Wiryawan 1997 RIRDC New Protein Layers

Rural Industries Research and Development Corporation
Egg Industries Research and Development Committee
PLEASE NOTE: THIS REPORT IS NOT PUBLISHED IN HARD PRINTED FORM
FINAL REPORT FOR PROJECT : UQ-21E
NEW VEGETABLE PROTEIN FOR LAYERS
Supervisor : Dr. J. G. Dingle
Researcher : Mr. K.G. Wiryawan
Department of Animal Production

The University of Queensland
Gatton, 4345
December 1997
SUMMARY
Main Title: NEW VEGETABLE PROTEIN FOR LAYERS
Objectives: To find rapid methods of assessing the protein and energy value of ten grain
legumes and to test methods designed to improve the nutritional value of grain
legumes for poultry.
Background: About 20 out of possible 10,000 grain legumes are cultivated for human food
some of which is available for animal feed. However, despite having good
amino acid and energy contents, grain legumes are not used very much at
present for animal feed because of uncertainty about their possible negative
effect due to anti-nutritional factors. Therefore information about the nutritive
value of grain legumes and about methods of neutralising their anti-nutritive
factors is necessary to give animal feed buyers confidence to use them in
poultry feeds.
Results: Laboratory and chick trials were conducted to analyse the grain legumes
chemically, physically and nutritionally.
Outcomes: Nine raw grain legumes and processed soybean meal could be classified into
high, medium, and low protein quality. Methionine supplementation and
protease enzymes and heating in an autoclave were highly beneficial and grain
legumes produced better growth rates than soybean meal did. Then nine grain
legumes could be grouped into high, medium and low for true metabolizable
energy. Carbohydrase enzymes tended to improve the ME values for all grain
legumes.
Implications: As the price of regular feed ingredients increases it is important to have
alternative feed supplies available. Grain legumes are good alternative sources
of feed although some species of grain legumes require further treatment or
supplementation to enable their nutrients to be available.
Publications:
1. Dingle, J.G. and Wiryawan, K.G. (1994). Proc.Qld.Poul. Sci. Sym. 3: 73-82.
2. Wiryawan, K.G. and Dingle, J.G. (1995a). Br. J.Nutr. 74: 671-679.
3. Wiryawan, K.G. and Dingle, J.G. (1995b). Proc. Nutr. Soc. Aust. 19. p. 203.
4. Wiryawan, K.G. and Dingle, J.G. (1995c). Proc. Aust. Poult. Sci. Sym. 7: 214.
5. Wiryawan, K.G. and Dingle, J.G. (1996). In New Crops, New Products, New
Opportunities for Australian Agriculture Volume 2, pp. 73-83
6. Wiryawan, K.G., Dingle, J.G. and Creswell, D. (1997). Recent Advances in Animal
Nutrition in Australia, p. 231.
7. Wiryawan, K.G., Dingle, J.G., Kumar, A., Gaughan, J.B. and Young, B.A. (1995).
Recent Advances in Animal Nutrition in Australia, p. 196.
8. Wiryawan, K.G., Kumar, A. and Dingle, J.G. (1997) .Proc.Qld. Poult. Sci. Sym. 6:
110-119.
9. Wiryawan, K.G. and Dingle, J.G. (1998). Proc. Aust. Poult. Sci. Sym. (submitted).
Project details:
RIRDC Project No: UQ21E
Researchers: Dr. J.G. Dingle and Mr. K.G. Wiryawan
Organisation Name: Department of Animal Production
Address: The University of Queensland Gatton College, QLD. 4345
Phone: (07) 54601250, Fax :(07) 54601444
Email: jgd@warigal.uqg.uq.oz.au
GRAIN LEGUMES FOR POULTRY
by
I Ketut Gede Wiryawan

S.Pt. (Bachelor in Animal Science, Universitas Mataram, Indonesia)
M.Agr.Sc. (University of Melbourne, Australia)
A thesis submitted to The University of Queensland in

fulfilment of the requirements for the degree of
Doctor of Philosophy
DEPARTMENT OF ANIMAL PRODUCTION
December, 1997
DECLARATION OF ORIGINALITY
The work reported in this thesis is to the best of the author’s knowledge and
belief, original, except as acknowledged in the text and has not been
submitted previously for a degree at any University
----------------------------------
K.G. Wiryawan
i
ABSTRACT
A series of experiments was conducted to evaluate the quality of ten grain legumes; black
gram, chickpea cv. Desi, chickpea cv. Kaniva, faba bean, field pea, green gram, lentil, lupin,
pigeon pea and solvent-extracted soybean meal (SBM) for poultry in a temperature -controlled
room at 31+0.5oC, except experiment 1a at 28+0.5oC. Young broiler chickens were fed
isoenergetic and isonitrogenous diets containing nominally 100 g kg-1 crude protein supplied by
legume meals or one isoenergetic nitrogen-free diet. Precision feeding method was adopted for
determination of true metabolizable energy (TME) and digestibility of nutrients.
The analyzed nutrient compositions indicated that grain legumes are potential sources of
protein and energy. The protein contents were between 180 and 460 mg g-1 and the average
gross energy content of grain legumes was 15.69 + 0.6 MJ kg-1. Compared with SBM, the
legume proteins, with the exception of lupin, were rich in lysine. The grain legumes contained
less tryptophan than SBM. Black gram, chickpeas, pigeon pea and SBM contained comparable
amounts, but other legumes contained less sulphur containing amino acids.
A comparison of three screening tests, Modified Limiting Amino Acid Score (MLAAS), Net
Weight Gain (NWG) and Net Protein Ratio (NPR), showed that MLAAS, NWG and NPR
methods could distinguish legume proteins of high, medium and low feed values. The MLAAS
calculation could be used as a reasonable estimate of the relative protein quality of most grain
legumes, but NWG and NPR were better methods as they detected limiting factors other than
limiting amino acids in raw and processed legumes.
The NPR value and NWG of chickens fed legume meals as the sole source of protein
increased (P<0.001) by methionine supplementation. There was no difference in the
regression of NWG on Methionine Intake (MI) within chickens fed chickpea cv. Desi,
chickpea cv. Kaniva or lentil diets nor within faba bean, field pea, green gram, lupin or pigeon
pea diets. The pooled response of NWG to MI of chickens fed the supplemented chickpeas,
lentil and soybean meal diets was: NWG(g)= 5.84 + 89.39 MI(g), SEb=8.68, R 2 =0.53,
P<0.001, which had a greater (P<0.05) slope than the pooled regression for field bean, field
pea, green gram, lupin and pigeon pea: NWG(g)= -7.43 + 85.26MI(g), SEb=6.08, R2 =0.63,
P<0.001, but the slope was not as steep (P<0.05) as the regression for the black gram diet:
NWG(g) = -35.73 + 118MI(g), SEb=8.89, R2 =0.90, P<0.001. The protein quality of all ten
grain legumes for poultry was improved by methionine supplementation, however the rate of
improvement for each legume varied in one of three different ways.
ii
The TME, True Protein Digestibility (TPD), Lipid Digestibility (LD) and Starch Digestibility
(SD) of chickpea cv. Kaniva and chickpea cv. Desi were generally higher and those of black
gram and pigeon pea were significantly lower than faba bean, green gram, lentil, lupin, field
pea and SBM . Positive correlations were obtained between TME and SD (r = 0.70, P<0.001)
and between TME and TPD (r = 0.57, P<0.001). The TME of grain legumes was reduced
when protein digestibility and starch digestibility were reduced and that the amount of
reduction depended on the concentration of tannin and Neutral Detergent Fibre (NDF) in each
grain legume.
The quality of low value legumes (black gram and lupin) can be improved by autocalving or
enzyme supplementation. The response of black gram and lupin to autoclaving was different.
The NPR value of lupin increased by 10% and the TME value by 6.5% when lupin meal was
autoclaved for 5 min but black gram did not give significant positive response. Excessive
heating reduced the quality of both legumes.
Supplementation of a 1 g kg-1 multi-enzyme product containing xylanase, .-amylase and

protease increased (some to a significant extent) the NPR value of grain legumes. The
improvement was positively correlated (r = 0.64; P<0.05) with the NDF content. Generally
legumes with a greater cell wall content showed a better response to mixed enzyme
suppplementation than those with a lower amount of cell wall content. The TME, protein and
amino acid digestibilities of black gram, lupin and SBM responded positively to
supplementation with xylanase, protease and .-amylase. The response was specific as each
legume gave a response in one chick production parameter more with one enzyme than the
others. Lupin, for almost all parameters responded most to xylanase, and SBM, for almost all
parameters, responded most to protease. The TME and protein digestibility of chicks fed
black gram did not respond to xylanase or protease but only responded to the multi-enzyme
mixture which also contained amylase in addition to xylanase and protease.
Therefore, with the exception of black gram, eight grain legumes were found to be valuable
new sources of protein and energy for poultry. Raw chickpea cv. Kaniva, chickpea cv. Desi
and lentil (dehulled) can substitute SBM. Other grain legumes can be used either after heat
treatment or when used with dietary supplements of xylanase and other feed enzymes.
iii
LIST OF ADDITIONAL PUBLICATIONS AND
CONFERENCE PRESENTATIONS
The publications and presentation listed below are relevant to this thesis.
Refereed Scientific Papers
Wiryawan, K.G. and Dingle, J.G. 1995a. Screening tests of the protein quality of grain
legumes for poultry production. British Journal of Nutrition 74: 671-679.
Refereed Conference Papers
Dingle, J.G. and Wiryawan, K.G. 1994. Protein value of grain legumes. Proceedings of
Queensland Poultry Science Symposium 3: 73-82.
Kumar, A., Dingle, J.G., Creswell, D. and Wiryawan, K.G. 1997. Enzymes for improved
nutritional value of layer diets. Proceedings of Queensland Poultry Science Symposium
6: 66-77.
Wiryawan, K.G. and Dingle, J.G. 1995b. Protein quality of grain legumes for chickens:
Effect of methionine supplementation. Proceeding of The Nutrition Society of Australia
19. p. 203.
Wiryawan, K.G. and Dingle, J.G. 1995c. Simple techniques for evaluation of protein quality
of grain legumes for poultry. Proceedings of Australian Poultry Science Symposium 7:
214.
Wiryawan, K.G. and Dingle, J.G. 1996. Nutritive value of grain legumes for monogastric
animals. In New Crops, New Products, New Opportunities for Australian Agriculture
Volume 2 (Eds. B.C. Imrie, R.A. Bray, I.M. Wood and R.J. Fletcher). Proceedings of the
First Australian New Crops Conference held at The University of Queensland Gatton
College 8 - July 1996, pp. 73-83
Wiryawan, K.G., Dingle, J.G., Kumar, A., Gaughan, J.B. and Young, B.A. 1995. True
metabolisable energy content of grain legumes: effects of enzyme supplementation.
Recent Advances in Animal Nutrition in Australia (Eds. Rowe, J.B. and Nolan, J.V.).
University of New England, Armidale. p. 196.
iv
Wiryawan, K.G., Dingle, J.G. and Creswell, D. 1997. Enzyme supplementation improves
protein quality of grain legumes for poultry production. Recent Advances in Animal
Nutrition in Australia (Eds. Corbett, J.L., Choct, M., Rowe, J.B. and Nolan, J.V.).
University of New England, Armidale. p. 231.
Wiryawan, K.G., Kumar, A. and Dingle, J.G. 1997. Treatments to improve the nutritive
value of grain legumes. Proceedings of Queensland Poultry Science Symposium 6: 110-
119.
Conference Presentations
• The University of Queensland Gatton College;
Second Annual Conference, 14/10/1994
‘Screening test of the protein quality of grain legumes for poultry production’
• The University of Queensland Gatton College the
Third Annual Conference, 04/10/1995
‘Feed enzyme supplement increases true metabolizable energy value of grain legumes’
• The University of Sydney, NSW;
Australian Poultry Science Symposium, 07/02/1995
‘Simple techniques for evaluation of protein quality of grain legumes for poultry’
• The University of New England, Armidale
Recent Advances in Animal Nutrition in Australia, 03/07/1995
‘True metabolisable energy content of grain legumes: effects of enzyme

supplementation’
• Hilton Hotel, Melbourne
Nutrition Society of Australia, 24/09/1995
‘Protein quality of grain legumes for chickens: Effect of methionine

supplementation’
v
• The University of Queensland Gatton College
First Australian New Crops Conference, 08/07/1996
‘Nutritive value of grain legumes for monogastric animals’
• The University of New England, Armidale
Recent Advances in Animal Nutrition in Australia, 30/06/1997
‘Enzyme supplementation improves protein quality of grain legumes for poultry

production’
• The University of Queensland Gatton College

Queensland Poultry Science Symposium
‘Treatments to improve the nutritive value of grain legumes’
vi
ACKNOWLEDGMENTS
The research reported in this thesis was supported by EIRDC and Department of Animal
Production and AusAID. The study of the effects of enzyme supplementation was partly
supported by Finnfeed International Ltd. UK.
Sincere appreciation is extended to my supervisor, Dr. John Dingle for his continuous
encouragement, advice, guidance and patience throughout this study. Support and
encouragement from Professor Bruce Young, my associate supervisor and Head of
Department of Animal Production is gratefully acknowledged.
I also extend my gratitude to Mr. A. Lisle, for his advice on statistical analysis, Mr. C. Palmer
(Queensland Department of Primary Industries, Animal Research Institute, Yeerongpilly) for
his excellent technical assistance in amino acid analysis, Dr. Arun Kumar and my wife, Rai,
for their assisstance in hand feeding of the chickens. Ms. K. Vockenson and Mr. F. Gorbacx
are thanked for their technical assistance in nitrogen and fibre analyses.
Finally, I sincerely thank Professor Dr. Mulyanto, the Rector of The University of Mataram,
my wife and my parent for their encouragements, and my daughters Intan and Mira for their
patience and sacrifice that they have made enabling the completion of this thesis.
vii
TABLE OF CONTENTS
Page
Declaration of Originality i
Abstract ii
List of Additional Publications and Conference Presentations iv
Acknowledgment vii
Table of Contents viii
List of Tables xi
List of Figures xiii
Chapter 1 GENERAL INDRODUCTION 1

Chapter 2 REVIEW OF LITERATURE 6
2.1. Nutritional Value of Grain Legumes for Monogastric 6
Animals
2.1.1. Inroduction 6
2.1.2. The Effects of Feeding Raw Grain Legumes to Poultry 7
and Pigs
2.1.3. Nutrient Content of Grain Legumes 8
2.1.4. The Nutritional Significance of Antinutrional Factors 13
in Grain Legumes
2.1.4.1. Protease inhibitors 13
2.1.4.2. Lectins 17
2.1.4.3. Tannins 18
2.1.4.4. Non-starch polysaccharides 19
2.1.5. Improving the Nutritional Value of Grain legumes 21
2.2. Rapid Biological Methods for Evaluating Nutritional Value 27

of Grain Legumes
2.2.1. Introduction 27
2.2.2. Nutrient Composition 27
2.2.3. Protein Quality Tests 28
2.2.3.1. General overview of existing protein 28
quality tests
2.2.3.2. Modified limiting amino acid score 30
viii
2.2.3.3. The limiting amino acid growth assay 31
2.2.3.4. Supplementary amino acid slope assay 32
2.2.4. Metabolizable Energy 35
2.2.4.1. Estimating ME values 35
2.2.4.2. Direct ME assays 36
Chapter 3 PRELIMINARY STUDY. THE COMPOSITION OF GRAIN 39

LEGUMES
3.1. Introduction 39
3.2. Chemical Analysis 39
3.2.1. Proximate composition, energy, amino acid 39
and starch contents
3.2.2. Cell wall component, tannin and trypsin inhibitor 40
contents
3.3. Results and Discussion 43
3.4. Conclusion 49
Chapter 4 EXPERIMENT 1. SCREENING TESTS OF THE PROTEIN 50

QUALITY OF GRAIN LEGUMES FOR POULTRY
PRODUCTION
4.2. Materials and Methods 51
4.2.1. Trial 1 51
4.2.2. Trial 2 54
4.2.3. Statistical analysis 54
4.4. Conclusion 59
Chapter 5 EXPERIMENT 2. THE EFFECTS OF METHIONINE 61
SUPPLEMENTATION ON THE PROTEIN QUALITY OF
GRAIN LEGUMES
5.4. Conclusion 71
Chapter 6 EXPERIMENT 3. EVALUATION OF TRUE METABOLIZABLE 72
ENERGY AND DIGESTIBILITY OF PROTEIN AND STARCH
OF TEN GRAIN LEGUMES FED TO YOUNG CHICKENS
ix
6.4. Conclusion 79
Chapter 7. EXPERIMENT 4. IMPROVING THE QUALITY OF LOW 80
VALUE LEGUMES: EFFECTS OF AUTOCLAVING
7.4. Conclusion 88
Chapter 8. EXPERIMENT 5. IMPROVING THE QUALITY OF LOW 89

VALUE LEGUMES: EFFECTS OF ENZYME
SUPPLEMENTATION
8.2.1. Trial 1 90
8.2.2. Trial 2 90
8.4. Conclusion 102
Chapter 9. GENERAL DISCUSSION AND CONCLUSIONS 103

9.1. Rapid biological assay for evaluating the protein quality of 103
grain legumes
9.2. Effect of methionine supplementation on the protein quality 105
of grain legumes
9.3. Energy and nutrient utilisation 106
9.4. Improving the quality of grain legumes 109
9.5. Conclusions 111
9.6. Implications and possible future research 112
References 114
x
LISTS OF TABLES
Table 2.1. Basic nutrient composition of some grain legumes 10
Table 2.2. Metabolisable and digestible energy values of some untreated 11
grain legumes
Table 2.3. Essential amino acid composition of some grain legumes compared 12
with poultry and pig requirements.
Table 2.4. Antinutritional factors (ANF) in grain legumes 14
Table 2.5. Effect of heat treatments on inactivation of ANF in Phaseolus 23
vulgaris beans
Table 2.6. Effect of heat-treatments on ANF of wing bean and on weight gain, 24
True Protein Digestibility and Biological Value for rats
Table 3.1. Proximate composition, gross energy and starch content of grain 44
legumes
Table 3.2. Essential amino acid (EAA) composition of some grain legumes 45
Table 3.3. Cell wall components, tannin and trypsin inhibitor contents of 48
grain legumes
Table 4.1. The composition of dietary treatments for Experiment 1 52
Table 4.2. Calculated and proximate nutrient content of the diets 53
Table 4.3. The order of protein quality of grain legumes on the basis of 56
modified limiting amino acid score (MLAAS), and net weight
gain (NWG) and net protein ratio (NPR)
Table 5.1. Ingredients and calculated composition of diets for studying effects 62
-1
of methionine supplementation (g kg air-dry weight)
Table 5.2. Effects of graded levels of methionine supplementation on Feed 66
Intake, NWG and NPR values in young chickens fed legume meal
diets.
Table 5.3. Linear relationship between NWG(g) and MI (g) of chickens fed 68
methonine supplemented legume diets
Table 5.4. The relative NWG of chickens fed legume diets supplemented with 70
graded level of methionine.
Table 6.1. True metabolizable energy, true nitrogen digestibility, lipid 76
digestibility and starch digestibility of some grain legumes in
growing chickens.
xi
Table 7.1. Dietary ingredients and nutrient composition 82
Table 7.2. Effect of autoclaving time on feed intake, NWG and NPR value of 85
black gram and lupin
Table 7.3. Effect of autoclaving time on AME, TME and APD value of black 88
gram and lupin in young chickens
Table 8.1. Composition of the diets for experiment 1 91
Table 8.2. The effects of adding 1 g kg-1 Avizyme 1500 on FI, NWG and NPR 94
values of ten grain legumes fed to young chickens
Table 8.3. True metabolizable energy (TME) and apparent protein digestibility 96
(APD) and true protein digestibility (TPD) of black gram, lupin and
soybean meal in growing chickens
Table 8.4. Effects of enzyme supplements on the apparent digestibility of 98
amino acids (AAAD) of black gram, lupin and SBM in growing
chickens
Table 8.5. Effects of enzyme supplements on the true digestibility of amino 99
acids (TAAD) of black gram, lupin and SBM in growing
chickens
xii
LISTS OF FIGURES
Figure 2.1. Mode of action of trypsin inhibitor 16
Figure 2.2. Possible response curves to dietary supplements with limiting amino 34
acid.
Figure 3.1. Variation in size and shape of grain legumes. 41
Figure 4.1. Concentration of amino acids required by chickens and those 58
provided by diet containing chickpea (Cicer arietinum cv. Kaniva)
or lupin (Lupinus angustifolius) at a level providing 100 g
protein/kg diet.
Figure 5.1. Regression of net weight gain (NWG) on methionine intake of 69
chickens fed legume diets supplemented with methionine.
Figure 6.1. Non-metabolized energy as percent of gross energy of ten grain 79
legumes when fed to growing chickens
Figure 7.1. Effect of lysine supplementation on NWG (A) and NPR (B) values 87
of autoclaved lupin
Figure 8.1. The NDF content and improvement of NPR value (%) of grain 95
legumes after supplementation with an enzyme product containing
xylanase,.-amylase and protease.
Figure 8.2. Effect of different enzyme supplements on mean apparent (A) and 100
true (B) digestibility of amino acids in black gram, lupin and SBM
for growing chickens
Figure 8.3. True digestibilities of amino acids of black gram, lupin and SBM in 101
growing chickens.
Figure 9.1. Relationship between the mean NPR and TME values in ten grain 107
legumes a measured in young chickens
xiii
Chapter 1. General Information
Chapter 1
General Introduction
1.1. Background
Conventional sources of protein like soybean meal (SBM) and fish meal for
monogastric animals are increasing in cost (FAO, 1995) and alternative sources may
have to be used in future (D’Mello, 1982). Grain legumes are potential substitute for
SBM because of similarities in their energy contents and amino acid profiles (Evans,
1985; Visitpanich et al., 1985a,b; Johnson and Eason, 1990; Ravindran and Blair,
1992; Igbasan and Guenter, 1996).
Grain legumes grow naturally in many tropical areas and have been cultivated as a
source of human food for centuries in both industrially developed and developing
countries (Farrington, 1974). The utilisation of grain legumes by the feed industry,
however, is limited because of uncertainty about their nutritional quality. Although
the analysed total amino acid and energy contents of grain legumes are quite similar,
their protein qualities and metabolizable energy values are quite variable and possibly
influenced by the content of a number of antinutritive factors (ANF) such as protease
inhibitors, lectins, tannins and non-starch polysaccharides (NSP). Evaluation of the
true nutritional quality in contrast to the analysed nutritional content of a range of
grain legumes would therefore be of economic interest to both the grain and livestock
industries in planning for future feed supplies.
The amount of ANF in grain legumes varies between and within legumes (Grant et al.
1983; Jansman et al. 1993; Alitor et al. 1994; Gatel, 1994). It has been well
established that ingestion of a significant amount of ANF by poultry or pigs reduces
weight gain and increases the feed to gain ratio (Moran et al. 1968; Batterham, et al.
1993; Brenes et al. 1993a, 1993b; Igbasan and Guenter, 1996). The mechanisms by
which the ANF induce their negative effects have not been clearly understood, but it
is agreed that they interfere with normal digestive functions (Liener, 1979; Huisman
and Tolman, 1992; van Kempen, 1993; Gatel, 1994).
Heat, chemical and enzyme destruction have been used to inactivate ANF. Heat
treatment is the most widely used method, however, excessive heating reduces the
1
availability of some amino acids (Amos et al. 1975; Almas and Bender, 1980; Yanez
et al. 1986; Metebe, 1989; Dhurandar and Chang 1990; Barneveld et al. 1993).
Therefore the most effective heat treatments depend upon the careful control of the
temperature and duration of heating. Enzyme supplementation may be the method of
choice for overcoming the effects of ANF and improving the nutritional value of grain
legumes in the future, but there has been little investigation into the type of enzyme
and how the enzyme addition would improve the nutritional value of grain legumes.
1.2. Research problem

The main research problem to be answered in this research is:
What is the nutritional value of grain legumes for poultry production?
This is elaborated into:
• Which methods should be used for evaluating the protein quality of grain
legumes?
• Does methionine supplementation improve the protein quality of grain legumes?
• What are the true metabolizable energy (TME) values and the digestibilities of
nutrients of grain legumes?
• What treatments can be applied to improve the quality of low value legumes?
To address these problems, a series of experiments was conducted in which broiler

chickens were fed diets having legume meals as the sole source of protein. The first
experiment was a screening test to determine overall protein quality of a range of raw
grain legumes for poultry. Generally poor growth was observed. Total methionine
appear to be deficient, therefore, the second experiment was an attempt to examine
the effect of methionine supplementation on the protein quality of grain legumes. The
third experiment was an evaluation of the utilization of major nutrients in grain
legumes by growing chickens. Using these values as a base, attempts were made to
improve the quality of low value legumes by heat treatment (Experiment 4) and by
enzyme supplementation (Experiment 5).
Essentially I argue that the protein quality and the metabolizable energy (ME) value
of grain legumes for chickens do not differ substantially except as modified by ANF
2
and that methionine supplementation, autoclaving and enzyme supplementation

overcome most of the effects of ANF leaving the nutritional value of grain legumes
essentially similar and hence of approximately equal replacement value. I do propose
however, new research areas that still need to be investigated to more clearly define
the nutritional value of grain legumes for poultry production.
1.3. Justification of research

Many different approaches have been taken to reduce the negative effects of the ANF
in grain legumes. Most published works have been based on feeding different levels
of legume meals, which may be acceptable when the aim is to established the safe
level of feeding. This method is, however, expensive to run. In addition researchers
have usually used a mixture of protein sources so that the true effects of feeding
legumes may have been masked by the presence of other sources of protein. There is a
need to develop a relatively cheap and rapid screening test to be used for evaluating
the nutritional value of a range of feedstuffs.
The results of this research are designed to produce procedures to evaluate the protein
quality and the TME value of grain legumes and secondly, to produce treatments that
can be applied to improve the nutritional value of grain legumes for the best results
for poultry production. Results are also expected to complement the existing data on
the nutritive value of grain legumes for poultry production.
1.4. Methodology chosen

