Use of Pectinases to Improve the Nutritive Value of Lupins for
Poultry
by
Ahmed Ali
B. Sci. Agric., Grad. Dip. Agric. Sci.
Sweet lupins (Lupinus angustifolius) grown in Western Australia
This thesis is presented for the degree of
Doctor of Philosophy
2009
The University of Western Australia
Faculty of Natural and Agricultural Sciences
School of Animal Biology
i
Declaration
The work presented in this thesis is the original work of the author, and none of the
material in this thesis has been submitted either in full, or part, for a degree at this or
any other university or institution. The study design and manuscript were carried out
by myself after discussion with my supervisors, Dr Ian H. Williams and Professor
Graeme B. Martin.
Ahmed Ali February 2009
ii
Summary
Australia produces 87% of the world’s lupins (Lupinus angustifolius) which have the
potential to be an excellent source of protein and energy in animal diets. However,
feed manufacturers and poultry producers cannot use more than about 5% lupins in
broiler and 7% in layer diets. The main reason is because 34% of the lupin grain
comprises complex cell-wall polysaccharides that are indigestible. The main
component of cell walls in lupins is pectin (33%).
Poultry cannot digest pectin because they don’t secrete the appropriate enzymes so
their ability to use lupins is limited. Undigested pectins increase the viscosity of
digesta in the bird’s digestive tract, which in turn reduces the digestibility of dry
matter and efficiency of feed utilisation. Pectins also increase water-holding capacity,
a characteristic directly related to water intake and wet droppings.
In this thesis, I tested the general hypothesis that breakdown of cell walls and pectins
will improve the nutritive value of lupins for broilers and layers and reduce wet
droppings. This hypothesis was tested in six experiments by treating lupins with
specific exogenous enzymes (pectinases) or mechanical-heat treatment (expansion)
plus pectinase.
In the first experiment, attempts to break down the cell walls and pectins using four
doses of pectinase, specifically polygalacturonase (PG), succeeded in improving the
nutritive value of whole and dehulled lupins for egg layers. The lowest dose, 0.6g/kg
diet, was the most effective dose for reducing water intake, wet droppings, the
viscosity of the digesta and the number of soiled eggs. This dose was also the most
effective for increasing the digestion of dry matter, the metabolisable energy of the
diet, feed conversion efficiency, and egg yield and egg-shell thickness. While the
higher doses, 0.8 and 1.0g/kg diet improved several measured variables, they had
some detrimental effects, particularly on wet droppings. Even using an optimal dose of
PG, the amount of lupins that could be included in the diet was limited. Increasing the
amount of lupins from 10 to 20% in the diet slightly increased water intake, wet
droppings and soiled eggs and reduced metabolisable energy of the diet even with PG
iii
treatment. This was because PG could not break down pectin chains by more than
11% within complex cell-wall lattices, suggesting that PG requires an external factor,
such as mechanical and/or heat process, to destroy these thick cell walls and help it
better access its target substrates.
As a result, I conducted a second experiment to test whether a mechanical-heat
treatment, such as expansion, might enhance the effectiveness of PG by increasing the
surface area available to the enzyme. Consequently, this should allow high (20%)
inclusion of lupins in the diet. Expansion alone had no beneficial effect on production.
Surprisingly, the combination of expansion + PG caused a greater breakdown of cell
walls and pectin but this was not reflected in performance of the birds. Heat generated
during expansion may have solubilized some of the insoluble fibres, inducing Maillard
reactions and increasing water-holding capacity. PG alone was beneficial, as already
shown in the earlier experiment with layers. It reversed the adverse effects, more so
for birds fed 10% whole and dehulled lupins than for 20% lupin inclusion. While these
results highlight the significance of PG treatment of lupins for broilers, PG failed to
improve growth performance or the metabolisable energy of the diet, or reduce wet
droppings at 20% lupin inclusion. Thus, it has become clear that PG is unable to
overcome the significant losses in productivity or increases in wet droppings with 20%
lupins in broiler and layer diets.
The second main question addressed in this thesis was why PG could not eliminate the
problems with 20% lupins in the diet, and what limited the breakdown by PG of pectin
chains in lupins to 11%? The main problem likely arises from the presence of methyl
ester radicals along the pectin chains that not only block the binding sites of PG to
glycosidic bonds along the pectin chains, but also themselves increase the viscosity
and water-holding capacity of pectin.
Therefore, an in vitro (Experiment 3) was conducted to test whether the breakdown of
methyl esters by a specific pectinase, pectin methyl esterase (PME), followed by PG
treatment will give a more complete breakdown of pectin and reduction of the water-
holding capacity and viscosity of lupins than PG on its own. This hypothesis was
iv
tested by incubating dehulled lupins with PG, PME and a combination of PG and
PME. The results showed that the combination of PG and PME broke down much
more of the pectin (50%) and cell walls (27%) than PG on its own (21% and 13%)
compared to control. In addition, the molecular weight of pectin was more than halved
and the length of the pectin chains was reduced 3-fold. Surprisingly, the combination
of PG + PME did not reduce the viscosity and water-holding capacity more than PG
on its own, as anticipated. This was likely because of the high dose (1400 unit) of
PME employed in combination with PG. The high dose of PME required large
volumes of water to convert methyl esters into methanol, and produced more non-
methylated pectin chains that cross-linked with neighbouring pectin polymers, leading
to an increase in water-holding capacity and viscosity of pectin.
As a consequence, I conducted another in vitro experiment (Experiment 4) to
determine the most appropriate dose of PME that should be used in combination with
PG. Six levels of PME (0, 200, 400, 600, 800 and 1000 units) in combination with PG
(1400 units) were incubated with dehulled lupins. The lowest dose of PME (200 units)
and PG (1400 units) tested induced a good breakdown of cell walls (27%) and pectin
(37%), and reduced the viscosity by 18% and water-holding capacity by 14%
compared with no pectinases. Indeed, a high dose of PME caused excessive
breakdown of methyl esters and kept water-holding capacity and viscosity high, not
only with dehulled lupins, but also with other legumes such as lathyrus, faba bean and
field pea.
Finally, I examined the effectiveness of the “optimum combination” of PME and PG
in two in vivo experiments, one with broilers (Experiment 5) and another with layers
(Experiment 6), to see if these pectinases would allow dehulled lupins to be
incorporated into the diet up to a level of 30%.
When broilers or layers were fed diets containing 10% dehulled lupins, PME and PG
were very effective at increasing digestibility of dry matter by 15%, feed conversion
efficiency by 6% and the metabolisable energy of the diet by 8%. Breakdown of pectin
was increased 5-fold in broilers and 7-fold in layers. Similarly, cell walls were reduced
v
by 2.3-fold in broilers and 2.6-fold in layers. In broilers, water intake was reduced by
10%, wet droppings by 11%, and feed conversion efficiency increased by 6% and
metabolisable energy by 6%. Equivalent figures for layers were 14, 15, 5 and 8%,
indicating that the pectinases were slightly more effective in layers than broilers.
For diets containing 20% dehulled lupins, pectinases were also very effective at
breaking down both pectin and cell walls to release nutrients and, concomitantly,
reducing water intake and wet droppings, but the magnitude of the responses was
slightly less than with the 10% dehulled lupin diets.
For diets containing 30% dehulled lupins, although the pectinases again were effective
at breaking down pectin and cell walls and reducing viscosity, they did not reduce
water intake or wet droppings. This might be due to the large amounts of non-
methylated pectic polysaccharides, which make up two thirds of the cell walls, by
increasing water-holding capacity particularly when dehulled lupins are included in
the diet at high levels (up to 30%). These polysaccharides might be broken down by
appropriate enzymes. This hypothesis is worth testing in the future.
Overall, the results of my study supported the general hypothesis. These in vivo results
are conclusive and consistent. They show that an optimum combination of PME and
PG is capable of including dehulled lupins up to 20% in broiler and layer diets without
any nutritional or hygienic problems. The strategies I developed have proven very
useful for breaking down the cell walls and pectins, improving the nutritive value of
lupins for broilers and layers, and reducing wet droppings. By using the optimum
combination of two pectinases, it should be possible to make substantial
improvements in the nutritive value of lupins for broilers and layers, most importantly
by reducing excessive water intake and wet droppings associated with feeding
dehulled lupins. Without pectinases, the amount of dehulled lupins used in poultry
diets is fairly small (7%), but if pectinases are used, this upper limit can be lifted to
20%.
vi
Acknowledgments
This thesis would not have been possible without the assistance of many people and
the generosity of research and funding organisations. I would like to take this
opportunity to thank them all for their help and inspiration during the course of my
PhD study and my employment at UWA.
First, I take pleasure to express my sincere appreciation to my major supervisor, Dr
Ian H. Williams, for his keen and dedicated supervision, invaluable help, constructive
criticism, and financial support throughout the project. I feel very proud being one of
Ian’s students at UWA. I must thank him not only for teaching me professional
scientific writing but also for his sense of humour and fun throughout the project.
I am also deeply indebted to my co-supervisor, Professor Graeme B. Martin for his
keen support, encouragement, critical discussion and guidance. Graeme has always
fulfilled all my requests satisfactorily whenever I asked him. I have unbounded respect
for Ian and Graeme as leading research scientists at UWA.
I would like to give a “big” thank you to Dr John Milton for his invaluable support,
instructive discussions and genuine interest in my research work. I’m very grateful to
Dr Milton and his wife, Dr Nui Milton, who helped me personally by offering me
casual work and employment, with their research projects at UWA and with their
private business, when my scholarship had finished.
Special thanks must go to Dr Krystyna Haq of the UWA Graduate Research School
for her reading of the various drafts of this thesis, critical review, valuable suggestions
and fruitful discussions. Words alone cannot express how grateful I am to Krystyna.
I extend my appreciation to all staff of the School of Animal Biology, in no particular
order: Emeritus Prof. David Lindsay, for his excellent teaching in scientific writing
and methodology, as well as his support, and Mr John Beesley, for being personal
support and for his technical help. Special thanks go to Mr Rob Hurn and Mrs Kerry
Knott for their administrative help and continuous support. I would like to thank all
vii
my fellow post-graduate students: Rob Creasy, Aprille Chadwick, Travis Murray,
Andrew Williams, Peter Hutton, Chris Mayberry and Felicity Donaldson for their
friendship and support. Peter is thanked for reminding me that there is more to life
than a PhD, and painting a house can sometimes be more exciting and profitable than
studying a PhD!
I would like to acknowledge the valuable assistance of Dr David Harris, Chemistry
Centre of WA, for help with the chemical analyses of lupins. Without David’s
guidance, a number of the analyses would have not been accomplished easily.
I record my deep gratitude to Daniel Goussac (Wesfeeds Pty Ltd) and Javed Hayat
(Poultry Farmers of WA Coop Ltd) for their invaluable technical assistance, diet
formulation, and kind donation of broiler and egg-layer diets. Also, thanks are
extended to Craig Menzie (Bartter Poultry Pty Ltd), Peter Bell (AAA Egg Pty Ltd)
and Bob Henley (Inghams Pty Ltd) for their donation of broilers and layers throughout
the project.
Financial support of this study by RIRDC (Rural Industries Research & Development
Corporation) and AECL (Australian Egg Corporation Limited), and by UWA through
its Completion Scholarship, are gratefully acknowledged. Without this support, I
would not have been able to complete my PhD study or work at UWA.
Many thanks go to Mrs Sofia Sipsas, Department of Agriculture & Food (Western
Australia), and Mr Mark Tucek, CBH Pty Ltd (Perth) for their financial and in-kind
support throughout the project. Many thanks to all industry partners for their research
collaboration and valuable support.
The enzyme manufacturers kindly donated pectinase samples and technical support
throughout the project: AB Enzymes GmbH (Darmstadt, Germany), Amano
Pharmaceutical Co Ltd (Nagoya, Japan), Orba Biochemistry San. Tic. A. S. (Istanbul,
Turkey), and Lyven Enzyme Industries (Caen, France).
viii
Finally, I am very grateful to my wife, Sahar, who encouraged me and also helped me
to keep my perspective. This thesis is a gift to her, and to my young daughters, Nura
and Sara, for their many smiles and for putting up with me while I worked long hours
throughout my project. Thanks to my parents, Kamal and Aydin, for their love and
unwavering support throughout the ups and downs of this whole process. At last, I can
tell them it is finished!
ix
Publications The publications and presentations listed below are relevant to this thesis. Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. 2009. Pectinases break down cell
walls in legumes. Proceedings of the Australian Poultry Science Symposium. 20, 45.
Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. 2009. Pectinases allow broilers to
perform well on diets with 20% dehulled lupins. Proceedings of Australian Poultry Science Symposium. 20, 46.
Williams, I. H., Ali, A. and Sipsas, S. 2008. Legumes for poultry. Improvement of
lupins and lathyrus for broilers and egg layers by enzyme treatment. Report for AECL-RIRDC (Australian Egg Corporation Limited -Rural Industries Research and Development Corporation). Project No. PRJ-000449. Barton, ACT, Australia. pp. 1 – 34.
Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. 2006. The optimal dose of
pectinase in lupin-based diets for laying hens. Proceedings of the Australian Poultry Science Symposium. 18, 218 – 221.
Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. 2006. Expansion and pectinase
treatment of lupins for broilers. Proceedings of the Australian Poultry Science Symposium. 18, 62 – 65.
Williams, I. H., Ali, A. and Sipsas, S. 2005. Lupins for poultry. Report for RIRDC
(Rural Industries Research and Development Corporation). Project no. WAU-1A. Barton, ACT, Australia. pp. 1 – 29.
Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. 2005. Hydrolysis of lupin pectin
by pectinases for broilers. Proceedings of the Australian Poultry Science Symposium. 17, 219 – 222.
Ali, A. 2005. The use of enzyme technology to improve the nutritional value of
Australian and Indonesian feedstuffs for poultry. 3rd National Seminar on Indigenous Poultry. World Poultry Science Association, Indonesian Branch. Diponegoro University, Semarang, Indonesia. pp. 1–8.
Ali, A., Williams, I. H., Martin, G. B. and Harris, D. J. 2001. Enzymatic pre-treatment
of lupins for broiler diets. Proceedings of the Australian Poultry Science Symposium. 13, 199 – 203.
Ali, A., Williams, I. H., Martin, G. B. and Harris, D. J. 2000. Important properties of
dehulled lupins in poultry diets. Proceedings of the Australian Poultry Science Symposium. 12, 206.
Ali, A. 1997. Enzyme treatment to increase digestibility of non-starch polysaccharides
(NSPs) in lupin-based diets fed to broiler chickens. Graduate Diploma in Science in Agriculture. Thesis by research. Animal Science. The University of Western Australia.
x
Table of contents Page Declaration i
Summary ii
Acknowledgements vi
Publications ix
List of Abbreviations xiii
Chapter 1 General Introduction 1
Chapter 2 Review of the Literature 4
2.1 Introduction 4
2.2 History of lupins in Australia 4
2.3 Feeding lupins to poultry – advantages and disadvantages 5
2.4 Heating, dehulling and removing oligosaccharides from lupins are ineffective 7
2.5 Reasons: complex cell walls and pectin in lupins 8
2.6 Anti-nutritive effects of pectins on poultry 10
2.7 Role of enzymes in breaking down cell-wall polysaccharides 14
2.8 Pectinase in poultry diets 16
2.9 Previous work on enzymes and lupins for poultry 17
2.9.1 Multi-enzyme preparations 18
2.9.2 Specific-enzyme preparations 21
2.9.3 Polygalacturonase for lupins 22
2.10 Determining the optimal dose of PG 24
2.11 Breakdown of complex cell walls by mechanical-thermal processing plus PG 25
2.12 Breakdown of pectins by combination of PG and pectin methyl esterase 26
2.13 Research Objectives and Hypotheses 30
2.13.1 Objectives 30
2.13.2 Hypotheses 32
Chapter 3 General Materials and Methods 33
3.1 In vitro experiments 33
3.1.1 Filtration rate of solution 33
3.1.2 Measurement of viscosity 33
3.1.3 Water-holding capacity 34
3.1.4 Quantification of cell-wall materials 34
3.1.5 Pectins 35
xi 3.1.6 Concentrations of galacturonic acid and polygalacturonic acid 35
3.1.7 Molecular weight of pectin 36
3.1.8 Pectin chain length 36
3.1.9 Methyl ester content of pectin and methanol production 37
3.2 In vivo experiments with broiler and egg layer chickens 38
3.2.1 Digestibility of dry matter and excreta moisture 38
3.2.2 Apparent metabolisable energy of the diet 38
3.2.3 Collection of digesta for viscosity measurement 39
3.2.4 Animal ethics 39
3.3 Statistical analysis 39
Chapter 4 The optimal dose of pectinase in lupin-based diets for laying hens 41
4.1 Introduction 41
4.2 Materials and Methods 42
4.3 Results 45
4.4 Discussion 50
4.5 Conclusions 52
Chapter 5 Mechanical expansion and pectinase as treatments to improve 53
the nutritional value of lupins for broilers
5.1 Introduction 53
5.2 Materials and Methods 56
5.3 Results 58
5.4 Discussion 62
5.5 Conclusions 64
Chapter 6 Complete breakdown of lupin pectin by pectinases in vitro 65
6.1 Introduction 65
6.2 Materials and Methods 66
6.3 Results 67
6.4 Discussion 68
6.5 Conclusions 70
Chapter 7 Optimum dose of pectinases for complete breakdown of 71
legumes in vitro
7.1 Introduction 71
7.2 Materials and Methods 72
7.3 Results 73
7.4 Discussion 74
7.5 Conclusions 75
xii Chapter 8 Pectinases for broiler diets based on dehulled lupins 81
8.1 Introduction 81
8.2 Materials and Methods 83
8.3 Results 84
8.4 Discussion 87
8.5 Conclusions 89
Chapter 9 Pectinases for laying hens fed diets based on dehulled lupins 90
9.1 Introduction 90
9.2 Materials and Methods 91
9.3 Results 93
9.4 Discussion 97
9.5 Conclusions 99
Chapter 10 General Discussion 100 References 106
xiii
List of abbreviations
abs. Absorbance AECL Australian Egg Corporation Limited AME Apparent metabolisable energy ANOVA Analysis of variance C6 Carbon six CP Crude protein CWP Cell-wall polysaccharides COOH Carboxylic acid group EDTA Ethylenediamine tetra acetic acid Endo-PG Endo-polygalacturonase g Gravity GLM General Linear Model HSD Tukey’s Honestly Significant difference IU International Unit k.N/m2 kilo Newton per square meter l Litre L. angus. Lupinus angustifolius LSD Least significant difference M Molarity m.Pas. Millipascal m2 Square metre mg Milligram MJ Megajoule ml Millilitre mm Millimetre MW Molecular weight N Normality nm Nanometer NSP Non-starch polysaccharides OCH3 Methyl ester radical P Probability PG Polygalacturonase (endo) PME Pectin methyl esterase psi Pound per square inch RIRDC Rural Industries Research and Development Corporation rpm Rotation per minute SCA Standing Committee on Agriculture sec. Second sem Standard error of mean t Tonne µg Microgram var. Variety
1
Chapter 1
General Introduction
In Australia, poultry producers, feed manufacturers and poultry nutritionists are all seeking
cheaper ingredients to replace expensive, imported protein meals such as soybeans. The
price of soybean meal has increased two- to three-fold in recent years, following the
banning of animal protein meals from use in ruminant diets. Lupins, the most important
legume produced in Australia, are an excellent source of protein and energy and,
importantly, cost one-third the price of soybean meal. While the theoretical nutritional
value of lupins and soybean are comparable, the metabolisable energy of lupins for broilers
is 30% lower than that of soybean meal. As a consequence, there is only a limited use of
lupins (~5%) in broiler and layer diets.
The main reason that lupins have a low metabolisable energy is that they contain a high
proportion of cell-wall materials, mainly pectins, in both the hull and kernel. Pectins
increase the viscosity of digesta in the intestinal tract and the high viscosity inhibits the
digestion of the nutrients. This leads to slow growth, poor feed conversion and low
utilisation of dietary nutrients (Annison et al, 1996; Hughes et al, 2000; Kocher et al, 2000;
Ali et al, 2006ab). In addition, pectins increase water intake because of their high water-
binding capacity. This causes ‘wet droppings’ (Ali, 1997; Annison et al, 1996; Hughes et
al, 2000; Kocher et al, 2000), leading to wet litter, odour problems and predisposition of
coccidiosis.
However, pectin can be broken down by an enzyme, pectinase, specifically
polygalacturonase (PG), which hydrolyses the glycosidic bonds between the basic units
(galacturonic acid) of the pectin chains (Ali, 1997; Ali et al, 2001, 2006ab). PG treatment
has been shown to give small but consistent improvements in the nutritive value of whole
and dehulled lupins for broilers. However, in lupins, pectins occur within a complex cell-
wall network so their hydrolysis by PG is limited to 11% (Ali et al, 2001). Furthermore,
although PG reduces viscosity, it does not prevent excessive water intake or wet droppings,
particularly at a high inclusion rate of lupins in broiler diets (Ali, 1997; Ali, et al, 2001).
2
We therefore sought ways to improve the action of PG to increase the proportion of lupins
in poultry diets.
The first concept that we tested was to increase the surface area of the cell-wall materials
by subjecting lupins to mechanical and/or thermal processes such as extrusion or
expansion. In addition to making pectinase more effective at hydrolysis of cell walls,
extrusion or expansion may destroy anti-nutritional factors in lupins, further increasing feed
intake, digestion and feed conversion efficiency.
Another major problem in hydrolysis of pectins with PG is the presence of methyl ester
radicals attached to carbon atom six of the galacturonic acid units in the pectin chain. The
methyl esters block the binding sites for PG that are located next to the glycosidic bonds.
The methyl ester radicals can only be removed by another specific pectinase, pectin methyl
esterase (PME). Therefore, a second aim of this project was to investigate whether a more
complete breakdown of pectin could be achieved by using a combination of the two
enzymes (PG + PME) rather than just PG alone. Numerous studies indicate a synergistic
interaction between PG and PME that leads to substantial breakdown of cell walls and
pectins, with a better reduction of water-holding capacity and viscosity than with PG or
PME on their own (Jansen & MacDonnell, 1945b; Demain & Phaff, 1957; Watkins, 1964;
Endo, 1965a; Rexova-Benkova & Markovic, 1976; Chesson, 1987; Christgau et al, 1996;
Kollar, 1998). It seems logical to extend this concept to lupin grain.
If the combinations of treatments, Expansion+PG or PG+PME, are effective in the live bird
then it should be possible to substantially improve the nutritive value of lupins for broilers
and layers, and also substantially reduce the excessive water intake and wet droppings that
are typically associated with inclusion of lupins in the diet. Clearly, these improvements
would benefit feed manufacturers and poultry producers because they would be able to
increase the lupin content from the current limit of 7% to 20%, or even 30%, in broiler and
egg layer diets without compromising production performance or increasing the incidence
of wet droppings. The cost savings would be substantial.
3
In this thesis, I tested the general hypothesis that breakdown of cell-wall constituents, in
particular pectins, would improve the nutritive value of lupins for broilers and egg layers. I
expect that successful breakdown of pectins would reduce viscosity and water-holding
capacity and, as a consequence, reduce water intake and wet droppings. The breakdown of
pectins would release digestible nutrients, increasing dietary metabolisable energy, growth
and feed conversion efficiency, with considerable financial benefits for the industries and
welfare benefits for the birds.
4
Chapter 2
Review of the Literature
2.1 Introduction
The bans on the use of antibiotics in poultry diets and on the use of animal-protein meals
for ruminants, and the recent drought, have left the Australian poultry producers to cope
with poor poultry production and performance and an increased cost of production. If the
poultry industry is to survive, researchers and nutritionists have to find an alternative way
to solve the major problems. It is easy to think that local plant proteins such as lupins can
replace imported, expensive soybean meal but poultry cannot digest lupins because they are
unable to break down complex carbohydrates present in lupins into simple sugars because
they don't secrete the relevant enzymes. Addition of microbial enzymes into the diet could
allow poultry to digest lupins efficiently and reduce the cost of the diet for poultry
producers.
This review will cover the nutritional advantages and main problems of plant-protein feed,
in particular lupins, when it is fed to poultry. It will also describe the main anti-nutritive
effects of cell-wall polysaccharides, such as high digesta viscosity and wet droppings,
associated with lupins and the consequent poor feed conversion efficiency of poultry.
Finally, the review will show how enzyme supplements should be able to offset the anti-
nutritive effects of cell-wall polysaccharides in vitro and in vivo in broilers and egg layers
and, at the same time, reduce wet droppings and improve feed conversion efficiency.
2.2 History of lupins in Australia
Lupins are the leading legume crops cultivated in the Mediterranean region of Australia.
Lupins were unknown in Australia until the seed was introduced from Germany into
Western Australia in the 1920s. They were then naturalised in the Geraldton region and
north coast of Perth (George et al, 1997; Smith, 2003). They were introduced because they
had several agronomic benefits. They are well adapted to the Western Australian
5
environment by their high tolerance of poor, acidic and deep sandy soils, which may be not
suitable for other high-producing crop plants (Gladstones, 1970,1974; Farrington, 1974;
Hill, 1977). They are an excellent crop for breaking disease cycles in cereals and for their
ability to fix atmospheric nitrogen into ammonia, thereby benefiting subsequent cereal
crops grown on the same area of land.
Traditionally, lupins were fed to ruminants only and were considered unsuitable as a feed
for monogastrics such as poultry and pigs because of their high content of naturally
occurring alkaloids that give a bitter taste. However, breeding programs in Western
Australia produced some varieties of Lupinus angustifolius and Lupinus albus with alkaloid
content as low as 0.01% (Gladstones, 1982; Johnson et al, 1986; Williams, 1986). Since
then, ‘sweet lupins’ have become widely used in the feed industry as a source of protein
and energy in a range of monogastric diets for poultry, pigs, fish and rabbits (Taverner,
1975; Pearson & Carr, 1977; Yule & McBridge, 1976; Ballester et al, 1980; Inborr, 1990;
Glencross et al, 2004). Two varieties, Borre (L. angustifolius) and Weeko III (L. luteus),
were produced commercially and used for the first time as a protein feed by broiler
producers in Western Australia (Underwood & Gladstones, 1979).
Since 1990, production of lupin cultivars has increased to the extent that Australia has
become the world main producer (85%) and exporter of lupins to the livestock feed market
in Europe, Asia and Middle East (NHMRC, 1991; Qureshi, 1993; ABARE, 2008). Western
Australia is now the largest lupin producer in Australia and accounts for 83% of total
national production of lupins.
2.3 Feeding lupins to poultry – advantages and disadvantages
Feed manufacturers, nutritionists and poultry producers in Australia are seeking cheaper
ingredients that can replace expensive, imported protein meals such as soybean meal. The
price of soybean meal has increased 2-3-fold since animal protein meals were banned from
use in ruminant diets in the last decade. As a result, Australia has increased its annual
imports of soybean meal to >250,000 tonnes from Brazil and the USA, of which 50% are
used for poultry, costing the poultry industry over $66 million (ABARE, 2008).
6
As alluded to earlier, Australia is the world’s leading producer of lupins with an annual
production of lupins exceeding 1 million tonnes (four times that of imported soybean meal,
ABARE, 2008). The nutrient content of lupins is somewhat different to that of soybean
meal: for example, protein content is lower (34 vs. 46%) but there is more fat (6 vs. 4%)
and energy (18 vs. 16 MJ/kg). Table 1 provides a comparison of the nutritive value and
price of the most common poultry feeds. Importantly, lupins cost approximately half the
price of soybean meal ($320 vs. $530 per tonne) so could be a cheaper alternative source of
protein and energy for poultry. However, the metabolisable energy of lupins for poultry is
about 30% lower than that of soybean meal. In addition, using lupins in poultry diets leads
to poor digestion of nutrients and low feed conversion efficiency. Moreover, lupins increase
water-holding capacity, a characteristic directly related to excessive water intake and
concomitant wet droppings of poultry, as well as increased number of soiled eggs from
layers. As a consequence, the Australian feed manufacturers and poultry producers have
limited their use of lupins in wheat-based diets to 5% for broilers and 7% for egg-layers. Table 2.1. The nutritive value of common ingredients fed to poultry.
Nutrients (%DM) Wheat1 Barley2 Corn1 Soybean meal1 Lupins1
Moisture 8.9 10.0 10.4 8.5 9.1
Protein 11.8 10.5 8.8 46.3 34.0
Fat 2.1 2.1 4.2 7.0 5.9
Ash 2.0 2.2 7.8 3.0 2.9
Fibre 4.6 6.5 10.0 6.5 13.7
Nitrogen-free extract 70.9 70.9 60.4 28.7 35.9
Gross energy (MJ/kg)3 19.1 16.7 17.1 16.1 17.8
Metabolisable energy (MJ/kg diet)4 12.8 13.2 15.9 13.1 9.7
Price ($/tonne)5 380 330 440 530 320
1Choct et al, (1995), Choct (2006); Petterson & Mackintosh (1994), Ali (1997); 2PGH (2007); 3Kim et al,
(2003); 4Graham & Balnave (1995), Leeson & Atteh (1995), Annison et al, (1996); 5ABARE (2008).
