Physico chemical and structural characterization of germinated
and cooked lentils (Lens culinaris Medic).
By
Mashair Ahmed Sulieman
B.Sc. (Agric.), 1999, Faculty of Agriculture, University of Khartoum
M.Sc. (Food Science), 2002, Faculty of Engineering and Technology,
University of Gazira.
A thesis submitted to University of Khartoum in fulfillment for the
requirements of the degree of philosophy doctorate (Agric).
Supervisor: Professor: Abdullahi Hamid ElTinay
Co supervisor: Professor: Elfadil Elfdal Babiker
Department of Food science and Technology
Faculty of Agriculture
University of Khartoum
October 2007
1
DEDICATION
To My:Parents
Husband
Kids
Sisters
Brothers
Friends
2
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to Professor Abdullahi Hamid
ElTinay for his helpful, persistent encouragement, continuous support and advices
and faithful guidance during the entire course of this work
Sincere thanks are to professor Elfadil Elfadl Babiker Co supervisor.
for his advices, support and follow-up of my study .
My sincere thanks are due to all colleagues and lab technicians of the
Department of Food Science and Technology, Faculty of Agriculture, University
of Khartoum for their cooperation and friendship during the course of
investigation
I wish to acknowledge the assistance given by Mohammed Elmotasim Eltayb for
the statistical analysis of data.
My deep thanks to my husband Mohamed Elamir for his un limited help and
support.
Thanks and gratitude are extended to DAAD scholarship for financing this study
and giving me opportunity to do part of this study in Germany.
I wish to acknowledge Professor Ortwin Simon, Dr. Schaefer and Paula. Free
University, [Berlin] for their help and faithful guidance during working in their
institute.
Finally I wish to record my indebtedness and my most sincere gratitude to my
beloved family.
Above all my special praise and unlimited thanks to Allah who helped and gave
me health to complete this work.
.
3
TABLE OF CONTENTS
DEDICATION…………………………………………………………………..
ACKNOWLEDGEMENTS……………………………………………………..
TABLE OF CONTENTS………………………………………………………….
LIST OF TABLES…………………………………………………………………..
LIST OF FIGURES…………………………………………………………………
ABSTRACT……………………………………………………………………….
ARABIC ABSTRACT………………………………………………………………
CHAPTER ONE: INTRODUCTION……………………………………………
CHAPTER TWO: LIETERATURE REVIEW……………………………………
2.1
2.2
2.3
2.4
2.5
The chemical composition of lentil……………………………………
2.1.1
Crude protein content……………………………………….
2.1.2
Lipids……………………………………………………………
2.1.3 Fibre and ash………………………………………………….
2.1.4 Total carbohydrates………………………………………….
Functional properties……………………………………………………
2.2.1 Protein solubility……………………………………………..
2.2.2 Effect of pH on nitrogen solubility…………………………
2.2.3 Water absorption capacity……………………………………
2.2.4 Fat absorption capacity………………………………………
2.2.5 Bulk Density (BD)…………………………………………….
2.2.6 Foaming properties……………………………………………...
2.2.6.1 Foaming capacity…………………………………..
2.2.6.2 Foaming stability…………………………………..
2.2.7 Emulsification properties………………………………………
2.2.7.1 Emulsion capacity (EC)……………………………
2.2.7.2 Emulsion stability…………………………………..
2.2.8 Gelation…………………………………………………….
2.2.9 Dispersibility…………………………………………………..
Phytase………………………………………………………………..
2.3.1 Phytates………………………………………………………
2.3.2 Formation of phytic acid………………………………………
Germination……………………………………………………………
2.4.1 Effect of germination on the chemical composition and other
nutrients:………………………………………………………
2.4.2 Effect of germination in phytase activity………………………
Interaction of phytic acid with minerals…………………………………
2.5.1 Zinc and phytic acid………………………………………….
2.5.2 Iron and phytic acid………………………………………….
2.5.3 Calcium and phytic acid……………………………………..
2.5.4 Magnesium and phytic acid………………………………….
4
Page
i
ii
iii
v
vi
vii
xi
1
4
4
4
4
5
5
6
7
8
8
9
10
10
10
11
11
11
12
12
12
13
13
14
15
16
17
18
18
19
19
20
2.6
2.7
2.8
2.5.5 Copper and phytic acid……………………………………..
2.5.6 Binding of phytic acid to proteins
Amino acids content
In vitro protein digestibility
2.7.1 Effect of cooking on protein digestibility
Protein fractions
CHAPTER THREE: MATERIALS AND METHODS
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
Materials…………………………………………….
Methods of analysis……………………………….
3.2.1 Proximate analysis…………………………
3.2.1.1 Moisture content……………………………….
3.2.1.2 Ash content……………………………….
3.2.1.3 Fat content………………………………….
3.2.1.4 Crude fibre…………………………
3.2.1.5 Crude protein
3.2.1.6 Carbohydrates
3.2.2 Nitrogen solubility
3.2.3 Nitrogen solubility as a function of NaCl concentration
3.2.4 Water absorption capacity
3.2.5 Fat absorption capacity
3.2.6 Bulk density
3.2.7 Foaming capacity and foam stability
3.2.8 Emulsification activity (EA) and emulsion stability (ES)……...
3.2.9 Gelation……………………..
3.2.10 Dispensability……………………………
Determination of phytase activity……………………
Analysis of the inositol phosphates…………………….
HCl extractable minerals………………………………….
Amino acid analysis………………………………………..
In vitro protein digestibility……………………………………..
Protein fractionation due to solubility…………………
Electrophoresis
CHAPTER FOUR: RESULTS AND DISCUSSION……………………………
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
Chemical composition
Nitrogen solubility as affected by pH
Nitrogen solubility at different sodium chloride concentration:
Water and oil absorption capacity………………………………..
Bulk density………………………………………………….
Emulsifying properties …………………………………………….
Foaming properties ……………………………………….
Gelation capacity……………………………………………….
Dispersibility…………………………………………………….
Effect of germination on chemical composition and energy value of lentil
cultivars……………………………………………………………
4.11 Effect of germination on phytase activity, phytate phosphorus and non-phytate
phosphorous………………………………………………
4.12 Total and extractable minerals of germinated lentil cultivars…………..
4.13 Effect of germination on the amino acid content……………………….
5
20
20
21
21
22
23
25
25
26
26
26
27
27
28
28
29
30
31
31
31
32
32
32
33
33
34
36
37
37
38
39
41
45
45
45
48
50
50
51
52
52
53
56
57
60
65
4.14 Effect of cooking on in vitro protein digestibility………………………
4.15 Effect of cooking on protein fraction……………………………………
4.16 Effect of cooking on SDS-PAGE electrophoresis of lentil
fractions………………………………………………………………….
70
73
protein
CONCLUSIONS ………………………………………………………………………79
RECOMMENDATIONS………………………………………………………………81
REFERENCE…………………………………………………………………………82
6
LIST OF TABLES
Table 1 Proximate composition of four cultivars of lentil……………………...
Table 2: Functionality of lentil protein isolate (LPI)………………………..
Table 3:
Page
46
52
Foam capacity (FC%) and effect of time on foam stability(%) of lentil 52
protein……………………………………………………….
Table 4: Effect of pH on least gelation concentration of lentil protein isolate...
Table 5 : Effect of pH on despersibility of lentil protein isolate………………..
54
54
Table 6: Proximate composition and energy value of lentil cultivars as affected by
germination………………………………………………….
56
Table 7: Effect of germination on phytic acid, phosphorus, phytase phosphorus and non
phytate phosphorus of lentil cultivars …………
60
Table 8 Effect of germination on total and extractable phosphorus and calcium
61
(mg/100g) in lentil cultivars………………………………………
Table 9: Effect of germination on total and extractable iron and magnesium (mg/100g)
62
in lentil cultivars………………………………………..
Table 10: Effect of germination on total and extractable copper and zinc (mg/100g) in
63
lentil cultivars…………………………………….
Table 11 : Effect of cooking on in vitro protein digestibility (IVPD) of lentil cultivars
using pepsin and pepsin with pancreatin…………………………….
71
Table 12 :Effect of cooking on protein fractions content(%) of lentil cultivars..
7
72
LIST OF FIGURES
Page
Fig.1:Effect of pH on nitrogen solubility of lentil protein
47
Fig 2 Effect of NaCl on nitrogen solubility of lentil cultivars
49
Fig. 3: Amino acid content of Selaim cultivar as affected by germination.
66
Fig. 4 : Amino acid content of Rubatab cultivar as affected by germination.
67
Fig. 5 : Amino acid content of Nadi cultivar as affected by germination.
68
Fig 6:
75
SDS-PAGE pattern of globulin fraction of cooked and uncooked
lentil cultivars.
. Fig 7: SDS-PAGE pattern of Albumin fraction of cooked and uncooked
76
lentil cultivars
Fig 8 SDS-PAGE pattern of Prolamin fraction of cooked and uncooked
77
lentil cultivars
Fig 9: SDS-PAGE pattern of Glutelin fraction of cooked and uncooked
lentil cultivars
8
78
ABSTRACT
Legumes such as lentil contain a high concentration of proteins,
carbohydrates and make an important contribution to human diet in
many countries. Lentil is a nutritious food legume cultivated for its
seeds which have a relatively higher content of protein and calories
compared to other legumes, it is a protein\ calorie crop.
Four lentil cultivars were used in this study, three of which are
Sudanese (Rubatab,Nadi and Selaim),obtained from Elhudaiba
Research Station ,the fourth one (Indian) is obtained from Khartoum
North market. Proximate composition showed moisture content
ranging from 6.4-10.3%, ash 2.7-3.7% ,fat 1.9-2.4% ,fibre 1.2-4.1%
and protein 32.4-35.6%.Solubility of lentil protein was determined at
pH values1-12 and sodium chloride concentrations 0.2-1N; minimum
protein solubility was observed at pH 5.0.The protein was coagulated
at the isoelecteric pH(5.0).Protein isolate were obtained for Nadi and
Selaim.The isolates had 80% protein content and functional properties
of lentil protein isolates were studied. Lentil protein has good
functional characteristics with emulsification activity of 75.3-62.94%
and emulsion stability was 41.4-46.3% and has lower foaming
capacity with higher stability. The total protein was highly dispersible
with a water absorption capacity of 1.9-2.2 ml H2O/g protein. Oil
absorption capacity of 1.9-2.0 ml oil/g protein and bulk density of 1.4
g/ml.
Lentil with high protein content has acceptable functional properties
which makes it a promising protein source in food systems and its
high nitrogen solubility and less than 1% fat content a characteristic
9
generally needed for textured product like meat. Also, lentil can be
incorporated in beverages and infant formula.
Sudanese lentil cultivars (Rubatab, Nadi and Selaim) were germinated
for 3 and 6 days. The germinated seeds were dried and milled.
Proximate composition, phytic acid content, phytase activity and
hydrocholoric acid (HCl) extractability of minerals were determined
.During germination crude fat and fiber increased, whereas nitrogen
free extract (NFE) and food energy decreased. Phytic acid content
decreased significantly (p≤ .05) with an increase in germination time
Germination resulted in a decrease in total phytate phosphorus with
correspondingly marked increase in non-phytate phosphorus. Total
and extractible mineral elements estimated in this study (phosphorus,
calcium, iron, magnesium) were positively correlated with duration of
germination except copper and zinc. Germination for 3 days increased
phytase activity significantly (p≤ 0.05) for all cultivars. Phytase
activity of Rubatab cultivar continued to increase up to 6 days
germination, however for Nadi and Selaim cultivars it slightly
decreased. In order to obtain lentil seeds with high phytase activity
low phytic acid and high mineral extractability, germination for not
more than 3 days is recommended. In studying the effect of
germination on the amino acid content results indicate that
germination increase partly of or all essential and nonessential amino
acids with slight variation between cultivars. In Selaim cultivar,
germinating seeds for 3 days increased the proportion of all essential
amino acids except methionine; increasing the period of germination
to six days decreased amino acid content, this was observed for
histidine, lysine and arginine. This result was also observed for
10
Rubatab cultivar in which the content of essential amino acids was
increased due to germination except methionine and lysine. In Nadi
cultivar germination for three days increased the essential and
nonessential amino acids. Generally all lentil cultivars were low in
their content of sulfur amino acids : methionine and cystine.
