Results indicated that IVPD of globulin of cv.Rubatab and albumin and Gi- glutelin for both cultivars decreased significantly (P < 0.05) due to debittering. The globulin , prolamin and G2- glutelin for cv.Dongola increased significantly (P < 0.05) . IVPD of G>- glutelin increased significantly (P < 0.05) for both cultivars as a result of debittering .

CHAPTER I INTRODUCTION

The genus lupinus is a member of the family Drgiiminacae subfamily Papilionaceae. Species of the genus lupinus are widely cultivated throughout temperate climate zones in both the Southern and Northern hemispheres, ranging from Russia and Poland to the Mediterranean countries and from Western Australia to Southern Chile and South Africa (Aguilera, and Trier, 1978). Cultivation of lupinus 1 practiced in Egypt as early as 200 B.C. Where, they were known as termis. Lupinus were also well known to as Tahuri (Campos and Eldash, 1978). The Greek word for Lupin albus was thermos. Other names for the plant throughout the Mediterranean area appear to be derived from this: termis (Egypt), turmus (Arabic), altramuz (Spain). This may indicated that the plant was first cultivated as a crop in Greece (Aguilera and Trier, 1978). Lupins can be grown in sandy and acid soils that are unsuitable for most other crops. Lupins have a high capacity for the absorption of phosphate that other plant do not use, and they can be harvested easily with combines (Compos and Eldash, 1978). Lupin tolerate a wide variation of soilsandofclimateconditions.lt was grown as an aesetival crop in cold temperate areas and as a winter crop in temperate and warm temperate ones (Gladstones, 1970). Since antiquite lupins have been known to improve soils, this is now known to stem from their ability to fix nitrogen and for this reason the lupins were used in crop rotation with cereals and pasture, and also for green manuring (Aguilera and Trier, 1978). The oldest agricultural species is L. albus; White lupin was known to the ancient Greeks and Romans (Hackbarth, 1961). In recent years, sweet varieties of L. angustifolus have been grown in different parts of the USA, where they are used principally for late winter cattle forage (Gladstones, 1970). Lupin mutabilis has recently attracted an increasing amount of scientific attention in Germany, Peru, Chile and England, and sweet varieties are being developed (Tello, 1976; Gross and von Baer, 1975; Masefield, 1975 and Ortiz, etaJL, 1975). The modern expansion of lupin growing can be said to have started in 1780, when King Frederick II of Prussia had seed of L. albus imported from Italy and personally supervised experiments on their cultivation (Hackbarth, 1961). The two species and Lupimis mutabilis have been cultivated for grains for 300 years or more in the Mediterranean basin and in the high lands of South America. The main interest in lupins for food arid feed is related to its high content of protein and relatively high oil content in some varieties. Tannous and Cowan (1967) tested samples at a bitter lupin from the mediterranean region after submitting then debittering process of prolonged steeping and boiling in water. The.average PER values for rats were elevated from 0.57 to 1.06 when the diet was supplemented with methionine as compared with 3.05 for casein standard. The low PER could have been the result of incomplete detoxification of the seeds or damage of the protein during the debittering process. Alkaloids-free lupins flour was reported to have been used as a protein additive in bread making (Ballester et al., 1984). Lupinus tennis (syn. L. ajbus) is commonly grown in the Middle East. For the most part, the plant is grown as a green manure on poor soils, however, the seeds are consumed to a certain extent. (Tannous et al., 1968; Grindley and Akour, 1955). Objectives of this Studv:- The objectives of the present work were to study differences in chemical composition of lupin cultivars cultivated in Sudan. Also to investigate the effect of traditional debittering processes on lupin proteins, the in vitro protein digestibility and to examine the presence of anti nutritional factors present in lupin seeds.

CHAPTER II LITERATURE REVIEW

2.1 Nutritional value of lupin seeds: Food are grown and used for food in nearly all parts of the world. About 20% of the protein currently available to man is derived from these legumes and they comprise nearly all of the dietary protein for up to 50% of the population in many developing countries. Of more than 13000 species of legumes, only about 20 are commonly consumed by humans (Haytowitz, et al., 1981). The western states contribute about 40% of the U.S Crop of dry beans, 30% of the processed bean and all of the seeds of dry and green beans (Salunkhe, et al., 1984). Lupin seed has been found suitable as a supplement for pig, poultry and stock feeding; for poultry feeding lupin can be usefully supplemented with methionine. The increasing shortage of protein feed for human and animal consumption and the rising cost of energy have drawn attention to legumes as an important source of protein and calories. A promising seeds are lupins, especially for areas in the world were soya does not grow with its two tolds advantage, which includes a high amount of both protein and oil. Lupins present a valuable food crop to combat malnutrition and a promising cash crop to provide income for small subsistence farmers (Shoeneberger, et al-, 1987). Population in many parts of Europe, Africa and South America have learned through ages to remove the water soluble alkaloids from lupin grains by cooking and soaking the seed in water for several days (Hatzold, et a|., 1983 and Lucisano, et al., 1982). In Chile and Peru, efforts are currently under way to promote the use of lupin as a cheap protein source especially for small subsistence farmers. Lupin flour may also be used to supplement common foods such as bread (Eldash and Compas, 1982). Other studies indicated lupins compare very favourably with as a source of human foods. As a consequence, lupin products are increasingly being incorporated into food stuffs as an economic alternative to bean products. For example the incorporation of full fat lupin flour into bread increases the protein content and improves the ammo acid balance (Eldash and Sgarbieri, 1980 and Ballester, et al., 1984). Similarly the addition of 2-6% lupin flour to macaroni improves both the cooking and colour characteristics of the product (Morad, et a]., 1980). In Peru the addition of lupin flour from L. albus is permitted in bread, biscuits, sauces, soups and noddies. In Chile upto 10% lupin flour can be added to bread, cakes, pastry and biscuits (Gross and von Baer, 1977). Liquid and powdered imitation milks was prepared from sweet lupin (Lupinus albus cv. Multolupa) by three stages grinding of blanched bean followed by homogenization and spray drying. Lupinus angustifolius and L. luteus appear to be able to replace meal in wheat-based poultry rations for chicks, laying hens or intensive broiler production in Western Australia (Hill, 1977). The protein of food legumes is relatively rich source of lysin and tryptophane but is low in sulfur amino acids (Bressani and Elias, 1974). 23_Characteristics of the seed: Seeds of lupin varieties of interest have shapes that range from almost round and flat to nearly spherical. Low-alkaloid varieties give an overall colour impression white to green. Cotyledons of mature seed are yellow. Some bitter varieties are mottled, which are dark gray to dark brown. Lupin seeds are larger than soybean and carry a substantial seed coat that ranges from 15% in L. albus to over 25% in L. luteus on whole dry seed basis. Soybeans has 9% hull on the average (Aguilera and Trier, 1978). 2.4 Chemical composition of lupin seed: As expected there is a considerable variation in composition among different species, superimposed on environmental factors that may affect the chemical make-up of the grain (Hill, 1977). Hove (1974) Aguilera and Trier (1978) reported that the chemical composition of lupins seeds varied considerably according to variety as well as location, legumes lupin seeds are characterized by a higher protein content range from 34.8 up to 62.9% (Aguilera and Trier, 1978). It should be noted that since the hull content of lupins is high the protein and oil contents of dehulled seeds is enhanced significantly. Tims, a full fat flour from L. luteus may contain ahnost 60% protein and defatting can accomplish additional concentration of protein. Lupin seed has a high protein content (25-44%) and contains upto 24% oil (Aguilera and Trier, 1978). Two species of sweet lupin: L. albus and L. luteus. were analyzed. Both species were good sources of protein 34 to 39%. Lipid content measured as ether content was 10.9% for L. albus and 4.7 for L. luteus. Both The concentration of total alkaloids in various ecotypes?? ofLupinus mutabilis cultivated in Peru amount to 3.3% (Hatzold, et aL, 1980). these compound must be removed by water extraction before consumption by man. Lupin crops are being planted in W. Australia for grazing sheep and cattle. A serious problem sometimes arising when grazing sheep on lupin develop lupinosis. A degenerative liver condition of mycotoxins origin (Warmelo, et al, 1970; Hill and Arnold, 1975). the classical symptoms of lupinosis may cause death in less than 48 hr. Biochemical indicators of lupinosis in the circulatery system have been identified (Aguilera and Trier, 1978). 2.2 Physical properties of lupin seeds: Physical properties such as dimensions, density bulk density and porosity are important in the engineering, handling and shortage of legumes. Correlations between physical and nutritional composition may be useful in plant-breeding work to predict content from such simple parameter as grain size, shape and weight. Meager information is available however, on physical properties and their relationships with chemical composition of dry beans. A study was therefore undertaken to investigated these aspects, of ten cultivars of dry beans commonly grown in the U. S. (Deshpande, et al., 1984). According to Hlynka and Busuk (1959) frictional properties of rice and paddy also play an important role in their degree of packing in a bulk i.e its porosity and hence the bulk density. The specific and bulk volumes of the beans, indicate their storage space requirements and handling volumes. Actual storage space requirement of beans, however, is indicated by their bulk volume (Deshpande, et al., 1984). legumes had high crude fibre content of more than 10% and low alkaloid contents (0.05 - 0.09%) (Ballester, et al., 1980). Lupin generally contains about twice the protein found in legumes normally consumed by man. Additionally lupin yields 1000-2000 kg/ha compared to 580-620 kg/ha for bean, or 760-870 kg/ha for chickpeas (Jalil, 1972). The initial protein content of the lupin seed was modified by dehulling and losses during blanching. Crude fibre decreased sharply after dehullmg because the hull was 18% of the seed weight. The hull to very high in dietary fibre (65-80%) (Feldhium and Wisker, 1986). The protein content of lupin (L. albus cv. multolupa) averages 42%: on dry basis. The oil content measured as etherextract was about 12% and the crude fibre was about 3%, that for whole seed is 11.3% (Yanez, et al., 1986). Full-fat sweet lupin flour (FFSL) (L. albus cv Multolupa) containing 35.6% protein was used to enrich cookies give to a group of school children. Full-fat sweet lupin replaced 5, 10, 15, 20 or 25% of wheat flour in the formula. The protein content of the cookies increased by 8.9% upto 55.7% for the 5% and 25% FFSL respectively (Ballester, et al., 1986). 2.4.1 Protein content: The true seed protein content varied from 30% to 45%. Protein quality and digestibility compares favourably with those of soybean. Protein yield data can be confusing, as they may be quoted for whole seed or for dehusked material or for flour and in lupin the husks represent a high proportion of whole dry seed ranging from 15% in L. albus to 25% in L. luteus fCerlctti andDuranti, 1979). Protein content in sweet lupin hull was 3.86% and in bitter hull was 2.77%.

10 Legumes have a high protein content ranging from 17-25% in the dry form. Protein content of the edible protein of legume seeds is double that of cereals and is slightly higher than that of meat, fish and eggs (Watt and Merrill, 1963). Legumes contribute 8-10% of the world protein supplies. Over 90% of the nitrogenous content in pulses are likely to be protein or protein derivatives capable of being utilized for protein (Patwardhan, 1962). Full fat sweet lupin flour (SLF), which is rich in protein 39.7% and has a fairly high concentration of the indispensable amino acids: lysine, leucine and thrconine was used to fortify bread at 7 and 10% level (on flour basis), the protein efficiency ratio (PER) of bread fortified with 7% (SLF) was 0.9 which is slightly higher than that of control (0,81). The PER of bread fortified with 10% SLF showed on increase of 58% over that of the control (EL dash and Sgarbieri, 1980). Sgarbieri and Galeazzi (1978) reported the protein in L. albus cv. multolupa ranged between 38-40%. Karara (1982)/Hammam (1977) found that the protein content of lupinu tennis seed varied according to both cultivar and location and it ranged between 43-50%. Preliminary results on lupin enriched bread (Ballester, etal., 1984) obtained using the standard baking method (AACC, 1976) has shown that 9% full-fat lupin flour (FFLF) increased the nutritional quality of bread without affecting its baking quality. In Chile two types of breads namely: Marraqueta and Hallulla, dominate the market and the effect of incorporation 6, 9 or 12% FFLF on baking properties and nutritional value of these bread been investigated (Ballester, etal, 1988).

11 Geervani and Theophilus (1980) reported 'that moist heat treatment enhances protein quality of legumes to a greater extent than dry heat. 2.4.2 Oil and Fatty acid: Lupin can be grown as an arable leguminous oil seed crop in temperate climates, though the oil yield as a proportion of dry seed is somewhat low in comparison with soybeans. The oil is readily extractable and can be refined by conventional processes to yield a pale edible oil in good yield. Owing to the presence of linolenic acid, lupin seed oil, like soybean oil has limited stability in ambient storage conditions but its quality compares favourably with that of soybean oil or raptseed oil (Fleetwood and Hudson, 1982). Hudson, et al. (1976) drew attention to the possibilities of lupin as an arable food crop for temperate climate and briefly noted a range of technically and nutritionally important features. The lipid content of 10.6% (L. albus cv. Multolupa) of sweet lupin was range of values in the literature for other species and varieties (Hove, 1974; Q) Junge, 1973). Crude lupin seed oil contains, in addition to triglycerides and small amount of free fatty acids (0.6-3.0%), small amounts of phospholipids, which are present in insignificant quantities to demand a specific degumming step in the refining sequence. Such a step can be included but offers little, if any, advantage. Lupin seed oil contains 1.0 to 1.5% ofnon-saponifiable matter, which includes in addition to the , , sterols and triterpen alcohols. Analysis of lupin seed oil for component fatty acids was carried out. A part from the five main fatty acids, small quantities of 16:1, 20:0, 20:1, 22:0, 22:1 and 24:0 were also detected, especially in L. albus and L. luteus. 22:1

12 may be enicic acid, but this has not yet been confirmed. It is absent in L. mutabilis. Fatty acid compositions of lupin seed and soybean oil are similar, but that there is marked inter-specific variation in the case of lupin seed oil (Fleetwood and Hudson, 1982). However, there is considerable variation between species with respect to oil content; L. albus and L. mutabilis being the most promising. A prime objective of breeding programmes is to raise the oil yield to levels which have already been achieved by similar procedure in the case of soybean (Hudson, etal-, 1976). Lupin oil usually contains a small amount of erucic acid (22:1) which is now considered an undesirable component. However, the amount never exceeds 2% and this acid is absent in L. mutabilis. Linolenic acid (18:2), the principle essential fatty acid, is always an important component. Lupin seed oil can be considered nutritionally good (Hudson, et al., 1976). the first recorded component fatty acid analysis of lupin seed oil was that of L. term is by Grindly and Akour (1955). Subsequently, recorded analyses for L. albus and L. luteus for L. mutabilis (Gross and von Baer, 1975) and for L. angustilfolius (Hasen and Czochanska, 1974). Mature seed of four lupinus species, L. albus, L. angustifolius. L. luteus and L. mutabilis have been shown to be possible sources of edible oil. The most promising are L. mutabilis and L. albus in which cultivars have been identified containing upto 21% and 13% respectively of oil in whole seed. Three of the species contain significant quantities of 11 component fatty

13 acid 16:0, 16:1, 18:0, 18:1, 18:2, 18:3,20:0,20:1,22:0, 22:1, and 20:0 but 20:1,22:1 and 24:0 are absent from L. mutabilis. It is possible to distinguish clearly between oil from the four species. Of the unsaponifiable components the sterols comprise -sitosterol, campesterol, estigmasterol and cholesterol. Though 4-methyl sterols cann't be detected. L. luteus seed oil contains primarily - but also a-, -tocopherols (Hudson, et al., 1983). 2.4.3 Dietary fibre: Legumes are good sources of dietary fibre (Walker, 1982; Shutler, et a]., 1987) whose high consumption has been correlated with decreased incidence of the so-called "diseases of affluence" (i.e diverticular diseases, colon cancers, obesity, coronary heart diseases, diabetes, dental caries, etc). According to Burkitt and Trowell (1975) such diseases of affluence are not commonly observed in developing countries where legumes and other high fibre plant food are common staples. Dietary fibre is reported to have a hypo-cholesterdemia effect (Shutler, et aL, 1987) as well as hypoglycemic effect (Jensen and Jepsen, 1982). It leads to a decrease in intestinal transit time and also increases faecal bulk, it binds bile acids, and degrades it to short chain fatty acids in the large intestine; it increases viscosity and slows digestion (Passmor and Eastwoord, 1986). Water soluble fibre is particularly effective in lowering serum cholesterol, while water insoluble fibre provides bulk, pushing food through the digestive system at a faster rate (Uzogara, et al., 1992).