7
2.4 Heating, dehulling and removing oligosaccharides from lupins are ineffective
Various methods have been tested to reduce the main anti-nutritional effects of lupins and
to improve the growth and feed conversion efficiency of broilers and layers. For example, it
was first believed that heat treatment could destroy the anti-nutritional factors in lupins
because lupins contain at least nine anti-nutritional factors (alkaloids, saponins, tannins,
trypsin and chymotrypsin inhibitors, phytate, glycosyl flavanol, oligosaccharides and uric
acid). However, heat or mechanical treatments such as extrusion, autoclaving, micronising,
heating, cooking, flaking and rolling lupins have only marginally increased feed quality and
did not reduce wet droppings (Smetana & Morris, 1972; Boldaji et al, 1986; Watkins &
Mirosh, 1987; Watkins et al, 1988; Perez-Escamilla et al, 1988; Bishop, 1989; Qureshi,
1993; Olver & Jonker, 1997). Too much heating of lupins caused a depression in weight
gain in broilers (Perez-Escamilla et al, 1988), and a marked reduction in amino acid
utilisation due to Maillard reactions (Larbier & Leclercq, 1994). Treatment such as
autoclaving and gamma irradiation also were found to cause a marked reduction in growth
of broilers and in the metabolisable energy of diets based on cereals (Vohra & Kratzer,
1964; Heinz & Poppe, 1975ab; Maga, 1978; Patel et al, 1980; Verma & McNab, 1982;
Classen et al, 1985; Guevara et al, 1999). Similar responses were noticed when lupins were
heated or autoclaved for pigs (Batterham et al, 1986ab, 1993).
These problems led to speculation that the lupin hulls were responsible for the detrimental
effects on growth and efficiency of feed conversion in monogastric animals (Bailey et al,
1974; Taverner et al, 1983; Champ et al, 1990; Juskiewicz & Zdunczyk, 1997; Dunshea et
al, 2001). Lupins consist of 25% hulls that are low in digestibility because they consist
mainly of cell-wall polysaccharides such as cellulose, hemicellulose and pectin (Bailey et
al, 1974; Brillouet & Roichet, 1983; Evans et al, 1993; Hughes et al, 1997). However,
removing the hulls increased the nutritive value of lupins by increasing protein and energy
content, but did not eliminate all cell-wall polysaccharides or their anti-nutritional effects.
Consequently, the kernel was still not regarded as the best protein source for poultry
because it still depressed production performance and caused wet droppings.
Next, the non-starch polysaccharides, in particular the oligosaccharides, were thought to be
the main cause of the growth depression when they were fed to poultry or pigs (Rackis,
8
1975; Wiggins, 1984; Saini & Gladstones, 1986; Champ et al, 1990; Leske et al, 1993;
Gorecki et al, 1997). Whole lupins contain a high proportion (up to 8%) of an α-galactose-
containing oligosaccharide, which is not broken down in the small intestine because the
intestinal mucosa does not have a specific enzyme, α-galactosidase, for cleaving α-linked
galactose units to galactose and sucrose. These oligosaccharides were believed to induce
flatulence from microbial fermentation that produced carbon dioxide, hydrogen and
methane in the large intestine. However, when α-galactosidase was added to the diet in an
attempt to break down the oligosaccharide, growth performance was not improved
(Veldman et al, 1993; Slominski et al, 1994; Bryden et al, 1994; Irish et al, 1995).
Removing the oligosaccharides from lupins by ethanol extraction also failed to improve
bird performance (Hughes et al, 1997; Kocher et al, 1999). Likewise, supplementing broiler
diets with oligosaccharide extracted from lupins did not reduce the adverse effects (Brenes
et al, 1989). So it was concluded that oligosaccharides were not the primary cause of
growth depression associated with lupins in poultry diets.
2.5 Reasons: complex cell walls and pectins in lupins
The main cause of the anti-nutritional effects in lupins in poultry is a high proportion of
indigestible cell-wall material (34%), commonly known as cell-wall polysaccharides or
non-starch polysaccharides. These cell-wall polysaccharides are the prime factor
responsible for poor growth and feed conversion efficiency, low metabolisable energy of
the diet and high wet droppings for poultry. This will be explained in detail below.
Eighty percent of cell-wall polysaccharides exist in the lupin kernel and its content is
strikingly higher than in other feeds including cereals (Table 2.2). The main component of
cell-wall polysaccharides of the lupin kernel is pectin (Bailey et al, 1974; Belo & de
Lumen, 1981; Carre & Leclercq, 1985; Carre et al, 1985; Ali, 1997; Carre, 1997).
Pectin is one of the most important components of the cell wall of plants, in particular
legumes. It is synthesised to provide firmness to the structure of the cell walls and middle
lamella by cementing the intracellular matrices together and adhering one cell to another
9
(Figure 2.1, Worth, 1967; Rees & Wight, 1969; Jarvis, 1984; Matsura & Hatanaka, 1990;
van Buren, 1991; Albersheim et al, 1996).
Table 2.2 Cell wall and pectin contents of common ingredients fed to poultry.
Parameters Wheat Barley Corn Soybean
meal
Lupin
seed
Lupin
kernel
Cell-wall polysaccharide (%DM) 11.4 16.7 8.1 19.2 28.2 34.2
Pectin (%DM) 1.2 2.2 0.2 6.1 8.1 12.2
Water-holding capacity (g:g) 2.0 2.3 1.6 3.2 3.8 7.1
Intrinsic viscosity (m.Pas/sec.) 5.4 3.1 1.0 1.6 5.1 6.9
Choct (2006); Ali (1997); Evans (1994); Bailey et al, (1974); Brillouet & Roichet (1983); Carre et al, (1985);
Carre (1997); Cheung (1991); Giger-Reverdin (2000); McNab & Shannon (1974,1975).
Figure 2.1 The main chemical structure of the plant cell wall and its pectin content (diagram taken from Wikipedia, 2008).
10
Chemically, pectin consists of a long linear chain of galacturonic acid units that are usually
linked to each other by beta type 1→4 glycosidic bonds (Fig. 2.2, Kertesz, 1951; Worth,
1967; Albersheim, 1974; BeMiller, 1986; Rolin & de Vries, 1990). Pectic materials can be
divided broadly into two groups; pectic acid, a smooth polymer of galacturonic acid units,
and the other is pectin, which is a polymer of galacturonic acid whose carboxyl groups are
methyl esterified (Fig. 2.2).
Figure 2.2 The molecular structure of pectin showing the linear galacturonic acid units, glycosidic bonds,
and the carboxyl (COO–) or methyl ester (CH3COO–) groups attached to the carbon atom six along the pectin
chain.
2.6 Anti-nutritive effects of pectins on poultry
The cell-wall polysaccharides are the prime anti-nutritional factors in lupins that are
responsible for poor growth and feed conversion efficiency, low metabolisable energy of
the diet and wet droppings for poultry. This is mainly because poultry cannot break down
the complex cell-wall lattices to release the digestible nutrients, which are entrapped within
the lattices and neutralise their anti-nutritional effects. Pectins, which are important for
maintaining the integrity of the cell-wall lattice, remain largely intact during the digestion
process in the bird’s intestine. The inability of birds to break down pectin poses several
nutritional and hygienic problems through three direct mechanisms:
1) Pectins increase viscosity of digesta
Viscosity is one of the most distinctive characteristics of pectin, is a result of the
dissolution of large molecular weight and soluble fractions of pectin from the cell walls.
The molecular weight of pectin ranges from 10 to 400 kilo daltons (McCready & McComb,
11
1953; Pilnik & Voragen, 1970; Rombouts & Thibault, 1986; Sakai et al, 1993) because the
chain of galacturonic acid units ranges from 30 to 300 units (Zitko & Bishop, 1966; Barash
& Eyal, 1970; McNeil et al, 1984; Chambat & Joseleau, 1980; Fry, 1988; Thibault et al,
1993; Kravtchenko et al, 1993). Moreover, up to 30% of pectins are water-soluble
(Rombouts & Pilnik, 1980; Rexova-Benkova et al, 1977; Sakai et al, 1993).
Thus, the combination of large molecular weight, long chains and soluble fractions allows
pectin to form large solutes that interfere with the free movement of other solutes in an
aqueous medium (Kertesz, 1951; Rees & Wight, 1969; Brillouet & Carre, 1983; Carre et al,
1985; Sakai & Ozaki, 1988; Carre, 1991; Sakai et al, 1993). As a result, these viscous
solutes limit diffusion of nutrients and hinder the effective interaction between nutrients
and digestive enzymes at the mucosal surfaces of a bird’s intestine, leading to reduced
nutrient absorption through the intestinal wall (Grammer et al, 1982; Erdman et al, 1986;
Ikeda & Kusano, 1983; Fengler & Marquardt, 1988). As viscosity increases, the rate of
diffusion of solutes is reduced, the ability of the of bird’s gut to physically mix the digesta
contents is severely compromised, and fewer nutrients are utilised by the bird. This is why
Choct & Annison (1991ab, 1992) and Bedford & Classen (1991,1992) were able to
demonstrate a negative correlation between the viscosity of digesta (based on arabinoxylan,
the main cell-wall polysaccharides of wheat) and digestion of nutrients, body weight gain
and feed conversion ratio in broilers.
In lupins, the high molecular weight (165 kilo daltons), relatively high percentage of
soluble fractions (15%), and long pectin chains, in addition to cross linkages of methyl
esters with neighbouring pectin polymers, generate a large numbers of matrices which, in
turn, increase the viscosity (Brillouet, 1984; Cheung, 1991; Ali, 1997; Ali & Harris,
unpublished results). This could explain why lupin-based diets are 100 to 700% more
viscous than diets based on other cereals and legumes by (Table 2; Evans et al, 1993; Ali,
1997).
2) Pectins increase water-holding capacity, water intake and wet droppings
The second most distinctive characteristic of pectin is its high water-holding capacity.
Pectins absorb water like a sponge because their basic molecules, galacturonic acids, are
12
hydrophilic in nature and contain ionisable negative carboxylic groups that bind positively
charged molecules (Fig. 2.2, James et al, 1978). Another distinctive characteristic of pectin
is that it is able to form a gel through cross linking via divalent C++ ions with corresponding
C++ ions of neighbouring pectic polymers (Powell et al, 1982; Morris et al, 1982; Manunza
et al, 1997, 1998). These cross linkages generate a large number of matrices and form gels,
which, in turn, increase the water-holding capacity and viscosity of pectin. Pectin
molecules require a large volume of water to support the three dimensional solid gel.
These two characteristics are most likely to be main causative factors of excessive water
intake and concomitant high incidence of wet droppings in birds fed diets containing
significant amount of pectins (Wagner & Thomas, 1977ab; Ricke et al, 1982; Lacassange,
1983; Drochner et al, 1990,1993; Carre et al, 1995ab; Langhout & Schutte, 1996; Ali,
1997). Increased water intake not only causes wet droppings, but also causes several
problems such as wet litter, unpleasant odours in poultry sheds, outbreaks of coccidiosis,
breast blister in broilers, and increases in the number of soiled eggs from layers.
Lupins have a 2.0 to 4.5-fold higher water-holding capacity than other feeds (Table 2.2)
and at least 3.7-fold (8.4 vs. 2.3 k.N/m2) higher gel-forming ability (Krause et al, 2001;
Kiosseoglou et al, 1999; Mavrakis et al, 2003; Drakos et al, 2007). This could explain why
lupins are associated with excessive water intake (330 vs 280 ml/bird/day) and wet
droppings of poultry more than other feeds (73 vs 66%, Vukic Vranjes & Wenk, 1995; Ali,
1997; Hughes et al, 1998, 2000; Kocher et al, 1999, 2000; Farrell et al, 1999).
3) Pectins reduce rate of feed passage, digestibility, growth and feed conversion
By increasing the viscosity of digesta, pectin slows down the mobility of food particles
through the digestive tract of birds. This reduced rate of feed passage means increased time
of feed retention in the digestive tract (Wagner & Thomas, 1977ab, 1978; Day & Thomas,
1980; Ali, 1997; Salih et al, 1991; Classen & Bedford, 1991). This may appear to be
beneficial for poultry by some researchers because better digestion of nutrients is achieved
by longer retention and exposure of food particles to endogenous enzymes and absorptive
surfaces of the intestine. However, retaining food particles leads to proliferation of
undesirable micro-organisms in the large intestine. For example, when nutrients pass
13
through the small intestine undigested, colonic bacteria multiply and compete effectively
with the host for starch and proteins. Gas and volatile fatty acids are produced because of
microbial fermentation, leaving the intestine inflated (Wagner & Thomas, 1977ab, 1978;
Day & Thomas, 1980; Drochner et al, 1993).
In addition, some of the intestinal bacteria produce acid-degrading enzymes, such as the
bile salt cholytaurine hydrolase, that reduces the ability of the bird to digest lipids (Feighner
& Dashkevicz, 1987,1988). The deconjugation of bile salts by hydrolase partitions fat into
the gel phase after disrupting the micelles. This makes emulsification and absorption of fats
less effective (Coates et al, 1981; Campbell et al, 1983ab; Judd & Truswell, 1985). For this
reason, birds may be exposed to deficiencies of the fat and fat-soluble vitamins that are co-
absorbed with the micelles, for example, vitamins A, E and D as well as B12 (Patel et al,
1980, 1981; Bishawi & McGinnis, 1984; Erdman et al, 1986; MacAuliffe et al, 1976ab;
Cullen & Oace, 1979; Schaus et al, 1985). In addition, some reports highlighted pectin
interference with mineral absorption (such as Mg, Ca, P, Na, K and Zn) via the strong
binding capacity of the ioinised carboxyl groups of galacturonic acids (James et al, 1978;
Aar et al, 1983; Bagheri & Gueguen, 1985; den Hartog et al, 1988).
It is worthwhile to note that longer digesta retention in the intestine might cause a
depression of feed intake by invoking the sensation of satiety via cholecystokinin, a
polypeptide hormone released into the duodenum and jejunum in response to the entrance
of digesta into the intestine (Dockray, 1977, 1979; Moran & McHugh, 1979; Savory &
Gentle, 1980; Wittert et al, 1997). Another factor that may also play a role in depressing
feed intake is the increase in water intake because of pectin levels, as alluded to earlier. In
turn, water intake increases at the expense of feed intake (van Kampen, 1983; Williams,
1996; Manning et al, 2007).
Consequently, feed intake is depressed, digestion of nutrients impaired, growth lowered,
feed conversion efficiency reduced and the metabolisable energy of the diet decreased for
both broilers and layers (Vohra & Kratzer, 1964; Erdman et al, 1986; Langhout & Schutte,
1996; Langhout et al, 1999, 2000). Based on these findings, it is not surprising that lupins
reduce digestibility, feed conversion efficiency and the metabolisable energy of the diet for
14
broilers and layers, since they delay feed passage through the digestive tract by 35 minutes
and depress feed intake by 8% (Ali, 1997). Similar responses were consistently noticed
when cell-wall polysaccharides, such as arabinoxylan in wheat and β-glucan in barley, were
present in broiler and layer diets (Campbell et al, 1983; Fengler & Marquardt, 1988; Gohl
& Gohl, 1977; Salih et al, 1991; van der Klis & van Voorst, 1993).
2.7 Role of enzymes in breaking down cell-wall polysaccharides
Poultry must first break down cell walls to obtain digestible nutrients from plant materials.
They do this mechanically in the gizzard, a process that is relatively inefficient. Enzymes
are better than mechanical treatment for breaking down cell walls, but poultry have no
endogenous cell-wall enzymes. If poultry are to break down the cell walls, an exogenous
enzyme must be added to the diet.
Enzymes can be produced by microorganisms that are used for microbial fermentation
processes by fermenting machines. Thus feed enzymes have been made available as
supplements to poultry diets for more than forty years. Initially, they were expensive
because of low supply and high costs (>$15/kg), but advances in the biotechnological
sector have driven down the cost of poultry enzymes to $3 – 10/kg. In addition,
improvements have been made in stability. Thus, dry-powder enzymes can now withstand
the 90°C heat typical of pelleting machines without loss of activity, and resist wide ranges
of pH typical of the digestive tract of birds.
How can enzymes be used to improve the nutritive value of feed for poultry?
Enzymes can be selected that will rupture the cell wall membrane by cleaving the covalent,
non-covalent, ester or glycosidic bonds among the cell-wall polysaccharides in a random
manner, resulting in formation of shorter chains of poly-, oligo- and mono-saccharides
(Bauer et al, 1973; Bailey & Kauss, 1974; Selvendran, 1985; Wilkie, 1985). This leads to
the collapse of the entire cell-wall network, releasing the nutrients entrapped within cell
wall matrices and increasing the cell wall permeability to the digestive pancreatic and
15
intestinal enzymes of the bird which can then access the nutrients. Most importantly, the
collapse of cell wall networks into shorter, single chains results in a significant drop in
viscosity and water-holding capacity of the substrate.
Three important points should be noted for rapid, effective breakdown of cell walls. First,
reducing one component of the cell walls by enzymatic hydrolysis may provide additional
benefits because it may help to collapse the entire structure of the cell walls since these
components are all linked to each other in chain-like groups (Knee et al, 1975; Wallner &
Bloom, 1977; El-Rayah & Labavitch, 1980; Themmen et al, 1982; van Buren, 1991).
Second, enzymes need only to cleave a few linkages in the cell-wall polysaccharide chains
to substantially reduce viscosity (Pressey & Avants, 1971; Thibault & Mercier, 1978;
Yamaguchi et al, 1994). Third, a maximum breakdown of any polysaccharide can be
achieved by using endo-acting enzymes that favour binding and cleaving bonds toward the
centre, rather than the ends of the polysaccharide molecules. Exo-acting enzymes, which
cleave bonds at the ends of chain molecules, cannot break more than 7% of total bonds of
cell-wall polysaccharides (Endo 1964c; Rombouts & Pilnik, 1980; Kester et al, 2000).
As a consequence, enzymatic breakdown can increase the digestion of nutrients and
metabolisable energy of a diet, and increase growth and feed conversion efficiency, and
reduce water intake and therefore wet droppings. There are many examples of this for
poultry. In cereals such as wheat, triticale and rye, breakdown of arabinoxylans can be
accomplished by endo-xylanase activity, which cleaves 1,4-glycosidic bonds of the xylan
backbone (Wong & Saddler, 1992; Kormelink et al, 1993). This breakdown reduced the
viscosity of digesta by 30%, increased growth rate by 12%, increased feed conversion
efficiency by 10%, increased digestion of dry matter by 18%, and increased the
metabolisable energy of the diet by 9% for broilers and layers fed diets based on these
cereals (Pettersson & Aman, 1988; Bedford & Classen, 1992; van der Klis, 1993; Choct et
al, 1995).
Similar responses were found by breaking down β-glucans, the main anti-nutritive, cell-
wall polysaccharides in barley and oats, by endo-β-glucanase for broilers (Gohl et al, 1978;
Classen et al, 1988; Edney et al, 1986; Pettersson & Aman, 1989). Phytase, an enzyme
16
designed for breaking down phytates to release phytate-bound phosphorus in wheat, corn,
sorghum and soybean-based diets, has also been very successful in eliminating the anti-
nutritive factors of phytate, thus enabling broilers and layers to utilise these feeds optimally
(Ravindran et al, 1995,1999a,b; Um & Paik, 1999; Selle et al, 2000).
2.8 Pectinase in poultry diets
The glycosidic bonds of a pectin chain can be broken down by a pectinase specifically
called endo-polygalacturonase, PG (EC 3.2.1.15), which is produced by micro-organisms
found in the rumen and also by fungi and yeast (Demain & Phaff, 1954; Clarke et al, 1969;
Cooke et al, 1976; Coleman et al, 1980; Bonhomme, 1990). PG randomly cleaves the
internal glycosidic bonds among the galacturonic acid units of the pectin chain, producing
shorter chains of poly-, oligo- and mono-galacturonic acid units (Fig. 2.3, Reid, 1950,1952;
Endo, 1964abcd; Cooke et al, 1976; Thibault & Mercier, 1978; Rombouts & Pilnik, 1980;
Polizeli et al, 1991). This cleavage led to reduction of cell-wall contents by 4-fold, pectin
by 7-fold, viscosity of pectin by 60%, water-holding capacity by 40%, pectin chain by 2-
fold, and molecular size of pectins by 60%, and increase in filtration rate of the viscous
solution by 70% in vitro (Kertesz, 1951; Endo, 1964; Thibault & Mercier, 1978; Liu &
Luh, 1980; Kollar, 1998; Ali, 1997; Daas et al, 1998; Benen et al, 1999; Ali et al, 2001).
Figure 2.3 Enzymic degradation of beta-glycosidic bonds of pectin in a random manner by PG. Cleavage of only a few linkages in the pectic polymers leads to rapid shortening of pectic
chains and a consequent large reduction in viscosity (Kertesz, 1951; Luh & Phaff, 1954ab;
McCready & McComb, 1954ab; Pressey & Avants, 1971; Thibault & Mercier, 1978;
Yamaguchi et al, 1994). For example, cleavage of 5% of the glycosidic bonds by PG has
17
been noticed to produce short chain products of oligo-galacturonic acids and a few tri-, di-
and mono-galacturonic acids and, at the same time, reduced the viscosity of pectin solution
by 50% (Reid, 1952; Endo, 1964a; English et al, 1972; Liu & Luh, 1980; Blanco et al,
1994; Kollar, 1998).
Burnett (1966) first found that supplementing diets with commercial pectinase, pectozyme
(PG), reduced excessive water intake by 36% and completely eliminated the sticky
droppings for broilers. In addition, adding commercial pectinase (mainly PG) to rye-based
diets (rye contains 8% pectins) reduced the viscosity effect by 20%, enhanced growth by
15% and reduced the incidence of sticky and wet droppings by 15% of broiler chicks (Patel
et al, 1980; Grammer et al, 1980,1982; Broz, 1987). Likewise, supplementing faba bean-
based diets with a commercial pectinase (PG at 2.5 g/kg diet) gave a 5% improvement in
feed conversion efficiency (Castanon & Marquardt, 1989). Similarly, treatment of diets
containing 4 – 6% pectins with pectinase increased weight gain by 26% and feed
conversion efficiency by 18% (Vohra & Kratzer, 1964; Kirstein & McGinnis, 1979; Patel
et al, 1980, 1981; Rassmussen & Petterson, 1997). Pectin is the major fraction (26%) of
cell-wall polysaccharides in dehulled peas (Reichert, 1981; Brillouet & Carre, 1983).
Supplementing dehulled pea-based diets with pectinase (mainly PG) increased feed intake
by 6% and growth rate by 7% of broilers, although it did not increase the metabolisable
energy of the diet (Igbasan & Guenter, 1996; Daveby et al, 1998).
2.9 Previous work on enzymes and lupins for poultry
The enzyme manufacturers have designed two types of feed enzymes to break down the
cell-wall polysaccharides in plant-protein meals, multi-enzyme preparations and single
specific enzymes. The effects of these enzymes on lupin utilisation by poultry are outlined
below.
2.9.1 Multi-enzyme preparations
Most studies have investigated commercially available multi-enzyme preparations for
improving the nutritive value of lupins for poultry. These preparations were composed of
18
several enzymes and different enzyme activities that were aimed at targeting several
linkages in the cell-wall polysaccharides. However, the results for lupin-based diets are
inconsistent and inconclusive. The main reason was that the multi-enzyme preparations did
not target the major components of the cell walls in lupins. For example, Brenes et al,
(1993, 2002, 2003) showed that supplementing whole or dehulled lupin-based diets with
three enzyme preparations, Energex, Bio-Feed Pro and Novozyme SP-230, significantly
improved the growth performance of broilers.
However, the case supporting these commercial enzymes for improving the nutritive value
of lupins is unconvincing for six reasons.
i) These enzymes do not appear to be appropriate for lupins. For example, Energex was
comprised of 75 units of β-glucanase, 150,000 units of hemicellulase, 10,000 units of
pectinase, and 400 units of endoglucanase; Bio-Feed Pro was comprised of 150,000 units of
protease; and Novozyme was comprised of 500 units of galactosidase, 2,500 units of
inulinase and 24,000 units of cellulase. None of these enzymes, except pectinase, target
cell-wall components. Also, the hemicellulase content of Energex was not only too high but
did not target the main anti-nutritive factors in lupins. Perez-Escamilla et al, (1988) found
that hemicellulase supplementation of lupin-based diets did not improve the growth of
broilers. Furthermore, it is questionable whether any benefit can be gained from the
glucanase and endoglucanase of the Energex or Bio-Feed Pro products since there is no
glucan in lupins or in other dietary ingredients. Similarly, Bio-Feed Pro and Novozyme
enzymes are not target enzymes for lupins. Thus, Hughes et al, (2004), Alloui et al, (1994a)
and Steenfeldt et al, (2003) did not find any improvement of growth from using Energex or
Bio-Feed Plus in lupin and faba bean-based diets for broilers.
ii) It was assumed that the pectinase enzyme of Energex was endo-polygalacturonase (PG).
If so, the dose of PG was far higher than that required for breakdown of pectin. It is well
known that a high dose of PG causes increased osmotic pressure in the gut due to excessive
breakdown of pectic chains and hence the release of excessive amounts of hydrophilic
galacturonic acid units (Kertesz, 1951; Gupta, 1962; McCready, 1970; Griffiths &
19
Kennedy, 1988; Schejter & Marcus, 1988; Sakai et al, 1993). These hydrophilic units
increase water intake and wet droppings of birds (Ali et al, 2005, 2006b).
iii) Brenes et al, (1993, 2002, 2003) did not measure water intake and concomitant wet
droppings, the two most important parameters that are directly associated with lupins. The
primary focus of the poultry producers and feed manufacturers is to reduce these two
factors when lupins or dehulled lupins are included in poultry diets. Also, they did not
measure the viscosity of digesta in any lupin-enzyme experiments.
iv) It is doubtful that any enzyme product allows poultry diets to include up to 70% whole
lupin or 50% of dehulled lupins as claimed by Brenes et al, (1993, 2002, 2003), because
these high levels of lupin inclusion are impractical in the poultry industry. Except for
soybean meal, feed manufacturers have restricted inclusion of most legumes to a maximum
30% in poultry diets with enzyme supplementation because of their high content of anti-
nutritive factors or because of the problem of wet droppings (Hoxey, Potts and Hayat, pers.
comm. 2005).
v) Brenes et al, (2003) showed that a combination of α-galactosidase (Novozyme SP-230,
an enzyme which breaks down oligosaccharides), Energex and Bio-Feed Pro improved the
growth performance of broilers. However, they showed earlier that the diets supplemented
with lupin oligosaccharide extract did not impair broiler performance (Brenes et al, 1989).
Bryden et al, (1994), Alloui et al, (1994ab), Slominski et al, (1994), Keller & Jeroch (1997)
and Igbasan et al, (1997) observed minimal or no response in supplementing lupins, canola
meal or pea based diets with α-galactosidase for broilers.
vi) Brenes et al, (1993, 2002, 2003) did not make any reference to breakdown of cell-wall
polysaccharides in lupin-based diets to indicate if the mentioned enzymes tested in their
experiments were the main factor for improving broiler growth and feed conversion
efficiency of broilers. Instead, they measured weight of liver and gizzard, and length of
intestinal sections; these two measurements give little indication of the breakdown of lupin
cell walls.
20
Unlike Brenes et al, (1993, 2002, 2003), Annison et al, (1996) demonstrated an advantage
of multi-enzyme preparations, Energex, Bio-Feed Pro and Novozyme SP-230, in lupin-
based diets for broilers by providing appropriate measurements (eg. viscosity of digesta,
wet droppings and breakdown of cell-wall polysaccharides) and adequate explanations.
Hughes et al, (2000) also demonstrated minor improvements with two multi-enzyme
preparations, Bio-Feed Plus and Energex, but Kocher et al, (2000) failed to demonstrate an
effect of three other multi-enzyme preparations for lupins. Part of the reason that they
found relatively small effects with enzymes may be that they used very small doses of
enzymes, 0.15 g/kg diet. In addition, they reported some curious results and demonstrated
an increase in viscosity of digesta with the addition of Energex (Kocher et al, 2000). They
assumed that the increase in viscosity was attributed to the pectinase content of the Energex
that solubilised the components of the non-starch polysaccharides. It is well established that
the primary function of pectinase is to decrease viscosity. However, there is one possible
reason why viscosity might have increased with Energex: if hemicelluloses were
solubilised, viscosity could be increased and, since the Energex consisted mainly of
hemicellulase activity (15,000 units/g in Enzyme) and relatively little pectinase (3017
units/g) at the inclusion rate of 0.15 g/kg diet, there was every chance that hemicelluloses
rather than pectins were broken down and solubilised. Annison et al, (1996) themselves
considered these results “perplexing”.