The effect of cooking on protein digestibility, protein fractions and
their electrophoretic characteristic for lentil cultivars was investigated.
Cooking significantly (p ≤ 0.05) reduced the protein digestibility using
pepsin and/or pancreatin. Pepsin digestibility of raw seeds ranged
from 44.6 to 52.1% and that of both pepsin and pancreatin ranged
from 81.8 to 99.9%. Cooking reduced the protein digestibility of the
cultivars and was found to range from 22.3 to 19.7% when pepsin was
used and ranged from 77.1 to 88.2% when both pepsin and pancreatin
were used. The major protein in lentil was albumin followed by
globulin. Cooking significantly (p ≤ 0.05) decreased the albumin
fraction. This decrease was accompanied by significant increment in
the glutelin fractions. SDS–PAGE electrophoresis of cooked lentil
protein fractions showed that lentil protein was altered quantitatively
and qualitatively due to cooking. Numbers of subunits of total protein
in lentil cultivars before cooking ranged from 17 to 19 bands.
However, after cooking they decreased and ranged from 13 to 16
bands. The effect of cooking was most pronounced in the prolamins
fractions and its subunits were reduced from 4 to 2 with a high
molecular weight of 56.0 kDa.
11
‫ﺧﻼﺻﺔ اﻻﻃﺮوﺣﺔ‬
‫اﻟﺒﻘﻮﻟﻴﺎت آﺎﻟﻌﺪس ﺗﺤﺘﻮي ﻋﻠﻰ ﻧﺴﺒﺔ ﻋﺎﻟﻴﻪ ﻣﻦ اﻟﺒ ﺮوﺗﻴﻦ واﻟﻜﺎرﺑﻮهﻴ ﺪرات اذ ﺗﻌﺘﺒ ﺮ ﻣ ﻦ ﺑ ﺪاﺋﻞ‬
‫اﻟﻠﺤ ﻮم اﻟ ﺸﺎﺋﻌﺔ اﻻﺳ ﺘﻌﻤﺎل ﻓ ﻰ اﻟ ﺪول اﻟﻨﺎﻣﻴ ﻪ‪،‬واﻟﻌ ﺪس ﻳﻄﻠ ﻖ ﻋﻠﻴ ﻪ ﻣ ﺼﻄﻠﺢ ﻣﺤ ﺼﻮل‬
‫اﻟﺒﺮوﺗﻴﻦ\ﻃﺎﻗﺔ‪.‬‬
‫اﺳ ﺘﺨﺪﻣﺖ ﻓ ﻰ ه ﺬﻩ اﻟﺪراﺳ ﻪ ارﺑﻌ ﻪ اﺻ ﻨﺎف ﻣ ﻦ اﻟﻌ ﺪس ﺛﻼﺛ ﺔ ﻣﻨﻬ ﺎ ﺳ ﻮداﻧﻴﻪ)ﺳ ﻠﻴﻢ‪ ،‬ﻧ ﺪى‬
‫ورﺑﺎﻃﺎب( وهﺬﻩ اﺳﺘﺠﻠﺒﺖ ﻣ ﻦ ﻣﺤﻄ ﺔ اﺑﺤ ﺎث اﻟﺤﺪﻳﺒ ﺔ واﻟ ﺼﻨﻒ اﻟﺮاﺑ ﻊ)هﻨ ﺪى ( ﺗ ﻢ اﺳ ﺘﺠﻼﺑﻪ‬
‫ﻣﻦ ﺳﻮق اﻟﺨﺮﻃﻮم ﺑﺤﺮى‪ .‬ﺑﻌﺪ اﻟﺘﺤﻠﻴﻞ وﺟﺪ ان اﻟﻌﺪس ﻳﺤﺘﻮى ﻋﻠﻰ رﻃﻮﺑ ﻪ ﺗﺘ ﺮاوح ﻣ ﻦ‪-6.4‬‬
‫‪،%10.3‬رﻣﺎد‪ ، %2.7-3.7‬دهﻦ‪،%1.9-1.2‬اﻟﻴﺎف‪ %4.1-1.2‬وﺑﺮوﺗﻴﻦ‪. %35.6-32.4‬‬
‫اﺟ ﺮى اﺧﺘﺒ ﺎر اﻟﺬوﺑﺎﻧﻴ ﺔ ﻟﺒ ﺮوﺗﻴﻦ اﻟﻌ ﺪس ﻓ ﻰ اوﺳ ﺎط اس هﻴ ﺪروﺟﻴﻨﻰ ﻣ ﻦ ‪12-1‬وﻓ ﻰ اوﺳ ﺎط‬
‫ذات ﺗﺮاآﻴ ﺰ ﻣﺨﺘﻠﻔ ﺔ ﻣ ﻦ ﻣﻠ ﺢ اﻟﻄﻌ ﺎم ‪N1.0-0.2‬اﻗ ﻞ ذوﺑﺎﻧﻴ ﺔ ﻟﻠﺒ ﺮوﺗﻴﻦ ﻟﻮﺣﻈ ﺖ ﻋﻨ ﺪ اس‬
‫هﻴﺪروﺟﻴﻨﻰ‪ 5.0‬وﺑﺎﻻﺳﺘﻔﺎدﻩ ﻣﻦ ﻧﻘﻄﺔ اﻟﺘﻌﺎدل اﻟﻜﻬﺮﺑ ﻰ ﻟﻠﺒ ﺮوﺗﻴﻦ ﺗ ﻢ ﻋ ﺰل اﻟﺒ ﺮوﺗﻴﻦ ﻟ ﺼﻨﻔﻴﻦ‬
‫ﻣﻦ اﻟﻌﺪس ﻧﺪى وﺳﻠﻴﻢ وآﺎﻧﺖ ﻧﺴﺒﺔ اﻟﺒﺮوﺗﻴﻦ ﻓﻰ اﻟﺒ ﺮوﺗﻴﻦ اﻟﻤﻌ ﺰول‪.%80‬اﻟﺨ ﻮاص اﻟﻮﻇﻴﻔﻴ ﺔ‬
‫ﻟﺒﺮوﺗﻴﻦ اﻟﻌﺪس اﻟﻤﻌﺰول ﺗﻤ ﺖ دراﺳ ﺘﻬﺎ ووﺟ ﺪ ان ﻟﻠﻌ ﺪس ﺧ ﻮاص وﻇﻴﻔﻴ ﺔ ﻋﺎﻟﻴ ﺔ ﺣﻴ ﺚ ان ﻟ ﻪ‬
‫ﻧ ﺸﺎط اﺳ ﺘﺤﻼﺑﻰ‪ 62.94%‬وﺳ ﻌﺔ رﻏﻮﻳ ﺔ ﻣﻨﺨﻔ ﻀﺔ ذات ﺛﺒﺎﺗﻴ ﺔ ﻋﺎﻟﻴ ﻪ‪ .‬اﻟﺒ ﺮوﺗﻴﻦ اﻟﻤﻌ ﺰول ﻟ ﻪ‬
‫ﻗﺎﺑﻠﻴ ﺔ ﻟﻼﻧﺘ ﺸﺎر ﻋﺎﻟﻴ ﺔ وﺳ ﻌﺔ ﻻﻣﺘ ﺼﺎص اﻟﻤ ﺎء‪2.2-1.9‬ﻣ ﻞ ﻣ ﺎء\ﺟ ﺮام ﺑ ﺮوﺗﻴﻦ وﺳ ﻌﺔ‬
‫ﻻﻣﺘﺼﺎص اﻟﺪهﻦ‪2.2-1.9‬ﻣﻞ دهﻦ\ﺟﺮام ﺑﺮوﺗﻴﻦ وآﺜﺎﻓﺔ‪ 1.4‬ﺟﺮام\ﻣﻞ‪ .‬ﺧﻠ ﺼﺖ اﻟﺪراﺳ ﺔ اﻟ ﻰ‬
‫ان ﻟﻠﻌ ﺪس ﺑ ﺮوﺗﻴﻦ ﻋ ﺎﻟﻰ ﻟ ﻪ ﺧ ﻮاص وﻇﻴﻔﻴ ﺔ ﻣﻘﺒﻮﻟ ﺔ ﻣﻤ ﺎ ﻳﺠﻌﻠ ﻪ ﻣ ﺼﺪر ﻟﻠﺒ ﺮوﺗﻴﻦ اﻟﻤﻮﻋ ﻮد‬
‫ﺑﺎﻟﺘﻄﺒﻴﻖ ﻓﻰ ﻣﺠﺎل ﺻﻨﺎﻋﺔ اﻻﻏﺬﻳﺔ ﻓﺎﻟﺬوﺑﺎﻧﻴﺔ اﻟﻌﺎﻟﻴﺔ ﻟﻠﺒﺮوﺗﻴﻦ ﻣﻊ ﻧ ﺴﺒﺔ اﻟ ﺪهﻦ اﻟﻤﻨﺨﻔ ﻀﺔ ﺗﻠ ﻚ‬
‫اﻟﺼﻔﺎت ﻣﺮﻏﻮﺑﺔ ﻓﻰ ﺻﻨﺎﻋﺔ ﻣﻨﺘﺠﺎت اﻟﻠﺤﻮم واﻟﻤﺸﺮوﺑﺎت واﻏﺬﻳﺔ اﻻﻃﻔﺎل‪.‬‬
‫اﺻﻨﺎف اﻟﻌ ﺪس اﻟ ﺴﻮداﻧﻴﺔ ﺗ ﻢ ﺗﻨﺒﻴﺘﻬ ﺎ ﻟﻔﺘﺭﺍﺕ ﺯﻤﻨﻴﺔ ‪ 3‬ﻭ‪ 6‬ﺃﻴﺎﻡ‪ .‬ﺍﻟﺒﺫﻭﺭ ﺍﻟﻤﻨﺒﺘﺔ ﺠﻔﻔﺕ‬
‫ﻭﻁﺤﻨﺕ ﻭﺃﺨﻀﻌﺕ ﻻﺨﺘﺒﺎﺭﺍﺕ ﺍﻟﺘﺤﻠﻴل ﺍﻟﻜﻴﻤﻴﺎﺌﻲ ﻭﻤﺤﺘﻭﻱ ﺤﻤﺽ ﺍﻟﻔﺎﻴﺘﻴﻙ ﻭﻨـﺸﺎﻁ ﺃﻨـﺯﻴﻡ‬
‫ﺍﻟﻔﺎﻴﺘﻴﺯ ﻭﺍﻹﺘﺎﺤﺔ ﺍﻟﺤﻴﻭﻴﺔ ﻟﻸﻤﻼﺡ‪ .‬ﻭﺠﺩ ﺃﻨﻪ ﻓﻲ ﺃﺜﻨﺎﺀ ﻋﻤﻠﻴﺔ ﺍﻟﺘﻨﺒﻴﺕ ﺍﺭﺘﻔﻌﺕ ﻨـﺴﺒﺔ ﺍﻟـﺩﻫﻥ‬
‫ﻭﺍﻷﻟﻴﺎﻑ ﺒﻴﻨﻤﺎ ﺍﻨﺨﻔﻀﺕ ﻨﺴﺒﺔ ﺍﻟﻜﺎﺭﺒﻭﻫﻴﺩﺭﺍﺕ ﻭﺍﻟﻁﺎﻗﺔ ﻜﺫﻟﻙ ﺍﻨﺨﻔـﺽ ﺤﻤـﺽ ﺍﻟﻔﺎﻴﺘﻴـﻙ‬
‫ﻤﻌﻨﻭﻴﹰﺎ ﻤﻊ ﺯﻴﺎﺩﺓ ﻓﺘﺭﺓ ﺍﻟﺘﻨﺒﻴﺕ‪ .‬ﺃﺩﻱ ﺍﻟﺘﻨﺒﻴﺕ ﺇﻟﻲ ﺍﻨﺨﻔﺎﺽ ﻤﻌﻨﻭﻱ ﻓـﻲ ﺍﻟﻤﺤﺘـﻭﻱ ﺍﻟﻜﻠـﻲ‬
‫ﻟﺤﻤﺽ ﺍﻟﻔﻴﺘﻴﻙ ﺍﻟﺫﻱ ﺘﺯﺍﻤﻥ ﻤﻊ ﺯﻴﺎﺩﺓ ﻭﺍﻀﺤﺔ ﻓﻲ ﺍﻟﻔﺴﻔﻭﺭ ﺍﻟﻼﻓﻴﺘﻴﻜﻰ‪ .‬ﺍﻹﺘﺎﺤـﺔ ﺍﻟﺤﻴﻭﻴـﺔ‬
‫‪12‬‬
‫ﻷﻤﻼﺡ )ﺍﻟﻔﺴﻔﻭﺭ‪ ،‬ﺍﻟﻜﺎﻟﺴﻴﻭﻡ‪ ،‬ﺍﻟﺤﺩﻴﺩ‪ ،‬ﺍﻟﻤﺎﻏﻨﺯﻴﻭﻡ‪ ،‬ﺍﻟﻨﺤﺎﺱ ﻭﺍﻟﺯﻨﻙ( ﺍﺯﺩﺍﺩﺕ ﻁﺭﺩﻴﹰﺎ ﻤﻊ ﻓﺘﺭﺓ‬
‫ﺍﻟﺘﻨﺒﻴﺕ ﻤﺎ ﻋﺩﺍ ﺍﻟﺯﻨﻙ ﻭﺍﻟﻨﺤﺎﺱ‪ .‬ﺍﻟﺘﻨﺒﻴﺕ ﻟﻔﺘﺭﺓ ‪ 3‬ﺃﻴﺎﻡ ﺯﺍﺩ ﻤﻥ ﻨﺸﺎﻁ ﺃﻨـﺯﻴﻡ ﺍﻟﻔـﺎﻴﺘﻴﺯ ﻟﻜـل‬
‫ﺍﻷﺼﻨﺎﻑ ﺇﺫ ﺍﻨﻪ ﻜﺎﻥ ﻴﺘﺭﺍﻭﺡ ﻤﻥ‪ FTU/kg68.3-29.3‬ﻗﺒل ﺍﻟﺘﻨﺒﻴﺕ ﻟﻴﺼل ﺇﻟـﻰ‪132-82‬‬
‫‪ FTU/kg‬ﻓﻲ ﺍﻟﻌﻴﻨﺎﺕ‬
‫ﺯﻴﺎﺩﺓ ﻨﺸﺎﻁ ﺃﻨﺯﻴﻡ ﺍﻟﻔﺎﻴﺘﻴﺯ ﻓﻲ ﺍﻟﺼﻨﻑ ﺭﺒﺎﻁﺎﺏ ﻤﺎﺯﺍل ﻤﺭﺘﻔﻌﺎ ﻤﻊ‬
‫ﺯﻴﺎﺩﺓ ﻓﺘﺭﺓ ﺍﻹﻨﺒﺎﺕ ل ‪ 6‬ﺃﻴﺎﻡ ﺒﻴﻨﻤﺎ ﻓﻲ ﺍﻟﺼﻨﻔﻴﻥ ﻨﺩﻱ ﻭﺴﻠﻴﻡ ﻁﺭﺃ ﺍﻨﺨﻔﺎﺽ ﻁﻔﻴﻑ ﻓﻲ ﻨـﺸﺎﻁ‬
‫ﺇﻨﺯﻴﻡ ﺍﻟﻔﺎﻴﺘﻴﺯ ﺒﻌﺩ ﻭﺼﻭل ﺍﻟﺘﻨﺒﻴﺕ ﻟﻴﻭﻤﻪ ﺍﻟﺴﺎﺩﺱ‪.‬‬
‫ﻓ ﻰ دراﺳ ﺔ اﺛ ﺮ اﻟﺘﻨﺒﻴ ﺖ ﻋﻠ ﻰ ﻣﺤﺘ ﻮى اﻟﺤﻤ ﻮض اﻻﻣﻴﻨﻴ ﺔ دﻟ ﺖ اﻟﻨﺘ ﺎﺋﺞ ﻋﻠ ﻰ ان اﻟﺘﻨﺒﻴ ﺖ ﻳﺰﻳ ﺪ‬
‫ﺟﺰﺋﻴ ﺎ او آﻠﻴ ﺎ ﻣ ﻦ ﻣﺤﺘ ﻮى ا اﻟﺤﻤ ﻮض اﻻﺳﺎﺳ ﻴﻪ وﻏﻴ ﺮ اﻻﺳﺎﺳ ﻴﻪ ﺑﺎﺧﺘﻼﻓ ﺎت ﺑ ﺴﻴﻄﻪ ﺑ ﻴﻦ‬
‫اﻻﺻﻨﺎف ‪.‬اﻟﺼﻨﻒ ﺳﻠﻴﻢ ﻋﻨﺪ ﺗﻨﺒﻴﺘﻪ ﻟﻔﺘ ﺮة ‪3‬اﻳ ﺎم ارﺗﻔﻌ ﺖ ﻓﻴ ﻪ ﻧ ﺴﺒﺔ ا اﻟﺤﻤ ﻮض اﻻﺳﺎﺳ ﻴﻪ ﻋ ﺪا‬
‫اﻟﻤﺜﻴﻮﻧﻴﻦ وﻣﻊ زﻳﺎدة ﻓﺘﺮة اﻟﺘﻨﺒﻴﺖ ﺑﻌﺾ اﻟﺤﻤﻮض اﻻﻣﻴﻨﻴﺔ اﻧﺨﻔ ﻀﺖ ﻟ ﻮﺣﻆ ذﻟ ﻚ ﻓ ﻰ ﻣﺤﺘ ﻮى‬
‫اﻟﻬ ﺴﺘﺪﻳﻦ ‪،‬اﻟﻼﻳ ﺴﻴﻦ واﻻرﺟﻨ ﻴﻦ‪.‬ه ﺬﻩ اﻟﻨﺘ ﺎﺋﺞ ﻟﻮﺣﻈ ﺖ اﻳ ﻀﺎ ﻋﻠ ﻰ اﻟ ﺼﻨﻒ رﺑﺎﻃ ﺎب ﺣﻴ ﺚ‬
‫ارﺗﻔﻌﺖ ﻓﻴﻪ ﻧﺘﻴﺠﺔ ﻟﻠﺘﻨﺒﻴﺖ آﻞ اﻟﺤﻤﻮض اﻻﺳﺎﺳﻴﺔ ﻋﺪا اﻟﻤﺜﻴﻮﻧﻴﻦ واﻟﻼﻳ ﺴﻴﻦ ‪.‬ﻓ ﻰ اﻟ ﺼﻨﻒ ﻧ ﺪى‬
‫اﻟﺘﻨﺒﻴﺖ ﻟﻔﺘﺮة ‪ 3‬اﻳﺎم زاد ﻣﻦ ﻧﺴﺒﺔ اﻟﺤﻤﻮض اﻻﻣﻴﻨﻴﺔ اﻻﺳﺎﺳﻴﺔ وﻏﻴﺮ اﻻﺳﺎﺳ ﻴﺔ‪ .‬ﺑ ﺼﻮرة ﻋﺎﻣ ﺔ‬
‫ﻟ ﻮﺣﻆ ان اﻟﻌ ﺪس ﺑﺎﺻ ﻨﺎﻓﻪ اﻟﻤﺨﺘﻠﻔ ﺔ ﺿ ﻌﻴﻒ ﻓ ﻰ ﻣﺤﺘ ﻮاﻩ ﻣ ﻦ اﻟﺤﻤ ﻮض اﻻﻣﻴﻨﻴ ﺔ اﻟﻜﺒﺮﻳﺘﻴ ﺔ‪:‬‬
‫اﻟﻤﺜﻴﻮﻧﻴﻦ واﻟﺴﺴﺘﻴﻦ‪.‬‬
‫ﺗﻤﺖ دراﺳﺔ اﺛﺮ اﻟﻄﺒﺦ ﻋﻠﻰ ﻣﻌﺎﻣ ﻞ ه ﻀﻤﻴﺔ اﻟﺒ ﺮوﺗﻴﻦ وﺗﺠﺰﺋﺘ ﻪ وﺧﻮاﺻ ﻪ اﻟﻜﻬﺮﺑﻴ ﻪ‪ ،‬وﺟ ﺪ ان‬
‫اﻟﻄ ﺒﺦ ﻳﻘﻠ ﻞ ﻣﻌﻨﻮﻳ ﺎ ﻣ ﻦ ﻣﻌﺎﻣ ﻞ ه ﻀﻢ اﻟﺒ ﺮوﺗﻴﻦ ﺑﺎﺳ ﺘﺨﺪام اﻟﺒﺒ ﺴﻴﻦ و\او اﻟﺒﺒ ﺴﻴﻦ واﻟﺒﻨﻜﺮﻳ ﺎﺗﻴﻦ‬
‫ﻣﻌﺎ‪.‬اذ ان ﻣﻌﺎﻣﻞ هﻀﻢ اﻟﺒﺮوﺗﻴﻦ ﺑﺎﺳ ﺘﺨﺪام اﻟﺒﺒ ﺴﻴﻦ ﻳﺘ ﺮاوح ﺑ ﻴﻦ ‪ %44.6,52.1‬ﻟﻠﻌﻴﻨ ﺎت ﻏﻴ ﺮ‬
‫اﻟﻤﻄﺒﻮﺧ ﺔ وﺑﺎﺳ ﺘﺨﺪام اﻟﺒﺒ ﺴﻴﻦ واﻟﺒﻨﻜﺮﻳ ﺎﺗﻴﻦ آﺎﻧ ﺖ ﺗﺘ ﺮاوح ﺑ ﻴﻦ ‪ %81.8,99.9‬اﻟﻄ ﺒﺦ ﻗﻠ ﻞ‬
‫ﻣﻌﻨﻮﻳﺎ ﻣﻦ هﺬﻩ اﻟﻬﻀﻤﻴﺔ ﻟﺘﺼﻞ اﻟﻰ ﻣﺎ ﺑﻴﻦ‪ %19.7، 22.3‬ﺑﺎﺳﺘﺨﺪام اﻟﺒﺒﺴﻴﻦ و‪%88.2,77.1‬‬
‫ﺑﺎﺳﺘﺨﺪام اﻟﺒﺒﺴﻴﻦ واﻟﺒﻨﻜﺮﻳﺎﺗﻴﻦ‪ .‬اﻟﺒﺮوﺗﻴﻦ اﻟﺴﺎﺋﺪ ﻓﻰ اﻟﻌﺪس هﻮ اﻻﻟﺒﻴﻮﻣﻴﻦ ﻳﻠﻴﻪ اﻟﻘﻠﻮﺑﻴﻮﻟﻴﻦ وﺟﺪ‬
‫ان اﻟﻄﺒﺦ ﻳﻘﻠﻞ ﻣﻌﻨﻮﻳﺎ ﻣﻦ ﻣﺤﺘﻮى اﻻﻟﺒﻴ ﻮﻣﻴﻦ ه ﺬا اﻻﻧﺨﻔ ﺎض آ ﺎن ﻣ ﺼﺤﻮﺑﺎ ﺑﺰﻳ ﺎدة اﻟﻘﻠ ﻮﺗﻠﻴﻦ‪.‬‬
‫اﻟﺘﺤﻠﻴ ﻞ ﺑ ﺎﻟﻬﺠﺮة اﻟﻜﻬﺮﺑﻴ ﺔ ﻻﺟ ﺰاء اﻟﺒ ﺮوﺗﻴﻦ اﻟﻤﺘﺤ ﺼﻞ ﻋﻠﻴﻬ ﺎ ﻗﺒ ﻞ وﺑﻌ ﺪ اﻟﻄ ﺒﺦ دﻟ ﺖ ﻋﻠ ﻰ ان‬
‫اﻟﺒﺮوﺗﻴﻦ ﻳﺘﻐﻴﺮ آﻤﺎ وﻧﻮﻋﺎ ﻧﺘﻴﺠ ﺔ ﻟﻠﻄ ﺒﺦ ﺣﻴ ﺚ ان ﻋ ﺪد ﺗﺤ ﺖ اﻟﻮﺣ ﺪات ﻟﺒ ﺮوﺗﻴﻦ اﻟﻌ ﺪس اﻟﻜﻠ ﻰ‬
‫ﻗﺒ ﻞ اﻟﻄ ﺒﺦ آﺎﻧ ﺖ ﺗﺘ ﺮاوح ﻣ ﻦ‪ 17‬اﻟ ﻲ ‪ 19‬وﺣ ﺪة وﺑﻌ ﺪ اﻟﻄ ﺒﺦ اﻧﺨﻔ ﻀﺖ ﻟﺘ ﺼﻞ اﻟ ﻰ ﻣ ﺎﺑﻴﻦ‬
‫‪13‬‬
‫‪13,16‬وﺣﺪة ووﺟﺪ ان اﺛﺮ اﻟﻄﺒﺦ آﺎن واﺿﺤﺎ ﻋﻠﻰ ﺑﺮوﺗﻴﻨﺎت اﻟﺒﺮوﻻﻣﻴﻦ ﺣﻴ ﺚ اﻧﺨﻔ ﺾ ﻋ ﺪد‬
‫ﺗﺤﺖ اﻟﻮﺣﺪات ﻣﻦ ‪4‬اﻟﻰ‪ 2‬وﺣﺪةﻧﺘﻴﺠﺔ ﻟﻠﻄﺒﺦ ﺑﺎوزان ﺟﺰﻳﺌﻴﺔ ﻋﺎﻟﻴﻪ ﺗﺼﻞ اﻟﻰ‪56‬آﻴﻠﻮداﻟﺘﻮن‪.‬‬
‫‪14‬‬
CHAPTER ONE
INTRODUCTION
Food legumes, distinctively termed "poor man's meat", appear
to be the most potential solution to overcome the crisis of proteincalorie malnutrition in the less developed countries. The term implies
a meat substitute associated with poverty. Being the case in poor
societies in addition to population pressure, it is more suitable to
supply protein directly from plants to humans without the energyintensive conversion of plant protein into animal proteins in the food
chain.
Lentils (Lens culinaris Medic) were among the earliest
cultivated for crops. L. orientalis is thought to be the progenitor of
cultivated L. culinaris (Zahary, 1972). It was domesticated in the
eastern Mediterranean region.
Lentil is probably the oldest grain legumes to be domesticated
(Bahl et al., 1993). It is now cultivated in most subtropical and also in
the northern hemisphere such as Canada and Pacific Northwest
regions.
In Sudan, lentils are incorporated into many dishes, e.g. tamia,
soup, stew. A survey in the urban area of Khartoum (1982 – 1983)
showed that lentils were the main substitute for faba beans and the per
capita consumption was 0.41 kg/ month (Ali et al. 1984).
Legumes such as lentil contain a high concentration of proteins,
carbohydrates and dietary fibre and make an important contribution to
human diet in many countries. Lentil is a nutritious food legume
cultivated for its seeds which have a relatively higher content of
protein and calories compared to other legume.
15
Lentil is a protein calorie crop, its protein content amounting to
22 – 35%. Lentil is deficient in the amino acid methionine and cystine,
it is an excellent supplement to cereal grain diet (Oplinger et al.,
1990).
About 90% of lentil protein is found in the cotyledons with
albumins and globulins being the major fractions. Digestibility
coefficients for lentil are relatively high and ranged from 78 – 93%,
while biological values range from 32 – 58% (Hulse, 1990).
Legumes have to be processed prior to consumption due to their
content of anti-nutritional factors such as trypsin inhibitor, phytic acid
and galactosides (Vidal-Valverde et al., 2002).
Plant protein products are gaining interest as ingredients in food
systems throughout many parts of the world; the final success of
utilizing plant protein additives depends greatly upon the favorable
characteristics that may impart to foods. Therefore, the relationship of
protein quality with processing parameters that affect the functional
performance of protein products is worthy of extensive investigation
(Wang and Kinsella, 1976).
Objectives of this study:
The aims of this study were:
1.
To quantify (isolation of lentil protein) and qualitatively (the
functional properties of lentil protein in formulated foods).
2.
The effect of germination on mineral availability and amino
acid content through activation of endogenous phytase.
3.
To study the effect of cooking on the in vitro protein
digestibility and the characterization of lentil proteins.
16
CHAPTER TWO
LITERATURE REVIEW
17
2.1 The chemical composition of lentil:
2.1.1 Crude protein content:
Legumes have been considered as leading candidates in protein
supply to the malnourished areas of the world. The seeds contain 2 to
3 times more proteins than cereals on a whole seed basis; lentil crude
protein is comparable with faba bean and is higher than chick pea, but
lower than soybean and lupin. However, the combination of legumes
and cereals provides a good balance of amino acids (Osman, 1990).
When a world collection of 1688 accessions of lentil was tested for
protein content, a variability of 23.4 – 36.4% was found (Hawtin
et al., 1977). Seed protein content varies considerably among varieties
and plant breeders could utilize this information to produce cultivars
yielding high protein content (Savage, 1988). On the other hand,
Krober et al. (1970) in their survey of the protein content of lentils
grown in widely differing locations in India showed that there were
significant differences in the crude protein of lentils grown in different
locations. Lentil protein was found to be deficient in methionine,
cystine and tryptophan (Bhatty and Slinkard, 1979).
2.1.2 Lipids:
Glycerol esters of fatty acids which make up to 99% of the
lipids of plant and animal origin (Nawar, 1996), have been
traditionally called oils and fats. Lipids in food exhibit unique
physical and chemical properties that are important to their functional
properties. In addition to their role as vitamin carriers, dietary lipids
frequently provide a significant proportion of calories. Dietary lipids
supply essential fatty acids (Sinclaire, 1961). Hundred grams of dried
lentil seeds contain 0.6 gram fat (Adsule et al., 1989; Muehlbauer
18
et al., 1985). Hulse (1990) reported that decorticated lentil seeds
contain 1.8 gram fat/100 gram decorticated seeds.
2.1.3 Fibre and ash:
Dietary fibre is considered to be plant-cell skeletal remains that
are resistant to digestion. While the term crude fibre is defined as the
washed, dried organic residue remaining after acid and alkaline
hydrolysis of a defatted material. It is composed largely of cellulose
(50 – 80%), hemicellulose
(~20%) and 10 – 50% of lignin. Fibres become of considerable
interest in human nutrition because of some beneficial attributes,
along their adverse effects (Jansen, 1980). Lentil decorticated seeds
were found to contain crude fibre of value of 0.9 g per one hundred
grams as reported by Huisman and Vanderpoel (1994); Hulse (1990).
Ash is a residue remains after ignition of organic matter, is used
as starting point for determination of elemental composition of food
material. Minerals are recognized to perform essential functions in
human nutrition. They are required by human body for good health
and growth. Lentils were found to contain 3.7 g ash per one hundred
grams of decorticated lentil seeds (Huisman and Vanderpoel, 1994;
Hulse, 1990).
2.1.4 Total carbohydrates:
Carbohydrates are the chief source of energy by providing 70 –
80% of the calories in the human diet (BeMiller and Whistler, 1996).
Most of the natural carbohydrates are in the form of oligomers or
polymers of simple and modified sugars. The simple sugars
(monosaccharide) are recognized as poly hydroxy aldehydes or
19
ketones or which yield them on hydrolysis (Maynard and Loosli,
1962). However, most of the monosaccharides and some of the
oligosaccharides are water-soluble. Hence, they constitute the percent
soluble carbohydrates fraction.