14 Ballester, et al. (1984) reported that the fibre content was 1.9% in iupjnus albus cv multolupa. Baliester, et al. (1980) reported that the fibre content was 11.7% in L. albus and 16.8% in L. luteus.

Lupin seed of all species is low in calcium and it appears that this element is concentrated in the testa rather than the kernel. Phosphorous appears to be concentrated in the kernel. Hove (1974) was unable to detect any phosphorous in the testa of L. angustifolius or L. luteus. A comparison of the manganese content of 33 samples of seeds from two species of lupin grown under field conditions in Victoria, Australia, indicated that lupinus angustifolius had a much lower Mn content (6 ly-g/g) than L. albus (1316yWg/g). In L. albus 80% of the Mn content of the seed was concentrated in the endosperm, while in L. angustifolius only 40% of the Mn was present in the endosperm. Whole flour and spray dried powder produced from L. albus seed with Mn content of 4479/-

15 important changes compared to previous methods of grain protein fractionation. The use of aqueous alcohol plus reducing agent after the aqueous alcohol extraction and a final extraction with basic buffer containing SDS plus reducing agent.'These changes resulted in much improved protein extraction (Taylor, et aj., 1984). Protein composition in terms of solubility fractions changes with maturity of the grain. In kernels analyzed shortly after pollination, free amino acids and salt soluble proteins predominate, whereas in nature kernels zein and glutelin are the major proteins. At the milky stage used for food, an intermediate composition occurs, these changes in composition during the grain maturation were reflected in decreased available lysine content in the older grain (Luk yanenko, 1977; Pukrushpan, et a]., 1977).

In a comparative study Sathe and Salunkhe (1981a ) found that, the protein concentrates and isolates were viscous than both albumin and globulin fraction at a certain concentration. Cerletti, et al. (1978) and Sathe, etal. (1982) reported that, the lupin protein fractionation by SDE-PAGE resulted in 13 or 12 protein sub-units. Moreover, lupin albumin fractions appear to contain at least five protein sub- units. While globulin fractions contain at least seven proteins sub-units. Oomah and Bushuk (1983) investigated the electrophoretic patterns of protein fractions of lupin Lupinus albus and L.angustifoHus and found them to be similar, however, the patterns of globulin fractions are variety dependent. Protein isoelectric precipitate (PIP) and protein micellasmass (PMM) both obtained from raw lupin flour had 10 and 8 protein subunits, respectively. In case of PI1M from debittered lupin flour the number detected

16 in the PIP from the same material. This considerable variation was mainly due to the different conditions used in extraction and precipitation of the two protein preparations. These findings are in good agreement with those reported by Blagrove and Gillespie (1979) who observed that the electrophoretic patterns of water-soluble and salt-soluble lupin protein on SDS-PAGE were found to be completely different. Data in the literature, though based in incomplete resolution, indicate that seed globulins consist of several fractions with similar properties in different lupin species. The most detailed information has been obtained on L. albus by Cerletti, et al. (1978). Globulins are 84% of total protein of dehusked seed which were divided into six major fractions with M.W 430000, 330,000, 300,000,/235,00, 187,000, which all contain a . One has isoelectric point 7.9, all the others have values in the acidic range between 5.7 and 6.2. AH dissociate into several different sub-units; most associations depend on rather loose ionic interactions, but some are due to disulfide bridges. Some sub-nuits of different fractions, appear highly similar (Cerletti, etal., 1978). Three of the fractions do not contain any cysteine or methionine or have only traces of them; three have both these amino acids at a higher level than soy protein. Fraction 1 represent only 5.7% of total globulins. The two other fractions with sulfur-containing amino acid are very much like in sub-unit composition and together make up 45% of total globulins; cysteine is present in apparently identical sub-units (Cerlett, et aj., 1978).

The albumin component of seed has a better amino acid composition than globulins but is scarce and contains a large number of different molecules species.

17 Results obtained on L. angustifolius suggest that bio-synthetic regulation affects the globulin fractions as separate entities, through their changes are interrelated. Tndeed deficiency of sulfur in the nutrient medium decreases two sulfur-rich protein components, which move in electrophoresis like fraction 1 and 7 respectively, and increases a component poor in sulfur with same electrophoretic behaviour as fraction 4, 5 and 6 (Gillespie, et a]., 1976). The selective amino acid distribution in separate molecules and the possibility of shifting their ratios give hope that a genetic approach may be effective in improving the quality of globulins (Cerletti and Duranti, 1979). Gopalakrishna et al. (1977) concluded that Xand^ fractions correspond to legumin and vicilin respectively on the basis of acid precipitation. The fraction resembles the conglutin tf fraction of lupinus reported by Blagrove and Gillespie (1975), in having higher amount of sulfur amino acids. 2.6 Classification of lupin protein fractions:- Osborne (1924) reported that globulins are the major storage proteins of legumes and they require appreciable salt concentration for solubilization and they account for 50 - 75% of the total seed proteins. Other studies on legume proteins have reached the same conclusion (Romero et al. 1975; Chakraborty et a]- 1979). Corn grain proteins can be separated into five fractions by a selective extraction method (Landry and Moureaux, 1970). I- Albumin, globulin, free amino acids and small peptides fragments and any other saline soluble compounds. II- The prolamin and zein.

18 m- Contains zein-like proteins that are soluble in alcohol after the disulfide bonds in the protein have been reduced with 2-ME. (G). IV Proteins that have some of the characteristics of glutelin - G2. V- True glutelin (G3), which is a complex high molecular weight

mixture of proteins which can be solubilized only by treatment with reducing agent and a detergent (SDS) at alkaline PH (Walt and Paulis, 1978). Because each fraction tends to have different characteristics and amino acids composition, the relative proportion of each, strongly affects the level of particular amino acid in the total grain protein (Johnson and Lay, 1974). When these fractions were analyzed for their amino acids content, the zein fraction was shown to be very low in Lysine content and lacking in tryptophan, since these zein fractions make up more than 50% of the kernel protein, it follows that they are also low in these two amino acids. The albumin, globulin and glutelin fractions, on the other hand, certain relatively high levels of lysine and tryptophan. Another important factor of the zein fraction is their very high content of leucine, an amino acid implicated in isoleucine deficiency (Patterson et al., 1980). 2-6-1 Albumins and globulins:- It is noteworthy that albumins and globulins represent the major proportion of legume protein. Fox et al. (1964) shoWtbat albumins and globulins form always the major fraction in comparison to prolamins and glutelins in leguminous seeds. Albumins and globulins are very heterogeneous and differed in polypeptide composition. Extraction with water and then with NaCl to solubilize what is termed albumins and globulins respectively. These

19 descriptions are inaccurate since water also extracts the lower molecular weight nitrogen (LMWN) as well as the albumin. In addition because of endogenous salt in the grain some globulins are also extracted (Wilson et al, 1981). El-Habbal et al. (1987) showed that during germination albumins and globulins increased significantly and reached their maximal values (58.94 and 23.18%) at the third and fourth day respectively. Gradual reduction was observed in both fractions as the time of germination progressed to reach 51.26 and 12.98% at 7 days after germination. Prolamins decreased significantly after one day germination followed by statistically stable amount. Meanwhile, gluteliris were generally and significantly decreased as germination after 6 and 7 days. El-Habbal et al. (1987) showed that as germination period advanced non-soluble protein was significantly increased and took place at the expense of albumins and globulins. Similar findings were obtained by Abdel-Hamid and Shadi (1981). The scanning electron microscopic observations indicated that albumin had rod-like structures with relatively smooth surface topography while globulins consisted of different shapes and sizes with relatively rougher surfaces than albumins. (Satlie and Saiunkhe, 1981 a). Viscosity appeared to be a function of not only the solids concentration but also of the type of proteins. Albumins registered comparable viscosity to that of globulins at corresponding concentration. Protein concentrate and protein isolate were more viscous than both the albumins and globulins at a particular concentration, suggesting that when albumins and globulins were

20 together, they behaved in different manner than when they were separate

(Sathe and Salunkhe, 198 la). The difference in water absorption by albumins and globulins (3.18 and 2.77 g/g dry sample, respectively) may have been due to the protein concentration and possibly the conformational characteristics of these proteins. (Sathe and Salunkhe, 1981). 2.6.2 Alcohol extractable proteins (AEP): AEP can be defined as a portion material extracted at room temperature by aqueous alcohol; free of reductant and salt, deprived of lipids and salt soluble proteins. Zein (AEP) proteins are synthesized in the developing endosperm where they from protein bodies-within the rough endoplasmic reticulum because they account for more than half of the total seed protein (Landry and Moureaux, 1981). Zein contained major polypeptides with average mol. wt. of 25000 and 21800 daltons (Misra etal., 1972) zein are devoid of lysine and tryptophan, an essential amino acids and certain much higher levels of leucine, proline and glutamic acid than the other com protein fractions (Paulis, 1982). The AEP content ranges from 3.1 to 6.9% of the total seed proteins in chickpea seed (Dhawan et aj., 1991). 2.6.3 Glutelins:- Glutelins are defined as including those proteins that are either soluble in dilute aqueous alkali or insoluble in neutral aqueous, saline solutions or alcohol (Osborne, 1924). After albumin, globulin and zein proteins were removed from corn, it was found that the addition of 2-ME to 70% ethanol -

21 0.5% sodium acetate solvent removed protein from corn (Paulis and Walt, 1969). This protein was thought to be zein-Iike in solubility but later it was termed alcohol-soluble reduced glutelin.based on the definition that all proteins remaining after remove 1 of salt and alcohol proteins were glutelins (Paulis and Wall, 1971). Glutelins consists of several different polypeptides chains linked by disulfide bonds to from an insoluble three- dimensional matrix (Nielson et al., 1970). Glutelins through non covalent bonding; they consist mainly of two categories linked by disulfide bonds. Alcohol soluble and alcohol insoluble glutelins are two types of polypeptides deposited in different subcellular structures. The alcohol soluble polypeptide resembles prolamins but have significant structural differences (Paulis, 1982). Corn grain contains three glutelin subgroups called G], G2 and G3 glutelin (Landry and Moureaux,

1970). 2.6.3.1 G^ -glutelins:-

G] - glutelin (zein - like) has an amino acid composition somewhat similar to zein, but with high levels of glycine, methionine, histidine and proline and lower levels of aspartic acid leucine and isoleucine, Gi - glutelin was previously recovered as a part of glutelin fraction, zein appears to be cross-linked to glutelin through disulfide bonds (Paulis and Walt, 1969). G\ -glutelin contains major polypeptide of molecule weight 26000,

23000 and 18000 daltons (Misra et al, 1972). 2.6.3.2 G2-glutelins;-

G -glutelin (glutelin - like) isolated at PH 10, can be fiactioned on the basis of their extractability at PH 3, contains major polypeptides of molecular

22 weight 61000, 58000, 25700 and 19000 daltons (Misra etal., 1972). Acid soluble G2 - glutelin exhibits some general characteristics of cereal prolamines. They are rich in proline, in glutamic acid or glutamine and typified by a high histidine and poor lysine and aspartic acid or asparagine. Moreover, they may be extracted both by acidic and alcoholic media. Indeed, amino acid composition of acid soluble G2 - glutelins and water soluble, alcohol soluble - glutelins are nearly identical (Landry and Moureaux, 1981). Consequently, the acid insoluble G - glutelins are not removed by alcoholic extraction. Because of their extractability and amino acid composition, especially their relatively high lysine content, they may be regarded as being similar to G - glutelins. Thus fraction IV contains both acid-soluble and acid-insoluble. G2 - glutelins, rich in histidine, which might be called prolamin-like and glutelin-like respectively. A mount of G2 -

i$*ufcj «fc d<'f ( 5) depends on conditions used earlier to extract salt- soluble and alcohol-soluble proteins (Landry and Moureaux, 1981). 2.6.3.3 G^ - glutelins:- They are polypeptides which do not separate clearly on SDS polyacrylamide gel (Misra et a]., 1972). Moreover, G3-glutelins having an amino acid composition similar to that of salt soluble proteins (though richer than them in hydrophobic residues and with lower cysteine content than other glutelin subgroups) appear to be non-exrractable by 2-ME in a saline or alcoholic medium or by their combination. The inability to extract G3- glutelins with such media may be related to non-covalent interpolypeptide bonds (Landry and Moureaux, 1981). %

23 G3-glutelins exist at the earliest stages of grain development before

zein accumulation so they may consist of membrane proteins from cell organelles such as mitochondria or ribosomes (Londry and Moureaux, 1979). This hypothesis lends support to the existence of non-covalent bonds in glutelins since hydrophobic interactions stabilize tliese membranes and their multimeric enzymes. This is also a close parallel between the decrease in the amount per grain of salt soluble protein and the increase of G3-glutelins observed during grain maturation. Therefore G3-glutelins include membrane- bound protein. As G3-glutelins could contain membrane proteins, all the more as detergent is necessary to its dissolution (Landry and Moureaux, 1981). Alkali-soluble protein fraction was 11% in legume (Deshpande and Nielsen, 1987). In six chickpeas analyzed for their protein fractions glutelins content ranged from 19.38 - 24.4% (Dhawan, et aj., 1991). The glutelins had molecular weight 45000 - 17000. The glutelins and prolamins had low molecular weight polypeptides and isoelectric points mainly in the acidic range. (Dhankher et al., 1990). 2.6.4 Insoluble protein:- Insoluble protein (residue) was not extracted because it was linked to cell wall (Wall and Paulis, 1978). The residue may be unextracted glutelin plus variable amounts of globulins and albumins associated with and cell debris (Sodek and Wilson, 1071). Moreover, a small amount of nitrogen remains in soluble after all these extraction procedures. This residue consist mainly of protein from previously defined groups becoming insoluble due to interaction with lipids,

24 carbohydrates, or polyphenols via oxidation process (Landry and Moureaux, 1981). 2.7 In vitro protein digestibility of lupin seeds:- Characteristics'bften used to define protein quality of a feed or food are its ammo acid composition and its protein digestibility (Hahn etaL, 1981). Protein digestibility primarily determines the availability of its amino acids (Hahn et al., 1981). Historically, protein digestibility has been determined bybioassays using rats or microorganisms. These procedure share the disadvantages of being time-consuming and expensive (Hahn et al., 1981). Saunders et al. (1973) have developed a pepsiti-trypsin system for the measurement of protein digestibility. The method involves a comparison of the protein content of a sample before digestion with the protein remaini% after digestion. They report that with this jn vitro system they obtained a high degree of correlation with in vivo digestibility for 14 different alfalfa protein concentrates dried by freeze-drying, air drying and high temperature drying, with a range in protein digestibility of 80 - 99% The lupin proteins digestibility for different species was found about 95% compared with 92% for soybean protein (Hudson et al., 1976). Moreover. Hammam (1977) found no significant difference between raw and debitter lupin L. termis flours in terms of their protein digestibility. Schoeneberger et al. (1983) observed that the debittered lupin has a protein digestibility «/ 90.9% compared to that of casein (91.9%).