These results are not surprising since several reports have shown that some commercially
available multi-enzyme preparations do not improve growth performance of broilers (Irish
& Balnave, 1993; Gerendai et al, 1997; Sherif et al, 1997ab; Rebole et al, 1999).
Taken together, the studies by Annison et al, (1996), Hughes et al, (2000) and Kocher et al,
(2000) discussed above show that finding an appropriate commercial multi-enzyme
preparation that matches the cell-wall polysaccharides in lupins is not an easy task. Hughes
et al, (2000) concluded that what was needed was development of appropriate exogenous
enzyme products to better target lupin’s cell-wall polysaccharides.
Finally, some of the inconsistencies in experimental outcomes might be explained by
impurities in the multi-enzyme preparations. For example, Chesson (1987) reported that
21
inconsistencies in the response of poultry to enzymes such as amylase supplementation
could be interpreted in terms of unrecognised levels of important ‘contaminating’ activities,
notably endo-β-glucanase and pentosanase, which possibly make the test of hypotheses
incorrect. He suggested to preferably use purified and single enzymes of defined activity in
the diets for the observed responses in feeding trials and to avoid confounded results.
Likewise, Bedford & Classen (1992,1993) acknowledged the difficulty in interpreting the
results if all enzyme sources used in the poultry trials are crude products and contain
several “contaminating activities” such as β-glucanase, α-amylase or arabinofuranosidase.
2.9.2 Specific-enzyme preparations
Due to the inconsistent and inconclusive effects of multi-enzyme preparations mentioned
above, it seems reasonable to use specific enzymes to target the main components of the
cell wall in lupins in poultry diets. The use of specific enzymes with defined activity
requires the definition of the site of enzyme action on the substrate. This should give clear
results of enzyme hydrolysis and its impact on bird performance.
Finding a specific target enzyme for lupin pectin can be a very hard task for two reasons.
First, the pectin structure is highly complex. The pectin chain in lupins is mainly composed
of galacturonans, rhamno-galacturonans and galactans, which are all entangled with
arabinans and a few xylan side-chains (Fig. 2.4).
Polygalacturonan Rhamno-galacturonan ↓ ↓ A-(1→2)-β-L-Rhap-(1→4)-α-D-Galp–A-(1→2)-β-L-Rhap-(1→4)-α-D-Galp-A-(1→2)-β-L-Rhap-(1→ 4 4 4 4 ↑ ↑ ← Endo-xylan → ↑ ← Endo-galactanan → ↑ 1 1 1 1 α-L-Araf D-Xylp β-D-Gal p β-D-Gal p 5 6 4 ↑ ← Endo-arabinan ↑ ↑ 1 ↓ 1 1 α-L-Araf -(2→1)-α-L-Araf– (5→1)-α-L-Araf-β-D-Gal p β-D-Gal p Rahp = rhamnose, Galp = galacturonic acid, Gal p = galactose, Araf = arabinose, Xylp = xylose Figure 2.4 Schematic representation of the complex structure of pectin in dehulled lupins (compiled from
proposed structures demonstrated by Tomoda & Kitamura, 1967; Brillouet, 1984; Carre et al, 1985; Cheung,
1991; Evans, 1994; Ali & Harris unpublished results).
22
This complexity of pectin structure highlights the difficulty of breaking down the cell-wall
polysaccharides of lupins by commercially available multi-enzyme preparations mentioned
earlier.
Second, there is no commercial enzyme available to break down these main and side chains
of lupin pectin. For example, rhamno-galacturonanase and galactanase have never been
commercialised, and it was not possible to obtain a pilot enzyme (500 g) for research
purposes because of the high cost involved in extracting these enzymes commercially (B.
McCleary, 1996; McCleary Pty Ltd, pers. comm.). Kocher (2000) tested galactanase in
soybean meal-based diet for broiler but did not mention the source of enzyme. The only
target enzyme found was a commercial pectinase (mainly polygalacturonase), an important
enzyme for breaking down pectin, since the main skeleton of the pectin chain in lupins is
polygalacturonans as shown above. Any breakdown of galacturonic acid units by PG might
lead to a collapse of the entire network of lupin cell walls. Pectinase is a broad spectrum of
polygalacturonase (PG, 2200 - 1800 units activity/g) with traces of side activity of
galactanase and arabinosidase. It is currently commercially available at cost-effective prices
($5 -10/kg) in both powder and liquid forms from several manufacturers: Amano
Pharmaceutical Co. Ltd, Nagoya, Japan; Lyven Enzyme Industries, Caen, France; AB
Enzymes GmbH, Darmstadt, Germany; and Orba Biochemistry Pty Ltd, Istanbul, Turkey.
2.9.3 Polygalacturonase (PG) for lupins
In contrast to the multi-enzyme preparations, preliminary in vitro and in vivo work
conducted by Ali (1997) and Ali et al, (2000, 2001), developed in consultation with poultry
researchers and nutritionists, feed manufacturers and lupin researchers, showed that it was
possible to achieve better results with lupins by using a more targeted enzyme such as PG.
For example, an initial in vitro experiment showed that incubation of lupins with PG
resulted in a breakdown of the cell wall by 24% and pectin by 33%, and a reduction of
viscosity of solution by 10% and filtration rate of supernatant by 11%. The breakdown of
cell walls and pectins by PG was evident by reduction of the concentration of the acidic
(galacturonic acid) and neutral (galactose, arabinose, rhamnose and xylose) sugars by
cleavage of glycosidic bonds present among the galacturonic acid units, and also cleavage
23
of glycosidic bonds of galacturonic acid units associated with these neutral sugars. The
breakdown of pectins by PG was evident from the shorter lengths of pectin chains and
lower molecular weight of pectin than in the control (Ali et al, 2001).
This in vitro work prompted us to test the beneficial effects of PG in live broilers.
Supplementing 10% whole lupins or dehulled lupins-based diets with 1.0 g PG/kg diet led
to a breakdown of the cell wall by 11% and pectin by 40%, reduction of viscosity of digesta
by 11%, increase of digestion of dry matter by 7%, and hence improvement of growth by
5% and feed conversion efficiency by 4%. However, PG was unable to reduce water intake
or wet droppings in broilers (Ali, 1997). This was because a high PG dose (1.0 g PG/kg diet
@ 3100 units/g product) was employed in the experiment leading to excessive release of
hydrophilic galacturonic acid units. Excessive releases of galacturonic acid units will
increase the osmotic pressure in the gut and this will increase water in the digestive tract
and lead to an increase in wet droppings. Evidence from several studies shows that low
levels of enzyme outperform higher levels in both layers and broilers (Petersen & Sauter,
1968; Patel & McGinnis, 1985; Boling et al, 2000; Lazaro et al, 2003). Therefore, an
optimal dose of PG must be determined if high water intake and wet droppings are to be
reduced for broilers fed whole or dehulled lupin-based diets.
In this thesis, three strategies have been developed to increase the efficiency of PG activity
for improving the nutritive value of whole or dehulled lupins for broilers and layers,
reducing high water intake and wet droppings, and increasing inclusion rate of whole or
dehulled lupins from the current limit of 7% to 20 or 30% in the diet. These strategies are:
1) To determine the optimal dose of PG;
2) To break down cell walls by mechanical-thermal process plus PG;
3) To break down pectins by a combination of PG and pectin methyl esterase.
The rationale behind these strategies is outlined below.
24
2.10 Determining the optimal dose of PG
Supplementing poultry diets with an inappropriate dose of enzymes produces variable
results. For example, if an enzyme is supplemented below the required dose, it may induce
little or no breakdown of substrate and consequently may not improve growth performance.
This may explain one of the reasons for failure of enzyme supplementation for improving
the growth, feed conversion efficiency or metabolisable energy of the diet for broilers or
layers (White et al, 1981; Hesselman et al, 1982; Ritz et al, 1995; Igbasan & Guenter,
1997; Daveby et al, 1998). At the same time, if the enzyme dose exceeds the required dose,
it will not only be costly, but also may not produce further improvements, and can even
reduce feed intake and feed conversion efficiency (Moran & McGinnis 1968; Friesen et al,
1991; Bedford & Classen, 1992).
As a result, it is essential to determine an optimal and economic dose of enzyme. Ali (2001,
unpublished results) conducted a dose-response experiment to determine the optimal dose
of PG for broilers fed 10 and 20% whole and dehulled lupin-based diets. Briefly, a dose of
0.8g PG (1400 units)/kg diet was found to be optimal because it reduced the viscosity of
digesta by 12%, reduced the water intake by 5% and wet droppings by 4%, and was better
than other doses tested (0, 0.4, 0.6 and 1.0g/kg diet). Additionally, it increased digestibility
by 9%, growth by 7%, feed conversion efficiency by 5%, and metabolisable energy by 4%
for broilers fed 10% whole and dehulled lupin-based diets. Strangely, the optimal dose of
PG could not break down more than 11% the glycosidic bonds of pectin, and the level of
wet droppings remained high (70%). In addition, while PG had no positive effects in diets
containing 20% lupins, birds showed poorer growth performance and had higher wet
droppings compared to those fed diets with 10% lupins. This indicated an inability of PG to
induce beneficial effects at high inclusion rates of whole or dehulled lupins in broiler diets.
This was likely to be caused by two factors: complexity of cell-wall lattices, in particular
pectin, and the presence of the methyl ester radicals in the pectin chains. The mechanisms
and potential strategies for overcoming these factors led to the next two strategies.
25
2.11 Breakdown of complex cell walls by mechanical-thermal processing plus PG
Polygalacturonase cannot break down the pectin chain of lupins by more than 11%
probably because pectins occur within a complex cell-wall network in lupins (Brillouet,
1984; Carre et al, 1985; Cheung, 1991; Evans et al, 1993; Evans, 1994; Harris & Smith,
2006). One way to increase the response to PG is to destroy the complex cell-wall lattices
outside the digestive tract of the bird by subjecting lupins to a mechanical-heat treatment.
This should increase the surface area available for PG to degrade the pectins in cell walls,
and lead to a great reduction of viscosity and water-holding capacity.
Another advantage of this approach is that pre-destruction of cell walls will allow the
endogenous enzymes of the bird itself to better meet their target substrates, hence helping
the bird to digest the nutrients more efficiently. The overall improvements could benefit
feed manufacturers and poultry producers because they will be able to include more than
10% whole or dehulled lupins in broiler and egg layer diets without compromising
production performance or increasing the incidence of wet droppings.
A mechanical-thermal treatment such as extrusion is effective at breaking down cell walls
but, because of the extreme pressures and low throughput, it is a high-cost operation and is
unlikely to become economic. A better alternative is expansion, often called low-cost
extrusion and, if it has a similar efficiency to extrusion for breaking down cell-wall
networks, it is likely to be cost effective. Expansion may have other benefits besides
making PG more effective at breaking down cell walls of lupins. It may destroy anti-
nutritional factors. There are at least nine anti-nutritional factors (alkaloids, saponins,
tannins, trypsin and chymotrypsin inhibitors, phytate, glycosyl flavanol, oligosaccharides
and uric acid) in lupins that may reduce feed intake and possibly digestion (Mercedes et al,
1993; Sipsas, 1994; Petterson & Mackintosh, 1997). These anti-nutritional factors should
be destroyed by heat, pressure and shear force of the expander, a beneficial outcome if
lupins are to be used at high inclusion rates (20%) in broiler diets. Furthermore, expansion
is a less aggressive process so it is less likely to damage heat-sensitive nutrients such as
vitamins (A, C, D, E, thiamine and folic acid) and amino acids than pelleting and extrusion
(Pipa & Frank, 1989; Coelho, 1994; Leeson & Summers, 1997). It is possible to expand
lupins despite its low content of starch by modifying the processing method, such as
26
changing the configuration of the screw setup and conditioning system in the expanded
barrel and changing the particle size of expanded lupins at the outlet of the expander (Ross
Maas, 2004; Department of Agriculture and Food, Western Australia, pers. comm.).
It is worthwhile to note that expansion or mechanical-heat processing of feed, in the
absence of supplemental enzymes, will break down cell-wall networks but produce little or
no benefit for broilers or layers. This is because the heat of these processes can increase the
viscosity of digesta and increase wet droppings because of solubilisation of the insoluble
fibres of cell-wall polysaccharides (Fadel et al, 1988; Bishop, 1989; Armstrong, 1993;
Vukic-Vranjes et al, 1994,1995; Fancher et al, 1996; Fasina et al, 1997; Liebert & Wecke,
1998). For lupins fed to layers, extrusion alone did not improve feed conversion efficiency
and egg yield or quality, yet slightly increased wet droppings (Watkins & Mirosh, 1987;
Bishop, 1989). Similarly, expansion lowered the glucosinolate (anti-nutritive factor)
content of canola but did not improve growth performance or metabolisable energy of diets
over the unprocessed canola for broilers (Fasina et al, 1997). Other studies also found no
effect of expansion on growth, feed conversion efficiency and metabolisable energy of diets
for broilers (Liebert, 1995; Douglas & Parsons, 2000).
On the other hand, enzyme supplementation of broiler diets without expansion treatment
produces improvements but they are smaller than those produced by a combination of
enzyme and expansion (Fancher et al, 1996; Fasina et al, 1997; Scott et al, 1997; Liebert &
Wecke, 1998). These findings lead to the conclusion that expansion and enzymes act
synergistically in breaking down the cell-wall constituents, reducing viscosity of digesta
and wet droppings, increasing growth rate and feed conversion efficiency of broilers and
increasing metabolisable energy of diets.
2.12 Breakdown of pectins by combination of PG and pectin methyl esterase
Methyl esters limit PG action during breakdown of pectin
Pectins are made up of repeating units of galacturonic acid joined together by glycosidic
bonds between carbons 1 and 4. On carbon 6, there are methyl ester radicals that form cross
links with neighbouring polymers of pectin via divalent ions such as Ca++ or Mg++ (Fig.
27
2.5), and also cross link with polymers of other sugars as side chains, for example,
arabinose, galactose and rhamnose (Fig. 2.4).
Figure 2.5 Cross links of pectic polymers with neighbouring polymers via methyl ester and Ca++ ions.
These methyl esters protect the glycosidic bonds that join the galacturonic acid units
together and therefore make pectin difficult to break down. For example, when pectins are
treated with PG, only 11% of the glycosidic bonds are broken down (Jansen &
MacDonnell, 1945ab; Endo, 1964ab; English et al, 1972; Rombouts & Pilnik, 1980; Kollar,
1998; Ali et al, 2001). Methyl esters block many of the binding sites of PG in two ways.
First, the methyl esters decrease breakdown of pectin by lowering the affinity of PG to its
substrate molecules by blocking the binding sites of PG to the glycosidic bonds (Rexova-
Benkova et al, 1977; Rombouts & Thibault, 1986). Second, PG requires at least two
adjacent carboxyl groups free of methyl esters in order to break down the glycosidic bonds
(McCready & Seegmiller, 1954; Watkins, 1964; Thibault & Mercier, 1978; Rombouts &
Pilnik, 1980; Rombouts & Thibault, 1986). Thus, breakdown activity of PG decreases with
increasing degree of methyl esterification of pectin (Pilnik et al, 1973; Rombouts & Pilnik,
1980; Christgau et al, 1996).
Consequently, while PG breaks down some of the pectin, the breakdown is limited. This is
the case for lupins, since lupin kernels contain 11% pectins within the cell-wall lattices and
the majority of these, between 80-90%, of the galacturonic acid units contain methyl esters
(Konovalov et al, 1999).
28
Methyl esters reduce growth performance and increase wet droppings of poultry
The methyl esters not only protect the glycosidic bonds from breakdown by PG but,
through their cross links with neighbouring polymers, are directly responsible for several
properties of pectin: high water-holding capacity, viscosity, gel formation, molecular
weight, and long chains (Northcote, 1958; Grant et al, 1973; Jarvis, 1984; Pilnik et al,
1973; Fishman et al, 1984; BeMiller, 1986; May, 1990; Willats et al, 2001). The greater the
number of methyl esters, the higher the water-holding capacity, gel formation and viscosity
of pectin, leading to high water intake and an increase in wet droppings in broilers and
layers. A high viscosity of pectin is implicated in poor digestion of nutrients and,
consequently, depressed weight gain and feed utilization (Vohra & Kratzer, 1964; Erdman
et al, 1986; Langhout & Schutte, 1996; Langhout et al, 1999, 2000). In fact, Langhout &
Schutte, (1996) and Langhout et al, (1999, 2000) found that supplementing broiler diets
with only a small amount of methyl-ester pectin, 1.5-3%, increased water-binding capacity
by 42%, water intake by 12% and viscosity of digesta by 74%, and depressed weight gain
by 29% and efficiency of feed conversion by 22%.
The anti-nutritive effects of methyl esters are not only restricted to poultry. In ruminants, a
metabolic disorder, bloat, is produced by feeding cattle fresh legumes. There is evidence to
suggest that the high methyl-ester pectin content of fresh legumes is the main factor
involved. High methyl-ester pectin binds to rumen fluids, producing a sticky and stable
foam and releasing copious amounts of gas (Conrad et al, 1958, 1960; Head, 1959; Gupta
& Nichols, 1962; Nichols & Deese, 1966; Penn et al, 1966; Chesson & John, 1982; Willats
et al, 2001). Furthermore, ruminal contents from bloated cows fed fresh alfalfa have been
found to be much more viscous than cows not fed fresh alfalfa (Nichols et al, 1957; Conrad
et al, 1958,1960; Nichols & Deese, 1966; Chesson & John, 1982).
Synergistic interaction between PG and pectin methyl esterase
Methyl ester radicals can be removed from pectin by a specific enzyme called pectin
methyl esterase (PME, EC 3.1.1.11). PME breaks down these radicals along the pectin
chain into methanol and hydrogen ions. When PME does this, many of the branches are
destroyed, leaving mainly smooth, linear chains of galacturonic acid units which are then
29
much more susceptible to attack from PG. Breakdown of glycosidic bonds by PG can be
increased by 4-10 fold after treatment of pectin with PME (Jansen & MacDonnell, 1945ab;
Demain & Phaff, 1957; Watkins, 1964; Endo, 1965a; Rexova-Benkova & Markovic, 1976;
Chesson, 1987; Christgau et al, 1996; Kollar, 1998).
There are currently more than 20 published reports that indicate a synergistic interaction
between PG and PME. These reports show that when these enzymes attack pectin together
at least one or more of the following changes to the pectin chain takes place. Viscosity,
water-holding capacity, molecular weight of pectin and chain length of pectin all decrease
more with a combination of PG and PME than they do when acted upon by PG or PME
individually (Jansen & MacDonnell, 1945b; Demain & Phaff, 1957; Watkins, 1964; Endo,
1965a; Rexova-Benkova & Markovic, 1976; Chesson, 1987; Christgau et al, 1996; Kollar,
1998). The first in vitro experiment with PG and PME (Jansen & MacDonnell, 1945ab)
tested the hypothesis that PG action depends on prior action of PME and found three
interesting results. First, addition of PME to PG increased the hydrolysis of the glycosidic
bonds of pectin over that observed in the absence of PG by 60-fold. This hydrolysis was
primarily due to the removal of methyl esters of pectin chains. Second, removing PME
from PG by acid treatment (destruction of PG activity only) of a crude pectinase
preparation decreased the breakdown of glycosidic bonds by 100-fold. Third, when PME
was used to remove all methyl esters from the pectin chains, then PG alone rapidly and
completely hydrolysed the glycosidic bonds of the pectin chains.
Examination of the literature shows that PG acts in synergy with PME for complete
breakdown of pectin in plant cell walls (Pressey & Avants, 1982) and complete breakdown
of pectin during fruit ripening (Huber, 1983; Proctor & Peng, 1989; Sethu et al, 1996) and
fruit softening (Awad & Young, 1979; Barrett & Gonzalez, 1994). This synergism is not
unique to PG and PME and has been reported for other combinations: polygalacturonase
and arabinosidase, polygalacturonase and xylanase; esterase and cellulase; esterase and
glucuronidase; esterase and xylanase, xylanase and mannanase; xylanase and arabinosidase.
In all cases, a more extensive breakdown can be achieved by a combination of enzymes
than by the action of a single enzyme (Wood & McCrea, 1979; Greve et al, 1984; Biely et
al, 1986; Lee & Forsberg, 1987; Joseleau et al, 1992; de Vries et al, 2000).
30
For lupins, our work (Ali, 1997; Ali et al, 2001) demonstrated that PG cannot break down
more than 11% the pectin chain regardless of dose. The most likely reason for this
relatively poor breakdown is the high incidence of methyl esters (80-90%, Konovalov et al,
1999). There is every chance that treatment of lupins with PME will allow PG to break
down lupin pectin much more effectively than PG on its own and, at the same time,
significantly reduce water-holding capacity and viscosity of lupins. If the combination of
these two enzymes is effective in the live bird then it may be possible to substantially
increase the lupin content of poultry diets, up to 20%, and control not only excessive water
intake and wet droppings but also increase the utilisation of nutrients, feed conversion
efficiency and metabolisable energy of the diet. Clearly, these improvements will benefit
feed manufacturers and poultry producers because they will be able to include more than
the current limit of 7% of lupins in broiler and egg layer diets without compromising
production or increasing the incidence of wet droppings.
The synergistic interaction between PG and PME should be initially tested in vitro by
incubating lupins with PG and PME at optimum pH and temperature. If successful, large-
scale experiments (in vivo) with broilers and layers can follow to quantify the benefits of
enzymatic treatment. If the positive effects are repeated in the digestive tract of birds there
is every reason to believe that lupins could become a valuable feed and included up to a
level of 20 or 30% in broiler and layer diets.
2.13 Research Objectives and Hypotheses 2.13.1 Objectives
The primary objective of this research is to provide the poultry industry with suitable
alternatives to imported, expensive soybean meal. To do this, we first need to improve the
nutritive value of locally-grown legumes such as lupins. Second, we need to reduce the
problem of wet droppings associated with lupins. Third, we need to destroy the thick cell
walls and their main anti-nutritional factor, pectic substances, in lupins to avoid the
problem associated with high viscosity and water-holding capacity. By achieving these
31
objectives, we should be able to increase the proportion of whole or dehulled lupins from
the current level of 7% to 20 or 30% in diets for both broilers and layers without significant
losses in productivity or increasing wet droppings of bird.
To meet these objectives, I conducted six experiments to evaluate the potential of specific
pectinases and expansion (mechanical-heat treatment) for breakdown of pectin and hence
the lattice that constitutes the cell walls of lupins. In the first experiment, I investigated
whether polygalacturonase (PG) would allow lupins to be better used by egg layers. I had
already established the optimal dose of PG (1400 units, 0.8g/kg diet) for broilers, so I
needed to test whether the same optimal dose applied to layers. PG is likely to be of greater
benefit in layers than broilers since the digestive system of adult hens is more developed
and mature than that of young broilers. This may allow even greater digestibility of lupins
and greater proportions of lupins to be safely included in layer than in broiler diets.
In the second experiment, I studied whether cell walls and pectins may be further degraded
by mechanical-thermal treatment such as expansion. I predicted that expansion of lupins
would enhance PG action and allow a greater breakdown of pectin through an increase in
the surface area of lupins.
In the third experiment, I tested whether we could further improve the action of PG by
using another enzyme, pectin methyl esterase (PME). I initially used in vitro techniques for
treating dehulled lupins with combination of PG and PME, before testing these enzymes in
diets for live birds. I obtained positive results as well as undesirable results with the in vitro
experiment. The combination of PG and PME substantially reduced cell walls and pectins,
but the high dose of PME tended to increase water-holding capacity and viscosity of
dehulled lupins. These undesirable effects urged me to conduct another in vitro
(Experiment 4) to determine the optimal dose of PME in combination with PG. After the
optimal dose was determined in vitro, I conducted two in vivo experiments to quantify how
the optimum combination of PG and PME would improve the nutritive value of dehulled
lupins for broilers and layers (Experiments 5 and 6).
32
2.13.2 Hypotheses
The experiments described in this thesis were designed to test the general hypothesis that
breakdown of cell walls and pectins of lupins by combination of a mechanical-thermal
process and pectinase, or by combination of PG and PME, would improve the nutritive
value of lupins for broilers and layers. Five specific hypotheses were tested:
1. Egg layers will benefit more than broilers from pectinase treatment of lupins because
their digestive tract is more developed; they will also respond better to different levels of
pectinase.
2. The expansion of lupins will improve the activity of PG by increasing the surface area
available for PG to break down the pectins in cell walls. Consequently, this will increase
the metabolisable energy content of the diet and improve the growth of broilers.
3. A combination of two pectinases, PG and PME, will break down pectin more and reduce
the water-holding capacity and viscosity of dehulled lupins more than PG on its own.
4. There is an appropriate dose of PME in combination with PG that will give a substantial
breakdown of pectin in lupins and, at the same time, reduce water-holding capacity and
viscosity of pectin.
5. The optimum combination of PME and PG added to diets containing dehulled lupins will
reduce viscosity of digesta and wet droppings and increase the growth of broilers. For
layers, the optimum combination of PME and PG is also expected to reduce the viscosity of
digesta, reduce wet droppings and soiled eggs, and increase the feed conversion efficiency.
33
Chapter 3
General Materials and Methods
To test the general hypothesis, in vitro and in vivo experiments were conducted. The
general materials and methods used in the experiments are presented below, and specific
materials, methods, treatments and details of chemical and statistical analyses are presented
in the experimental chapters where applicable.
3.1 In vitro experiments Two experiments were conducted in vitro:
Experiment 3: tested the synergistic effect of PG and PME at a single dose.
Experiment 4: tested the effects of different doses of PME in combination with PG to
determine an optimal dose.
3.1.1 Filtration rate of solution
The filtration rate was calculated by measuring the volume of supernatant filtered through
filter paper (8 µg, no. 41, Whatman) divided by the filtration time (µl/sec.). After filtration,
the solutions were taken to measure the viscosity.
3.1.2 Measurement of viscosity
The viscosity of the pectin solution was measured with a low coefficient of variation (2 –
3%) using a viscotester (HAAKE, PK 100, VT 550). Only 0.25 – 0.50 ml of supernatant
was needed for the viscosity measurement. Before measurement, the supernatant was
filtered through cheese cloth to remove any floating particles. Viscosity was determined
using a cone plate PK5 at a shear rate of 4802/sec. and speed rate of 800/min. at a
temperature of 22°C, as described in the manufacturer’s handbook. The supernatant of
samples did not exhibit Newtonian flow behaviour, i.e. shear thinning (viscosity decreases
34
with increasing shear rate). Hence, the viscosity of the solution was a function of the shear
rate.
3.1.3 Water-holding capacity
Water-holding capacity was measured according to the method described by Robertson &
Eastwood (1981ab) and modified by Armstrong et al, (1993). After the samples were
centrifuged at 15000 g for 15 min., the supernatants were removed for the viscosity
measurement as indicated earlier. They were then weighed, freeze-dried, and the dried
weights were taken. The dry material was subjected to extraction of the cell-wall materials
and pectins. Water-holding capacity was expressed as gram of water per gram of organic
matter (g:g).
3.1.4 Quantification of cell-wall materials
Two carbohydrate components, cell-wall materials and pectic substances, were analysed by
chemical, enzymatic and gravimetric methods according to the procedure of Carre et al,
(1985) with small modifications. Cell-wall materials were extracted from freeze-dried
samples as follows. Lipid and pigments of the samples were removed by successive
treatment with 5 volumes of propanol : hexane (2:3) in a crucible with a magnetic stirring
for 4 hours. The samples then were washed with 1 volume of methanol-chloroform (1:2) on
filter paper under vacuum. After air and oven drying, the defatted samples were suspended
in 200 ml of phosphate buffer (0.1 M pH 7.5) containing 0.02% streptomycin sulfate as a
bactericide, and the suspension was homogenised by a polytron. Proteolysis was then
started by addition of pronase (20 mg) of Streptomyces griseus and allowed to proceed at
30°C for 16 hours. The medium was centrifuged and the residue was retreated with pronase
(10 mg) for a further 6 hours for complete removal of protein. After centrifugation, the
residue was heated in 100 ml of water at 95°C for 5 min. to allow gelatinisation, and the
temperature was adjusted to 50°C. Amylolysis was then started by addition of α-amylase of
Bacillus lichneiformis (1.25 mg in 100 ml 0.2 M acetate buffer, pH 5.6) and allowed to
proceed for 3 hours. The residue was recovered by centrifugation, extensively washed with
35
water and dried by solvent exchange through ethanol, acetone and diethyl ether. The
resulting white powder was considered as purified, cell-wall materials.