Adsule et al. (1989) and Muehlbauer et al. (1985) reported that
100 gram of dried lentil seeds contain 65.0 gram total carbohydrates.
Husle (1990) reported that 100 grams of decorticated lentil seed
contain 58.89 gram total carbohydrates.
2.2 Functional properties:
Functionality of food proteins defined as those physical and
chemical properties, which affect the behavior of proteins in food
systems during processing, storage, preparation and consumption
(Fennema, 1996). A functional property is any non-nutritional
property of a food or food additive that affects its utilization (Rhee,
1985). The functional properties of proteins, however, are found to be
influence by and vary according to the source of protein, method of
preparation, concentration, environmental conditions (i.e. temperature,
pH and ionic strength) and modification i.e. physical, chemical or
enzymatic (Kinsella, 1979).
The range of desirable and attractive functional properties that
should be looked for is almost as abroad as the range of foods
themselves. For example, if one is considering producing a beverage,
two desirable functional properties should be considered solubility and
suitable viscosity. For bread, the need is for a protein that is
compatible with gluten. For various meat systems, desirable qualities
would include water-binding emulsifying properties and the ability to
be formed into fibres. For other purposes the properties of gel
20
formation, whippability adhesiveness and thickening might be
considered beneficial (Mattil, 1971).
2.2.1 Protein solubility:
The functional properties of proteins are often affected by
protein solubility and those most affected are foaming, emulsification
and gelation. The solubility of a protein is the thermodynamic
manifestation of the equilibrium between protein-protein and protein
solvent interactions (Damodaran, 1996). Solubility characteristics,
under various conditions, are very useful in selecting optimum
conditions for extracting proteins from their natural sources (Wang
and Kinsella, 1976). In addition, solubility behaviour provides a good
index of the potential applications of protein in food system. It imparts
information useful in the optimization of processing conditions as well
as in determining the effect of heat treatments which might affect
potential application (Kinsella, 1976). The major interactions that
influence the solubility characteristics of proteins are hydrophobic and
ionic in nature. Hydrophobic interactions promote protein-protein
interactions and result in decreased solubility, whereas ionic
interactions promote protein-water interactions and result in increased
solubility (Fennema, 1996). Nitrogen solubility is influenced by
several solution conditions, such as pH, ionic strength, temperature
and the presence of organic solvents (Fennema, 1996).
2.2.2 Effect of pH on nitrogen solubility:
Nitrogen solubility profiles over a range pH values are being
used increasingly as a guide for protein functionality, since this relates
directly to many important properties. Most food proteins have a Ushaped pH-solubility curve and due to the fact that most food proteins
21
are acidic in nature, they exhibited minimum solubility of pH 4 – 5
(i.e. near their isoelectric pH's). The high net charge acquired at both
acid and alkaline pH's caused arise in solubility due to unfolding of
protein molecules, with the degree of unfolding being greater at
alkaline than the acid pH (Damodaran, 1996). Fan and Sosulski (1974)
reported minimum extractability of nitrogenous constituents from
mung bean flour to be found in the range of 4 – 5. Furthermore, Hang
et al. (1970) concluded that several bean proteins, namely, mung bean,
and kidney bean, had a common point of minimum dispersion at pH 4.
The low protein extractabilities at pH values of 4 – 6 were essentially
attributed to the intermolecular attraction of protein molecules in the
isoelectric zone (Molina et al., 1974). Hsu et al. (1982) reported that
the lowest solubility of the protein from yellow pea, lentils and faba
bean were at pH 4.5 – 5.
2.2.3 Water absorption capacity:
Water retention is defined as the ability of the food material to
hold water against gravity (Hansen, 1978). In food applications, water
holding capacity of a protein preparation is more important than water
binding capacity. Water absorption capacity refers to the ability of the
protein to imbibe water and retain it against gravitational force within
a protein matrix such as protein gels or beef and fish muscle. The
WAC is the sum of bound, hydrodynamic and physically entrapped
water (BeMiller and Whistles, 1996). Water retention has been used as
a criteria for selection of protein additives for food systems especially
meat products (Lin and Zayas, 1987). The ability of protein to entrap
water is associated with juiciness and tenderness of comminuted meat
22
products and desirable textural properties of bakery and other gel-type
products (Fennema, 1996).
2.2.4 Fat absorption capacity:
Oil absorption is mainly attributed to the physical entrapment,
of oil and is related to the number of nonpolar side chains on proteins
that bind hydrocarbon chain of fats (Kinsella, 1979 and Lin et al.,
1974). The ability of proteins to bind fat is an important functional
property for food applications such as meat replacers and extenders,
principally because it enhances flavour retention and improves mouth
feel (Kinsella, 1976). Hutton and Campbell (1981) have explained the
role of protein-lipid interactions, in the absorption capacity of protein.
They suggested that, noncovalent bonds, such as hydrophobic,
electrostatic and hydrogen bonding significantly contribute in
stabilization of protein-lipid complexes. Contrast to its situation in the
aqueous media, hydrogen bonding was found to be of a secondary
importance
in
lipoprotein
complexes
(Karel
et
al.,
1975).
Hydrophobic bonding is likely to play a major role in stabilizing the
interactions of both polar and a polar lipids with proteins (Ryan,
1977). Therefore surface hydrophobicity was suggested to be the
major determinant of fat binding capacity (Voutsinas and Nakai,
1983).
2.2.5 Bulk Density (BD):
Bulk density depends on interrelated factors including intensity
of attractive, interparticle forces, particle size, number of contact
points (Peleg and Bagley, 1983). It also depends on type of solvent
23
used to extract the protein products (Wang and Kinsella, 1976) and on
method of drying (Bryant, 1988). However, low BD is a desirable
property when powdered food materials are to be packed in a limited
space. In addition, high BD also finds use where they can be
incorporated into light snack food.
2.2.6 Foaming properties:
The foaming property of a protein refers to its ability to form a
thin tenacious film at gas-liquid interfaces so that large quantities of
gas bubbles can be incorporated and stabilized (Fennema, 1996). The
factors affecting foam formation and stability are environmental
factors (pH, sugars, lipids and protein concentration). McWatters and
Cherry (1977) observed that protein solubility was more closely
related to the type of foam produced than the increase in volume.
Soybean and peanut flour suspensions contained higher levels of
soluble protein than those of field pea and pecan forming foams of
much thicker consistency and small air cells.
2.2.6.1 Foaming capacity:
The foaming capacity of a protein refers to the amount of
interfacial area that can be created by the protein (Fennema, 1996).
2.2.6.2 Foaming stability:
Foam stability refers to the ability of protein to stabilize foam
against gravitational and mechanical stresses (Fennema, 1996).
Foam stability is important since the usefulness of whipping
agents depends on their ability to maintain the whip as long as
possible (Lin et al., 1974).
24
2.2.7 Emulsification properties:
Emulsion is a dispersed system consisting of two immiscible or
sparingly soluble liquids, termed the oil phase and the water phase,
separated by a third component termed an emulsifier which may be
solid. The interphase between the two phases is very large and its
integrity is critical to the stability of the whole system (Friberg and
Venable, 1983). Emulsification properties play a significant role in
many food systems including meat products, batters and dough and
salad dressing (Betschart et al., 1979). The emulsifying properties of a
protein depend on two phenomena (i) the ability to reduce interfacial
tension because of its adsorption to the interface and (ii) the ability to
foam a film which would act as an electrostatic structural and
mechanical barriers. Emulsion formation relies on a fast adsorption
unfolding and reorientation, while stability is determined by the
interface free energy and by the film rheological properties
(Damodaran, 1996).
2.2.7.1 Emulsion capacity (EC):
Emulsion capacity is the volume (ml) of oil that can be
emulsified per gram of protein before phase inversion occurs
(Fennema, 1996). The EC of protein is affected by several factors
such as type of protein, pH, salts, sugars and presence of low
molecular weight surfactants (McWatters and Holmes, 1979).
2.2.7.2 Emulsion stability (ES):
The ES, which reflects the ability of proteins to impart strength
to emulsion for resistance to stresses (Patel and Kilara, 1990), is
commonly measured in terms of oil and/or cream separating from an
25
emulsion during a certain period of time at stated temperature and
gravitational field (Acton and Saffle, 1970). The time required for
specified degree of breakdown to occur.
2.2.8 Gelation:
Interactions between protein molecules when induced by
heating may give rise to protein aggregates. When such heat-induced
aggregation takes place at a protein concentration high enough to
entrap water in the three-dimensional matrices of the aggregates,
gelation or coagulation will result (Kim et al., 1990). Gelation may be
defined as a protein aggregation in which polymer-polymer or
polymer solvent interactions as well as attractive and repulsive forces
are so balanced that a tertiary network or matrix is formed. Such a
matrix is capable of immobilizing or trapping large amount of water
(Schmidt, 1981). Factors affecting protein gelation include: method of
protein preparation, it's concentration, rate and duration of heating,
cooling conditions and the presence of salts, lipids, thiol and sulfites.
2.2.9 Dispersibility:
The dispersibility of a mixture in water indicates it's reconstitutability. The higher dispersibility is the better (Kulkarni et al., 1991).
The major factors affecting dispersibility are temperature, ionic
composition, pH and degree of agitation of the solvent. Soy protein
isolate and casein have good water dispersion characteristics at neutral
pH (6.5 – 7) (Johnson, 1970). Dispersibility of sesame protein isolate
was significantly higher at neutral and alkaline pH than acidic pH
(Khalid et al., 2003). Higher dispersibility enhances the emulsifying
and foaming properties of proteins, which was observed during
making of bread, macaroni and cookies (Kinsella, 1979).
26
2.3 Phytase:
Phytase (meso-inositol hexaphosphate phosphohydrolase, EC
3.1.3.8) is widely distributed in plants, animal and fungi (Cosgrove,
1980). Phytase dephosphorylates free inositol phosphates. The phytase
acts on inositol hexaphosphate to yield inositol and orthophosphate,
via inositol penta – to monophosphate as intermediary products. Plant
seeds are rich in phytate and both phytate and phytase are present in
most plant seeds. Phytase activity usually increases on germination
(Peers, 1953; Mayer, 1958; Kuvaeva and Kretovich, 1978). The mode
of action of phytase has remained controversial. Maiti et al. (1974)
reported that degradation of phytate by phytase occurs in a stepwise
manner starting with dephosphorylation from position 6 followed by
removal of phosphorus from position 5 and 4, 1 and 3, or 1 and 4; the
phosphate at position 2 being stable.
2.3.1 Phytates:
Occurrence:
Phytates constitute about 1 to 2% by weight of many cereals
and oilseeds, although amounts as high as 3 to 6% have been reported
for some particular varieties (Cheryan, 1980). An analysis of over 20
varieties of soybean showed phytic acid contents of 0.72 to 2.18% by
weight of defatted meal (Anon, 1984) and 1 to1.47% by dry weight of
15 varieties of soybean seeds (Lolas and Markakis, 1977). It has long
been recognized that phytic acid and its salts were one of the major
forms of phosphorus in many plant seeds (Posternak, 1903 and
Webster, 1928). Typically about 60 to 90% of all the phosphorus in
these seeds is present as phytic phosphorus.
27
O'Dell et al. (1972) found that 90% of the phytate in corn was
concentrated in the germ. In the case of wheat and rice, most of the
phytate was in the outer layers, the pericarp and aleurone. In other
oilseeds, such as peanuts (Dieckert et al., 1962), cotton seeds (Lui and
Altschul, 1967) and sunflower (Saio et al., 1977). Phytate appears to
be concentrated with crystalloid-type substructures or globoids, which
might serve as storage sites.
Phytate-degrading enzymes (phytases) catalyze hydrolysis of
phytate (myo-inositol hexa phosphate, 1P6), the major storage form of
phosphorus in plant kingdom, phytase belong to special group of
phosphatases, which are capable of hydrolyzing phytate to a series of
lower phosphate esters of myo-inositol and phosphate (Frias et al.,
2003).
2.3.2 Formation of phytic acid:
The ripening process of plant is characterized by the
accumulation of substances in the seeds. The important mineral
nutrient phosphorus is no exception to this accumulation process. The
active transport of phosphorus to seeds from leaves and roots is an
important part of the ripening. In the rice plant, at the end of the
ripening about 60% of the phosphorus in the whole plant is found in
the seeds (Kasai and Asada, 1959). Most of the phosphorus thus
transported in the seeds exists in the form of phytic acid, inositol
hexaphosphate. It has been generally supposed that phytic acid
functions as storage form of phosphorus and is utilized as a source of
phosphorus at germination. The occurrence of transphosphorylation
between phtyic acid and adenosine diphosphate has been suggested
(Morton and Raison, 1963). Further the occurrence of an enzyme
28
catalyzing the transphosphorylation between phytic acid and
guanosine diphosphate was established in mung bean (Biswas and
Biswas, 1965). The phosphorylation of inositol in the formation of
phytic acid does not occur in a sequential fashion through phosphorrylated inositol, but occurs through a hypothetical phosphorylated
inositol derivative (Asada et al., 1969).
2.4 Germination:
Seed germination is a primary step to generate a new plant.
During germination, a series of active and complex biochemical and
physiological reactions are taking place, which results in extensive
changes in composition and/or morphology. Intensive investigations
on compositional changes of plant seed during germination are
important because of the necessity of understanding the compositional
changes and relevant functions from the view point of plant science.
When the seeds are destined for food use, an understanding of the
compositional changes resulting from germination in relation to food
quality is also important (Chioce et al., 1997).
In Egypt, it is common to germinate some legume seeds which
are rich in protein (20- 50%) such as termes (Lupin termes), broad
bean (Vicia faba) and chick pea (Cicer arietinum), before direct
eating, cooking or used in salad dressing. Germination improves the
nutritional value of proteins which are hydrolyzed into easily
assimilable polypeptides and essential amino acids and decreases
trypsin inhibitor (Ahmed et al., 1995).
2.4.1 Effect of germination on the chemical composition and other
nutrients:
29
Legume seeds have made a significant contribution to the
human diet since ancient times. They are a good and inexpensive
source of dietary proteins, carbohydrates, vitamins and minerals.
However legumes contain large amount of anti-nutritional factors (e.g.
trypsin inhibitors, alpha-galactasides inositol phosphates) in the raw
seeds this need to be reduced by processing before consumption
(Augustin and Klein, 1989).
Germination of legume seeds for human consumption has been
a common practice in the orient for centuries and appears to be a
simple and effective processing method for achieving desirable
changes in nutritional quality. At present, germinated legumes are
becoming an increasing proportion of the total consumption of food
legumes in the world (Ghorpade and Kadam, 1989), and they are also
used to produce flours of high nutritional value (Doughty and Walker,
1982).
Many species including legumes are now offered in the markets
and in health food shops. These including alfalfa (Medicago sativa
L.), lentil (Lens culinaris M.), mung bean (Vigna radiate L.), soy bean
(Glycine max L.),Merril, pea (Pisum sativum L.), adzuki bean (Vigna
amngularis Ohwi et Ohashi), pinto bean (Phaseolus vulgaris cv pinto)
and chick pea (Cicer arietinum L.).
Germination causes important changes in the biochemical,
nutritional and sensory characteristics of legume seeds. Extensive
breakdown of seed storage compounds and synthesis of structural
proteins and other cell components takes place during this process.
Fats and carbohydrates, that are often at surplus levels in western diets
are broken down while dietary fibre, that is mostly at a sub-optimal
30
level, increase. Vitamins and secondary compounds, many of which
are considered beneficial as antioxidants, often are altered,
dramatically during germination. Phytic acid and dietary fibre both
affects the uptake of micro-nutrient in the digestive tract and these
compounds change differently during the germination process. Other
anti-nutrient factors, such as the flatulence – producing α
galactosidase, trypsin and chymotrypsin inhibitors, which affect the
digestion of proteins are also reduced after germination (Frias et al.
1995; Vidal-Valverde et al., 1994).
2.4.2 Effect of germination in phytase activity:
A rapid increase of phytase activity was monitored in plant
seeds during their germination (Chen and Pan, 1979). Generally, it is
assumed that during seed germination phytate, after decomposition by
phytase, is utilized in the form of phosphate and inositol (Asada et al.,
1969). Some authors described the rise in phytase activity to
denovophytase synthesis during germination (Satirana and Bianchetti,
1967; Mandal and Biswas, 1970; Meyer et al., 1971), while other
attributed it to arise of an already existing phytase activity (Eastwood
and Laidman, 1971). Nevertheless, it was concluded that seeds contain
both constitutive and germination-inducible phytase (Nayini and
Markakis, 1986). Germination is the most effective process for the
reduction of phytic acid in legumes. These losses may be attributed to
the activity of the enzyme phytase. Phytic acid serves as important
reserve of phosphorus generated by the action of phytase during seed
germination in developing seedling (Vidal-Valverde et al., 1994).
2.5 Interaction of phytic acid with minerals:
31
Phytic acid occurs naturally in many foods derived from plants,
principally in cereals and legumes, and it is considered to be an
important reserve material in the germination and growth of plants
(Cosgrove, 1980). Six phosphate groups in the molecule of IP6 make
a strong chelating agent, which binds minerals such as Ca+2, Mg+2,
Fe+3 and Zn+2. Under gastrointestinal pH conditions, insoluble metal,
phytate complex are formed (Gifford and Clydesdale, 1990; Plate and
Clydesdale, 1987) which make the metal unavailable for absorption
from the intestinal trace of animals and humans (Kratzer and Vohra,
(1986; Nelson, et al., 1989).
Because of its high density of negatively charged phosphate
groups, phytate forms mixed salts with mineral cations which are
assumed to play an important role in mineral storage. These salts
called phytins contain predominantly K and Mg, whereas other metals
such as Ca, Zn, Fe or Cu are found in much smaller amounts (Lopez
et al., 2002). In many plants species, 90% of the phytin is localized in
the aleurone layer and only 10% in the embryo. In contrast, in maize,
most of the phytin is found in the germ and only small fraction is
found in the aleurone (O'Dell et al., 1972).
2.5.1 Zinc and phytic acid:
Zinc is an essential trace element involved in the immune
function, in the activation of many enzymes and in the growth (Lopez
et al., 2002).
32
The availability of Zn for intestinal absorption and body
utilization is the net effect of absorption inhibiting and promoting
components of the diet. The amount of phytic acid, the type and
amount of protein and the total Zn content have a major impact on the
amount of Zn absorbed from foods.
Phytic acid is the major determinant of Zn absorption,
especially for diets with low animal protein content. Phytic acid
strongly binds Zn in the gastrointestinal tract and reduces its
availability for absorption and reabsorption (Flanagan, 1984).
2.5.2 Iron and phytic acid:
The single most prevalent deficiency on a worldwide scale is
iron (Fe) deficiency anemia, affecting an estimated 30% of the world's
population (Lopez et al., 2002).
Sandberg and Svanberg (1991) reported that phytic acid
decrease Fe solubility and the inhibition of Fe absorption is closely
related to the content of phytate in bread (Brune et al., 1992). The
inhibitory effects of phytic acid on Fe can be counteracted by Fe
absorption enhancers such as protein (Reddy et al., 1996) or organic
acids (Gilboly et al., 1983). Ascorbic acid is the most effective
enhancer of non-haem Fe absorption.
2.5.3 Calcium and phytic acid:
In humans, phytic acid decrease Ca absorption (Reinhold et al.,
1976) and the phytic acid breaks down improves Ca availability.
Although the literature has frequently reported an inhibitory effect of
phytic acid on Ca absorption (Lönnerdal et al., 1989; Rimbach et al.,
33
1995), some researchers failed to observe any negative effect of phytic
acid on Ca absorption on rat model (Miyazawa et al., 1996; Nickel
et al., 1997). The reason for the decreased phytic acid degradation
caused by Ca may be the formation of insoluble Ca phytate complexes
which are poor substrate for phytase (Sandberg et al., 1993).
2.5.4 Magnesium and phytic acid:
Plants rich in phytic acid (such as whole cereal products,
legumes or oilseeds) contain large amounts of Mg. Unrefined foods
remain the major sources of Mg. Less purified products such as whole
wheat flour are rich in phytic acid but they provide five fold more Mg
than white wheat flour. Thus, in comparison with white wheat flour,
the consumption of whole wheat flour can contribute to improve Mg
balance in rats (Levrat-Verny et al., 1999).
2.5.5 Copper and phytic acid:
Relationship between copper availability and phytic acid is still
confusing. Some authors claim that, phytic acid had no effect on Cu
absorption in men (Turnlund et al., 1985). The effect of phytic acid on
Cu absorption seems to be modulated by several factors, especially the
Zn level in the diet (Lopez et al., 2002). Phytic acid can indeed
enhance Cu absorption because of its ability to bind Zn, thus
counteracting its capacity to compete with Ca at the intestinal
absorption sites (Champagne and Hinojosa, 1987).
2.5.6 Binding of phytic acid to proteins:
The strong phytate-protein interaction at acidic pH is the reason
why protein isolates prepared by isoelectric precipitation may contain
as much as 60 to 70% of the original phytic acid of the raw soy bean
34
(Churella, 1976; Okubo etal ,1975; Omosaiye and Cheryan, 1979). At
alkaline pH, the nature of the protein-phytate interaction is some what
uncertain, although above the isoelectric point of pH5 both proteins
and phytic acid have a net negative charge (Cheryan, 1980).
2.6 Amino acids content:
Plant protein represents the primary source of food protein for
humans and animals. More than two third of these primary food
proteins come from cereal and legume seeds. Together with proteins,
legume seeds provide a high proportion of carbohydrates, and fibres.