25 The apparent digestibility of lupin protein is better than that of other legumes such as peas and bean, for which Hove and king (1978) found values lower than 80%. 2.8 Antinutritional factors:- Of the various antinutritional factors that are found in grain legumes, irypsin and chymotrypsin inhibitors, amylase inhibitors, polyphenol (commonly refereed to as tannins) oligosaccharide and phytic acid (Singh, 1988). Food legumes are well known for causing flatulence when consumed in large quantities. This property is mostly attributed to high levels of oligosaccharides: stachyose, faffinose, and verbascose. Studies of flatulence include experiments with intestinal microflora animals (principally rats and clogs), and man (Cristofaro, et al. 1974). Finally it has been reported by RISAT (1991) that phytic acid interferes with mineral utilization. Also cooking and germination reduce the levels of phytic acid in pigeon pea. 2.8.1 Phytic acid Defimtion:- Phytate, phytin and myoinositol hexaphosphate are synonymous terms for an organic phosphate defined by most workers as phytic acid (Garcia- villanova eta]., 1982). The term phytin implies a calcium - magnesium salt of phytic acid, where as phytate would mean the mono to dodeca anion phytic acid. However commercially available phytic acid or phytate often contains lower phosphate derivatives than hexaphosphate (Cosgrove, 1963) Cosgrove (1966)

26 reported that phytic acid can be dephosphorylated by phosphatase enzymes commonly called phytases and also by heating in acid or alkali. Its hydrolysis produces inosito (-2-P and five molecules of orthophosphoric acid (Graf, 1982; Graf, 1983; loewus and Loewus, 1983). Phytates have been found in cereal grains and legumes up to a level of approximately 5% by weight (de Boland et al., 1975). Phytic acid has also been implicated in the prevention of dental caries through its ability to bind with tooth calcium in the formation of resistant enamel (Maga, 1982). The interaction of phytic acid with proteins has been studied mainly in soybean and other legumes. Many factors were found to affect the protein phytate complexes and their solubility such as protein matrix (Carnovale et al., 1988) and nature of these complexes (Carnovale et al.., 1988). Phytate is of great importance as it acts as an antinutritonal factor when consumed in excess (Parrish et al., 1990). It has the ability to chelate Ca, Mg, Fe and Zn (Huls et al, 1980; Rendleman, 1982; TahaetaL, 1987;

Parrish et a]., 1990) in addition to Mo (Lolas and Markakis? 1975). It is well known that phytate decreases the in vitro digestibility of casein by pepsin (Knuckles et al., 1989). 2.8.2 Tannins:- Definition:- Tannin are polymeric phenols of higher molecular weight (mol. Wt. 500- 5000) containing sufficient phenolic hydroxyl groups to permit the formation of stable cross links with proteins (Swain, 1965).

27 Classification of tannins:- Plant tannins have been classified by Freudenberg (1920) into two main groups based on structural types: the hydrolyzable tannins and the condensed tannins. This is the most widely accepted classification proposed so far (Haslam, 1966; Swain, 1965; White, 1957) for the two groups. • Hydrolysable tannins:- The building units for this type are gallic acid and sugar or sugar esters of gallic acid. Polyester types hydrolysed by acid or enzymes into simpler fragments such as gallic acid or ellagic acid plus glucose (Haslam, 1966). • Condensed tannins:- The main building unit is catechin which is flavan-3-ol and it's isomers. Condensed tannins have no carbohydrates core. Condensed tannins are more complex and heterogeneous. Structures for various condensed plant tannins were proposed by several workers (Drews et a]., 1967; Thompson etal., 1972). According to Tamir and Alumot (1969), tannins have the ability to bind proteins forming insoluble complexes through the formation of hydrogen bonds between hydroxyl groups of tannin polyphenols and the carbonyl groups of peptide bonds of proteins (Millie and Stojanovik, 1972). In other studies, tannins reduced crude protein and precipitated blood protein (Bate- Smith, 1973). Tannins are phenolic polymers which through hydrogen bonding with peptide linkages, precipitate proteins from aqueous solution rendering plant proteins relatively indigestible and reducing enzyme activity (Goldstein and Swain, 1965; van Sumere etal, 1975).

28 Tannin bind certain proteins veiy strongly (liagerman and Bulter, 1981) and thus diminish the digestibility and nutritional value of high tannin sorghum grain (Price et al., 1979). A negative correlation was found between tannin content and protein digestibility (Mclead, 1974).

CHAPTER HI MATERIALS AND METHODS

3.1 Materials 3.1.1 Ten kilograms of lupin seeds were purchased from local market: 3.1.2 Debittering of lupin seeds: Seeds were debittered following a traditional procedure Lupin seeds Cleaning Boiling for 30 and 60 min

Debittering in tap water forthree days

dehulling Drying (60 C) Milling

Screening

Full-fat lupin flour

Defatting

defatted lupin flour

Flow diagram for preparation of debittered flour. 3.2 Methods: 3.2.1 Physical properties: Physical properties of lupin seeds were determined according to Salunkheetal. (1984).

31 The length (L) breath (g) and thickness were measured on 25 randomly selected seeds for each cultivar using a vernier caliper. Weights were determined by weighing 100 randomly selected seeds. 3.2.1.2 Density: Density (g/cc) was determined by displacement of xylene. Hundred seeds selected for weight determination were placed in a 250 ml measuring cylinder containing a known volume of xylene. The difference between the initial and the final reading gave the volume of the 100 seeds of known weight. Each measurement performed in triplicate. 3.2.1.3 Bulk density: The bulk density (g/cc) was determined was determined by placing known weight of seeds in a measuring cylinder. The cylinder was tapped 25 times for each sample by dropping it from a height of 4 - 6 inches and the volume recorded. All samples were tested to triplicate. 3.2.1.4 Porosity: * The porosity (the fraction of void space is a bulk of seeds) was calculated from the following relationship: Porosity = Density - bulk density x 100 Density 3.2.1.5 Specific volume and bulk volume: The specific and bulk volume of seeds were calculated as follows: Specific volume = 1 /density cc/g Bulk volume = 1/bulk density cc/g

32 SHAPES OF TWO CULTIVARS

A:RUBATAB B: DONGOLA 3.2.2 Defatting of lupin seed flour: Lupin flour was defatted at room temperature. The flour was placed in a container n-hexane was added. The solvent to flour ratio was 10:1. The mixture was stirred for 16 hours with magnetic stirrer and then filtered. The flour was washed again with n-hexane to remove traces of oil and filtered. The oil free flour was dried in open air at room temperature and stored in a cool dry place. 3.2.3 Proximate analysis: Proximate analysis of raw and roasted full fat lupin flour was carried out according to AOAC (1984). 3.2.4 Carbohydrates: Carbohydrates were determined by differences. Carbohydrates = 100 - [moisture% + ash% + fibre% +oil% +crude protein%. 3.2.5 Determination of fattv acids profile: Gas chromatography: According to the method of Bertram et al. (1983). Methyl esters of the components fatty acids were prepared by transesterification with 0.5 ml sodium methoxide in methanol. * The mixed methyl esters were analyzed on Pye 104 gas chromatograph fitted with a flame ionization detector. A glass column (1.5 m long by 0.4 cm internal diameter) packed with 10% polyethylene glycol succinate was operated at 185-195 C with nitrogen carrier gas at 30 ml/min. A sample of 10 - 20 g mixed methyl easier dissolved in 1 1 hexane was injected.

34 3.2.6 Determination of antinutritional factors: 3.2.6.1 Tannins: Quantitative estimates of tannin was carried out using the vanillin-HCl in methanol and 1% vanillin in methanol (Price, ct al, 1978). The reagent was prepared daily by mixing equal volumes of 1% vanillin in methanol and 8% concentrated HO in methanol. It was discarded if a trace of colour appeared. Dm catechin was used to prepare the standard curve. This was done by adding 100 mg of D-catechin to 50 ml of 1% vanillin in methanol. From this stock solution various dilutions were prepared. Five ml of vanillin-HCl reagent (0.5%) were added to 2 ml of each dilution. The absorbance was read using spectrophotometer model WAP SI01 at 500 nm after 20 minutes at 30 C from addition of reagents. The absorbance was plotted against catechin concentration. Preparation of sample: Grains are ground to pass 0.4 mm screen. 0.2g of the ground grain was placed in a test tube then 10 ml of 1% concentrated HO in methanol was h added. The test tube was capped and continuously shaken for 20 minutes and then centrifuged at 5000xg for 5 minutes. One ml of the supernatant was pietted into each of the tubes and proceeding as was described for the standard curve above. For zero setting 1 ml distilled water (blank) was mixed with 5 ml 4% concentrated HO and 5 ml vanillin reagent in a test tube and incubated for 20

35 I annin Standard Curve

i I 0.22-) I !

\ | 0.1 S-i i

o j / |

•8 ^ < J / |

/• • 0.08-

0 06-

0.04 0.02 0.04 0.06 0.C Concentration (mg/!) min. at 30 C (blank). Absorbatice at 500 nrn was read using spectrophotottieter. Tannin concentration was expressed as catechin equivalent (CE) as follows: CE = (C x 10 x 100)/(1000 x W) C = Concentration corresponding to the optical density. 10 = Volume of extract (ml); it was 100 ml for protein fractions. 1000 = To convert from g to mg. W = Weight of sample. 3.2.6.2 Phvtic acid: Phytic acid was determined by the method of Wheeler and Ferrel (1971) with slight modification. Principle: Phytic acid was extracted in low acid medium, then precipitated as ferric phytate by addition of FeCl3 which was converted to Fe(OH)3 by addition of NaOH. The ferric hydroxide converted to Fe(NC>3)3 through addition of HNO3 was measured optically. The phytate phosphorous was calculated assuming a 4:6 iron:phosphorous ratio.

3.2.6.2.1 Reagents:

Three percent trichloroacetic acid (TCA), FeCl3 (2 mg Fe+3 ion/ml

TCA), 1.5NNaOH,3.2NHNO3, 1.5MKSCN,Fe(NO5)3. 3.2.6.2.2 Apparatus: 1-centrifuge 2- Shaker 3- Water bath 4- Spectrophotometer 3.2.6.2.3 Procedure: Two grams of milled dried sample were weighed in 125 ml conical flask. Fifty ml of 3% TCA were added to the flask, then placed in a mechanical shaker for 3 hours, the suspension was centrifuged for 5 min. 10 ml aliquot of the suspension were transferred to 40 ml tube. Four ml of FeCl3

solution were added by pipette (FeCl3 solution containing 2 mg Fe+3 ion/ml

TCA). The tube was heated in boiling water bath for 45 min., then cooled and centrifuged for 10-15 min. The clear supernatant was decanted, the precipitate was washed twice by dispersing well in 25 ml 3% TCA and was heated for 10-15 min. in a boiling water bath and was centrifuged. Washing was repeated once using water; the washed precipitate was dispersed in water and 3 ml of 1.5N NaOH were added with mixing. Water was added till 30 ml volume, then the tube was heated in a boiling water bath for 30 min. and hot filtered using Whatman No. 2; The precipitate was washed with hot water. The washings were decanted, and the precipitate was dissolved from the paper with 40 ml hot 3.2 N HNO3 into 100 ml volumetric flask. The paper was washed with water and the washings were collected in the same flask then completed to volume. 0.5 ml aliquots were taken from the above solution and transferred into 10 ml volumetric flask. Two ml of KSCN were added and completed to volume by adding distilled w*iter then immediately within 1 min the absorpance was read using a spectrophotometer at 480 nm.

38 .6,2,4 Calciilations;- Staiidard curve of different Fe (NO3)3 concentrations was plotted linst absorbency to calculate the Fe^ concentration. The phytate

Dsphorous was calculated from Fe+3 concentration assuming 4:6 Iron: Dsphorous molar ratio. Hhytic Acid Standard Curve

0.45-!

0.4-i

E c 0.35- o CO 0.3- 13 c o 0.25- "5. o 0,2-

0.15-

0.1 "!

0.05 r- 1 1 ;— 0.5 1.5 £5 3 3.5 4 Concentration (mg/l) Ashing was carried out according to the AOAC (1984) method. To the ash obtained about 20 ml of the 2N HCI added and the mixture was brought to boiling for one minute to dissolve the minerals in HCI. The mixture was filtered in a conical flask and the volume competed to 100 ml using distilled water. Minerals were determined using atomic absorption spectrophotometer for Fe, Zn, Pb, Ca and Mg. 3.2.7.1 Sodium and potassium: Sodium and potassium were determined according to the AOAC (1984) using EEL flame photometer. One ml of the extract was taken and diluted to 50 ml with distilled water. A standard solution of NaCl was prepared by dissolving 2.54g of NaCl powder in one litre of distilled water. Ten mi of solution were diluted to one litre with distilled water giving 10 mg/1 concentration. Standard solution of KC1 was prepared by dissolving 2.54g of KC1 powder in one litre distilled water, then 10 ml of solution were taken and diluted with 1000 ml distilled water to give 10 mg/1 concentration. The flame photometer was adjusted to zero using distilled water and to 100 transmission using the prepared standard solution (NaCl and KC1), then the sample reading was recorded and the percentage of minerals was calculated as follows: Mineral0/© = FRxDFxlOOOx 100 10 x wt of sample x 100 x 2 where: FR = Flame reading.