3.1.5 Pectins
The pectic substances were extracted using the procedures described by Aspinall et al,
(1967ab) and Carre et al, (1985) with some modifications during tubing dialysis of the
solution. Extracted cell-wall materials were suspended in 2% of chelating agent, Na2EDTA
(pH 5.0), at 100°C for 4 hours. After centrifugation, the residue was retreated 3 times under
the same conditions. The supernatants from the successive extractions were pooled,
extensively dialysed against water (15 days until the conductivity reading reached 1.29
µs/cm), and precipitated by adding 2.3 volumes of ethanol. The cream-coloured fluffy
precipitate pectins were recovered by centrifugation, washed with ethanol and dried by
solvent exchange.
3.1.6 Concentrations of galacturonic acid and polygalacturonic acid
The concentrations of galacturonic acid and polygalacturonic acid (µg/g dehulled lupins)
were measured by spectrophotometric absorption using a multi-cell system with 250 µl/cell
(Beckman DU 640) at absorption of λ = 520 nm according to the method of Blumenkrantz
& Asboe-Hansen (1973), modified as described by El-Rayah & Labavitch (1977). P-
hydroxydiphenyl and sodium tetraborate + sulfuric acid were used as reagents in the assay
solution. Several concentrations of D-galacturonic acid monohydrate were dissolved in
deionised water and used as a standard (Fig. 3.1).
Polygalacturonic acid was dissolved in sulfuric acid and used as a standard to construct the
standard curve of polygalacturonic acid with 3 replicates per standard (Fig. 3.1). The only
modification made to the original procedure was reduction of the dilution volume before
adding tetraborate reagent, explained as follows. At the final stage of the preparation, the
volumes of standard and experimental samples were assigned to 12.5 ml instead of 15.5 ml
36
to produce a reproducible straight-line standard curve of valid range between 0.1 – 1.1 (Fig.
3.1).
Figure 3.1 Calibration curves of galacturonic acid and polygalacturonic acid using spectrophotometric
absorption.
3.1.7 Molecular weight of pectin
The molecular weight of pectin was determined according to the method described by Kim
et al, (2000) using high performance size exclusion chromatography (column: TSK Gel
3000PW, Tosoh Corp., Tokyo, Japan). The temperature of the column was maintained at
35°C and refractive index detector at 50. The mobile phase (0.15 M NaNO3 plus 0.02%
sodium azide) was filtered through a 0.1 µm cellulose acetate filter and degassed overnight
before use. The flow rate of the mobile phase was 0.4 ml/min. and the injection volume was
500 µl. Output voltages of refractive index were recorded to calculate the molecular weight
of pectin.
3.1.8 Pectin chain length
The pectin chain length (degree of polymerisation of polygalacturonic acids) was calculated
using the equation below based on the viscosity and molecular weight of the pectic
polymer. This was followed by the hypoiodite method for measurement of the
concentration of galacturonic acid units per pectin chain (Barash & Eyal, 1970).
37
DP =
!
"
0.49 #10-4
$
% &
'
( )
0.82
# C
100
Where DP is degree of polymerisation
η is intrinsic viscosity of polygalacturonic acids (m.Pas/sec. measured at 22°C in
250 µl), divided by the viscosity coefficient, 0.49×10-4
C is concentration in terms of galacturonic acid (µg/250 µl).
3.1.9 Methyl ester content of pectin and methanol production
The methyl ester content of pectin, and the release of methanol as a by-product of methyl
ester breakdown by PME, were determined by spectrophotometric methods according to
Wood & Siddiqui (1971) with a slight modification as follows. The methyl ester samples
(0.08 g) were weighed into tubes, into which were added 2 ml 4.0 M NaOH, and 2 ml de-
ionised water. The tubes were cooled in an ice-water bath for 20 min., held at 5°C for 4.5
hours, 5.5 ml cold 6 N H2S04 was added, and the mixture then was transferred to a 25 ml
volumetric flask and diluted to volume with water. Aliquots were centrifuged and 1.0 ml
was put into test tubes. The tubes were held in an ice-water bath for 5 min., 0.2 ml 2%
KMnO4 was added and mixed; after 15 min., 0.2 ml 0.5 M sodium arsenite (in 0.12 N
H2SO4) and 0.6 ml water were added and mixed. After 1 hour, 2 ml 0.02 M pentane-2,4-
dione (in a 50:50 mixture of 4.0 M ammonium acetate and 0.l M acetic acid) were added,
mixed, and the tubes held at room temperature for 1 hour. Absorbance at 420 nm was
measured and the concentration of methanol was calculated from a standard curve prepared
with concentrations of methanol, ranging from 0.2 to 1.1 µM. The percentage of methyl
ester content was calculated by the following formula:
Methyl ester =
!
Molar concentration of galacturonic acid g methyl ester sample
Molar concentration of methanol g methyl ester sample " 100
38
3.2 In vivo experiments with broiler and egg layer chickens
Four in vivo experiments were conducted:
Experiment 1: tested the optimal dose of PG in layer diets. Whole and dehulled lupins were
added into the diet and treated with four doses of PG, and fed to layers for 10 weeks.
Experiment 2: tested the synergistic effect of expansion and PG for broilers. Whole and
dehulled lupins were expanded, added into the diet, treated with PG and fed to broilers for
14 days.
Experiments 5 and 6: tested the optimal dose of PG+PME for broilers and layers. Dehulled
lupins were added to the diet, treated with PG and PME and fed to broilers for 14 days and
layers for 10 weeks.
3.2.1 Digestibility of dry matter and excreta moisture
The percentage digestibility of dry matter of diet (DDM) was determined using the
following formula:
DDM =
!
Feed DM intake - Excreta DM output
Feed intake " 100
Total excreta were collected by placing a plastic sheet beneath each individual cage twice
daily every two days for broilers and once every week for layers. Excreta samples were
weighed, stored at –20°C, freeze-dried and reweighed for determination of percentage
water content.
3.2.2 Apparent metabolisable energy (AME) of the diet
The freeze-dried samples of excreta and diets were ground to pass through 0.7 mm screen
to determine the content of cell walls and pectin as described earlier, and also to determine
the metabolisable energy of the diet. The excreta and feed samples were compressed into a
pellet form. The pellet was ignited using a ballistic oxygen bomb calorimeter (Gallenkamp-
CB 330) to measure the amount of energy released (kilocalorie/g sample) for calculating
the apparent metabolisable energy (AME):
39
AME =
!
(Feed intake " Diet GE) - (Excreta output " Excreta GE)
Feed intake
where GE is gross energy
3.2.3 Collection of digesta for viscosity measurement
At end of the experiments, the birds were killed by cervical dislocation, except in
Experiment 2 (expansion and PG) where the broilers were killed by carbon dioxide gassing
(Chapter 5). Intestinal digesta were collected from the ileum for the measurement of
viscosity. All digesta samples were then frozen at –20°C. The digesta were thawed, pooled
and approximately 1.5 g were centrifuged at 6000 × g at 15°C for 15 min. The supernatant
was collected for measurement of viscosity using a viscotester (HAAKE, PK 100, VT 550)
as described above (3.1.2).
3.2.4 Animal ethics
The Animal Ethic Committees (AEC) of the University of Western Australia and the
Department of Agriculture and Food, Western Australia, approved the broiler and layer
experiments (RA/3/100/296; RA/3/100/ 663) conducted at the Medina Research Station.
Health and husbandry practices complied with the Code of Practice for Poultry in Western
Australia issued by the Department of Local Government and Regional Development,
March 2003.
3.3 Statistical analysis
The design of the experiments reported in this thesis is described separately for each
experiment. The basic statistical calculations and analyses were carried out using Microsoft
Excel. Statistical analysis of the data was carried out by analysis of variance (ANOVA) or a
General Linear Model (GLM) procedure using the statistical package Genstat (Release 9.2,
Lawes Agricultural Trust, IACR Rothamsted). Transformation of the data was carried out
40
where required. Statistical significance was accepted at P < 0.05. Based on previous
experience of the known variation in each measured variable, the number of replicates per
treatment was calculated as necessary to detect a significant difference of 5% between the
treatments. If ANOVA of any measured parameter was significant statistically, differences
between two means were tested using the Least Significant Difference (LSD) or Tukey’s
Honestly Significant Difference (HSD), depending on the design of the experiment. Data in
the tables and graphs are expressed as means + standard error of the mean (sem).
41
Chapter 4
Experiment 1: The optimal dose of pectinase in lupin-based diets for
laying hens*
4.1 Introduction
Despite lupins being a rich source of protein and energy and being locally available
throughout the year, feed manufacturers in Australia are unable to incorporate more than
7% of whole or dehulled grain in egg layer diets. This is because lupins contain
considerable amounts (34%) of indigestible and complex cell-wall carbohydrates known as
non-starch polysaccharides (Evans et al, 1993; Chesson, 1993; Annison & Choct, 1993).
These polysaccharides mainly consist of pectic substances that cannot be digested because
poultry lack the specific enzymes that can hydrolyse them into simple sugars.
In addition to their poor nutritional value, the undigested pectins increase the viscosity of
the digesta in the intestine and this interferes with digestion and absorption of nutrients (see
the Literature Review). They also increase the water-holding capacity of the digesta and
increase water intake by the bird. The combined outcome is poor weight gain, low
metabolisable energy of the diet, wet droppings and dirty eggs.
However, treating lupins with pectinase, specifically polygalacturonase (PG), can reverse
most of these effects primarily by breaking down the pectin chain (Ali, 1997, 2003). A few
reports have also shown that pectinases improve the nutritional value of feed for layers but
the optimal dose has not been determined (Burnett, 1966; Patel & McGinnis, 1980). The
optimal dose of PG (0.8 g/kg diet) has been established for lupin-based diets for broilers
(Ali, 2001, unpublished) but not for layers. PG is likely to be of greater benefit in layers
than broilers because the digestive system of adult hens is more developed and mature than
that of young broilers. This may allow greater amounts of lupins to be incorporated in layer
than in broiler diets.
*A report of this study has been presented in the Australian Poultry Science Symposium, 2006, 18, 218 - 221.
42
Two hypotheses were tested in this experiment:
1. Egg layers will benefit from dietary pectinases (PG) but, because their digestive tract is
mature, the optimum level may differ from that seen in young, growing broilers (see
Chapter 2.10).
2. PG should allow feed manufacturers to use 20% whole and dehulled lupins in layer
diets without compromising production performance or the dry-litter condition of the
layers.
4.2 Materials and methods
4.2.1 Experimental Design
Whole and dehulled lupins were treated with PG (AB Enzymes GmbH, Darmstadt,
Germany) at one of four concentrations (0, 0.6, 0.8 and 1.0 g/kg diet). The effectiveness of
treatment was tested by measuring feed and water intake, feed conversion ratio,
digestibility of dry matter, apparent metabolisable energy of the diet, viscosity of digesta,
moisture content of excreta, egg yield and quality (egg weight, egg-shell weight and
thickness, yolk colour, Haugh unit and numbers of soiled eggs).
4.2.2 Housing, diet formulation, feeding and data collection
Two hundred and forty hens, 20-weeks old (brown Hy-Line), were sourced from Swan
Valley Egg Farm and housed in metabolism cages in an environmentally-controlled room
at Medina Research Station, Medina, Department of Agriculture and Food, Western
Australia. The hens were randomly distributed to individual cages (0.4 x 0.4 x 0.4 m3 per
hen). During the experimental period, the temperature was maintained at 22°C, the relative
humidity at 55% and photoperiod was controlled by providing artificial light from 04.00 to
20.00 hours. All the cages were fitted with individual feeders and drinkers. Feed and water
were supplied ad libitum throughout the experiment and the diets were fed in mash form.
43
The experimental diets were fed to the hens for 11 weeks (1 week adaptation to the cages +
10 weeks experimentation). The diets were formulated to be isocaloric and isonitrogenous
to meet all nutrient requirements of laying hens on the basis of the standards of the
Standing Committee on Agriculture, Australia (SCA, 1987, Table 4.1).
Table 4.1 Ingredient and nutrient composition of layer diets used in Experiment 1.
Ingredients Whole lupins (%) Dehulled lupins (%)
10 20 10 20
Wheat 13% CP 64.3 58.0 66.8 61.5
Lupins 10.0 20.0 10.0 20.0
Soybean meal 46% CP 4.7 3.0 3.0 1.0
Meat & bone meal 48% CP 10.4 8.0 10.1 5.5
Vegetable oil 1.3 1.9 0.5 1.05
Limestone fine 8.7 8.5 9.0 9.7
Di-calcium phosphate 0.0 0.0 0.0 0.6
Salt (iodised) 0.1 0.1 0.1 0.2
Sodium bicarbonate 0.1 0.1 0.1 0.1
DL-Methionine 0.2 0.2 0.2 0.2
Choline chloride 75% 0.1 0.1 0.1 0.1
Commercial layer premix# 0.3 0.3 0.3 0.3
Calculated analysis of dietary nutrients and apparent metabolisable energy (AME)
AME (MJ/kg)2 11.6 11.6 11.6 11.6
Crude protein 17.0 17.4 17.0 17.0
Calcium 4.3 4.0 4.4 4.3
Available phosphorus 0.5 0.4 0.5 0.4
Methionine + cysteine 0.7 0.7 0.7 0.7
Lysine 0.8 0.8 0.8 0.7
# Pectinase was added to each diet at levels 0, 0.06, 0.08 and 0.1%. Xylanase was added 0.02% to each experimental diet. Vitamin-mineral premixes provided per kg diet: Vitamin A, 9,000 IU, vitamin D3, 2,750 IU; vitamin E, 50 IU; menadione 2.5 mg, vitamin B1, 2.5 mg; vitamin B2, 6.6 mg, vitamin B12, 0.025 mg; niacin 45 mg choline chloride, 500 mg; d-pantothenic acid, 12 mg; pyridoxine, 5 mg; biotin 0.2 mg; folic acid, 2 mg; ethoxyquin, 100 mg; manganese oxide, 62.6 mg; zinc oxides, 5 mg; ferrous sulfate 68.2 mg; copper sulfate, 4.4 mg; potassium iodine, 1.1 mg sodium selenite 0.10 mg. Each hen was weighed at the start of the experiment and weekly thereafter. After the first
week of adaptation, feed and water intake, egg weight, egg-shell weight, shell thickness,
yolk colour, Haugh unit and number of soiled eggs were recorded weekly. The number of
44
eggs produced per bird was recorded daily. Feed spillage was collected to adjust feed intake
per replicate per day. Feed conversion ratio was calculated by dividing the net weight of
feed consumed by the weight of egg produced per replicate. The amount of water
evaporated from each drinker was subtracted from daily water consumption to estimate net
water intake by the bird. Egg-shell thickness was measured using an electronic micrometer
screw gauge (µm) on three points at the equatorial region of the egg, and expressed as an
average value. Yolk colour was scored against a Roche Yolk Colour Fan (F. Hoffman-La
Roche Ltd., Switzerland). Haugh units were measured by adjusting egg weight and height
of the albumen using an Ames micrometer (Model S-8400, Ames Co., Waltham, MA).
The details of measurements of viscosity of digesta, excreta moisture, digestibility of dry
matter, metabolisable energy of the diet and content of cell wall and pectin were described
in Chapter 3.
4.2.3 Enzyme inclusion in the diet
Powder pectinase (PECLYVE CP), obtained from Lyven Enzyme Industries (Caen,
France), mainly consisted of polygalacturonase (PG) activity of 2200 unit/g, with traces of
pectin methyl esterase and pectin lyase. Before it was added to the experimental diets, PG
activity was assayed using spectrophotometric absorption according to the method
described by Gross (1982). One unit of PG activity was defined as the amount of enzyme
that releases 1 µmole of D-galacturonic acid from pectin chain per minute under the assay
conditions.
Xylanase (RovabioTM xylanase Excel AP, 2000 units/g, Adisseo Asia Pacific Pte Ltd,
Singapore) was added at 0.2 g/kg to all experimental diets to break down the xylan in
wheat, the main cereal used, and to ensure there was no confounding effect of wheat xylans
on the experimental results, as suggested by the committee of Rural Industries Research
and Development Corporation (RIRDC).
45
4.2.4 Statistical Design
The experiment consisted of a 2 × 2 × 4 complete factorial arrangement of treatments with
two levels of lupins (10 and 20%), two types of lupins (whole and dehulled) and four doses
of pectinase (0, 0.6, 0.8 and 1.0 g/kg diet). Sixteen experimental treatments and 15
replicates per treatment were analysed for each measured variable. Data were analysed by
Analysis of Variance (ANOVA) using the statistical package Genstat (Release 9.2, Lawes
Agricultural Trust, IACR Rothamsted). If the ANOVA revealed significant effect of
treatments, means were separated by Tukey’s Honestly Significant Difference (HSD).
4.3 Results
4.3.1 Viscosity
PG reduced the viscosity of the digesta of hens fed all diets (10 and 20% whole and
dehulled lupins) by 11%. There were no differences in viscosity among the levels of PG
(Table 4.2).
4.3.2 Water intake and excreta moisture
PG reduced the water intake of hens on all diets by the same extent (7%). Excreta moisture
was reduced by a similar extent (6%) but this happened only at the two lower doses of PG
(0.6 and 0.8 g). When the amount of lupins in the diet was increased, excreta moisture
increased. However, PG reduced the excreta moisture of 20% lupin diets back to the same
level as the 10% lupin diets and this response was consistent for both whole and dehulled
lupins.
4.3.3 Soiled eggs
The lowest level of PG reduced soiled eggs by 8% (Table 4.2). There were no significant
interactions between enzyme dose and level of lupin inclusion.
46 Table 4.2 Responses of laying hens to lupin-based diets supplemented with PG.
PG dose Whole lupins Dehulled lupins
g/kg diet 10% 20% 10% 20%
Mean
Viscosity (m.Pas/sec.) 0 4.48 5.91 4.21 5.88 5.12a 0.6 4.02 5.28 3.77 5.29 4.59b 0.8 3.98 5.23 3.72 5.26 4.55b 1.0 3.87 5.23 3.72 5.27 4.52b s.e.m. 0.15 0.19 0.15 0.22 0.18
Water intake (ml/hen/day) 0 206 218 184 226 209a 0.6 188 203 167 209 193b 0.8 189 210 174 209 195b 1.0 192 211 169 211 195b s.e.m. 4 7 6 7 6
Excreta moisture (%)
0 72.9 75.4 69.5 73.6 72.9a 0.6 67.8 70.1 62.3 69.2 67.4b 0.8 69.4 72.8 66.6 70.7 69.9ab 1.0 69.8 73.3 67.7 71.0 70.5ab s.e.m. 1.8 1.9 2.1 2.1 2.0
Soiled eggs (%) 0 5.53 5.80 5.11 5.53 5.49a 0.6 5.07 5.35 4.59 5.21 5.05b 0.8 5.10 5.45 4.82 5.33 5.18ab 1.0 5.21 5.54 5.02 5.25 5.26ab s.e.m. 0.18 0.19 0.21 0.12 0.18
Breakdown of cell-wall polysaccharides (%)
0 1.24 1.01 1.61 1.12 1.25a 0.6 1.35 1.06 1.76 1.19 1.34b 0.8 1.36 1.08 1.78 1.19 1.35b 1.0 1.36 1.07 1.80 1.19 1.36b s.e.m. 0.04 0.04 0.05 0.04 0.04
Breakdown of pectin (%) 0 2.22 2.03 2.36 2.09 2.18a 0.6 2.68 2.36 2.93 2.47 2.61b 0.8 2.67 2.39 2.96 2.50 2.63b 1.0 2.67 2.36 2.94 2.51 2.62b s.e.m. 0.19 0.20 0.21 0.21 0.20 Length of pectin chain (number of galacturonic acid unit/pectin chain)
0 60.6 61.7 62.8 63.9 62.3a 0.6 53.8 55.1 55.6 56.9 55.4b 0.8 53.6 55.0 56.2 56.7 55.4b 1.0 53.5 55.2 55.8 56.8 55.3b s.e.m. 2.7 2.9 2.6 2.9 2.8
Means within columns with different superscripts differ significantly (P<0.05).
47
4.3.4 Breakdown of cell wall-polysaccharides and pectin The lowest dose of PG (0.6 g) increased the breakdown of cell-wall polysaccharides by 9%
and pectin by 24%, and reduced the chain length of pectin by 11%. There were no
differences in breakdown of cell-wall polysaccharides, pectin or chain length of pectin for
any of these variables among the levels of PG (Table 4.2). When the amount of lupins in
the diet was increased, the amount of breakdown of cell walls decreased by 32% and pectin
decreased by 16%. No significant interactions were observed between enzyme dose and
level of lupin inclusion.
4.3.5 Feed intake and feed conversion ratio (FCR) PG had no effect on the feed intake of hens fed lupin-based diets. Only the lowest level of
PG reduced FCR by 4% and, again, there were no interactions (Table 4.3). Table 4.3 Performance of laying hens fed lupin-based diets supplemented with PG.
PG dose Whole lupins Dehulled lupins
g/kg diet 10% 20% 10% 20%
Mean
Feed intake (g/hen/day) 0 121 122 114 119 119 0.6 119 121 112 118 118 0.8 120 121 113 118 118 1.0 119 120 113 117 118 s.e.m. 2 2 2 2
Feed conversion ratio (g feed : g egg) 0 2.02 2.04 1.89 1.95 1.97a 0.6 1.94 2.01 1.82 1.92 1.92b 0.8 1.96 2.00 1.85 1.89 1.93ab 1.0 1.96 1.99 1.87 1.90 1.93ab s.e.m. 0.03 0.02 0.02 0.02 0.02
Digestibility of dry matter (%)
0 57.4 55.4 59.6 57.7 57.5a 0.6 61.2 59.5 63.8 60.5 61.3b 0.8 60.7 57.6 60.8 59.3 59.6ab 1.0 60.0 56.8 59.4 59.0 58.8ab s.e.m. 1.7 1.8 2.2 1.4 1.8
Apparent metabolisable energy (MJ/kg DM) 0 10.4 9.7 10.8 10.1 10.4a 0.6 11.0 10.1 11.4 10.5 10.8b 0.8 10.8 10.0 11.2 10.3 10.6ab 1.0 10.8 10.0 11.1 10.3 10.5a s.e.m. 0.1 0.1 0.1 0.1 0.1
Means within columns with different superscripts differ significantly (P<0.05).
48
4.3.6 Digestibility of dry matter and apparent metabolisable energy
The lowest dose of PG (0.6 g) increased the digestibility of dry matter by 7% and increased
metabolisable energy of the diet by 4%. Both the higher doses had no effect and there were
no interactions.
4.3.7 Egg production and egg weight
The lowest level of PG (0.6 g) increased egg production by 3%. The higher levels of PG
had no significant effect.
4.3.8 Haugh unit, yolk colour and shell weight and thickness
PG had no effect on yolk colour, Haugh units or shell thickness but the lowest level
increased the weight of the egg shell by 2% (Table 4.4).
49 Table 4.4 Egg characteristics of laying hens fed lupin-based diets supplemented with PG. PG dose Whole lupins Dehulled lupins
g/kg diet 10% 20% 10% 20%
Mean
Egg production (egg/100 hens/day) 0 89.2 89.0 89.2 87.5 88.7a 0.6 91.8 91.5 91.8 89.7 91.2b 0.8 90.8 89.9 91.0 89.2 90.2ab 1.0 90.5 88.7 91.0 89.2 89.8ab s.e.m. 0.9 0.9 0.8 1.0 0.9
Egg weight (g) 0 60.3 60.4 61.0 61.4 60.8 0.6 61.5 60.9 62.2 61.9 61.6 0.8 61.6 61.0 61.3 62.4 61.6 1.0 61.4 60.9 61.1 62.0 61.3 s.e.m. 0.7 0.9 0.9 0.8 0.8
Haugh unit 0 87.3 86.7 87.7 87.8 87.4 0.6 87.5 86.8 88.1 87.7 87.5 0.8 87.5 86.9 87.2 88.1 87.4 1.0 87.3 86.8 87.1 87.6 87.2 s.e.m. 0.6 0.7 0.6 0.6 0.6
Yolk colour (Roche scale) 0 5.8 5.8 5.8 5.7 5.8 0.6 5.9 5.8 5.9 5.8 5.9 0.8 5.9 5.9 5.8 5.8 5.9 1.0 5.9 5.8 5.8 5.8 5.8 s.e.m. 0.1 0.1 0.1 0.1 0.1
Shell weight (g) 0 6.07 6.09 6.11 6.02 6.07a 0.6 6.22 6.14 6.25 6.08 6.18b 0.8 6.22 6.15 6.15 6.11 6.16ab 1.0 6.20 6.14 6.12 6.08 6.14ab s.e.m. 0.05 0.06 0.05 0.05 0.05
Shell thickness (µm)
0 358 355 358 356 357 0.6 359 357 359 356 358 0.8 359 357 356 358 358 1.0 358 356 357 357 357 s.e.m. 3 3 2 4 3
Means within columns with different superscripts differ significantly (P<0.05).
50
4.4 Discussion
The first hypothesis was accepted because treatment of diets containing whole or dehulled
lupins with PG significantly reduced water intake, wet droppings, viscosity of the digesta
and number of soiled eggs. PG had no effect on feed intake but reduced feed conversion
ratio slightly. PG also slightly improved digestibility of dry matter and metabolisable
energy of the diet. There was a small increase in egg yield and egg-shell weight but no
effect on egg weight, shell thickness, yolk colour or Haugh unit score.
The second hypothesis was also accepted because 0.6 g PG/kg diet allowed layers to handle
20% whole or dehulled lupins without compromising their production performance or
increasing their wet droppings to levels that would be considered too high. PG reduced wet
droppings (excreta moisture) of layers eating 20% lupins to levels normally seen on diets
containing 10% lupins.
By breaking down the lupin pectins, PG was able to reduce the viscosity of digesta. This
breakdown released nutrients confined within the cell-wall lattices and allowed them to be
digested and absorbed. As a consequence, digestibility of dry matter and metabolisable
energy of the diet were increased and feed conversion ratio (g feed : g egg) decreased. The
increase in egg production and egg-shell weight were most likely due to the increase in
digestibility of dry matter and metabolisable energy of the diet. The decrease in percentage
of soiled eggs was likely due to the decrease in water intake and wet droppings.
The most effective dose of those tested was the lowest one, 0.6 g PG/kg. This dose
produced all the significant results outlined above. The highest level, 1.0 g PG/kg diet, was
also effective but, in some cases, did not give the anticipated results. For example, both the
lowest and highest levels reduced viscosity and water intake significantly but it was only
the lower level that significantly reduced excreta moisture and soiled eggs. Several studies
have confirmed that low levels of enzyme outperform higher levels in both layers and
broilers (Petersen & Sauter, 1968; Patel & McGinnis, 1985; Boling et al, 2000; Lazaro et
al, 2003) but a few studies have been less conclusive (Francesch et al, 1995; Scott et al,
1999), probably due to differences in source and activity of the enzyme employed.
51
Dose level is particularly important and there is likely to be a compromise between having
sufficient breakdown of pectin to release nutrients but not so much to release large numbers
of small, indigestible molecules, mono-galacturonic acid units. For example, 0.6 g PG/kg
diet significantly reduced excreta moisture from 60.9 to 56.3% but higher doses of PG
reduced it less. The 1.0 g PG/kg diet reduced excreta moisture to 58.6%. Small units of
mono-galacturonic acid are not only poorly metabolised by poultry and other monogastrics
(Longstaff et al, 1988; Longstaff & McNab, 1986; Yule & Fuller, 1992), but they are also
hydrophilic, increasing water-holding capacity (Kertesz, 1951; Gupta, 1962; McCready,
1970; Griffiths & Kennedy, 1988; Schejter & Marcus, 1988; Sakai et al, 1993). Breakdown
of pectic chains to release excessive numbers of galacturonic acid units will increase the
osmotic pressure in the gut, increasing water in the digestive tract and leading to an
increase in wet droppings (Rick Carter, 2001, Kemin Pty Ltd, pers. comm.).
Despite the improvements, PG was less effective in reducing the water intake and wet
droppings of birds fed high (20%) inclusion rate of lupins compared to 10% inclusion.
Also, PG broke down less cell walls and pectins than anticipated when lupin content was
increased from 10 to 20% in the diet. Furthermore, PG could not break down the pectin
chains more by than 11%, regardless of the dose used. These results suggest that PG alone
has a limited ability in breaking down the pectin chains within complex cell-wall lattices.