With reference to the recommended daily allowance of human as well
as of monogastric domestic animals and poultry, the amino acid
composition of legumes is unbalanced, 80% of their proteins being
specific (Barasi, 1997). Protein quality is affected by essential amino
acid composition, amino acid imbalance, digestibility and biological
availability of the amino acids, and by the anti-nutritional activity of
some components of the seeds (Deshpande and Domodaran, 1990). In
general, legumes are rich in lysine, but deficient in sulphur containing
amino acids (methionine and cystine). However, with small increase
in one of these two amino acids, tryptophan would become the next
limiting amino acid in legume seeds. The amino acid composition of
plant proteins is related to their genetic characteristics as reported by
Mosse and Baudet (1983). Sotelo et al. (1995) found more crude
protein in wild species of beans than in cultivated ones, but the
content of essential amino acids and the chemical score were higher in
cultivated beans. For lentil it is known from the literature that they
contain a number of unidentified amino acids (Bhatty, 1988). The
35
concentration of unidentified amino acids is greater in cultivated
lentils than in wild lentils (Bhatty, 1986).
2.7 In vitro protein digestibility:
The digestibility of legume protein is dependent on protein
structure (Deshpande and Damodaran, 1989) and the presence of antinutritional factors such as trypsin inhibitor, phytates and tannins
(Nielson, 1984). Various processing and cooking methods could
improve the protein and starch digestibility of legume seed by
decreasing the level of some anti-nutrients (Kataria and Chauhan,
1988; Bishnoi and Khetarpaul, 1994). Moreover, an increase in
digestibility after thermal treatments may be attributed to some other
factors such as disruption of protein structures and cell wall
encapsulated starch, starch gelatinization and physical disintegration
of the legume seeds (Tovar et al., 1991).
Low nutritional value of legume proteins has long been ascribed
to both the presence of a limiting amount of essential amino acids
methionine and cysteine and the poor digestibility of proteins (Evans
and Bauer, 1978; Sarwar and Peace, 1986).
The biological utilization of a protein is primarily dependent on
its digestibility by proteases. Heat treatment may alter the protein
structure leading to changes in the digestibility. An in vitro method
was used to assay digestibility. In comparison with in vivo methods,
these are reliable, rapid, simple and could be used commercially for
monitoring protein quality (Swaisgood and Catignani, 1991).
2.7.1 Effect of cooking on protein digestibility:
Heating is responsible for protein denaturation, eventually
followed by aggregation of the unfolded molecules which results in
36
loss in solubility. The mechanism of thermal aggregation of the
algometric storage proteins, the main components of legume seeds has
been investigated by several techniques in model system with isolated
11S and for 7S globulins. Thermal denaturation involves an initial
stepwise dissociation of subunits and subsequent reassociation of only
partially unfolded molecules with formation of either soluble or
insoluble complexes (Kinsella et al., 1985).
The cooking process affects in vitro protein digestibility (IVPD)
legume seed. Kataria et al. (1989) reported that IVPD was higher
when soaked seeds of mung beans were cooked than when unsoaked.
Bressani (1984) using Phaseolus vulgaris observed that increased
cooking time reduced the IVPD of the whole and the hulled seeds.
Arbab and El Tinay (1997) reported that cooked sorghum has less
IVPD than the uncooked control. Fageer and El Tinay (2004) reported
that after cooking, pepsin digestibility significantly decreased in corn.
Reduction in protein digestibility was reported by Yousif (2000) and
was attributed to formation of disulphide bonding resulting in the
folding of the protein molecule and hence decreasing its susceptibility
to digestive enzymes. The negative effect of cooking on the IVPD
observed by Abdel Rahim (2004) in faba bean.
2.8 Protein fractions:
Based on solubility behavior in various solvents, proteins have
been classified into fractions (Osborne and Mendel, 1914). Therefore,
four main protein fractions are well known, these are (i) albumins: are
those soluble in water at pH 6.6, (ii) globulins which are soluble in
dilute salt solutions at pH 7, (iii) glutelins, are those soluble only in
acid (pH2) and alkaline (pH 12) solutions, and prolamines are those
37
soluble in 70% alcohol (Damodaran, 1996). A small amount of protein
remain insoluble after all these extraction steps, that found to consist
many of proteins from previously defined groups becoming insoluble
due to interaction with lipids, carbohydrates or polyphenols via
oxidation processes (Sodek and Wilson, 1971).
38
CHAPTER THREE
MATERIALS AND METHODS
3.1 Materials:
Three Sudanese lentil cultivars (Nadi, Selaim and Rubatab)
used in this study were obtained form Elhudaiba Research Station, the
fourth cultivars (decorticated) which is Indian was obtained from
Khartoum North market. These samples were carefully cleaned and
freed from foreign materials. Sudanese cultivars were decorticated to
remove hulls by using laboratory dehuller [TADD model 4E -220
(ANADA)] for different times depending on thickness of the hull
(Selaim and Rubatab for 1.5 minutes, Nadi for 2 minutes), then they
were ground to pass 0.4 mm screen by using a laboratory milling
machine (Type 120 No. 69444 – FINLAND).
For cooking, the flour was suspended in water (1:10 w/v) and
boiled in a boiling water bath for 20 min., the cooked gruel was then
dried at 65oC and reground to pass through 0.4 mm screen.
For germination a bulk of healthy and clean seeds (Nadi, Selaim
and Rubatab) each cultivar bulk was divided into three equal portions,
the first portion was reserved as a control (ungerminated seeds), the
second portion was allowed to germinate for 3 days and the third
portion was allowed to germinate for 6 days. Prior germination, seeds
were soaked at room temperature (30oC) in distilled water for 2 hours.
Germination was carried out in sterile Petri dishes lined with wet filter
paper for 3 and 6 days at 4oC. At the end of respective germination
period samples were dried at room temperature, ground to pass a 0.4
mm screen.
39
Preparation of lentil protein:
The flour was defatted and desolvenilized in an open air. The
protein of defatted flour was extracted in an alkaline medium as
follows: a sample of defatted flour (100g) was extracted once with
0.01 M phosphate, buffer pH8 containing 10 mM 2-mercaptoethanol
(2-ME) at 25oC for 2 hours using a rotary shaker, then centrifuged at
8000 x g for 20 min. and the supernatant was acidified to pH5 with 2N
(HCl) and then centrifuged at 8000 x g for 20 minute, the precipitate
was allowed to dry at room temperature for 5 hours then milled to
pass 9.04 mm screen.
3.2. Methods of analysis:
3.2.1 Proximate analysis:
3.2.1.1 Moisture content:
Moisture content was determined according to the AOAC
(1990) as follows:
Two grams of sample were weighed using a sensitive balance in
clean dry and pre-weighed crucible and then placed in an oven at
105oC and left overnight. The crucible was transferred to a desiccators
and allowed to cool and then weighed. Further placements in the oven
were carried out until approximately constant weight was obtained.
Moisture content was calculated using the following formula:
MC (%) =
Where
MC=
W1 =
W2 =
W3 =
(W2 – W1) – (W3 – W1)
W2 – W 1
Moisture content
Weight of empty crucible
Weight of crucible with the sample
Weight after drying
40
X 100
3.2.1.2 Ash content:
Ash content of the sample was determined according to the
method of AOAC (1990) as follows:
Two grams of sample were placed in a clean dry pre-weighed
crucible, and then the crucible with its content ignited in a muffle
furnace at about 550oC for 3 hours or more until light gray ash was
obtained. The crucible was removed from the furnace to a desiccators
to cool and then weighed. The crucible was reignited in the furnace
and allow to cool until constant weight was obtained. Ash content was
calculated using following equation (calculated on dry matter bases):
AC (%) =
Where
AC=
W1 =
W2 =
Ws =
W2 – W 1
X 100
Ws
Ash content
Weight of empty crucible
Weight of crucible with ash
Weight of sample
3.2.1.3 Fat content:
Fat was determined according to the method of AOAC (1990)
using soxhlet apparatus as follows:
An empty clean and dry exhaustion flask was weighed. About 2
gram of sample was weighed and placed in a clean extraction thimble
and covered with cotton wool. The thimble was placed in an extractor.
Extraction was carried out for 8 hours with petroleum ether. The heat
was regulated to obtain at least 15 siphoning per hour. The residual
ether was dried by evaporation. The flask was placed in an oven at
105oC till it dried completely and then cooled in a desiccators and
weighed. The fat content was calculated using the following equations
(calculated on dry matter bases):
41
:
FC (%) =
Where
FC=
W1 =
W2 =
Ws =
W2 – W 1
X 100
Ws
Fat content
Weight of extraction flask
Weight of extraction flask with fat
Weight of sample
3.2.1.4 Crude fibre:
Crude fibre was determined according to AOAC (1990). Two
grams of defatted sample were treated successively with boiling
solution of H2SO4 and KOH (0.26 N and 0.23 N, respectively). The
residue was then separated by filtration, washed and transferred into a
crucible then placed into an oven adjusted to105oC for 18 – 24 hours.
The crucible with the sample was weighed and ashed in a muffle
furnace at 500oC and weighed. The crude fibre was calculated using
the following equation (calculated on dry matter bases):
:
CF (%) =
Where
CF=
W1 =
W2 =
Ws =
W1 – W 2
X 100
Ws
Crude fibre
Weight of crucible with sample before ashing
Weight of crucible with sample after ashing
Weight of sample
3.2.1.5 Crude protein:
The crude protein was determined by using the micro-Kjeldahl
method according to AOAC (1990) as follows:
42
Digestion:
0.2 gram of the sample was weighed and placed in small
digestion flask (50 ml). About 0.4 gram catalyst mixture (96%
anhydrous sodium sulphate and 3.5% copper sulphate) was added, 3.5
ml of approximately 98% of H2SO4 was added. The contents of the
flask were then heated on an electrical heater for 2 hours till the colour
changed to blue-green. The tubes were then removed from digester
and allowed to cool.
Distillation:
The digested sample was transferred to the distillation unit and
20 ml of 40% sodium hydroxide were added. The ammonia was
received in 100 ml conical flask containing10 ml of 2% boric acid
plus 3 – 4 drops of methyl red indicator. The distillation was
continued until the volume reached 50 ml.
Titration:
The content of the flask were titrated against 0.02 N HCl. The
titration reading was recorded. The crude protein was calculated using
the following equation (calculated on dry matter bases):
:
CP (%) =
Where
CP =
T =
B =
N =
Ws =
1000=
(T – B) x N x 14 x 100 x 6.25
Ws x 1000
Crude protein
Titration reading
Blank titration reading
HCl normality
Sample weight
To convert to mg
43
3.2.1.6 Carbohydrates:
Carbohydrates were determined by difference according to the
following equation:
Carbohydrate = 100 – (MC + AC + FC + CF + CP)
Where
MC =
AC =
FC =
CF =
CP =
Moisture content
Ash content
Fat content
Crude fibre
Crude protein
3.2.2 Nitrogen solubility:
In order to determine the iso-electric point of lentil protein,
nitrogen solubility of defatted lentil flour was measured by using
Beuchat et al. (1975) method at different pH levels ranging from 1 –
12. The water soluble nitrogen in the defatted four was extracted by
rotary shaking with distilled water at 1:10 solute: solvent ratio for an
hour at room temperature (30oC) after pH adjustment. The slurry was
centrifuged at 3000 g for 30 minute. Nitrogen content of supernatant
was determined following micro-Kjeldahl method and expressed as
percentage of the total N.
Calculation:
NSI =
% water soluble nitrogen
X 100
% of total nitrogen
Soluble nitrogen =
T x N x TV x 14 x 100
a x b x 1000
Where
NSI =
T
=
N =
TV =
14 =
a
=
b
=
1000=
Nitrogen solubility index
Titer reading
Normality of acid (HCl – 0.02 N).
Total volume of a liquor extracted
Each ml of hydrochloric acid is equivalent to 14 mg nitrogen
Number of ml of a liquor taken for digestion
Number of mg sample flour extracted
No. of mg in one gram
44
3.2.3 Nitrogen solubility as a function of NaCl concentration:
Nitrogen solubility of defatted lentil flour was determined at
different NaCl concentrations by procedure of Hagenmair (1972) as
described by Quinn and Beuchat (1975) with slight modification. 0.2
gram material were dispersed in 10 ml NaCl solutions ranging from
0.2 – 1.0 M and mechanically shaken for 1 hour at room temperature
(30oC) centrifuged at 3000 xg for 2 minute at room temperature.
Soluble nitrogen in the supernatant was estimated by micro-Kjeldahl
method. Nitrogen solubility was expressed as a percent of the nitrogen
content of the sample.
3.2.4 Water absorption capacity:
Water absorption capacity (WAC) was estimated for lentil
protein by the method of Lin et al. (1974) with a modification
described by Wang and Kinsella (1976). 10% protein suspension was
stirred in 50 ml centrifuge tube using a glass rod for 2 minutes at room
temperature. After 30 minutes shaking the tube was centrifuged for 25
minutes at 3700 rpm. The freed water was carefully decanted in a
graduated measuring cylinder and the volume recorded. WAC was
expressed as ml water retained by one gram protein.
3.2.5 Fat absorption capacity:
The fat absorption capacity (FAC) of the sample was measured
by the modified method of Lin et al. (1974). Two grams of the protein
were treated with 20 ml of refined corn oil in 50 ml centrifuge tube.
The suspension was stirred with a glass rod for 5 minutes and the
content were allowed to equilibrate for a further 25 minutes at room
temperature (30oC). The suspension was centrifuged for 20 minutes at
45
5000 rpm at room temperature. The freed fat was decanted into a 10
ml graduated cylinder and the volume was recorded. FAC was
expressed as ml oil bound by 100 gram dry matter.
3.2.6 Bulk density:
Bulk density of sample was determined by the method of Wang
and Kinsella (1976). Ten grams of lentil protein were placed in a 25
ml graduated cylinder and packed by gently tapping the cylinder on
bench top (10 times). The volume of the sample was recorded. The
bulk density was expressed as g/ml of the sample.
3.2.7 Foaming capacity and foam stability:
Foam measurements were determined as described by Venktesh
and Prakash (1993), following the method developed by Lawhon et al.
(1972). One hundred millitres of distilled water were added to 3 grams
protein. The mixture was homogenized for 5 minutes in a Moulinex
blender set at high speed. At room temperature and then transferred to
a 250 ml measuring cylinder. The volume of foam at 30 second was
calculated and the volume increase is expressed as percent foam
capacity.
FC (%) =
Volume after whipping – volume before whipping
Volume before whipping
X 100
The foam stability (FS) was determined by measuring the
decrease in volume of foam as a function of time up to a period of 120
minutes. The stable foam volumes were recorded at time intervals of
10, 30, 60 and 120 minutes.
FS (%) =
Foam volume after time (t)
Initial foam volume
46
X 100
3.2.8 Emulsification activity (EA) and emulsion stability (ES):
The procedure described by Volkert and Kelin (1979) was used
for both emulsification activity and emulsion stability. Emulsions
were prepared with 1 gram protein, 50 ml distilled water at room
temperature (~ 25oC) and 50 ml of corn oil. The mixture was
emulsified for 30 minutes. Each emulsified sample was divided
equally into 50 ml centrifuge tubes. Content of one tube was directly
centrifuged at 3000 ×g for 30 minutes, while the other centrifuged
under the same conditions after heating in a water bath at 80oC for 30
minutes and cooling to 15oC. The height of the emulsified layer, as a
percentage of the total height of material in the untreated tubes was
used to calculate the emulsification activity and stability using the
following formulae:
EA (%) =
ES (%) =
Height of emulsion
X 100
Height of whole layer
Height of emulsion layer after heating
X 100
Height of whole layer
3.2.9 Gelation:
Least gelation concentration of the sample was measured by the
method of Coffman and Garcia (1977) with slight modification.
Appropriate sample suspension of 2, 4, 6, 8 and 10% were prepared in
10 ml of distilled water. The test tubes containing this suspension
were then heated for one hour in a boiling water bath following by
rapid cooling under running cold tap water. The test tubes were further
cooled for 3 hours at (4oC). The least gelling concentration was
determined as that concentration which did not fall down or slip when
the test tubes were inverted.
47
3.2.10 Dispersability:
The dispersability of the protein at selected pH levels (3, 7 and
10) was measured according to the method of Kulkarni et al. (1991).
Three grams of the protein were dispersed in distilled water in a 250
ml stoppered measuring cylinder and the desired pH was adjusted by
addition of drops of dilute HCl or NaOH solution. Then distilled water
was added to reach a volume of 30 ml, the mixture was stirred
vigorously and allowed to settle for three hours. The volume of settled
particles was subtracted from 30 and multiplied by 100 and reported
as percentage dispensability.
3.3 Determination of phytase activity:
Estimation of phytases activity was based on the estimation of
inorganic orthophosphate (P) released by hydrolysis of phytic acid,
following the method described by Engelen et al. (1994).
Reagents:
(a) Buffer solution:
18.1 gram sodium acetate anhydrous and 0.147 gram CaCl2 2H2O the dissolved in 900 ml water, 1.68 ml acetic acid (100%) was
added. The pH adjusted to 5.50 with acetic acid and the volume
completed to 1 L with water.
(b) Substrate solution:
0.84 gram sodium phytate dissolved in 90 ml buffer solution,
the pH adjusted to 5.50 with acetic acid and completed to 100 ml with
water, this solution is prepared fresh as required.
(c) Nitric acid solution: were
Seventy ml nitric acids (65%) were added to 130 ml water.
(d) Ammonium heptamolybdate stock solution:
48
Hundred grams ammonium heptamolybdate dissolved in 900 ml
water and 10 ml ammonia (25%) added then diluted to 1 L with water.
(e) Ammonium monovandate stock solution:
2.35 grams ammonium monovandate were dissolved in 400 ml
water at 60oC while stirring slowly 20 ml nitric acid (No. c) were
added then cooled to room temperature and diluted to 1 L with water.
(f) Colour stop mix:
250 ml ammonium heptamolybdate stock solution and 250 ml
ammonium monovandate were mixed then 16 ml nitric acid (65%)
were added, cooled to room temperature and diluted to 1 L with water
(prepared fresh daily).
Preparation of standard solution:
A commercial phytase (5240 FTU/g, Novozymes) was selected
as standard. 0.0477 gram phytase standard was weighed in 100 ml
volumetric flask, dissolved with buffer solution with Triton X-100,
then 1.5 ml of this solution was transferred into a volumetric flask and
filled up to 50 ml with buffer. Serial dilutions were made to final
concentration of 0.0075, 0.01050, 0.030, 0.045, 0.060 and 0.0675
FTU/ml phytase standards this was prepared in duplicate (standard
and blank) for phytase activity assay.
Preparation of sample:
Five grams of sample were extracted in 100 ml conical flask
with 50 ml sodium acetate buffer solution (5.5 pH) for 1 hour by using
a shaker at room temperature, the supernatant was filtered into 50 ml
GREINER®-tube, then centrifuged for 10 minutes at 3000 rpm. An
amount of the supernatant was pipetted into test tubes and diluted with
buffer solution with Triton X-100 to phytase activity of 0.01 – 0.07
FTU/2 ml.
49
Phytase activity assay:
Sample and standard solution tubes were incubated in a water
bath, each tube equilibrate for 5 minutes, then 4 ml substrate solution
was added at 37oC and mixed at time = 65 minutes. The incubation
was terminated by adding 4 ml colour-stop mix. For blank tubes were
placed in the water bath to equilibrate for 5 minutes, then 4 ml colour
stop mix and 4 ml substrate solution were added to all blank tubes and
mixed. All tubes were centrifuged for 5 minutes at 3000 rpm, and then
the absorbance was measured at 415 nm using a spectrophotometer
after zeroing the instrument with water. The corrected absorbance was
calculated by subtracting absorbance blank from that of corresponding
sample standard solution.
The computer was used to plot the absorbance difference of the
standard solution against the corresponding exactly calculate activity
(FTU/2 ml to be analyzed), the best fitting cure through the origin was
drawn. The enzyme concentration was determined by reading the
corrected absorbance difference for the sample from the line
produced.
3.4 Analysis of the inositol phosphates:
The inositol phosphate IP5 and IP6 were determined (germinated
and ungerminated samples) as described by Sandberg (1986) using
HPLC apparatus. 0.5 gram of the sample were placed in 50 ml
beaker, 10 ml of HCl 0.5 M were added and stirred with magnetic
stirrer (500 rpm) at room temperature for 2 hours, freezed overnight at
(-20oC), stirred for 1 hour at room temperature (20oC), then
centrifuged at 14.000 rpm for 8 minutes and the supernatant was taken
in microcon YM-30 centrifuge in eppendorf-centrifuge (14000 rpm
50
for 25 minutes at room temperature), the clear supernatant (0.5 ml)
was taken for HPLC analysis.. Phytic acid was expressed as a total of
IP5 and IP6.
3.5 HCl extractable minerals:
HCl extractable minerals of germinated and ungerminated lentil
cultivars were estimated according to the method described by
Mahajan and Chauhan (1988). Samples were extracted with 0.03 N
HCl by taking 2 grams in a 50 ml conical flask, 20 ml of 0.03 N HCl
were added and shaked at 37oC for 3 hours. The extract was filtered
through ash less filter paper. The clear extract obtained after filtration
was oven dried and then placed in a muffle furnace at 550oC for 4
hours. Samples were cooled and 5 ml of 5N HCl were added and
boiled gently for 10 minutes using sand bath then cooled, diluted to
volume (100 ml) with distilled water and taken for mineral
determination. Ca, Fe, Mg, Cu and Zn were determined by atomic
absorption
spectrophotometry
phosphorus
was
determined
calorimetrically using the phosphovanadomolybdate method of
AOAC (1980). Phytate phosphorus was
determined
by
using
Reddy (1982) relationship
. Phytate phosphorus
(mg/100)=
A x 28.18 Where A is phytic acid
100
Non- phytate phosphorus was calculated by difference between the
total phosphorus and phytate phosphorus.