41 DP = Dilution factor.

Phosphorous determination was carried out according to Vanado Molybdate method AOAC (1970). 3.2.7.2.1 Apparatus: 1- Electrophotocolorimeter 2- Volumetric flask 100 ml. 3.2.7.2.2 Reagents: A. Vanado-molybdate: Twenty grams NH4-molybdate (NH4)6M02O24-4H2O were dissolved in 200 ml hot water and were cooled. Separately lg of NH4-metavandate was dissolved in 120 ml hot water, it was cooled and 140 ml concentrated HNO3 were added, under a fume hood. Molybdate solution gradually was added to the vanadate solution and diluted to 1 litre. B. P-standard solution (100 ml) P-dissolved: 0.4394g dry aniiydrous KH2PO4 were dissolved in distilled water and the volume made to 1 litre. The solution was stored in a dark brown pyrex glass bottle in a cool place. C. P-standard solution (25 mg/I): The 100 mg/1 P-stock solution was diluted 4 times. Fresh solution was prepared periodically to ensure high accuracy. 3.2.7.2.3 Procedure: Five ml of sample were pipetted from wet digest into 100 ml volumetric flasks and 25 ml distilled water were added. Within 5 min. 20 ml of vanado-

42 molybdate reagent were added, diluted to volume, before mixed and left to stand for 10 min. and then the determination of % transmission at 400 iim. Standard curve for P: Zero, 2, 4,5, 10, 15 and 20 ml of 25 ml mg/1 P standard solution was pipetted into a series of 100 ml volumetric flasks. The colour was developed according to the procedure outlined above. Optical density was plotted against concentration. Phosphorous Standard Curve .12-

0.11- /' i i i

£ 0.09- c 008- 40 0 c 0.07- o

rp t 01 06- IT' oCO .Q < 0.05- /

0.04- w.

0,03- / 0.02 5 10 15 20 Concentration (mg/i) 3.2.8 Fractionation of lupin protein on the basis of solubility carried out by two methods: 3.2.8.1 Mendel-Osborne (1914) technique: Triplicate samples were taken in plastic bottle provided with screw cap. The sample was extracted twice with 60 ml of distilled water. Extraction was carried out for half an hour with continuous shaking on a shaker. The extract was separated from the residue by centrifugation. The clear supernatant liquids were collected. The residue was then extracted successively in a similar manner with 1.0M NaCl solution, 70% ethanol and 0.2% NaOH solution and the extracts collected in the same way as described above. The residues after these successive extractions with four solvents were the insoluble residues. The protein content of the four extracts and the residues was determined by the micro-kjeldahl method, Nitrogen was assayed by the micro-kjeldahl method. Lupin defatted flour

I I Water shaking, centrifuging 2x Albumin * Residue r 1M NaCl shaking, centrifuging 2x Globulin Residue

I \ 70% EtOH shaking, centrifuging 2x Prolamin Residue

I I 0.2% NaOH shaking, centrifuging 2x Glutelin Residue Schematic diagram for protein fractionation, from lupin flour; 2x means the material is extracted twice with the particular solvent and the supernatants from the two extractions are combined.

45 3.2.8.2 Landrv and Moureaux: Lupin grain protein can be separated into six fractions by a selective extraction method of Landry and Moureaux (1970). 3.5g of defatted sample were kept in suspension with 35 ml of extractants by magnetic stirring in 50 ml centrifuge tubes. The duration and number of extractions with each solvent and the identification of protein fraction are shown in Table 1. 3.2.8.2.1 Sample (3.5g) was mixed with 35 ml of 0.5M NaOH for 3 extraction times (60, and 30 min.) at 4 C the total volume collected was 105 ml. 3.2.8.2.2 The same sample was mixed with 35 ml of distilled water for two extraction times (15, 15 min.) at 4 C, the total volume was 70 ml. The protein fractions of step 1 represented the globulins and step 2 represented the albumins. 3.2.8.2.3 The sample was mixed with 35 ml of ethanol (60%) at 20 C for the extraction time (30, 30 min.) and with 35 ml of ethanol (60%) for 30 min. at 60 C. The sample was mixed with isopropanol (55%) for 3 extraction times (60, 30, and 15 min.) at 20 C. The total volume collected was 210 ml. The protein fractions extracted represents prolamins. 3.2.8.2.4 The sample was mixed with 35 ml of 60% ethanol (v/v) in the presence of 0.6% 2-mercaptoethanol (2-ME) (0.21 ml/35 ml) or sodium metabisulphate (0.25M) for two extraction times

46 Table 1. Protein extraction procedure for sequence Ao and DQ

Step Sequence Extractant Volume Extraction Fraction Protein (ml) time (min) groups

I A0D0 NaCl 0.5M (4 C) 105 60, 30, 30 I Globulin 2 A<)Do Water (4 C) 70 15, 15 II Albumin

3 Ao EtOH 60% (20 C) 105 30, 30, 30 and then at 60 C III Prolamin E>0 2-prOH 55% (20 C) 105 60, 30, 15 Ao EtOH, 60% + 2-ME 4 0.6% (v/v) (20 C) 70 30,30 DO 2-ProH 55% + 2- IV Gi-glutelin ME 0.6% (v/v) (20 70 30,30 C).

5 AoDO NaCl, 0.5M, pHIO 105 60, 30, 30 V + 2-ME, 0.6% (v/v) G2-glutelin (20 C). 6 AoDo NODOdso4, 0.5, pHIO + 2-ME, 105 60, 30, 15 VI G3-gIuteIin 0.6% (v/v) (20 C).

Insoluble AODQ * protein

(30, 30 min.) at 20 C. After this the sample was mixed with 35 ml of isopropanol (55%) with 2-ME (0.6%) for two extraction times (30, 30 min.) at 20 C. The total extraction volume was 140 ml representing Gj-glutelin.

4.7 2.8.2.5 The sample was mixed with 35 ml of borate buffer (pH 10) in the esence of 0.6% 2-ME and 0.5M NaCl for 60, 30 and 30 min. at 20 C. The tal volume extracted was 105 ml representing G2-ghiteIins. 2.8.2.6 The sample was mixed with 35 ml of borate buffer (pH 10) with 6% 2-ME and 0.5% sodium dodecyt sulfate (NaDOd SO4) (w/v) for 60, 30 id 15 min. at 20 C. The total volume extracted was 105 ml representing G3- utelins. 2.8.2.7 The solid material was isolated from extractants by centrifugation id 3000 xg for 15 min. For each solvent supernatants were combined to give ie extract. The extract obtained with water contained minute amount of trogen. (< 1% of total nitrogen of sample) and was dissolved . Nitrogen was ;sayed with the micro-kjeldahl method. orate buffer: (0.0125M Na2Bo7.10H2O and 0.02M NaOH). ,6g od sodium borate were dissolved in 250 ml distilled water and 0.4g of )dium hydroxide in 250 ml distilled H2O were added and mixed together, id was adjusted to pH 10 using either NaOH, or HC1. Calculations: oluble protein% =TFxNxTVxl4x 6.25 x 100 =x axbx 1000 rotein fraction% = Soluble protein (x) Total protein of the sample

48 where: TF = Titre reading N = Normality of HCI TV = Total volume of aliquot extracted 14 = Each ml of HC1 is equivalent to 14 mg nitrogen a = No. of ml of aliquot taken for digestion = (10 ml) b = Weight of sample extracted = 3.5g. 1000 = To convert from g to mg. 3.2.9 In vitro protein digestibility with pepsin: In vitro protein digestibility (IVPD) was carried out by the method of Maliwal (1983) as modified by Manjula and John (1991). A known weight of the sample containing 16 mg nitrogen was taken in triplicate, and incubated with 1 mg pepsin in 15 ml of 0.1 M HCl at 37 C for 18 hr. The reaction was terminated by the addition of 15 ml of 10% trichloroacetic (TCA). The mixture was then filtered quantitatively through Whatman No. 1 filter paper and the TCA soluble fraction assayed for nitrogen. Protein digestibility = N in supernatant - N in blank x 100 N in sample N in blank = N in pepsin enzyme and reagents.

CHAPTER IV RESULTS AND DISCUSSION

4.1 Physical properties of lupin seeds: The physical properties of''two lupin cultivars are shown in Table 2. 4.1.1 Seed weight, density and volume: The weight of 100 seeds for lupinus termis for cv. Dohgola was 49.76g and were higher than those of cv. Rubatab (31.47g). These findings were similar to those reported by Deshpande et al. (1984) for dry beans (Phaseolus vulgaris L.) which showed a wide variation (15.03 - 5b.33g). The density was 1.15 and 1.22 g/cc for cv. Dongola and cv. Rubatab respectively. The bulk density of cv. Dongola was 1.13 g/cc and for cv. Rubatab was 1.20 g/cc. Porosity of cv. Rubatab was 1.64% and of cv. Dongola was 1.57%. Desphande et al. (1984) reported density within a narrow, range of 1.18 - 1.36 g/cc and bulk density of 0.68 - 0.75 g/cc and porosity of 40.7 - 48.5%. Specific volume (cc/g) of cv Dongola and of cv. Rubatab was 0.87 and 0.82 cc/g respectively. Bulk volume of cv. Dongola and cv. Rubatab was 0.88 and 0.83 cc/g respectively. 4.1.2 Seed dimensions: The length of cv. Dongola was 1.14 mm and was higher than that of cv. Rubatab which was 0.83 mm. Seed thickness was found to be greater in cv. Rubatab than for cv. Dongola which was 0.52 and 0.55 mm respectively. The length/breadth (L/B)

50 Table 2 Physical properties of Lupinus terrois cultivars. Physical properties Sample Rubatab Dongoia Weight of 100 seed (g) 31.43(±2.02) 49.76(±1.3) Density (g/cc) 1.22(±0.01) U5(±0.00) Bulk density (g/cc) 1.20(±0.00) 1.13(±0.00)' Porosity (%) 1.64(±0.01) 1.57(±0.00) SpeciiBc volume (cc/g) 0.82(±0.00) 0.87(±0.00) Bulk volume (cc/g) 0.83(±0.00) 0.88(±0.00) Length (cm) 0.83(±0.01) 1.14(±0.01) Breadth (mm) 0.74(±0.02) 1.03(±0.01) Length/breadth (L/B) 1.12(±0.0l) 1.11(±0.01) Thickness (mm) 0.55(±0.00) 0.52(±0.00) Breadth/thickness (B/T) 1.35(±0.00) 1.98(±0.00)

Means of length, breadth and thickness were measured from 25 seeds. Values are means (±SD) ratio was identified for the two cultivars (1.1) and was less than the ratio of 1.51-1.65 observed by Desphande et al. (1984). The breadth/thickness (B/T) ratio was 2.0 for cv. Dongoia and 1.35 for cv. Rubatab. Desphande etal. (1984) obtained BAT in the range 1.17-1.65. 4.2 Chemical composition of lupin seeds: The chemical composition of two varieties cultivated in Sudan is shown in Table 3. The results are expressed as on dry matter basis (DMT).

51 4.2.1 Moisture: The moisture content for cv. Rubatab of unroasted whole seeds was 4.5% and for whole seed debittered for. 30 and 60 min. were 4.9 and 4.6 respectively. Moisture content of debittered kernel for 30 min. and 60 min. were 5.2 and 5.4% respectively. Moisture content for cv Dongola for raw whole seed, debittered whole seed (30, 60 min.) and debittered kernel (30, 60 min.) were 4.4, 4.5, 4.8, 4.6, 4.4, 5.0 and 5.1% respectively. Satheetal. (1982) observed that moisture content for L. mutabilis was 6.9% and 7.7% for L. albus cv. Multolupa. According to Ballester, et al. (1984) moisture content was 6.8% for L. albus cv. multolupa. 4.2.2 Fibre content: Fibre content of cv. Rubatab was 10.5,10.9 and 10.4% for raw whole seed and debittered whole seed for 30 and 60 min. respectively. Fibre content of debittered kernels for 30 and 60 min. was 0.9 and 1.1% respectively. Fibre content for 1*5.1* Jek'ilterrJ--fir 3

52 Table 3 Chemical analysis of raw and debittered cultivars of LujSSHS tennis. Variety Cooking time Moisture (%) Fibre (%) Ash(%) (min) Rubatab Whole seed uncooked 4.48(±0.00) 10.5(±0.00) 1.36(±0.03) Whole seed 30 4.94(±0.01) 10.86(±0.00) 1.51 (±0.00) Whole seed 60 4.58(±0.03) 10.41 (±0.00) 1.52(±0.00)

Kernel 30 5.17(±0.00) 0.89(±0.00) 1.37(±0.00) Kernel 60 5.44(±0.00) 1.12(±0.00) 1.29(±0.00)

Testa 30 5.30(±0.00) 45.00(±0.08) 1.86(±0.00) Testa 60 5.68(±0.03) 45.65(±0.04) 1.79(±0.00)

Dongola Whole seed uncooked 4.44(±0.00) \ 9.52(±0.07) 1.48(±0.02) Whole seed 30 4.46(±0.01) 9.76(±0.00) 1.43(±0.00) Whole seed 60 4.79(±0.00) 8.82(±0.00) 1.44(±0.00)

Kernel 30 4.55(±0.02) ' \-f (±0.02). . 1.29(±0.00) Kernel 60 4.4.44(±0.03) 1.78(±0.00) 1.26(±0.00)

Testa 30 5.64(±0.02) Lbrf (±0.02) 1.79(±0.00) Testa 60 5.5. ll(±0.03) 7.25(±0.00) 1.86(±0.00)

53 Table 3 (contd.) Variety Cooking Fat (%) Protein (%) NFE (%) Calories time (min) Rubatab Whole seed unroasted 10.44(±0.08) 49.88(±1.38) 23.33(±0.20) 3S6.80 Whole seed 30 10.02(±0.00) 47.14(±0.01) 25.53(±0.04) 380.86 Whole seed 60 9.92(±0.00) 47.27(±0.06) 26.3(±0.01) 383.56

Kernel 30 12.12(±0.01) 59.33(±0.10) 21.12(±0.68) 430.88 Kernel 60 12.34(±0.00) 56.91(±0.00) 23.10(±0.00) 430.30

Testa 30 0.95(±0.00) 9.21(±0.00) 37.68(±0.00) 196.11 Testa 60 1.06(±0.00) 9.29(±0.01) 36.53(±0.00) 192.82

Dongola

Whole seed * unroasted 10.12(±0.01) 50.37(±0.62) 24.07(±0.00) 388.84 Whole seed 30 10.51(±0.00) 50.37(±0.01) 23.48(±0.79) 389.90 Whole seed 60 10.87(±0.01) 49.34(±0.01) 24.74(±0.04) 394.15

Kernel 30 11.54(±0.01) 57.42(±0.06) 23.38(±0.01) 427.06 Kernel 60 12.54(±0.00) 56.03(±0.02) 23.94(±0.00) 432.74

Testa 30 0.62(±0.08) 5.61(±0.00) 39.63(±0.74) 186.54 Testa 60 0.83(±0.00) 7.79(±0.01) 37.16(±0.01) 187.27