The chemical composition of grain legumes was determined by using standard
procedures to provide a base level of knowledge about the legumes. For evaluating
the protein quality of a range of grain legumes, net protein ratio (NPR), modified
limiting amino acid score (MLAAS) and net weight gain (NWG) were employed. The
rapid assay of Sibbald (1976) was modified for determination of TME value. The
effects of autoclaving and enzyme addition are assessed in terms of NPR and TME
value and protein and amino acid digestibilities using young broiler chickens.
Detailed methodologies are described for each experiment. The data are analysed by
using the General Linear Model (GLM) procedure of SAS (SAS Institute. 1990) and
the treatment means are compared using the least significant difference or the
Duncan’s new multiple range test.
3
1.5. Outline of the thesis

This thesis describes the nutritional evaluation of grain legumes for poultry
production. Following the general introduction is the review of literature. The focus of
this review is the nutritive value of grain legumes for monogastric animals and rapid
biological methods for evaluating the nutritive value of a feedstuff or a diet. In
Chapter 3, the chemical composition of grain legumes utilised in these studies was
described. Methods for evaluating the protein quality of grain legumes are discussed
in Chapter 4. Methionine is the first limiting amino acid in grain legumes. The effect
of methionine supplementation on the protein quality of grain legumes is determined
in Chapter 5. In Chapter 6, the True Metabolizable Energy (TME) content of grain
legumes is evaluated. Methods for improving the quality of grain legumes for poultry
production are described in Chapter 7 and 8. In the general discussion (Chapter 9),
results of the experiments are discussed and a consideration of implications of the
results and the possibilities for further research are highlighted.
1.6. Delimitation of scope and key assumptions
Although the objectives of this research were to establish the analytical methods, and
the actual analysis of, the nutritional quality of grain legumes for poultry, all
experiments were conducted using young meat chickens. Young meat chickens
provide a faster and more sensitive assessment of the adequacy of nutrient provision
than other types of poultry or other livestock. Although the results are likely to be
applicable to some degree to other types of poultry and livestock, the conclusions
drawn are ultimately limited to young meat chickens. Therefore results need to be
tested before their application to other types of poultry and livestock..
4
1.7. Abbreviations and Definitions

The following abbreviations and definitions are followed throughout this thesis.
AA Amino acid
AAAD, TAAD Apparent and true amino acid digestibilities
AAI, AAF Amino acid intake and amino acid in faeces
AME, TME Apparent and true metabolizable energy; without nitrogen (N) correction
ANF Anti nutritional factors: e.g. trypsin inhibitors (TI), lectins, and tannin and
non-starch polysaccharides (NSP)
APD Apparent digestibility of protein
CF, NDF, ADF Crude, neutral detergent and acid detergent fibre
CP Crude protein calculated as N x 6.25
EAAL, EEL, EPL Endogenous amino acid, energy and protein loss
EI, FI, PI Energy, feed and protein intake
FE Faecal energy
GE Gross energy: the energy released after a complete combustion of material in
megajoule (MJ)
LAAS, MLAAS Limiting and Modified limiting amino acid score: the lowest ratio of mg
AA/g protein in legume to mg of AA/g standard protein or dietary protein
allowance
ME, DE Metabolizable energy and Digestible energy value of a diet or a feedstuff in
MJ kg-1
NFE Nitrogen-free extract: 1 - (fat + CP + CF + ash)
NPR Net protein ratio: calculated as NWG of chicken under test diet plus weight
lost of chicken fed N-free diet divided by protein intake
NSP Non-starch polysaccharides
NWG Net weight gain: calculated as final body weight minus initial body weight
2
r Multiple correlation coefficient: indicates the proportion of the total
variability in the dependent variable accounted for by an equation
SBM Solvent extracted soybean meal
SEM Standard error of the mean
TI Trypsin inhibitors
TUI Units of trypsin inhibited per milligram sample
5
Chapter 2. Review of Literature
Chapter 2
Review of Literature
2.1. Nutritive Value of Grain Legumes for Monogastric Animals
2.1.1. Introduction
Grain legumes play an important role among vegetable materials commonly used in
human and animal diets. World legume production has steadily increased during
recent decades (FAO, 1994). Production of soybean, field peas, chickpeas, broad
beans and lentils in 1993 was 111011, 16032, 6641, 4028, and 2698 metric tonnes
respectively. The USA is the world’s largest producer of soybean (FAO, 1994) but
over 60% of world production of legumes is from developing countries. Developed
countries, however, are also increasing their production of grain legumes to
substitute for soybean imports from the USA (Gatel, 1994). In Asia about 20 grain
legumes out of a possible 10,000 or so (Farrington, 1974; Ravindran and Blair, 1992)
are cultivated, mainly for human consumption. Seed rejected for human consumption
is available for stockfeed industries. Increasing the use of grain legumes in poultry
and pig feeds may give economic benefits to these industries and to regional
communities.
Soybean meal and fish meal currently occupy a central role in the feeding of
monogastric animals in many developed countries. However, increasing human
demand for protein in developing countries, coupled with a relatively high cost of
imported ingredients has turned the attention of animal nutritionists to the exploitation
of non-conventional raw materials and by-product which the region has in abundance
(D’Mello, 1982; Bourne, 1997).
Grain legumes are potential substitutes for soybean meal because of the similarity in
their amino acid and energy profiles (Table 2.2). Although the use of grain legumes in
pig and poultry production is regarded primarily as supplying a source of protein
(Wiseman and Cole, 1988), they are also potentially good sources of energy
(Visitpanich et al. 1985a; Evans, 1985; Johnson and Eason, 1990; Miller and Holmes,
1992) as most contain over 60% carbohydrate (mainly starch) (Reddy et al. 1984) (see
NFE values in Table 2.1).
6
The potential utilisation of grain legumes as a source of protein and energy for
monogastric animals is however governed not only by their content of essential
amino acids and metabolisable energy (ME), but also by the possible presence of
anti-nutritional factors (ANF). Therefore the extent to which grain legumes are used
in commercial poultry and pig production is still limited because of uncertainty about
their effective nutritional quality. Investigation of the factors affecting the nutritional
value of grain legumes would yield information useful in deciding the most efficient
way to utilise available resources for poultry and pig diets.
The first section of this chapter is focused on the evaluation of grain legumes as
sources of nutrients (mainly amino acids and energy) for poultry. The significant
effects of ANF and methods that have been used to improve the nutritional value of
grain legumes are also discussed. A brief account of methods used for evaluating
nutritional value is presented in the second section.
2.1.2. The effects of feeding raw grain legumes to poultry and pigs
Feeding raw legumes to chickens generally resulted in lower growth rate and reduced
feed efficiency compared with feeding soybean meal or processed legumes. Results of
studies with raw faba beans (Ilian et al. 1985; Tortuero et al. 1988; Wurzner et al.
1988; Jansman et al. 1989 and De Castro et al. 1992) showed that raw faba bean can
be included up to 30% in broiler diets without significant negative effects on bird
performances. Recent studies with raw faba bean (cv. fiord), field pea (cv. glenroy),
lupin (cv. Gungurru) and chickpea (cv. amethyst) showed that the first three legumes
can be included up to 36% in broiler starter diets, while inclusion of 36% chickpea
significantly reduced weight gain and increase feed conversion (Perez-Maldonado et
al. 1997a). These reductions were not significant when a diet containing 36% raw
chickpea was fed during the finisher period (Perez-Maldonado et al. 1997b).
Previously, Miller and Holmes (1992) reported that increasing the level of raw
chickpea and mung bean from 10 to 40% in the diets of broilers reduced their weight
gain by 14%, while feeding the same levels of raw field peas resulted in weight gains
similar to that produced by a commercial diet. Similarly, the feed conversion ratios
(FCR) of meat chickens fed diets containing 10 to 30% pigeon pea were not
significantly different from those fed a control diet. However FCR was increased by
up to 14 and 19% when the level of pigeon pea was increased to 40 and 50%
respectively (Tangtaweewipat and Elliot, 1989). Thus, the extent of the negative
7
effects of feeding raw legumes depends on the type of legumes, the levels of inclusion
and the age of the birds.
Different cultivars of the same legumes produced different responses. Igbasan and
Guenter (1996) showed that the AME value and starch digestibility for diets
containing raw “Impala” and “Radly” cultivars were similar but higher than the values
obtained when “Sirius” cultivars were added to the diets, and the apparent
digestibilities of protein among the three cultivars were also significantly different.
Comparative studies on pigs fed diets in which 37% of dietary protein was
contributed by either raw chickpea or raw pigeon pea (Visitpanich et al. 1985a)
showed that chickpea produced growth performance similar to heated soybean meal.
Feeding pigeon pea resulted in lower growth rate and lower dressing percentage than
feeding chickpea or heated soybean meals. The effects of feeding raw legume meals
appear to be different in pigs from those in poultry. It is difficult, however, to
compare the severity of the effect of the inclusion of raw legumes in pig diets with
those in poultry diets because the same levels of legumes were not used nor would
the base diets be identical. In addition the legumes used might not have been the same
cultivars. Nevertheless the reaction of poultry and pigs to ANF has been found to
differ (Liener, 1979; Birk, 1989; Huisman and Tolman, 1992) and this may explain
some of the differences in the response of poultry compared to pigs when fed raw
grain legumes.
2.1.3. Nutrient content of grain legumes

The nutrient content and amino acid compositions of a range of grain legumes
analysed by several authors are shown in Table 2.1, 2.2 and 2.3. These unprocessed
legumes are potential sources of energy and protein for poultry and pigs. The protein
content of grain legumes is between 20 and 38% (Table 2.1) and varies between and
within species.
The metabolisable energy value measured in chickens varied, from as low as 8 MJ kg-
1
for pigeon pea and high-fibre chickpea to as high as 13.8 MJ kg-1 for the low-fibre
chickpea (Table 2.2). The range of digestible energy measured in pigs was 13.5 to
15.87 MJ kg-1. Assuming that the ME value for pigs is approximately 94% of DE
value (Visitpanich et al. 1985b), then the ME value of grain legumes for pigs is higher
than for chickens. The possible explanation of this is that pigs may be able to use
8
products of fermentation in the hind gut as sources of energy more efficiently than
poultry can.
The amino acid composition of legume proteins has been widely studied. Although
there are differences in amino acid concentrations between legumes, there may be
equally as much variation between samples of one legume (Evans, 1985). The amino
acid content of grain legumes varies according to cultivar and environment (Wyckoff
et al. 1983; Barampama and Simard, 1993; Igbasan and Guenter, 1996).
Compared with soybean meal protein, legume proteins, with the exception of lupin,
faba bean and lentil, are rich in lysine. Grain legumes contain more threonine but less
tryptophan and sulphur containing amino acids than soybean meal (Table 2.3).
When the amino acid requirements of broiler chickens, layer chickens (NRC, 1994)
and growing pigs (NRC, 1988) are compared with the amino acid content (expressed
as mg amino acid per gram protein) of all legumes, grain legumes are generally good
sources of lysine but deficient in both methionine and cystine while tryptophan is
marginal. Provided that the diet is supplemented with methionine, grain legumes are
potential substitutes for soybean meal in the diets of monogastric animals. Untreated
grain legumes, however, contain ANF which are inherently developed for growth and
protection of the legume (Gatehouse et al. 1987; Bond and Smith, 1989) but depress
production of pigs and poultry when ingested in excessive amounts.
9
Table 2.1. Basic nutrient composition of some grain legumes (g kg-1 DM)
Crude Crude Crude Ash NFE Ca P

protein fat fibre
Black gram 260 15 50 40 635 2.0 4.0
Chickpeas 220 40 90 30 620 1.0 4.0
Common bean 240 20 40 40 660 2.5 4.0
Cowpeas 250 15 40 30 665 1.0 4.0
Faba bean 230 20 70 40 640 1.0 3.0
Field peas 250 15 60 40 635 1.0 3.0
Green gram 240 15 50 40 635 2.0 4.0
Lentils 250 30 20 40 660 1.0 3.0
Lupins* 322 60 145 31 442 2.2 3.3
Pigeon peas 200 20 80 40 660 1.5 3.0
SBM 498 7 53 71 371 2.5 8.6
Winged bean 380 150 120 40 310 3.0 3.0
Sources: Ravindran and Blair (1992)

*Evans (1985)
10
Table 2.2. Metabolisable and digestible energy values of some untreated grain
legumes
Legume References AME DE

(Chickens) (Pigs)
-1
MJ kg MJ kg-1
Chickpea (low fibre) Johnson and Eason (1990) 12.5 -

Visitpanich et al. (1985b) - 16.2
Miller and Holmes (1992) 13.8
Chickpea (high fibre) Johnson and Eason (1990) 10.3 -
Visitpanich et al. (1985a) - 14.9
Miller and Holmes (1992) 8.1 -
Cowpea Evans (1985) 12.26 13.5
Fialho et al. (1985) - 14.59
Dolichos Evans (1985) 11.68 13.5
Faba bean Johnson and Eason (1990) 11.0 -
Perez-Maldonado et al.(1997b) 10.57 -
Field pea Evans (1985) 12.96 14.2
Davies (1984) - 14
Johnson and Eason (1990) 10.82 -
Green gram Evans (1985) 11.70 15.5
Creswell (1981) 12.7 -
Wiryawan et al.(1997) - 15.87
Lupin Evans (1985) 9.75 14.2
Navy bean Evans (1985) 9.75 14.2
Pigeon pea Tangtaweewipat and Elliot (1989) 8.0 -
Fialho et al. (1985) - 14.28
Visitpanich et al. (1985b) - 15.1
Soybean meal* Evans (1985) 10.67 14.2
Johnson and Eason (1990) 9.46 -
* Solvent extracted
11
Table 2.3. Essential amino acid (EAA) composition of some grain legumes (mg AA g-1 protein) compared with poultry and
pig requirements. *
p p
q
Amino acid Black Green Chick Pigeon Field Lentils Faba Lupins Soybean Broilers Layers Pigs
gram gram peas peas peas bean meal 0-3 weeks 20-50 kg
Arginine 67 63 88 59 72 80 86 103 83 54 47 17
Histidine 22 29 33 38 26 30 24 26 29 15 11 15
Isoleucine 46 41 44 38 44 43 44 37 56 35 43 31
Leucine 74 80 74 70 67 79 64 64 82 52 55 40
Lysine 76 78 74 68 72 70 62 46 68 48 46 50
Methionine + 17 19 22 20 14 13 21 20 24 39 39 27
Cysteine
Methionine 11 12 12 10 8 7 8 6 14 22 20 -
Phenylalanine + 84 78 95 80 72 68 77 68 91 58 55 44
Tyrosine
Phenylalanine 59 52 59 44 44 43 43 34 49 31 31 -
Threonine 35 40 31 32 40 30 37 34 28 35 31 32
Tryptophan 10 12 10 8 9 12 10 9 14 9 11 8
Valine 50 38 68 43 49 44 37 38 46 39 47 32
*
All data for legumes are from Ravindran and Blair (1992) except for lupins which are derived from Evans (1985).
p
Calculated from NRC (1994) amino acid requirement of broiler chickens and layers.
q
Calculated from NRC (1988) amino acid requirement for swine based on ration containing 15% protein.
- Data was not available.
12
2.1.4. The nutritional significance of ANF

A major constraint on the use of grain legumes and other nontraditional feed
ingredients is that they contain a number of naturally occurring substances known as
antinutritional factors (ANF) that depress poultry performance. Distribution and
physiological effects of ingestion of ANF are presented in Table 2.4. The main ANF
in grain legumes are protease (trypsin and chymotrypsin) inhibitors, lectins and
tannins (Wiseman and Cole, 1988). Other ANF which may play an important role in
decreasing the nutritive value of grain legumes are non-starch polysaccharides (Chang
and Satterlie, 1981; Brillouet and Carre, 1983; Semino et al. 1985). Because of
genetic differences, each legume species or cultivar is likely to have different amounts
of ANF (Grant et al. 1983; Alitor et al. 1994; Gatel, 1994). This raises the possibility
of improving the nutritional value of grain legumes through genetic manipulation
(Bond and Duc, 1993) but makes the selection of a single method for eliminating
ANF seem unlikely.
2.1.4.1. Protease inhibitors

Protease inhibitors (trypsin and chymotrypsin inhibitors) are proteins of wide
distribution in the plant kingdom, and they are common constituents of particular
legume seeds. They are readily denatured by heat, acid and alkali.
Active trypsin inhibitors have been shown to have a double detrimental effect on the
utilisation of legume proteins because they inhibit trypsin or chymotrypsin activities
and they ‘lock in’ a relatively high proportion of cysteine which is already in short
supply (Kakade et al. 1969).
13
Table 2.4. Distribution and physiological effects of antinutritional factors (ANF) in

grain legumes
ANF Distribution Physiological Effect
Protease inhibitors most legumes depressed growth, pancreatic

hypertrophy/hyperplasia, acinar
nodules, interference with protein
digestion
Tannins most legumes interference with protein and starch

digestion
Lectins most legumes depressed growth, death
Amylase inhibitors most legumes interference with starch digestion
Glycosides
oligosaccharides most legumes flatulence
non-starch polysaccharides most legumes depress digestibility of protein, starch
and fat
(NSP)*
saponins most legumes affect intestinal permeability
cyanogen lima bean respiratory failure
vicine/convicine faba bean haemolytic anaemia
Phytate most legumes interference with mineral availability
Alkaloids lupins reduce palatability, depressed growth
Adapted from Liener (1989). *Smits and Annison (1996)
14
The physiological responses of animals to the same level of trypsin and chymotrypsin
inhibitors in different legumes may vary. Pigs, for example can tolerate dietary levels
of at least 4.7 and 4.5 mg g-1 of trypsin and chymotrypsin inhibitors respectively from
chickpea meals (Batterham et al. 1993). However, in one experiment the growth of
pigs fed diets containing a pigeon pea meal which contained a lower level of these
two protease inhibitors, was severely depressed. The alternative explanation is that
there may be other unidentified inhibiting factors in pigeon pea meal that have a
greater effect than the trypsin and chymotrypsin inhibitors. Nevertheless, it is
important to know both the levels of trypsin and chymotrypsin inhibitor in different
legumes and their threshold level for the safe use of each legume in practical
situations.
Trypsin inhibitors interfere with normal protein digestion in the intestinal tract, as
shown in Figure 2.1. Both trypsin inhibitor and chymotrypsin inhibitor primarily
inactivate proteolytic enzymes, i.e., trypsin and chymotrypsin, secreted by the
pancreas, through the formation of inhibitor-enzyme complexes (Liener, 1994). As a
result, a greater quantity of enzymes is secreted by the pancreas. This leads to an
enlargement of the pancreas in chickens and rats, but not in pigs (Liener, 1979; Birk,
1989). This may be due to an inverse relationship between the size of the pancreas
and the sensitivity of the response (Liener and Kakade, 1969). Van der Poel et al.(
1990a; 1990b), after an intensive study of variation among species of animal in
response to the feeding of heat-processed beans (Phaseolus vulgaris) concluded that
the piglet is more sensitive to the negative effects of the ANF present in beans than
the chick and rat, and that the chick is more sensitive than the rat.
Pancreatic secretion is mediated by both secretin and cholecystokinin in rats

(Wantanabe et al. 1992). An increased pancreatic secretion gives rise to the depletion
of body tissues of methionine and cystine to meet the demand for the synthesis of
these sulphur-containing enzymes (Kakade et al. 1969). This loss of amino acid
serves to exacerbate an already critical situation with respect to methionine and
cystine supply in legumes (Table 2.3). It is not surprising therefore that methionine or
cystine supplementation effectively counteracts much of the growth depression in
rats when fed raw soybean (Liener and Kakade, 1969; Gumbmann and Friedman,
1987), beans or field peas (Evans and Bandemer, 1967). Growth of chickens,
15
Trypsinogen CCK
(Pancreas) (Mucosa)
Dietary TI
Protein
Trypsin
(Intestine)
Amino acid Trypsin-TI
Figure 2.1. Mode of action of trypsin inhibitor (TI).
CCK=cholecystokinin (After Liener, 1994)
16
likewise, was markedly improved when raw peas were supplemented with methionine
(Moran et al. 1968; Reddy et al. 1979). The question that remains is how much
methionine needs to be added when a legume is the sole source of dietary protein?
Growth depression from feeding grain legumes is usually due to more than one
factor. Kakade et al. (1973) estimated that trypsin inhibitors from raw soybean
account for about 40% of the enlargement of the pancreas and 40% of the growth
retardation. The extent to which trypsin inhibitors from other legumes inhibit the
growth of chickens and pigs, however, has not been clearly understood. Other growth
inhibiting substances such as lectins, tannins and non-starch polysaccharides in raw
soybeans and other legumes are probably responsible for the rest of the impairment
of the animals’ performances.
2.1.4.2. Lectins
Lectins, otherwise referred to as phytohaemagglutinins, are glycoproteins capable of

agglutinating red blood cells in vitro, and of binding to receptors on the epithelial
cells of the intestinal mucosa, thereby interfering with digestion (Baintner, et al. 1993;
Gatel, 1994). Legume seeds, especially in the cotyledons (Marquardt et al. 1975), are
particularly rich in this sugar-binding protein (Grant et al. 1983). Lectins are heat
sensitive proteins but they are resistant to gut proteolysis (Nakata and Kimura, 1985).
Lectins isolated from a number of legumes including soybean and several varieties of
Phaseolus vulgaris have been shown to inhibit growth when incorporated in the diet,
and to be toxic when injected into animals. In rats for example, the incorporation of
0.8% soybean lectins in the diet caused a 25% growth inhibition (Liener, 1974).
Feeding rats a diet containing more than 0.5% pure Phaseolus vulgaris lectins
resulted in loss of weight and at times death (Hanovar et al. 1962) suggesting that
lectins from Phaseolus vulgaris are more toxic than lectins from soybeans.
The result of a survey conducted by Grant et al. (1983) on the nutritional and
haemagglutination properties of legumes in the United Kingdom showed that the
lectins in lentils (Lens culinaris), peas (Pisum sativum), chickpeas (Cicer arietinum),
black-eyed peas (Vigna sinensis), pigeon peas (Cajanus cajan), green gram
(Phaseolus aureus), faba bean (Vicia faba) and adzuki bean (Phaseolus angularis),
17
were non-toxic. Growth depression observed after feeding these legumes therefore
appears to be due to other effects than direct toxicity.
Lectins exert their effect through damage to the gastrointestinal tract. Lectins bind to
epithelial cells and react with different functional compartments of the small intestine
according to their sugar specificity (Etzler and Branstrator, 1974; Pusztai, 1989). This
leads to extensive damage to the brush border followed by disruption of the functional
formation of the brush border membrane (Nakata and Kimura, 1985) which in turn
causes interference with the normal secretory and absorptive functions of that region.
The net effects are an appreciable decrease in the digestion and absorption of nutrients
and the loss of endogenous nitrogen due to an increased sloughing of the cells into the
lumen of the gut (Pusztai, 1989). In consequence of the above, lectins stimulate a
thickening of the wall of the intestine and an overgrowth of Escherichia coli (Pusztai
et al. 1993) leading to further inhibition of nutrient uptake.
Lectins are powerful chemicals inhibiting the growth of chickens and other animals
through their ability to combine with and disrupt the absorbtive surface of the
intestinal mucosa.
2.1.4.3. Tannins
Tannins are defined as water-soluble phenolic compounds, widely distributed in the
plant kingdom and having high protein binding capacity (Marquardt, 1989). Most of
them reside in the seed coat [outer and inner integuments (Deshpande et al. 1982;
Singh, 1993)]. Jadhav et al. (1989) reported that among grain legumes, black gram,
faba bean, green gram, horse gram, kidney bean, moth beans, peas and pigeon peas
have the highest tannin content. The white-flowered or the white seeded cultivars for
both beans and peas have negligible amounts of tannin (Griffiths, 1981; Bos and
Jetten, 1989). It is therefore important to state whether the grain legumes tested in
animal trials include or exclude their testa and to measure the tannins in the testa and
in the decorticated grain.
Most nutritional reports on tannins have been based on work with faba bean (Vicia
faba) due to the greater negative effect of tannin than other ANF in this legume
(Griffiths, 1981). Like other ANF, ingestion of a sufficient amount of dietary tannin
reduced daily gain and impaired feed efficiency due to decreased digestibility of
18
protein, in rats (Moseley and Griffiths, 1980; Alzueta et al. 1992), chickens
(Santidrian and Marzo, 1989; Longstaff and McNab, 1991) and pigs (Jansman et al.
1993).
Similar responses were reported from feeding a dark-flowered cultivar of peas.