PG may require pre-destruction of these complex lattices by a mechanical and/or thermal
process so it can further access its target glycosidic bonds. If this access could be provided,
PG might allow inclusion of 20% lupin in poultry diets without increasing water intake or
wet droppings. This concept will be investigated in next experiment.
As anticipated, layers performed better when the lupins were dehulled, but the difference
was not great. This suggests that lupin hulls do not act simply as a diluent because they are
responsible in their own right for increases in viscosity, water intake, excreta moisture and
soiled eggs. So, the dehulling process may improve the nutritive value of lupins for layers.
52
4.5 Conclusions
Four conclusions can be made from the present study:
1. PG improves the nutritive value of whole and dehulled lupins for laying hens. Of the
doses tested, 0.6 g PG/kg diet is required in a layer diet containing either 10 or 20%
whole and dehulled lupins.
2. 0.6 g PG/kg should allow feed manufacturers to include 20% lupins in layer diets. It is
possible that doses even lower than 0.6 g might elicit an even better response.
3. Increasing the dose of PG above 0.6 g/kg diet might be detrimental and increase wet
droppings and hence soiled eggs.
4. Enzyme supplementation can reduce the production costs because more lupins can be
substituted for expensive soybean meal while allowing the problem of wet droppings to
be managed.
53
Chapter 5
Experiment 2: Mechanical expansion and pectinase as treatments to
improve the nutritional value of lupins for broilers*
5.1 Introduction
Feed manufacturers and the grain industry in Western Australia are seeking a new strategy
to break down the indigestible cell-wall polysaccharides of lupins to increase their
nutritional value for poultry. Lupins are locally grown, cheap and have the potential to
become nutritionally comparable to that of imported soybean meal which is expensive.
The nutritional value of lupins can be improved by treatment with pectinase, an enzyme
that is capable of degrading the cell-wall polysaccharides. By treating lupins with pectinase,
growth performance of broilers can be increased and the incidence of wet droppings
reduced (Ali, 1997, 2003). However, the extent to which pectinase can break down the cell
walls is governed by several factors such as the complexity of the cell-wall
polysaccharides, the retention time of digesta, and extremes of pH throughout the digestive
tract that may inactivate the enzyme. As a consequence, treatment of lupins with only
pectinase can increase nutritional value but the response can be variable.
One possible way to reduce the variability and increase the response to enzyme treatment is
to subject lupins to mechanical and thermal processes such as extrusion. While extrusion
can effectively destroy the cell-wall network by shear force, pressure and temperature, the
process by itself only marginally improves the nutritional value and wet droppings remain
high (Bishop, 1989). So, for best results, extrusion must be combined with enzyme
treatment. However, extrusion is a high-cost operation because of the extreme pressures
and the low throughput, hence an alternative must be sought. Expansion is often called low-
cost extrusion and has several advantages over extrusion. It requires less energy and
maintenance input, has a large throughput of feed, and allows inclusion of high levels of fat
*A report of this study has been presented in the Australian Poultry Science Symposium, 2006, 18, 62 - 65.
54
and other liquid ingredients without compromising the quality of the pellet. Expanded feed
is easy to pellet and this less aggressive process causes less damage to heat-sensitive
vitamins and amino acids than extrusion.
Like extrusion, expansion enhances feed intake by 4%, feed conversion ratio by 3%,
nitrogen retention by 7%, and metabolisable energy by 11% of wheat- and barley-based
diets (Plavnik & Sklan, 1995; Fancher et al, 1996; Scott et al, 1997; Fasina et al, 1997).
Expansion effectively eliminates harmful microbes and destroys anti-nutritional factors
(ANFs) that exist in many feeds. This is an important factor because lupins contain toxic
fungi such as phomopsins and several ANFs, namely, alkaloids, saponins, tannins, trypsin
and chymotrypsin inhibitors, phytate, C-glycosyl flavanol, oligosaccharides and uric acid
(Sipsas, 1994; Petterson & Mackintosh, 1997). These ANFs can depress feed intake so it
would be an advantage to destroy them. Edwards & Tucek (2000) have suggested that
expansion of lupins may prove beneficial but this has yet to be investigated.
Most importantly, expansion can destroy cell-wall lattices of wheat and barley and might
do the same in lupins. For example, Chesson et al, (2002) have shown that expansion
reduces the β-glucan content of barley by 12-fold and the arabinoxylan content of wheat by
5-fold. Destruction of cell walls reduces particle size and increases the surface area of the
feed. This allows a) greater release of the digestible nutrients that were previously
encapsulated within the cell-wall matrices and makes them available to the bird’s own
digestive enzymes, b) better access for exogenous enzymes like pectinase to better target
their substrates in lupins, and c) greater breakdown of indigestible cell-wall structures
which may help to reduce ANFs. Overall, there should be a reduction in viscosity and
water-holding capacity of expanded lupins and, as a consequence, wet droppings should be
reduced while growth and metabolisable energy are improved.
If expansion was used on its own without pectinase, it could be detrimental because
viscosity and water-holding capacity might increase if too much insoluble non-starch
polysaccharide was solubilized by the heat of process (Fancher et al, 1996; Liebert &
Wecke, 1998; Chesson et al, 2002). For example, there are two reports that demonstrate
that expansion per se has little effect on improving growth and metabolisable energy (1.5 –
55
4.0%; Vest, 1996; Plavnik & Sklan, 1995), and there are several reports where expansion of
wheat, wheat bran, and barley has increased the viscosity of the digesta (Fadel et al, 1988;
Vukic-Vranjes et al, 1994; Fancher et al, 1996; Fasina et al, 1997; Liebert & Wecke,
1998).
However, where expansion has been used in combination with enzyme treatment
improvements have been forthcoming. For example, expansion + enzyme treatment (eg.
xylanase or β-glucanase) counteracted the increase in viscosity (34 vs. 14.3%) and gave
increased weight gain (7.1 vs. 4.4%), better feed conversion ratio (3.3 vs. 2.3%) and higher
metabolisable energy (11 vs. 8.5%) than either expansion or enzyme treatment on their own
(Scott et al, 1997; Liebert & Wecke, 1998). Chesson et al, (2002) reported that thermal-
mechanical treatment such as expansion perforated plant-cell walls which allowed the
release of readily digested proteins, oils and polysaccharides contained within them, and the
entry of host enzymes caused a reduction in viscosity and digestion to take place within the
cells. Therefore, for lupins, it seems that enzyme treatment must be used in concert with
expansion to counteract detrimental effects such as an increase in viscosity of digesta.
In reviewing the literature there are only two papers that show an interaction between
expansion and enzyme treatment. Complete degradation of cell-wall structures of lupins, in
particular the pectins, by combined expansion (thermal and mechanical process) and
enzymatic treatments has not been studied but deserves close examination as this may be
one way to improve the nutritional value of lupins for broilers.
The hypothesis tested in this experiment was that a combination of expansion and pectinase
has a synergistic effect on the breakdown of cell-wall polysaccharides of lupins and,
consequently, will increase the metabolisable energy content of the diet and give a
significant improvement in the growth of birds.
56
5.2 Materials and methods
5.2.1 Experimental Design
Whole and dehulled lupins were expanded, added into the diet at either 10 or 20%, treated
with and without PG (0 and 0.8 g/kg diet) and then fed to broilers for 14 days. The
effectiveness of expansion and PG and their combination were tested by measuring feed
and water intake, weight gain, feed conversion ratio, digestibility of dry matter, apparent
metabolisable energy of the diet, viscosity of digesta in the intestine, wet droppings, cell-
wall polysaccharides, and pectin.
5.2.2 Housing, diet formulation, feeding and data collection
Five hundred and seventy six mixed-sex broilers (Cobb strain) aged 3 weeks were sourced
from Inghams Pty Ltd. The birds were randomly distributed into metabolism cages (3 birds
per 0.6 x 0.5 x 0.8 m3 cell) in an environmentally-controlled room at Medina Research
Station. During the experimental period, the temperature was maintained at 24 – 26°C and
the relative humidity was kept at 55%. The lighting regime was kept at 23 hours light : 1
hour dark throughout the experiment.
The diets were fed to the birds for 18 days (4 days for adaptation to the cages and
environment plus 14 days of experimentation). The diets were formulated to be isocaloric
and isonitrogenous to meet all nutrient requirements of broiler chicks on the basis of the
standards of Standing Committee on Agriculture, Australia (SCA, 1987, Table 5.1). The
birds had free access to feed and water throughout the experiment and the diets were fed in
a pellet form using a commercial pelleting machine (Lister Junior, UK).
During pelleting, the temperature of the pelleting machine was kept below 50°C to avoid
the enzyme denaturation. The pectinase preparation consisted mainly of polygalacturon-
ase, PG, at 1,800 units/g, and was added at 0.8 g per kg diet (1400 unit/kg). PG activity was
assayed in the final, pelleted diets and found to be 1,823 units/kg diet as measured by
spectrophotometric absorption according to the method described by Gross (1982).
57
Xylanase was added to all experimental diets to break down the wheat xylan in as for egg
layers described in the previous experiment.
Table 5.1 Composition of the experimental diets for broiler chickens.
Ingredients Whole lupins (%) Dehulled lupins (%) 10 20 10 20 Wheat 11% CP 65.0 59.4 68.3 66.0 Lupins 10.0 20.0 10.0 20.0 Soybean meal 47% CP 15.8 10.5 13.6 6.2 Meat & bone meal 48% CP 3.8 3.8 4.0 3.9 Canola oil 4.4 5.3 3.1 2.8 Salt (iodised) 0.3 0.3 0.3 0.3 Sodium bicarbonate 0.35 0.35 0.35 0.35 Lysine 0.1 0.2 0.2 0.2 Choline chloride 75% 0.05 0.05 0.05 0.05 Commercial broiler premix1 0.1 0.1 0.1 0.1 Calculated analysis of dietary nutrients and apparent metabolisable energy (AME) AME (MJ/kg) 13.2 13.2 13.2 13.2 Crude protein 20.0 20.0 20.0 20.0 Calcium 0.6 0.6 0.6 0.6 Avail. phosphorus 0.3 0.3 0.4 0.4 Methionine + cysteine 0.7 0.7 0.7 0.7 Lysine 0.9 0.9 0.9 0.9
1The premix provided vit. A 8,000 IU, vit. D3 2,400 IU, vit. E 8 mg, vit. K 0.3 mg, niacin 20 mg, riboflavin 4 mg, calcium pantothenate 6 mg, vit. B12 10 µg, pyridoxine 0.5 mg, folic acid 0.5 mg, biotin 30 µg, cobalt 0.2 mg, iodine 1 mg, copper 12 mg, iron 20 mg, manganese 75 mg, selenium 0.1 mg and zinc 50 mg.
Feed intake, weight gain and water intake per replicate were recorded seven times (every 2
days) throughout the 14-day experiment. Feed and water intake was measured for the layers
described in Chapter 4.3. Feed conversion ratio was calculated by dividing the feed intake
by weight gain for each replicate. The details of measurement of viscosity, excreta
moisture, digestibility of dry matter, metabolisable energy of the diet and content of cell
wall and pectin, have been described in Chapter 3.
5.2.3 Expansion of lupins
Specifications for expansion of lupins including the configuration of the screw of the
expander were as follows. The barrel temperature of the expander was approximately
58
110°C throughout the length of the chamber (47.5 cm). The temperature at four heating and
cooling zones of the barrel was 90, 100, 110 and 120°C, respectively, and 126°C through
the 2 mm die hole (exit gap). The residence time of lupins in the expander was
approximately 30 seconds. The moisture level of the expanded lupins in the barrel was
approximately 36% due to the high water-holding capacity of lupins. The die pressure was
approximately 118 – 122 psi, speed of the screw was 175rpm and screw motor torque was
32%. Expanded lupins were immediately cooled and dried by spreading as a thin layer on a
metal table in a cooling room for 2.5 hours. After drying at 60°C, the expanded lupins were
ground and mixed with other dietary ingredients. These conditions were designed to cope
with the small amounts of starch (approximately 1%) in lupins.
5.2.4 Statistical Design
The experiment consisted of a 2 × 2 × 2 × 2 complete factorial arrangement of treatments
(no expansion or expansion, whole or dehulled lupins, 10 or 20% lupins, 0 or 0.8 g PG/kg
diet). Sixteen experimental treatments and 12 replicates per treatment were analysed for
each measured variable. Data were analysed by Analysis of Variance (ANOVA) using the
statistical package Genstat (Release 9.2, Lawes Agricultural Trust, IACR Rothamsted). If
the ANOVA of any measured variable revealed significant effect of treatments (P<0.05),
differences between two means were tested using Tukey’s Honestly Significant difference
(HSD).
5.3 Results
5.3.1. Feed intake, digestibility, weight gain, feed conversion and apparent metabolisable
energy (AME)
PG on its own had no effect on feed intake but it increased digestibility by 10%, increased
weight gain by 5% and improved feed conversion efficiency by 5% and AME by 5% across
all lupin diets. By contrast, expansion alone increased feed intake by 3% across all lupin
59
diets. But it had no effect on weight gain, digestibility, feed conversion or AME. The
combination of expansion and PG was the same as expansion on its own (Table 5.2).
Table 5.2 Influence of expansion of lupins and PG supplementation on the growth performance of broilers
(21 to 39 d post-hatch). Treatment Whole lupins Dehulled lupins
10% 20% 10% 20%
Mean
Feed intake (g/bird/day) Control 130 123 137 130 130a PG 131 124 137 131 131a Expansion 133 129 140 135 134b Expansion+PG 133 130 140 134 134b s.e.m. 1 2 1 1 1
Weight gain (g/bird/day) Control 47.3 42.3 55.7 49.6 48.7a PG 50.2 44.1 58.9 51.8 51.3b Expansion 47.4 42.7 55.7 48.9 48.7a Expansion+PG 48.7 43.2 56.7 48.7 49.3a s.e.m. 1.1 0.9 1.2 1.1 1.1
Feed conversion ratio (feed g : gain g)
Control 2.75 2.93 2.46 2.64 2.70a PG 2.61 2.81 2.34 2.53 2.57b Expansion 2.83 3.02 2.53 2.78 2.79a Expansion+PG 2.75 3.01 2.47 2.77 2.75a s.e.m. 0.03 0.04 0.03 0.04 0.04
Digestibility of dry matter (%) Control 51.7 49.6 53.5 51.6 51.6a PG 57.9 52.6 60.7 55.0 56.6b Expansion 50.1 48.3 51.6 51.0 50.3a Expansion+PG 51.3 49.4 55.0 52.2 52.0a s.e.m. 1.1 1.1 1.2 0.9 1.1
Apparent metabolisable energy (MJ/kg) Control 11.0 10.2 11.3 10.4 10.7a PG 11.6 10.6 11.9 10.8 11.2b Expansion 10.7 10.0 11.0 10.0 10.4a Expansion+PG 11.1 10.1 11.5 10.3 10.7a s.e.m. 0.2 0.3 0.2 0.2 0.2
Means within columns with different superscripts differ significantly (P<0.05).
60
Increasing lupins from 10 to 20% reduced feed intake by 4%, reduced digestibility by 5%,
reduced weight gain by 11% and reduced the efficiency of feed conversion by about 8%.
Broilers consuming dehulled lupins ate 5% more feed, digested them 5% better, grew 16%
faster and converted feed into live weight gain 10% better than broilers consuming whole
lupin diets.
5.3.3 Viscosity, water intake, excreta moisture and breakdown of cell-wall
polysaccharide (CWP) and pectin
PG on its own reduced the viscosity of digesta by 14%, water intake by 6% and excreta
moisture by 6% across all lupin diets. Expansion worked in the opposite direction and
increased viscosity by 10%, water intake by 3% and excreta moisture by 3%. So a
combination of expansion and PG cancelled each other out and the combination of the two
had little effect on any of these parameters (Table 5.3).
PG had a large effect on cell walls. It more than doubled the breakdown of cell-wall
polysaccharides and increased the breakdown of pectin 4-fold. Expansion was even more
effective than PG because it increased the breakdown of cell-wall polysaccharides by 3-
fold. However, it only increased pectin breakdown by 66%.
There was a synergistic effect of PG plus expansion on the breakdown of cell walls. When
applied together PG and expansion increased breakdown of cell-wall polysaccharides by
3.5-fold and pectin by 5.5-fold.
Increasing lupins from 10 to 20% increased viscosity by 32%, water intake by 10% and
excreta moisture by 5%. In addition, breakdown of cell-wall polysaccharides was reduced
by 20% and pectin by 28% when the proportion of lupins in the diet was increased. But PG
reduced the excreta moisture of 20% lupin diets to the same extent as diets with 10%
lupins. Dehulled lupins induced a higher viscosity (16%), increased water intake by 6% and
excreta moisture by 3% compared with whole lupins. There was also more breakdown of
cell-wall polysaccharides (26%) and pectin (5%) in dehulled lupin diets than diets with
whole lupins.
61
No interactions between the treatments were detected for any of the measures in the
experiment. Table 5.3 Response of broilers to expansion of lupins and PG supplementation. Treatment Whole lupins Dehulled lupins
10% 20% 10% 20%
Mean
Viscosity (m.Pas/sec.) Control 5.11 6.91 6.30 7.93 6.56a PG 4.08 6.42 4.73 7.45 5.67b Expansion 5.72 7.95 6.71 8.57 7.24c Expansion+PG 5.16 7.02 6.43 8.04 6.66a s.e.m. 0.2 0.2 0.2 0.2 0.2
Water intake (ml/bird/day) Control 305 330 321 350 327a PG 283 319 299 336 309b Expansion 314 342 333 363 338c Expansion+PG 311 337 328 356 333d s.e.m. 2 2 2 2 2
Excreta moisture (%)
Control 65.4 68.7 68.5 70.3 68.2a PG 60.1 66.6 62.7 68.2 64.4b Expansion 67.4 71.1 70.6 72.9 70.5a Expansion+PG 65.7 69.8 67.5 71.7 68.7a s.e.m. 1.2 1.1 1.0 1.1 1.1
Breakdown of cell-wall polysaccharides (%)
Control 3.2 2.1 4.6 3.0 3.2a PG 7.3 5.8 9.2 7.3 7.4b Expansion 9.7 7.4 12.4 9.6 9.8c Expansion+PG 10.6 9.4 13.6 10.3 11.0c s.e.m. 1.2 1.3 1.3 1.4 1.3
Breakdown of pectin (%) Control 2.5 2.2 2.5 2.3 2.4a PG 11.1 8.1 12.1 8.9 10.1b Expansion 5.3 3.0 4.6 3.1 4.0a Expansion+PG 15.0 11.2 16.3 11.2 13.4c s.e.m. 1.2 1.3 1.4 1.3 1.3
Means within columns with different superscripts differ significantly (P<0.05).
62
5.4 Discussion
The hypothesis was rejected because expansion did not increase the efficacy of the PG,
increase the growth performance or reduce wet droppings of the birds. However, expansion
was very effective at breaking down both cell walls and pectin. The question is, why wasn’t
this reflected in better efficacy of PG and in better performance of the broilers?
Expansion may have had a negative influence on the nutritive value of lupins and there are
three possibilities. First, the heat of expansion may have solubilised the insoluble fractions
of the cell-wall polysaccharides which, in turn, may have increased viscosity (Fadel et al,
1988; Vukic-Vranjes et al, 1994). Any increase in viscosity reduces digestion, growth, feed
conversion efficiency and metabolisable energy of the diet. This negative effect was more
pronounced with whole than dehulled lupins because whole lupins contain 25% fibrous
hulls of which 85-95% is insoluble fibre (Harris & Jago, 1985; Cheung, 1991; Evans et al,
1993).
Second, expanded lupins had a higher absorptive capacity for water because of the
“puffing” effect which increased porosity of the final product. Expanded lupins swell more
than unexpanded lupins and this would be expected to lead to increased water intake by the
birds and increased wet droppings.
Third, the heat generated during expansion could have damaged some of the heat-sensitive
nutrients such as lysine, due to the Maillard reaction. For example, Fadel et al, (1988)
found extrusion increased Maillard products by 63% in barley-extruded diets fed to pigs. In
addition, perhaps some of the endogenous enzymes (amylase and proteases) may have been
destroyed (Batterham et al, 1986ab).
There are several reports indicating that broilers fed expanded/extruded feed grow slower,
use feed less efficiently and have digesta of higher viscosity than birds fed unprocessed
feed (Adams & Naber, 1969; Vukic-Vranjes et al, 1994; Nissinen, 1994; Vukic-Vranjes &
Wenk, 1995; Edwards et al, 1999). There are some reports that indicate otherwise (Plavnik
& Sklan, 1995; Fancher et al, 1996; Scott et al, 1997; Fasina et al, 1997). In addition, some
reports suggest that a combination of extrusion/expansion and enzyme supplementation
63
does not improve broiler growth more than that of the enzyme on its own (Vukic-Vranjes et
al, 1994; Vukic-Vranjes & Wenk, 1995; Scott et al, 1997).
However, Chesson et al, (2002) believes that expanding or extruding feeds may be
beneficial. They suggest that pre-conditioning of a feedstuff before expansion significantly
lowers the viscosity compared to conditioning with water during expansion. This is an
interesting concept and might be worthy of further investigation.
PG on its own was very effective at reducing viscosity of digesta, water intake and wet
droppings. It increased the digestibility of dry matter and, as anticipated, increased growth
rate by 5% and metabolisable energy of diet by 5% but only at the 10% inclusion rate of
lupins, not the 20% rate, except for digestibility of dry matter. The reason there was no
improvement at the 20% level was that PG reduced the viscosity of digesta at 10% lupin
inclusion much more than that at 20% lupin inclusion (22 vs. 7%).
All the above enzymatic improvements were greater with inclusion of dehulled than with
whole lupins in the diet, except for water intake and wet droppings. This suggests that
dehulling improves the nutritive value of lupins with/without PG supplementation and may
reduce any detrimental effects of hulls on the growth and efficiency of feed conversion of
broilers (Ali, 1997; Hughes et al, 1998).
As with layers, increasing the inclusion rate of lupins from 10 to 20% led to increases in
viscosity, water intake and wet droppings, and also reduced growth performance, feed
conversion efficiency and the metabolisable energy of the diet, with and without PG
treatment. Breakdown of cell-wall polysaccharides and pectins was reduced. This suggests
that the complex pectin in lupins contains a large amount of methyl ester radicals
(Konovalov et al, 1999). Numerous studies suggest that these radicals not only block the
binding sites for PG on the glycosidic bonds of pectin chains, but also have a high ability to
form gels, increasing the viscosity and water-holding capacity of pectin (Jansen &
MacDonnell, 1945ab; Endo, 1964ab; English et al, 1972; Powell et al, 1982; Morris et al,
1982; Janssen & Carre, 1985; Liners & van Cutsom, 1992; Kim & Carpita, 1992; Manunza
et al, 1997,1998). In addition, methyl ester radicals appear to contribute to the anti-nutritive
64
effects of lupins because their presence of more than 3% in broiler diets leads to a reduction
of growth by 29%, feed conversion efficiency by 22% and dietary metabolisable energy by
13% (Vohra & Kratzer, 1964; Erdman et al, 1986; Langhout & Schutte, 1996; Langhout et
al, 1999, 2000). For these reasons, the methyl ester radicals must be removed. This
hypothesis was tested in the next in vitro experiment and may also be worth testing in
broilers.
5.5 Conclusions
1. There does not appear to be any synergistic interaction between PG and expansion as
hypothesised. Although expansion seems to break down cell walls this benefit is not
translated into improved growth rate. Perhaps expansion exerts negative effects on
nutritive value such as increased viscosity, water-holding capacity and Maillard
reactions
2. PG on its own can improve the nutritive value of lupins for broilers and reduce wet
droppings, particularly in diets containing 10% whole or dehulled lupins.
3. PG seems unable to increase weight gain when lupins make up 20% of the diet.
However they can lower wet droppings to levels seen in birds consuming 10% lupins.
4. Dehulling improves the nutritive value of lupins and growth performance and efficiency
of feed conversion for broilers.
5. Enzymatic treatment by PG alone can lift the level of lupins from the current 5% in
diets to 10% without increasing wet droppings. This level can be lifted to 20% without
much loss in productivity if it is considered that a suitable level of wet droppings is that
seen at the10% inclusion rate.
65
Chapter 6
Experiment 3: Substantial breakdown of lupin pectin by pectinases
in vitro* 6.1 Introduction
A major stumbling block for the degradation of cell walls by any pectinase is the existence
of pectin methyl ester radicals attached to the carbon 6 atom of galacturonic acid units
along the pectin chain. The pectinase, polygalacturonase (PG), specifically targets the
glycosidic bonds that join the galacturonic acid units together. But when cell walls are
treated with PG alone, only 11% of the bonds are broken because the methyl ester radicals
block the binding sites of PG to the glycosidic bonds along the pectin chain (Endo, 1964ab;
English et al, 1972; Christgau et al, 1996; Kollar, 1998; Ali et al, 2001).
In addition, the methyl esters through their cross links with neighbouring polymers via
divalent ions such as Ca++ and Mg++ are directly responsible for the properties of water-
holding capacity and viscosity (Northcote, 1958; Grant et al, 1973; Jarvis, 1984). Increased
water-holding capacity is the main cause for an increase in water intake and hence an
increase in wet droppings. Higher viscosity is also undesirable as it is implicated in poor
digestion of nutrients and consequently, depressed weight gain and poor feed utilization
(Erdman et al, 1986; Langhout & Schutte, 1996; Langhout et al, 1999, 2000).
However, these radicals can be removed by a specific enzyme, pectin methyl esterase
(PME) which strips off these radicals along the pectin chain and hydrolyses them into
methanol and hydrogen ions. When PME does this, many of the branches are destroyed,
leaving mainly smooth, linear chains of galacturonic acid units that are 4 to 10 times more
susceptible to attack from PG (Jansen & McDonnell, 1945ab; Endo, 1961ab; Christgau et
al, 1996).
As a consequence, PME improves the hydrolytic activity of PG for complete degradation of
pectin and reduces the viscosity and water-holding capacity of feedstuffs containing pectin.
*A report of this study has been presented in the Australian Poultry Science Symposium, 2005, 17, 219 - 222.
66
Lupins seems a particularly good target for the action of PME because lupin kernel contains
8 – 11% pectin within cell-wall lattices, and the majority of this, between 80 – 90%, is
methyl esterified (Konovalov et al, 1999).
Therefore, a hypothesis was tested that incubation of dehulled lupins with a combination of
two pectinases, PG and PME, should give a more complete breakdown of pectin and
reduction of water-holding capacity and viscosity than PG on its own.
6.2 Materials and methods
6.2.1 Experimental Design
Dehulled lupins were incubated with PG and PME. The extent of breakdown of pectin was
estimated by measuring the changes in viscosity, water-holding capacity, filtration rate,
content of cell wall, content of pectin and its molecular weight, chain length and methyl
ester, and methanol production.
6.2.2 Incubation of dehulled lupins with PG and PME
One hundred grams of dehulled lupin grain (Lupinus angustifolius, var. Quilinock) were
incubated with 1,400 units of either PG, PME (AB Enzymes GmbH, Darmstadt, Germany),
PG+PME or without enzyme. The incubation mixture was prepared by dissolving dehulled
lupin flour in 700 ml acid buffer (420 g citric acid + 2800 ml 1M NaOH + 2000 ml 1M
CaCl2, pH = 3.9) according to the assay method described by the enzyme manufacturer.
The pH was adjusted by addition of citric acid (0.1 M). The mixture was incubated at 38°C
and shaken at 150 rotations per min. for one hour. All samples were centrifuged at 15000 g
for 10 min. at 20°C for complete precipitation. The supernatant was collected and measured
for viscosity, water-holding capacity, pH, and filtration rate. Before measurement, the
supernatants were filtered through cheese cloth and the fresh weights of filtrates were
recorded before freeze-drying for determination of water-holding capacity.
67
6.2.3 PG and PME activity assays
PG and PME activities were measured by spectrophotometric absorption according to the
methods described by Gross (1982) and Hagerman & Austin (1986). One unit of PG or
PME activity was defined as the amount of enzyme that releases 1 µmole of D-galacturonic
acid or methyl ester from polygalacturonic acid units of pectin chain per minute under the
assay conditions.
6.2.4 Statistical Design
Analyses of the data were carried out by using an ANOVA in a complete randomised
design (PG, PME, control (no enzyme) and PG+PME). Twelve replicates per treatment
were analysed for each measured variable using statistical package Genstat (Release 9.2,
Lawes Agricultural Trust, IACR Rothamsted). If ANOVA of any treatment was statistically
significant (P<0.05), orthogonal contrasts were used to compare the differences among
their means as follows: 1) control vs. enzymes, and 2) PG+PME vs. PG and PME.