3.6 Amino acid analysis:
500 milligrams of the pulverized sample (0.5 mm) was acid
hydrolyzed (6M HCl) for about 24 hours in a closed bottle, then the
hydrolyzed sample was distributed into two bottle one of them was
51
oxidized (hydrogen peroxide/formic acid, 24 hour, chilled), the other
was left without oxidation, then the pH was adjusted to 2.2 with
NaOH and filled to 100 ml with citrate buffer pH 2.2. Two ml were
then filtered (membrane filter) and used for analyzing amino acid
using analyzer/ion exchange chromatography.
All amino acids were detected at 570 nm, except proline which
was detected from a separate detector channel at 440 nm.
3.7 In vitro protein digestibility:
In vitro protein digestibility was determined for cooked and
uncooked lentil samples by using pepsin enzyme alone or by using
pepsin and pancreatin.
a) In vitro protein digestibility by using pepsin:
The method described by Maliwal (1983) was used as modified
by Monjula et al. (1991) for determination of in vitro protein
digestibility. A known weight of the sample containing 16 mg
nitrogen was taken in triplicate and hydrolyzed with 1 milligram
pepsin in 15 ml of 0.1 M HCl at 37oC for 2 hours. The reaction was
terminated by addition of 15 ml of 10% w/v trichloroacetic acid
(TCA). The mixture was filtered quantitatively through Whatman No.
1 filter paper. The TCA soluble fraction was assayed for nitrogen
using the micro-Kjeldahl method. Digestibility was calculated using
the following formula:
Protein digestibility =
N in supernatant – N in blank
X 100
N in sample
N in blank= N in pepsin enzyme and reagent.
52
b) In vitro protein digestibility by using pepsin-pancreatin
In vitro protein digestibility of cooked and uncooked lentil
samples was measured according to the method of Saunders et al.
(1973), in which is based on double digestive pepsin-pancreatin
system. About 250 mg sample was suspended in 15 ml of 0.1 N HCl
containing 1.5 mg pepsin, the mixture was incubated at 37oC for 3
hours then neutralized with 0.5 N NaOH and treated with 4 milligram
pancreatin in7.5 ml 0.2 M phosphate buffer (pH 8.0) containing 0.005
M sodium azide. Then the mixture was incubated at 37oC for 24
hours. 10 ml of 10% tirchloroacetic acid (TCA) was added to stop the
reaction then centrifuged at 5000 rpm for 5 minutes. Five ml from the
supernatant were taken for nitrogen determination using the microKjeldahl method.
Calculation:
Protein digestibility %=
(T – B) x N x 14 x 100 x TV
x ×a
Where :N =
T =
B =
a =
14 =
250 =
CP%=
Normality of HCl
ml of titer
ml of blank
Number of ml of a liquor
Equivalent weight of nitrogen
Sample weight in mg
Percent crude protein
250 x Cp%
X
=
100×6.25
TV = Total volume of the liquor
3.8 Protein fractionation due to solubility:
The Mendel-Osborne (1914) technique for protein fractionation
was used in this study with slight modification as follows:
53
Determination of salt soluble proteins:
A sample of 2grams of cooked and uncooked lentil was taken
for fractionation of total protein. To this amount of the flour, 2
volumes of 20 ml NaCl (1 M) were added and the mixture was shaken
for 30 minutes using a mechanical shaker, then centrifuged at 3000
rpm for 20 minutes to separate the insoluble part from the liquor.
About 10 ml of the solvent were taken for protein estimation
according to the micro-Kjedhal method. The following formula was
used for calculating percentage of golublin.
T.F x N xTV x 14 x 6.25 x 100
a x b x 1000
Soluble protein %=
Where:
T.F =
N =
TV =
14 =
a =
b =
1000=
Titer figure
Normality of HCl
Total volume of the liquor extracted
Equivalent weight of nitrogen
Number of ml of liquor taken for digestion (10 ml)
Number of g sample extracted (2 g)
To convert from g to mg
Soluble fraction % =
Soluble protein x 100
Total protein of sample
Determination of water soluble proteins:
The insoluble part obtained after extraction of globulin was reextracted with 2 volumes of 20 ml distilled water for 30 minutes with
continuous shaking. The mixture was then centrifuged at 3000 rpm for
20 minutes to separate the insoluble part. The peptized liquor was
collected and about 10 ml of the liquor were taken for estimation of
soluble protein by micro-Kjeldhal method. Albumin was calculated
using the above formula.
54
Determination of alcohol soluble proteins:
The insoluble part obtained after extraction of albumin was reextracted in the same manner using 70% ethanol to determine
prolamin faction.
Determination of alkali-soluble proteins:
The insoluble part obtained after extraction of prolamin was reextracted in the same manner using (0.2%) NaOH to determine
glutelin fraction.
Determination of insoluble protein:
The residues remaining after successive extraction represents
the insoluble protein, 0.2 gram was taken for protein determination
using micro-Kjeldahl method.
3.9 Electrophoresis:
The determination of protein bands and molecular weight
pattern of protein fractions of cooked and uncooked lentil samples
were carried out according to the method described by Laemmli
(1970) using sodium dodecyle sulfate polyacrylmide gel electrophoresis (SDS-PAGE).
Reagent:
-
Monomer solution: 60 grams acrylamide and 1.6 gram
bisacrylamide were dissolved in 100 ml distilled water, stored
at 4oC in the dark.
-
Resolving gel buffer: (1.5 M tris-HCl pH 8.8) 36.6 grams tri
was dissolved in 50 ml distilled water, then pH was adjusted
with HCl to 8.8.
55
-
Stacking gel buffer. (0.5 M tris HCl pH 6.8) 3 grams tri was
dissolved in 50 ml distilled water then pH was adjusted with
HCl to 6.8.
-
Ten percent sodium dodecyle sulfate: 10 grams SDS dissolved
in 100 ml distilled water.
-
Ten percent ammonium per sulfate: prepared freshly by
dissolving 0.1 gram in 1 ml distilled water.
-
Treatment buffer: (0.125 M tris, 4% SDS, 20% gram glycerol,
10% 2-mercapto ethanol and 0.02% bromophenol blue) 2.5 ml
stacking gel buffer, 4 ml 10% SDS, 20 ml 20% glycerol, 1 ml
10% 2-mercapto ethanol and 2 grams bromophenol blue then
the volume was made with distilled water up to 10 ml.
-
Tank buffer: (0.025 M tris, 0.192 M glycine, 0.1 SDS, pH 8.3)
15.14 grams tris, 74.06 grams glycine and 5 grams SDS were
dissolved in 5 liter distilled water.
-
Staining solution: (0.1% commassie brilliant blue, 40%
methanol and 10% acetic acid). 0.2 gram commassie brilliant
blue were added to 400 ml methanol and 70 ml acetic acid then
volume was made up to 1 litre with water.
-
TEMED
-
70% ethanol
-
Molecular weight protein marker as
standard consists of protein
Glactosidase
Phosphorylaseb
Bovine serum albumin
Alcohol dehydrogenate
Carbonic anhydrase
Lysozyme
56
Molecular weight
Kilo Dalton(kDa)
116
97.4
66.2
37.6
28.5
14
Preparation of separating or resolving gel 12%:
1.3 ml monomer solution, 3.1 distilled water, 2.5 ml resolving
gel buffer 8.8 pH, 100 µl sodium dodecyle sulfate 100 µl ammonium
per sulfate, 10 µl TEMED.
Sample preparation:
Two ml of each fraction were mixed with 100 µl treatment
buffer, albumin and globulin fraction were mixed on vortex mixer for
5 minutes. The prolamine fraction was vortexes for a total of 20
minutes at separate intervals, between each two intervals it was placed
at about 26oC for a total extraction period of 1 hour glutelin and
residue were shaked for 2 hours at left at room temperature (not more
than 35oC for 2 hours).
Procedure:
Two glass plates with two sides spacers were assembled and
camped, the assembly was placed upright using clamp as supports on
glass plate. The resolving gel (described above) was poured between
two glass plates, 70% ethanol was poured onto the resolving gel to
ensure a flat surface and exclude air, after gel polymerized the ethanol
was discarded. The stacking gel (described above) were poured on the
top of the resolving gel, combs were inserted into the stacking gel then
removed after gel polymerized 10 µl of the ample and the standard
were applied to the stacking gel, the tank buffer (described above) was
used as electrode buffer, the sample were run using vertical
electrophoresis system at 100 voltage for 2 hours.
Gels were stained overnight with commasiae brilliant blue and
distained with distaining solution till protein bands were clearly
visible.
57
Molecular weight determination:
The approximate molecular weights of subunit of each fraction
were calculated from the values of the protein markers used.
58
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Chemical composition:
The proximate composition of lentil cultivars is illustrated in
Table 1. The moisture content ranged from 6.4 to 10.4, which is
similar to that reported by Muehlbauer et al. (1985), but lower than
that reported by Adsule et al. (1989). The ash content ranged from 2.7
to 3.7% which is similar to that reported by Adsule et al. (1989) and
Muehlbauer et al. (1985). The protein content ranged from 32.3 to
35.7 which are the same as that reported by Adsule et al. (1989). The
oil content ranged from2.0 to 2.4% which is lower than that reported
by Duke (1981), but similar to the value reported by Husle (1990).
The fibre content ranged from 2.0 to 4.1% which is lower than that
reported by Duke (1981), but higher than that reported by Husle
(1990).
4.2 Nitrogen solubility as affected by pH:
Figure 1 shows the variation in nitrogen solubility at different
pH levels of lentil protein. The minimum nitrogen solubility was
found to range from 8.1 to 23.6% for the Selaim and Nadi cultivars
respectively, at pH 5 indicating the isoelectric point of lentil protein.
Hsu et al. (1982) found that the lowest solubility of the protein from
yellow pea, lentils and faba bean were at pH 4.5 – 5.0. On the other
side of pH 5 there was a sharp increase in solubility of lentil protein,
while at pH 2 it was 63 to 58% for Indian and Nadi cultivars
respectively.
59
Table 1.Proximate composition of four cultivars of lentil
Cultivars
Seliam
Nadi
M.C. (%)
Indian
Ash
(%)
Oil (%)
Total
carbohydrates (%)
32.380
1.950
51.49
2.430
(± 0.84)c
(± 0.16)b (± 0.68)a (± 0.31)a (± 0.32)b
(± 1.39) a
10.370
4.130
47.04
a
(± 0.45)
3.780
Protein
(%)
7.976
(± 0.16)
Rubatab
Fibre
(%)
2.700
a
(± 0.26)
33.390
b
(± 1.20)ab
(± 0.26)c (± 0.28)b (± 0.26)a (± 0.34)ab
(± 1.52)a
6.420
1.170
51.39
(± 0.07)
3.030
c
(± 0.16)
2.050
(± 2.47)b
1.300
(± 0.69)
32.340
(± 0.11)
ab
8.733
c
2.950
(± 1.56)
2.100
a
35.560
b
(± 0.22)
52.63
2.440
a
(± 0.17)
a
(± 0.31)a
Values are means ± SD
Means not sharing a common superscript letter in a column are significantly different at
(P< 0.05) as assessed by Duncan's multiple range test.
60
Selaim
90
Nadi
80
Rubatab
70
Indian
N it r o g e n s o l u b il i ty i n d e x ( % )
100
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
pH
Figure 1. Effect of pH on nitrogen solubility of lentil protein
61
10
11
12
At alkaline region (pH 12), solubility was increased to reach its
maximum value (88%). Sgarbieri et al. (1978) reported that higher
pH, although more efficient in extracting the proteins, should be
avoided due to possibility of disruption of the protein structure and
degradation of certain amino acids. Lentil protein showed good
solubility in both acid and alkaline pH regions, which is an important
characteristic for food formulation (Idouraine etal, 1991). Parakash
and Norasinga (1986) reported similar results.
4.3 Nitrogen solubility at different sodium chloride concentration:
The solubility of lentil protein at different NaCl concentrations
is shown in Figure 2. In dilute NaCl (0.2 M) solution, the solubility of
lentil protein was lower than in water. However, protein solubility
increased up to 0.6 M (salting-in) and thereafter started to decrease
(salting-out). Schut (1976) suggested that NaCl causes a shift in the
isoelectric point to more acidic pH as a result of specific ion binding
effects. Since inorganic anions are bound to protein more strongly
than inorganic cations due to heir smaller hydrated radii, anion are
able to attain a closer proximity to the protein molecule and are able to
"screen" the charged groups of the protein more effectively than
cations. Thus, with the addition of NaCl and selective binding of the
chloride anions, the protein would have an excess of negative chares
at the pH of original isoelectric point and more acid is, therefore
needed to reach the new isoelectric point. This was observed also by
Lin and Hung (1998) who reported that high concentration of NaCl
(0.15 M) reduced chickpea protein solubility.
62
Nitrogen solubilityindex (%)
Selaim
Nadi
Rubatab
Indian
50
45
40
35
30
25
20
15
10
5
0
0.0
0.2
0.4
0.6
0.8
1.0
NaCl concenteration(N)
Figure 2. Effect of NaCl concentration on nitrogen solubility of lentil protein
63
4.4 Water and oil absorption capacity:
As shown in Table 2 lentil protein isolate had a water
absorption capacity of 1.9 and 2.0 ml H2O/g protein for Nadi and
Selaim, respectively which is within the range of commercial values
of protein concentrates (1.9 – 2.2 ml H2O/g protein) as reported by Lin
and Zayas (1987). It has been reported that the protein concentrate
exhibits poor water-binding capacity compared to that of the isolate
this is likely due to the fact that the protein isolate has great ability to
swell, dissociate and unfold exposing additional binding sites, whereas
the carbohydrates and other components of protein concentrate may
impair it (Kinsella, 1979).
The oil absorption capacity of lentil protein isolate (LP1) was
1.9 – 2.0 mg oil/g protein for Nadi and Selaim, respectively. LP1
showed higher oil absorption capacity than chickpea (Marina, 1986).
Kinsella (1979) explained the mechanism of absorption as physical
entrapment of oil; several authors have related the oil absorption
capacity to the interaction of nonpolar side chain of the protein as well
as the conformation features. Results obtained in this study suggested
that LP1 had both good water and oil absorption capacities.
4.5 Bulk density:
Lentil protein isolate (LP1) had bulk density of 1.4 g/ml for
both Nadi and Selaim cultivars. This value is higher than that of
cowpea as reported by Ragab et al. (2004). Bulk density depends on
combined effects of interrelated factors such as intensity of attractive
inter particle forces, particle size and number of contact points (Peleg
and Balgey, 1983). Higher bulk density is desirable since it helps to
reduce the paste thickness which is an important factor in
convalescent and child feeding (Padmashree et al., 1987).
64
4.6 Emulsifying properties:
As shown in Table 2 lentil protein has emulsifying activity of
75.3 and 62.9% for Nadi and Selaim, respectively and emulsion
stability of 41.4 and 46.3% for the cultivars, respectively. Results of
emulsifying activity obtained in this study are higher than that
reported by Bora (2002) who found that emulsification activity of
Native lentil globulin was 45.1%. The data obtained is lower than that
obtained by Khalid et al. (2003) who reported that emulsion stability
of sesame protein isolate at neutral pH was high. Elzialde et al. (1991)
reported that the emulsion stability is enhanced by high protein and oil
concentrations and these factors are highly interrelated. They also
reported that emulsion stability depends primarily upon the water and
oil absorption capacity.
4.7 Foaming properties:
As shown in Table 3 lentil protein isolated has a lower foam
capacity (22.7 and 28.3) for Nadi and Selaim respectively, but its
foam was highly stable when compared with soy protein as reported
by Soetrisno and Holnes (1992).
Turner (1969) reported that partially hydrolyzed protein
component needed to increase foam volume and larger component are
required to obtain stable foam. It is generally thought that low
molecular weight proteins increase foamability but are unable to
stabilize foams (Petruccelli and Anon, 1994). In addition, the
hydrolyzed proteins may undergo resynthesis of peptide bonds by
microbial proteases, producing products different from the original
protein, that display altered foaming properties (Damodaran, 1996).
65
Table 2. Functionality of lentil protein isolate (LPI)
Cultivars
WAS
FAC
BD
EA
ES
Nadi
1.9(±0.12) 1.9(±0.12) 1.4(±0.00) 75.3(±4.12) 41.4(±1.22)
Seliam
2.0(±0.00) 2.0(±0.00) 1.4 (±0.00 62.9(±8.51) 46.3(±3.21)
WAC: Water absorption capacity, FAC: Fat absorption capacity, BD:
Bulk density, EA: Emulsification activity, ES Emulsification stability.
Table 3. Foam capacity (FC %) and effect 0f time on foam
stability (%) of lentil protein
cultivar
Nadi
Seliam
FC
22.5
28.3
Foam stability Time (min.)
0
15
30
60
120
100.00
77.66
62.34
56.48
52.95
±0.00a
±6.11a
±8.85c
±6.11d
±0.00e
100.00
75.02
68.77
62.52
56.26
±0.00
a
±0.00
b
±10.8
c
±10.8
d
±0.00e
Values are means ±SD
Means not sharing a common superscript letter in a row are significantly different
at (P< 0.05) as assessed by Duncan's multiple range test.
66
4.8 Gelation capacity:
The least gelation concentration of LP1 is shown in Table (4).
LP1 formed a weak gel at 6% w/ml, while a strong gel was formed at
8% and very strong gel at 10%. No gel was formed at 2 and 4%. This
observation agrees with the finding of Osman (2004) who reported
that chickpea protein has higher content of starch which induces
gelation due to starch-starch and/or starch-protein interaction. The
important initial step in heat-induced gelation of globular proteins is
denaturation which exposes the functional groups; so that the
intermolecular network could be activated through disulfide bond
formation which increases water holding capacity and increasing gel
hardness (Soetrison and Holnes, 1992).
4.9 Dispersibility:
As shown in Table 5 lentil protein isolate had a higher
dispersibility at pH 7 (76.3 and 53.3 for Selaim and Nadi,
respectively). At pH 10 it was 73.9 and 40.7% for the two cultivars,
respectively. At pH 3 it was 53.3 and 23.3% respectively. This
observation agrees with Khalid et al. (2003) who reported that
dispersibility of sesame protein was significantly higher at neutral and
alkaline pHs than acidic pHs. Higher dispersibility enhances the
emulsifying and foaming properties of the protein, which occurs
during making of bread, macaroni and cookies (Kinsella, 1979).
67
Table 4. Effect of pH on least gelation concentration of lentil
protein isolate
pH
Concentration of lentil protein
2%
4%
6%
8%
10%
3
-
-
+
++
+++
7
-
-
+
++
+++
10
-
-
+
++
+++
- : No gel
++: Strong gel.
+: Weak gel
+++ : Very strong gel.
Table 5. Effect of pH on despersibility of lentil protein isolate
Cultivars
pH
7
3
10
Nadi
23.33(±0.00)c 53.33(±0.00)a
40.66(±0.00)b
Seliam
53.33(±0.00)c 76.66(±0.00)a
73.33(±0.00)b
Values are means± SD
Means not sharing a common superscript letter in a row are significantly
different at (P< 0.05) as assessed by Duncan's multiple range test.
68
4.10 Effect of germination on chemical composition and energy
value of lentil cultivars:
As shown in Table 6 there were remarkable changes in
proximate composition and food energy values of lentil due to
germination. Crude fat and crude fibre increased slightly for the three
cultivars as germination progressed. Ash content was slightly
increased for Selaim cultivar while for Rubatab and Nadi it slightly
decreased with the germination time. The protein content of Nadi and
Selaim cultivars slightly increased while that of Rubatab decreased
with germination. The nitrogen free extract (NFE) and food energy for
all cultivars decreased with the germination time. The slight increment
in fat content during germination compared to other constituents for
all cultivars may be due to the fact that fat contains about twice the
food energy values of protein and carbohydrate (Osborne and Voogt,
1978), the reduction in food energy value of the germinated seeds
might be attributed to the very slow increment in fat content with
increasing germination period. Nielson and Liener (1984) and Shastry
and John (1991) attributed the reduction of both nutrients (NFE and
protein) to their utilization during germination. Reduction of some
storage nutrients of lentil seeds resulted in a concomitant increase in
other nutrients.
Changes in nutrient and in anti-nutrient factors occurring during
germination depend on the type of legume and on the sprouting
conditions (i.e. time, temperature, light cycle) (Frias et al., 1995). That
clearly indicated potential for optimization. Kuo et al. (2003) who also
reported that the growth conditions during the germination process
can have important effects on the composition of secondary
metabolites of nutritional importance.
69
Table 6. Proximate composition and energy value of lentil cultivars as affected by germination
Cultivars
Rubatab
Nadi
Selaim
Germinatin Crud fibre
(Days)
(%)
Ash
(%)
Crude
protein (%)
Fat (%)
Nitrogen free
extract (%)
Energy
(kcal/100g)
0
3.7 (±1.0)b
2.5 (±0.03)a
28.2 (±0.03)a
0.6 (±0.0)b
67.5 (±0.01)a
388.2 (±0.10)a
3
5.2 (±0.5)a
2.5 (±0.005)a
27.0 (±0.34)a
0.6 (±0.01)b
64.7 (±0.02)b
372.2 (±0.15)b
6
5.8 (±0.07)a
2.4 (±0.01)b
27.6 (±0.54)a
0.67 (±0.0)a
63.5 (±0.208)c 370.7 (±0.208)a
0
1.9 (±0.1)b
3.9 (±0.06)b
24.5 (±0.00)b
0.65 (±0.06)c
70.0(±0.136)a
383.0 (±0.10)a
3
5.7 (±0.15)a
3.1 (±0.07)a
26.5 (±1.60)a
0.80 (±0.01)b
64.0 (±0.15)c
368.9 (±0.20)c
6
5.8 (±0.1)a
3.0 (±1.0)b
25.7 (±0.04)a
0.97 (±0.01)a
64.6 (±0.01)b
369.6 (±0.15)b
0
1.6 (±0.03)b
2.80 (±1.0)b
27.9 (±0.15)a
0.45 (±0.01)c
67.3 (±0.15)a
384.9 (±0.15)a
3
5.0 (±0.11)a
3.0 (±1.0)b
28.1 (±0.13)a
0.52 (±0.05)b
63.4 (±0.20)c
370.4 (±0.15)c
6
6.4 (±0.076)a
3.0 (±1.0)b
28.4 (±0.08)a
0.75 (±0.01)a
64.5 (±0.10)b
378.4 (±0.15)b
Values are means (± SD)
Means not sharing same superscript letter in a column are significantly different at (P< 0.05) as assessed by Duncan's multiple range test.