54 Fibre content of cv Dongola was 9.5. and 9.8% for raw whole seed and debittered whole seed for 30 min. Fibre content decreased significantly (P< 0.05) in debittered whole seed at 60 min. to 8.82%. The fibre content of debittered kernel for 30 and 60 min. was 1.8 and 1.8%. High fibre values were found in debittered testa for 30 and 60 min. (46.7 and 47.3%). Camacho, et al. (1988) obtained similar results of crude fibre: 11.5% for whole seed and 2.3% for dehulled seed of L. albus. Similar results were also reported by Ballester, et a]. (1988) who obtained 3.7% crude fibre for L. albus. Crude fibre of lupin flour roasted at 80-90 C for 10, 20, 30 and 40 min. were 3.9, 3.5, 3.2 and 3.3% respectively as was reported by Ballester et al. (1986). These results were higher than those obtained in this study. Dehulling decreased the seed fibre content as reported by Zduncyk et al. (1996). Schoeneberger et al. (1987) reported fibre content in debittered lupin flour from L. mutabilis as 10.7%, his results were identical to our results. Crude fibre of two species of lupin: L. albus and L. luteus were 11.7 and 16.8% respectively in raw whole seed (Ballester, etal. 1980). These results are relatively similar to the results obtained in this study. Crude fibre obtained by Hove (1974) for two species L. angustifolius cv Unwhite for whole seed, seed coat and kernel were 16.8, 54.9 and 3.5% respectively. For L. luteus cv. Weiko HI whole seed, seed coat and kernel

55 were 17.7, 56.5 and 4.4% respectively (Hove, 1974) these values were higher than those obtained in this study. 4.2.3 Ash content: Ash content for the two cultivars studied ranged from 1.3-1.9%. Hove (1974) reported that the ash content of L. arigustifolius cv Unwhite as 3.5, 3.1 and 3.7% for whole seed, seed coat and kernel respectively. For L. luteus cv Weiks in ash content was 4.0,2.2 and 4.4% for whole seed, seed coat and kernel respectively. These values were higher than the values obtained in this study. Ash content of two species of lupin: L. albus and L.- luteus were 3.2 and 3.5% respectively in whole seed (Ballester, et a]. 1980). Schoeneberger et al. (1987) reported ash content in debittered lupin flour and raw of L. mutabilis as 2.5 and 3.6% respectively. Ash content of whole seed and dehulled seed was 3.7 and 3.8% respectively (Camacho et al. 1988). These results were lower than those reported by Yanez et al. (1986) who observed that the ash content was 3.5% for lupin flour roasted for 30 mm. in species L. albus. The results were also lower than those reported by Bailey et al. (1974) who reported values od ash as 2% for L. angustifolius. Our results were lower than those reported by Aguilera and Trier (1978) who reported that the ash content was 3.4 and 3.2% for lupin seed, 2.4 and 3.4% for dehulled seed flours in L. ajbiis. In L. luteus ash content was 3.5% for lupin whole seed and in the range of 2.7 - 4.4% for dehulled seedfAguilera and Trier J978).

56 4.2.4 Fat content: Fat content for cv Rubatab was 10.4, 10.0 and 9.9% for raw whole seed, debittered whole seed for 30 and 60 min respectively, and was 12.1 and 12.3% for debittered kernel for 30 and 60 min. respectively. These results were similar to those obtained by Ballester et al. (1984) who reported that oil content was 12.7% for L. albus. Eldash and Sgabieri (1980) reported that lipid content was 12.2% for L. albus. Hudson et al. (1976) reported that the oil content was 5.1-11.9% for L. albus and 7.2-14.3% for L. mutabilis and 9.5% for L. termis. Oil content in debittered testa for 30 and 60 min was 0.95 and 1.0% respectively for cv Rubatab. Oil content of cv Dongola was relatively the same as that for cv Rubatab, 10.1,10.5 and 10.9% for raw whole seed and debittered whole seed for 30 and 60 min respectively. It was 11.5 and 12.5% for debittered kernel for 30 and 60 min. For debittered testa for 30 and 60 min. was 0.62 and 0.83% respectively. These results were lower than those reported by Schoeneberger et al. (1987) who reported oil content of 25.7% for debittered lupin flour for L. mutabilis. and for raw lupin flour was 20.1%. The results obtained in this study were similar to those obtained by Camachoetal. (1988) who reported oil content of 9.2% for whole seed and 12.1% for dehulled seed of L. albus. Ballester et aj. (1988) reported that the ether extract was 12.5% for full fat lupin flour from L. albus. while Yanez et al. (1986) reported that the ether extract in roasted lupin flour from L. albus was 11.4 and 13.3% for raw and roasted (30-90 C for 40 min.) respectively. These results were similar to the results obtained by Ballester etal (1986)

57 who reported that ether extract was 11.2% for L. albus. Aguilera and Trier (1978) gave oil content as 10% for L. albus and 5.5-6.7% for L. angustifolius. Hove (1974) reported that oil content was 6.5, 1.0 and 7.2% for seed, seed coat and kernel respectively for L. agnustifolus. It was 5.2, 1.0 and 6.1% for whole seed, seed coat and kernel respectively for L. luteus. The results of oil content obtained by Schoeneberger et al. (1982) were higher than those obtained in this study. For L. mutabih's it was 15.0,20.1 and 26.9% for semi-sweet raw, bitter raw and lupin seed cooked-water-extracted respectively. Debittering increased the oil content. Hudson et al. (1983) reported that the oil content was 21.0 and 13.0% for L. mutabilis and L. albus respectively. Hudson et al. (1976) reported that lipid content of lupin bean as 12.0% oil in kernel, 1.2% oil in husk and 10.2% for whole seed. These results were relatively similar to those obtained in our study. High content of oil is sufficiently encouraging to make lupin seed as a good source of edible oil. 4.2.5 Fatty acids: The fatty acid composition of lupin seed oil is shown in Table 4. The predominant fatty acids were oleic acid (18:1) linoleic acid (18:2) and palmitic acid (16:0) for the two cultivars studied. There was no differences between raw and debittered whole seed. The percent of myristic acid (14:0) was 0.3% for cv Rubatab and in cv Dongola it ranged from 0.22-0.3%. These results were similar to those reported by Schoeneberger etal. (1982) who reported 0.3% for this fatty acid. i Palmitic acid ranged from 10.2-10.4% for cv Rubatab and was 9.9-11,2% for cv Dongola. These results were relatively similar to those obtained by Hudson et al. (1976): 8.3,12.7, 8.5,13 and 8.1% for L. albus, L. angustifolius. L. luteus. L. mutabilis and L. termis respectively. Fleetwood and Hudson (1982) observed that the content of palmitic acid was 7.7, 12.7, 7.1 and 12.4% for L. albus. L. angustifolius. L. luteus and L. mutabilis respectively. The percent of palmitic (16:0) for L. mutabilis was 9.8% (Schoeneberger et al. 1982). These results were similar to the results for cv Dongola (10.2-10.4%). Palmitic acid was 8.3,10.2 and 10.7% for L. albus. L. angustifolius respectively (Aguilera and Trier, 1978).

59 Table 4 Fatty acids composition of Lupinus tennis seed oil.^f ncllyi e-skr) Variety Cooking Laurie Myristic Palmitic Palmtoleic Largeric time 12:0 14:0 16:0 16:1 17:0 (min) Rubatab Whole seed uncooked Trace 0.31 10.30 0.57 — Whole seed 30 Trace 0.33 11.16 0.60 — Whole seed 60 Trace 0.31 10.44 0.57 —

Kernel 30 Trace 0.31 10.32. 0.58 __ Kernel 60 Trace — 10.16 0.51 —

Dongola Whole seed uncooked Trace 0.27 10.91 0.66 0.18 Whole seed 30 Trace 0.25 10.93 0.60 — Whole seed 60 Trace 0.22 9.87 0.60 0.18

Kernel 30 Trace 0.26 10.92 0.65 0.17 Kernel 60 Trace 0.32 11.22 0.66 Trace

60 Table 4 (contd.) Variety Cooking Stearic Oleic Linoleic Linolenic Arachidic time (min) 18:0 1.8:1 18:2 18:3 20.:0 Rubatab Whole_seed uncooked 2.45 55.62 18.87 6.82 5.06 Whole seed 30 2.41 53.14 18.99 8.55 4.82 Whole seed 60 2.48 52.37 19.09 9.66 5.08

Kernel 30 2.42 52.70 19.06 9.47 5.14 Kernel 60 2.64 54.24 18.30 9.34 4.81

Doneola Whole_seed uncooked 2.07 55.97 16.63 8.20 5.11 Whole seed 30 2.66 55.38 16.58 8.35 5.24 Whole seed 60 2.20 55.85 17.94 7.97 5.18

Kernel 30 3.06 53.98 16.97 8.17 5.61 Kernel 60 2.91 55.30 16.12 8.58 4.89

Palmitic (16:Q) had a low percentage of 0.6 for Rubatab and Dongola. These results were relatively similar to those obtained by Schoeneberger et al. (1982) (0.4%) but were higher than those obtained by Hudson etal. (1983) (0.4, 0.2, 0.2 and 0.3% for L. ajbus, L. agnustifolus, L luteus and L. mutabilis respectively). Largeric acid (17:0) was nil! in cv Rubatab and was 0.1% forcv Dongola. Stearic acid (18:0) ranged from 2.4-2.6% for cv Rubatab and 2.1-3.1% for cv Dongola. These results were similar to those obtained by Hudson et al. (1976), (2.1, 1.7 and 2.7% for L. termis, L. albus and L. luteus respectively). The results for stearic acid obtained in this study were similar to those reported by Aguilera and Trier (1978) who reported 2.5 and 2.4% for L. albus and L. luteus respectively. Stearic acid was K8 and 2.6% for L. albus and L. luteus respectively (Fleetwood and Hudson, 1982). In L. angustifolius it was 6.0% and L. mutabilis was 7.5% (Fleetwood and Hudson, 1982). These values were higher than our results. Oleic acid (18:1) is a major fatty acid in lupin seed oil. Oleic acid was 55.6, 53.1 and 52.4% for raw whole seed and debittered whole seechfor 30 and 60 min. It was 52.7 and 54.2% for debittefecfkernePfor 30 and 60 min7 respectively. Oleic acid in cv Dongola ranged from 54-56.0. These results were similar to those obtained by Hudson et aj. (1976) who reported oleic acid percentage as 54.4 for L. albus then for L. termis it was 46.2% which is lower than the levels reported in this study. Other results confirmed our findings showing similar values for oleic acid as in L. albus and L. luteus (55.0 and 50.9% respectively) (Fleetwood and Hudson, 1982). Oleic acid values for L. mutabilis was 53.9% (Schoeneberger, et al. 1982).

62 Linoleic acid (18:2) ranged from 18.3-19.1% for cvRubatab. For cv Dongola it ranged from 16.1-17.9%. Those levels were lower than those obtained by Schoeneberger etal. 1982), who reported values of 25.9%. The value of linoleic acid obtained in this study was relatively similar to that reported by Fleetwood and Hudson (1982) who obtained 18.2 and 19.2% for L. albus and L. tennis respectively. Hudson et al. (1983) also reported similar results for L. albus (18.0%). Aguiler and Trier (1978) reported linoleic levels of 17.7% for L. albus. Higher levels of linoleic acid were reported by Aguilera and Trier (1978): 28.1, 43.5 and 49.4% for L. mutabilis, L. angustifolus and L. luteus respectively. LinoWcacid (18:3) ranged from 6.8-9.5 for cv Rubatab and 8.0-8.5% for cv Dongola. Hudson et al. (1983) obtained similar results 9.0% for L. albus and for L. luteus it was 6.4%, but lower results for L. angustifolus. L. mutabilis (4.7 and 2.3% respectively). Aguilera and Trier (1978) reported 9.1 and 8.3 linole«u acid for L. albus and L. luteus respectively which confirms the results obtained in this study. Hudson et aj. (1976) reported 9.2% linolenic acid for L. tennis and 8.5% for L. albus. Arachidic acid (20:0) in cv Rubatab ranged from 4.8-5.1%. For cv Dongola it ranged from 4.9-5.6%. These results were higher than those reported by Hudson et al- (1983) who reported 1.0% for L. albus and 2.3% for L. luteus. Also, Aguilera and Trier (1978) reported 2.8% for L. albus and 2.0% for L. luteus. Levels of arachidic acid found in the present study were much higher than those obtained by Schoeneberger et al. (1982) who reported 0.6%.

63 The oil content in lupin seed was about 10-12% . Fatty acids profile for lupin seed oil showed presence of the following fatty acids : Myristic ( 14:0 ) , largeric (17:0 ), palmitolic and linolenic ( 18:3 ). Okie acid was the major fatty acid in lupin as in ground nut. Linoleic acid content lupin seed was lower than in;r . nut ( 16 -19% and 26% respectively). Palmitic acid values were higher than in groundnut (11 and 8.3 % respectively ) . Arachidic acid is also higher than in groundnut ( 5 and 2.8 % respectively ) . Stearic acid content in lupin seed was comparable to that in groundnut (2.5-3 and 3.1 % respectively). 4.2.6 Antinutritional factors: 4.2.6.1 Phytic acid content: Phytic acid content of cv Rubatab was 0.9% for raw whole seed, it decreased significantly (P < 0.05) due to debittering process. Debittered whole seed for 30 and 60 min. contained 0.37 and 0.23% phytic acid respectively. Phytic acid content in debittered kernel for 30 and 60 min. was 0.6 and 0.47% respectively. Phytic acid content of cv Dongola was 0.83% for raw whole seed and

0.37 and 0.15% for debittered whole seed for 30 and 60 minvrespectively. It decreased significantly (P< 0.05) due to debittering. It decreased significantly (P ^ 0.05) due to debittering. The content of phytic acid was 0.15 and 0.08% for debittered kernel for 30 and 60 minvrespectively. These results are in agreement with those reported fortencowpea cultivars; the average of phytic acid for raw whole beans was 0.31% and it decreased in cooked and soaked beans to 0.29% and 0.23% respectively. Soaking decreased phytic acid content of the seeds by 19-28% and cooking

64 by 8-12% (Ologhobo and Fetuga, 1984). Khan et at. (199.1) found the loss of phylic acid varies from 18.0 to 47.-% for fresh maize and from 12 to 53% for dry maize. 4.2.6.2 Tannin content: Tannin content of all samples was very low (Table 5). Tannin decreased from 0.03 to 0.02% in whole seed and kernels. These results are lower than those obtained by Rahma and Roa (1984) who found tannin content of 0.86 and 0.11% for raw and debittered L. tennis seed respectively.