6.3 Results
The combination of enzymes, PG+PME, reduced cell walls by 27%, pectin nearly 50%,
molecular weight of pectin by 56% and the length of pectin chains by 65% more than PG,
PME and control treatments. Also, the combination reduced methyl esters by a similar
amount (64%) and increased methanol by 116%. This combination reduced viscosity by
7% and water-holding capacity by 15% (Table 6.1).
When PME was used alone, methyl esters were reduced and methanol was increased more
than the PG and control. However, PME increased both the viscosity and water-holding
capacity and decreased filtration rate of the supernatant (Table 6.1).
68
PG used alone induced similar changes compared with the combination of pectinases but
the extent of change was much less with the exception of viscosity and filtration rate. PG
had no effect on methanol or methyl esters.
Table 6.1 Effect of PG, PME and PG+PME on physical-chemical properties of dehulled lupins in vitro
(mean+sem).
#Least significant differences (LSD) were applied to the single degree of freedom orthogonal contrasts at the 5% level of probability as follows: 1) control vs enzymes, and 2) PG and PME vs PG+PME. §Number of galacturonic acid units per pectin chain. 6.4 Discussion The evidence unequivocally supported the hypothesis because the combination of
pectinases was superior to either pectinase alone. PG and PME together were very effective
at breaking down pectin (50% reduction) and reducing cell walls (27%). Using just PG
alone, we expect a breakdown of between 10 and 20%. Other parameters measured
confirmed the effectiveness of the combination of enzymes. For example, molecular weight
of pectin was more than halved and the length of pectin chains was reduced 3-fold. As
anticipated, the methyl esters on the pectin were reduced by a similar magnitude (3-fold)
and they were converted to methanol which more than doubled.
Surprisingly, the breakdown of pectins and cell walls by PG+PME did not change filtration
rate and the reductions in viscosity and water-holding capacity were smaller than
anticipated. There are at least four possible reasons. First, removal of methyl esters from
Parameters Control PG PME PG+PME LSD#
Cell-wall polysaccharides (% DM) 23.4±0.9 20.3±1.2 23.5±1.0 17.1±0.9 2.8
Pectin (% DM) 10.9±0.8 8.6 ±0.3 9.1±1.0 5.8±0.3 1.9
MW of pectin (kilo daltons) 135±5 101±5 128±6 59±3 14
Length of pectin chain§ 63.0±2.8 56.1±2.6 61.2±2.2 22.3±1.6 6.7
Methanol (µg/ml incubation media) 8.8±0.8 9.6±0.8 14.6±1.5 19.0±1.8 3.7
Methyl esters of pectin chain (%) 20.1±0.9 18.6±1.0 9.7±0.6 7.3±0.5 2.2
Viscosity (m.Pas/sec.) 1.69±0.01 1.48±0.01 1.91±0.03 1.57±0.03 0.09
Water-holding capacity (g:g) 3.56±0.17 3.19±0.15 4.10±0.21 3.04±0.13 0.48
Filtration rate (µl/sec.) 69.4±2.6 79.0±3.6 47.0±1.6 63.2±2.7 7.9
69
the cell-wall matrix by PME gives more non-methylated galacturonic acid chains and these
can be cross-linked via their carboxyl groups in the presence of divalent ions such as Ca++
and Mg++. In turn this leads to gel formation and an increase in water-holding capacity and
viscosity (Penn et al, 1966; Nichols & Deese, 1966; Gupta & Nichols, 1962; Willats et al,
2001).
Second, PME itself requires large amounts of water to convert methyl esters (CH3COO)
into methanol (CH3OH) and H+. This also increases water-holding capacity of the pectin.
Third, PME converts insoluble to soluble pectin, causing the coagulation of soluble pectin
to form a gel. This leads to a large retention of water for formation of the gel network
(Kertesz, 1951; Oi & Satomura, 1965; Ben-Arie & Lavee, 1971).
Fourth, the result could be dose dependent. Just enough PME is required to expose the right
number of sites on pectin so that PG can break down the maximum number of glycosidic
bonds. Too much PME could be detrimental if it produces too many radicals because the
radicals themselves are responsible for water-holding capacity (Endo, 1964ab; Pressey &
Avants, 1982). Therefore, had we chosen a lower dose of PME, the reductions in viscosity
and water-holding capacity may have been greater because the production of methyl ester
radicals may have been less. Before this in vitro work is tested in broilers the appropriate
combination of PG and PME needs to be determined more precisely. This is the topic of
Chapter 7.
The combination of PG and PME used did not achieve complete breakdown of pectin
which was contrary to expectations. There is a PME that can be extracted from plants and it
removes blockwise nearly all of the methyl esters from the pectin chain. The common PME
comes from microorganisms and this only removes a portion of the radicals because it acts
randomly (Sajjaanantakul & Pitifer, 1991; Kester et al, 2000; van Alebeek et al, 2000). We
used the commercial PME from microbial origin which can only hydrolyse about half of
the methyl esters. Complete breakdown of pectin requires other pectinases such as exo-PG,
pectin lyase and rhamnogalacturonase for cleavage of the terminal, methylated and
70
rhamnogalacturonan glycosidic bonds of the pectin chain, respectively (Rombouts &
Pilnik, 1980; Sakai et al, 1993).
6.5 Conclusions
1. The two pectinases are much more effective than a single pectinase for breaking down
cell walls. First, PME is required to remove the methyl ester radicals to expose the
glycosidic bonds of pectin. Second, PG is then required to break down the glycosidic
bonds to destroy the pectin lattice.
2. This synergistic action between PME and PG could allow greater inclusion of dehulled
lupins, up to 20%, into broiler diets without an increase in wet droppings.
71
Chapter 7
Experiment 4: Optimum dose of pectinases for substantial breakdown of
legumes in vitro* 7.1 Introduction A complete breakdown of pectin in plant cell walls requires a combination of pectinases,
polygalacturonase (PG) and pectin methyl esterase (PME). PME removes the methyl ester
radicals attached to the carbon 6 atom of galacturonic acid units of the pectin chain
(Kertesz, 1951; Oi & Satomura, 1965; Kester et al, 2000). When these radicals are
removed, PG can attack the glycosidic bonds and break down the main pectin chain. Either
enzyme used on its own is not effective.
Our recent in vitro work has demonstrated that PG (1400 units/g) used on its own breaks
down about 11% of the cell walls in dehulled lupins. Importantly, a combination of PG plus
PME gave a much greater breakdown of cell walls (27%), pectin (50%) and length of
pectin chains (65%) (Ali et al, 2005). However, the particular combination of enzymes
used did not reduce viscosity or water-holding capacity any further than when PG was used
on its own. We think this was because the dose of PME was too high (1400 units). High
doses of PME attract large volumes of water to convert insoluble to soluble pectins. In
addition, high doses of PME produce too many hydrophilic methyl ester radicals, thereby
leading to increased viscosity and water-holding capacity (Kertesz, 1951; Oi & Satomura,
1965; Ben-Arie & Lavee, 1971). High viscosity and water-holding capacity are detrimental
for poultry as they depress feed utilisation and weight gain and increase wet droppings,
particularly in birds fed dehulled lupins (Ali et al, 2006ab).
If the combination of enzymes is to be used commercially, a precise dose of PME in
combination with PG must be determined so that we can achieve a substantial breakdown
of cell walls and, at the same time, keep viscosity and water-holding capacity to minimal
levels.
*A report of this study has been presented in the Australian Poultry Science Symposium, 2009, 20, 45.
72
Lupins, lathyrus, field peas and faba beans are locally-grown legumes that have
considerable potential for use in the Australian poultry industry because potentially, they
could supply energy and protein. However, all legumes are limited in their amounts that can
be added to a diet because they have high water-holding capacity which leads to wet
droppings. If they can be treated with enzymes which break down pectins, water-holding
capacity might be reduced which will reduce wet droppings. There are also likely to be
additional benefits of reduced viscosity of digesta and increased release of nutrients which
will stimulate growth and improve feed conversion ratio.
The main objective of this experiment was to test a hypothesis that there is an appropriate
dose of PME (less than 1400 units) in combination with PG (1400 units) that will give a
substantial breakdown of pectin in legumes and, at the same time, reduce water-holding
capacity and viscosity.
7.2 Materials and methods
7.2.1 Experimental design
Dehulled lupins, lathyrus, faba bean and field pea were incubated with six doses of PME in
combination with PG. The extent of breakdown of pectins and the extent of reduction of
water-holding capacity and viscosity were estimated by measuring the changes in water-
holding capacity, viscosity, filtration rate, content of cell wall, content of pectin and its
molecular weight, chain length and methyl ester, and methanol production.
7.2.2 Incubation of legumes with PME and PG
One hundred grams of dehulled lupins of narrow leaf (Lupinus angustifolius, var.
Quilinock) and lathyrus (Lathyrus cicera, var. Chalus) flours were incubated with 1400
units of PG and six doses of PME (0, 200, 400, 600, 800 and 1000 units) for 1 hour in the
acid media. Faba bean (Vicia faba var. Fiord) and field pea (Pisum sativum var. Kaspa)
were used as control legumes. The details of the incubation mixture, analytical procedures
and the measurements made are described in the previous chapter.
73
7.2.3 Statistical design
The Analysis of variance (ANOVA) was performed by the General Linear Model (GLM)
procedure using statistical package Genstat (Release 9.2, Lawes Agricultural Trust, IACR
Rothamsted). Twelve replicates per treatment were analysed for each measured parameter.
If ANOVA of any measured parameter was statistically significant (P<0.05), differences
between two means were tested using the Least Significant Difference (LSD).
7.3 Results
The lowest dose of PME (200 units/g) in combination with PG (1400 units/g) not only
broke down cells walls by 27% but also reduced both water-holding capacity (14%) and
viscosity (18%) of dehulled lupins. For lathyrus, the response was less. Cell walls were
broken down by 17% and water-holding capacity was reduced by 11% and viscosity by 7%
(Fig. 7.1, 7.7 and 7.8).
The breakdown of cell walls was indicated by all parameters measured. For example, for
dehulled lupins, pectin was reduced by 37% (Fig. 7.2), molecular weight by 53% (Fig. 7.3),
chain length of pectin by 63% (Fig. 7.4) and methyl esters by 52% (Fig. 7.5). Methanol was
increased by 38% (Fig. 7.6). The response for lathyrus was similar but the changes were
smaller (Fig. 7.3, 7.4, 7.5 and 7.6). There was no additional benefit achieved by increasing
the concentration of PME from 200 up to 1000 units.
As alluded to above, the lowest level of PME (200 units) in combination with PG (1400
units/g) reduced viscosity and water-holding capacity but, as the level increased up to 1000
units, these parameters were increased (Fig. 7.7, 7.8 and 7.9).
74
7.4 Discussion
The lowest dose of PME (200 units) we used in combination with PG (1400 units) gave a
satisfactory outcome because there was a substantial breakdown of cell walls and pectin
and a substantial reduction in both water-holding capacity and viscosity. For example in
dehulled lupins, cell walls were reduced by 27%, water-holding capacity by 14% and
viscosity by 18%. Higher doses of PME (above 200 units) + PG (1400 units) increased both
viscosity and water-holding capacity. We didn’t fully test our hypothesis because we didn’t
test a dose of PME of less than 200 units. We thought that 200 units would be the minimum
dose required to achieve a worthwhile breakdown of cell walls. In hindsight, it now seems
as though doses below 200 units should be tested to determine the optimal dose.
A 27% breakdown of cell walls of dehulled lupins using 200 units of PME and 1400 units
of PG was a substantial breakdown and was similar to that achieved in our previous work
where we used equal amounts of PME (1400 units) and PG (1400 units) (Ali et al, 2005).
However, what was most important was that by reducing the amount of PME, viscosity and
water-holding capacity were also reduced substantially while the extent of cell-wall
breakdown was maintained. In our previous in vitro work (Ali et al, 2005) excellent
breakdown of cell walls was achieved but viscosity and water-holding capacity remained
high and at an unacceptable level. The results of this experiment clearly show that too much
PME causes excessive breakdown of methyl esters and keeps water-holding capacity and
viscosity high.
Apart from water-holding capacity and viscosity, there are other reasons to limit the amount
of PME because potentially toxic products can be generated. As the dose of PME increased,
the number of free methyl ester radicals increased and these were converted to the
breakdown product, methanol. For example, methanol increased by 43% as PME increased
from 200 to 1000 units. Excessive release of methanol by high doses of PME could be toxic
to the health of birds and/or detrimental to productivity.
Removal of methyl esters from pectin chains and conversion into methanol is not the only
reason for increased viscosity and water-holding capacity. Removing large numbers of
methyl esters from the pectin chain with high doses of PME produces more non-methylated
75
galacturonic acid chains. These chains cross link with the neighbouring pectic polymers via
Ca++ and Mg++ ions as alluded to in the previous chapter. In turn, these cross-links lead to
gel formation and an increase in water-holding capacity and viscosity (Penn et al, 1966;
Nichols & Deese, 1966; Gupta & Nichols, 1962; Willats et al, 2001).
Although the combination of 200 units of PME and 1400 units of PG appears to give a very
satisfactory response, further refinement may give even better results. We did not test doses
of PME below 200 units which might reduce water-holding capacity and viscosity even
further. Further investigation is warranted to determine if doses of PME of less than 200
units can reduce water-holding capacity and viscosity further and still maintain a similar
level of breakdown of cells walls.
Surprisingly, lathyrus did not respond to PME like dehulled lupins. We expected that the
proportional breakdown of cell walls would be similar for both legumes but this was not the
case. The combination of PME and PG we used broke down 27% of the cell walls in
dehulled lupins but only 17% in lathyrus. The most plausible explanation is that the
structure of the pectin chain differs between these legumes. Lupins have more methyl esters
per pectin chain than lathyrus (24 vs. 14%) and they have longer chains because there are
more galacturonic acid units per chain (65 vs. 37).
Although the response of lathyrus, faba beans and field peas to a combination of PME and
PG was less than that of dehulled lupins, perhaps half to three quarters, enzyme treatment
of these legumes still appears beneficial.
7.5 Conclusions
1. The lowest levels of pectinase tested of 200 units of PME and 1400 units of PG was
most satisfactory because it gave sufficient breakdown of cells walls and reduced both
water-holding capacity and viscosity of dehulled lupins. Responses by the other
legumes tested were less but still significant so enzyme treatment might be worthwhile.
76
2. Treatment of legumes with pectinases could lead to a considerable increase in the
amount of energy and protein for poultry and allow much higher inclusion rates in
poultry diets, perhaps to levels of 30% without compromising productivity or
increasing wet droppings.
77
78
79
80
81
Chapter 8
Experiment 5: Pectinases for broiler diets based on dehulled lupins* 8.1 Introduction If locally-grown legumes such as lupins are to become viable alternatives to animal protein
and imported soybean meal in poultry diets, four major problems need to be addressed and
overcome. First, the cell walls of these legumes, composed primarily of pectin, are complex
and difficult for the bird to degrade so that nutrients within cells are not released for
digestion. Second, the pectin in these cell walls is associated with high water-holding
capacity, a characteristic directly related to wet droppings. Third, pectins increase viscosity
of digesta, which in turn reduces digestion of nutrients. Fourth, poor digestibility of lupins
leads to more nutrients being excreted into the environment. These problems might be
solved by more effective breakdown of pectin and, because birds do not secrete pectin
enzymes of their own, exogenous pectinases added to the diet may offer potential.
Pectins can be difficult to break down because methyl esters block the glycosidic bonds
between the repeating units of galacturonic acid that make up the pectin chain (Jansen &
MacDonnell, 1945ab; Endo, 1964ab; English et al, 1972; Rexova-Benkova et al, 1977;
Rombouts & Thibault, 1986; Ali et al, 2005). Thus, when pectins are treated with
polygalacturonase (PG), only 11% of the glycosidic bonds are broken down (Ali et al,
2006b). This is thought to be caused by methyl ester radicals blocking the bonds between
the units of galacturonic acid and preventing the action of PG. Methyl ester radicals can be
removed by a specific enzyme, pectin methyl esterase (PME), destroying many of the
branches of pectin and leaving mainly smooth, linear chains of galacturonic acid units
which are then much more susceptible to attack by PG.
Methyl esters are directly responsible for the water-holding capacity and viscosity of pectin
(Kertesz, 1951; Morris et al, 1982; Powell et al, 1982; Janssen & Carre, 1985; Liners &
van Cutsom, 1992; Kim & Carpita, 1992). Increased water-holding capacity is the main
causative factor for an increase in water intake and hence an increase in wet droppings in
*A report of this study has been presented in the Australian Poultry Science Symposium, 2009, 20, 46.
82
broilers (Lee & Campbell, 1983; Erdman et al, 1986; Langhout & Schutte, 1996; Ali et al,
2006a). High viscosity is implicated in poor digestion of nutrients and, consequently,
depressed weight gain and feed utilization by broilers (Vohra & Kratzer, 1964; Erdman et
al, 1986; Longstaff & Schutte, 1996; Langhout & Schutte, 1996; Langhout et al, 1999,
2000).
In 2005, we tested this concept by treating dehulled lupins in vitro with a combination of
pectinases (PG plus PME) and found several improvements in both physical and chemical
properties (Ali et al, 2005). There was a substantial improvement in the breakdown of cell
walls but, on the negative side, water-holding capacity and viscosity were both increased.
More recently, we studied other combinations of pectinases and found that, if we reduced
the amount of PME, we could still obtain a satisfactory breakdown of cell walls and, at the
same time, reduce water-holding capacity and viscosity (Chapter 7). If the best in vitro
combination of these two enzymes (200 units of PME and 1400 units of PG) is effective in
diets for the live bird, then it may be possible to avoid the problems with water intake and
wet droppings and also substantially increase the inclusion rate of dehulled lupins in broiler
diets. This would make dehulled lupins a valuable and cheaper alternative source of protein
with which to replace animal or soybean meals.
In this experiment we tested two hypotheses:
1. A combination of PME and PG (200 units PME plus 1400 units of PG) added to diets
containing dehulled lupins will reduce viscosity of digesta and wet droppings and
increase the growth of broilers.
2. The combination of PME and PG will allow feed manufacturers to include a greater
percentage of dehulled lupins (up to 30%) in broiler diets without compromising the
production performance or increasing wet droppings.
83
8.2 Materials and methods
8.2.1 Experimental design
Dehulled lupins were added to the diet at one of four levels: 0, 10, 20 or 30%, treated with
or without PME (200 units) + PG (1400 units) and fed to broilers for 14 days. The
effectiveness of the combination of PME and PG was tested by measuring feed and water
intake, weight gain, feed conversion ratio, digestibility of dry matter, apparent
metabolisable energy of the diet, viscosity of digesta in the intestine, wet droppings, cell-
wall polysaccharides, and pectin. The methods have been described in detail in Chapter 3.
8.2.2 Housing, diet formulation and feeding
Two hundred and eighty eight, mixed-sex broilers (ROSS 308 strain), aged 3 weeks, were
randomly distributed among metabolism cages. The details of housing, diet formulation,
feeding, management, and data and sample collections have been described in Chapter 5.3.
The ingredients and calculated nutrient content of the experimental diets are shown in
Table 8.1.
Powdered pectinases were added at rates of 1,400 units of PG and 200 units of PME per kg
of diet. PG and PME activities in the final, pelleted diets were found to be 1,394 and 210
units/kg diet as measured by spectrophotometric absorption according to the methods
described by Gross (1982) and Hagerman & Austin (1986). Xylanase was added to all
experimental diets as described for the previous egg layer and broiler experiments.
8.2.3 Statistical Design
The experiment consisted of a 4 x 2 complete factorial arrangement of treatments with four
levels of dietary inclusion of dehulled lupins (0, 10, 20 and 30%) and two levels of PME +
PG (minus and plus enzyme). Twelve replicates per treatment were analysed for each
measured parameter. If the ANOVA of any measured variable revealed significant effect of
84
treatments (P<0.05), differences between two means were tested using Least Significant
Difference (LSD).
Table 8.1 Composition of the experimental diets for broiler chickens.
Dehulled lupins (%) Ingredients (%)
0 10 20 30 Wheat (11% CP) 74.4 71.2 67.6 59.8 Dehulled lupins (40% CP) 0 10 20 30 Soybean meal (46% CP) 9.7 5.2 0 0 Meat & bone meal (48% CP) 12.0 10.0 8.8 3.6 Canola oil 3.0 2.5 2.2 3.2 Limestone fine 0 0 0 0.65 Di-calcium phosphate 0 0 0.3 1.5 Salt 0.13 0.16 0.18 0.27 DL-Methionine 0.23 0.26 0.29 0.31 L-Lysine 0.12 0.19 0.26 0.30 L-Threonine 0.12 0.14 0.16 0.17 Choline chloride 0.06 0.06 0.06 0.06 Premix1 0.25 0.25 0.25 0.25 Calculated analysis of dietary nutrients and apparent metabolisable energy (AME) AME (MJ/kg) 13.0 13.0 13.0 13.0 Crude protein (%) 20 20 20 20 Calcium (%) 1.2 1.1 1.0 1.0 Available Phosphorus (%) 0.6 0.5 0.5 0.5 Methionine + cysteine (%) 0.7 0.7 0.7 0.7 Lysine (%) 1.0 1.0 1.0 1.0
1The premix contained (/g) vit. A 8,000 IU, vit. D3 2,400 IU, vit. E 8 mg, vit. K 0.3 mg, niacin 20 mg, riboflavin 4 mg, calcium pantothenate 6 mg, vit. B12 10 µg, pyridoxine 0.5 mg, folic acid 0.5 mg, biotin 30 µg, cobalt 0.2 mg, iodine 1 mg, copper 12 mg, iron 20 mg, manganese 75 mg, selenium 0.1 mg and zinc 50 mg.
8.3 Results
8.3.1 Feed intake, weight gain, feed conversion ratio, digestibility and metabolisable
energy
The combination of PME + PG changed all parameters except feed intake in diets with 10
and 20% dehulled lupins. Weight gain increased by 6%, feed conversion ratio decreased by
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6%, digestibility of dry matter increased by 15% and metabolisable energy of diet increased
by 6%. The enzymes had no effect when 30% dehulled lupins was used in the diet (Table
8.2). Table 8.2. Response of broilers to PME + PG supplementation.
Dehulled lupins (%)
0 10 20 30
Feed intake (g/bird/day) None 139 141 133 124 PME + PG 140 141 134 126 s.e.m. 5 5 3 3
Weight gain (g/bird/day) None 63.5 62.7a 58.4a 53.6 PME + PG 63.4 66.7b 61.4b 55.4 s.e.m. 1.4 1.5 1.2 1.2
Feed conversion ratio (feed g : gain g)
None 2.28 2.33a 2.34a 2.39 PME + PG 2.27 2.20b 2.23b 2.34 s.e.m. 0.07 0.05 0.04 0.05
Digestibility of dry matter (%) None 57.3 52.4a 50.1a 47.6 PME + PG 56.9 60.2b 55.5b 50.2 s.e.m. 1.2 1.3 1.3 1.3
Apparent metabolisable energy (MJ/kg) None 12.3 11.8a 11.1a 10.6 PME + PG 12.4 12.5b 11.6b 10.8 s.e.m. 0.2 0.3 0.2 0.3
a,bMeans within columns with different superscripts differ (P<0.05).
8.3.2 Viscosity, water intake, excreta moisture, cell-wall polysaccharides and pectin
PME + PG reduced the viscosity of digesta by 29%, water intake by 10% and excreta
moisture by 11%. Breakdown of cell-wall polysaccharides was increased by 2.5-fold and
pectin by 6-fold. Again, the changes were less for diets that contained 30% dehulled lupins
(Table 8.3).
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8.3.3 Proportion of dehulled lupins in diet
Increasing the proportion of dehulled lupins from 10 to 30% reduced feed intake by 11%,
reduced weight gain by 16%, increased feed conversion ratio by 5%, reduced digestibility
by 13% and reduced metabolisable energy of the diet by 12% irrespective of enzymes. In
addition, viscosity increased by 74%, water intake increased by 24% and excreta moisture
increased by 12%. Breakdown of cell-wall polysaccharides and pectin was reduced by
49%. There were no significant interactions between enzyme supplementation and level of
lupin inclusion.
Table 8.3. Response of broilers to PME + PG supplementation.
Dehulled lupins (%)
0 10 20 30
Viscosity (m.Pas/sec.) None 5.11 6.38a 7.78a 9.61a PME + PG 5.06 4.54b 6.82b 8.99b s.e.m. 0.08 0.10 0.12 0.15
Water intake (ml/bird/day) None 296 326a 353a 388 PME + PG 294 294b 332b 379 s.e.m. 4 5 5 5
Excreta moisture (%) None 62.6 67.9a 70.1a 72.8 PME + PG 63.0 60.4b 64.4b 71.0 s.e.m. 1.1 1.2 1.2 1.2
Breakdown of cell-wall polysaccharides (%) None 4.2 4.8a 3.5a 3.1a PME + PG 4.2 11.3b 6.3b 4.2b s.e.m. 0.2 0.3 0.3 0.2
Breakdown of pectin (%) None 2.3 2.5a 2.1a 1.9a PME + PG 2.3 14.9b 6.7b 3.8b s.e.m. 0.1 0.5 0.3 0.2
Means within columns with different superscripts differ (P<0.05).
87
8.4 Discussion The combination of PME and PG was highly successful at breaking down the cell walls of
dehulled lupins and, therefore, making more nutrients available to broilers. Most
importantly, wet droppings were reduced and the viscosity of the digesta was also reduced,
allowing better digestion of the released nutrients. These results fully supported our
hypothesis.
PME + PG had the greatest effects on diets containing 10% dehulled lupins. The
metabolisable energy content of the diet increased by 6% which allowed a 6% increase in
growth and increase in feed conversion efficiency. Cell walls were reduced substantially (3-
fold) and there were large reductions in viscosity (29%) and wet droppings (11%). Enzyme
treatment of diets containing 20% dehulled lupins was not as spectacular as for the diets
containing 10% lupins but, nevertheless, the changes were still substantial and very
worthwhile. Metabolisable energy was increased by 5%, growth by 5% and feed
conversion efficiency by 5%. Cells walls were reduced by 80%, viscosity by 12% and wet
droppings by 8%.
Surprisingly, the enzyme combination had much less effect than anticipated on the diet
containing 30% dehulled lupins. There were no differences in metabolisable energy, growth
or feed conversion efficiency. Cells walls were reduced by 36% and pectin by 2-fold, and
the viscosity fell 7% but the proportion of wet droppings didn’t change. Hence, our second
hypothesis was partially supported because the enzyme combination was very successful in
diets containing 10 and 20% dehulled lupins but of marginal value in diets containing 30%
dehulled lupins.
It is curious why the addition of PME and PG to diets containing 30% dehulled lupins that
broke down cell walls and pectin and reduced the viscosity of digesta but did not reduce
water intake or wet droppings. There are at least two plausible reasons. First, as the
proportion of lupins in the diet increases, so too do the non-pectic polysaccharides,
substances that also influence water-holding capacity and viscosity. Cell walls in dehulled
lupins are very complex and are made up of one third pectins and two thirds non-pectic
polysaccharides, such as cellulose and hemicellulose, galactan, arabinan, rhamno-
88
galacturonan, and xylan (Brillouet & Riochet, 1983; Carre et al, 1985; Cheung, 1991;
Cheetham et al, 1993; Harris & Smith, 2006). Pectins are soluble and increase water-
holding capacity and viscosity. The non-pectic polysaccharides and their side branches,
particularly the soluble fractions, also increase water-holding capacity and viscosity. So,
when lupins are treated with pectinases, the pectins are broken down and their influence on
water-holding capacity and viscosity is much reduced. However, as the proportion of lupins
in the diet was increased, non-pectic polysaccharides became the dominant influence on
water-holding capacity and viscosity.
A second reason why pectinases were less effective in diets with 30% lupins may involve
anti-nutritional factors that may reduce feed intake and increase water intake. For example,
as we increased the level of dehulled lupins in the diet from 10 to 30%, feed intake was
reduced by 11% and water intake rose by 24%. Any increase in water intake will increase
wet droppings (El Defrawi & Hudson, 1979; Kim & Madhusudhan, 1988; Sipsas, 1994;
Ruiz et al, 1995; Olver & Jonker, 1997; Karunajeewa & Bartlett, 1985; Perez-Escamilla et
al, 1988; Hughes et al, 1998, 2000). So it seems as though any reduction in wet droppings
arising from the action of PME + PG on pectins was opposed by an increase driven by a
higher water intake by birds eating diets with 30% dehulled lupins. Anti-nutritional factors
can be inactivated by heating but it must be carefully controlled. Overheating can increase
water intake, wet droppings and viscosity of digesta because of the solubilisation of the
insoluble fibre fraction of non-starch polysaccharides (Fancher et al, 1996; Liebert &
Wecke, 1998; Chesson et al, 2002; Ali et al, 2006a).