70
5.11
Effect of germination on phytase activity, phytate
phosphorus and non-phytate phosphorous
Table 7 shows phytase activity, phytate, phosphorus, phytate
phosphorus and non-phytate phosphorus of germinated lentil cultivars.
Phytase activity of ungerminated lentil seeds was found to be 29.30
34.70 and 68.30 FTU/kg for Rubatab, Nadi and Selaim cultivars,
respectively. Germination of the seeds for 3 days increased phytase
activity significantly (P< 0.05) to 82.0, 132.0 and 108.0 for the
cultivars, respectively. For Rubatab cultivar, phytase activity
continued to increase progressively with an increase in the period of
germination to 6 days. Results reported by Viresos et al. (2000)
showed that phytase activity in soybean and peas are 32 and 86
FTU/kg, respectively. Different authors reported that plant seeds are
rich in phytate and both phytate and phytase are present in most plant
seeds. Phytase activity usually increases with germination (Peers,
1953; Mayer, 1958; Kuvaeva and Kretovich, 1978).
Honke et al. (1998) and Kozlowska et al. (1996) reported that
processes, such as soaking and germination; activate the endogenous
phytases, which were able to hydrolyze IP6 releasing lower inositol
phosphates.
Phytic acid content of ungerminated lentil seeds were found to
be 848.9, 1487.4 and 941.7 mg/100g for Rubatab, Nadi and Selaim
cultivars, respectively, for all cultivars germination of the seeds for 3
days reduced by 26 to 41% of the total phytate of the seeds. Further
reduction (46 – 75%) in phytate of the cultivars was observed when
the seeds were germinated for 6 days. Results showed that
germination is an effective process that causes significant (P< 0.05)
loss of more than 50% of total phytate of the seeds. Similar results
71
were observed by Duhan et al. (2002) who found that after 48 hours
germination a loss up to 45% was noticed in pigeon pea. Loss of
phytic a cid during germination was hydrolytic activity of phytase,
which is reported to be present in various plant foods (Lolas and
Markakis, 1975).
About 61.3 to 90.3% of the total phosphorus was present as phytate
phosphorus in the ungerminated seeds of the cultivars. Germination
resulted in a significant (P< 0.05) decrease in total phytate phosphorus
with correspondingly marked increase in non-phytate phosphorus HCl
extractable phosphorus. Cleavage of phosphorus from phytic acid may
explain the increase in the level of non-phytate phosphorus.
Phytic acid was reduced by 44 and 66% for lentil cultivars, the
reduction of phytic acid is also affected by the time taken in
germination. Kataria et al. (1988) reported that germination for 24
hours of soaked black gram seeds reduced phytic acid by 32%; after
60 hours of germination the reduction was 54%. Alonso et al. (2000)
reported that germination of faba bean for 24 and 72 hours resulted in
a reduction in phytic acid by 53 and 60.8%, respectively.
4.12 Total and extractable minerals of germinated lentil cultivars:
Tables 8, 9 and 10 show the effect of germination of lentil
cultivars on total and extractable minerals. For all cultivars P content
increased with germination time with a maximum value (502.2
mg/100g) obtained for Nadi cultivar after germination for 6 days
(Table 8). Extractable P for all cultivars also significantly (P< 0.05)
increased with the germination time with a maximum extractable
72
value (398.0 mg/100g) recorded for Nadi cultivar which represented
about 79% of the total P of the cultivar.
Total and extractable Ca content for all cultivars were significantly
(P< 0.05) increased with germination time with maximum value of
119.5 mg/100g obtained for Nadi cultivar after germination for three
days and out of this amount about 87% was found to be extractable.
Total and extractable Fe content for all cultivars were significantly
(P< 0.05) increased with germination time with maximum value of
13.5 mg/100g and extractable value of 6.0 mg/100g observed for
Selaim cultivar germinated for 6 days (Table 9). Total and extractable
Mg for all cultivars followed a trend similar to that obtained for Fe.
However, Cu and Zn content were fluctuating with the germination
time. Turnlund et al. (1985) reported that phytic acid had no effect on
copper absorption in men, the effect of phytic acid on copper
absorption seems to be modulated by several factors especially zinc
level in the diet (Lopez et al., 2002).
The results obtained clearly indicated that germination of lentil
seeds for a certain period greatly improved the availability of
minerals. The increment in both total and extractable minerals after
germination also indicated the strong correlation between phytate
content and minerals extractability. Similar results were observed by
Oloya (2004) who found that germination of pigeon pea seeds for up
to 4 days resulted in significantly higher content of iron, calcium,
magnesium and phosphorus. Abelrahman et al. (2004) reported that
germination of pearl millets greatly reduced phytic acid content with a
concomitant increase in extractable minerals.
73
Table 7. Effect of germination on phytic acid, phosphorus, phytase phosphorus and non phytate phosphorus
of lentil cultivars
Cultivars
Germination
(Day)
Phytase activity FTU/Kg
Phytic acid
Total
phosphorus
Phytate phosphorus
Total
Rubatab
0
3
6
Nadi
0
3
6
Selaim
0
3
6
29.3
(± 3.2)c
82
(± 4.40)b
128
(± 13.50)a
34.7
(± 4.20)c
132
(± 12.10)a
106.3
(± 5.50)b
68.3
(± 4.10)
108.3
(± 9.00)c
80
(± 8.20)b
848.9
(± 0.01)a
617.1
(± 0.00)b
413.8
(± 0.00)c
1487.4
(± 0.00)a
1106.8
(± 0.06)b
806.2
(± 0.00)c
941.7
(± 0.00)a
548.6
(± 0.00)b
234.7
(± 0.06)c
363.3
(± 1.53)a
377.3
(± 22.5)b
388
(± 0.00)a
464.0
(± 0.00)b
497.3
(± 5.51)a
502.3
(± 4.51)a
432.7
(± 4.51)b
435
(± 2.00)b
450
(± 1.00)a
239.3
(± 0.10)a
137.9
(± 0.00)b
116.6
(± 0.05)c
419.1
(± 0.00)a
311.7
(± 0.20)b
227.3
(± 0.00)c
265.4
(± 0.00)a
164.8
(± 0.00)b
66.1
(± 0.30)c
% of total
phosphorus
65.8
(± 0.29)a
46.5
(± 0.27)b
301
(± 0.00)c
90.3
(± 0.00)a
62.7
(± 0.70)b
45.2
(± 0.40)c
61.3
(± 0.66)a
37.9
(± 0.20)b
14.71
(± 0.01)c
Non phytate
phosphorus
Total
% of total
124.1
(±1.51)c
203.6
(± 22.0)b
271.4
(± 0.00)a
44.9
(± 0.00)a
185.6
(± 0.29)c
275.0
(± 5.51)b
167.3
(± 4.51)c
270.2
(± 2.00)b
383.9
(± 1.00)a
34.2
(± 0.46)c
54.0
(± 6.00)b
70
(± 0.00)a
9.7
(± 0.00)c
37.4
(± 1.10)b
54.8
(± 0.90)a
38.6
(± 1.00)c
62.1
(± 0.45)b
66.1
(± 1.00)a
Values are means (±SD)
Means not sharing a common superscript letter in a column are significantly different at (P< 0.05) as assessed by Duncan's
multiple range test.
74
Table 8. Effect of germination on total and extractable phosphorus and
calcium (mg/100g) in lentil cultivars
Cultivars
Rubtabe
Time
germination
(Days
0
3
6
Nadi
0
3
6
Selaim
0
3
6
P
Ca
Total
Extractable
Total
Extractable
363.3
(±1.53)b
377.3
(±22.5)b
380.0
(±0.00)c
464.0
(±0.00)b
497.3
(±5.51)b
502.3
(±4.51)a
432.7
(±4.51)b
435.0
(±2.00)b
450.0
(±1.00)a
246.6
(±0.00)c
269.0
(±0.00)b
287.5
(±0.00)a
354.5
(±0.00)b
395.5
(±0.00)a
398.0
(±0.00)a
331.0
(±0.00)c
349.0
(±0.00)b
363.0
(±0.00)a
63.5
(±9.70)b
107.3
(±1.50)a
111.0
(±7.00)a
72.0
(±7.30)b
119.5
(±2.50)a
118.0
(±4.00)a
68.6
(±0.00)b
100.3
(±1.80)a
104.0
(±1.00)a
41.5
(±0.45)a
86.2
(±0.10)b
93.2
(±1.20)a
48.6
(±4.60)b
104.2
(±2.20)a
98.6
(±2.60)b
65.5
(±2.60)b
83.1
(±3.10)a
83.2
(±2.20)a
Values are means (±SD)
Means not sharing a common superscript letter in a column are significantly different at
(P< 0.05) as assessed by Duncan's multiple range test.
75
Table 9. Effect of germination on total and extractable iron and
magnesium (mg/100g) in lentil cultivars
Cultivars
Rubtabe
Time germination (Days
0
3
6
Nadi
0
3
6
Selaim
0
3
6
Fe
Mg
Total
Extractable
Total
Extractable
5.4
(±0.05)b
11.0
(±0.00)a
11.2
(±2.30)a
5.5
(±0.70)b
8.9
(±0.08)a
8.9
(±0.03)a
6.5
(±0.00)c
10.3
(±0.00)b
13.5
(±0.06)a
3.1
(±0.10)c
5.1
(±0.60)b
5.6
(±0.60)a
3.0
(±0.00)b
4.4
(±0.20)a
4.4
(±0.70)a
2.7
(±0.30)c
5.6
(±0.06)b
6.0
(±0.00)a
87.9
(±0.60)c
110.0
(±7.00)b
118.8
(±0.00)a
99.9
(±10.00)b
115.0
(±0.00)a
118.0
(±0.00)a
95.8
(±0.50)c
112.5
(±0.05)b
119.0
(±1.00)a
80.0
(±5.00)c
97.0
(±4.00)b
106.0
(±2.00)a
91.5
(±1.50)b
109.3
(±2.30)a
107.0
(±3.00)a
86.3
(±2.30)b
100.9
(±0.90)a
103.1
(±2.10)a
Values are means (±SD)
Means not sharing a common superscript letter in a column are significantly different at
(P< 0.05) as assessed by Duncan's multiple range test.
76
Table 10. Effect of germination on total and extractable copper and
zinc (mg/100g) in lentil cultivars
Cultivars Time
germin-
Cu
Zn
ation (Days)
Rubtabe
0
3
6
Nadi
0
3
6
Selaim
0
3
6
Total
Extractable
Total
Extractable
1.5
(±0.02)b
1.4
(±0.20)b
1.8
(±0.00)a
0.9
(±0.00)a
1.0
(±0.00)a
1.3
(±0.00)a
5.30
(±3.00)a
5.60
(±0.30)a
5.50
(±0.10)a
5.34
(±1.40)a
5.18
(±1.80)a
5.00
(±0.00)a
2.8
(±1.50)a
2.1
(±0.26)b
1.7
(±0.00)c
1.6
(±0.00)a
1.8
(±0.00)a
1.2
(±0.00)b
6.10
(±0.00)a
6.15
(±0.60)a
5.80
(±0.90)a
5.80
(±0.30)a
5.80
(±0.01)a
5.00
(±0.10)a
2.0
(±0.08)a
1.8
(±0.14)a
2.0
(±0.11)a
1.5
(±0.00)a
1.2
(±0.00)a
1.5
(±0.00)a
6.80
(±0.10)a
7.03
(±0.30)a
6.88
(±0.07)a
6.44
(±0.20)a
6.60
(±0.00)a
6.17
(±0.20)b
Values are means (±SD)
Means not sharing a common superscript letter in a column are significantly different at
(P< 0.05) as assessed by Duncan's multiple range test.
77
4.13 Effect of germination on the amino acid content:
The germination of lentil affects the content of amino acid depending
on cultivar. In Selaim cultivar (Fig 3) germination produced high increase of
all essential amino acids except methionine which was 0.6 g/kg in raw
sample, thereafter it decreased to 0.4 g/kg after six days germination. At
three days germination some of amino acid increased, but they decreased
after six days germination these were threonine, histidine, lysine and
arginine. Dramatic increase was seen for the essential amino acid valine,
isoleucine, leucine, phenyalanine from 8.5, 7.2, 16.4 and 11.1 g/kg,
respectively to 10.5, 8.6, 16.8 and 12.2g/kg, respectively in Selaim
germinated for six days. There was increment in Aspartic acid and proline
which was also detected by Kuo et al. (2003) in germinated lentil.
In Rubatab cultivar (Fig 4) the increment of most amino acids was
observed after three days germination, these were threonine, valine,
isoleucine, phenyalanine and histidine which had content of 10.0, 10.9, 9.6,
13.0 and 8.0 g/kg, respectively in raw sample to reach up to 10.2, 11.0, 9.8,
13.0 and 8.0 g/kg, respectively in Rubatab germinated for three days. Three
of the essential amino acid of this cultivar were decreased due to
germination for six days these were methionine, lysine and proline.
In Nadi cultivar (Fig 5) germination produced increase in amino acids
content mainly observed at three days germination, these results were
observed
for
threonine,
valine,
methionine,
isoleucine,
leucine,
phenylalanine, lysine, arginine and proline. There was decrease in some
amino acids in this cultivar due to germination thee were aspartic acid,
78
tyrosine and glycine which was also observed by Kuo
et al. (2003) in
germinated lentil.
The increment of amino acids during germination may be attributed to
that seedlings are the site of high amino acid biosynthetic activity, resulting
in high content of free amino acid (Sutcliffe and Byrant, 1977). Also storage
proteins can undergo proteolysis and contribute to the increase of free amino
acids (Lea and Joy, 1983).
4.14 Effect of cooking on in vitro protein digestibility:
The effect of cooking on in vitro protein digestibility (IVPD) of lentil
cultivars is shown in Table 11. When the cultivars flour was digested with
pepsin, uncooked flour gave a range of 44.6-51.9. Cooking of the flour
significantly (P ≤ 0.05) decreased the IVPD and it was found to range from
19.7 to 24.0%. However, when pepsin together with pancreatin was used
the IVPD of raw flour was significantly (P ≤ 0.05) increased and was found
to range from 81.8% to 99.7%. Cooking significantly (P ≤ 0.05) decreased
the IVPD and was found to range from 77.1% to 81.60%. It was clear that
the IVPD obtained was significantly (P ≤ 0.05) affected by cooking even
when the flour was digested with both pepsin and pancreatin.
Similar
results were observed by Carbonaro et al. (1997) who attributed the lack of
improvement in digestibility of faba bean and lentil to be related in part to
protein aggregation that is a consequent to the thermal treatment. Carbonaro
et al. (1993) suggested formation of aggregated protein on heat treatment
through oxidation of sulfhydryl groups and through interactions between
79
la
ni
ne
80
uc
in
e
in
e
Figure 3. Amino acid content of Selaim cultivar as affected by germination.
Ly
si
ne
ist
id
in
e
Pr
ol
in
e
A
rg
in
in
e
H
Ty
ro
si
ne
Ph
en
yl
al
an
in
e
Amino acid
Le
Is
ol
eu
c
SD6
et
hi
on
in
e
40
al
in
e
SD3
V
Cy
sti
ne
A
ac
id
in
e
ly
cin
e
ic
Se
r
G
lu
tam
id
Concentration (g/kg US)
45
M
G
ac
on
in
e
rti
c
Th
re
Sp
a
SD0
35
30
25
20
15
10
5
0
ac
id
in
e
la
ni
ne
al
in
e
81
Pr
ol
in
e
RD6
A
rg
in
in
e
35
Ly
si
ne
RD3
Ty
ro
si
ne
Ph
en
yl
al
an
in
e
H
ist
id
in
e
40
uc
in
e
RD0
Le
et
hi
on
in
e
Is
ol
eu
ci
ne
V
Cy
sti
ne
A
id
ly
cin
e
ic
Se
r
G
lu
tam
M
G
ac
on
in
e
rti
c
Th
re
Sp
a
Concentration (g/kg US)
45
30
25
20
15
10
5
0
Amino acid
Figure 4. Amino acid content of Rubatab cultivar as affected by germination
on
82
Amino acid
Figure5. Amino acid content of Nadi cultivar as affected by germination.
in
e
ne
ol
in
e
rg
in
in
e
Pr
A
Ly
s
ist
id
i
ne
in
e
la
lan
i
H
en
y
in
e
cin
e
Ty
ro
s
Le
u
e
in
e
leu
c
eth
i
ali
n
Concentration (gl/kgUS)
35
Is
o
M
ne
sti
ne
V
Cy
lan
i
ly
ci
ne
A
G
ac
id
Se
rin
e
ni
ne
ca
ci
d
lu
ta
m
ic
Ph
G
ar
ti
Th
re
o
Sp
40
NDO
ND3
ND6
30
25
20
15
10
5
0
acidic and basic residues and would be more resistant to proteases as
reported by Darcy, (1984) and Desrosiers et al. (1987). Moreover, Otterburn
et al. (1977) suggested the formation of a three dimensional network on
severe heating of proteins, as a result of Ca+2 mediated electrostatic bonds,
hydrophobic interactions and the involvement of cross links, preventing
enzyme penetration or masking the sites of the enzyme attack. The negative
effect of cooking on the IVPD also observed by Abdel Rahim (2004) for
faba bean and for corn as reported by Yousif (2000) who attributed the
reduction in IVPD to the formation of disulphide bonds resulting in folding
of protein molecule and hence decreasing its susceptibility to digestive
enzymes.
4.15 Effect of cooking on protein fractions:
Table 12 shows the effect of cooking on total protein fractions of
lentil cultivars. The total protein of lentil was fractionated on the basis of
solubility for each cultivar into albumins, globulins, prolamins and glutelins.
For all cultivars the albumins content of uncooked flour ranged from 56.26
to 64.00% and when the cultivars were cooked it decreased significantly (P
≤ 0.05) and was found to range from 30.19 to 39.87%. Similar results were
obtained by Yagoub (2003) for cooked karkade seed, who attributed that
loss on cooking to high susceptibility of albumin to heat treatment. For all
cultivars globulins content of uncooked flour ranged from 26.28 to 29.50%
and after cooking it ranged from 22.77 to 29.22%. The prolamins content of
uncooked flour ranged from 1.43 to 1.96 and after cooking it ranged from
1.00 to 1.64% for all cultivars. Glutelins content of uncooked cultivars
ranged from 2.10 to 3.50 and it was significantly (P ≤ 0.05) increased after
cooking and was found to range from 20.70 to 27.65%. It was clear that
83
cooking increased glutelins fraction by about more than 10 fold. The
increment in glutelin after cooking was reported in cereals by Fageer and El
Tinay (2004), Arbab and El Tinay (1997) and Yousif (2000).
4.16 Effect of cooking on SDS-PAG electrophoresis of lentil protein
fractions:
Figures 6-10 show the SDS-PAGE pattern of lentil protein fractions before
and after cooking of four cultivars. For all cultivars the number of bands of
globulin fraction ranged from 2 to 4 for cooked and uncooked samples,
respectively with low molecular weight ranging from 12 – 45.5 kDa (Figure
1). Number of bands for the prolamin fractions was greatly affected by
cooking for the cultivars Indian, Selaim and Rubatab they were found to
have four bands before cooking and after cooking showing two bands
reaching a molecular weight of ~56.0 kDa (Figure 2). Gultelin fractions had
bands with high molecular weight reaching 71 kDa. The number of bands of
this fraction ranged from 3 to 6 (Figure 3). Protein residue showed 4 – 5
bands with high molecular weight reaching 78.4 kDa. Within one cultivar,
the number of bands (subunits) of the total protein was affected by cooking.
Uncooked samples showed a high number of bands of total protein fractions
reaching 19, 17, 17 and 17 bands for cultivars Indian, Selaim, Rubatab and
Nadi, respectively. Cooking decreased these numbers to 16, 15, 16 and 13
for the cultivars Indian, Selaim, Rubatab, Nadi, respectively (Figure 4).
Albumin had 1 – 3 bands for cooked and uncooked samples with a
molecular weight ranging from 10.6 – 38.5 kDa (Figure 5). Results obtained
in this study are similar to those reported by Ahmed et al. (1995) on three
types of legumes. They found that chickpea seed protein containing 19bands
84
Table 11. Effect of cooking on in vitro protein digestibility (IVPD) of
lentil cultivars using pepsin and pepsin with pancreatin.
Cultivars Treatments
Nadi
Rubatab
Seliam
Indian
IVPD (%)
Pepsin
Pepsin with pancreatin
Uncooked
48.61 (± 0.60)a
81.76 (± 1.69)a
Cooked
20.93 (± 1.21)b
77.05 (± 1.42)b
Uncooked
50.21 (± 1.74)a
94.46 (± 1.06)a
Cooked
24.02 (± 0.53)b
81.55 (± 1.31)b
Uncooked
44.57 (± 2.11)a
99.88 (± 1.19)a
Cooked
22.32 (± 0.16)b
88.16 (± 2.60)b
Uncooked
51.96 (± 0.82)a
99.72 (± 2.74)a
Cooked
19.74 (± 0.29)b
88.65 (± 1.02)b
Values are means (±SD).