Hlodversson (1987) reported that incorporation of 35% peas (Pisum arvense) in pig
diets reduced apparent digestibility of protein and energy by 21.2 and 13.6%
respectively compared with the same amount of a white-flowered cultivar (Pisum
sativum). In humans, the true digestibility, biological value and net protein utilisation
of low-polyphenolic pigeon pea cultivars were higher than for those of high-
polyphenolic cultivars (Singh, 1993). In line with those findings, Buraczewska et al.
(1989) found that the methionine digestibilities of isonitrogenous diets with coloured-
flowered peas were lower than those with white-flowered peas (58 versus 80%).
These results, however, cannot be fully accounted for by the effect of tannins. It is
possible that the two cultivars were different in their nutrient composition, or other
ANF may have been involved.
Tannins have a high protein binding capacity. Tannin-protein complexes, which are
extremely hydrophobic (Hagerman and Butler, 1980; Mitaru et al. 1984), are partly
responsible for low protein digestibility and low amino acid availability and thus
increased faecal nitrogen excretion in rats (Glick and Joslyn, 1970; Moseley and
Griffiths, 1980), pigs (Hlodversson, 1987) and chickens (Ortiz et al. 1993). Addition
of tannic acid to a heated soybean diet for chickens also reduced the rate of in vivo
intestinal absorption of galactose and leucine (Santidrian and Marzo, 1989). Tannins
may bind not only the dietary protein but also the enzyme protein as in the case of
trypsin inhibitors. The activities of trypsin, α-amylase and lipase reduced after
chickens were fed high-tannin faba bean hulls (Longstaff and McNab, 1991; Jansman
et al. 1994). Therefore, supplementation by both tannase and normal digestive
enzymes, are likely to improve the protein value of tannin-containing grain legumes.
2.1.4.4. Non-starch polysaccharides (NSP)

The major components of non-digestible carbohydrates of a feedstuff are non-starch
polysaccharides (NSP) made up of water insoluble cellulose and water soluble gums,
hemicelluloses, pectic substances and mucilages (Prosky and deVries, 1992). Grain
19
legumes have been shown to contain relatively high concentrations of NSP (Brillouet
et al. 1988; Smits and Annison, 1996; Choct, 1997). These substances are resistant to
endogenous enzymic digestion in the alimentary tract (Classen and Bedford, 1991)
and their presence decreases the rate of gastric emptying and increases small intestinal
transit time ( Potkins et al. 1991; Roberfroid, 1993).
Pectins and gums are especially effective in increasing the viscosity in the intestinal
tract (Choct et al. 1995) and thereby reducing the digestive and absorptive
mechanisms in the lumen of the gut (Classen and Bedford, 1991). Viscous materials
reduce the availability of all nutrients. They appear to act as a molecular filter
because the severity of their effect varies with the molecular size of each nutrient
(Dingle, 1979; Dingle and Wiryawan, 1994; Smits and Annison, 1996). Hence the
absorption of fats, fat soluble vitamins and cholesterol will be most affected as the
bile salt micelles, used for transporting these substances in the gut, are large
molecules compared with the size of sugars, amino acids, vitamins and minerals
(Scott et al. 1976). In addition, gums and pectins may cause gumminess of the ration
on ingestion, making it unpalatable and causing an increase in water intake and
excretion by poultry. Consequently health problems for poultry and unpleasant
working conditions may arise from the accumulation of wet droppings.
Choct et al. (1995) showed that the addition of 40 g kg-1 NSP from a wheat milling
by-product, to a commercial broiler diet, decreased weight gain, feed efficiency and
AME by 28.6, 27 and 21.2% respectively. These decreases are associated with
decreases in “ileal digestibility” of starch (Knudsen et al. 1993), protein and lipid
(Choct and Annison, 1992) found to be caused by increased levels of dietary NSP.
The exact mode of action by which the NSP’s of legume origin affect growth and
digestibility in poultry has not yet been investigated, however it is possible that they
act in the same manner as the NSP’s of cereal origin. That is, they may increase the
viscosity of the digesta in the lumen of the gut, resulting in a reduction in the rate of
hydrolysis of starch, protein and fat and a reduction in the transport and uptake of the
products of starch, protein and fat digestion from the gut.
20
2.1.5. Improving the nutritional value of grain legumes
2.1.5.1. Plant breeding

Some promising results have been shown by plant breeders in their efforts to
eliminate ANF (Bond and Duc, 1993). Reduction of condensed tannin content
through genetic manipulation of faba bean, for example, has resulted in increased
digestibility of dry matter and nitrogen in pigs (van der Poel et al. 1992) and a 2.5%
increase in feed conversion in broiler chicks (Helsper et al. 1996). Plant breeding is,
however, a long-term process and the results, at least for the removal of trypsin
inhibitors in peas (Pisum sativum), have not been consistent. Some cultivars had
higher trypsin inhibitor activity than one of their parents, and cultivars derived from
the same cross showed different trypsin inhibitor activities, suggesting that the
hereditary transmission was not systematic (Leterme et al. 1992).
2.1.5.2. Physical treatments

The most widely used methods for the reduction of the negative effects induced by
ANF are physical treatments. They can be grouped into mechanical treatments and
heat treatments. Decortication is an effective method for reducing tannin content of
grain legumes since most tannins reside in the testa (Deshpande, et al. 1982; Rao and
Prabavati, 1982; Barroga et al. 1985; Singh, 1993). Weight gain, feed-to-gain ratio,
apparent protein digestibility (APD) and apparent metabolizable energy (AME) were
improved by 5.7, 7.0, 16.4 and 13.9% respectively when growing chickens were fed
dehulled-high-tannin peas compared with those fed an untreated pea diet (Brenes et
al. 1993b). This improvement may have occured not only because of the reduction of
tannin content but also because of the reduction of fibre content associated with
removal of the testa. On the other hand, lectins and trypsin are more concentrated in
the cotyledon than in the hull (Marquardt et al. 1975; Valdeouze et al. 1980), so that
dehulling is not an effective method for reducing lectins and proteinase inhibitors.
2.1.5.2. Heat treatments

Heat treatment, which is based on heat denaturation of the proteinaceous inhibitors, is
a good method of decreasing the activity of lectins, and trypsin and chymotrypsin
inhibitors, generally improving the nutritional value of grain legumes (Hanovar et al.
1962; Kadam et al. 1987; van der Poel, 1990; van der Poel et al. 1990a,b; Estevez et
21
al. 1991). Several different heat treatments can be applied to reduce the ‘heat-labile’
trypsin inhibitor activity and lectin activity (Table 2.5). Brenes et al. (1993b) reported
that inclusion of 47.5% autoclaved (121oC for 20 min) high-tannin peas (Pisum
sativum) into a corn-soy basal diet increased the apparent metabolizable energy
(AME) and apparent protein digestibility (APD) by 21 and 11% respectively,
compared with inclusion of the same amount of unprocessed peas. Application of the
same technique to lupin (Lupinus albus) increased weight gain by 11% and reduced
feed-to-gain ratio by 10% but failed to reach a significant level (Brenes et al. 1993a).
The results of these two studies suggest that the effect of autoclaving is specific to
each legume, and may depend on the concentration of the heat-labile ANF.
Comparative studies in rats (Kadam et al. 1987) showed that infrared radiation,
autoclaving and boiling in water are effective means for reducing trypsin inhibitor
activity and lectin activity of wing bean (Psophocarpus tetragonolobus). Weight gain,
protein digestibility and biological value were dramatically increased when rats were
fed heat treated wing bean diets (Table 2.6). The biological value of diets containing
autoclaved, infrared treated and boiled winged bean was not significantly different.
Application of infrared radiation to peas fed to young chickens also resulted in a
significant increased in apparent metabolizable energy (AME), apparent protein
digestibility (APD) and starch digestibility (Igbasan and Guenter, 1996). These
treatments are potential methods for improving the nutritional value of grain legumes.
The choice of the treatment will, therefore, depend on the availability of facilities and
the economic considerations.
22
Table 2.5. Effect of heat treatments on inactivation of ANF in Phaseolus vulgaris beans
Process Heating Conditions Legume Reduction ANF Reference

Temp. (O C) Time In Trypsin In Lectin
(min) Inhibitor Activity (%)
Activity (%)
Autoclaving 121 5 Navy bean 82 100 Kakade and Evans, 1965
121 30 Navy bean 88 100 Kakade and Evans, 1965
121 15 Red bean 100 100 Myer and Froseth, 1983
121 10 Wing bean 72 nm Tan et al. 1984
Extrusion 145 16 Red bean 78 98 Myer and Froseth, 1983
Heating, 88 60 Navy bean 95 99 Dhurandar and Chang, 1990

presoaked
100 10 Navy bean 95 100 Dhurandar and Chang, 1990
100 10 Red bean 95 100 Dhurandar and Chang, 1990
100 15 Beans nm 100 Thomson et al. 1983
80 120 Beans nm 100 Thomson et al. 1983
Steam heating 105 20 Beans 94 95 van der Poel et al. 1990

105 40 Beans 97 98 van der Poel et al. 1990
100 15 White bean 65 100 Rodriguez and Bayley, 1987
100 75 White bean 97 98 Rodriguez and Bayley, 1987
Toasting nm 40 Beans 94 94 Huisman and van der Poel, 1989
nm = not measured
23
Table 2.6. Effect of heat-treatments on ANF of wing bean and on weight gain, True Protein Digestibility (TPD) and
Biological Value for rats*
Treatment Trypsin Inhibitor Lectin Tannins Phytate Weight Gain True Biological
(mg g-1) (Units mg-1) (mg g-1) (mg g-1) (g/7 days) Digestibity of Value (%)
Protein (%)
Oven heated 17.4 192 7.5 6.1 -12.6 30.7 -102.1
Untreated 17.7 200 7.6 6.1 -12 50.5 -73.7
Microwave 17.9 196 7.4 5.9 -14 37.3 -69.9
Autoclave 0.70 nd 5.1 5.9 2.6 72.2 53.2
Infrared 0.66 10 6.7 5.8 9.2 84.4 70.5
Boiling water 0.36 nd 2.5 6.1 10.4 84.3 70.8
* Adapted from Kadam et al. (1987).
24
The effect of heat depends on the temperature and duration of processing (Ichihara et
al. 1994; Qin et al. 1996; van der Poel et al. 1990; Wu et al. 1996). Excessive heat
treatment can result in the reduction of protein solubility and may destroy certain
amino acids (Almas and Bender, 1980; Metebe, 1989; Dhurandhar and Chang, 1990,
van Barneveld et al. 1993). It is therefore important to find the exact conditions of
heating which maximise the destruction of the ANF and minimise damage to the feed
protein.
2.1.5.3. Chemical treatments

More recently some chemical treatments (such as the addition of exogenous enzymes)
have been developed to destroy ANF without affecting the composition of the dietary
nutrients. The inactivation of trypsin inhibitors, chymotrypsin inhibitors and lectins of
raw soybean by incubation with microbial proteases (Huo et al. 1993) and extensive
hydrolysis of raffinose-family oligosaccharides of ground cow peas by microbial α-
galactosidase (Somiari and Balogh, 1993) have been demontrated. This finding open
up the possibility to employ enzymes to enhance the quality of grain legumes.
Although most evidence concerning enzymatic inactivation of ANF comes mainly
from in vitro research, they are a good indication of in vivo digestion.
Enzyme supplements that digest NSP are likely to increase the metabolisable energy
and protein values of grain legumes in the same way they do in wheats (Choct and
Annison, 1992; Choct et al. 1995; Hew et al. 1995). Bryden et al. (1994) found that
adding a feed enzyme, ‘Lupinase’ (β-galactanase and minor α-galactosidase), to
lupin diets increased the apparent metabolisable energy (AME) value by 10.7%.
Similarly, the addition of 0.5 to 0.75 g kg-1 feed enzyme containing hemicellulase,
xylanase, pentosanase and cellulase activities to diets containing dehulled-lupins
increased the AME value by 14% (Annison et al. 1995). The ideal ratio of enzymes
needed to improve the nutritional value of grain legumes may be specific for each
legume. However, using an enzyme cocktail containing a mixture of proteases and
carbohydrases may be more economical.
In summary, grain legumes are potential sources of energy and protein for
monogastric animals. Their use in poultry production is limited by low methionine
levels in the protein and the presence of ANF. The low methionine levels can be
25
overcome by methionine supplementation, however the exact mode of action of ANF

in grain legumes has not been clearly understood. There is evidence that they produce
their negative effects on animals through their interference with normal digestive
functions, resulting in a reduction in the digestibility of dietary energy, protein/amino
acids, and other nutrients. Heat treatments are the most widely used techniques for
inactivating ANF, however, as well as potentially destroying nutrients, this method
increases the operating cost for a feed company. Enzyme supplementation is likely to
be a safer, more effective and cheaper method of reducing the negative effects of
antinutritional factors in grain legumes. The best enzymes to use and the extent of
their effectiveness in improving animal performance need to be elucidated.
26
2.2. Rapid Biological Methods for Evaluating the Nutritional Value of Grain
Legumes
2.2.1. Introduction
Conventional sources of protein for monogastric animals may, in future, be in short
supply because of their requirement for human food. The routine utilisation of new
and nonconventional sources of protein (i.e., grain legumes and leaf proteins) will
ultimately need rapid tests of quality because they are more variable (and hence less
predictable) in nutritional quality than traditional feed sources.
The suitability of a feedstuff for meeting animal requirements is indicated by its
nutrient composition, therefore a general overview of the methods of determination of
nutrient content is presented in this section. Because of the significant role of grain
legumes in providing amino acids and energy for poultry and pigs, emphasis is placed
on the methods for determination of protein quality and metabolizable energy. New
rapid methods are also suggested.
2.2.2. Nutrient Composition

The method commonly used in determining nutrient content of a feedstuff or of a diet
is the Proximate System of Analysis developed by Henneberg and Stohmann at the
Weende Experiment Station, Germany in 1862 (Close et al. 1986). The Weende
system measures moisture, ash, crude protein (CP), crude fibre(CF), and lipids. The
nitrogen-free extract (NFE) which is calculated as : 1 -(ash + CP + CF + lipids),
represents the carbohydrate component. Some refinements have been developed,
however the basic concept is the same. Although these constituents represent rather
heterogenous groups of nutrients, they still give valuable information for comparison
between feedstuffs. Additional determinations can be done for specific nutrients as
more analytical methods become available but these are not pursued here.
Nutrient composition, however, is just an indicator of potential nutritive value.

Further tests are still needed for a more accurate definition of the nutritive value of a
feedstuff or of a diet.
2.2.3. Protein Quality Tests

A large number of protein quality tests measuring different aspects of protein quality
are available. Dingle (1972) identified 33 in vitro and 61 in vivo tests.
27
The in vitro tests predict protein quality of a feedstuff through laboratory studies.
Most in vitro assays attempt to predict protein quality through measurement of
protein digestibility using either single (Johnston and Coon, 1979; AOAC, 1984) or
multienzyme techniques (Hzu et al. 1977; Satterlee et al. 1979; Parsons, 1991; Oshodi
et al. 1995) and amino acid digestibility by using microbial assay (Ford, 1981). In
vitro assays are rapid and inexpensive. Their results often correlate well with one of
the in vivo assays (Parson, 1991). However, they may be of limited value unless they
show a high correlation with in vivo assays using the target animal. This means that
the in vivo assays are still needed. Furthermore, the problems may become more
complicated when ANF influence protein quality test in vitro and in vivo to different
extents.
The in vivo tests invariably compare the production that results from feeding equal
amount of test protein. About fifty percent of standard chick growth assays and most
rat assays use 9 to 10% crude protein in the diet. The amount of the test material
included in diets containing 10% protein varies depending on the protein
concentration of the test material, but the greatest difference between protein sources
will be shown if all the dietary protein consists of the test protein.
2.2.3.1. General overview of existing protein quality tests

Some protein quality tests, such as weight gain (Calet, 1967) and Protein Efficiency
Ratio (PER) originally proposed by Osborne et al. (1919), [as cited by Hegsteg and
Chang (1965)], are relatively easy to understand and perform and have been used for
a long time. However these methods have been criticised for assuming that all the
protein consumed by the animals is utilised for growth, because a certain proportion
of the protein is used for maintenance. Bender and Doell (1957) improved the PER
method by using a zero-protein group. The index was termed Net Protein Ratio (NPR)
which was claimed to give more accurate results. An examination of cereals, grains
legumes and high-protein food products, conducted by Lachance et al. (1977) showed
that NPR was best for screening high-quality legume foods compared with PER or
Net Protein Utilisation and was very useful for evaluating proteins that do not
promote growth.
Biological Value (BV), as determined by the method of Mitchell (1924), is a direct

measure of the proportion of the nitrogen retained in the body, and it is generally
28
considered as the method of choice. However, this method is difficult to understand

and perform.
For acceptance and use by the feed industry a protein quality test:
• needs to be rapid and low in cost in terms of labour and facilities,
• needs to be able to be used as a screening test for a large numbers of samples in a

short test period,
• must be able to provide a quantitative nutritive value of a test protein as a single
source of protein,
• should show a relative potency compared with a reference protein,
• would be able to be conducted without the necessity of complicated practical and
laboratory analysis, so that it can be used both in developed and developing
countries.
The Ad Hoc Working Group on Protein Quality Measurements for the Codex
Committee on Vegetable Protein(CCVP), after evaluating previous protein quality
tests devised the Protein Digestibility-Corrected Amino Acid Score. This was
recommended to be the most suitable routine method for predicting protein quality of
vegetable protein products for humans (Sarwar and McDonough, 1990). In its pure
form this test would require nitrogen (N) and amino acid analyses of the test materials
and determination of true protein digestibility by the rat balance method. In its applied
form, the test assumes that the amino acid composition and the amino acid
requirements can be taken from published values, and that the digestibility of each
amino acid is very close to the protein digestibility. This still leaves N content and N
digestibility of test materials to be measured, however most laboratories are capable
of these measurements. This protein quality test seems to correspond reasonably well
with how protein is used in the body (Sarwar and McDonough, 1990).
This test can be simplified more when applied to poultry and pigs. The amino acid
composition of feedsuffs and animal requirements for respective amino acids are
available from published works. Most feedstuffs have high protein digestibilities in
poultry and pigs. The range of nitrogen digestibility values for oil seed meals, for
example, is 80-95%, and the bioavailabilities of most amino acids are in the vicinity
29
of the nitrogen digestibility (Bodwell, 1986). The nitrogen digestibility of most grain
legumes (measured at the terminal ileum), varies from 78 to 95% and 74 to 80% for
poultry and pigs respectively (Rhone Poulenc Animal Nutrition, 1989). However the
digestibility of the protein in most grain legumes is not a good predictor of the
digestibility of limiting amino acids (Sarwar et al. 1989). Therefore the Protein
Digestibility-Corrected Amino Acid Score may not accurately predict biological
quality of legume proteins for monogastric animals, unless it is adjusted for the
digestibility of methionine/cystine (Marletta et al. 1992; Wu et al. 1996b). The
additional work required to derive the actual protein and methionine/cystine
digestibility values may not be justified when the aim is to find a rapid and simple
technique.
For grain legumes, Dingle and Wiryawan (1994) proposed three new protein quality
tests that can be conducted rapidly and simply. They are the Modified Limiting
Amino Acid Score (MLAAS), the Limiting Amino Acid Growth Assay (LAAG) and
the Limiting Amino Acid Slope Assay (LAAS).
2.2.3.2. Modified Limiting Amino Acid Score (MLAAS)

Many protein quality tests use a standard reference protein such as casein, egg
albumen or soy protein. Egg albumen is likely to be closer to the amino acid
requirement of poultry especially chicks, than is casein, so would be a preferable
standard. However, a standard protein is a substitute for a real requirement, therefore
soybean meal may be used as a practical reference protein since it is widely used both
in poultry and pig rations and is considered as a good quality protein. The Modified
Limiting Amino Acid Score (MLAAS) uses a different basis for selecting the standard
protein. This new test uses the amino acid composition of the test protein (eg., Evans,
1985; Ravindran and Blair, 1992) in comparison with the requirement of amino acid
by the test animal [eg., NRC (1994) for poultry and NRC (1988) for pigs]. The
MLAAS combines the concepts used in the modified Chemical Score of Mitchell and
Block (1946) and Bender (1958) and the Protein Score of FAO (1957). It is
calculated on the basis of the following formula.
amino acid percent of test protein

MLAAS = X 100
required amino acid percent of required protein fortest animal
30
The lowest ratio of amino acid of the test protein is the MLAAS value for a defined
animal. Assuming that the true digestibility of legume proteins is 82% (Sarwar and
McDonough, 1990) and the amino acids released are fully available, the potential
growth and production of the animal at that protein level will be restricted to a
MLAAS of 82%.
2.2.3.3. The Limiting Amino Acid Growth Assay (LAAG)

The final objective of all protein quality tests is the protein production of target
animals, measured either as egg production, net weight gain or carcass gain. Since
poultry have a relatively constant body composition irrespective of variations in
diet composition (Low, 1991) net weight gain can become a sensitive means for
measuring protein quality of different feedstuffs. It gives comparable results to
nitrogen retention and is much easier to measure (Sibbald, 1987).
This method may not be a good measure of protein utilisation by pigs because pigs
are more sensitive than poultry to variation in protein quality (Low, 1991). Pigs fed a
diet of low quality protein tend to deposit more fat than those fed a high quality
protein diet. Therefore net weight gain may not be an acccurate indicator of protein
quality of feedstuffs for pigs. In this case, balance trials have to be conducted.
It has been pointed out that grain legumes contain limited amounts of sulfur
containing amino acids and they contain antinutritional factors that interfere with
normal digestive and absorptive function in the gut. Their combined effect results in
a particular growth response of chickens to dietary protein. A perfect protein
(reference protein) with balanced amino acid profile and containing no inhibitors (i.e.
with 100% digestibility and availability) would produce 100% growth. The ratio
between the net weight gain of chickens fed the test protein to those fed the reference
protein is “The Limiting Amino Acid Growth Assay (LAAG)”. In the absence of a
perfect protein, any other protein source can be substituted as a reference point.
Assuming that little variation occurs in protein intake, the LAAG may be a good
indicator of protein quality.
2.2.3.4. Supplementary Amino Acid Slope Assay (SAAS)

For any molecular sieve (such as the viscous contents that form in an animal gut after
consuming soluble non-starch polysaccharides), the greater the concentration of a
31
penetrating molecule the greater the number that pass through the sieve, and the
smaller the proportion that pass through the greater is the inhibitory effect. Similarly,
ANF such as tannins that combine with or cause precipitation of protein in the gut
also have a linear concentration effect (Ortiz et al, 1993). Therefore, an attempt can
be made to quantify the relative inhibitory effect of the combined set of ANF by using
graded supplements of one or more nutrients.
The third new protein quality test, the Supplementary Amino Acid Slope Assay
(SAAS), attempts to provide a single measure of the overall inhibitory effect on the
availability of amino acids by using graded supplements of the most limiting amino
acid. The slope (regression coefficient) of the line joining the level of supplements in
the diet and the response (i.e., net weight gain) produced by each level of
supplementation is a direct measure of efficiency of protein utilisation and will give
clues about the effects of antinutritional factors (Figure 1).
Lines parallel to that of the reference protein would be shown when the
antinutritional factors in the test materials induce a constant inhibitory effect at
various levels of supplements. If the limiting amino acid is equal in each test protein
and there are no antinutritional factors in the diet, then the response (eg. growth) of
the chickens fed the test proteins would be equal. Any variation to the slope of the
lines would be an indicator of the presence and type of antinutritional factor(s).
If there were no inhibition of absorption of free amino acid the response to graded
levels of supplementation with the first limiting amino acid would be as in curve 1.
The growth will not reach 100% (maximum) if there is a second limiting amino acid
(curve 1a). The level it reaches will be a measure of adequacy of the second limiting
amino acid.
The response at the maximum growth end of the curve is of particular interest.
Growth curves 1, 3, 4 and 5 in figure 1 reach their maximum at 100% of the limiting
amino acid requirement in the diet. Curve 2 however does not reach maximum growth
until 110% limiting amino acid in the diet.
The interpretation of curve 2 is that this is the situation where there is an inhibition of
release or absorption of amino acid in the intestine, but additional supplement
overcomes the inhibitory effect. In curve 3, maximum growth is not achieved because
32
of an inhibitory effect and adding more of the limiting amino acid than 100% of the
requirement fails to elicit growth.
In curve 4, additional amino acid beyond the required concentration in the diet
reduces growth due to amino acid interaction inhibiting the utilisation of other
essential nutrients.
In curve 5, there is a sudden increase in growth rate in the middle of the curve,
because of saturation of some antinutritional factors which at that point have been
inactivated. In the case shown, further increase in level of supplements beyond the
100% requirement level, fails to increase the response.
The Supplementary Amino Acid Slope Assay (SAAS) is a model proposed for use as
a rapid protein quality assay.
The three protein quality tests described above, although relying on some possibly
difficult theoretical concepts, are simple and easy to carry out. They require only:
33
100
1a
80
Percent of
maximum
response 60 CURVE 1
(eg. growth)
40 2
20 4
0 25 50 75 100 125
Percent of limiting amino acid

requirement in the diet
Figure 2.2. Possible response curves to dietary supplements

with limiting amino acid.
34
a. The test materials, (for example, grain legumes),

b. A reference protein source, (for example egg protein or other high quality protein
such as soy bean meal),
c. Apparatus for measuring total nitrogen, (for example Kjeldahl apparatus),
d. A source of pure amino acid to supplement the first limiting amino acid in the test
protein, (for example DL-Methionine),
e. Equipment for keeping and weighing animals.
These tests can therefore be conducted by a feed miller or poultry farmer in a

developed or a developing country. They are suitable for use in screening a large
number of samples in a relatively short time. It takes only 1 to 4 weeks. The
reproducibility of these tests for evaluating the quality of vegetable protein such as
grain legumes, however needs to be tested before they can be applied in practical
situations.
2.2.4. Metabolisable Energy

Data on the metabolizable energy (ME) value of some grain legumes for poultry has
been reported (Table 2.2). It should be noted that variation exists not only among
legumes but also between bird type (Slinger et al. 1964; Bourdillon et al. 1990b) and
between assay procedures (Sibbald, 1980; Askbrant, 1988) suggesting that routine
tests for ME content should be done for every ingredient entering the feed industry
when high efficiency is to be maintained. This is important because feed energy
contributes up to 70% of feed or about 40% of on farm cost (Sibbald, 1982). The
purpose of this section is to compare procedures for establishing the ME for a
feedstuff or a diet.
2.2.4.1. Estimating ME values