We found no evidence that by-products of pectin breakdown, methanol or galacturonic acid
units, had any adverse effects on growth, water consumption or mortality of broilers as has
been suggested by some researchers. No inflation of the small intestine by methanol
production was observed during collection of digesta from the ileum. The galacturonic acid
units, which cannot be metabolised by monogastrics, were probably excreted through the
digestive tract of the bird (Longstaff & McNab, 1986,1989; Longstaff et al, 1988; Yule &
Fuller, 1992). This suggests that the PME and PG can be safely used for broilers.
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8.5 Conclusions
1. The combination of PME (200 units per kg) and PG (1400 units per kg) significantly
improved the nutritive value of dehulled lupins for broilers. Growth performance is
increased and wet dropping is reduced.
2. By using PME + PG, feed manufacturers and broiler producers should be able to
include dehulled lupins up to 20% without compromising performance, or increasing
wet droppings. This should reduce feed costs substantially.
3. Although PME + PG reduces the viscosity of diets containing 30% dehulled lupins, this
benefit is not translated into improved growth rate. Research is needed to test whether
factors other than pectin, for example, non-pectin polysaccharides and anti-nutritional
factors, are coming into play at this high level of lupin inclusion and opposing the
beneficial effects of the pectinases.
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Chapter 9
Experiment 6: Pectinases for laying hens fed diets based on dehulled
lupins 9.1 Introduction As feed prices rise, the Australian feed manufacturers and egg producers need to replace
imported soybean meal or meat meal with the locally-grown lupins, a cheaper alternative
protein and energy source for egg layers. However, the proportion of lupins added to layer
diets is limited to about 7% because of poor efficiency of feed conversion, high wet
droppings and an increased number of soiled eggs. This is because lupins contain large
amounts (34%) of cell-wall polysaccharides that consist mainly of pectins (Evans et al,
1993; Chesson, 1993; Annison & Choct, 1993). Pectins increase water-holding capacity
and viscosity which, in turn, increase water intake and concomitant wet dropping of birds
(Northcote, 1958; Grant et al, 1973; Jarvis, 1984; Bishop, 1989; Evans, 1994; Carre, 1997;
Ali et al, 2001, 2005). High viscosity of digesta interferes with the digestion and absorption
of nutrients, thereby lowering growth and feed conversion efficiency of birds (Erdman et
al, 1986; Langhout & Schutte, 1996; Langhout et al, 1999, 2000).
Pectins can be broken down by polygalacturonase (PG), but the breakdown is limited to
11% because methyl ester radicals block the binding sites of PG to the glycosidic bonds of
the pectin chain (Endo, 1964ab; English et al, 1972; Rexova-Benkova et al, 1977;
Rombouts & Thibault, 1986; Ali et al, 2005). Fortunately, there is another pectinase, pectin
methyl esterase (PME), that can remove these methyl ester radicals leaving smooth, linear
pectin chains, thereby giving PG much greater access to the glycosidic bonds. However,
caution must be exercised in using too much PME as excess PME in combination with PG
may lead to increased water-holding capacity and viscosity of digesta (Endo, 1964ab;
Pressey & Avants, 1982; Ali et al, 2005).
In Chapter 7, we used an in vitro technique to determine the optimal dose of PME which, in
combination with PG, will give sufficient breakdown of cell walls and pectin in dehulled
lupins to release nutrients and, at the same time, reduce viscosity and water-holding
91
capacity. We have tested this optimal combination of PME + PG in broiler diets with up to
30% added dehulled lupins and found that it improved the nutritive value of a diet
containing 20% dehulled lupins. Growth rate of birds increased by 6%, feed conversion
efficiency by 6% and metabolisable energy of the diet by 6%. Water intake was reduced by
10% and wet droppings by 11% (Chapter 8). Despite these excellent improvements for
diets containing 20% dehulled lupins, PME + PG did not improve the performance of birds
fed 30% dehulled lupins. However, the situation might be different for egg layers because
being older they have a more developed digestive system than young broilers and might be
able to cope better with a high content of dehulled lupins in their diet. As a result, PME +
PG might allow perhaps 30% dehulled lupins to be included in egg layer diets without
depression of feed conversion efficiency or an increase in wet droppings.
The main objective of this experiment was to test two hypotheses:
1. A combination of PME and PG added to diets containing dehulled lupins would reduce
the viscosity of digesta, wet droppings and soiled eggs and increase the feed conversion
efficiency of laying hens.
2. The combination of PME and PG should allow up to 30% dehulled lupins in layer diets
without compromising production performance or increasing wet droppings or soiled
eggs.
9.2 Materials and methods
9.2.1 Experimental design
Dehulled lupins were added into the diet at one of four levels: 0, 10, 20 or 30%, treated
with or without a combination of PME and PG and fed to hens for 10 weeks. The
effectiveness of PME and PG was tested by measuring feed and water intake, weight gain,
feed conversion ratio, digestibility of dry matter, apparent metabolisable energy of the diet,
viscosity of digesta, moisture content of excreta, egg yield and quality (egg weight, shell
weight and thickness, yolk colour, Haugh unit, and numbers of soiled eggs).
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9.2.2 Housing, diet formulation and feeding
One hundred and sixty hens (18-week-old) of brown Hy-Line were sourced from Altona
Hatchery Pty Ltd and housed in metabolism cages in an environmentally-controlled room
at Medina Research Station. The details of housing, diet formulation, feeding, and data and
sample collections have been described in Chapter 4.3. The detail of enzyme inclusion rate
was described in Chapter 8.3, except in the present experiment the diets were fed in the
form of mash. The ingredient and nutrient composition of the experimental diets are shown
in table below.
9.1 Composition of the experimental diets for laying hens.
Dehulled lupins (%) Ingredients (%) 0 10 20 30
Wheat (11% CP) 71.8 69.3 63.5 54.6 Dehulled lupins (40% CP) 0 10 20 30 Soybean meal (48% CP) 8.6 2.9 0 0 Meat & bone meal (51% CP) 7.1 7.3 5.3 2.0 Canola oil 2.2 1.2 1.2 2.0 Limestone fine 8.8 8.8 9.3 9.7 Di-calcium phosphate 0 0 0 1.0 Salt 0.19 0.19 0.22 0.28 DL-Methionine 0.17 0.19 0.21 0.23 L-Lysine 0 0 0.02 0.01 L-Threonine 0.97 0 0 0 Choline chloride 0.06 0.06 0.06 0.06 Premix1 0.25 0.25 0.25 0.25 Calculated analysis of dietary nutrients and apparent metabolisable energy (AME)
AME (MJ/kg) 11.6 11.6 11.6 11.6 Crude protein (%) 17.0 17.0 17.0 17.0 Calcium (%) 4.0 4.0 4.0 4.0 Available Phosphorus (%) 0.6 0.6 0.6 0.6 Methionine + cysteine (%) 0.7 0.7 0.7 0.7 Lysine (%) 0.7 0.7 0.7 0.7
1The vitamin-mineral premix provided per kg diet: Vitamin A, 9,000 IU, vitamin D3, 2,750 IU; vitamin E, 50 IU; menadione 2.5 mg, vitamin B1, 2.5 mg; vitamin B2, 6.6 mg, vitamin B12, 0.025 mg; niacin 45 mg choline chloride, 500 mg; d-pantothenic acid, 12 mg; pyridoxine, 5 mg; biotin 0.2 mg; folic acid, 2 mg; ethoxyquin, 100 mg; manganese oxide, 62.6 mg; zinc oxides, 5 mg; ferrous sulfate 68.2 mg; copper sulfate, 4.4 mg; potassium iodine, 1.1 mg sodium selenite 0.10 mg.
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9.2.3 Collection of digesta for viscosity measurement In the middle of the experiment (at the end of Week 5), five hens per treatment were killed
by cervical dislocation to collect digesta from the ileum to measure the viscosity of digesta,
as suggested by the research committee of the Australian Egg Corporation Limited
(AECL). The remaining birds were killed by cervical dislocation for collecting the digesta
for the measurement of viscosity at the end of the experiment. Details of the analytical
procedures of viscosity samples remained same as described in Chapter 3.2.6.
9.2.4 Statistical design
The experiment consisted of a 4 x 2 complete factorial arrangement of treatments with four
levels of dietary inclusion of dehulled lupins (0, 10, 20 and 30%) and two levels of PME +
PG (minus and plus enzyme). Each experimental treatment had 15 replicates and each
replicate had one bird per cage. However, the number of replicates per treatment was
increased by an additional five birds per treatment to measure the viscosity of digesta in the
middle of the experiment for the same reason as described above. The data were analysed
by ANOVA, and if any measured parameter was statistically significant (P<0.05),
differences between two means were tested using the Least Significant Difference (LSD).
9.3 Results
9.3.1 Feed intake, feed conversion ratio, digestibility and metabolisable energy
The combination of PME and PG reduced the feed conversion ratio by 5%, and increased
the digestibility of dry matter by 11% and metabolisable energy of the diet by 8% for hens
fed diets containing 10 and 20% dehulled lupins. However, PME + PG had no effect on
feed intake. At 30% dehulled lupins, PME + PG had no effect on growth parameters except
that it reduced feed conversion ratio by 2% (Table 9.2).
94 Table 9.2 Production performance of hens fed diets based on dehulled lupins and supplemented with
PME + PG.
Dehulled lupins (%)
0 10 20 30
Feed intake (g/hen/day)
None 105 105 109 111 PME + PG 104 103 108 110 s.e.m. 3 3 3 3
Feed conversion ratio (g feed : g egg) None 1.88 1.90a 2.02a 2.09a PME + PG 1.87 1.81b 1.95b 2.04b s.e.m. 0.02 0.02 0.02 0.02
Digestibility of dry matter (%)
None 66.2 59.9a 58.3a 56.4 PME + PG 66.3 66.4b 61.8b 58.5 s.e.m. 1.6 1.5 1.6 1.5
Apparent metabolisable energy (MJ/kg DM) None 11.4 10.7a 10.1a 9.7 PME + PG 11.5 11.6b 10.7b 10.0 s.e.m. 0.2 0.2 0.2 0.2
Means within columns with different superscripts differ (P<0.05). 9.3.2 Viscosity, water intake, excreta moisture, soiled eggs, cell-wall polysaccharides and pectin PME + PG reduced the viscosity of digesta measured at the middle and end of the
experiment by 19%, water intake by 14% and excreta moisture by 15%. Breakdown of cell-
wall polysaccharides was increased by 2.6-fold and pectin by 6.5-fold for diets based on 10
and 20% dehulled lupins. Again, the changes were less for diets containing 30% dehulled
lupins and there was no effect of PME + PG on water intake or excreta moisture. There was
also no difference in the viscosity of digesta collected at the middle or end of the
experiment (Table 9.3).
95 Table 9.3 Response of hens to physical and chemical properties of diets based on dehulled lupins and
supplemented with PME + PG.
Dehulled lupins (%)
0 10 20 30
Viscosity (m.Pas/sec.) at week 5 None 3.62 4.88a 6.93a 9.56a PME + PG 3.56 3.96b 6.09b 9.08 b s.e.m. 0.05 0.05 0.09 0.12
Viscosity (m.Pas/sec.) at week 10 None 3.61 4.83a 6.92a 9.59a PME + PG 3.51 3.98b 6.13b 9.13b s.e.m. 0.02 0.04 0.04 0.05
Water intake (ml/hen/day) None 176 182a 217a 247 PME + PG 177 156b 193b 236 s.e.m. 6 7 8 6
Excreta moisture (%)
None 62.4 74.6a 79.9a 83.2 PME + PG 62.2 63.3b 71.7b 81.8 s.e.m. 0.9 1.0 0.8 0.9
Soiled egg (%) None 5.21 6.62a 7.22a 8.04a PME + PG 5.20 5.32b 6.46b 7.49b s.e.m. 0.19 0.20 0.21 0.23
Breakdown of cell-wall polysaccharides (%) None 4.4 4.8a 3.6a 2.6a PME + PG 4.5 12.6b 7.1b 3.7b s.e.m. 0.2 0.5 0.3 0.2
Breakdown of pectin (%) None 2.6a 2.8a 2.4a 2.2a PME + PG 2.6 18.2b 9.8b 5.0b s.e.m. 0.2 0.5 0.3 0.2
Means within columns with different superscripts differ (P<0.05). 9.3.3 Egg production and quality PME + PG increased egg production by 4% and egg weight and egg-shell weight by 3% for
hens fed diets containing 10 and 20% dehulled lupins. At 30% dehulled lupins, PME + PG
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increased only egg production by 3%. However, PME + PG had no effect on Haugh unit,
yolk colour or egg-shell thickness (Table 9.4).
Table 9.4 Response of egg quantity and quality to PME + PG supplementation.
Dehulled lupins (%)
0 10 20 30
Egg production (eggs/100 hens/day) None 74.6 75.2a 75.0a 74.8a PME + PG 75.1 78.0b 77.5b 76.9b s.e.m. 1.1 1.1 1.0 1.0
Egg weight (g) None 56.5 55.8a 54.6a 53.6 PME + PG 55.8 57.4b 55.8a 54.2 s.e.m. 0.5 0.5 0.5 0.5
Haugh unit
None 87.8 88.0 87.4 87.4 PME + PG 88.1 87.9 87.9 87.5 s.e.m. 0.7 0.6 0.7 0.6
Yolk colour (Roche scale)
None 5.8 5.9 5.8 5.9 PME + PG 5.8 5.8 5.8 5.8 s.e.m. 0.1 0.1 0.1 0.1
Shell weight (g) None 5.89 5.90a 5.86 5.80 PME + PG 5.88 6.06b 5.98 5.87 s.e.m. 0.05 0.05 0.06 0.07
Shell thickness (μm) None 359 359 356 352 PME + PG 358 361 358 352 s.e.m. 3 3 4 4
Means within columns with different superscripts differ (P<0.05). 9.3.4 Proportion of dehulled lupins in diet Increasing the inclusion of dehulled lupins from 10 to 30% in the diet increased feed intake
by 7%, reduced feed conversion efficiency by 11%, digestibility by 9% and metabolisable
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energy of the diet by 12% with or without enzyme treatment. In addition, the viscosity
increased by 2.2-fold, water intake by 44% and excreta moisture by 29%. Breakdown of
cell-wall polysaccharides was reduced by 59% and pectin by 47%. There was no effect on
egg production or quality. When we collected ileal samples, we saw no evidence that there
was any distension of the gut that may indicate excessive production of methanol or
galacturonic acid units. No interactions between the treatments were detected for any of the
measures in the experiment.
9.4 Discussion
The first hypothesis of the experiment was accepted because the combination of PME and
PG reduced the viscosity of digesta by 19%, wet droppings by 15% and the number of
soiled eggs by 19%, for the hens fed diets containing 10 and 20% dehulled lupins. In
addition, feed conversion ratio was improved by 5%, digestibility was increased by 11%
and metabolisable energy by 8% and there was no effect on feed intake as expected. There
was a small increase in egg production (4%) and egg and shell weight (3%) but no effect on
shell thickness, yolk colour or Haugh unit score.
It appears that the substantial breakdown of cell-wall lattices, in particular the long pectin
chains by PME + PG led to a significant drop in the viscosity of digesta. This allowed the
digestible nutrients that were previously confined within the cell-wall lattices to be released
and made available for digestion and absorption in the digestive tract of bird. As a
consequence, the digestibility of dry matter and metabolisable energy of the diet were
increased and feed conversion ratio was decreased. The increase in total eggs produced, egg
weight, and egg-shell weight were most likely due to the increase in digestibility of dry
matter. The decrease in the percentage of soiled eggs was most likely due to the decrease in
water intake and wet droppings.
The second hypothesis was partially accepted because PME and PG allowed the laying
hens to handle 30% dehulled lupins in their diet. The results for the high inclusion rate
(30%) of dehulled lupins were surprising because, as expected, PME + PG broke down the
cell walls and pectins, reduced the viscosity of digesta and the number of soiled eggs, and
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increased the feed conversion efficiency. However, PME + PG did not reduce water intake
or wet droppings. Feed conversion efficiency was improved slightly but digestibility and
metabolisable energy remained unchanged. This might be due to the large amounts of non-
pectic polysaccharides, which make up two thirds of the cell walls, because when dehulled
lupins are present in high amounts (up to 30%) of the diet they exert a substantial effect
(Brillouet & Riochet, 1983; Carre et al, 1985; Cheung, 1991; Cheetham et al, 1993; Harris
& Smith, 2006). High amounts of these polysaccharides could increase water-holding
capacity within cell-wall lattices and thereby increase wet droppings. In previous broiler
work, we found that the PME and PG combination did not improve lupins sufficiently so
they could be included at 30% in a diet but hypothesised that the more developed digestive
tract of layers might handle lupins up to 30%. However, this was clearly not the case.
It has been suggested that the by-products of pectin breakdown, methanol and galacturonic
acid units, might be detrimental to growth, increase water intake and may increase
mortality. There was no evidence in previous broiler work of any detrimental effects. Nor
was there any evidence in the current layer work of any detrimental effects as has been
suggested by some workers (Mingan Choct, pers. comm. 2006). No inflation of the small
intestine by methanol production was observed during digesta collection from the ileum.
This suggests that PME and PG can be safely used for hens.
PME + PG consistently reduced the viscosity of digesta measured at the middle and end of
the experiment by 19%. As a result, no difference was detected in the viscosity of digesta
collected at the middle or end of the experiment. This finding dismissed the concerns that
variation may take place in the viscosity values at the middle and end of the experiment, as
suggested by the AECL committee.
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9.5 Conclusions
1. The combination of PME and PG significantly improved the nutritive value of dehulled
lupins for layers. PME + PG was very effective at reducing the viscosity, wet droppings
and soiled eggs associated with dehulled lupins, and improving the feed conversion
efficiency, egg yield and weight and egg-shell weight.
2. The combination of PME + PG will allow inclusion of dehulled lupins up to 20% in
layer diets and, at the same time, completely control wet droppings.
3. The high level of wet droppings might be reduced for hens fed 30% dehulled lupins in
diets treated with PME + PG if the non-pectic polysaccharides can be broken down by
appropriate enzymes.
4. This synergistic action between PME and PG could make inroads towards greater
inclusion of other legumes into hen diets since they contain similar, problematic cell-
wall polysaccharides to those of lupins.
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Chapter 10
General Discussion
The general hypothesis that breakdown of cell walls and pectins of lupins improves the
nutritive value of lupins for broilers and layers was supported. By using a combination of
two pectinases, PG and PME, the viscosity and water-holding capacity of lupins were
reduced (Chapter 7). As a consequence, water intake was reduced and so wet droppings
were reduced (Chapters 8 and 9). Furthermore, the enhanced breakdown released the
nutrients for digestion, leading to an increase in production performance and feed
conversion efficiency and an increase in the metabolisable energy of the diet.
Based on the present results, the lowest dose (0.6g/kg diet) of PG gave the best results in
egg layer diets in terms of an increase in feed conversion efficiency, in the digestibility and
metabolisable energy of the diet, and in egg yield and egg-shell weight (Chapter 4). A PG
dose higher than 0.6g/kg diet cannot be recommended because it tended to slightly increase
water intake and wet droppings for layers.
Although the optimal dose of PG alone gave good results when lupins comprised up to 20%
of the diet, an inclusion of more than 10% of lupins cannot be recommended. Even at the
optimal dose of PG (0.6g/kg diet), inclusion of more than 10% lupins was associated with a
high level of water intake and wet droppings, an increased number of soiled eggs, and
reductions in the digestibility and metabolisable energy of the diet. This was probably
because PG had a limited ability in breaking down the pectin chains (no more than 11% of
pectin chains were broken) and reducing high viscosity of the digesta at the 20% inclusion
rate of lupins, regardless of the dose. Similar responses were found in our previous broiler
and in vitro experiments (Ali, 1997; Ali, 2001, unpublished; Ali et al, 2001). These results
suggest that PG alone is not likely to allow the inclusion of more than 10% lupins in
poultry diets.
As a result, we attempted to improve PG activity in two ways: pre-treatment of lupins with
expansion before treatment with PG (Chapter 5), and treatment of lupins with a
combination of two pectinases, PG and PME (Chapters 6).
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Expansion of lupins followed by PG treatment was deemed to be unsuccessful because it
produced more undesirable than desirable results. Expansion increased PG activity for
breaking down the cell walls and pectins, as was anticipated. However, this breakdown was
not reflected in improved growth performance of broilers, because the expansion, instead of
reducing the viscosity of digesta, water intake and wet droppings of birds, increased them
even more than the control treatment. The main aim of this experiment was to reduce, not
increase, the high level of water intake and wet droppings associated with lupins. These
undesirable effects stemmed from solubilisation of insoluble fibres in lupins by the
expansion physical conditions, as explained in detail in Chapter 5. The undesirable effects
were more obvious with whole lupins than dehulled lupins because 25% of whole lupins is
fibrous hulls, of which 85-95% is insoluble fibre (Harris & Jago, 1985; Cheung, 1991;
Evans et al, 1993). Based on these results, feed manufacturers and poultry producers are
advised not to expand lupins and treat them with enzyme for broilers, albeit that this advice
may serve against the interests of expander or extruder manufacturers.
The most exciting results in this thesis were from using a combination of PG and PME. The
initial in vitro experiment (Chapter 6) showed promising results that it was possible to
achieve a substantial breakdown of cell walls and pectins and a substantial reduction in
pectin chain length by treating dehulled lupins with PG and PME. It is clear that the methyl
ester radicals along the pectin chain were the main barriers for breakdown of pectin by PG
alone because removing them with PME was an essential step. However, the amount of
enzyme was important. The PME dose used (1400 units) was too high and introduced
problems such as increased water-holding capacity and viscosity of dehulled lupins.
Therefore, our next step was to establish the optimal dose of PME to be used in
combination with PG in vitro (Chapter 7) for breakdown of cell walls and pectin while at
the same time reducing water-holding capacity and viscosity of dehulled lupins. After the
optimal dose (200 units) of PME in combination with PG (1400 units) was established, we
tested it in vivo. PG+PME proved to be very effective in preventing the adverse effects of
pectins and improving the nutritive value of dehulled lupins up to 20% in the diets for both
broilers and layers (Chapters 8 and 9). These were another exciting results observed in this
102
thesis because the in vitro results clearly reflected the in vivo situation. These results
suggest that dehulled lupins up to 20% can successfully replace soybean meal without
significant losses in productivity or increases in wet droppings.
However, the enzyme treatment did not reduce water intake or wet droppings of broilers
and layers fed 30% dehulled lupins in the diet, although there was a slight increase in feed
conversion efficiency and egg production, and a slight reduction in soiled eggs with layers
(Chapters 8 and 9). Besides, as dehulled lupins was increased from 10 to 20 or 30% in
broiler and layer diets, the production performance, feed conversion efficiency, digestibility
of dry matter and metabolisable energy of the diet all were reduced, and viscosity of
digesta, water intake and wet droppings all were increased. Surprisingly, PG+PME broke
down the cell walls and pectins in broiler and layer diets, and also reduced the viscosity of
digesta but the viscosity values at 30% lupin inclusion were almost double those at 10%
inclusion. This suggests that the non-pectic polysaccharides, the factors likely responsible
for increasing viscosity and water-holding capacity, should be broken down by appropriate
enzymes in the presence of PG+PME if 30% of dehulled lupins are to be included in broiler
and layer diets. This concept may be worthy of further investigation.
Overall, these experiments show that specific enzymes targeting specific bonds and radicals
in cell-wall pectins lead to an improvement in the digestion of lupins for commercial
broiler and layer production. This research demonstrated the importance of selecting
targeted enzymes at optimal doses to maximize the benefits and minimize the problems
associated with lupins in poultry diets. At the same time, the optimal dose of these specific
enzymes allows dehulled lupins to be included up to 20% without significant losses in
productivity or increases in wet droppings.
Future work
Considering the complexity of lupin pectins and the present findings, I propose the
development of a multi-enzyme preparation that comprises six enzymes: PG, PME and
xylanase (for wheat, the main ingredient of broiler and layer diets, and for few xylan side-
103
branches in lupins), arabinosidase, and also galactanase and rhamnogalacturonase if they
become available commercially (Fig. 10.1).
PG + PME Rhamnogalacturonase ↓ ↓ A-(1→2)-β-L-Rhap-(1→4)-α-D-Galp–A-(1→2)-β-L-Rhap-(1→4)-α-D-Galp-A-(1→2)-β-L-Rhap-(1→ 4 4 4 4 ↑ ↑ ← Endo-xylanase ↑ ← Endo-galactanase → ↑ 1 1 1 1 α-L-Araf D-Xylp β-D-Gal p β-D-Gal p 5 6 4 ↑ ← Endo-arabinosidase ↑ ↑ 1 ↓ 1 1
-α-L-Araf (2→1)-α-L-Araf– (5→1)-α-L-Araf
β-D-Gal p β-D-Gal p Figure 10.1 Proposed mechanism of complete breakdown of the complex pectin structure of lupins and the
main sites of cleavage by enzymes.
This combination should be tested first in vitro and then in vivo and should allow lupins to
be used up to a level of 30% in broiler and layer diets without significant losses in
productivity or increases in wet droppings.
Future work should also consider transferring the enzyme technology to other legumes such
as lathyrus, field pea and faba bean for poultry since these legumes contain large amounts
of problematic cell-wall polysaccharides similar to those of lupins. Canola meal also seems
to be a good target feed because it contains large amounts of pectin (15%, Slominski &
Campbell, 1990).
Conclusions
The nutritive value of lupins can be substantially improved if they are treated with a
combination of two pectinases, PG and PME. The combination of PG and PME at optimal
doses (1400 unit PG/g and 200 unit PME/g) gives maximum responses for increasing feed
conversion efficiency, increasing digestibility of dry matter, and increasing the
metabolisable energy of diets and completely eliminating wet droppings for both broilers
104
and layers. In layers, egg production and quality are improved, and the number of soiled
eggs is reduced.
These improvements should allow inclusion rates of dehulled lupins in diets to be lifted
from below 7% up to at least 20% in broiler and egg layer diets. Poultry producers looking
for lower feed costs can save about $34 per tonne of diet if they replace imported soybean
meal with 20% dehulled lupins plus PME + PG. These improvements should allow poultry
producers to meet the challenge of current spiraling feed costs.
The synergistic action between PG and PME could make inroads towards greater inclusion
of other local legumes, which also contain large amount of pectins, into broiler and layer
diets.
The key to including 30% dehulled lupins in broiler and layer diets using PG+PME would
be to find other enzymes that break down non-pectin polysaccharides. This investigation
may prove beneficial but this has yet to be conducted.
In view of the achievable improvements, a commercial preparation of pectinases should be
developed for future application to broiler and layer diets.
Original contributions of the study
I believe that I have made original and substantial contributions to science in each field of
the study chosen in this thesis. In my in vitro and in vivo studies, I have contributed to our
current understanding of the underlying mechanisms behind the poor utilisation of lupins
by broilers and layers, which can be summarised in four steps.
First, I identified the main problems involved in feeding lupins to poultry for causing poor
feed conversion efficiency, low metabolisable energy of diet and high wet droppings of
broilers and layers. I used a very systematic approach to break down the complex structure
of pectin within cell-wall lattices of lupins based on a clear understanding of pectin
biochemistry and the results of my previous in vitro and in vivo experiments.
105
Second, I devised ways to solve these problems using appropriate enzymes and
mechanical-thermal process to destroy the problematic cell walls and their main
constituents, pectins.
Third, past work with mutli-enzyme preparations showed very little success in breaking
down the cell-wall polysaccharides because the enzyme components and their activities of
those preparations did not match their target substrates in lupins. Consequently, I selected
specific, purified enzymes with known activities such as pectinases, polygalacturonase
(PG) and pectin methyl esterase (PME), to target specific bonds and radicals (methyl esters)
of the pectin chains in lupins. Thus, this study considered the details of pectin structure and
targeted the specific bonds with appropriate enzymes. The initial tests of my in vitro
experiments showed promising results for substantial breakdown of pectins as well as
reduction of viscosity and water-holding capacity, as hypothesised.
Finally, I tested the combination of PG and PME in broiler and layer diets and found this
approach very successful in leading to breakdown of pectin and therefore increasing the
nutritive value of lupins for both broilers and layers.