Means not sharing a common letter in a column are significantly different at (P≤ 0.05) as
assessed by Duncan multiple range test
85
Table 12. Effect of cooking on protein fractions (%) of lentil cultivars
Cultivar
Nadi
Rubatab
Seliam
Indian
Treatment Globulin
Albumin
Prolamin
Glutelin
Insoluble
Protein rec-
protein
overy (%)
Uncooked
56.26 (±1.03)a
56.26 (±1.03)a
1.96 (±0.08)a
2.53 (±0.06)b
8.23 (±0.00)b
97.15
Cooked
22.7 (± 0.00)b
35.23 (±2.03)b
1.64 (±0.19)b
27.35 (±1.52)a
12.2 (±0.01)a
99.19
Uncooked
26.47 (±0.00)a
64.00 (±0.00)a
1.50 (±0.00)a
3.50 (±0.00)b
7.89 (±0.00)b
103.36
Cooked
24.59 (±0.00)b
37.95 (±0.00)b
1.31 (±0.09)b
26.94 (±0.46)a
12.37 (±0.00)a
103.16
a
a
a
1.64 (±0.00)
2.10 (±0.00)
8.78 (±0.01)
100.67
61.87 (±2.23)
b
b
Uncooked
29.22 (±0.01)
Cooked
26.28 (±0.01)b
39.87 (±1.54)b
1.1 (±0.00)b
27.2 (±0.63)a
12.28 (±0.00)a
103.21
Uncooked
29.50 (±0.02)a
61.50 (±0.46)-a
1.43 (±0.12)a
3.20 (±0.26)b
7.10 (±0.01)b
102.73
Cooked
26.72 (±0.01)b
30.19 (±1.10)b
1.00 (±0.00)b
27.65 (±0.50)a
13.33 (±0.00)a
98.89
Values are means (±SD).
Means not sharing a common letter in a column are significantly different at (P≤ 0.05) as assessed by Duncan multiple range test
86
with a molecular weight ranging from 12 to 89 kDa. Faba bean
contained 25 bands with a molecular weight ranging from 12 to 78
kDa and terms seeds contained 16 bands with a molecular weight
ranging from 12 to 98 kDa. Chiou et al. (1997) reported that the
molecular weight of peanut cultivars proteins (untreated samples)
ranged from 14 to 67 kDa.
87
116
97.4
66.2
37.6
28.5
14.0
MW marker
1
2
3
4
5
6
7
8
Figure 6. SDS-PAGE pattern of globulin fraction of cooked and uncooked lentil
cultivars. Lane 1, Uncooked Indian; lane 2, Cooked Indian; lane 3, Uncooked
Selaim; lane 4, Cooked Selaim ; lane 5, Uncooked Rubatab; lane 6, Cooked
Rubatab; lane 7, Uncooked Nadi; lane 8, Cooked Nadi.
MW marker: 14.0, 28.5, 37.6, 66.2, 97.4 and 116 kDa..
88
116
97.4
66.2
37.6
28.5
14.0
MW marker
1
2
3
4
5
6
7
8
Figure 7. SDS-PAGE pattern of albumin fraction of cooked and uncooked lentil
cultivars. Lane 1, Uncooked Indian; lane 2, Cooked Indian; lane 3, Uncooked
Selaim; lane 4, Cooked Selaim ; lane 5, Uncooked Rubatab; lane 6, Cooked
Rubatab; lane 7, Uncooked Nadi; lane 8, Cooked Nadi.
MW marker: 14.0, 28.5, 37.6, 66.2, 97.4 and 116 kDa..
89
116
97.4
66.2
37.6
28.5
14.0
MW marker
1
2
3
4
5
6
7
8
Figure 8. SDS-PAGE pattern of prolamin fraction of cooked and uncooked lentil
cultivars. Lane 1, Uncooked Indian; lane 2, Cooked Indian; lane 3, Uncooked
Selaim; lane 4, Cooked Selaim ; lane 5, Uncooked Rubatab; lane 6, Cooked
Rubatab; lane 7, Uncooked Nadi; lane 8, Cooked Nadi.
MW marker: 14.0, 28.5, 37.6, 66.2, 97.4 and 116 kDa..
90
116
97.4
66.2
37.6
28.5
14.0
MW marker
1
2
3
4
5
6
7
8
Figure 9. SDS-PAGE pattern of glutelin fraction of cooked and uncooked lentil
cultivars. Lane 1, Uncooked Indian; lane 2, Cooked Indian; lane 3, Uncooked
Selaim; lane 4, Cooked Selaim ; lane 5, Uncooked Rubatab; lane 6, Cooked
Rubatab; lane 7, Uncooked Nadi; lane 8, Cooked Nadi.
MW marker: 14.0, 28.5, 37.6, 66.2, 97.4 and 116 kDa..
91
116
97.4
66.2
37.6
28.5
14.0
MW marker
1
2
3
4
5
6
7
8
Figure 10. SDS-PAGE pattern of insoluble fraction of cooked and uncooked
lentil cultivars. Lane 1, Uncooked Indian; lane 2, Cooked Indian; lane 3,
Uncooked Selaim; lane 4, Cooked Selaim ; lane 5, Uncooked Rubatab; lane 6,
Cooked Rubatab; lane 7, Uncooked Nadi; lane 8, Cooked Nadi.
MW marker: 14.0, 28.5, 37.6, 66.2, 97.4 and 116 kDa..
92
CONCLOSIONS
Results of this study indicated that the four lentil cultivars showed
variations in moisture, fat, fibre and ash contents but no variation
observed in total protein content; however the Indian cultivar had the
highest value of protein.
Minimum nitrogen solubility was observed at pH 5 indicating the
isoelecteric point of lentil protein. On the other side of pH 5 there was
a sharp increase in solubility of lentil protein, the nitrogen solubility in
sodium chloride extracts was lower than in water
Lentil has high protein content with acceptable functional properties
which makes it a promising protein source in food applications.
Germination resulted in remarkable changes in proximate composition
and food energy values of lentil and increased significantly phytase
activity while phytic acid decreased.
Germination resulted in significant decrease in total phytate
phosphorus with correspondingly marked increase in nonphytate
phosphorus. Total and extractable mineral elements (phosphorus,
calcium, iron, magnesium) were positively correlated with duration of
germination except copper and zinc.
Lentil was low in sulphur-containing amino acids (methonine,
cystiene) germination caused improvement in content of essential and
nonessential amino acids.
93
Cooking resulted in significant reduction in IVPD using pepsin or
pepsin with pancreatin and also reduced albumin fraction content
while glutelin content increased. The major protein in lentils was
albumin followed by globulin.SDS-PAGE of cooked and uncooked
proteins fractions showed that lentil protein was altered quantitatively
and qualitatively due to cooking. this effect was most pronounced in
prolamin fractions.
94
RECOMMENDATIONS
The findings of this study helped to establish the food utilization of
lentil because of its high content in protein with acceptable functional
properties which makes it a promising protein source in food system.
However further studies are required to relate potential functionality
of lentil protein to performance in specific food systems.
Germination could be applied to make highly nutrition meal: in total
and extractable minerals and amino acids content with low phytic
acid. Hence germination prior to consumption is recommended
Lentil can be used in cereal based foods that it raised its lysine
content.
The practice of consumption of germinated lentil by people in
developing countries should be encouraged in an attempt to reduce the
incidence of prevailing mineral deficiencies, lowering the phytic acid
content and improving content of essential amino acids.
Germinating lentil seeds for three days should be recommended.
Further studies are needed in studying the effect of the competitive
inhibition between food components (other than phytic acid) with
minerals and protein.
95
REFERENCES
Abdel Rahim, S.I. (2004). Effect of processing on anti-nutritional
factors and in vitro protein digestibility of faba bean (Vicia
faba). M.Sc. Thesis, Faculty of Agriculture, University of
Khartoum, Sudan.
Abdelrahman, S.M. (2004). Effect of malt pretreatment and
fermentation on anti-nutritional factors and mineral
bioavailability of pearl millet (Pennisetum glaucum L. ) Ph.D.
Thesis, University of Khartoum, Sudan.
Acton, J.C. and Saffle, R.I. (1970). Stability of oil in water emulsions.
I. Effects of surface tension, level of oil, viscosity and type of
meat protein. J. Food Sci. 35: 852-855.
Adsule, R.N.; Ladam, S.S. a nd Leung, H.K. (1989). Lentil in CRC
Handbook of World Food Legumes (eds. D.K. Salukehe and
S.S. Kadam) Boca Raton, Florida, USA, CRC Press.
Ahmed, F.A.; Abdel-Rahim, E.A.; Abdel Fatah, O.M.; Erdmann, V.A.
and Lippman, C. (1995). The changes of protein patterns during
one week of germination of some legume seeds and roots. J. of
Food Chemistry 52: 433-437.
Ali, A.E.; Ali, A.M. and Nordblom, T.L. (1984). Lentil consumption
in the Khartoum area. LENS Newsletter 1(11): 5-6.
Alonso, R.; Aguirne, A. and Marzo, F. (2000) Effect of extrusion and
traditional proce.
Anon (1984). Removal of phytic acid in soybean protein to increase
the utilization of trace elements in soybeans and also the
solubility of soybean protein, Report of the North Regional
Research Center, Agricultural Research Service, U.S.
Department of Agriculture, Washington, D.C. 11.
. AOAC (1990) Official Methods of Analysis of the Association of
Official Analytical Chemists, 15th edn. (Helrich K., ed)
Arlington, VA
96
Arbab, M.E. and El Tinay, A.H. (1997). Effect of cooking and
treatment with sodium bisulphate or ascorbic acid on the in
vitro protein digestibility of two sorghum culivrs. Food
Chemistry, 59: 339-343.
Asada, K.; Tanakak,; Kasai, Z.. (1969). Formation of phtyic acid in
cereal grains. Ann. N.Y. Acad. Sci. 165: 801-814.
Augustin, J. and Klein, B.P. (1989). Nutrient composition of raw,
cooked canned and sprouted legumes. In: Matthews, R.H.
Editor, 1989. Legumes chemistry, technology and human
nutrition, Marcel Dekker, New York, pp. 187-217.
Bahl, P.N., S.Lal and B.M.Sharma. (1993). An overview of the
production and problems in South East Asia p.110.In.W.Erskine and M.C. Saxena (eds),Lentil in South Asia.
Proceedings of the seminar on lentils in South Asia.ICARDA,
Aleppo, Syria.
Barasi, M.E. (1997). Human nutrition. A health perspective, New
York, Oxford University Press, 328 p.
BeMillers, J.N. and whistler, R.L. (1996). Carbohydrates. In: Owen,
R., Fennema (ed) food chemistry, 3rd edn. Marcel Dekker, Inc.
270 Madison Av. New York, 10016, USA, pp. 158-91.
Betschart, A.A.; Fong, R.Y. and Hanamoto, M.M. (1979). Safflower
protein isolates: Functional properties in simple systems and
breads. J. Food Sci.44: 1022-1035.
Beuchat, L.R.; Chjerry, J.B. and Quinn, M.R. (1975).
Physicochemical properties of peanut flour as affected by
proteolysis. J. of Agricultural and Food Chemistry 23: 616-620.
Bhatty, R.S. (1986). Protein subunits and amino acid composition of
wild lentil. Phytochemistry, 25 3: 641-644.
Bhatty, R.S.(1988). Composition and quality of lentil (Lens culinaris
medic): A review. Canadian Institute of Food Science and
Technology Journal,21: 2, 144-160.
Bhatty,R.S.;and Slinkard, A.E. (1979). Composition, starch properties
and protein quality of lentils. Canadian Institute of Food
Science and Technology Journal, 12: 88-92.
97
Bishnoi, S. and Khetarpaul, N. (1994). Protein digestibility of
vegetable and field peas (Pism sativum): Varietal differences
and effect of domestic processing and cooking. Plant Foods
Hum. Nutr. 49: 71-76.
Biswas, S. and Biswas, B.B. (1965). Enzymatic synthesis of
guanosine triposphate from phytin and guanosine diphosphate.
Biochm.Biophys. Acta, 108: 710.
Bora P.S. (2002). Functional properties of native and succinated lentil
(Lens Culinars) globulin. J. Food Chem. 171-176.
Bressani, R.; Eias, L.G. and Braham, J.E. (1984). Efecto
deldescascarado sorbe el volor proteiico y complementario del
frijol comun. INCAP Informe Annual p. 62-63 (cited in
Berssani).
Brune, M.; Rossander-Hulten, L.; Hallberg, L.; Gleerup, A. and
Sandberg, A. (1992). Iron absorption from bread in human:
inhibiting effects of cereal fibre phytate and inositol phosphates
with different numbers of phosphate groups. Journal of
Nutrition, 122: 442-449.
Bryant, L.A.; Montecalvo, J.; Morey, J.R.; and Loy, K.S. (1988).
Processing functional and nutritional properties of okra seed
products. J. Food Sci.l 53: 616-620.
Carbonaro, M.; Cappelloni, M.; Nicoli, S.; Lucarini, M. and
Carnovale E. (1997). Solubility-digestibility relationship of
legume proteins. Journal of Agricultural and Food Chemistery,
45, 3387 – 3394.
Carbonaro, M.; Carnovale, E. and Vecchini, P.(1993). Protein
solubility raw and cooked beans (Phaseolus vulgaris): Role of
the basic residues. J. Agric. Food Chem., 41: 1169-1175.
Champagne, E.T. and Hinojosa, O. (1987). Independent and mutual
interaction of copper (II) and zinc (II) ions with phytic acid.
Journal of Inorganic Biochemistry, 30: 15-22.
Chen, L.H.; Pan, S.H. Cited in J.V. Erdman Jr. : Oilseed phytates:
Nutritional implications. Jam. Oil Chem. Soc.l56: 739-7410
(1979).
98
Cheryan, M. (1980). Phytic acid interactions in food systems. CRC.
Crit. Rev. Food Sci. Nutr. 13: 297.
Chioce, R.Y.; Ku,K.L. and Chen, W.L. (1997). Compositional
characterization of peanut kernels after subjection to various
germination times. J. Agric. Food Chem. 45: 3060-3064.
Chiou, R.Y.Y.; Ku, K.L and Chen,W.L.(1997). Compositional
characterization of peanut kernels after subjection to various
germination times. Journal of Agricultural and Food
Chemistery, 45,3060 – 3064.
Churella, H.R. (1976). Phytic acid level in infant soy protein isolate
formulans and its effect on mineral availability to the rat, Ph.D.
Thesis, Ohio State University, Columbus.
Coffman, C. and Garcica, V.V. (1977). Functional properties and
amino acid content of protein isolate frommugbean flo9ur.
Journal of Food Technology, 12:473-478.
Cosgrove, D.J.(1980). Inostiol phosphates their chemistry,
biochemistry and physiology. Amsterdam: Elsevier Scientific
Publishing Company.
Damodaran, S. (1996). Amino acids peptides and proteins. In: Owen,
R., Fennema (ed) food chemistry, 3rd edn. Marcel Dekker, Inc.,
270 Madison Avenue. New York, 10016, USA. pp. 322-425.
Darcy, B. (1984). Availability of amino acids in monogastric animals.
Variations of digestive origin. Cited by Desrosiers et al. J. Food
Sci. 1987, 52: 1525-1528.
Deshpande, S.S. and Damodaran, S. (1989). Structure – digestibility
relationship of legume proteins. J. Food Sci., 54: 108-113.
Deshpande, S.S. and Damodaran, S. (1990). Food legumes: Chemistry
and technology. In: Advances in cereal science and technology
(ed. Pomeranz, Y.) St. Paul, American Association of Cereal
Chemists, 147-241.
Desrosiers, T.; Bergeron, G. and Savoie, L. (1987). Effect of heat
treatment in vitro digestibility of delactosed whey protein as
determined by the digestion cell technique. J. Food Sci.,52:
1252-1528.
99
Dieckert, J.W.; Snowden, J.E.; Jr., Moore, A.T.; Heinzelman, D.C.
and Altschul, A.M. (1962). Composition of some sub-cellular
fractions from seed of Arachis hypogaea, J. Food Sci., 27: 321.
Doughty, J. and Walker, A. (1982). Las leguminosas en nutricion
humana, FAO, Organicion de las Naciones Unidas para la
Agriculturay la Alime ntacion, Rome Italie.
Duhan, A.; Khetarpaul, N. and Bishnoi, S. (2002). Content of phytic
acid and HCl-extractability of calcium, phosphorus and iron as
affected by various domestic processing and cooking methods.
Food Chem. 78: 9-14.
Duke, J.A. (1981). Handbook of legumes of world economic
importance Plenum Press. New York, p. 52-57.
Eastwood, D. and Laidman, D.L. (1971). The metabolization of
macro-nutrient element in the germination wheat grain. Phytochemistry 10: 1275-1284.
Elizalde, B.E.; Pilosof, A.M.R.; Bartholomi, G.B. (1991). Prediction
of emulsion instability from emulsion proteins. J. Food Sci., 56:
116-119.
Engelen, A.J.; Vander Heeft, F.C.; Randsddrop, P.H.G. and Simt,
E.L.C. (1994). Simple and rapid determination of phytase
activity. J. AOAC Inter 77: 760-764.
Evans, R.J. and Bauer, D.H.(1978). Studies of the poor utilization of
the rat of methiomine and cystine in heated dry bean seed
(Phaseolus vulgaris). J.Agric.Chem. 26: 779-784.
Fageer, A.S.M. and El Tinay, A.H. (2004). Effect of genotype, malt
pretreatment and cooking in in vitro protein digestibility and
protein fractions of corn. Food Chemistry 84: 613-619.
Fan, T.Y. and Sosulski, F.W. (1974). Dispersibility and isolation of
proteins from legume flour. Can. Inst. Food Si. Technol. J.7:
256.
Fennema, R.O. (1996). Food chemistry, (3rd edition). pp 365-396.
Marcel Dekker, Inc. New York. Basel, Hong Kong.
100
Flanagan, P.R. (1984). A model to produce pure zinc deficiency in
rat and its use to demonstrate that dietary phytate increase in the
excretion of endogenous zinc. Journal of Nutrition, 114:
493-502.
Frias, J.; Diaz-Pollan, C.; Hedley, C.L. and Vidal-Valverde, C. (1995).
Evolution of trypsin inhibitor activity during germination of
lentils. Journal of Agriculture and Food Chemistry, 43, pp.
2231-2234.
Frias, J.; Doblado, R.; Antezana, J.R. and Vidal-Valverde, C. (2003).
Inositol phosphate degradation by the action of phytase enzyme
in legume seeds. J. Food Chem. 81: 233-239.
Friberg, S.E. and Venable, R.V. (1983). Microemulsion. In
"Encycopedia of emulsion technology" Vol. 1, (ed.) Becher, pp.
287. Marcel Dekker, Inc. New York.
Ghorpade, V.M. and Kadam, S.S. (1989). In: Salunke, D.K. and
Kadam, S.S., Editors, 1989. CRC handbook of world food
legumes: Nutritional chemistry, processing technology and
utilization. Vol. 111 CRC, Boca Raton, FL, pp. 165-206.
Gifford, S.R. and Clydesdale, F.M. (1990). Interactions among
calcium, zinc and phytate with three different sources. Journal
of Food Science, 55: 1720-17**.
Gilboly, M.; Bothwell, T.H.; Torance, J.D. et al. (1983). The effect of
organic acids, phytates and polyphenols on iron absorption from
vegetables. British Journal of Nutrition, 49: 331-342.
Hagenmair, R. (1972). A research note: Water binding to some
purified oilseed proteins. J. Food Sci., 37: 965-966.
Hang, Y.D.; Steinkraus, K.H. and Hackler, L.R. (1970). Comparative
studies on the nitrogen solubility of mung beans, pea beans and
red kidney beans. J. Food Sci., 35: 318-320.
Hansen, J.R. (1978). Hydration of soybean protein: Effect of isolation
method and various other parameters on hydration. J. Agric.
Food Chem. 26: 301-304.
Hawtin, G.C.; Rachie, K.O. and Green, J.M. (1977). Breeding strategy
for the nutritional improvement of pulses. In: J.H. Hulse, K.O.
101
Rachie and L.W. Billingsley (Editors) Nutritional standards and
methods of evaluation for food legume breeders. IDRC, ttawa,
Ont. pp 43-51.
Honke, J.; Kozlowska, H.; Vidal-Valverde, C.; Frias, J.; Gorecki, R.
(1998). Changes I quantities of inositol phosphates during
maturation of legume seeds. Zeitschrift. Fülebensmittle
Untersuchungund Forschung 2006: 279-283.
Hsu, D.L.; Leang, H.K.; Morad, M.M.; Finney, P.L. and Leung, C.T.
(1982). Effect of germination on electrophoretic, functional and
bread baking properties of yellow pea, lentil and faba bean
protein isolate. Cereal Chemistry, 58: 344-350.
Huisman, J. and Vanderpoel, A.F.B. (1994). Aspects of the nutritional
quality and use of cool season food legumes in animal fed p.
53-76. In: F.J. Muehlbauer and W.J. Kaiser (eds), Expanding
the production and use of cool season food legumes. Kluwer
Academic publishers, Dordrecht, The Netherlands.
Hulse, J.H. (1990). Nature, composition and utilization of grain
legumes. p. 11-27. in: ICRISAT. Uses of tropical grain
legumes: Proceedings of consultants method, 27-30 Mar, 1989.
ICRISAT Center, India, Patancheru, A.P. 502324. India:
ICRISAT.
Hutton, C.W. and Campbell, A.M. (1981). In: Protein functionality in
foods, J.P. Cherry (ed.), American Chem. Soc. Washington,
DC, ACS Symps. Ser. No. 147, p. 177.