4.2.7 Mineral content: Mineral content in lupin seed flour for two cultivars is shown in Table 6. 4.2.7.1 Calcium content: Calcium content of cv Rubatab of whole unroasted seed was 0.08%, it was 0.1% for debittered whole seed for 30 and 60 min. Calcium content was 0.09% for debittered kernel for 30 min and was 0.1% for debittered kernel for 60 min. The slight increase in Ca could be attributed to Ca uptake from soaking water during debittering process. Calcium content of cv Dongola for raw whole seed was 0.1%, it increased as a result ofdebitteringO.il in debittered kernels (30 and 60 min.). The increase was 20% in debittered whole seed for cv Rubatab and 10% in debittered kernel. vsiw Table 5 Phytate/tannin ofLupinus tennis cultivars. Variety Cooking time Phyticacid (%) Tannin (%) (min) - Rubatab Whole seed uncooked 0.90(±0.00) a 0.03(-±0.01) a Whole seed 30 0.37(±0.01)b 0.03(±0.01) a Whole seed 60 0.'23(±0.00) c 0.03(±0.01) a

Kernel 30 0.60(±0.00) a 0.02(±0.01) a Kernel 60 0.47(±0.00) a 0.03(±0.01)a

Dongola Whole seed uncooked 0.83(±0.00) a 0.03(±0.02) a Whole seed 30 0.37(±0.00)b 0.03(±0.01) a Whole seed 60 0.15(±0.00)b 0.03(±0.01)a

Kernel 30 0.15(±0.01)a * 0.02(±0.01) a Kernel 60 0.08(±0.00) a 0.02(±0.01)a

Values are means (± SD) Means not sharing a common letters in column are significantly different at P < 0.05 as assessed by Duncan's Multiple Range Test (DMRT).

66 Table 6 Mineral;? in Lupinus termis seeds. Cooking time P% Fe% Mg% K% Ca% (min.) Rubatab Whole seed uncooked 0.21 0.03 0.20 0.48 0.08 Whole seed 30 0.17 0.03 0.12 0.45 0.10 Whole seed 60 0.19 0.03 0.19 0.40 0.10

Kernel 30 0.26 0.03 0.17 0.52 0.09 Kernel 60 0.21 0.03 0.22 0.66 0.10

Dongola Whole_seed uncooked 0.30 0.02 0.18 0.47 0.10 Whole seed 30 0.21 0.02 0.17 0.38 0.11 Whole seed 60 0.17 0.03 0.17 0.43 0.11

Kernel 30 ' 0.12 .0.02 0.15 0.49 0.07 Kernel 60 0.20 0.03 0.21 0.71 0.10

67 Table 6 (contd.) Cooking Na Zn Mn Pb time (min.) pg/g Hg/g "g/g u/g Rubatab Whole seed uncooked 56.3(±3.50) 126.9(±2.2)a 4.86(±1.93) 13.9(±0.41) Whole seed 30 58.62(±1.53) 82.29(±1.3)b ft <&&>) 13.1(±2.06) Whole seed 60 65.51(±2.02) 94.13(±1.2)b 6.25(±0.01) 17(±0.89)

Kernel 30 62.07(±2.21) 135.4(±2.1)a b'l (±0.64) 9.29(±1.19) Kernel 60 64.77(±0.25) 107.0(±2. )a 3.93(±0.64) 12.40(±1.2)

Doneola Whole.seed uncooked 67.22(±2.89) 100.4(±3.2)a 6.02(±1.12) 17.8(±1.79) Whole seed 30 63.22(±2.02) 112.7(±3.1)a 6.25(±1.42) 10.8(±0.02) Whole seed 60, 72.41 (±0.25 ) 98.60(±2.3)a 6.25(±1.44) 15.4(±0.12)

Kernel 30 57.47(±3.26) 117.1(±2.3)a 5.55(±0.73) 10.8(±0.02) Kernel 60 68.96(±0.83) 120.3(±2.4)a 5.32(±2.58) 12.4(±1.44)

Values are means (± SD) Means not sharing a common letters in column are significantly different at P< 0.05 as assessed by Duncan's Multiple Range Test (DMRT).

68 In cv Dongola whole seed the increase was up to 30% in debittered kernels. These results were lower than those reported by Hove (1974) who reported calcium content of 0.25, 0.77 and 0.13% for whole seed, seed coat and kernel for L. angustifolius cv Unwhite respectively. For L. angustifolius cv Weiko III calcium content was 0.27% for whole seed, 0.5% for seed coat and 0.2% for kernel. The results obtained in this study were higher than those obtained by Camacho et al (1988) who reported calcium content of 0.05% for L. albus. Our results were relatively similar to those determined by Hill (1977) who reported the levels of Ca in whole seed in the range of 0.03-0.24% and for kernel 0.02% then for L. angustifolius the calcium content ranged from 0.19-0.26% for whole seed and 0.13-0.19% for kernel. Calcium levels in L. luteus ranged from 0.06-0.27% for whole seed and was 0.56% for testa (Hill, 1977). High values of calcium were concentrated in whole seed compared to the kernel. 4.2.7.2 Potassium content:

Content of K in cv Rubatab: raw whole seed and debittered whole seed for 30 and 60 min were 0.48, 0.45 and 0.40% respectively. In debittered kernel for 30 and 60 min it was 0.56 and 0.66% respectively. Potassium content, slightly decreased with debittering due to leaching by water. Potassium values in cv Dongola were 0.47, 0.38 and 0.43 for raw whole seed, debitterd whole seed for 30 and 60 min respectively. There was slight decrease due to debittering. The content of K in debittered kernel for 30 and 60 min was 0.49 and 0.71% respectively. These results were significantly lower than those obtained by Hill (1977) who reported K content of 1.06% for whole seed of L. albus. 0.85-1.15% for whole seed and 0.85% for kernel of L. angustifolus, and 1.07% for whole seed of L. hiteus ind 1.63% for whole seed of L. mutabilis. 4.2.7.3 Magnesium content: Magnesium content of cv Rubatab was 0.2% for raw whole seed, 0.12% for debittered whole seed for 30 min and 0.19% for debittered whole seed for 60 min. The Mg content of debittered kernel for 60 min. was 0.22%. Magnesium content ranged from 0.15-0.21% for cv Dongola, Mg content was 0.18, 0.17 and 0.17% for raw whole seed, debittered whole seed for 30 and 60 min. Magnesium in debittered kernel for 30 and 60 min. was 0.15 and 0.22% respectively. These results were similar to those reported by Hill (1977) who found Mg content in the range from 0.12-0.26% for whole seed in L. albus and for L. angustifolus it ranged from 0.13-0.31% for seed and 0.16-0.17% for kernel. 4.2.7.4 Iron content: Iron content of cv Rubatab was 0.03% for all samples as well as for cv Dongola. These results were significantly higher than those obtained by Hill (1977) who reported Fe levels of 0.01% for whole seed and kernel for L. albus. In L. angustifolius it was also 0.01% for whole seed and for kernel. 4.2.7.5 Phosphorous content: Phosphorous content was 0.21, 0.17% and 0.19% for whole seed debittered whole seed for 30 and 60 min respectively for cv Rubatab. Phosphorous in debittered kernel for 30 min was 0.26 and at 60 min was 0.21%. Phosphorus in cv Dongola was 0.3, 0.21 and 0.17% for raw whole - . f...... seed and debittered whole seed for 30 and 60 min. In the kernel it was 0.12

70 and 0.20% for debittered kernel for 30 and 60 min respectively. These results were significantly lower than those obtained by Camacho, etaj. (1988) who reported P content of 0.35% for L. albus. Hill (1977) reported higher levels of P compared to those reported in this study. Phosphorous content ranged from 0.18-0.58% of whole seed and 0.59% for kernel of L. albus. For L. luteus P content ranged from 0.36-0.79% for whole seed and was 0.54-0.56% for kernel. Hill (1977) reported higher levels for P in L. mutabilis: 0.88% for whole seed. These results were somewhat similar to those obtained by Hove (1974) who reported 0.15, 0.09 and 0.16% for whole seed, seed coat and kernel of L. angustifolius cv Unwhite respectively. For L. luteus P levels were 0.21,0.15 and 0.24% for whole seed, seed coat and kernel respectively (Hove, 1974). The kernel had more phosphorous than whole seed, which is in agreement with the findings of Hove (1974). 4.2.7.6 Zinc content:

Zinc content in cv Rubatab was 126.9 jig/g for raw whole seed, 82.3 ug/g for debittered whole seed for 30 min and 94.1 fig/g for debittered whole seed for 60 min. The effect of debittering was to decrease the zinc content in cv Rubatab; but in cv Dongola it had no effect. Zinc content in cv Dongola ranged from 98-120 ug/g. These results were higher than those reported by Hove (1974) who obtained 38, 37, 38 ug/g zinc for whole seed, seed coat and kernel respectively for L. angustifolius cv Unwhite. For L. luteus cv Weiko III zinc content was 54, 20 and 68 ug/g for seed. Seed coat and kernel respectively (Hove, 1974). The results obtained in this study were relatively similar to those obtained by Hill (1977) who reported 50-120 and 120 ug/g for whole seed and kernel

71 respectively for L. luteus. Zinc content was 58-150 ug/g for whole seed and was 87-220 ug/g for kernel for L. liiteus (Hill, 1977). Other lupin species had low content cf zinc such as L. angustifolius and L. mutabjlis as was reported by Hill (1977) who gave 55-75 ug/g for whole seed and 68-71 ug/g for kernel for L. angustifolus and for L. mutabilis it was 76 ug/g for whole seed. 4.2.7.7 Sodium content: Sodium content of cv Rubatab slightly increased after debittering. Sodium content was 56.3% ug/g for raw whole seed, 58.6 ug/g for debittered whole seed for 30 min and 65.5 ug/g for debittered whole seed for 60 min. Sodium content of debittered kernel for 30 min. was 62.1 ug/g and 64.8 ug/g for debittered kernel for 60 min. Sodium content for cv Dongola slightly increased after debittering; it was 67.2 u.g/g for raw whole seed 63.2 p.g/g for debittered whole seed for 30 min. and 72.4 ng/g for debittered whole seed for 60 min. Sodium content in debittered kernel for 30 and 60 min. was 57.5 and 69.0 u^g respectively. These results were significantly lower than those reported by Hill (1977) who obtained 0.95% Na for L. albus in whole seed, then for L. angustifolius whole seed it was 0.05 - 0.06% and 0.05% for kernel, also for L. mutabilis had high Na level (about 1.21%). 4.2.7.8 Manganese content: Manganese content in cv Rubatab increased after debittering for whole seed. Manganese content was 4.9 ug/g for raw whole seed which increased to 5.6 and 6.3 ug/g for debittered whole seed for 30 and 60 min. respectively. Manganese content of debittered kernel for 3p and 60 min. was 5.3 and 3.9 ug/g respectively.

72 Manganese content of cv Dongola was 6.0, 6.3 and 6.3 ug/g for raw whole seed and debittered whole seed at 30 and 60 min respectively. Manganese content of debittered kernel for 30 and 60 min was 5.6 and 5.3 ug/g respectively. These levels were lower than those obtained by Hill (1977) who reported 164-3397 ug/g for whole seed and 108-3390 ug/g for kernel. In L angustifolius manganese levels were 10-89 ug/g for whole seed 21-37 ug/g for kernel and 17 ug/g for testa. The result obtained in this study were lower than those reported by Hove (1974) who gave 40,17 and 37 ug/g for whole seed, seed coat and kernel respectively for L. angustifolus cv Uniwhite. Manganese content in our samples was very low compared to those obtained by Hung et al. (1987) who reported 61 ug/g for L. angustifolius and 1316 ug/g in L. albus. It was also lower than those obtained by Zdunczyk et al. (1996) who gave a range of 0.4-1.45 g/kg. 4.2.7.9 Lead content: The content of lead for cv Rubatab for raw whole seed was 14 ug/g, for debittered whole seed for 30 and 60 min was 13,1 and 17.0 ug/g respectively. Lead content of debittered kernel for 30 and 60 min was 9.3 and 12.4 ug/g respectively. Lead content of cv Dongola was 17.8, 10.8 and 15.4 ug/g for whole seed, debittered whole seed for 30 and 60 min respectively. Lead content was 10.8,12.4 ug/g for debittered kernel for 30 and 60 min respectively. 4.2.8 Total protein: Crude protein content is shown in Table 7. Protein content of cv Rubatab was 49.9% for raw whole seed, 47.2% for debittered whole seed for

73 T

30 min. and 47.3% for debittered whole seed for 60 min. The crude protein of debittered kernel for 30 min. was 59.3% which decreased significantly (P < 0.05) in debittered kernel for 60 rain. (56.7%). The higher values for protein in the kernel is due to dehulling as was reported by Zduncayk et al. (1996). The crude protein in cv Dongola was 50.4% for raw whole seed, 50.4% for debittered whole seed for 30 min. and was 49.3% for debittered whole seed for 60 min. The protein content of debittered kernel for 30 and 60 min. were 57.4 and 56.0% respectively. These levels were higher than those of debittered whole seed in the two varieties. The results obtained in this study were higher than those reported by Sathe et al. (1982) who obtained protein content of 44.4% for L. mutabilis. Hudson et al. (1976) reported crude protein content L. mutabilis in the range of 40-46%, while Tello (1976) reported crude protein content of 43.3% for L. mutabilis sweet. Schoeneberger et al. (1982) reported crude protein content of 47.7% for L. mutabilis semi-sweet and 55.9% for L. mutabilis debittered; also Schoeneberger et al. (1987) obtained protein content of 48.6% for debittered L. mutabilis. The protein content of whole seed was higher than the protein content of 38% for L. tennis. 35% for L. ajbus, 31% for L. angustifolius and 44% for L. luteus (Hudson, et al., 1976). Results obtained by Hove (1974) showed that protein content of 29.6% for seed, 2.1% for seed coat and 38.1% for kernel of L. angustifolius cv Uniwhite and protein content for L. lutcus cv Weiko HI of 40.1, 3.0 and 50.5% for seed, seed coat and kernel respectively.

74 In this study the results of protein content in kernels rather than whole seed were in agreement with Zdunczyk et a]. (1994). The high protein levels of lupin seed enhances its nutritional value. 4.3 Protein fractions: 4.3.1 Mendel-Osborne method: The protein solubility fractions of lupin cultivars is shown in Table 7. 4.3.1.1 Albumin: Albumin extracted by Mendel and Osborne method for cv Rubatab was 24.8% for raw, whole seed, 3.3% for debittered whole seed for 30 min. and 3.2% for debittered whole seed for 60 min. Albumin content decreased significantly (P < 0.05) due to debittering. Albumin content debittered kernel for 30 min. was 5.7% and 3.7% for debittered kernel for 60 min. Albumin content of kernel decreased significantly (P < 0.05) due to debittering. Albumin content of cv Dongola was 18.9% for raw whole seed, it decreased due to debittering to 3.1 and 5.4% for debittered whole seed for 30 and 60 min. respectively. Albumin levels in debittered kernels were 3.4 and 5.5 for debittered kernel for 30 and 60 min. respectively. Singh and Jambunathan (1982) obtained albumin content for legume seeds ranging from 10-20% which is similar to that given by the Mendel- Osborae method in this study (18.9% for raw whole seed). The results obtained for debittered samples were in good agreement with the results obtained by Oomah and Bushuk (1983) who found albumin content ranging from 2-10% for L. albus and L. angustifolius.