There are several methods for evaluating the ME of a feedstuff for poultry. Sibbald
(1982) divided the methods into indirect and direct assays. Indirect assays are
designed to estimate ME content of a feedstuff when laboratory facilities are limited.
Most of them attempt to predict ME value of a poultry diet or of a feedstuff by
relating the observed variation in its nutrient composition and/or digestibility of fat,
protein and carbohydrates to the AME/nutrient ratio derived from direct assay.
35
Many equations for deriving the ME value of a diet or of a feedstuff from its nutrient
analysis have been published (Carpenter and Clegg, 1956; Titus, 1958; Fisher 1982
and Fisher and McNab, 1989). Although some of the prediction equations yield
precise estimates of ME value, their accuracy is doubted unless the equations are
tested with independent data (Sibbald, 1982). In addition, predictive ability of the
equations may be reduced by variability present in analyzed components (as most
equations are based on analytical data).
For grain legumes, it is also unlikely that those equations are applicable because most
grain legumes contain antinutritional factors such as trypsin inhibitors, tannins,
lectins and NSP which may interfere with energy utilisation. Misleading conclusions
about the ME values of a feedstuff or of a diet can be avoided by the application of
direct assays.
2.2.4.2. Direct ME Assays

The direct assay is basically a balance method in which the ME value of a feedstuff or
a mixed diet is derived by relating energy consumption to energy voided in excreta.
The value may be expressed as apparent metabolizable energy (AME), nitrogen
corrected apparent metabolizable energy (AMEn), true metabolizable energy (TME)
and nitrogen corrected true metabolizable energy (TMEn). They can be calculated
using the following equations (Sibbald, 1980):
( Fi x GEf ) − ( E x GEe)
AME/ g of feed = (1)
Fi
[( FixGEf ) − ( ExGEe)] − ( NRxK )

AMEn/g of feed = (2)
Fi
[( FixGEf ) − ( ExGEe)] + ( FEm + UEe)

TME/g of feed = (3)
Fi
[( FixGEf ) − ( ExGEe) − ( NRxK )] + [( FEm + UEe) + NRoxK )]

TMEn/g of feed = (4)
Fi
where Fi is the feed intake (g); E is the exreta output (g); GEf is the gross energy/g of
feed; GEe is the gross energy/g of excreta; NR is the nitrogen retention (nitrogen
intake minus nitrogen in excreta); K is a constant, usually either 34.4 or 36.5 kJ;
36
FEm is the metabolic faecal energy; UEe is the endogenous urinary energy; and NRo
is nitrogen retention for fasted birds.
There are three types of balance experiments for measuring ME value (Fisher and
McNab,1989).
• Conventional assay in which birds are accustomed to the test diet for a few days to
establish equilibrium conditions, followed by 3-5 days collection. In most cases,
complete diets are used and substitution methods are used for ingredients.
• Rapid assays, in which birds are starved before and after feeding a known amount
of test diet to control carryover effect, but birds have free access to feed. Complete
diets and substitution methods are used.
• Rapid assays, in which birds are starved followed by tube-feeding or precision

feeding. The test ingredient can be directly introduced into the bird’s crop, so the
necessity of substituting the test ingredient can be avoided.
Most existing data on requirements and feed composition (NRC, 1977; 1984; 1994,
Evans, 1985) are expressed as AME, as determined by the so called ‘conventional
balance procedure’ developed by Hill and Anderson (1958). In this assay test
material is substituted for part of the basal diet or ingredient of known ME value. The
birds are accustomed to the diet and level of feeding for 4 days then a total collection
of excreta is made for 5 days. Farrell et al. (1989) after comparing several method of
measurements, and Bourdillon et al. (1990a) from their interlaboratory tests,
concluded that AMEn is highly reproducible and widely accepted as a means of
establishing ME for poultry. However, some experiments suggest that it less reliable
for determining the ME value of a test material (Bourdillon et al. 1990b). McNab
(1996) suggested that variation of ME values of wheat was not associated with
different varieties or environment where they were grown, but associated with the
procedures. Thus, there has not been a conclusive agreement on which system should
be the defined one for ME in poultry.
The rapid TME assays (type 3) as pioneered by Sibbald (1976) has been clearly
described (Sibbald, 1980), critically reviewed ( Farrell, 1981; Sibbald, 1982; Fisher,
37
1982; Fisher and McNab, 1989), and futher developed by McNab and Blair (1988).
The characteristics of the assay compared with AME methods are:
• TME assay makes a correction on metabolic faecal and urinary energy lost (EEL),
so TME values are independent of variability associated with feed intake.
• Effect of palatability encountered in AME assay is avoided by introducing the test
material directly into the crop,
• The strongest arguments for selecting TME are:
• The assay is simple,
• It requires much less material,
• Results can be expected in 48h after receiving the samples, assuming that the
birds are ready at any time.
Because of these characteristics, it is likely that in time, the definition of the energy
content of a poultry diet will be based on the TME assay. NRC (1994), for example,
has included for some feedstuffs, including SBM, field peas, and faba bean, both the
AME and the TME values. The TME value of other feedstuffs still needs to be
established.
38
Chapter 3. Composition of Grain Legume
Chapter 3
Preliminary Study: The Composition of Grain Legumes
3.1. Introduction
It is well established that the composition of grain legumes varies between and within
species. In order to ascertain their suitability to meet diet specifications and evaluate of
their nutritional quality it is necessary to determine their nutrient and antinutrient
contents. The purpose of this chapter is to determine and to discuss the analytical
value of the major nutrients and several ANF of ten grain legumes including solvent-
extracted soy bean meal (SBM) used throughout this study.
3.2. Chemical analyses
3.2.1. Proximate composition, energy, amino acid and starch contents

The ten grain legumes evaluated in this study were: Black gram (Phaseolus mungo),
Chickpea cv. Desi (Cicer arietinum), Chickpea cv. Kaniva (Cicer arietinum), Faba
bean (Vicia faba), Field pea (Pisum sativum), Green gram (Phaseolus aureus), Lentil
(Lens culinaris), Lupin (Lupinus angustifolius), Pigeon pea (Cajanus cajan) and
SBM (Figure 3A-J).
The SBM and all raw legumes were obtained from local (Queensland, Australia)
suppliers. They were hand-cleaned to remove wastes, then ground to pass a 1 mm
screen. The content of dry matter (DM), ash, ether extract (EE), and crude fibre (CF)
were determined following standard procedures (AOAC, 1984). Nitrogen (N)
contents were analysed using Kjedhal method and/or in an automatic nitrogen
analyser using the combustion method (Sweeney, 1989). Crude protein (CP) content
was calculated as N x 6.25. Organic matter (OM) was calculated as dry matter minus
ash, whereas nitrogen free extract (NFE) was calculated as dry matter minus (ash plus
crude protein plus ether extract plus crude fibre).
The gross energy (GE) was determined with a Parr Oxygen Bomb Calorimeter, using
benzoic acid as standard and correcting for total acid production as determined by
titration with 0.0709 N sodium carbonate solution.
Quantitative analyses of individual amino acids were carried out using HPLC after
hydrolysis with 6M HCl for 18 h at 110oC (Cohen et al. 1989). Because cystine and
39
methionine are destroyed by acid hydrolysis, they were oxidised to cysteic acid and
methionine sulphone prior to hydrolysis. Tryptophan is destroyed by the presence of
hydrochloric acid. Therefore sodium hydroxide was used to hydrolyse the protein for
tryptophane analysis.
Starch content of the legume meals was calculated from the amount of glucose
released after enzymatic hydrolysis by amyloglucosidase (EC. 3.2.1.3) as described
by Longstaff and McNab (1991). Duplicate portions of finely ground samples (50
mg) were gelatinised in 9 ml 0.2 M-sodium acetate buffer (pH 4.5), and placed in an
oven at 100oC for 3 h. The tubes were cooled down to below 50oC followed by
incubation with 1 ml sodium acetate buffer (pH 4.5) without or with 0.1 mg/ml
amyloglucosidase in a shaking water-bath at 50oC for 16 h. After cooling, 0.2 ml
supernatant fractions were diluted to 10 ml with distilled water and reducing sugars
were measured photometrically after their reaction with p-hydroxybenzoic acid
hydrazide according to Lever (1972).
3.2.2. Cell wall component, tannin and trypsin inhibitor contents

Acid detergent fibre (ADF) and neutral detergent fibre (NDF) were determined
according to the methods of Van Soest et al. (1991). Heat stable α-amylase (AOAC
approval number A3306 ; Sigma Chemical Co., St. Louis, MO) was used in ADF and
NDF determinations.
Vanillin assay (Price et al. 1978) was employed to determine the condensed tannin
content of all grain legumes used throughout this study. Approximately 200 mg finely
ground sample was extracted (within a day) with 10 ml methanol in rotating capped
tubes for 20 min and centrifuged in a desk top centrifuge (1500 rpm for 15 min). Five
millilitres vanillin reagent were added at 1 min interval to 1 ml aliquots of the
samples. The blanks were prepared by adding 5 ml of 4% concentrated HCl in
methanol to 1 ml aliquot of the same sample. Absorbance at 500 nm was read in
‘Shimadzu UV-120’ spectrophotometer after 20 min incubation in a waterbath
maintained at 30oC.
40
Note: Photographs (Figure 3.1 A – J) not available electronically
A B
C D
Figure 3.1. Variation in size and shape of grain legumes. (A) Black gram ; (B)
Chickpea cv. Desi; (C) Chickpea cv. Kaniva; (D) Faba bean ; (E) Field pea; (F)
Green gram; (G) Lentil; (H) Lupin; (I) Pigeon pea; and (J) Soybean.
41
Figure 3E. Field pea Figure 3F. Green gram
Figure 3G. Lentil Figure 3H. Lupin
Figure 3I. Pigeon pea Figure 3J. Soybean
42
Absorbance of the blank was subtracted from the absorbance with vanillin. A
standard curve was developed by pipetting 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 ml of
100 mg catechin in 100 ml methanol into a volumetric flask made up to 10 ml with
methanol.
The procedure used to determine trypsin inhibitor activity was that of Kakade et al.
(1974) as modified by Valdebouze et al. (1980). One gram of finely ground sample
was suspended by constant stirring for 30 min in 100 ml of water adjusted to pH 2.9
with hydrochloric acid.
Portions of 0, 0.6, 1.0, 1.4 ml of sample suspension were pipetted into graduated test
tubes and adjusted to 2 ml with water. To each tube, 2 ml of trypsin solution and 5 ml
BAPNA solution (prewarmed at 370C) were added respectively. The tubes were
incubated in a water bath at 37oC. Exactly 10 min later, the reaction was terminated
by adding 1 ml 30% acetic acid, mixed and filtered (Whatman No. 3). The absorbance
of the filtrate was measured at 410 nm. against a reagent blank prepared by adding 1
ml of 30% acetic acid to a test tube containing 2 ml water and 2 ml trypsin solution.
Trypsin inhibitor activity (TIA) was expressed in units of trypsin inhibited (TUI) per
milligram sample. All analyses were done in duplicate.
3.3. Results and discussion

There is not much variation in the dry matter (DM) content of grain legumes. The
average DM content of ten grain legumes analysed was 900.7 ± 9.5 mg g-1 (Table 3.1).
Proximate composition of grain legumes reported here was in the range of published
values (Evans, 1985; Ravindran and Blair, 1992; NRC. 1994). Most of grain legumes
analysed did not contain appreciable amounts of lipid. Chickpeas and lupin however,
contained 40 to 60 g kg-1 lipid.
Grain legumes are potential source of energy for poultry and pigs. There was not much
variation in gross energy (GE) content of grain legumes. The average GE content of
grain legumes was 15.69 + 0.6 MJ kg-1. This may be asociated with a relatively high
content of nitrogen-free extract (NFE) mainly sugars and starch (Reddy et al. 1984). The
starch contents of field pea, green gram, lentil and pigeon pea were twice as much as
43
Table 3.1. Proximate composition, gross energy (GE) and starch content of grain legumes (g kg-1 air-dry weight)
Legumes Dry Ether Crude Ash NFE GE Starch

Matter Extract Protein (MJ Kg-1)
Black gram (Phaseolus mungo) 903.7 11.3 249.9 34.6 564.9 15.27 347.5
Chickpea,cv Desi (Cicer arietinum) 903.4 44.7 193.2 34.6 531.9 15.91 226.5
Chickpea,cv Kaniva (Cicer arietinum) 902.6 57.7 188.9 26.1 593.2 16.49 314.0
Faba bean (Vicia faba) 908.6 12.3 234.7 31.3 542.8 15.85 372.1
Field pea (Pisum sativum) 906.1 12.0 223.6 43.7 569.0 15.23 416.4
Green gram (Phaseolus aureus) 899.3 14.1 223.5 47.9 570.3 15.43 444.3
Lentil (Lens culinaris) 893.9 10.5 239.0 19.8 610.5 15.16 422.2
Lupin (Lupinus angustifolius) 916.4 50.4 313.5 34.0 371.5 16.61 -
Pigeon pea (Cajanus cajan) 885.1 19.0 181.4 37.9 572.1 14.84 405.0
Soybean meal (Glycine max)
p
888.0 15.2 454.5 61.8 303.0 16.13 -
1
Solvent extracted
44
Table 3.2. Essential amino acid (EAA) composition of some grain legumes (mg AA g-1 protein)
Amino Black Chickpea Chcikpea Faba Field Green Lentil Lupin Pigeon SBM
acid gram cv.Desi cv. Kaniva bean pea gram pea
Arg 71.13 94.08 92.32 89.66 96.73 72.55 86.14 120.49 64.41 68.85
His 29.15 25.34 24.70 23.73 24.19 29.05 21.39 26.54 37.52 24.38
Ieu 45.02 41.66 41.52 38.95 39.75 44.26 39.23 42.79 39.33 43.59
Leu 84.51 73.51 73.63 69.95 70.51 82.32 68.97 69.80 76.85 72.82
Lys 77.17 73.46 72.34 68.37 79.22 80.31 70.16 53.76 77.82 63.11
Met 16.35 12.30 12.47 6.66 7.62 12.07 6.25 4.91 11.32 11.29
Met+Cyst 24.56 27.64 26.57 17.65 20.99 19.92 15.08 14.85 24.20 24.40
Phen 64.01 57.84 58.82 42.24 48.10 63.94 47.31 42.77 104.32 50.86
Phen+Tyr 93.37 82.62 83.76 70.38 76.95 92.23 72.59 77.41 129.89 82.67
Thre 35.01 36.43 37.46 35.27 37.61 36.09 35.23 36.19 39.33 38.10
Try 9.76 7.09 6.55 6.44 6.94 9.22 6.36 8.82 5.06 21.91
Val 55.04 44.98 45.73 43.86 47.82 56.49 45.14 41.98 47.28 44.88
45
those of chickpea cv. Desi. The starch content of SBM and lupin, however, were not
reported because the values obtained by the method employed in this study were not
consistent. This might be associated with their high protein contents (Lever, 1972).
Limited studies showed that the starch content of SBM is relatively low i.e., 0.3 to
1.1% and lupin (dehulled) is about 2% air dry weight (Brillouet et al. 1988; Annison et
al. 1996).
Protein contents were between 180 and 460 g kg-1. Apart from SBM, lupin contained
the greatest and pigeon pea the least protein. It has been reported that winged bean
(Psophocarpus tetragonolobus) also contains a high level of protein (Table 2.1). It is
worth noting that variability in nutrient composition exists within species. Reichert and
McKenzie (1982) and Monti (1983), for example, showed that the protein content of
field peas varies from 155 - 397 g kg-1.
The essential amino acid contents of the legumes tested are presented in Table 3.2.
Compared with SBM, the legume proteins, with the exception of lupin, were rich in
lysine. The grain legumes contained less tryptophan than SBM. Black gram, chickpeas,
pigeon pea and SBM contained comparable amounts, but other legumes contained less
sulphur containing amino acids. Overall, an inverse relationship (r = -0.63; P<0.05)
was observed between lysine and protein content. However, when SBM was excluded
from the calculation, the negative correlation was observed not only for lysine (r = -
0.72; P<0.05) but also for the sulphur containing amino acids (r = -0.73; P<0.05).
These results suggest that proportion of these amino acids decrease when the protein
content increases. This is in agreement with the negative correlations previously
reported in field peas and beans (Evans and Boulter, 1982) and field peas, faba beans
and lupin (Mosse, 1990). Gatel (1994), after reviewing the relationships between
protein and amino acid contents in grain legumes concluded that any increase in
protein content will decrease the protein quality in term of amino acid profile.
Although there are differences in amino acid concentrations between legumes, there
may be equally as much variation between samples of the one legume (Evans, 1985).
The amino acid content of grain legumes may vary according to cultivar and
environment (Wyckoff et al. 1983; Barampama and Simard, 1993). When the amino
acid requirements of broiler chickens, layer chickens and growing pigs (see Table 2.3.)
are compared with the amino acid content of all legumes, grain legumes are generally
46
good sources of lysine but deficient in both methionine and cystine while tryptophan is
marginal (Dingle and Wiryawan, 1994). Provided that the diet is supplemented with
methionine, grain legumes appear to be potential substitutes for soybean meal in the
diets of monogastric animals.
The cell wall components, (CF, NDF and ADF), tannin and trypsin inhibitor (TI)
contents are shown in Table 3.3. For all analytical values of fibre fractions, lupin had
the highest and chickpea cv. Kaniva the lowest. Carre and Brillouet (1989) reported
that the NDF content of white-flowered peas and dehulled-lupin were 90.4 and 65.1 g
kg-1 respectively. It was also reported that the cell wall material of white lupin (Lupinus
albus) cotyledon is characterized by a low content of cellulose but a high content of
pectic substances (Carre and Leclercq, 1985). Differences in NDF especially for lupin
in this study, may be accounted for by different varieties and/or by a high level of cell
wall in the seed coats.
Reddy et al. (1985) showed that among grain legumes, black gram, faba bean, green
gram, horse gram, kidney bean, moth beans, peas and pigeon peas have the highest
tannin content. Results of analyses of grain legumes in this study showed a similar
order of tannin content. Black gram contained significantly higher tannin levels than
other legumes, while lupin and SBM contained the least amount. Overall, however,
tannin contents measured in this study were lower than the range reported by Reddy et
al. (1985). These differences might be associated with storage conditions. The legumes
were analysed for their tannin content around 10 mo after harvesting and were kept at
air temperature. Sievwright and Shipe (1986) revealed that there was a 26 - 37%
decrease in measurable condensed tannins when black beans (Phaseolus vulgaris)
were kept at 30oC with 80% relative humidity for 6 mo.
The TI contents of black gram (5.44 TUI mg-1), chickpea cv Desi (5.32 TUI mg-1) and
chickpea cv. Kaniva (4.45 TUI mg-1) were higher than the TI of other legumes. Lupin,
however contained a negligible amount of TI. The TI contents of the legumes assayed
47
Table 3.3. Cell wall components, tannin and trypsin inhibitor contents of grain
legumes (g kg-1 air-dry weight)
CF ADF NDF Tannin TI

(TUI mg-1)
Black gram 43.0 96.3 148.4 16.22 5.44
Chickpea cv. Desi 99.0 159.1 234.2 0.62 5.32
Chickpea cv .Kaniva 36.7 61.9 108.5 0.51 4.45
Faba bean 87.5 150.3 209.6 2.53 2.52
Field pea 57.8 98.5 177.6 0.61 2.57
Green gram 43.5 92.9 139.9 2.10 3.68
Lentil* 14.1 43.7 138.2 <0.1 2.10
Lupin 47.0 203.7 239.4 1.53 <1
Pigeon pea 74.7 140.2 195.7 2.64 3.33
SBM 53.5 147.8 164.9 0.49 2.51
*Dehulled lentil, NDF = Neutral Detergent Fibre, ADF= Acid Detergent Fibre,
CF = Crude Fibre, TI = Trypsin Inhibitors, TUI = trypsin units inhibited
48
in this study were in the range of published values (Valdebouze et al. 1980; Saini and
Batterham, 1988).
Lectin contents of grain legumes studied herein was not analysed because they were
assumed to be non toxic. The result of a survey conducted by Grant et al. (1983) on the
nutritional and haemagglutination properties of legumes in the United Kingdom
showed that the lectins in lentils (Lens culinaris), peas (Pisum sativum), chickpeas
(Cicer arietinum), black-eyed peas (Vigna sinensis), pigeon peas (Cajanus cajan),
green gram (Phaseolus aureus), faba bean (Vicia faba) and adzuki bean (Phaseolus
angularis), were non-toxic.
3.4. Conclusion
Grain legumes are potential energy sources for poultry and pig production as they
contained relatively high levels of NFE mainly starch. As sources of protein, they are
rich in lysine but deficient in methionine and cysteine.
For all analytical values of fibre fractions of the legume meals, lupin had the highest
and chickpea cv. Kaniva the lowest. In term of tannin content, black gram contained
significantly higher tannin levels than other legumes, while lupin and SBM contained
the least amount.
49
Chapter 4. Screening Tests
Chapter 4
Experiment 1. Screening Tests of the Protein Quality of Grain

Legumes for Proultry Production
(The report of this experiment was published in British Journal of Nutrition 74: 671- 679)
4.1. Introduction
Conventional sources of protein for animals such as fish meals and soybean meals are
often in short supply and expensive. Other grain legumes offer an alternative to fat
extracted soybean meal (SBM) because they have a similar amino acid profile
(Ravindran & Blair, 1992) and are often cheaper. At the present time however, the
utilisation of grain legumes as sources of protein for poultry is limited due to
uncertainty about their nutritional quality. The variation in quality of grain legumes
appears to be a combination of variable protein quality and variable amount of
antinutritional factors (ANFs). The availability of a rapid protein quality test would
give feed manufactures a greater ability to select and use grain legumes and hence
build confidence in the market.
Dingle (1972) identified 61 in vivo and 33 in vitro methods for the evaluation of
protein quality. For the purpose of identifying a screening test which could be used to
rank the protein quality of various sources of protein, limiting amino acid score,
LAAS (Bender, 1958), growth rate or net weight gain (NWG), and net protein ratio
(NPR) (Bender & Doell, 1957), were investigated because of their simplicity and short
test time. In protein quality tests, diets containing 10% protein are usually applied,
since more consistent and differential result have been obtained (Bressani, 1977).
The objectives of the present study were to develop and evaluate the above protein
quality test methods for their rapidity and repeatibility in estimating the protein quality
of some grain legumes for poultry production.
4.2. Materials and methods

Two trials were conducted to compare three methods for the evaluation of protein
quality of grain legumes for poultry production.
50
4.2.1. Trial 1
4.2.1.1. Diets
The SBM and all raw legumes were obtained from local (Queensland, Australia)
suppliers. They were hand-cleaned to remove wastes, then ground to pass a 1 mm
screen. As the sole source of dietary protein, each of the legume meals contributed 10
percent dietary protein calculated from published values. Cornstarch, dicalcium
phosphate, limestone , and vitamin and mineral premixes were added to each diet
according to least cost formulation (UFFF, 1986). All diets were made isoenergetic (13
MJ ME per kg) by adding sunflower oil and were made equal in fiber content by
adding rice hull meal (Table 4.1 and Table 4.2). Ingredients for each diet were mixed
in a single batch for approximately 25 min.
4.2.1.2. Chickens and experimental procedures

Eighty unsexed commercial broiler chickens were used in the assay. During day 1 to
day 6 post hatching, the chickens were kept in a group and fed a commercial starter
diet, and on day 7 allocated to the dietary treatments (Table 4.1). The chickens were
housed individually in a four-tier double sided battery brooder. Each treatment had
eight replicates and two replicates of each treatment were distributed randomly in each
tier. The room temperature was maintained between 28o to 29oC and light was
provided continuously. The chickens had ad libitum access to feed and water for 14
days. They were weighed at 7 days and 21 days after an overnight fast. Feed
allowance, feed refusal and feed spills were measured.
51
Table 4.1. The composition of dietary treatments for Experiment 1
Composition (g) N-Free Soybean Black Chickpea Chickpea Faba Field pea Green Lupin Pigeon
meal gram cv. Desi cv. Kaniva bean gram pea
Soybean meal 222.20

Black gram (Phaseolus mongo) 427.4
Chickpea (Cicer arietinum) 487.1
cv. Desi
Chickpea (Cicer arietinum) 511.5
(cv.Kaniva)
Faba bean (Vicia faba) 446.4
Field pea (Pisum sativum) 448.4
Green gram (Phaseolus aureus) 418.4
Lupin (Lupinus angustifolius) 346.0
Pigeon pea (Cajanus cajan) 500.0
Maize starch 851.2 676.0 486.9 400.6 418.6 463.2 498.6 498.0 588.0 372.4
Sunflower oil - - 15.0 48.8 5.0 15.0 - 26.2 25.3 88.0
Limestone 16.5 20.0 19.9 16.0 16.0 19.9 17.9 22.6 18.3 19.9
Dicalcium phosphate 26.1 18.2 17.6 22.9 22.9 19.2 19.1 12.9 20.3 17.8
Rice-hull meal 100.0 61.5 31.2 20.5 22.1 34.1 13.9 20.0 - -
Vitamin and mineral premix* 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
TOTAL 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0
*Per tonne of diet the vitamin and mineral premix contained; 8000 iu vitamin A, 3000 iu vitamin D3, 5 g vitamin E, 300 g vitamin K, 3 g Calcium
pantothenate, 3 g Riboflavin, 15 g Niacin, 10 mg Vitamin B12, 5 mg Biotin, 100 mg Choline, 200 mg Cobalt, 500 mg Iodine, 5 g Copper, 20 g Iron,
80 g Manganese, 50 g Zinc, 20 g Ethoxyquin, 100 mg Selenium, and 200 mg Molybdenum.
52
Table 4.2. Calculated and proximate nutrient content of the diets (g kg-1 air-dry weight)
Diets N-Free SBM Black Chickpea Chickpea Faba Field pea Green Lupin Pigeon pea
gram cv. Desi cv.Kaniva bean gram
Calculated values:
ME (MJ/kg) 13 13 13 13 13 13 13 13 13 13
Crude Protein - 100 100 100 100 100 100 100 100 100
Crude Fat - 8.6 29.9 34.6 34.6 23.9 39.3 29.9 43.6 96.8
Crude Fiber 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0
Ca 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0
P 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7
Amino Acids:
Arginine - 7.1 6.6 - 9.6 6.0 8.9 6.6 10.3 5.9
Histidine - 2.6 2.1 - 2.6 3.8 2.2 2.9 2.6 3.8
Isoleucine - 4.4 4.5 - 4.2 5.8 4.6 4.1 3.6 3.8
Leucine - 7.4 7.4 - 7.4 7.2 8.2 7.4 6.4 7.0
Lysine - 6.0 7.5 - 6.8 6.8 7.1 7.2 4.6 6.8
Methionine - 1.2 1.1 - 1.0 0.7 1.1 1.1 0.5 0.9
Phenylalanine - 4.9 5.9 - 5.6 5.4 4.9 5.9 3.4 4.4
Threonine - 3.6 3.4 - 3.3 4.0 3.8 3.5 3.3 3.2
Tryptophan - 1.6 1.0 - 1.8 1.0 0.8 1.8 0.9 0.8
Valine 5.2 5.0 - 3.9 4.7 4.9 4.8 3.4 4.3
Analysed value
Dry matter 893.0 894.1 892.7 900.6 894.7 894.7 890.4 890.9 898.2 898.2
Crude protein 4.4 99.8 10.7 95.4 95.7 113.6 100.7 92.0 117.4 97.0
Crude fat - 3.4 19.8 70.6 34.5 20.5 5.4 32.1 42.7 97.5
Crude fiber 3.4 3.3 2.9 5.5 2.6 5.1 3.1 2.5 5.1 3.7
- Data not available
53
The chickens’ response to dietary protein was assessed in term of NWG and NPR.
NPR was calculated as [ weight gain of the chickens fed the test diet + weight loss of
the chickens fed protein-free diet] divided by protein intake (Bender & Doell, 1957).
The Modified Limiting Amino Acid Score (MLAAS) used in these trials was based on
the lowest ratios of mg amino acid per g protein in the legume to mg of the same
amino acid per g dietary protein allowances recommended by NRC (1994) as proposed
by Dingle and Wiryawan (1994). The protein quality of grain legumes was ranked
according to the descending value of their Modified Limiting Amino Acid Score
(MLAAS) , NWG and NPR scores The distribution of the scores was further ranked
into high, medium and low.
4.2.2. Trial 2
This was a repetition of the first experiment with the following modifications: a) the
analysed protein content (Table 3.1) of each legume was used to calculate dietary
protein; b) room temperature was increased to 31 ± 0.5o C; c) the chickens were
placed in the cages and fed a mixture of commercial and SBM diet in the ratio of 3 :
1 for two days prior to the start of the feeding trial, and d) body weights were measured
at day 14 also.
4.2.3. Statistical analysis

Statistically significant differences between the mean NWG, protein intake and NPR
value, of chickens given each dietary treatment, and for each experiment, were
calculated by using the statistical analysis systems procedure General Linear Model
(SAS Institute, Inc. 1990).