Idouraine, A.; Yenesen, S.B. and Weber, C.W. (1991). Teparybeen
flour, albumin and golublin fractions isolated. J. Food Sci., 56:
1316-1318.
Jansen, G.R. (1980). A consideration of allowable fibre levels in
weaning foods. Food Nutr. Bull. 2: 38-47.
Johnson, D.W. (1970).Oilseed proteins properties and applications.
Food prod. Devel. 3, 8, 78.
Karel, M.; Schaich, K. and Roy, B.R. (1975). Interaction of
peroxidizing methyl linoleate with some proteins and amino
acids. J. Agric. Food Chem., 23: 159-163.
102
Kasai, Z. and Asada, K. (1959). The behaviour of mineral nutrients
absorbed by crops. V. The behaviour of phosphorus absorbed at
each stage of growth in ripening process of rice plant. Mem.
Res. Inst. Food Sci. Kyoto Univ. Kyoto, Japan, 18: 22.
Kataria, A. and Chauhan, B.M. (1988). Content and digestibility of
carbohydrates of mug eans (Vigna radiate L.) ass affected by
domestic processing and cooking. Plant Foods Hum. Nutr. 38:
51-59.
Kataria, A.; Chauhan, B.M. and Puria, D. (1989). Anti-nutrients and
protein digestibility (in vitro) of Mung bean as affected by
domestic processing and cooking. Food chemistry, 32: 9-17.
Khalid, E.K.; Babiker, E.E. and El Tinay, A.H. (2003). Solubility and
functional properties of sesame seed proteins as influenced by
pH and/or salt concentration. J. Food Cehm. 82: 361-366.
Kim, S.Y.; Park, P.S.W. and Rhee, K.C. (1990). Functional properties
of proteolytic enzyme modified by spy protein isolate, J. Agirc.
Food Chem., 38: 651-656.
Kinsella, J.E. (1976). Functional properties of proteins I foods. A
survey CRC Crit. Rev. Food Sci. Nutri. 7: 219-280.
Kinsella, J.E. (1979). Functional properties of soy proteins. J. Am. Oil
Chem. Soc., 56: 242-257.
Kinsella, J.E.; Damodaran, S.; German, B. (1985). Physicochemical
and functional properties of oilseed proteins with emphasis on
soy proteins. In new protein foods, Altschul, A.M., Wikke,
H.L., Eds.; Academic Press, New York, Vol. 5, pp. 107-179.
Kozlowska, H. Honke, J. Sadowska, J. Frias, J. and Vidal-Valverde,
C. (1996). Natural fermentation of lentils. : Influence of time,
concentration and temperature on the kinetics of hydrolysis of
inositol phosphorus. J. of Food and Agric. 71: 367-375.
Kratzer, F.H. and Vohra, P.(1986). Role of phytic acid and other
phosphorus as chelating agents. In F.H. Kratzer and P. Vohra
(eds), chelates in nutrition. Boca Raton, Fl: CRC Press.. 49-61.
103
Krober, O.A.; Jacob, M.K.; Lai, R.K. and Kashkary, V.K. (1970).
Effects of variety and location o the protein content of pulses.
Ind. J. Agric. Sci., 40: 1025-1030.
Kulkarni, D.K.; Kulkarni, D.N. and Ingle,U.M.(1991). Sorghum maltbased weaning food formulations: Preparation, functional
properties and nutritive value. Food and nutrition Bulletin,13:
324-327.
Kuo, Y.H.; Rozan, P.; Lamein, F. and Vidal-Valverde, C. (2003).
Effect of different germination conditions on the contents of
free protein and non protein amino acids of commercial
legumes.
Kuvaeva, E.B. and Knetovich, V.L. (1978). Phytase of germinating
pea seeds. Sov. Plant Physiol. (Engl. Transl.) 25: 290.
Laemmli, U.K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature,227: 680-685.
Lawhon, J.T.; Cater, C.M. and Mattil, K.F. (1972). Whippable extract
from glandless cottonseed flour. J. Food Sci., 37: 317-321.
Lea, P.J. and Joy, V. (1983). Amino acid inter-conversion in
germinating seeds. In: Nozzolillo, c. Lea, P.J. and Loewus, F.A.
Editors, 1983. Recent advances in phytochemistry Vol. 17,
Plenum, New York, pp 77-109.
Levrat-Verny, M.A. and Coudray, C.; Bellanger, J. et al. (1999).
Whole wheat flour ensures higher mineral absorption and
bioavailability than white wheat flour in rats. British Journal of
Nutrition, 82: 17-21.
Lin, C.S. and Zagas, J.F. (1987). Functionality of corn germ proteins
in a model system: Fat binding capacity and water retention. J.
Food Si. 52: 1308.
Lin, L.H. and Hung, T.V. (1998). Functional properties of acetylated
chickpea proteins. J. Food Sci., 63: 331-336.
Lin, M.J.Y.; Humbert, E.S. and Sosulski, F.W. (1974). Certain
functional properties of sunflower meal products. J. Food Sci.,
39: 368-370.
104
Lolas, G.M. and Markakis, P. (1975). Phytic acid and other
phosphorus compound of beans (P. vulgris L.). J. of Agric. And
Food Chem. 23: 13-15.
Lolas, G.M. and Markakis, P.(1977). The phytate of navy bean
(Phaseolu vulgaris). J.Food Sci. 42: 1094.
Lönnerdal, B.; Sandberg, A.S.; Sandstrom, B. and Kunz, C. (1989).
Inhibitory effects of phytic acid and other inositol phosphates
on zinc and calcium absorption in suckling rats. Journal of
Nutrition, 119: 211-214.
Lopez, H.W.; Leenhardt, F.; Conjdray, C. and Remesy, C. (2002).
Minerals and phytic acid interaction: it is a real problem for
human nutrition? International Journal of Food Science and
Technology, 37: 727-739.
Lui, N.S.T. and Altschul,A.M. (1967). Isolation of globoids from
cottonse aleurone grain. Arch. Biochem. Biophys. 121: 678.
Maiti, I.B.; Majumdar, A.L. and Biswas, B.B. (1974). Purification and
mode of action of phytase from phaseolus vulgaris.
Phytochemistry,13: 1047.
Mahajan,S. and Chauhan,B.M.(1988).Effect of natural fermentation
on the extractability of minerals from pearl millet flour.53 (5)
1576-1577.
Maliwal, B.P.(1983). In vitro method to assess the nutritive value of
leaf concentrate. J. of Agric. And food Chemistry, 31: 315-319.
Mandal, N.C. and Biwas, B.B. (1970). Metabolism of inositol
phosphates. I. Phytase synthesis during germination
incotyledons of mug beans, Phaseolus aureus plant Physiol. 45:
4-7.
Marina,C.B. 1986.Functional properties of drum-dried chickpea
(Cicer arietinum L) flours.J.Food Sci.,63:331-336.
Mattil, K.F. (1971). The functional requirements of proteins for foods.
J. Amer. Oil. Chem.. Soc., 48: 477-480.
Mayer, A.M. (1958). The breakdown of phytin and phytase activity in
germinating lettuce seeds. Enzymologia, 19: 1.
105
Maynard, L.A. and Loosli, J.K. (1962). The nutrients and their
metabolism. In: L.A. Maynard and J.K. Loosli, animal nutrition,
5th en, McGraw-Hill book Coo., Inc. The maple Press Co., New
York, pp 10-38.
McWatters, K.H. and Holmes, M.R. (1979). Influence of pH and salt
concentration on nitrogen solubility and emulsification
properties of soy and peanut flours. J. Food Sci. 44: 770-773.
McWattes, K.H. and Cherry, J.P. (1977). Emulsification, foaming and
protein solubility properties of defatted soybean, peanut, field
pea and bean flours. J. Food Sci., 42: 6, 1444-1447.
Meyer, H.; Mayer, A.M.; Harel, E. (1971). Acid phosphatases in
germinating lettuce – evidence for partial activation. Physiol.
Plant. 24: 95-101.
Miyazawa, E.; Iwabuchi, A. and Yoshida, T. (1996). Phytate
breakdown and apparent absorption of phosphorus, calcium and
magnesium in germ free and conventionalized rats. Nutrition
Research, 16: 603-613.
Molina, M.R.; Argueta, C.F. and Bressani, R. (1974). J. Agric. Food
Chem. 2: 309.
Monjula, S. and John, E. (1991). Biochemical changes and in vitro
protein digestibility of endosperm of germinating Dolichos
lablab. J. Sci. of Food and Agric. 55: 229-233.
Morton, R.K. and Raison, J.K. (1963). A complete intracellular unit
for incorporation of amino acid into storage protein utilizing a
deonsine triphosphate generated from phytate nature (London)
200: 429.
Mosse, J.I. Baudet, J. (1983). Crude protein content and amino acid
composition of seeds: Variability and correlations. In: plant
protein for human food, proceedings of a European.
Muehlbauer, F.J.J.I. Cubers and Summerfied (1985). Lentil (Lens
culinans medic) pp. 226-311 in R.J. Summerfield and E.H.
Roberts (eds), Grain legume crops. Collin and Graffon Street,
London, U.K.
106
Nawar, W.W. (1996). Lipids. In: Owen, R., Fennema (ed) food
chemistry, 3rd edn. Marcel Dekker, Inc., 270 Madison Avenue.
New York, 10016, USA. pp. 226-254.
Nayini, N.R. and Markakis, P. (1986). Phytases, in phytic acid:
Chemistry and Application. Pilatus Press, Minneapolis.
Nelson, R.L.; Yoo,k S.J.; Tanure, J.C.; Adrian-Opoulos, G. and
Misumi, A. (1989). The effect of iron in the experimental
colorectal carcinogenesis. Anticancer Research, 9: 1477-1480.
Nickel, K.P.; Nielsen, S.S.; Smart, D.J.; Mitchell, C.A. and Belury,
M.A. (1997). Calcium bioavailability of vegetarian diets in rats:
potential application in a bio-regenerative life support system.
Journal of Food Science, 62: 619-6231.
Nielson, S.S. and Liener, I.E. (1984). Degradation of the major
storage proteins of Phasedu vulgaris during germination. Role
of endogenus proteases and proteases inhibitor. Plant Physio.
74: 494-502.
O'Dell, B.L.; De Borland, A.R. and Koirtyohann, S.R. (1972).
Distribution of phytate and nutritionally important elements
among the morphological components of cereal grains. Journal
of Agricultural and Food Chemistry, 20: 718-721.
Okubo, K. and Waldrop, A.B.; Lacobucci, G.A. and Myers, D.V.
(1975). Preparation of low-phytate soybean protein isolate and
concentrate by ultra-filtration, cereal chem. 52: 263.
Oloya, R.A.(2004). Chemical and nutritional quality changes in
germinating seeds of Cajanus cajan L. Food Chem. 85:
479-502.
Omosaiye, O. and Cheryan, M.(1979). Low-phytate, full fat soy
protein product by ultra-filtration of aqueous extracts of whole
soybeans, cereal chem.. 56: 58.
Oplinger, E.S.; Hardman, L.L. Kaminiski, A.R.; Kelling,
K.A.(1990).Lentil,in Alternative crop Maual. University of
Minnesota, center for alternative plant and animal products. The
Minnesota Extension Services, St. Paul, Minnesota.
107
Osborne D.R. and Voogt, P. (eds) (1978). Calculation of calorific
value. In the analysis of nutrients in foods. New York,
Academic press.
Osborne, T.B. and Mendel, L.B. (1914). Nutritional properties of
maize kernel. Cited by Soch, L.V.; Shoup, F.K.; Bathurst, J. and
Liang, D.J. Cereal Chem. (1970), 47: 472-481.
Osman, E.A. (1990). Lentil quality attributes in relation to cultural
practices and genetic constitution of the crop. M.Sc. Thesis,
University of Khartoum, Sudan.
Osman, N.M. (2004). Effect of Autoclaving on solubility and
functional properties of Chick pea (Cicer aretinum) flour.M.Sc.
thesis.faculty of Agriculture. University of Khartoum. Sudan.
Otterburn, M.; Healy, M. and Sinclair, W. (1977). The fermentation
isolation of importance of isopeptides in heated proteins. In: M.
Friendman (ed), protein cross-linking, nutritional and medical
consequences, Plenum Press, New York, p. 239.
Padmashree, T.S.; Vijayakshmi, L. and Puttaraj, J. (1987). Effect of
traditional processing on functional properties of cowpea
(Vigna catjang). J. Food Sci. Techn. 324: 221-224.
Patel, M.T. and Kilara, A. (1990). Studies on whey protein
concentrates. 2. foaming and emulsifying properties and their
relationship with physicochemical properties. J. Diar. Sci., 73:
2731-2740.
Peers, G.F. (1953). The phytase of wheat. Biochem. J.53: 102.
Peleg, M. and Bagley, E.B. (1983). Physical properties of foods. Avi. :
West Prto, ct.
Petruccelli, S. and Anon, M.C.(1994). Relationship between the
method of abstention and the structural and functional
properties of soy protein isolate. 11. surface properties. J. Agric.
Food Chem. 42: 2170-2170.
Plate, S.R. and Clydesdale, F.M. (1987). Interaction of iron, alone and
in combination with calcium, zinc and copper, with phytate
rich, fibre rich fraction of wheat bran under gastrointestinal pH
condition. Cereal Chemistry, 64: 102-105.
108
Posternak, S. (1903). Serum nouveau principle phosphor-organique di
arigine vegetale, La phytine, C.R. Soc. Biol. 55, 1190.
Prakash, V. and Narasinga Rao, M.S. (1986).l Physiochemical
properties of oilseed proteins. CRC Crit. Rev. Biochem. 20:
265-363.
Quinn, M.R. and Beuchat, L.R. (1975). Functional property changes
resulting from fungal fermentation of peanut flour. J. Food Sci.
40: 475-478.
Ragab, D.M.; Babiker, E.E. and El Tinay, A.H. (2004). Fractionation,
solubility and functional properties of cowpea (Vigna
unguiculata) protein as affected by pH and/or salt
concentration. Food Chem. 84: 204-412.
Reddy, M.B.; Hurnel, R.F.; Juillerat, M.A. and Cook, J.D. (1996). The
influence of different protein sources on phytate inhibition of
nonheme-iron absorption in humans. American Journal of
Clinical Nutrtion,63:203-207.
Reddy, N.P.; Sathe, S.K. and Salunkhe, D.K. (1982). Phytase in
legume and cereals. Advances in Food Research, 28: 1-9.
Reinhold, J.; Faradji, B.; Abadi, P. and Ismail-Beigi, F. (1976).
Decreased absorption of calcium, magnesium, zinc and
phosphorus by humans due to increased fibre and phosphorus
consumption as wheat bead. Journal of Nutrition,106: 493-503.
Rhee, K.C. (1985). Peanuts (Groundnuts) in new protein foods, Vol.
5, seed storage proteins, Altschul, A.M., Wikle, H.L., ed.
Academic Press. New York. pp 359-391.
Rimbach, G.; Pallauf, J.; Brandt, K. and Host, E.(1995). Effect of
phytic acid and microbial phytase on Col accumulation Zn
status and apparent absorption of Ca,. P, Mg, Fe, Zn, Cu and
Mn in growing rats. Annals of Nutrtion and Metabolism, 39:
361-372.
Ryan, D.S. (1977). In: R.E. Feeney, and J.R. Whitaker (eds). Food
proteins improvement through chemical and enzymatic
modification, Washington, DC, p. 67.
109
Saio, K.; Gallant, D. and Petit, L. (1977). Electron microscope
research on sunflower protein bodies, cereal chem.. 54: 1171.
Sandberg, A.S. and Ahderinne, R. (1986). HPLC method for
determination of inositol tri-, tetra-, penta-, and hexa phosphates
in food and intestinal contents. J. of Food Sci. 51: 457-550.
Sandberg, A.S. and Svanberg, U. (1991). Phytate hydrolysis by
phytase in cereal effect of in vitro estimation of iron
availability. Journal of Food Science, 56: 30.
Sandberg, A.S.; Larsen, T. and Sandstrom, B. (1993). High dietary
calcium level decrease colonic phytate degradation in pigs fed a
rapeseed diet. Journal of Nutrition, 123: 559-566.
Sarwar, G.; Peace, R.W. (1986). Comparisonbetween true digestibility
of total nitrogen and limiting amino acids in vegetable proteins
fed to rats. J. Nutr. 116: 1172-1184.
Satirana, M.L. and Bianchetti, R. (1967). The effect of phosphate on
the development of phytase in the wheat embryo. Physiol.
Plant. 20: 1066-1075).
Saunders, R.M.; Connor, M.A.; Booth, A.N.; Bickoff, E.N. and
Koheir, C.O. (1973). Measurements of digestibility of alfalfa
protein concentrate by in vitro and in vivo methods. J. Nutr.,
103: 530-535.
Savage, G.P. (1988). The composition and nutritive value of lentils
(lens culinaris). Nutrition Abstracts and Review (series A), 5
(58): 320-343.
Schmidt, R.H. (1981). Gelation and coagulation page 13 lin: Protein
functionality in foods. ACS symp. Ser. 147. J.P. Cherry –ed.
Am. Chem. Soc., Washington, DC.
Schut, J. (1976). Meat emulsion, in: Friberg, S. (ed.) Food Emulsions.
Marcel Dekker Inc., New York, pp. 385-458.
Sgarbieri, V.C. and Galeazzik, M.A.M. (1978). Some physiochemical
and nutritional properties of sweat lupin (L. albus cv multolupa)
protein. J. Agric. Food Chem. 26 (6): 1438-1442.
110
Shastry, M. and John, E. (1991). Biochemical changes and in vitro
protein digestibility of the endosperm of germinating Doichos
lablab. J. of the Sci. of Food and Agric. 52: 529-538.
Sinclarie, H.M. (1961). The nutrients and their metabolism. Cited in:
L.A. Maynard and J.K. Loosli, Animal nutrition. 5th edn., 1962,
McGraw-Hill book co., Inc., New York, pp. 10-38.
Sodek, L. and Wilson, C.M. (1971). Amino acid composition of
protein isolated from normal, opaque-2, and floury-2 corn
endosperm by a modified Osborne procedure. J. Agric. Food
Chem. 19: 1144-1149.
Soetrison, U. and Holnes, Z.A. (1992). Protein yield and
characteristics from acid and salt coagulation on yellow pea
(Psim sativum L. Miranda) flour extraction. J. Agric. Food
Chem. 40: 975-980.
Sotelo, A.I.; Sousa, H.; Sanchez, M. (1995). Comparative study of the
chemical composition of wild and cultivated beans (Phaseolus
vulgaris). Plant Food for Human Nutrition, 47: 94-100.
Sutcliffe, J.F. and Bryant, J.A. (1977). Biochemistry of germination
and seedling growth. In: Sutctiffe, J.F. and Pate, J.S. Editors,
1977. The physiological of garden pea, Academic Press,
London, UK, p. 45-83.
Swaisgood, H.E and Catignani, G.L. (1991). Protein digestibility: in
vitro methods of assessment. Advances in Food Nutrition
Research 85: 185-236.
Tovar, J.; de Francisco, A.; Bajorck, I. and Asp, N.G. (1991).
Relationship between microstructure and in vitro digestibility of
starch in precooked leguminous seed flours. Food Struct. 10:
19-26.
Turner, J.R. (1969). Modified soy protein and the perpetration thereof.
Cited by Puski, g., Cereal Chem.., 1975, 52: 665-664.
Turnlund, J.R.; Kind, J.C.; Gong, B.; Keyes, W.R. and Michel, M.C.
(1985). A stable iso-tope study of copper absorption in young
men: Effect of phytate and α-cellulose. American Journal of
Clinical Nutrition, 42: 18-23.
111
Venktech, A. and Prakash, V. (1993). Functional properties of the
total proteins of sunflower (Helianthus annus L.) seed: Effect of
physical and chemical treatments. J. Agric. Food Chem. 41:
18-23.
Vidal-Valverde, C.; Frias, J.; Estrella, I.; Gorospe, M.J.; Ruiz, R. and
Bacon, J. (1994). Effect of processing on some anti-nutritive
factors of lentils. J. Agric. Food Chem. 42: 2291-2295.
Vidal-Valverde, C.; Frias, J.; Sierra, I.; Blazquez, I.; Lambeun, F. and
Kuo, Y.H. (2002). New functional legume food by germination
effect of nutritive value of beans, lentil and peas, c-u. Food Res.
Tech., 215: 472-476.
Viresos, A.; Centeno, C.; Brenes, A. Canales, R.; Lazano, A. (2000).
Phytase and acid phosphatase activities in plant feedstuffs. J. of
Agric. Chem. 48: 4009-4013.
Volkert, M.A. and Klein, B.P. (1979). Protein dispersibility and
emulsion characteristic of flour soy products. J. Food Sci., 44:
43-96.
Voutsinas, L.P. and Nakai, S. (1983). A simple tubidemetric method
for determining the fat binding capacity of proteins. J. Food
Sci., 48: 26-32.
Wang, J.C. Kinsella, J.E. (1976). Functional; properties of novel
protein alfalfa leaf protein. J. Food Sci. 41:286-292.
Webster, E. (1928). Phosphorus distribution in grains, J. Agric. Res.
37: 123.
Yaghoub, A.A. (2003). A biophysical study on total proteins of the
traditionally fermented Roselle (Hibiscuss sabdariffa L.) seed
"Furundu", Ph.D. Thesis, Faculty of Agriculture, University of
Khartoum, Sudan.
Yousif, N.E. (2000). Effect of fermentation and dry cooking following
fermentation on protein fractions and in vitro protein
digestibility of sorghum, corn and rice. Ph.D. Thesis, Faculty of
Agriculture, University of Khartoum, Sudan.
112
Zahary, D. (1972). The wild progenitor and place of origin of the
cultivated lentil (Lens culinaris M.). Economic Botany,236:
326-332.
113