75 The effect of soaking and heat treatment in this work reduced protein oiubility in agreement with the results observed by Chango et al. (1993). .3.1.2 Globulin: Globulin was the major protein fraction for raw whole seed. The jlobulin content for cv. Rubatab for raw whole seed was 41.1% and for lebittered whole seed for 30 and 60 min. was 16.5 and 10.6% respectively. Hie globulin fraction decreased significantly (P < 0.05) as the result of iebittering. The globulin content of debittered kernel for 30 and 60 min. was 29.3 and 26.7% respectively. Globulin content of cv. Dongola for raw whole seed was 50.3% and for debittered whole seed for 30 and 60 min. was 44.4 and 13.4% respectively. The globulin fraction decreased significantly as a result of debittering. The globulin content of debittered kernel for 30 and 60 min. was 39.3 and 16.5% respectively. Oomah and Bushuk (1983) showed that globulin content for two lupin species (L. albus and L. angustifolius) ranging from 53-85%. They also observed defatting of lupin seed meal affected protein solubility by decreasing the amount of globulins. Defatting altered the solubility of two protein component (26000 and 27000 daltons) whereby they became insoluble in water (Oomah and Bushuk, 1983). They observed defatting caused loss of three globulin bands (Oamah and Bushuk, 1983). In native globulins heat treatment destroy the high Mw polypeptides giving rise to smaller fragments (Semino and Cerletti, 1987).

76 Number of protein subunits of raw L. tennis flour were 12 subunits which decreased to 6 subunits in debittered lupin flour and were further reduced to 3 subunits in lupin powder as reported by Mohamed et aK (Unpublished). Singh and Jambunathan (1982) obtained about 70.1% water and salt- soluble protein fractions together in whole seed of pigeon pea. Singh (1981) found that 70.4% for albumins and globulins in decorticated seed of pigeon pea, and reported 17.4% of glutelin. Legume seed major proteins are usually 70% globulin, 10-20% albumin, 10-20% glutelin while prolamin is usually present in very small amount (Singh and Jambunathan, 1982). Varasundharosoth and Barnes (1985) found that globulin fraction ranges from 17% for L. cosnitimi to 46% for L. albus. The results in this study for raw whole seed were similar to those obtained by Osborae (1924) who reported that the globulins content ranged from 50 to 75% for total seed protein of legumes. They were relatively similar to results obtained by Maraurez and Lajolo (1981) who reported 52.3% globulins content in Phaseolus vulgaris. 4.3.1.3 Alcohol extractable protein (AEP): Alcohol extractable proteins of cv Rubatab for raw whole seed was 1.9% and for debittered whole seed'for 30 and 60 min. was 2.2 and 2.7% respectively. The alcohol extractable protein content of debittered seeds increased significantly (P < 0.05) compared to alcohol-extractable proteins of raw whole seeds.

77 The alcohol-extractable proteins were 3.0 and 2.9% for debittered kernel for 30 and 60 rain, respectively. The AEP content of cv Dongola was 2.0% for raw whole seed and 2.5 and 2.7% for debittered whole seed for 30 and 60 min. respectively. The alcohol-extractable proteins were 1.8 and 2.7% for debittered kernel for 30 and 60 min. The alcohol-extractble proteins increased due to the heat process for the two cultivars. These results were relatively similar to those obtained by Singh et al. (1981) who found the alcohol-extractable proteins content was 3.1% in decorticated seed of pigeon pea. These results were at variance with those obtained by Varasundharosoth and Barnes (1985) who found AEP were 1% in lupin seed. 4.3.1.4 Glutelins: Glutelins are defined as including those proteins that are either insoluble neutral aqueous solution, saline solution or alcohol but soluble in dilute alkali (Osborne, 1924). Deshpande and Nielsen (1987) reported that alkali-soluble protein fraction was 11% for legume seed. Glutelins extracted by Mendel-Osborne are shown in Table 7 for the two cultivars. The glutelins content of cv Rubatab

78 Table 7 Protein fractions of raw and debittered cultivars of Lupinus termis according to' Mendel-Osborne method (1914). Variety Cooking Actual Albumin (%) Globulin (%) Prolamin (%) time protein (min.) (%) Rubatab Whole seed uncooked 49.87a 24.79(±0.07)a 41.08(±2.13)a f-92(±0.01)b Whole seed 30 47.14b 3.3(±0.09)b 16.46(±0.16)b 2.16(±0.08)a Whole seed 60 47.27b 3.25(±0.02)b 10.63(±0.71)c 2.66(±0.03)a

Kernel 30 59.33a 5.66(±0.05)a 29.26(±L29)a 2.99(9±0.07)a Kernel 60 56.69b 3.7(±0.06)b 26.67(±1.09)a 2.85(±0.03)a

Dongola Whole seed uncooked 50.37a 18.9(±0.45)a 50.26(±2.68)a 1.99(±0.04)a Whole seed 30 50.37a 3.12(±0.04)c 44.36(±2.37)b 2.46(±0.12)a Whole seed 60 49.34b 5.37(±0.20)b 13.46(±0.15)c 2.71(±0.31)a

Kernel 30 57.42a 3.37(±0.30)b 39.26(±0.74)a !.83(±0.01)a Kernel 60 56.03b 5.46(±0.04)a I6.48(±0.02)b 2.71(±0.08)a

79 pn

Table 7 (contd.) Variety Cooking Glutelin(%) Residue (%) Total protein time (min.) recovered (%) Rubatab Whole_seed uncooked 13.19(±0.63)b 19.00(±0.51)a 99.98 Whole seed 30 57.23(±1.89)a 20.79(±0.01)a 99.93 Whole seed 60 62.75(±2.74)a 20.73(±0.09)a 100.03

Kernel 30 25.36(±0.88)b 31.86(±0.04)a 100.42 Kernel 60 37.66(±1.23)a 34.53(±1.63)a 99.98

Dongola Whole seed uncooked 14.03(±0.02)c 15.34(±l:05)b 100.52 Whole seed 30 35.37(±0.01)b 16.54(±0.10)b 101.85 Whole seed 60 50.07(±0.09)a 29.82(±0.001)a 101.43

Kernel 30 3t.57(±0.76)b 24.89(±1.03)a 100.92 Kernel 60 52.72(±0.14)a 22.86(±0.76)a 100.23 Values are means (±SD). Means not sharing a common letters in column are significantly

different at P< 0.05 as assessed by Duncan's multiple range test (DMRT).

80 r raw whole seed and for debittered whole seed for 30 and 60 min. was .2, 57.2 and 62.8% respectively. The amount of glutelm increased jnificantly (P <0.05) respectively. The amount of glutelm increased gnificantly due to debittering. The glutelin in cv Dongola was 14% raw whole seed and for debittered hole seed for 30 min and 60 min. was 35.4 and 50.1%. The glutelin content >r debitterd kernel for 30 and 60 min was 31.6 and 52.7% respectively. The lutelins increased significantly (P< 0.05) in all samples due to debittering. Glutelins levels of raw whole seed in this study were relatively similar d those obtained by Dhawan et al. (1991) who found chickpea glutelins anging from 19 - 24%. The glutelin fraction was 19.6% for pigeon pea seed Singh et al., 1981). Marqurez and Lajolo (1981) reported glutelm content of 12.4% for Phaseolus vulgaris. Varasundharosoth and Barnes (1985) found upin seed glutelin was 17%. 4.3.1.5 Insoluble protein: The content of insoluble protein for cultivar Rubatab for raw whole seed was 19% and for debittered whole seed for 30 and 60 min. was 20.8 and 20.7% respectively. Insoluble protein of debittered kernel for 30 and 60 min. was 31.9 and 34.5% respectively. The insoluble protein content of cv Dongola was 15.3% and for debittered whole seed for 30 and 60 min. was 16.5 and 29.8% respectively. Insoluble protein content of debittered kernel for 30 and 60 min. was 24.9 and 22.9% respectively. In view of the fact that the classical procedure of Mendei-Osborne (1914) extracted only 65-85% proteins which was reflected in the substantial

81 amount of protein in the residue, it was necessary to use the Landry and Moureaw (1970) procedure for fractionating lupin proteins. The latter method also fractionates the glutelin fractions into subtractions (G], Gj, and

G3 glutelins).

4.3.2 Landrv and Moureasux method: 4.3.2.1 Globulin: Globulin was the major protein fraction for raw whole seed. The globulin content for cv Rubatab for raw whole seed was 62.5% and for debittered whole seed for 30 and 60 min. was 30.7 and 28.1% respectively. The globulin fraction decreased significantly. The globulin fraction decreased significantly (P ^ 0.05) as a result of debittering. The globulin content debittered kernel for 30 and 60 min. was 41.5 and 39.9% respectively. Globulin content of cv Dongola for raw whole seed was 66.4% and for debittered whole seed for 30 and 60 min was 38.9 and 24% respectively. The globulin fraction decreased significantly (P< 0.05) as aresult of debittering. The globulin content of debittering kernel for 30 and 60 min. was 28.4 and 36.2% respectively. The globulin content generally extracted by this method were more than to those extended by the Mendel-Osborne method. Oomah and Bushuk (1983) showed that globulin content for two lupin species (L. albus and L. angustifolius) ranged from 53-85%. They also observed defatting of lupin seed meal affected protein solubility by decreasing the amount of globulins. Defatting altered the solubility of two protein components (26000, 27000 daltons) whereby they become insoluble in water (Oomah and Bushuk, 1983). It was also observed defatting caused loss of three bands of globulin (Oomah and Bushuk, 19b). The results in this study for raw whole seed were similar to those obtained by Osborae (1924) who reported that the globulins content ranged from 50 to 75% for total seed protein of legumes. In native globulin heat treatment destroy the high molecular weight polypeptides giving rise to smaller fragments (Semino and Cerletti, 1987). 4.3.2.2 Albumin: Albumin extracted by Landry and Moureaux method for cv Rubatab was 3.2% for whole seed and for debittered whole seed for 30 and 60 min. was 1.9 and 1.1% respectively. Albumin content decreased significantly (P <0.05) due to debittering. Albumin content of debittered kernel for 30 and 60 min. was 1.5 and 1.6% respectively. Albumin content of cv Dongola for raw whole seed and debittered whole seed for 30 and 60 min. was 3.1, 1.5 and 1.8% respectively . Albumin levels in debittered kernel for 30 and 60 min were 1.3 and 1.5% respectively. Generally, the values of albumins obtained by Mendel-Osborne method were higher than those obtained by Landry and Moureas method to attachment of subunits of proteins with globulins in the Landry and Moureaux method (1970). The results obtained in this study were lower than those obtained by Singh and Jambunathan (1982). They were in agreement with the results ed by Oomah and Bushuk (1983) who found albumin content ranging 1-10% for L. albus and L. angustifolius. The effect of soaking and heat treatment in this study reduced protein lity in agreement with the results observed by Chango et aj. (1993). Albumin and globulin were composed of at least 14 and 10 subunits ctively with corresponding apparent molecular weight ranging from 3-554000 and 193000-460000 respectively. The predominant MW of lits of albumin and globulin 266000 and 123000 dalton.(Sathe and ikhe, 1981a).

.3 Alcohol-extractable proteins: Alcohol-extractable proteins of cv Rubatab for raw whole seed were it and for debittered whole seed for 30 and 60 min. were 2.0 and 4.8% actively. Alcohol extractable proteins were 3.8 and 4.3% for debittered kernel 10 and 60 min respectively. Alcohol extractable proteins fractions of cv Dongola for raw whole I were 3.5% and for debittered whole seed for 30 and 60 min. were 4.7% 4.2% respectively. Alcohol-extractable proteins fractions were 3.1 and Vo for debittered kernel for 30 and 60 min. respectively. These results were relatively similar to those obtained by Singh et al. 81) who found the alcohol extractable protein fraction content was 3.1 % iecorticated seed of pigeon pea.

84 4.3.2.4 Glutelins: Glutelins were fractionated into subfractions (Gj, G2, and G3

glutelins) as is shown in Table 8.

Gj-glutelin content of cv Rubatab for raw whole seed, debittered whole seed for 30 and 60 mm. was 2.0, 1.1 and 1.5 respectively. The content of Gi-glutelin decreased significantly (P<0.05) due to debittering. Gi-glutelin of debittered kernel for 30 and 60 min. was 2.4,1.9% respectively. Gi-glutelin of cv Dongola was 2.0,2.8 and 1.2% for raw whole seed and debittered whole seed for 30 and 60 min. respectively. Levels of G\- glutelin in debittered kernel for 30 and 60 min. were 1.8 and 1.4% respectively. These results are relatively similar to those obtained byNugdellah (1996) who found G\ -glutelins of cowpea to decrease by heating process ranging from 1.0 to 3.0% of raw cowpea. 4.3.2.4.2 G2-glutelin:

G2-glutelin content of cv Rubatab was 14.6 for raw whole seed, it decreased significantly (P < 0.05) in debittered whole seed for 30 and 60 min. (3.9 and 2.1% respectively). In debittered kernef for 30 and 60 min. it was 1.0 and 3.1°/^ respectively.

85 Table 8 Protein fractions of raw and debittered cultivars of -Vnc tennis according tolLandry and Mouieaux method (1970). Variety Cooking Actual Globulin (%) Albumin (%) Prolamin(%) time protein (min.) (%) - Rubatab Whole seed uncooked 49.88a 62.53(±Ul)a 3.23(±0.6)a 2.45(±0.02)ab Whole seed 30 47.14b 30.73(±1.2)b 1.86(±0.61)b 2.01(±0.02)b Whole seed 60 47.27ab 28.13(±1.53)b 1.14(±0.25)b 4.83(±0.59)a

Kernel 30 59.33a 41.48(±1.80)a 1.51(±0.45)a 3.78(±0.17)a Kernel 60 56.69b 39.91(±2.13)a 1.58(±0.21)a 4.32(±0.15)a

Dongola Whole seed uncooked 50.37a 66.36(±1.67)a 3.12(±0.34)a 3.54(±0.13)a Whole seed 30 50.37a 38.91(±0.21)b 1.50(±0.28)c 4.66(±0.28)a Whole seed 60' 49.34b 23.98(d=1.33)c 1.77(±0.00)c 4.19(±0.05)a

Kernel 30 57.42a 28.36(±2.82)a 1.29(±0.12)a 3.06(±0.38)a Kernel 60 56.03b 36.24(±0.78)a 1.50(±0.56)a 3.27(±0.04)a

86 Table 8 (contd.)

Variety Cooking G|glutelin G2-glutelin G3-glutelin Residue (%) Total protein time (%) (%) (%) (min.)