There were some differences between calculated and analysed values of dietary protein,
fiber and fat for experiment 1 due to differences between published and analysed data.
These differences were small except in the case of crude fat in chickpea cv. Desi and
field pea. In the case of protein content, chickpea contained about 15% lower, while
faba bean and lupin were about 15% higher than the calculated values. Analysed
protein content of the nonprotein diet may be accounted for by residual amounts of
nitrogen in corn starch and the rice hull meal.
54
NWG and protein intake of chickens fed different legumes in experiment 2 were
significantly (P<0.001) higher than those in experiment 1, indicating that the modified
experimental conditions improved responses of chickens to the dietary proteins. The
small increase in room temperature may have been partly responsible for the
difference. Many chickens in experiment 1 showed signs of discomfort (chirping and
shivering) throughout the trial, indicated that that the temperature of 28.5 ± 0.5 o C
was not warm enough for chickens of 1 - 3 weeks old. The introduction of dietary
treatments, at the same time as the chickens were placed in the single cage
environment, caused some chickens to stop eating for some days, resulting in a low
total feed intake which caused a reduced weight gain. A two-day period of adaptation to
the cages and the mash diet was justifiable since the chickens accepted the experimental
mash diets immediately. The limitation of using mash diets was a relatively larger
amount of feed spill, especially with diets incorporating a large amount of starch,
compared with a crumbed diet. Careful recovery of feed spill increased the precision
of measuring feed intake.
There was no significant difference between blocks, indicating that, under the
conditions of our studies, tier height and differences in light intensity in the room did
not affect chickens’ performances.
Duncan's multiple range test showed that the NWG for chickens fed chickpea cv.
Kaniva in experiment 1, or both chickpeas’ diets in experiment 2, were similar to those
of chickens fed SBM and significantly (P<0.05) higher than the NWG of chickens fed
the other legumes (Table 4.3). This indicated that the nutritive values of SBM and
chickpeas were superior to those of other selected legumes.
55
Table 4.3. The order of protein quality of grain legumes on the basis of modified limiting amino acid score (MLAAS), and net weight gain
(NWG) and net protein ratio (NPR)
† †
Legumes MLAAS* Order of Protein Intake (g) NWG (g) NPR† Order of Protein Quality Growth
protein Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 MLAAS NWG NPR Test
content Exp.1 Exp.2 Exp.1 Exp.2 Category
55.2 1 ab ab ab a a a 1 2 2 2 1 high
SBM 19.06 21.52 8.65 27.20 3.01 3.53
Chickpea 54.4 7 bc ab bc a a a 2 4 2 2 1 high
16.14 21.89 1.06 27.62 3.09 3.49
cv. Desi
Chickpea, 54.4 8 a a a a a a 2 1 1 1 1 high
20.03 24.33 20.26 37.06 3.45 3.53
cv.Kaniva
47.0 6 bc b bc b ab b 4 5 5 4 4 medium
Green gram 16.28 19.34 -0.97 6.61 2.94 2.86
46.0 9 ab b ab b ab b 5 2 4 5 4 medium
Pigeon pea 19.74 20.11 8.67 9.12 2.91 2.88
42.0 3 c ab d bc c c 6 8 7 8 8 low
Black gram 14.85 22.29 -23.11 1.22 1.73 2.24
36.8 4 bc c c bc c b 7 7 8 7 7 low
Faba bean 15.57 14.74 -10.33 -7.54 2.47b 2.80
34.6 5 bc c c b ab bc 8 6 5 6 4 medium
Field pea 15.84 19.37 -7.30 6.98 2.62 2.88
27.6 2 c c d c c c 9 9 9 9 9 low
Lupin 14.72 15.00 -23.56 -15.54 1.71 2.22
- - 1.43 1.43 5.06 5.06 0.24 0.18

SEM
* Based on amino acid requirement of broiler chickens of 0 - 3 weeks old (Dingle & Wiryawan, 1994).
†
The values with common superscripts in the same column were not significantly different (P>0.05).
56
The greater NWG of chicks fed cv. Kaniva than those fed cv. Desi could partly be
explained by the lower fiber content of cv. Kaniva (2.6 vs 5.5%, Table 2). Although
SBM and cv. Kaniva have similar MLAAS values, and SBM was assumed to be free
from antinutritional factors, chickens fed SBM diet did not grow faster than chickens
fed cv. Kaniva in either experiment. This could partly be explained by differences in
total protein intake since the feed intakes of chickens were different but their NPR
values were similar (Table 4.3). It is possible that over-heating during processing may
have reduced the availability of certain amino acids from SBM (Rani and Hira, 1993;
van Barneveld et al. 1993) or there may have been a significant amount of residual
ANF. Irish and Balnave (1993) reported that chickens fed on diets in which SBM is
the sole source of protein showed poorer growth than those fed diets in which the
protein was supplied by a combination of SBM with any of the other sources of protein.
The low NWG from feeding legumes other than chickpeas and SBM, and the severe
weight losses of chickens fed lupin, were apparently due to a combination of
insufficient intake of sulfur-containing amino acids due to their limiting amounts in the
diets and to low feed intake possibly associated with the sticky nature of the lupin diet.
ANF may also have been responsible for some of the weight loss.
Figure 1 indicates the amino acids that are well supplied and those that are in short
supply for chickens’ requirements from chickpea cv. Kaniva and lupin. The amount
supplied by most amino acids in both experiments fulfilled the birds' requirements at
10% protein. However methionine, the first limiting amino acid in all diets supplied
only 27 to 55.2% of the requirement. Lupin, especially, was not only very deficient
in methionine but also contained insufficient lysine. Supplementation of lupin with both
methionine and lysine has improved its protein quality (Sarwar et al. 1978; Perez-Alba
et al. 1990). Threonine is the next limiting amino acid for both of these legumes.
There were negative correlations between the protein contents of the legumes and their
MLAAS (r = -0.80; P<0.01), NWG ( r = -0.87; P<0.01) and NPR (r = -0.76; P<0.01)
values. These indicate that the protein quality of unprocessed legumes with a high
protein content are poorer than those with a lower protein content.
57
Amino acid concentration (g/kg diet)
12
10
0
ARG LEU LYS VAL ISO THR PHE MET HIS TRY
Figure 4.1. Concentration of amino acids (g/kg diet) required by chickens ( )

and those provided by diet containing chickpea (Cicer arietinum cv. Kaniva; )
or lupin (Lupinus angustifolius; ) at a level providing 100 g protein/kg diet.
58
Grain legumes with a high protein content have been found to be associated with
relatively low concentration of lysine, sulphur amino acids, tryptophan and threonine
(Mosse, 1990; cited by Gatel, 1994). The legumes with moderate protein concentration
were sources of medium quality protein.
The MLAAS values were positively correlated with NWG values and NPR values.
The correlation coefficients were respectively 0.74 and 0.75 (P<0.02) in experiment 1
and respectively 0.90 (P<0.001) and 0.78 (P<0.01) in experiment 2. These suggest that
the protein quality of most grain legumes for broiler chickens could be reasonably
estimated from their analysed amino acid content relative to the dietary amino acid
requirement of the chickens.
Although the protein quality of some grain legumes, when based on NPR value, was
in most cases in a similar order to their theoretical value (MLAAS), in other cases
their order was higher or lower than expected (Table 4.3). The three protein quality
indices distinguished three low, three medium and three high nutritional value
legumes. The categories designated for black gram and field pea by MLAAS were
reversed in the NWG and NPR scores.
MLAAS and NPR methods showed a consistent result for SBM, chickpeas, faba bean,
green gram and lupin. However the range of NPR values was much narrower than the
range of MLAAS values (1.6 :1 vs 2:1, the highest to the lowest) and therefore
NPR may not be as useful as MLAAS for grading feed proteins into high, medium and
low value. However, these results should be interpreted with caution because only
small sample sizes were used.
4.4. Conclusion
The rapid screening tests MLAAS, NWG and NPR, could be used to grade grain
legumes into high, medium and low quality. On the basis of NPR values, the two
chickpea varieties and SBM were grouped together into sources of high quality protein
; green gram, pigeon pea and field pea were source of medium quality protein and
black gram, faba bean and lupin were sources of low quality protein. In categorising
the protein value of grain legumes into high, medium or low these rapid tests gave
reasonably repeatable results in two experiments. Any of these is probably an adequate
59
basis for purchasing and formulating decisions. The correlation between the three
screening tests was high, therefore the use of the Modified Limiting Amino Acid Score,
the simplest and the quickest method, employing calculation and no growth test, was a
reasonable estimate of protein quality for most grain legumes.
60
Chapter 5. Methionine supplementation
Chapter 5
Experiment 2. Protein Quality of Grain Legumes for Chickens: Effect of

Methionine Supplementation
5.1. Introduction
The presence of antinutritional factors and the limiting quantity and low bioavailability of
sulphur-containing amino acids (SAA) in grain legumes (Evans and Bauer, 1978; Prieto et
al. 1994) are the main factors responsible for their limited use in poultry feed. Fan et al.
(1994) reported that ileal digestibility of methionine and cystine of white-flowered peas
(Pisum sativum) in pigs were 67.8 - 75.1% and 58.5 - 65.9 % respectively. Ileal true
digestibility of methionine of peas, lupin and faba bean in poultry was 74, 89 and 75%
respectively (Fickler et al. 1996). The in vivo study in rats showed that the availability of
SAA was - 18.6% in raw and 40-68% in thermally processed bean (Wu et al. 1996).
Methionine supplementation to cereal-pea diets was shown to improve feed/egg ratio in
laying hens (Bertram et al. 1995), and feed/gain ratio in growing pigs (Zivkovic, 1987).
Smulikowska and Chibowska (1993) concluded for a broiler chicken diet containing 300g
kg-1 faba bean that methionine supplementation for broiler chickens was as effective as
soybean meal as a supplementary protein source in wheat-triticale diets.
The aim of this experiment was to study the effect of graded level of methionine
supplementation on the protein quality of the grain legumes in growing chickens measured
as NWG and NPR values.
The experimental procedures and the parameters measured were similar to those of
Experiment 1 (Trial 2), except that, following two days adaptation to single cages and diets,
seven days old unsexed broiler chickens were given free access for 14 days to drinking
water and to either an isoenergetic diet containing nominally 100 g kg-1 crude protein
supplied by
61
Table 5.1. Ingredients and calculated composition of diets for studying effects of methionine supplementation (g kg -1 air-dry weight)
Ingredients (5) Black gram Chickpea cv. Desi Chickpea cv. Kaniva Faba bean
Control 75% 100% Control 75% 100% Control 75% 100% Control 75% 100%
Black gram 394.4 394.4 394.4
Chickpea cv. Desi 517.6 517.6 517.6
Chickpea cv. Kaniva 522.7 522.7 522.7
Faba bean 426.1 426.1 426.1
Starch 465.2 464.3 463.5 396.9 396.6 395.2 357.3 356.9 354.8 527.1 525.8 525.5
Sunflower oil 46.2 46.5 46.9 42.0 42.4 42.8 23.9 24.1 24.6 7.0 7.6 7.7
Premix* 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Rice -hull meal 53.1 53.1 53.1 - - - 45.9 45.9 45.9 - - -
Di-calcium phosphate 22.3 22.3 22.3 25.0 25.0 25.0 25.0 25.0 25.0 19.0 19.0 19.0
Limestone 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 16.7 18.8 18.8 18.8
Methionine - 0.6 1.1 - 0.5 1.1 - 0.7 1.2 - - 1.4
Calculated content
TME (MJ kg-1) 13 13 13 13 13 13 13 13 13 13 13 13
Crude protein 100 100 100 100 100 100 100 100 100 100 100 100
Fat 50.6 51.0 51.3 65.0 65.2 65.8 53.7 54.2 54.4 12.2 12.8 12.9
Crude fibre 35.0 35.0 35.0 51.2 51.2 51.2 35.0 35.0 35.0 37.2 37.2 37.2
Calcium 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0
Available phosphorus 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7
Arginine 6.9 6.9 6.9 10.3 10.3 10.3 10.5 10.5 10.5 9.5 9.5 9.5
Histidine 2.2 2.2 2.2 2.7 2.7 2.7 2.8 2.8 2.8 2.4 2.4 2.4
Isoleucine 4.7 4.7 4.7 4.5 4.5 4.5 4.6 4.6 4.6 3.3 3.3 3.3
Leucine 7.6 7.6 7.6 2.7 2.7 2.7 2.8 2.8 2.8 5.9 5.9 5.9
Lysine 7.7 7.7 7.7 7.4 7.4 7.4 7.5 7.5 7.5 4.2 4.2 4.2
Methionine 1.1 1.6 2.2 1.0 1.6 2.2 1.0 1.6 2.2 0.8 1.6 2.2
Phenylalanine 6.0 6.0 6.0 6.0 6.0 6.0 6.1 6.1 6.1 3.1 3.1 3.1
Threonine 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.0 3.0 3.0
Tryptophan 1.0 1.0 1.0 1.9 1.9 1.9 2.0 2.0 2.0 0.8 0.8 0.8
Valine 5.1 5.1 5.1 4.1 4.1 4.1 4.3 4.3 4.3 3.4 3.4 3.4
*As in Table 4.1
62
Table 5.1. Continued..
Ingredient (%) Field pea Green gram Lentil Lupin

Control 75% 100% Control 75% 100% Control 75% 100% Control 75% 100%
Field pea 443.9 443.9 443.9
Green gram 441.8 441.8 441.8
Lentil 408.7 408.7 408.7
Lupin 31.90 319.0 319.0
Starch 458.9 456.7 455.8 419.9 419.3 419.0 400.6 398.9 398.0 630.5 628.6 627.6
Sunflower oil 30.1 30.8 31.2 51.3 51.6 52.0 63.4 64.1 64.5 8.2 9.0 9.8
Premix* 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Rice-hull meal 27.6 27.6 27.6 46.6 46.6 46.6 86.2 86.2 86.2 - - -
Di-calcium phosphate 18.0 18.0 18.0 21.9 21.9 21.9 19.3 19.3 19.3 24.5 24.5 24.5
Limestone 20.0 20.0 20.0 16.5 16.5 16.5 19.8 19.8 19.8 15.8 15.8 15.8
Methionine - 1.0 1.6 - 0.6 1.1 - 1.0 1.5 - 1.1 1.7
Calculated content
TME (MJ kg-1) 13 13 13 13 13 13 13 13 13 13 13 13
Crude protein 100 100 100 100 100 100 100 100 100 100 100 100
Fat 35.4 36.0 36.5 57.5 57.9 58.2 68.1 68.7 69.1 24.3 25.1 25.5
Crude fibre 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 46.9 46.9 46.9
Calcium 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0 12.0
Available phosphorus 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7
Arginine 7.1 7.1 7.1 7.0 7.0 7.0 8.1 8.1 8.1 10.3 10.3 10.3
Histidine 2.3 2.3 2.3 3.1 3.1 3.1 3.0 3.0 3.0 2.7 2.7 2.7
Isoleucine 3.2 3.2 3.2 4.4 4.4 4.4 4.4 4.4 4.4 4.5 4.5 4.5
Leucine 5.0 5.0 5.0 7.9 7.9 7.9 8.0 8.0 8.0 2.7 2.7 2.7
Lysine 6.5 6.5 6.5 7.6 7.6 7.6 7.1 7.1 7.1 4.7 4.7 4.7
Methionine 0.6 1.6 2.2 1.1 1.6 2.2 0.7 1.6 2.2 0.5 1.6 2.2
Phenylalanine 3.3 3.3 3.3 6.2 6.2 6.2 4.4 4.4 4.4 6.0 6.0 6.0
Threonine 3.0 3.0 3.0 3.7 3.7 3.7 3.0 3.0 3.0 3.6 3.6 3.6
Tryptophan 0.8 0.8 0.8 1.9 1.9 1.9 1.2 1.2 1.2 1.9 1.9 1.9
Valine 3.5 3.5 3.5 5.1 5.1 5.1 4.4 4.4 4.4 4.1 4.1 4.1
*As in Table 4.1
63
Table 5.1. Continued.
Ingredient (%) Pigeon pea SBM N-Free

Control 75% 100% Control 75% 100%
Pigeon pea 551.3 551.3 551.3
SBM 215.9 215.9 215.9
Starch 305.3 304.3 303.6 666.6 665.8 664.9 855.2
Sunflower oil 104.3 104.7 105.2 5.8 6.2 6.5 -
Premix* 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Rice-hull meal - - - 69.4 69.4 69.4 10.0
Di-calcium phosphate 16.9 16.9 16.9 23.8 23.8 23.8 26.1
Limestone 20.2 20.2 20.2 16.4 16.4 16.4 16.5
Methionine - 0.5 1.1 - 0.5 1.0 -
Calculated content
TME (MJ kg-1) 13 13 13 13 13 13 13
Crude protein 100 100 100 100 100 100 -
Fat 114.8 115.2 115.7 9.1 9.4 9.8 -
Crude fibre 41.2 41.2 41.2 35.0 35.0 35.0 3.5
Calcium 12.0 12.0 12.0 12.0 12.0 12.0 12.0
Available phosphorus 4.7 4.7 4.7 4.7 4.7 4.7 4..7
Arginine 6.5 6.5 6.5 7.1 7.1 7.1 -
Histidine 4.2 4.2 4.2 2.6 2.6 2.6 -
Isoleucine 4.2 4.2 4.2 4.4 4.4 4.4 -
Leucine 7.7 7.7 7.7 7.3 7.3 7.3 -
Lysine 7.5 7.5 7.5 6.0 6.0 6.0 -
Methionine 1.1 1.6 2.2 1.2 1.6 2.2 -
Phenylalanine 4.8 4.8 4.8 4.9 4.9 4.9 -
Threonine 3.5 3.5 3.5 3.6 3.6 3.6 -
Tryptophan 0.9 0.9 0.9 1.6 1.6 1.6 -
Valine 4.7 4.7 4.7 5.1 5.1 5.1 -
* As in Table 4.1
64
one legume meal only without methionine supplementation, or with methionine
supplementation to meet 75 and 100% of the chickens requirement for methionine, or an
isoenergetic protein free diet. The composition and the calculated nutrient contents of the
diets is presented in Table 5.1.
Differences between the mean of Net Weight Gain (NWG) and Net Protein Ratio (NPR)
values of chickens fed different diets and supplements were tested for significance using the
General Linear Model (GLM) procedure SAS (SAS Institute, 1990). The regression lines of
methionine intake on NWG of chickens fed each legume diets were tested for homogeneity
according to Mead et al. (1993).

The feed intake (FI), NWG and NPR values of chickens fed ten grain legumes as the sole
source of protein increased (P<0.01) as the level of dietary methionine increased (Table
5.2). Low feed intake of chickens fed unsupplemented diets may be a primary response of
chicken to methionine deficiency. Boorman (1979) after reviewing the effects of amino
acid imbalance on feed intake concluded that severe deficiency of amino acid caused large
decreases in the intake of a diet. Feed intake increased as the diets were supplemented with
the first limiting amino acid followed by an increase in NWG. Feed intake of all diets
supplemented to 75% methionine requirement was 19.5% higher than those of
unsupplemented diets and those supplemented to 100% requirement was 10% higher than
those of 75% requirement. Thus, the primary response of chickens to methionine
supplemented diets was to increase their FI.
The efficiency of protein utilisation was also increased as indicated by an increase in the
NPR values (some to a significant extent) as the level of methionine was increased to 100
% requirement. Overall, the increase of the NPR value of grain legume diets with graded
levels of methionine supplementation were linear (P<0.001). The regression equation
describing NPR as a function of levels of dietary methionine (LM) was: NPR = 2.15 +
0.01 LM. This suggests that methionine supplementation improves the protein quality of
grain legumes in a significantly linear relationship between the unsupplemented diets and
100% of the requirement for methionine of meat chickens.
65
Table 5.2. Effects of graded levels of methionine supplementation on Feed Intake, NWG and NPR values in young chickens fed legume meal
diets.
Feed Intake (g) NWG (g) NPR

No. 75 100 No. 75 100 No. 75 100
Suppl. Suppl. Suppl.
x xy z x y x x x y
Black gram (50)* cd119.8 e150.3 c188.6 g-20.78 e-4.37 d12.69 d1.71 c1.89 c2.47
x x x x x x x y y
Chickpea cv. Desi (45) a233.9 cd222.4 bc221.7 a37.66 ab43.85 abc47.03 ab3.02 ab3.49 a3.61
x xy y x y y x x x
Chickpea cv. Kaniva (45) a263.4 a309.2 a304.1 a42.51 a58.07 a61.92 a3.44 ab3.63 a3.70
x xy yz x y y x xy y
Faba bean (36) bc151.2 de189.6 bc219.7 ef-4.50 c26.14 c32.35 c2.41 ab3.16 ab3.37
x y y x y y x x x
Field pea (27) bc148.3 cd214.6 bc193.5 cd9.98 bc30.39 c29.57 abc2.94 ab3.06 ab3.26
x x y x xy y x xy xy
Green gram (50) b175.9 de174.1 b255.9 cd14.12 cd24.11 c36.33 bc2.65 ab3.25 bc2.84
x x y x y z x y y
Lentil*** (32) bc144.1 de199.6 b229.2 de3.51 bc36.62 ab55.90 bc2.60 a3.83 a3.4
x y y x y z x xy y
Lupin (23) d103.5 de187.9 bc235.6 fg-15.54 de8.87 c33.68 d1.74 c2.19 bc2.75
x x x x y xy x x x
Pigeon pea (50) a230.3 ab279.0 b252.6 bc21.28 bc38.45 c33.34 bc2.64 b2.96 bc2.88
x x x x x y x x x
Soybean meal (54) a231.7 bc237.1 b244.8 ab32.89 bc37.32 ab53.61 a3.23 ab3.40 a3.50
Overall 181.8x 217.3y 239.4z 12.12x 30.83y 38.52z 2.64x 3.12y 3.20y
* The values between brackets are percent of NRC (1994) methionine requirement supplied by unsupplemented diet containing 100g kg-1 crude protein.
** Values within columns under each heading with the same subscripts and in rows with the same superscript are not significantly different at 5% level of
probability.
*** Dehulled meals.
66
These responses support the hypothesis that for any molecular sieve (such as the viscous
contents that form in an animal gut after consuming soluble non-starch polysaccharides),
the greater the concentration of a penetrating molecule the greater the number of molecule
that pass through the sieve, and the smaller the proportion that pass through the greater is
the inhibitory effect. Similarly, ANF such as tannins that combine with or cause
precipitation of protein in the gut also have a linear concentration effect (Ortiz et al. 1993).
The equation, however, has a limited ability to predict the NPR of methionine
supplemented legume diets since the coefficient of determination was low (R2 = 0.09),
probably because of the wide variation between legumes (CV = 18.91) and the wide range
of supplementation level of dietary methionine.
The increase of NWG of chickens fed the methionine-supplemented legume diets can be
reasonably described by methionine intake (MI). Overall, the relationship between NWG
and MI was linear according to equation: NWG (g) = -8.769 + 101.31MI (g); R2 = 0.56.
Regression analysis for effect of MI on NWG for each legume is shown in Table 5.3. A
test for homogeneity of the regression slopes of NWG on MI of ten grain legumes showed
that there were three groups of results. There was no significant difference between the
regression slopes of NWG on MI within chickens fed the chickpea cv. Desi, chickpea cv.
Kaniva, or lentil diets (Group A) nor within chickens fed the faba bean, field pea, green
gram, lupin or pigeon pea diets (Group B). However, the mean regression factor of group
A was significantly greater than the mean regression factor of group B (Figure 5.1).
Methionine addition to group A and B legumes might produce similar effect on NWG,
however at each methionine concentration , group A gave better response than group B.
This indicates that concentration of methionine in group B legumes was lower than book
values or because of lack of availability of original methionine. Chickens fed the black
gram diet (Group C) had a significantly greater regression factor for NWG on methionine
intake than chickens in either Group A or Group B. Although black gram, chickpeas,
pigeon pea and SBM contained comparable amounts of sulphur containing amino acids
(Table 3.2), the availability of those amino acids in black gram might be significantly
lower than in other legumes even assuming that the supplemented methionine was readily
available.
The relative NWG (using NWG of chickens fed SBM at 100% methionine requirement as
a reference protein) of the chickens resulted from feeding different level of methionine are
67
Table 5.3 . Linear relationship between NWG(g) and MI (g) of chickens fed methionine
supplemented legume diets
Legume Inter- Parameter SEb Probability R2

cept Estimate
(MI)
Black gram -35.730 118.695 0.187 0.0001 0.904
Chickpea cv. Desi 15.911 73.848 22.330 0.0034 0.342
Chickpea cv. Kaniva 28.399 53.183 17.980 0.0073 0.285
Faba bean -7.938 82.550 12.801 0.0001 0.654
Field pea 1.232 74.350 10.686 0.0001 0.708
Green gram 3.048 63.435 13.043 0.0001 0.518
Lentil -8.076 122.877 11.361 0.0001 0.842
Lupin -23.356 110.720 12.727 0.0001 0.783
Pigeon pea 6.061 58.555 14.688 0.0007 0.431
SBM 8.199 69.102 20.317 0.0026 0.345
SEb = Standard Error of parameter estimate
68
(A)
100
y = 89.396x + 5.8375
80 R2 = 0.533; P<0.001
60
NWG (g)
40
20
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
-20
(B)
100
y = 85.258x - 7.426
80
R2 = 0.633; P<0.001
60
NWG (g)
40
20
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
-20
-40
(C)
100
y = 118.69x - 35.73
80 R2 = 0.9036; P<0.001
60
NWG (g)
40
20
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
-20
-40
Methionine Intake (g)
Figure 5.1. Regression of net weight gain (NWG) on methionine intake of chickens fed legume
diets supplemented with methionine. Group A(SBM, chickpea cv.Desi, chickpea cv. Kaniva and
lentil); Group B (faba bean, field pea, green gram, lupin and pigeon pea); Group C (black gram).
69
Table 5.4. The relative NWG of chickens fed legume diets supplemented with graded level of
methionine.
Legume* Percent Methionine in Diets

No. Supple- 75 100
mentation
Black gram (50) -0.39 0.01 0.21
Field pea (27) 0.19 0.57 0.55
Faba bean (36) -0.08 0.49 0.61
Pigeon pea (50) 0.40 0.72 0.62
Lupin (23) -0.29 0.16 0.63
Green gram (50) 0.26 0.45 0.68
Chickpea cv. Desi (45) 0.70 0.82 0.88
Soybean meal (54) 0.58 0.74 1.00
Lentil** (32) 0.06 0.69 1.05
Chickpea cv. Kaniva (45) 0.78 0.88 1.16
* The values between brackets are percent methionine requirement supplied by
unsupplemented diet.
** Dehulled meals.
70
shown in Table 5.4. Although the diets have been supplemented up to 100 percent
methionine requirement, growth of the chicks fed black gram, chickpea cv. Desi, field pea,
faba bean, green gram and pigeon pea diets did not equal that produced by the SBM diet.
This was an indicator that interference with normal digestive function caused by the
presence of ANF resulted in a reduction in availability of nutrients. The extent of the
negative effect of ANF varied. The chicks fed black gram were the most severely affected
while those fed chickpea cv. Desi were least affected. The NSP and tannin contents may
have been partly responsible for growth depression of chicks fed the black gram diet. It
was observed that chicks fed the black gram diet developed sticky feed-balls between the
beaks, reducing feed consumption and produced sticky droppings, indicating gumminess
of the diet.
At the stage at which dietary methionine met the theoretical requirement, NWG of the
chicks fed the chickpea cv. Kaniva and the lentil diets were in the vicinity of 16 and 5%
respectively, higher than those fed SBM. The reasons why the NWG of chicks fed SBM
(the reference protein) diet was lower than for chicks fed chickpea cv. Kaniva and lentil
was not clear. It is possible that over-heating during processing may have reduced the
availability of certain amino acids from SBM (Amos et al. 1975; Rani and Hira, 1993; van
Barneveld et al. 1993) or there may have been a significant amount of residual ANF. Irish
and Balnave (1993) reported that chicks fed on diets in which SBM is the sole source of
protein showed poorer growth than those fed diets in which the protein was supplied by a
combination of SBM with any of the other sources of protein. Soybean protein may not be
perfectly balanced for amino acids when used alone, but it complements the amino acid
concentration found in other common ingredients.
5.4. Conclusion
Methionine supplementation improved the protein quality of grain legumes in a
significantly linear way between unsupplemented and 100% of the requirement of meat
chickens. The rate of improvement varied between legumes, but they could be grouped as
fast (black gram) or moderate rate of improvement (9 legumes). When supplemented to
100% theoretical requirement, NWG of chickens fed chickpeas is better than the NWG of
chickens fed 5 legumes (i.e., black gram, field pea, faba bean, pigeon pea, green gram and
lupin), but not significantly different from the NWG of chickens fed SBM or lentil.
71
Chapter 6. TME value
Chapter 6
Experiment 3. Evaluation of true metabolizable energy and

digestibility of protein and starch of ten grain legumes fed to young
chickens
6.1. Introduction
Screening tests for ten grain legumes (Chapter 4) found that their protein quality was
high (chickpea, cv Kaniva, chickpea cv. Desi, and SBM) , medium (green gram , field
pea, pigeon pea) and low (lupin, faba bean and black gram). It was suggested that the
factors responsible for low quality in legumes were methionine deficiency and the
presence of anti nutritional factors. Results of the study on methionine
supplementation showed that NWG of chickens fed legume as the sole source of
protein increased (P<0.001) as the level of dietary methionine increased (Chapter 5).
When the methionine was present at 100% recommended allowance, the NWG of
chickens fed the chickpea cv. Kaniva and the lentil diets were in the vicinity of 16 and
5% higher than those fed SBM, whereas NWG of chickens fed the black gram,
chickpea cv. Desi, field bean, field pea, green gram, lupin and pigeon pea diets were
78, 12, 45, 32, 37 and 38% lower than those fed the soybean meal diet. It is likely
that the NWG was a result of the combined effect of variation in NPR values and
variation in metabolisable energy. Edwards and Campbell (1991) emphasised that the
response of poultry to dietary protein is limited by dietary energy.
The reported apparent metabolizable energy values of some grain legumes for
chickens varied from as low as 8 MJ kg-1 for pigeon pea and high-fibre containing
chickpea to as high as 13.8 MJ/kg for low-fibre containing chickpea (Table 2.2). It
should be noted that variation may exist not only among legumes but also between
bird type (Slinger et al. 1964) and assay procedures (McNab, 1996). Therefore the
ME of a feedstuff should be assayed using the target animals for which the feedstuff
is to be used. Variation in ME value may be associated with variation in starch
digestibility. Mollah et al. (1983) in their studies on ME of wheat, suggested that in
most cases, variation in AMEn is entirely accounted for by variation in starch and
protein digestibilities. In line with those finding, Longstaff and McNab (1987)
observed a good correlation between TME value of faba bean with starch
digestibility. This suggests that any component of the ingredient that reduces starch
72
digestibility would reduce the metabolizable energy value since about 50% of dietary
energy metabolized by poultry is derived from starch (Yuste et al. 1991).
The purpose of this study was to determine the true metabolizable energy (TME)
value in relation to the starch and nitrogen digestibilities and the tannin and Neutral
Detergent Fibre (NDF) contents of ten grain legumes in young chickens.
6.2. Materials and Methods
6.2.1. Test materials

The grain legumes studied herein were similar to those used in previous experiments.
All grain legumes were ground to pass a 1 mm screen.
6.2.2. Bird management and excreta collection

One hundred and ninety one-week-old commercial meat chickens which had been
reared in a group were randomly allocated to individual cages. The chickens were fed
a commercial starter diet up to day 20 then left for 24 h without feed to empty their
alimentary canals of feed residues. The rapid method of TME determination of
Sibbald (1976) was applied with modification since free feeding presented problems
such as in recovering feed lost in the faecal collection tray and drinking water.
Modification was made in that chickens were tube-fed a mixture of one portion of
legume meal to two portions of water using a plastic syringe. It required only about
10-15 seconds to fill the crop with the feed mixture. Each test material was fed to six
replicates of three chickens each. Four groups of three birds were left without feed to
derive a mean value of endogenous energy and nitrogen loss.
A plastic tray was placed under each cage and the time recorded. The precise amount
of test material consumed was the difference between the weight of the chicken
before and after feeding. The excreta was collected for 24 h after feeding, freeze-dried
and left at room temperature for 24 h, weighed and ground to pass a 0.8 mm screen
and kept for analyses of their gross energy, protein and starch contents.
The TME and TPD values for each replicate were calculated using the following
equations:
EI − FE + EEL
TME ( MJ / kg ) =
FI
73
PI − ( PF − EPL)
TPD (%) = X 100
PI
where EI = energy intake, FE = faecal energy, FI=feed intake and EEL = endogenous
energy lost of unfed birds, EPL= endogenous protein lost, PI= protein intake,
PF=faecal protein.
6.2.3. Analytical method

The chemical analysis was performed as described in Chapter 3, while the statistical
analysis was as described in previous chapters.

True metabolizable energy (TME) values, true protein digestibility (TPD) and starch
digestibility (SD) of grain legumes are presented in Table 6.1. Although their GE
content was not different, the TME values of grain legumes were significantly
different. TME values of pigeon pea, faba bean, lupin and black gram were
significantly (P<0.05) lower than the TME of other legumes. The TME values of
chickpea cv. Desi, field pea, green gram, lentil and SBM were higher than the TME of
pigeon pea, faba bean and lupin, but significantly lower than those of chickpea cv.
Kaniva. Among all grain legumes studied herein, chickpea cv. Kaniva was not only
the source of the highest quality protein (Chapter 4) but also the best source of
metabolizable energy.
Protein, lipid and starch digestibilities of grain legumes were also significantly
different (P<0.01). Over 80% of the protein of the chickpeas, green gram and SBM,
and between 70 to 76% of the protein of field pea, lentil, lupin and pigeon pea was
digestible by growing chickens, but less than 70% of the protein of black gram and
faba bean was digestible. The TDP of the chickpeas, green gram and SBM was in a
range of published values (Rhone Poulenc Animal Nutrition, 1989; Carre et al. 1991;
Brenes et al. 1993; Carre, 1997), but those of black gram, faba bean and lupin were
lower (P<0.05). TPD was not affected by the level of trypsin inhibitor in legume
meals, but tannin content may have significantly reduced protein digestibility.
Digestibility of protein in this study was negatively correlated with tannin content (r =
-0.31, P < 0.05). Condensed tannin in faba bean, field peas and chickpea cv. Desi
have been reported to decrease digestibility of protein and the extent of the negative
effect depended on dietary concentration of tannin (Carre, 1997). Ortiz et al. (1993)
showed that the digestion of faba bean protein by young chicks was significantly
74
affected by the level of tannin in the diet. Inclusion of freeze dried tannin at 1.6% into
a chick diet resulted in an 8% reduction of ileal digestibility of protein. Faba bean
tannin also significantly reduced the protein digestibility of piglets (Jansman et
al.1993).
Lipid digestibility (LD) varied from as low as 50% in faba bean to as high as 89% in
chickpeas. There was no negative correlation between LD and tannin or between LD
and NDF contents. A significantly positive correlation (r2 = 0.83; P<0.01) between
lipid content and LD suggests that the higher the lipid content of grain legumes the
higher the digestibility of their lipid. However, there was no clear evidence to explain
that relationship. Ingestion of legume meal of higher lipid contents may have
increased the secretion of bile acid, facilitating an increase in lipid digestibility (Juste
et al.1983).
Apart from starch digestibility (SD) of SBM and lupin, the SD’s of grain legumes
were significantly different from each other. Yuste et al. (1991) reported that the pea
and bean starch digestibilities in chicks were 94 and 78% respectively, which was
slightly lower than those obtained with cockerels. SD of black gram and pigeon pea
were significantly (P<0.05) lower than SD of other legumes. Over 93% of starches of
field pea, chickpea cv. Kaniva and lentil were digestible in young chickens. SD of
chickpea cv. Desi, green gram and faba bean were significantly lower than those of
chickpea cv. Kaniva but significantly greater than those of black gram and pigeon
pea. Correlation analyses indicated that reduced starch digestion was closely related to
tannin content (r= -0.62, P<0.001) and cell wall (NDF) contents (r = -0.30, P< 0.05).
Flores et al. (1994)
75
Table 6.1. True metabolizable energy (TME), true nitrogen digestibility (TND),
lipid digestibility (LD) and starch digestibility (SD) of some grain legumes in
growing chickens.
Legume* TME TPD LD SD Tannin

(MJ kg-1) (%) (%) (%) Content
mg g-1
Black gram 9.39d (10) 67.21e (9) 69.09c (6) 78.64e (8) 16.22 (1)
Pigeon pea 10.22d (7) 75.88abcd(5) 77.95b (4) 79.05d (7) 2.64 (2)
d e d bc
Faba bean 10.16 (8) 65.51 (10) 50.38 (10) 90.26 (4) 2.53 (3)
Green gram 13.74b (2) 78.10abc(4) 66.12c (7) 89.76bc (5) 2.10 (4)
Lupin 9.67d (9) 69.44de (8) 87.39a (3) nm 1.53 (5)
cd a a d
Chickpea cv. Desi 13.25 (4) 81.96 (1) 88.15 (2) 86.96 (6) 0.62 (6)
c cde bc ab
Field pea 12.01 (6) 70.88 (7) 72.83 (5) 93.52 (3) 0.61 (7)
Chickpea cv. Kaniva 15.09a (1) 80.29ab (2) 88.90a (1) 95.03a (1) 0.51 (8)
cd abc d
SBM 12.94 (5) 79.17 (3) 57.01 (9) nm 0.49 (9)
b bcde c ab
Lentil 13.38 (3) 71.88 (6) 65.10 (8) 93.62 (2) <0.1 (10)
SEM 0.42 2.61 2.61 1.32 -
* Value in brackets are the rank orders,
SEM = Standard error means,
nm = not measured,
abcdef
Different superscripts in the same columns are significantly different (P<0.05)
76
reported that the addition of tannin extract or tannin-containing faba bean hull always
decreased the digestibility and TMEn of semipurified starches when fed to young
chickens. The effects depends on the quantity of tannin ingested. The difference in SD
of chickpea cv. Desi and SD of chickpea cv. Kaniva might be due to differences in
their content of amylase inhibitor. Singh et al. (1982) reported that the level of
amylase inhibitors in chickpea cv. Desi was greater than those of chickpea cv. Kabuli,
and that was negatively correlated with in vitro starch digestibility.
The positive correlations obtained between TME and SD (r = 0.70, P<0.001) and
between TME and TPD (r = 0.57, P<0.01) suggest that any component in grain
legumes which inhibits digestibility of starch and/or protein would result in a decrease
in TME value. Tannin intake induced a greater negative effect on SD (r = - 0.64; P<
0.001) than on TPD (r = -0.31; P< 0.05), but not significantly reduced LD. However
SD, TPD and LD of grain legumes were not evenly affected by tannin intake. The
extent of the inhibition of SD, TPD and LD by tannin may be related to the relative
amount of the nutrients ie., starch was most affected, protein was affected to a lesser
extent and lipid was least affected. However, this theory is not supported by chickpea
cv. Desi which has a relatively low SD but high TPD and LD, although the SD was
still quite high (close to 87%). SD in some legumes such as field pea and lentil was
high but their TPD and LD were low, suggesting the presence of other ANF.
The possible mechanism by which tannin exerts its negative effects is through the
formation of insoluble complexes with carbohydrates and proteins and/or through
formation of complexes between tannin and digestive enzymes. The formation of
tannin-starch complexes has been shown to decrease the in vitro amylolysis of several
legumes and other starches (Deshpande and Salunkhe, 1982) and the tannin-protein
complexes, which are extremely hydrophobic (Hagerman and Butler, 1980; Mitaru et
al. 1984), were partly responsible for low protein digestibility. Intensive studies by
Longstaff and McNab (1991) on effect of tannin showed that diets with tannin-rich
hulls of faba bean caused a large reduction in the digestion of amino acids, starch and
lipid compared with the control diet mainly due to inactivation of digestive enzymes
by the formation of tannin-enzyme complexes in the digestive tract. Tannin
inactivated trypsin the most, alpha-amylase to a lesser extent and lipase the least and
therefore lowered the digestion of amino acids the most, starch to a lesser extent and
77
lipid the least. Tannin in most grain legumes studied herein may have been exerting
its additional antinutritional potency on substrates rather than enzymes.
The cell wall content (NDF) also contributed to a lesser extent to the decrease in SD
(r = - 0.30, P< 0.05) and TND (r = -0.23, P< 0.10). However, there was no significant
correlation between tannin and NDF with LD.
Figure 6.1. showed that about 40% of energy content of black gram and lupin and
about 30 to 35% of those from faba bean and pigeon pea were not metabolized by the
chickens. Regression analysis showed that the non-metabolized energy of grain
legumes was significantly increased by the presence of both NDF and tannin (R2 =
0.62; P<0.001) according to the following equation :
Non-metabolized energy (MJ/kg) = -14.161 + 15.804Tannin (%) + 1.886 NDF(%)
In line with these findings, Bjerg et al. (1988) reported that contents of tannin and of
insoluble and total dietary fibre were negatively correlated with quality as expressed
by digestible energy, protein digestibility and biological value of the faba beans. The
equation indicates that around 62% of variation of energy lost was accounted for by
variation in tannin and cell wall contents. This means that measuring the non-
metabolized energy is an indicator of the amount of ANF in a diet, and conversely
measuring the ANF (tannin and NDF in particular) is an indicator of non-
metabolizable energy or non-digestible protein and starch
It is difficult to compare the metabolizable energy value of legumes from different

reports because of the variation between and within legumes as demonstrated by the
differences shown between Spring and Winter cultivars (Carre et al. 1991) and
between Kaniva and Desi varieties of chickpea in these trials.
78
50
Non-metabolized energy
40
30
20
10
0
Faba bean
Chickpea
Lupin
Pigeon
Green
Field pea
Black
Lentil
SBM
cv.Kaniva
gram
Chickpea
gram
cv Desi
pea
Figure 6.1. Non-metabolized energy as % of gross energy of ten grain legumes when
fed to growing chickens
6.4. Conclusion
The TME, TPD, LD and SD of grain legumes were significantly different. The
chickpea cv. Kaniva was the best source of energy and protein but black gram, faba
bean and lupin showed the lowest values for chickens. Tannin and cell wall
components play a significant role in decreasing the TME, TPD and SD of grain
legumes. The non-metabolizable energy content of grain legumes can be predicted by
using their tannin and NDF contents and vice versa.
79
Chapter 7. Effect of autoclaving
Chapter 7
Experiment 4. Improving the quality of grain legumes: effect of

autoclaving.
7.1. Introduction
Apart from methionine deficiency, the presence of greater quantities of antinutritional
factors might be the cause of low Net Protein Ratio (NPR) value (see Chapter 4) and
low True Metabolizable (TME) value (see Chapter 6) of black gram and lupin
compared with other legumes.
A wide range of methods for the thermal inactivation of antinutritional factors of grain
legumes has been studied. Methods such as, autoclaving, toasting, and extrusion
cooking have improved the nutritive value of grain legumes. Studies with chickens
showed that autoclaving smooth peas (Pisum sativum) for 3 min at 1300C and 170 kpa
increased their AME value, and their protein and starch digestibility (Conan and
Carre, 1989). Studies with other legumes such as lupin, yellow peas and faba beans
(Boldaji, et al. 1986), pigeon pea, African yam bean and cowpea (Nwokolo and Uji
1985), jack beans (D’Mello et al 1985) and winged beans (D’Mello et al 1983) also
showed that autoclaving increased their ME contents. The weight gain of chicken fed
diets containing 75% autoclaved faba bean or autoclaved field peas increased by 8 or
4%, and the FCR decreased by 10 or 11%, compared with those fed raw beans or raw
peas (Ernest, 1984)
The effects of autoclaving, however, depend on both temperature and duration of

heating (Amos et al. 1975; Yanez et al. 1986). Extended steam heating of Phaseolus
vulgaris beans from 40 min to 80 min resulted in lower weight gain and higher feed-to-
gain ratio in piglets (Van der Poel et al. 1990a). Excessive heating may reduce the
availability of some amino acids, especially lysine. Van Barneveld (1993) reported that
26 to 40% of the lysine of peas was not available after heating from 135 to 150oC.
Therefore it is important to define the optimum heat treatment to maximise the
inactivation of ANF and minimise the loss of amino acid.
The aims of this experiment were to study the effect of autoclaving conditions
(temperature, pressure and time) on the nutritive quality of black gram and lupin for
80
growing chickens and to study whether lysine supplementation of autoclaved lupin

would have any effect on its NPR value.
7.2.1. Autoclaving and diet formulation

Black gram and lupin meals were spread to a depth of approximately 1.5 cm on
stainless steel pans and heated to 125oC and 180 kPa for 0, 5, 10 , 15 and 20 min in a
standard laboratory steriliser. The autoclaved meals were used as the sole sources of
protein in an isoenergetic diet containing nominally 100 mg g-1 crude protein.
Cornstarch, dicalcium phosphate, limestone , and vitamin and mineral premixes were
added to each diet according to least cost formulation (UFFF, 1986). Dietary fibre
content was adjusted with solka floc (Table 7.1). SBM meal was used to formulate an
isoenergetic and isoprotein control diet. To investigate whether over heating may
destroy lysine, another set of lupin diets was formulated with the addition of 3 g kg-1
lysine (i.e., approximately an extra 50% lysine content of lupin diet).
7.2.2. Bird management

One week old male-meat chickens were randomly allocated to individual cages (8
chickens per treatment) in a temperature controlled room (30 ± 0.5oC) and given
unlimited access to diet and water for 14 days. Eight chickens were fed an isoenergetic
protein-free diet as a negative control group. They were weighed at 7 days and 21 days
after an overnight fast. Feed allowance, feed refusal and feed spills were measured.
The chickens’ response to dietary protein was assessed in term of net weight gain
(NWG) and net protein ratio (NPR). NPR was calculated as [weight gain of the
chickens fed the test diet + weight loss of the chickens fed protein-free diet] divided by
protein intake (Bender & Doell, 1957).
7.2.3. Determination of TME and Apparent Digestibility of Protein (APD)

Upon conclusion of the 14 days feeding trial, the chickens were randomly reallocated in
the cages and fed a commercial diet for 3 days then left without feed for 24 h to empty
81
Table 7.1. Dietary ingredients and nutrient composition (g kg-1)
Protein- Black gram Lupin

free diet
Ingredients
Black gram 400.20 -
Lupin - 319.00
Starch 851.20 446.10 559.20
Sunflower oil - 73.10 41.00
Premix* 2.00 2.00 2.00
Solka floc 16.50 39.10 36.00
Di-Calcium phosphate 26.10 22.30 24.50
Limestone 100.00 16.70 16.50
Methionine - 0.60 1.70
Calculated content
TME (MJ kg-1) 13.00 13.00 13.00
Crude protein - 100.00 100.00
Fat - 77.60 57.10
Crude fibre 104.0 50.00 50.00
Calcium 120.00 120.00 120.00
Available phosphorus 47.00 47.00 47.00
Arginine 7.11 12.05
Histidine 2.91 2.65
Isoleucine 4.50 4.28
Leucine 8.45 6.97
Lysine 7.71 5.31
Methionine 2.20 2.20
Phenylalanine 6.40 4.28
Threonine 3.50 3.62
Tryptophan 0.97 0.71
Valine 5.00 4.20
*Per tonne of diet the vitamin and mineral premix contained; 8000 iu vitamin A, 3000 iu vitamin D3, 5 g
vitamin E, 300 g vitamin K, 3 g Calcium pantothenate, 3 g Riboflavin, 15 g Niacin, 10 mg Vitamin
B12, 5 mg Biotin, 100 mg Choline, 200 mg Cobalt, 500 mg Iodine, 5 g Copper, 20 g Iron, 80 g
Manganese, 50 g Zinc, 20 g Ethoxyquin, 100 mg Selenium, and 200 mg Molybdenum.
82
their alimentary canals. The true metabolizable energy (TME) value and apparent
digestibility of protein (APD) of the diets were determined according to method of
Sibbald (1979a) with slight modification. The modification was made in that chickens
were tube-fed a mixture of 1 portion of diet and 1 portion of water using a plastic
syringe. It required only about 10-15 seconds to fill the crop with the feed mixture. The
precise amount of test material consumed was the difference between the weight of the
chicken before and after feeding. Each test material was fed to four replicates of two
chickens each. A plastic tray was put under each cage and the time recorded. The
excreta was collected 24 h after feeding, freeze dried, left overnight at room
temperature, weighed and ground to pass 0.8 mm screen and kept for analyses of their
energy, nitrogen, and uric acid contents.
The TME value and APD for each group of birds were calculated according to equation
1 and equation 2 respectively:
EI − FE + EEL
TME ( MJ / kgDM ) = .............................................................(1)
FI
PI − PF
APD (%) = X 100 ...............................................................................(2)
PI
where EI = energy intake, FE = faecal energy, FI = feed intake and EEL = endogenous
energy lost of unfed birds, PI = protein intake, and PF = faecal protein.