Rubatab Whole seed uncpoked 1.97(±0.02)a 14.59(±0.43)a 5.26(±0.28)ab 11.32(±0.38)a 101.35 Whole seed 30 U4(±0.05)c 3.86(±0.07)b 56.84(±1.21)a 2.77(±0.0)b 99.21 Whole seed 60 1.49(±1.02)b 2.14(±0.93)b 60.2(±0.35)a 2.33(±0.22)b 100.26

Kernel 30 2.36(±0.06)a 0.96(±0.01)a 46.68(±1.51)a 1.76(±0.02)a 98.53 Kernel 60 1.94(±0.04)a 3.06(±0.03)a 45.59(±1.72)a 2.86(±0.03)a 99.26

Doneola

Whole seed unroasted 2.04(±0.02)a 13.55(±O.O5)a 6.95(±1.45)a 7.58(±0.75)b 103.15 Whole seed 30 2.78(±0.48)a 1.67(±0.01)c 48.61 (±1.72)b 4.03(±0.25)b 102.16 Whole seed 60 l.l(±0.06)a 2.18(±0.01)b 63.07(±1.49)a 3.17(±O.O3)c 99.54

Kernel 30 1.79(±0.07(a 2.90(±0.01)a 61.59(±l.38)a 2.34(±0.1)a 101.33 Kernel 60 1.42(±0.08)a 2.32(±0.04)a 52.42(±0.2)a 2.02(±0.15)a 99.19

Values are means (±SD). Means not sharing a common letters in column are significantly different at P< 0.05 as assessed by Duncan's Multiple Range Test (DMRT). G2-glutelin content of cv Dongola was 13.6% for raw whole seed, it decreased significantly for debittered whole seed for 30 and 60 min (1.8 and 2.2% respectively). G2-glutelins of debittered kernel for 30 and 60 min. were 2.9 and 2.3% respectively. The effect of debittering caused a decrease in the content of G2-glutelin for both cultivars. The amount of G2-glutelin for debittered samples (whole seed or kernels) were similar to those obtained by Nugdellah (1996) who found that G2-glutelin of cowpea ranging from 1.6-2.9%.

4.3.2.4.3

G3-glutelins content of cv Rubatab was 5.3% for raw whole seed which increased significantly (P< 0.05) to 56.$ and 60% for debittered whole seed for 30 and 60 min. respectively. G3-glutelin of debittered kernel for 30 and 60 min. was 46.7 and 45.6% respectively. G3-glutelin of cv Dongola was 7.0% for raw whole seed and was 48.6% and 63.1% for debittered whole seed for 30 and 60 min. respectively. The values of G3-glutelin in debittered kernel for 30 and 60 min. were 61.6 and 52.4% respectively. G3-glutelins increased due to debittering process for the two cultivars examined. The results obtained in this study were in good agreement with those obtained by Nugdella (1996) who found significant increases in G3- glutelins for cooked cow pea seeds. Oomah and Bushuk (1983) confirmed the occurrence of glutelin fractions with molecular weight ranging from 21000-130000 daltons. The increase in glutelin in the presence of sodium dedecysulfate(SDS) at high

88 pH level is consistent with the results obtained by Richard et ah (1987) who showed lupin protein extracted at pH 8.5 contained high molecular weight polypeptides. 4.3.2.5 Insoluble protein: The content of insoluble protein determined for cv Rubatab for raw whole seed was 11.4% and for debittered whole seed for 30 and 60 min. was 2.8 and 2.3% respectively. Insoluble protein of debittered kernel for 30 and 60 min. was 1.8 and 2.9% respectively. The insoluble protein content of cv Dongola was 7.6,4.0 and 3.2% for raw whole seed and debittered whole seed for 30 and 60 min. respectively. Levels of insoluble protein content of debittered kernel for 30 were 2.3% and 2.0% for debittered and kernel for 60 min. The values of insoluble protein obtained in the Mendel-Osborne method were much higher than those extracted by Landry and Moureaux method. Since the native quaternary and tertiary structure of lupin protein are modified at higher temperatures (well below 100 ) exposure of buried # substrate sites is a more likely explanation of our results (Bonomi et al. 1983). 4.4 Effect of dcbittcring on the in vitro protein digestibility of two lupin cultivars: 4.4.1 IVPD of protein: The m vitro protein digestibility of raw Lupimis termis cv Rubatab and cv Dongola was 73.9 and 73.0% respectively (Table 9). The digestibility of debittered samples for 30 min. was 75.7% for whole seed Table 9 Effect of debittering on IVPDf/iof lupin crude protein and protein fractions. Variety Cooking Crude protein Globulin Albumin Prolamin time (%) (%) (%) (%) (min.) Rubatab Whole seed uncooked 73.92(±0.32)b 97.03(±0.98)a 78.31(±0.46)a 57.14(±0.02)a Whole seed 30 75.71(±0.24)ab 78.64(±2.31)c 45.31(±1.55)b 56.27(±3.38)a Whole seed 60 77.74(±0.05)a 77.48(±0.27)c 33.85(±1.89)c 54.01(±1.86)a

Kernel 30 77.42(±0.18)b 96.54(±0.36)a 29.74(±0.82)a 82.39(±1.72)a : Kernel 60 81.02(±0.14)a 96.66(±0.74)a 29.17(±2.71)a 64.33(±2.33)b

Dongola Whole seed uncooked 72.96(±0.16)c 44.98(±0.87)c 96.04(±2.0)a 67.24(±0.81)c Whole seed 30 75.57(±0.47)b 57.01(±0.46)b 87.86(±0.77)b 79.70(±2.08)b Whole seed 60 79.24(±0.72)a 87.66(±0.38)a 76.56(±0.24)c 93.1 (±0.4 l)a

Kernel 30 76.13(±0.25)b 61.19(±0.24)b 92.41(±0.35)a 90.05(±[.0)a Kernel 60 80.0(±0.52)a 78.86(±1.13)a 69.69(±1.49)b 3L16(±0.04)b

90 Table 9 (contd.) Variety Cooking Gj-glutelin G2-glutelin G3-glutelin

time (min.) (%) . (%) (%) Rubatab i. Whole seed uncooked 64.38(±0.08)a 74.6(4±3.03)a 45.14(±2.02)c Whole seed 30 60.78(±2.01)b 76.6(±0.36)a 83.39(±0.39)b Whole seed 60 53.47(±0.01)c 79.7l(±2.86)a 88.50(±1.72)a

Kernel 30 55.49(±1.0)a 61.06(±1.28)a 94.04(±2.08)a Kernel 60 , 48.85(±1.46)a 57.03(±1.08)a 78.69(±0.08)b *

Dongola Whole seed uncooked 94.17(±0.34)a 63.73(±0.08)c 35.26(±1.05)c Whole seed 30 84.89(±0.34)b 76.49(±1.58)b 57.57(±1.03)b Whole seed 60 48.49(±0.77)c 91.03(±0.37)a 76.74(±0.63)a

Kernel 30 58.94(±0.08)b 58.42(±0.77)a 78.69(±1.48)a Kernel 60 74.16(±0.31)a 55.76(±0.46)a 74.96(±0.53)a

Values are means (±SD). Means not sharing a common letters in column are significantly different at P< 0.05 as assessed by Duncan's Multiple Range Test (DMRT). of cv Rubatab and 75.6% for cvDongola. Digestibility of debittered whole seed for 60 min. was 77.7% and 79.2% of cv Rubatab and cvDongola respectively (Table 9). Significant differences were observed in the digestibility of raw whole'seed and debittered whole seeds for 30 and 60 min. in cv Rubatab, but was only so for cv Dongola for raw whole seeds and debittered whole seed for 60 min. In vitro protein digestibility of debittered kernels for cv Rubatab was 77.4 and 81.0% for 30 and 60 min. respectively (Table 9). The IVPD for debittered kernel for cv Dongola for 30 and 60 min. was 76.1 and 80.0% respectively. Debittering resulted in improved protein digestion for the two lupin cultivars. These results were relatively similar to those obtained by Sathe et al. (1982) who reported that the in vitro digestibility of lupin flour was 71.1% which increased up to 77.6% by heating in a boiling water bath for 30 min. Improved digestibility on heating may have been due to denaturation of protein facilitating susceptibility towards enzymic attack. Improved in vitro proteins digestibility on heating observed in the present investigation is in agreement with that reported for digestibility of Phaseolus vulgaris (Chang and Safferlee, 1980) and in winged beans (Phaseolus tetragolobus (L.) DC) (Ekpenyong and Borchers, 1980). The apparent nitrogen digestibility of raw and debittered L. mutabilis was 80.0 and 81.2% respectively (Schoeneberger, et aJL 1982). The results obtained in this study were at variance with those reported by Pereira and Piom (1977) who found that cooking adversely affected the IVPD of lupin seeds.

92 Singh et al (1995) found that roasting caused minimal protein damage •md reported 37.7 and 44.7% for IVPD in raw and roasted lupin seed. Geerivani and Theophilus (1980) observed that heat processing (boiling the legumes for 30 min. at 100 C) improved the digestibility of four legumes studied: in red gram it increased from 51 upto 68%; in bengal it increased from 73 upto 81%, in black gram it increased from 82 up to 86%. The in vitro protein digestibility of full fat defatted and debittered lupin flours were 85.5,76 and 90.6% respectively as reported by Mohamed et al. (undated) Sathe and Salankhe (1981D) found that the protein digestibility of wingged bean increased after defalting as a result of reduction of tannins and inhibitors. Protein digestibility of L. albus and L. luteus were 74.9 and 74.1% respectively (Ballester et al., 1980). These findings are in agreement with the results obtained in this study. Ahmed and Nour (1990) reported that the protein digestibility of L. tennis was 55% which is lower than the results obtained in this study. 4.4.2 IVPD of protein fractions: The IVPD of protein fractions is shown in Table 9. 4.4.2.1 Globulins: The jn vitro digestibility of globulins in cv Rubatab decreased significantly (P < 0.05) with increasing debittering period. For raw whole seed, it was 97% and of debittered whole seed for 30 and 60 min. was 78.6 and 77.5% respectively. These results are in agreement with the result obtained by Marquez and Lajalo (1981).

93 The IVPD of globulins in debittered kernels for 30 and 60 min. war. 96.5 and 96.7% respectively. The IVPD of globulin in cv Dongola at raw whole seed and debittered whole seed (30 and 60 min.) was 45, 57.0 and 76.6% respectively. The IVPD of debittered kernels for 30 and 60 min. was 61.2 and 78.9% respectively. The importance of IVPD of globulins as a result of debittering is in good agreement with the results obtained by Marquez and Lajolo (1981) who showed improved IVPD of globulins due to heating. 4.4.2.2 Albumin: The effect of debittering on the IVPD of albumins is similar to that of globulins. The IVPD of albumins in cv Rubatab for raw whole seed debittcred whole seed for 30 and 60 min. were 78.3,43.3 and 33.3% respectively. The IVPD decreased significantly (P < 0.05) as a result of debittering. The IVPD of albumin in debittered kernels was 92.4%, it decreased significantly to 62.7% as a result of debittering. The results are in good agreement of those obtained by Marquez and Lajolo (1981). The digestibility of albumin of cv Dongola as raw whole seed, debittered whole seed for 30 and 60 min. was 96.0, 87.9 and 76.6% respectively. It decreased significantly (P< 0.05) as aresult of debittering. The IVPD for debittered kernels for 30 and 60 min. was 92.4% and 69.7 respectively, it decreased significantly (P < 0.05) as a result of roasting. The decrease in digestibility of albumins observed in tliis study is relatively similar to the results obtained by Marquez and Lajolo (1981) who showed

94 that the residue left after digestion of autoclaved albumin contained peptides with molecular weights of 14000 and 20000. Molecular weight of trypsin- jnhibitor in beans has been reported to be between 10000 and 15000 and with an abundance of disulphide bridges which helps to stabilize the structure of albumin (Pusztai, 1968). This may explain the low digestibility of peptides formed during heating of the albumins in both cultivars and for globulin of Rubatab. 4.4.2.3 Alcohol extractable proteins (AJEP): The FVPD of alcohol extractable proteins of cv Rubatab for raw whole seeds, debittered whole seeds (30 and 60 min.) was 57.1, 56.3 and 54% respectively. The FVPD in both debittered kernels (30 and 60 min.) was 82.4 and 64.3% respectively. There was a highly significant decrease as roasting time increased. The IVPD of AEP for cv Dongola raw whole seeds, debittered whole seeds (30 and 60 min.) increased significantly (P < 0.05) and were 67.2, 79.7 and 93.1% respectively. The IVPD of debittered kernels (30 and 60 min.) was 90.0 and 81.16% respectively. It decreased significantly (P < 0.05) due to debittering. IVPD of AEP in both cultivars decreased due to debittering. 4.4.2.4 Gj^-glutelins:

The IVPD of Gi-glutelin for Rubatab decreased significantly (P < 0.05) for raw whole seed and debittered whole seeds (30 and 60 min.), they were 64.4, 60.8 and 53.5% respectively. For debittered kernels (30 and 60 min) the IVPD was 55.5 and 48.9% respectively. For cv Dongola IVPD decreased significantly (P < 0.05) in raw whole seeds and debittered whole seeds (30 and 60 min.), they were 94.2, 84.9 and

95 48.5% respectively. The IVPD for debittered kernels (30 and 60 min.) increased significantly from 58.9 to 74.2%. 4.4.2.5 G2-glutelins: The IVPD of raw whole seeds and debittered whole seed (30 and 60 min.) was 74.6, 76.6 and 79.7% respectively for cvRubatab. The IVPD of G2~glutelins of debittered kernels (30 and 60 min.) decreased significantly

(P < 0.05) and was 61.1 and 57.0% respectively. The IVPD of G2~glutelins of cv Dongola raw whole kernels and debittered whole seeds (30 and 60 min.) increased significantly (P < 0.05) and were 63.7, 76.5 and 91.0% respectively. The IVPD for debittered kernels (30 and 60 min.) was 58.4 and 55.8% respecively. 4.4.2.6 Gv-glutelins:

Table 9 shows the IVPD of G3-glutelins of cvRubatab were 45.1, 83.4 and 88.5% for raw whole seeds and debittered whole seeds respectively. The increase in IVPD as a result of debittering was significant (P< 0.05). The IVPD of debittered kernels (30 and 60 min.) was 94 and 78.7% respectively. The IVPD of G3-glutelins for cv Dongola raw whole seeds and debittered whole seeds (30 and 60 min.) was 35.2, 57.6 and 76.7% respectively. The IVPD increased significantly (P < 0.05) as a result of debittering. The IVPD of G3-glutelins debittered kernels (30 and 60 min.) was 78.7 and 75.0% respectively.

96 The improvement in IVPD of G3-glutelin as a result of debittering process is in good agreement with the results reported by Marquezand Lajolo (1981) who observed an improvement in IVPD of glutelins as a result beating, for fifteen Brazilian varieties of Phaseolus vulgaris.

97 Summary and conclusions

en Lup><\ WC'"* In the present investigation/ the results indicated that cv. Dongola had higher weight and volume while cv.Rubatab had higher thickness and density . Debittered whole seeds both cuttivars had higher protein and carbohydratef. The debittered kernels in both cultivarx had higher protein , carbohydrate , oil and calorific value . The two varieties ( raw or debittered ) did not contain antinutritional factors. Fatty acid composition of lupin seed showed that oleic acid was the major fatty acid. The major minerals of lupin seeds were : k7, f, Mg, Ca and Fe . Fractionation of protein was carried out by two methods : Mendel- Osborne and Landry and Moureaux. Globulin was the major fraction for both fractionation procedures in raw whole seeds . After debittering^ glutelin was the major fraction for both methods

Debittering resulted in reduction of the albumin, globulin, Gi- glutelin and Gi - glutelin . The GJ - glutelin fraction increased as a result of debittering. In vitro protein digestibility ( IVPD ) of total protein was improved by debittering . IVPD of the G3 - glutelin fraction was increased due to debittering . In general debittering caused improvement in protein digestion for both cultivar.T, studied.

98

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