The feed intake (FI), net weight gain (NWG) and net protein ratio (NPR) are
presented in Table 7.2. A significant interaction between legumes and autoclaving time
on FI and NWG were observed. This suggests that chickens’ responses to the two
legumes autoclaved for different times were different. Although the NWG of chickens
fed B5 diet was significantly greater (P<0.05) than those fed BO and B20 diets, overall,
NWG, FI and NPR values were not significantly affected by autoclaving time. On the
other hand, a slight increase (c. 4.2%) in FI and a dramatic increase (c. 50%) in NWG
were observed when the chickens were given the L5 diet compared to the L0 diet, and
resulted in an increase in the NPR value.
Regression analysis showed that the effect of autoclaving time on NPR value of lupin
meal was curvilinear fitting the following equation (R2 = 0.49; P<0.001):
83
NPR = 3.86 + 0.05 x - 0.005x2
where x = autoclaving time (min).
This suggests that the protein quality of lupin was significantly (P<0.05) improved
when lupin meal was autoclaved for 5 min, but heating the meal longer than 5 min did
not improve response. In fact, FI, NWG and NPR value were significantly reduced
when lupin meals were autoclaved for 20 min, apparently due to severe damage of the
protein, because of NPR value was lower with respect to the raw lupin diet. Yanez et
al. (1986) reported 51% and 29% reduction of lysine and sulfur containing amino
acids respectively after roasting (80 - 90oC) lupin meals for 10 to 40 min. Furthermore,
it was reported that 26 to 40% of the lysine of peas was not available after heating
from 135 to 150oC respectively (Van Barneveld et al. 1993). Thus, inclusion of
excessively heated lupin meal in this experiment might have created an imbalance in
dietary amino acids, one of main reasons for the reduction of FI (Harper and Rogers,
1965; Boorman, 1979; D’Mello, 1994). This might be the main factor responsible for
the retardation of growth of chickens fed excessively heated lupin meals.
NWG and NPR value of lupin diets supplemented with 3 g kg-1 are shown in Figure
7.1. Lysine supplementation of L0 and L5 diets did not give positive responses to FI
and NWG. A significant decreased in FI of chicken fed L10, L15 and L20 was
overcome by lysine supplementation. The NWG of chicken fed supplemented L10 was
significantly greater than NWG of chicken fed L10 and not significantly different from
those of fed L5 suggesting that loss of lysine during autoclaving was replaced with
supplementation, assuming that concentration of other amino acids was not
significantly affected. Overall, lysine supplementation of autoclaved lupin improved
FI, but not the NPR value. The results presented in this figure seem to suggest that an
amino acid other than lysine may have become the second limiting amino acid in
heated protein.
84
Table 7.2. Effect of autoclaving time on feed intake, NWG and NPR value of black
gram and lupin
Legume Time Diet Feed Intake NWG NPR NPR
(min) (g) (g) (% SBM)
Black gram 0 B0 211.08bcde 40.84cd 3.75bcd 82.96
5 B5 265.46ab 69.56ab 4.04ab 89.38
bcd bc ab
10 B10 231.48 57.05 4.13 91.37
15 B15 229.56bcd 51.59bc 3.82ab 84.51
20 B20 208.43bcde 41.59cd 3.67ab 81.19
Lupin 0 L0 249.83abc 57.66bc 3.80ab 84.07

5 L5 294.51a 86.43a 4.17a 92.25
10 L10 196.45cde 36.55cd 3.79ab 83.85
15 L15 174.75de 25.94de 3.56b 78.76
20 L20 155.24e 8.10e 2.71c 59.95
SBM 0 - 260.40ab 78.07a 4.52a 100

SEM - 18.20 7.68 0.17 -
abcde
Different superscript in the same column are significantly different (P<0.05)
Standard error mean
85
The NPR value of black gram and lupin after autoclaving for 5 min were about 89 and
93% of the NPR SBM. These results indicate that short term autoclaved black gram and
lupin are potential alternates for SBM in chickens’ diet.
The effect of autocalving on metabolizable energy and protein digestibility is presented

in Table 7.3. Autoclaving did not significantly improve the TME value of black gram
diets, but the TME value of L5 was significantly (P<0.05) greater than the TME value
of the L0 diet. This might be due to improvement in starch digestion. Longstaff and
McNab (1987) reported that the digestibility of starch in heat-treated peas was slightly
better than that of raw peas. In line with Yanez et al. 1986), the APD measured in this
study was not improved by autoclaving. In fact, prolonged heating reduced (P<0.05)
the APD value of both legumes. Products of the Maillard reaction might have
contributed to higher nitrogen excretion in faeces.
Analysis of correlation show that NPR value and APD are correlated ( r = 0.75; P <
0.01) and that there is a tendency for the NPR value of these two legumes to be partly
explained by variation in their TME value (r = 0.54, P = 0.11). Edwards and Campbell
(1991) emphasised that response to dietary protein is limited by dietary energy.
There is no clear reasons to account for the lack of positive response of black gram
meal to autocalving. It is possible that there were no-heat-labile anti nutritional factors
in the sample of black gram meal used. Black gram has been reported to be devoid of
lectins (Reddy and Salunkhe, 1981). Although black gram contains relatively higher
concentration of trypsin inhibitors than other legumes (see Chapter 3), this particular
inhibitor may be resistant to heat treatment. An intensive study conducted by Padhye
and Salunkhe, (1980) showed that only 9% of the activity of the trypsin inhibitor in
black gram meal was lost when the meal was heated for 5 min at 100oC.
86
(A)
100.00
90.00 Lupin
80.00 Lupin+lys
70.00
NWG (g)
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0 5 10 15 20
4.50 (B)
Lupin
4.00
Lupin+lys
3.50
3.00
NPR
2.50
2.00
1.50
1.00
0 5 10 15 20
Autoclaving Time (min)
Figure 7.1. Effect of lysine supplementation on NWG (A) and NPR (B) values of
autolcaved lupin
87
Table 7.3. Effect of autoclaving time on AME, TME and APD value of black gram and
lupin in young chickens
Legume Time Diet TME APD

(min) (MJ kg-1) (%)
Black gram 0 B0 14.80ab 76.72a
5 B5 14.90ab 72.23a
10 B10 14.51ab 79.04a
15 B15 14.04bc 76.03a
20 B20 13.56c 69.28ab
Lupin 0 L0 14.23bc 72.89a
5 L5 15.15a 70.21ab
10 L10 14.31abc 68.71ab
15 L15 14.36abc 70.39ab
20 L20 14.01bc 60.97b
SEM - - 0.27 3.21
abc
Different superscript in the same column are significantly different (P<0.05)
7.4. Conclusion
Response of black gram and lupin to autoclaving was different. Black gram did not
give a positive response to autoclaving. The NPR and ME value of lupin increased after
autoclaved for 5 min. These results suggest that the nutritive value of lupin can be
improved by autoclaving and other method should be sought to improve the nutritive
value of black gram.
88
Chapter 8. Enzyme supplementation
Chapter 8
Experiment 5: Improving the quality of grain legumes: effect of

enzyme supplementation
8.1. Introduction
Grain legumes were shown to contain relatively high concentrations of non-starch
polysaccharides (NSP) which are resistant to endogenous enzymic digestion in the
alimentary tract (Brillouet et al. 1988; Smits and Annison, 1996; Choct, 1996; Choct,
1997). Feed enzyme supplements that digest NSP and proteins may be able to
increase the metabolisable energy and protein values of grain legumes in the same
way they do in wheats (Choct and Annison, 1992; Hew et al. 1995; Choct et al.
1995, 1996).
Studies with lupins showed that adding an enzyme cocktail containing carbohydrase,
α-galactosidase and a protease to a diet containing 48% lupin (Brenes et al. 1993)
resulted in an 11% increase in weight gain and a 9% increase in feed to gain ratio of
broiler chicks. Similarly, adding a crude enzyme preparation containing β-galactanase
and a minor α-galactosidase (Bryden et al. 1994), or a mixture of hemicellulase,
xylanase, pentosanase and cellulase (Annison et al. 1995), to lupin diets increased the
apparent metabolisable energy (AME) value by 10.7% and 14% respectively.
Application of a carbohydrase to a pea containing diet resulted in a 14% increase in
AME value (Jeroch et al. 1995). Grain legumes are high in carbohydrate and protein,
therefore application of an enzyme cocktail containing carbohydrases and protease
may improve their nutritional value.
The objectives of the present experiments were:

1. to measure the effect on chicken growth of adding a multi-enzyme product
containing xylanase, .-amylase and protease (Avizyme 1500, Finnfeeds
International Ltd. UK,) to ten grain legumes included as 30% to 55% of chicken
diets, and
2. to study the effect of three different enzyme preparations on the true
metabolizable energy (TME) value and the digestibility of protein and amino
acids of black gram, and lupin and to compare them with that of SBM, the most
commonly used source plant protein in poultry diets.
89
8.2. Materials and Methods

There were two trials in this experiment.
8.2.1. Trial 1
The effect of supplementation with a multi-enzyme product containing xylanase, .-
amylase and protease (Avizyme 1500, Finnfeeds International Ltd., UK ) on the
protein quality of the ten grain legumes was evaluated.
An isoenergetic N-free diet and ten isoenergetic legume diets containing nominally
100 g kg-1 crude protein with or without 1 g kg-1 enzyme supplementation (Table
8.1). were given to seven-day-old male meat-chickens for 14 days. The treatments
were arranged in a 2 x10 factorial design with 9 replicates each. The chickens were
caged in a temperature-controlled room at 30± 0.5oC and had ad libitum access to feed
and water. The chickens were weighed after overnight fasting at the beginning and
end of the observation period.
The chickens’ response to dietary protein was assessed in terms of NWG and NPR
value.
8.2.2. Trial 2
The NPR value of black gram and lupin was found to be low (Chapter 4). This may be
an indication of both low digestibility of protein and low metabolizable energy value.
Therefore, the effects of three different enzyme preparations, a xylanase ‘Avizyme
1300’ (EA), a multi-enzyme product ‘Avizyme 1500’ as in trial 1 (EB), and a
protease (EP), on the TME value, apparent digestibility of protein (APD) and amino
acid (AAAD) and the true digestibility of protein (TDP) and amino acids (TAAD) of
black gram and lupin were assayed according to the method of Sibbald (1979). SBM
was also assayed for comparison.
Seven-day-old male-broiler chickens were weighed and randomly allocated into
individual cages in a temperature-controlled room at 30 ± 0.5oC. They were fed a
90
Table 8.1. Composition of the diets for experiment 1 (g kg-1)
Black Chickpea Chickpea Faba Field Green Lentil Lupin Pigeon SBM N-Free
gram cv. Desi cv. pea gram pea
Kaniva
Black gram 400.2

Chickpea cv. Desi 442.5
Chickpea cv. Kaniva 499.0
Faba bean 426.1
Field pea 447.2
Green gram 447.4
Red lentil 418.4
Lupin 319.1
Pigeon pea 551.3
SBM 210.0
Corn starch 446.0 512.3 424.0 464.7 456.0 485.1 509.3 559.2 324.2 717.1 851.4
Vegetable oil 73.1 - 0.1 44.3 34.0 5.1 2.0 41.0 81.4 - -
Solka flock 39.1 0.4 32.3 38.0 38.0 21.0 40.0 36.0 - 29.4 104.0
Limestone 16.7 16.5 16.6 21.5 17.9 21.9 13.9 16.5 25.2 16.4 16.5
Dicalcium phosphate 22.3 25.1 25.0 3.8 3.4 16.5 12.6 24.5 14.8 23.9 26.1
Vitamin/Mineral mix*. 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
DL-Methionine 0.6 1.2 1.0 1.6 1.5 1.0 1.6 1.7 1.1 1.2 -
Total 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0 1000.0
*Per tonne of diet the vitamin and mineral premix contained; 8000 iu vitamin A, 3000 iu vitamin D3, 5 g vitamin E, 300 g vitamin K, 3 g Calcium pantothenate, 3
g Riboflavin, 15 g Niacin, 10 mg Vitamin B12, 5 mg Biotin, 100 mg Choline, 200 mg Cobalt, 500 mg Iodine, 5 g Copper, 20 g Iron, 80 g Manganese, 50 g
Zinc, 20 g Ethoxyquin, 100 mg Selenium, and 200 mg Molybdenum.
91
commercial starter diet for two weeks. After a 24 h fasting period, they were hand-fed
25 - 35g of black gram, lupin or SBM in a 1:1 slurry of water: legume meal without
or with 1 g kg-1 EA, EB or 0.45 g kg-1 EP, by using a plastic syringe to place the
slurry directly into the crop. A plastic tray under the cage collected excreta for the
following 24 h period. The excreta of two birds was pooled, stored at -20oC, freeze-
dried, left at room temperature for 24 h, and weighed and ground to pass 0.5 mm
screen for analyses. There were six replicates of two birds per treatment. Because
analysis of amino acids is expensive, two replicates of excreta samples were
proportionally pooled, so that three samples per treatment were analysed for amino
acids.
The endogenous and metabolic energy and nitrogen used in calculating TME and
TPD were obtained from three pooled samples, each collected from two chickens,
whilst the amino acid values used in calculation of TAAD was measured from a
pooled sample of four chickens as described for the precision-fed birds (Sibbald,
1979). The TME and digestibility were calculated according to the following
formulas:
EI − FE + EEL
TME ( MJ / kg ) = ...................................................................... (1)
FI
PI − PF
APD (%) = X 100 ................................................................................. (2)
PI
PI − ( PF − EPL)
TPD (%) = X 100 ..................................................................... (3)
PI
AAI − AAF
AAAD (%) = X 100 ......................................................................... (4)
AAI
AAI − ( AAF − EAAL)
TAAD (%) = X 100 .......................................................... (5)
AAI
where:
EI = energy intake PF = faecal protein
FE = faecal energy EPL = endogenous protein loss
FI = feed intake in kg AAI = amino acid intake
EEL = endogenous energy lost by unfed AAF = faecal amino acid and
birds
PI = protein intake EAAL= endogenous amino acid loss.
92
8.2.3. Chemical and data analyses

All chemical analyses were performed as described in Chapter 3 and the statistical
analysis was performed as described in earlier chapters.
8.3.1. Trial 1
The NWG and NPR values of each grain legume fed to young chickens are presented
in Table 8.2. The response of chickens to enzyme-supplemented legume diets was
significantly different (P<0.01). Overall, NWG and NPR increased (some to a
significant extent) when the multi-enzyme mixture product containing xylanase,.-
amylase and protease was added to the diet. The significant improvement of NPR
value for chickpea cv. Kaniva, faba bean and pigeon pea was an indication of an
enhancement in digestibility of nutrients.
There was a significant (P<0.05) positive correlation (r = 0.64) between percent

improvement in NPR values and the cell wall (NDF) content of grain legumes
(Figure 8.1). Generally legumes with a greater cell wall content showed a better
response to mixed enzyme supplementation than those with a lower amount of cell
wall content. The xylanase of the enzyme mixture may have reduced the antinutritive
activity of NSP by partial cleavage of the NSP (Choct, 1996) thereby increase the
accessibility of digestive enzymes to their substrates.
8.3.2. Trial 2
Table 8.3 shows the effects of enzyme supplementation on the true metabolizable
energy (TME), and the apparent digestibility (APD) and true digestibility (TPD) of
protein of the three legumes. There was no significant difference between the TME
values of SBM and lupin and both of these legumes had higher TME values (P<0.05)
than that of black gram.
93
Table 8.2. The effects of adding 1 g kg-1 Avizyme 1500 on NWG and NPR values of
ten grain legumes fed to young chickens
Legume NWG (g) NPR
Control Enzyme Control Enzyme

e e c d
Black gram x25.86 x27.86 x3.02 x3.16
bcd bc ab abc
Chickpea,cv Desi x46.20 x64.49 x3.65 x3.88
de cd bc abc
Chickpea,cv Kaniva x32.69 y54.33 x3.31 y3.76
bcd abc c bc
Faba bean x46.84 y65.98 x3.00 y3.58
ab a a ab
Field pea x58.39 y82.03 x3.77 x3.91
ab abc a ab
Green gram x55.79 x67.85 x3.86 x3.91
bc ab a a
Lentil x50.32 y70.56 x3.78 x3.95
cde cd c abc
Lupin x35.90 x54.31 x3.23 x3.49
a ab bc abc
Pigeon pea x66.68 x73.01 x3.28 y3.69
bc d ab ab
SBM x50.26 x47.78 x3.67 x3.94
Mean x46.89 y60.82 x3.46 y3.73
LSD 15.3 16.16 0.39 0.37

abcd
Values within column with different superscripts are significantly different
(P<0.05)
xy
Values within rows under a similar heading with different subscripts are
significantly different (P<0.05)
94
Increase of NPR value NDF

25.00
20.00
15.00
10.00
5.00
0.00
Faba bean
Pigeon pea
Chickpea
Lupin
SBM
Green gram
Lentil
Black gram
Field pea
cv.Kaniva
Chickpea
cv. Desi
Figure 8.1. The NDF content and improvement of NPR value (%) of grain legumes
after supplementation with an enzyme product containing xylanase,.-amylase and
protease.
95
Table 8.3. True metabolizable energy (TME) and apparent protein digestibility (APD)
and true protein digestibility (TPD) of black gram, lupin and soybean meal in
growing chickens
Legume Enzyme TME APD TPD

(MJ kg-1) (%) (%)
Black gram Control 9.33de 51.0ef 70.9d

EA 9.12e 48.8f 68.1d
EB 10.30cd 53.2def 72.4d
EP 8.56e 39.1g 58.6e
Lupin Control 11.18bc 54.8def 74.1cd

EA 12.68a 61.9cd 77.6bcd
EB 11.95ab 59.6cde 75.6cd
EP 11.18ab 56.7cdef 75.6cd
SBM Control 12.11ab 64.7bc 82.6abc

EA 12.28ab 70.9ab 86.2ab
EB 12.04ab 72.7ab 87.6a
EP 11.75ab 76.6a 90.1a
Pooled SEM - 0.367 2.9 3.1

abc....
Means in column bearing different superscripts are significantly different
(P<0.05)
EA = Enzyme A: xylanase (Avizyme 1300)
EB = Enzyme B: multi enzyme (Avizyme 1500)
EP = Enzyme P: protease (unnamed product of Finnfeeds International Ltd.)
96
Overall, enzyme addition increased (P<0.05) TME values, but the response was not
consistent, and there was a significant interaction (P<0.05) between legumes and
enzymes. The multi-enzyme product (EB) increased (P<0.05) the TME value of black
gram by 9 percentage unit, but did not improve the TME value of either SBM or
lupin. On the other hand, xylanase (EA) addition significantly (P<0.01) improved the
TME value of lupin, but not black gram and SBM. The positive effect of addition of
EA on TME value probably due to an increase in digestibility of the NSP component
of the lupin (Annison et al. 1996). All enzymes tested failed to improve the TME
value of SBM, possibly because of the highly digestible nature of the SBM protein,
starch and lipid by gastrointestinal enzymes.
There was a highly significant (P<0.001) difference between the three legumes
studied in both apparent protein digestibility (APD) and true protein digestibility
(TPD). The average APD and TPD for the non supplemented legumes were 64.7 and
82.6% for SBM, 54.8 and 74.1% for lupin and 51.0 and 70.9% for black gram .
Overall, enzyme supplementation increased APD by 6%, however only with the multi
enzyme product was the increase significant. The TPD values were not significantly
improved and in some cases were significantly decreased by enzyme
supplementation. For example, protease improved the APD (P<0.01) of SBM but
significantly depressed (P<0.01) the APD and the TPD of black gram. These results
suggest that the enzyme or enzyme mixture needed for enhancement of the
nutritional value of a particular grain legume is specific.
The apparent amino acid digestibilities (AAAD) and the true amino acid
digestibilities (TAAD) of black gram, lupin and SBM when fed to growing chickens
are shown in Table 8.4 and Table 8.5. Although the mean AAAD of SBM was higher
(P<0.05) than lupin and black gram, there was no significant difference between the
TAAD of lupin and SBM, but both of them were higher (P<0.01) than the TAAD of
black gram (Figure 8.2). The mean AAAD and TAAD coefficients were 59.4 and
69.4% for black gram, 73.7 and 85.3% for lupin and 77.4 and 85.5% for SBM
respectively. The differences in AAAD of SBM and lupin might be due to lower
endogenous amino acid excreted by the chickens fed SBM meal compared to those
fed lupin meal. Marked differences were observed between individual amino acids
(Figure 8.3). The least digestible essential amino acids were methionine for black
gram, methionine and
97
Table 8.4. Effects of enzyme supplements on the apparent digestibility of amino acids (AAAD) of black gram, lupin and SBM in growing
chickens (%)
Amino acid Black gram Lupin SBM Lsd

Control EA EB EP Control EA EB EP Control EA EB EP (n=3)
Alanine 41.5de 49.1cd 31.5e 46.1cde 59.5bc 71.7ab 70.7ab 66.5ab 66.4ab 67.8ab 73.8ab 76.6a 14.7
b b b b a a a a a a a a
Arginine 70.8 74.2 65.1 66.8 93.1 94.6 94.7 93.9 89.7 90.1 87.4 92.3 9.3
cd bc d d ab a a a a a a a
Aspartic acid 58.8 67.2 55.0 55.0 78.1 84.7 84.4 82.7 81.2 81.3 85.0 86.7 11.4
c bc cd d ab a a a a ab a a
Glutamic acid 74.0 76.7 71.9 59.5 89.2 92.5 90.6 92.5 89.5 88.7 90.5 91.3 12.8
cde bcd e de abc abc ab a abc ab ab ab
Glycine 53.7 61.0 38.8 45.6 67.3 69.4 72.3 79.0 69.9 73.5 78.1 77.8 17.6
cd bcd d d abc a a abc abc ab a a
Histidine 56.3 58.4 51.4 46.4 67.4 74.2 75.1 66.7 68.4 69.9 76.9 74.1 13.1
de bcd e cde abc ab a abc abc abc ab ab
Isoleucine 56.3 64.9 47.5 62.7 74.0 79.2 81.1 76.8 75.2 75.4 79.8 80.2 16.1
cd bc d cd ab a a a ab a a a
Leucine 59.4 65.8 52.5 56.4 76.3 83.7 84.1 80.0 77.7 79.2 82.2 85.7 12.7
bcd abcd cd d abcd a ab ab bcd abc ab a
Lysine 75.9 79.0 71.8 70.5 78.2 86.5 81.9 83.6 76.0 79.8 81.7 87.8 8.7
cd cd d cd cd cd bc cd a ab a a
Methionine 58.1 45.9 41.2 54.1 47.6 50.1 61.4 48.0 79.4 76.3 80.5 86.1 17.6
de cde e e bcd ab abc abc ab ab a a
Phenylalanine 61.1 66.6 56.5 55.8 71.4 81.5 75.3 77.8 78.9 81.2 84.0 85.4 11.3
bc b bc c a a a a a a a a
Proline 41.7 48.2 36.0 30.6 69.2 77.6 76.1 76.8 72.6 80.6 77.3 80.6 17.4
bc b c bc a a a a a a a a
Serine 53.0 61.9 47.3 49.5 75.7 82.0 82.4 77.9 77.7 78.4 80.8 85.3 11.1
Threonine 58.7bc 57.8bc 54.8cd 42.9d0 75.1a 79.3a 82.6a 76.8a 74.8a 77.7a 69.6ab 77.8a 14.8
bcd cd de e a a a a a abc ab ab
Tyrosine 73.7 71.1 61.3 55.3 89.2 88.3 91.9 88.3 88.4 83.0 85.8 86.3 13.0
def cde f ef bcd ab ab abc abc abc ab a
Valine 57.4 61.8 47.5 50.3 68.3 76.7 77.7 72.1 72.1 72.9 77.8 81.6 13.0
Mean 59.4d 63.1d 52.0e 52.9e 73.7c 79.5ab 80.1ab 77.5bc 77.4bc 78.5b 80.7ab 83.3a 4.5
abc
Means in rows bearing different superscripts are significantly different (P<0.05)
98
Table 8.5. Effects of enzyme supplements on the true digestibility of amino acids (TAAD) of black gram, lupin and SBM in growing
chickens (%)
Amino acid Black gram Lupin SBM Lsd

Control EA EB EP Control EA EB EP Control EA EB EP (n=3)
cd bc d cd ab a a a a a a a
Alanine 56.0 63.0 45.4 60.3 76.7 89.1 82.3 83.4 78.2 79.8 85.2 87.1 15.0
bc b c bc a a a a a a a a
Arginine 77.1 80.4 71.1 73.0 96.8 98.2 97.2 97.5 94.6 95.1 92.1 96.7 9.1
Aspartic acid 65.5bc 73.6b 61.4c 61.5c 85.7a 92.3a 89.5a 90.1a 86.4a 86.6a 90.1a 91.3a 11.4
c bc cd d0 ab a ab a ab ab ab a
Glutamic acid 79.1 81.6 76.8 64.6 93.4 96.7 93.4 96.6 93.0 92.3 93.9 94.4 12.8
bcd abc d cd ab ab ab a ab ab a a
Glycine 65.5 72.3 50.1 57.1 78.1 80.2 79.5 85.5 78.7 82.4 86.6 85.5 17.6
Histidine 67.0bc 68.6bc 61.6c 56.9c 78.8ab 85.7a 82.8a 77.9ab 77.9ab 79.5ab 86.2a 82.5a 12.8
cd abc d bcd ab a ab ab ab ab ab ab
Isoleucine 68.7 76.8 59.4 74.9 86.6 92.1 89.7 89.2 84.7 85.1 89.1 88.7 15.7
cd bc d cd ab a a a ab a a a
Leucine 66.9 73.1 59.7 63.8 85.2 92.7 90.1 88.6 84.2 85.8 88.6 91.5 12.6
Lysine 82.9def 85.8bcde 78.5ef 77.4f 88.1abcd 96.4a 88.6abcd 93.3ab 84.1cdef 88.0bcd 89.6abcd 91.9abc 8.4
cd d d cd bc abc abc bc ab ab ab a
Methionine 66.5 53.9 49.2 62.3 74.7 77.4 79.7 74.6 88.4 85.5 89.3 94.0 18.0
c bc c c a ab a a a a a a
Phenylalanine 69.4 74.6 64.5 64.0 93.8 83.5 89.8 86.7 89.2 91.6 91.6 92.3 11.8
bc b bc c a a a a a a a a
Proline 54.8 60.8 48.7 43.4 82.0 90.4 84.7 89.4 81.6 89.8 86.1 88.6 17.3
bc b c c a a a a a a a a
Serine 61.1 69.7 55.0 57.4 84.2 90.5 88.1 96.1 84.2 85.0 87.1 91.0 11.0
bc bcd cd d a a a a abc ab abc ab
Threonine 77.3 75.7 72.5 61.0 92.5 96.9 94.5 94.0 87.5 90.6 81.9 89.0 15.2
bc cd de e a ab a ab ab abc abc abc
Tyrosine 84.9 81.8 72.0 66.3 98.4 97.5 98.1 97.4 96.1 91.0 93.2 93.2 12.7
cd bc d cd ab a a a ab ab a a
Valine 67.4 71.3 57.0 60.1 81.0 89.5 86.3 84.5 81.2 82.1 86.7 89.6 12.9
d d e e c a abc abc c bc abc ab
Mean 69.4 72.7 61.4 62.7 85.3 91.2 88.0 88.9 85.5 86.7 88.6 90.5 3.9
abc
Means in rows bearing different superscripts are significantly different (P<0.05)
99
(A)
100
90 Control
80 EA
70
AAAD (%)
60 EB
50 EP
40
30
20
10
Black gram Lupin SBM
100
(B)
90 Control
80
EA
70
TAAD (%)
60 EB
50
EP
40
30
20
10
Black gram Lupin SBM
Figure 8.2. Effect of different enzyme supplements on mean apparent (A) and
true (B) digestibility of amino acids in black gram, lupin and SBM for
growing chickens
100
1 Black gram Lupin SBM
TAAD (mg/g) 0.9
0.8
0.7
0.6
0.5
0.4
Arg His Ieu Leu Lys Met Phe Thr Tyr Val
Amino acid
Figure 8.3. True digestibilities of amino acids of black gram, lupin and SBM
in growing chickens.
101
histidine for lupin and valine for SBM. This was in agreement with those reported by
Perez et al. (1993), Fan et al. (1994) and Fickler et al. (1996).
Overall, AAAD and TAAD were increased by enzyme supplementation (Figure 8.3).
The magnitude of the response, however, was not consistent and there was a
significant interaction between legume and enzyme and between legume and amino
acid. Both lupin and SBM showed positive responses in amino acid digestibility for
all enzymes tested, however, the greatest improvement (P<0.05) was observed after
addition of protease to SBM and after the addition of xylanase to lupin. Although the
TAAD of black gram increased after addition of xylanase (4.7%), this was not
significantly greater (P=0.095).
8.4. Conclusion
The TME, protein and AA digestibilities of lupin, SBM and black gram, responded
positively to supplementation with xylanase, protease and .-amylase. The response
was specific as each legume gave a response in one chick production parameter more
with one enzyme than the others. Lupin, for almost all parameters responded most to
xylanase, and SBM, for almost all parameters, responded most to protease. The TME
and protein digestibility of chicks fed black gram did not respond to xylanase or
protease but only responded to the multi-enzyme mixture which also contained
amylase in addition to xylanase and protease.
102
Chapter9. General discussion
Chapter 9
General Discussion
The conventional feedstuffs for monogastric animal production may be in short
supply in the future. Grain legumes are potential substitutes for SBM because of the
similarity in their amino acid and energy profiles (Chapter 3). Although the use of
grain legumes in poultry and pig production is regarded primarily as a source of
protein, they are also potential sources of energy as they contain over 15.7 MJ GE kg-
1
. Compared with SBM, the grain legumes selected in this study, with the exception
of lupin, are good sources of lysine but contained less tryptophan. Faba bean, field
pea, lentil, lupin and green gram also contained less sulphur-amino acids. A negative
correlation between the protein and lysine and sulphur amino acid contents suggests
that the quality of protein in term of amino acid profile decrease when the protein
content increases.
Limited use of grain legumes in commercial poultry and pig production is due to
uncertainty about their nutritional quality associated with the naturally occuring ANF.
The levels of ANF varied between and within legumes, even between the same
legume cultivated in different locations. Furthermore, determination of content of
ANF for each grain legumes is laborious and the values may not reflect the actual
inhibitory activities because of variability in physiological responses of animals
(Liener and Kakade, 1980; van der Poel et al. 1990b; Batterham et al. 1993). The
development of the rapid protein quality tests detailed in this thesis will enable feed
manufacturers to select and use grain legumes in diet formulation for their productive
performance prior to mixing diets.
9.1. Rapid biological assay for evaluating the protein quality of grain legumes
The Protein Digestibility-Corrected Amino Acid Score was recommended to be the
most suitable routine method for predicting protein quality of vegetable protein
products for humans (Sarwar and McDonough, 1990). This protein quality test seems
to correspond reasonably well with how protein is used in the body. However the
digestibility of the protein in most grain legumes is not a good predictor of the
digestibility of methionine/cystine (Sarwar et al. 1989; Marletta et al. 1992). The
mean digestibility of amino acid in heat treated beans in rats was 7% less than true
103
digestibility of protein (TDP), but, the difference between digestibility of

methionine/cystine and TDP was between 16 to 37% (Wu et al.1996b). Therefore the
Protein Digestibility-Corrected Amino Acid Score may not accurately predict the
biological quality of legume proteins for monogastric animals, unless it is adjusted
for the digestibility of methionine/cystine. The additional work required to derive the
actual protein and methionine/cystine digestibility values may not be justified when
the aim is to find a rapid and simple method.
The most rapid method for evaluating the protein quality of grain legumes or a
feedstuff is the Modified Limiting Amino Acid Score (MLAAS) as described in
Chapter 2. A comparison of three screening tests; MLAAS, NWG and NPR for
evaluating the protein quality of grain legumes for chickens (Chapter 4) showed that
MLAAS, Net Weight Gain (NWG) and Net Protein Ratio (NPR) methods could
distinguish grain legumes of high (chickpea cv. Desi, chickpea cv. Kaniva and SBM),
medium (field pea, green gram and pigeon pea) and low (black gram, faba bean and
lupin) feed values. This result suggests that the MLAAS calculation could be used as a
reasonable estimate of the relative protein quality of most grain legumes. MLAAS,
however, may not predict the actual protein quality of grain legumes with high level of
protease inhibitors and tannins. It cannot be used to estimate the protein quality of
new feedstuffs of unknown amino acid compositions.
The NWG and NPR are better methods as they detect limiting factors other than
limiting amino acids in raw and processed legumes. When feed intake between test
diets are not significantly different, the NWG alone is suitable for comparing the
protein quality of a range of feedstuffs or diets for poultry. In a situation wherein feed
intake among the test diets is variable the NPR index should be employed. After an
examination of the protein quality of cereals, grain legumes and high-protein food
products in rats using NPR, Protein Efficiency Ratio (PER) and Net Protein
Utilisation (NPU), Lachance et al. (1977) concluded that NPR was best for screening
high-quality legume foods compared with PER or NPU and was very useful for
evaluating proteins that do not promote growth.
NPR value has a good repeatability. The NPR value of ten grain legumes measured in
experiment 1, for example, was highly correlated with those of the control group from
104
experiment 2 (r = 0.95). In addition, the procedure is simple, without a necessity for

conducting complicated laboratory works.
The detailed procedure for measuring NPR value using chicken is as follow:
1. A test and a reference diets containing nominally 100 g kg-1 crude protein supplied
by the test material only and an isoenergetic protein-free diet are formulated.
2. One day old chicks are reared in group and fed commercial starter diet for 5 days.
On day 6, the chicks of similar body weight are allocated randomly to individual
cages and fed similar diets.
3. After an over night without feed, the birds are weighed and given unlimited access
to drinking water and the test diets for 14 d, left overnight without feed and
weighed. Feed allowance, feed left and feed spill are weighed.
4. The NPR is calculated as [weight gain of the chickens fed the test diet + weight loss
of the chickens fed protein-free diet] divided by protein intake.
By using these two protein quality indices, the protein quality of a range new
feedstuffs or diets for poultry can be assessed in 14 days. The simplicity of the
procedure, which is based on the precise measurement of feed intake and body weight
changes during the tests period, allows the test to be conducted on a farm.
9.2. Effect of methionine supplementation on the protein quality of grain legumes

The analysed amino acid compositions of the grain legumes proteins, when compared
with the amino acid requirements of poultry (NRC, 1994) indicated that overall the
ten grain legumes were deficient in methionine, threonine and, in some cases,
tryptophan. Promising results have been reported recently (Spencer et al.1997) that
concentration of methionine in lupin, can be improved through genetic engineering.
The methionine level of lupin was increased by 124%. However, improving the
quality of grain legumes through genetic manipulation is a long term process.
Furthermore, the benefit of feeding the transgenic legume meals to poultry has not
been established.
Methionine supplementation to cereal-pea diets was shown to improve feed/egg ratio

in laying hens (Bertram et al. 1995). Smulikowska and Chibowska (1993) concluded
that a methionine supplemented diet containing 300g kg-1 faba bean for broiler
chickens was as effective as SBM as a supplementary protein source in wheat-triticale
105
diets. In line with these reports, the efficiency of protein utilisation of ten grain
legumes (Chapter 5) was increased as indicated by increases in the NPR values as the
level of methionine was increased to 100 % of requirement. Overall, the order of the
protein quality of ten grain legumes after supplementation to 100% methionine
requirement was similar to those without supplementation, except for faba bean and
lupin which were in the low quality protein group in the screening tests but moved to
the group of medium quality protein after supplementation. There is no clear factor to
explain this changes. It may be due to their lower methionine contents. These results
suggests that, the level and the degree of the negative effect of ANF in each group of
legumes in this study were not significantly different. On the basis of results discussed
in Chapter 4 and 5, it can be concluded that raw chickpeas (chickpea cv. Kaniva and
chickpea cv. Desi), are potential substitute to SBM. Treatments to eliminate the
negative effect of ANF in other grain legumes are needed to enhance their nutritive
value.
9.3. Energy and nutrient utilisation

Although the GE content of grain legumes was not different, their TME values were
significantly different (Chapter 6). The TME of pigeon pea, faba bean, lupin and
black gram were significantly lower than the TME of other legumes. The TME
values of chickpea cv. Desi, field pea, green gram, lentil and SBM were higher than
the TME of pigeon pea, faba bean and lupin, but significantly lower than those of
chickpea cv. Kaniva. The positive correlations obtained between TME and SD (r =
0.70, P<0.001) and between TME and TPD (r = 0.57, P<0.01) suggest that any
component in grain legumes which inhibits digestibility of starch and/or protein
would result in a decrease in TME value.
Tannin and NDF were the main ANF that significantly reduced the digestibility of
both starch and protein. Tannin exerts its negative effects through the formation of
tannin-substrate complexes (Deshpande and Salunkhe, 1982; Hagerman and Butler,
1980; Mitaru et al. 1984) and tannin-enzyme complexes (Longstaff and McNab,
1991). The availability of the digestive enzymes after the ingestion of legume meals
may have been decreased by the formation of enzyme-fibre complexes. Schneeman
(1978) suggested that enzymes such as trypsin and chymotrypsin could be adsorbed
onto fibres such as solka fibre, xylan, cellulose acetate, wheatbran and rice bran.
106
It appears that the rank order of TME value of grain legumes follows the order of
their protein quality. Regression analysis of means of NPR value observed in
Experiment 1 and 2 and the TME value obtained in Experiment 3 indicates that the
relationship between NPR and TME value of ten grain legumes studied herein is
linear (Figure 9.1). The regression equation suggests that around 63% of the variation
in NPR value of grain legumes in this study was contributed by variation in the TME
value. Every MJ increase in TME value of grain legumes results in approximately 0.5
increase in the NPR value.
4.0
3.5 y = 0.2077x + 0.2873
R2 = 0.6303; P<0.01
3.0
2.5
NPR
2.0
1.5
1.0
0.5
0.0
5 7 9 11 13 15 17
TME (MJ/kg)
Figure 9.1. Relationship between the mean NPR and TME values in ten grain legumes
a measured in young chickens
It is recognised that failure to provide a precise estimate of TME content of a

feedstuff can lead to unexpected results. In addition, as in NPR value, the TME
content of a feedstuff (particularly that of plant origin) varied depending upon
cultivars and location. Therefore it is important to determine the TME value of a
new feedstuff before its inclusion into a poultry diet.
Methods for measuring metabolizable energy value of a feedstuff or a diet have been
discussed in the second section of Chapter 2. Among three balance methods for
measuring the energy content of a feedstuff or of a diet (Fisher and McNab, 1989), the
rapid method (type 3) pioneered by Sibbald (1976) was adopted because it is simple,
rapid and requires much less material. The assay outlined by Sibbald (1976) may be
simplified and adopted for routine use. The procedure would be as follows:
107
1. Three weeks old chickens or cockerels housed in individual cages, have free
access to drinking water, are left without feed for approximately 24 h.
2. A mixture of one portion of finely ground test material and one to two portion of
water (depending upon the moisture content of the meal) is prepared.
3. A bird is weighed, then 40-50 g feed mixture is introduced into the crop using a
plastic syringe fitted with a soft-plastic tube (3 7 mm and 20 cm ) and weighed
again. The precise amount of test material consumed is the difference between the
weight of the chicken before and after feeding.
4. The bird is placed back to its cage, a plastic tray is placed under the cage and the
time is recorded.
5. Step 2 and 3 are repeated to provide the number of replications.
6. The same number of birds of similar body weight were selected and placed in
cages without feeding.
7. Exactly 24 h after placement of the birds, the plastic trays are removed, the
feathers and scurf are removed using a dust baster, the excreta is collected
quantitatively, frozen, freeze-dried, leave over night and weighed.
8. Samples of the test material and excreta are ground to pass 0.8 mm screen and
analysed for their gross energy content.
9. The TME is calculated using the following formula:
EI − FE + EEL
TME ( MJ / kg ) =
FI
where EI = energy intake, FE = faecal energy, FI = feed intake (kg) and
EEL = endogenous energy lost of unfed birds.
For experienced person, it requires only about 10-15 seconds to fill the crop with the
feed mixture so that minimum harm is induced to the chickens. However caution
should be given especially to the inexperienced person, that overfilling the crop may
cause the bird to regurgitate and the mixture may cause a respiration problem.
Assuming that the birds are available at anytime, the TME value of a feedstuff can be
expected within a week of arrival of the material. In addition, the excreta may be
further analysed for measurement of digestibility of other nutrients.
108
9.4. Improving the quality of grain legumes

A major constraint on the use of grain legumes in poultry diets is that they may
contain ANF such as protease inhibitors, tannins, lectins, amylase inhibitors and
NSP, that depress poultry performances through interference with normal digestive
function.
A number of techniques for the elimination of ANF, thus improving the nutritional
value of grain legumes, is available. Some promising results have been shown by
plant breeders that ANF can be eliminated through plant breeding programs. Plant
breeding is, however, a long-term process and the results, at least for the removal of
trypsin inhibitors in field peas (Pisum sativum) have not been consistent (Leterme et
al. 1992).
The most widely used methods for the reduction of the negative effects induced by
ANF are physical treatments. Decortication is an effective method for reducing
tannins and fibre contents of grain legumes since both are mostly reside in the testa,
and heat treatment is a good method of decreasing the activity of lectins, trypsin
inhibitors and chymotrypsin inhibitors.
Infrared radiation and boiling in water are as effective as autoclaving in improving the
quality of raw legumes (Kadam et al. 1987). The efficacy of heat treatment, however,
depends on the conditions of heating (pressure, temperature and time). Studies on the
effect of autoclaving time (Chapter 7) on the protein quality of low value legumes
(black gram and lupin) indicate that autoclaving lupin meal at 125oC and 180 kPa for
5 min significantly improves NWG and NPR value, but no significant improvement
was observed for black gram, suggesting that the response of each legume to
autoclaving time is different. It is also apparent that the response of chickens to
legume meals autoclaved for different times was curvilinear, supporting the
established theory that prolonged or elevated heatings, reduces the availability of
some basic amino acids, such as lysine, for growth. Therefore, to use heat treatments
effectively the temperature and duration of processing have to be carefully
controlled.
Enzymes have been widely used to improve the nutritive value of poultry and pig
diets based on wheat and barley. There is no specific enzymes yet being produced to
enhance the nutritive value of grain legumes. As grain legumes contain relatively high
109
concentrations of NSP (Brillouet et al. 1988; Smith and Annison, 1996; Choct,
1997), enzyme supplements that digest NSP are likely to increase the metabolisable
energy and protein values of grain legumes in the same way they do in wheats (Choct
and Annison, 1992; Choct et al. 1995; Hew et al. 1995). A number of reports show
that the addition of β-galactanase and minor α-galactosidase (Bryden et al. 1994), and
feed enzyme containing hemicellulase, xylanase, pentosanase and cellulase activities
(Annison et al. 1995) improve the AME of lupin, open up the possibility to use
enzymes to improve the nutritional value of other grain legumes. The ideal ratio of
enzymes needed to improve the nutritional value of grain legumes may be specific for
each legume, however, using an enzyme cocktail containing a mixture of proteases
and carbohydrases may be more economical (Marquardt et al. 1993).
The mean NPR value of the ten grain legumes studied herein increased significantly
(P<0.001) after supplementation with a mixed-enzyme product containing xylanase,
.-amylase and protease (Chapter 8). A significant (P<0.05) positive correlation (r =
0.64) between percent improvement in NPR values following enzyme
supplementation and the NDF content suggests that the xylanase acts to break the cell
wall and the .-amylase and protease then assist the endogenous amylase and protease
to digest the exposed starch and protein. Protease may also reduce the activity of
trypsin inhibitors, chymotrypsin inhibitors and lectins possessed by some legumes as
demonstrated in in vitro studies by Huo et al. (1993). Therefore, the improvement in
NPR value may have been associated with an increase in TME and digestibility of
protein.
Application of three different enzyme products; a xylanase, a mixed enzyme

containing xylanase, .-amylase and protease, and a protease to black gram, lupin and
SBM showed that the response of these legumes to enzyme supplementations was
specific as each legume gave a response in one chick production parameter more with
one enzyme than with the others. Lupin, for almost all parameters responded most to
xylanase, and SBM, for almost all parameters, responded most to protease. The TME
and protein digestibility of chicks fed black gram did not respond to xylanase or
protease but only responded to the multi-enzyme mixture which also contained
amylase in addition to xylanase and protease. Differences in the degree of
110
improvement of NPR value after enzyme supplementation (Figure 8.1) also indicates
that responses of each legume to a multi enzyme mixture is specific.
Although the mean improvement in NPR value of black gram and lupin by
autoclaving for 5 min (10.53%) was higher than for enzyme supplementation
(6.56%), they were not significantly different. A combination of both autoclaving or
heat treatment and enzyme supplementation may result in better responses. Although
the economic analysis has not been shown, enzyme supplementation is likely to be a
safer, more effective and cheaper method for reducing the negative effects of ANF in
grain legumes.
9.5. Conclusions
Among the protein quality indices developed in this study, the Net Protein Ratio
(NPR) is the best for grading a wide range of grain legumes into high, medium and
low values. The NPR index and the modified Sibbald (1976) methods are simple and
rapid assay for determination the protein quality and the TME content of a range of
feedstuffs.
Ten grain legumes were shown to have similar amino acid compositions in their
proteins and similar gross energy. The analysed amino acid compositions of grain
legumes when compared with the amino acid requirements of poultry indicated that
overall the ten grain legumes were deficient in methionine, threonine and, in some
cases tryptophan. Growth of broiler chickens was significantly improved by
methionine supplementation of all grain legumes. Despite the above results, it was
found that growth of chickens fed equal quantities of protein of similar amino acid
composition from the ten grain legumes was not equal. The causes of the lower
growth were higher levels of lectins and protease inhibitors that could be destroyed
by heat treatment, and non-starch polysaccharides, especially hemicellulose, gums
and mucilages (as part of the NDF) that could be destroyed by exogenous xylanase.
After these treatments the grain legumes were of approximately equal value for
chicken growth, with the exception black gram, which was concluded to contain
additional factors such as pectins and tannins that were not affected by treatments
used.
Therefore, with the exception of black gram, eight grain legumes were found to be
valuable new sources of protein and energy for poultry. Raw chickpea cv. Kaniva,
111
chickpea cv. Desi and lentil (dehulled) can substitute SBM. Other grain legumes can
be used either after heat treatment or when used with dietary supplements of xylanase
and other feed enzymes.
9.6. Implications and possible future research

The work described in this thesis was aimed at developing techniques that could be
used to predict the relative protein quality and the true metabolizable energy value of
a wide range of grain legumes for poultry production and to complement the existing
data of the nutritive value of grain legumes.
The protein quality tests developed in this thesis may be adopted in practice as tools
to evaluate the relative protein value of a range of feedstuffs in relation to a standard
protein sources. The modified Sibbald (1976) method may also be adopted for
measuring the TME value of a feedstuff. The techniques are rapid and simple without
complicated laboratory works. Either heat treatment or enzyme supplementation is a
suitable method for improving the quality of low value legumes. The choice would
depend on the availability of facilities.
A wide range of other legumes and other non-conventional feed resources need to be
evaluated before recommendations can be made for their use in commercial poultry
production. The benefits of combining heat treatment and enzyme supplementation
also need to studied.
112
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Rural Industries Research and Development Corporation

Egg Industries Research and Development Committee

PLEASE NOTE: THIS REPORT IS NOT PUBLISHED IN HARD PRINTED FORM

FINAL REPORT FOR PROJECT : UQ-21E

NEW VEGETABLE PROTEIN FOR LAYERS

Supervisor : Dr. J. G. Dingle

Researcher : Mr. K.G. Wiryawan

Department of Animal Production

I Ketut Gede Wiryawan

A thesis submitted to The University of Queensland in

DEPARTMENT OF ANIMAL PRODUCTION

Supplementation of a 1 g kg-1 multi-enzyme product containing xylanase, .-amylase and

Refereed Scientific Papers

Refereed Conference Papers

• The University of Queensland Gatton College;

Second Annual Conference, 14/10/1994

• The University of Queensland Gatton College the

Third Annual Conference, 04/10/1995

• The University of Sydney, NSW;

Australian Poultry Science Symposium, 07/02/1995

• The University of New England, Armidale

Recent Advances in Animal Nutrition in Australia, 03/07/1995

‘True metabolisable energy content of grain legumes: effects of enzyme

• Hilton Hotel, Melbourne

Nutrition Society of Australia, 24/09/1995

‘Protein quality of grain legumes for chickens: Effect of methionine

First Australian New Crops Conference, 08/07/1996

‘Nutritive value of grain legumes for monogastric animals’

• The University of New England, Armidale

Recent Advances in Animal Nutrition in Australia, 30/06/1997

‘Enzyme supplementation improves protein quality of grain legumes for poultry

• The University of Queensland Gatton College

‘Treatments to improve the nutritive value of grain legumes’

Chapter 1 GENERAL INDRODUCTION 1

2.2. Rapid Biological Methods for Evaluating Nutritional Value 27

Chapter 3 PRELIMINARY STUDY. THE COMPOSITION OF GRAIN 39

Chapter 4 EXPERIMENT 1. SCREENING TESTS OF THE PROTEIN 50

Chapter 8. EXPERIMENT 5. IMPROVING THE QUALITY OF LOW 89

Chapter 9. GENERAL DISCUSSION AND CONCLUSIONS 103

1.2. Research problem

What is the nutritional value of grain legumes for poultry production?

This is elaborated into:

• Does methionine supplementation improve the protein quality of grain legumes?

To address these problems, a series of experiments was conducted in which broiler

and that methionine supplementation, autoclaving and enzyme supplementation

1.3. Justification of research

1.4. Methodology chosen

1.5. Outline of the thesis

1.6. Delimitation of scope and key assumptions

1.7. Abbreviations and Definitions

2.1. Nutritive Value of Grain Legumes for Monogastric Animals

2.1.3. Nutrient content of grain legumes

Crude Crude Crude Ash NFE Ca P

Chickpeas 220 40 90 30 620 1.0 4.0

Common bean 240 20 40 40 660 2.5 4.0

Cowpeas 250 15 40 30 665 1.0 4.0

Faba bean 230 20 70 40 640 1.0 3.0

Field peas 250 15 60 40 635 1.0 3.0

Green gram 240 15 50 40 635 2.0 4.0

Lentils 250 30 20 40 660 1.0 3.0

Lupins* 322 60 145 31 442 2.2 3.3

Pigeon peas 200 20 80 40 660 1.5 3.0

SBM 498 7 53 71 371 2.5 8.6