Overall, I have shown for the first time, that two pectinases can allow poultry producers
and feed manufacturers to include dehulled lupins up to 20% in broiler and layer diets
without causing any nutritional and hygienic problems. I have communicated these results
to poultry and feed industries in Western Australia and they are very willing to use
commercial pectinases in their diet formulations. Thus, I have made original contributions
from basic to applied research, and taken the research from the laboratory to the field.
106
References
Aar, P. J. van der., Fahey, G. C., Jr. Ricke, S. C. Allen, S. and E. Berger, L. L. 1983. Effects of dietary fibers on mineral status of chicks. Journal of Nutrition. 113, 653 – 661.
ABARE, 2008. Australian Bureau of Agricultural and Resource Economics. FEED GRAINS future supply
and demand in Australia. eReport, edited by Hafi, A. and Connell, P. prepared for the Grains Research and Development Corporation. Canberra, Australia.
Adams, O. L. and Naber, E. C. 1969. Effect of physical and chemical treatment of grains on growth of and
feed utilization by the chick. 1. The effect of water and acid treatments of corn, wheat, barley and expanded or germinated grains on chick performance. Poultry Science. 48, 853 - 858.
Albersheim, P., Darvill, A. G., O’Neill, M. O., Schols, H. A. and Voragen, A. G. J. 1996. An hypothesis: The
same six polysaccharides are components of the primary cell walls of all higher plants. pp. 47 – 55. In Pectins and Pectinases, Proceedings of an International Symposium, Wageningen, The Netherlands, December 3 - 7, 1995. Ed. Visser, J. and Voragen, A.G.J. Elsevier Science Publication. Amsterdam, The Netherlands.
Albersheim, P. 1974. The Primary cell wall and control of elongation growth. In Plant Carbohydrate
Biochemistry. pp. 145 – 164. Ed. Pridham, J. B. Academic Press Publication. New York, USA. Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. 2006a. The optimal dose of pectinase in lupin-based diets
for laying hens. Proceedings of the Australian Poultry Science Symposium. 18, 218 – 221. Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. 2006b. Expansion and pectinase treatment of lupins for
broilers. Proceedings of the Australian Poultry Science Symposium. 18, 62 – 65. Ali, A., Williams, I. H., Martin, G. B. and Sipsas, S. 2005. Hydrolysis of lupin pectin by pectinases for
broilers. Proceedings of the Australian Poultry Science Symposium. 17, 219 – 222. Ali, A. 2001. Unpublished results. The optimal dose of polygalacturonase for broiler chicken diets based on
dehulled lupins. Department of Animal Science. The University of Western Australia. Ali, A., Williams, I. H., Martin, G. B. and Harris, D. J. 2001. Enzymatic pre-treatment of lupins for broiler
diets. Proceedings of the Australian Poultry Science Symposium. 13, 199 – 203. Ali, A., Williams, I. H., Martin, G. B. and Harris, D. J. 2000. Important properties of dehulled lupins in
poultry diets. Proceedings of the Australian Poultry Science Symposium. 12, 206. Ali, A. 1997. Enzyme treatment to increase digestibility of non-starch polysaccharides (NSPs) in lupin-based
diets fed to broiler chickens. Graduate Diploma in Science in Agriculture. Thesis by research. Department of Animal Science. The University of Western Australia.
Alloui, O., Smulikowska, S., Chibowska, M. and Pastuszewska, B. 1994a. The nutritive value of lupin seeds
(L. luteus, L. angustifolius and L. albus) for broiler chickens as affected by variety and enzyme supplementation. Journal of Animal Feed Science. 3, 215 – 227.
Alloui, O., Chibowska, M. and Smulikowska, S. 1994b. Effects of enzyme supplementation on the digestion
of low glucosinolate rapeseed meal in vitro, and its utilization by broiler chicks. Journal of Animal Feed Science. 3, 119 – 128.
Annison, G., Hughes, R. J. and Choct, M. 1996. Effect of enzyme supplementation on the nutritive value of
dehulled lupins. British Poultry Science. 37, 157 - 172. Annison, G. and Choct, M. 1993. Enzymes in poultry diets. Proceeding of the 1st Symposium on Enzymes in
Animal Nutrition. Ed. Wenk, C. and Boessinger, M. Kartause Ittingen, Switzerland. 61 – 68.
107 Armstrong, E. F., Eastwood, M. A. and Brydon, W. G. 1993. The influence of wheat bran and pectin on the
distribution of water in rat caecal contents and faeces. British Journal of Nutrition. 69, 913 – 920. Armstrong, H., 1993. Nutritional implications of expanded feed. Feed Mix. 1, 24 - 27. Aspinall, G. O., Begbie, R., Hamilton, A. and Whyte, J. N. C. 1967a. Polysaccharides from soy-bean. Part III.
Extraction and fractionation of polysaccharides from cotyledon meal. Journal of Chemistry Society (Part C). 1065 – 1970.
Aspinall, G. O., Cottrell, W. I., Egan, S. V., Morrison, I. M. and Whyte, J. N. C. 1967b. Polysaccharides from
soy-bean. Part IV. Partial hydrolysis of the acidic polysaccharide complex from cotyledon meal. Journal of Chemistry Society (Part C). 1071 – 1980.
Awad, M. and Young, R. E. 1979. Postharvest variation in cellulase, polygalacturonase, and pectin methyl
esterase in avocado (Persea americana Mill, cv. Fuerte) fruits in relation to respiration and ethylene production. Plant Physiologyogy. 64, 306 – 308.
Bagheri, S. and Gueguen, L. 1985. Effect of wheat bran and pectin on the absorption and retention of
phosphorus calcium magnesium and zinc and by the growing pig. Reproduction Nutrition Development. 25, 705 – 716.
Bailey, R. W. and Kauss, H. 1974. Extraction of hydroxyproline-containing proteins and pectic substances
from cell walls of growing and non-growing mung bean hypocotyl segments. Planta. 119, 233 - 245. Bailey, R. W., Mills, S. E. and Hove, E. L. 1974. Composition of sweet and bitter lupin seed hulls with
observations on the apparent digestibility of sweet lupin seed hulls by young rats. Journal of the Science of Food and Agriculture. 25, 955 - 961.
Ballester, D., E. Yanez, R. Garcia, S. Erazo, F. Lopez, E. Harrot, S. Cornejo, A. Lpoez, J. Pokniak, and C. O.
Chichester, 1980. Chemical composition, nutritive value and toxicological evaluation of two species of sweet lupin (L. albus and L. luteus). Journal of Agriculture and Food Chemistry. 28, 402 - 405.
Barash, I. and Eyal, Z. 1970. Properties of a polygalacturonase produced by Geotricum candidum.
Phytopathology. 60, 27 – 36. Barrett, D. M. and Gonzalez, C. 1994. Activity of Softening Enzymes during cherry Maturation. Journal of
Food Science. 59, 574 – 577. Batterham, E. S., Andersen, L. M. and Baigent, D. R. 1993. Utilization of ileal digestible amino acids by
growing pigs: methionine. British Journal of Nutrition. 70, 711 – 720. Batterham, E. S., Andersen, L. M., Burnham, B. V. and Taylor, G. A. 1986a. Effect of heat on the nutritional
value of lupin (Lupinus angustifolius)-seed meal for growing pigs. British Journal of Nutrition. 55, 169 – 177.
Batterham, E. S., Andersen, L. M., Lowe, R. F. and Darnell, R. E. 1986b. The nutritional value of lupin
(Lupinus albus)-seed meal for growing pigs; availability of lysine, effect of autoclaving and net energy content. British Journal of Nutrition. 56, 645 – 659.
Bauer, W. D., Talmadge, K. W., Keegstra, K. and Albersheim, P. 1973. The structure of plant cell walls. II.
The hemicellulose of the walls of suspension-cultured sycamore cells. Plant Physiology. 51, 174 - 187.
Bedford, M. R. and Classen, H. L. 1993. An in-vitro assay for prediction of broiler intestinal viscosity and
growth when fed rye based diets in the presence of exogenous enzymes. Poultry Science. 72, 137-143.
108 Bedford, M. R. and Classen, H. L. 1992. The influence of dietary xylanase on intestinal viscosity and
molecular weight distribution of carbohydrates in rye-fed broiler chicks. In xylans and xylanases. Progress in Biotechnology. Vol. 7, 361 - 370. [ed.] J. Visser. Elsevier science publishers. Wageningen. The Netherlands.
Bedford, M. R. and Classen, H. L. 1991. Reduction of intestinal viscosity through manipulation of dietary rye
and pentosanase concentration is affected through changes in the carbohydrate composition of intestinal aqueous phase and results in improved growth rate and food conversion efficiency of broiler chicks. Journal of Nutrition. 122, 560 - 569.
Belo, P. S. Jr. and de Lumen, B. O. 1981. Pectic substance content of detergent-extracted dietary fibers.
Journal of Agriculture and Food Chemistry. 29, 370 - 373. BeMiller, J. N. 1986. An introduction to pectins: structure and properties. In Chemistry and function of
pectins. pp. 3 – 21. Ed. Fishman, M. L. and Joseph, J. J. American Chemical Society (ACS) symposium, series 310. Washington.
Ben-Arie, R. and Lavee, S. 1971. Pectic changing occurring in Elberta peaches suffering from woolly
breakdown. Phytochemistry. 10, 531 – 538. Benen, J. A., Kester, H. C. and Visser, J. 1999. Kinetic characterization of Aspergillus niger N400
endopolygalacturonases I, II and C. European Journal of Biochemistry. 259, 577 – 85. Biely, P., Mackenzie, C. R., Puls, J. and Schneider, H. 1986. Cooperativity of esterases and xylanases in the
enzymatic degradation of acetyl xylan. Biotechnology. 4, 731 - 733. Bishawi, K. O. and McGinnis, J. 1984. Studies on the action of pectin in depressing the growth of chicks.
British Poultry Science. 25, 519 – 528. Bishop, R. J. 1989. Egg research programme update. In Golden Egg Farms Newsletter - Western Australian
Marketing Board. 43, 14 – 22. Blanco, P., Sieiro, C., Diaz, A. and Villa, T. G. 1994. Production and partial characterization of an
endopolygalacturonase from Saccharomyces cerevisiae. Canadian Journal of Microbiology. 40, 974 – 977.
Blumenkrantz, N. and Asboe-Hansen, G. 1973. New method for quantitative determination of uronic acids.
Analytical Biochemistry. 54, 484 – 489. Boldaji, F., Geoger, Nakaue, H. S., Savage, T. F. and Arscott, G. H. 1986. Effect of autoclaving and cooking
on true metabolisable energy (TME) and n-corrected TMEn content of white lupin, yellow peas and faba beans. Nutrition Reports International. 34, 159 – 164.
Boling, S. D., Douglas, M. W., Shirley, R. B., Parsons, C. M. and Koelkebeck, K. W. 2000. The effects of
various dietary levels of phytase and available phosphorus on performance of laying hens. Poultry Science. 79, 535 – 538.
Bonhomme, A. 1990. Rumen ciliates: their metabolism and relationships with bacteria and their hosts. Animal
Feed Science and Technology. 30, 203 – 266. Brenes, A., Slominski, B. A., Marquardt, R. R., Guenter, W. and Viveros, A. 2003. Effect of enzyme addition
on the digestibilities of cell wall polysaccharides and oligosaccharides from whole, dehulled, and ethanol-extracted white lupins in chickens. Poultry Science. 82, 1716 – 1725.
Brenes, A., Marquardt, R. R., Guenter, W. and Viveros, A. 2002. Effect of enzyme addition on the
performance and gastrointestinal tract size of chicks fed lupin seed and their fractions. Poultry Science. 81,670 – 678.
109 Brenes, A., Marquardt, R., Guenter, W. and Rotter, B. A. 1993. Effect of enzyme supplementation on the
nutritional value of raw, autoclaved, and dehulled lupins (Lupinus albus) in chicken diets. Poultry Science. 72, 2281 – 2293.
Brenes, A., Trevino, J., Centino, C. and Yuste, P. 1989. The influence of b-galactosides extracted from lupin
seed (L. albus) on the digestion of dietary starch by growing chicks. In Recent Advances of Research in Antinutritional Factors in Legume Seeds. Ed. Huisman, J. van der Poel, A. F. B., Liener, J. E. Pudoc, Wageningen, Netherlands. pp. 374 – 377.
Brillouet, J.-M. 1984. Carbohydrates of lupin seed. Proceeding of the 3rd International Lupine Congress. La
Rochelle (France). 366 – 383. Brillouet, J. M. and Riochet, D. J. 1983. Cell wall polysaccharides and lignin in cotyledons and hulls of seeds
from various lupin (Lupinus L.) species. Journal of the Science of Food and Agriculture. 34, 861 – 868.
Brillouet, J.-M. and Carre, B. 1983. Composition of cell walls from cotyledons of Pisum sativum, Vicia faba
and Glycine max. Phytochemistry. 22, 841 – 847. Broz, J. 1987. Improvement of nutritive value of rye for broiler chickens by supplemental enzymes. Proc. 6th
European Symposium on Poultry Nutrition. Konigslutter, Germany. pp. 6 - 7. Bryden, W. L. R. J. Gill and Balnave, D. 1994. Feed enzyme supplement improves the apparent metabolisable
energy of lupins for broiler chickens. Proceedings of the Australian Poultry Science Symposium. 6, 115.
Burnett, G. S. 1966. Studies on viscosity as the probable factor involved in the improvement of certain barleys
for chickens by enzyme supplementation. British Poultry Science. 7, 55 – 75. Campbell, G. L., Campbell, L. D. and Classen, H. L. 1983a. Utilisation of rye by chickens: effect of microbial
status, diet gamma irradiation on sodium taurocholate supplementation. British Poultry Science. 24, 191 – 203.
Campbell, G. L., Classen, H. L. and Goldsmith, K. A. 1983b. Effect of fat retention on the rachitogenic effect
of rye fed to broiler chicks. Poultry Science. 62, 2218 – 2223. Carre, B. 1997. The qualities of grain legumes for poultry. Proceedings of the Australian Poultry Science
Symposium. 9, 46 – 53. Carre, B., Flores, M P. and Gomez, J. 1995a. Effects of pelleting, lactose level, polyethylene glycol 4000, and
guar gum compared to pectin on growth performances, energy values, and losses of lactose, lactic acid, and water in chickens. Poultry Science. 74, 1810 – 1819.
Carre, B., Gomez, J. and Chagneau, A. M. 1995b. Contribution of oligosaccharide and polysaccharide
digestion, and excreta losses of lactic acid and short chain fatty acids, to dietary metabolisable energy values in broiler chickens and adult cockerels. British Poultry Science. 36, 611 – 629.
Carre, B. 1991. The chemical and biological bases of a calculation system developed for predicting dietary
energy values: poultry model. Chapter 5. In Invitro digestion for pigs and poultry. pp. 67 – 85. Ed. Fuller, M. F. CAB international publication.
Carre, B. and Leclercq, B. 1985. Digestion of polysaccharides protein and lipids by adult cockerels fed on
diets containing a pectic cell-wall material from white lupine Lupinus albus cotyledon. British Journal of Nutrition. 54, 669 – 680.
Carre, B., Brillouet, J. M. and Thibault, J. F. 1985. Characterisation of polysaccharides from white lupin
(Lupinus albus L.). Journal of the Science of Food and Agriculture. 33, 285 – 292.
110 Castanon, J. I. R. and Marquardt, R. R. 1989. Effect of enzyme addition, autoclave treatment and fermenting
on the nutritive value of field beans (Vicia faba L.). Animal Feed Science and Technology. 26, 71 – 79.
Chambat, G. and Joseleau, J.-P. 1980. Isolation and characterisation of a homogalacaturonan in the primary walls of Rosa cells cultures in vitro. Carbohydrate Research. 85, C10 – C12.
Champ, M., Barry, J L., Bonnet, C., Berot, S., Delort, and Laval, J. 1990. The role of cell wall
polysaccharides and α galactosides in the flatus induced by the consumption of a legume seed lupine in the rat. Sciences des Aliments. 10, 317 – 324.
Cheetham, N. W. H., Cheung, P. C. K. and Evans, A. J. 1993. Structure of the principal non-starch
polysaccharide from the cotyledons of Lupinus angustifolius (cultivar Gungurru). Carbohydr. Polym. 22, 37 – 47.
Chesson, A., Fuller, M. F. and Alldrick, A. 2002. Wheat and barley in pig and poultry diets: effects of
physical processing and enzymes; assessment of nutritive value. The Home-Grown Cereals Authority (HGCA) Report. Rowett Research Institute, Aberdeen, Scotland.
Chesson, A. 1993. Feed enzymes. Animal Feed Science and Technology. 45, 65 – 79. Chesson, A. 1987. Supplementary enzymes to improve the utilization of pig and poultry diets. In Recent
Advances in Animal Nutrition. 1987. Butterworths, London, UK, 71 – 89. Chesson, A. and John, A. M. 1982. Legume pectic substances and their degradation in the ovine rumen.
Journal of the Science of Food and Agriculture. 33, 852-859. Cheung, P.C.-K. 1991. The carbohydrates of Lupinus angustifolius: a composite study of seeds and structural
elucidation of the kernel cell-wall polysaccharides of Lupinus angustifolius. Ph.D. Thesis. School of Chemistry, University of News South Wales.
Choct, M., Hughes, R. J., Trimble, R. P., Angkanaporn, K. and Annison, G. 1995. Non-starch polysaccharide-
degrading enzymes increase the performance of broiler chickens fed wheat low of apparent metabolisable energy. Journal of Nutrition. 125, 485 – 492.
Choct, M. 2006. Enzymes for the feed industry: past, present and future. Worlds Poultry Science Journal, 62,
2 – 6. Choct, M. and Annison, G. 1992. The inhibition of nutrient digestion by wheat pentosans. British Journal of
Nutrition. 67, 123 - 132. Choct, M. and Annison, G. 1991. Anti-nutritive activity of wheat pentosans in broiler diets. British Poultry
Science. 33, 811 - 821. Christgau, S., Kofod, L. V., Halkier, T., Andersen, L. N. Hockauf, M., Dorreich, K., Dalboge, H. and
Kauppinen, S. 1996. Pectin methyl esterase from Aspergillus aculeatus: Expression cloning in yeast and characterization of the recombinant enzyme. Biochemical Journal. 319, 705 – 712.
Clarke, R. T. J., Bailey, R. W. and Gaillard, D. E. 1969. Growth of rumen on plant cell wall polysaccharides.
Journal of General Microbiology. 50, 79 - 86. Classen, H. L. and Bedford, M. R. 1991. The use of enzyme to improve the nutritive value of poultry feeds. In
Recent Advances in Animal Nutrition. 95 - 116. Edited by Haresign and Cole. Archor press. Butterworths. UK.
Classen, H. L., Campbell, G. L. and Grootwassink, J. W. D. 1988. Improve feeding value of Saskatchewan-
grown barley for broiler chickens with dietary enzyme supplementation. Canadian Journal of Animal Science. 68, 1253 - 1259.
111 Classen, H. L., Campbell, G. L., Rossnagel, B. G. Bhatty, R. and Reichert, R. D. 1985. Studies on the use of
hulls barley in chick diets: deleterious effects and methods of alleviation. Canadian Journal of Animal Science. 65, 725.
Coates, M. E., Cole, C. B. Fuller, R., Houghton, S. B. and Yokota, H. 1981. The gut microflora and the
uptake of glucose form the intestine of the chick. British Poultry Science. 22, 289 - 294. Coelho, M. 1994. Comparison with pelleting and extrusion: vitamin stability in expanders. Feed Management
45, 10 - 15. Coleman, G. S., Sandford, D. C. and Beahon, S. 1980. The degradation of polygalacturonic acid by rumen
ciliate protozoa. Journal of General Microbiology. 120, 295 – 300. Conrad, H. R., Pounden, W. D., Fetter, A. W. and Ramseyer, R. D. 1960. Total pectic substances and uronic
acids in pasture plants and their relationship to incidence of bloat. Proceedings of the 5th Conference on Rumen Function. pp. 25. Chicago. USA.
Conrad, H. R., Pounden, W. D., Bentley, O. G., and Fetter, A. W. 1958. Production of gas in rumen
fermentations and stable mass formation from alfalfa fiber and Pectic substances. Journal of Dairy Science. 41, 1586.
Cooke, R. D., Ferber, C. E. M. and Kanagasabapathy, L. 1976. Purification and characterisation of
polygalacturonases from a commercial Aspergillus niger preparation. Biochimica et Biophysica Acta. 452, 440 – 451.
Cullen, R. W. and Oace, S. M. 1979. Methyl malonic acid and vitamin B-12 excretion of rats consuming diets
varying in cellulose and pectin. Journal of Nutrition. 108, 640 – 647. Daas, P. J. H., Arisz, P. W., Schols, H. A. De Ruiter, G. A. and Voragen, A. G. J. 1998. Analysis of Partially
Methyl-Esterified Galacturonic Acid Oligomers by High-Performance Anion-Exchange Chromatography and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Analytical Biochemistry. 257, 195 – 202.
Daveby, Y. D., Razdan, A. and Aman, P. 1998. Effect of particle size and enzyme supplementation of diets
based on dehulled peas on the nutritive value for broiler chickens. Animal Feed Science and Technology. 74, 229 – 239.
Day, R. M. and Thomas, O. P. 1980. Growth depression of chicks fed a crude rye extract containing pectic
substances. Poultry Science. 59, 2754 – 2759. de Vries, R. P., Kester, H. C. M., Poulsen, C. H., Benen, J. A. E. and Visser, J. 2000. Synergy between
enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides. Carbohydrate Research. 327, 401 – 410.
Demain, A. L. and Phaff, H. J. 1957. Cucumber curing, softening of cucumbers during curing. Journal of
Agricultural and Food Chemistry. 5, 60. Demain, A. L. and Phaff, H. J. 1954. Hydrolysis of the oligogalacturonides and pectic acid by yeast
polygalacturonase. Journal of Biological Chemistry. 210, 381 – 393. den Hartog, L. A., Huisman, J., Thielen, W. J. G., Van Schayk, G. H. A., Boer, H. and Van Weerden, E. J.
1988. The effect of including various structural polysaccharides in pig diets on ileal and fecal digestibility of amino acids and minerals. Livestock Production Science. 18, 157 – 170.
Dockray, G. J. 1977. Progress in gastroenterology. Molecular evolution of gut hormones: Application of
comparative studies on the regulation of digestion. Gastroentology. 72, 344 – 358. Dockray, G. J. 1979. Cholecystokinin-like peptides in avian brain and gut. Experientia. 35, 628 – 630.
112 Douglas, M. W. and Parsons, C. M. 2000. Effect of presolvent extraction processing method on the nutritional
value of soybean meal for chicks. Poultry Science. 79, 1623 – 1626. Drakos, A., Doxastakis, G. and Kiosseoglou,V. 2007. Functional effects of lupin proteins in comminuted
meat and emulsion gels. Food Chemistry. 100, 650 - 655. Drochner, W., Stadermann, and B. Yildiz, G. 1993. Influence of pectins on performance and metabolism of
poultry. Übers Tierernährung. 21, 121 – 180. [Abstract]. Drochner, W., Cerci, I. H., Stadermann, B. and Luders, H. 1990. Pair-feeding experiments concerning the
effects of increasing levels of low methylated pectins in the diet on the metabolism of laying hens. Archives of Animal Nutrition. 40, 431 – 442.
Dunshea, F. R., Gannon, N. J., van Barneveld, R. J., Mullan, B. P. Campbell, R. G. and King, R. H. 2001.
Dietary lupins (Lupinus anugustifolius and Lupinus albus) can increase digesta retention in the gastrointestinal tract of pigs. Australian Journal of Agricultural Research. 52, 593 – 602.
Edney, M. J., Classen, H. L. and Campbell, G. L. 1986. Application of simple radial gel diffusion assay of
endo-β-glucanase activity in dietary enzyme supplements. Poultry Science. 65, 72 – 77. Edwards, A. and Tucek, M. 2000. Lupins perform well. Asian Pork. 1 – 3. Edwards, J. M., Jr., Carlos, A. B., Kasim A. B. and Toledo, R. T. 1999. Effects of steam pelleting and
extrusion of feed on phytate phosphorus utilization in broiler chickens. Poultry Science. 78, 96 - 101. El Defrawi, E. A. and Hudson, B. J. F. 1979. A C-glycosyl-flavonol from the seeds of four Lupinus species.
Journal of Plant Foods. 3, 175 – 179. El-Rayah, A. A. and Labavitch, J. M. 1980. Cell wall metabolism in ripening fruit. I. Cell wall changes in
ripening 'Bartlett' pears. Plant Physiologyogy. 65, 1009 – 1013. El-Rayah, A. A. and Labavitch, J. M. 1977. A simplified method for accurate determination of cell wall
uronide content. Journal of Food Biochemistry. 1, 361 – 365. Endo, A. 1964a. Studies on pectolytic enzymes. VIII. Purification and properties of endo-polygalacturonase I.
Agricultural Biology and Chemistry. 28, 535 - 542. Endo, A. 1964b. Studies on pectolytic enzymes of molds. IX. Purification and properties of endo-
polygalacturonase II. Agricultural Biology and Chemistry. 28, 543 – 550. Endo, A. 1964c. Studies on pectolytic enzymes of molds. X. Purification and properties of endo-
polygalacturonase III. Agricultural Biology and Chemistry. 28, 551 – 558. Endo, A. 1964d. Studies on pectolytic enzymes of models. VII. Turbidimetry of apple juice clarification and
its application to determination of enzyme activity. Agricultural Biology and Chemistry. 28, 234 - 238.
Endo, A. 1961a. Purification and properties of endo-polygalacturonase III. Agricultural Biology and
Chemistry. 25, 551 - 558. Endo, A. 1961b. Studies on pectolytic enzymes of molds. III. General characteristics of pectolytic enzymes
produced by Coniothrium diplodiella. Agricultural Biology and Chemistry. 25, 543 – 550. English, P. D., Maglothin, A., Keegstra, K and Albersheim, P. 1972. A cell wall-degrading endopolygalac-
utornase secreted by Colletotricum linedmuthianum. Plant Physiologyogy. 49, 293 – 297.
113 Erdman, J. W., Fahey, G. C. and White. C. B. 1986. Effect of purified dietary fiber sources on β-carotene
utilization by the chick. Journal of Nutrition. 116, 2415 – 2423. Evans, A. J. 1994. The carbohydrates of lupins, composition and uses. Proceeding of the 1st Australian
Technical Symposium. Perth, WA. pp. 110 – 114. Evans, A. J., Cheung, P. C-K. and Cheetham, N. W. H. 1993. The carbohydrate composition of cotyledons
and hulls of cultivars of lupinus-angustifolius from Western Australia. Journal of the Science of Food and Agriculture. 61, 189 – 194.
Fadel J. G., Newman, C. W., Newman, R. K. and Graham, H. 1988. Effects of extrusion cooking of barley on
ileal and fecal digestibilities of dietary components in pigs. Canadian Journal of Animal Science. 68, 891 – 898.
Fancher, B. I., Rollins, D. and Trimbee, B. 1996. Feed processing using the annular gap expander and its
impact on poultry performance. Journal of Applied Poultry Research. 5, 386 – 394. Farrell, D. J., Perez-Maldonado R. A. and Mannion P. F. 1999. Optimum inclusion of field peas, faba beans,
chick peas and sweet lupins in poultry diets. II. Broiler experiments. British Poultry Science. 40, 674 – 680.
Farrington, P. 1974. Effect of planting time on growth and seed yield of lupins, peas and vetches on the Swan
Coastal Plain, Western Australia. Australian Journal of Experimental Agriculture and Animal Husbandry. 14, 539 - 546.
Fasina Y. O., Campbell, G. l. and Tyler R T. 1997. Whole canola/pea and whole canola/canola meal blends in
diets for broiler chickens. 1. Evaluation of steam-pelleting or expansion processing. Canadian Journal of Animal Science. 77, 185 – 190.
Feighner, S. D. and Dashkevicz, M. P. 1988. Effect of dietary carbohydrates on bacterial cholyltaurine
hydrolase in poultry intestinal homogenates. Applied and Environmental Microbiology. 54, 337 – 342.
Feighner, S. D. and Dashkevicz, M. P. 1987. Sub therapeutic levels of antibiotics in poultry feeds and their
effects on weight gain, feed efficiency, and bacterial cholyltaurine hydrolase activity. Applied and Environmental Microbiology. 53, 331 – 3 36.
Fengler, A. I. and Marquardt, R. R. 1988. Water-soluble pentosans from rye: II. Effects on rate of dialysis and
on the retention of nutrients by chick. Cereal Chemistry. 65, 298 - 302. Fishman, M. L., Pfeffer, E., Barford, R. A. and Doner, L. W. 1984. Studies on pectic solution properties by
high-performance size exclusion chromatography. Journal of Agriculture and Food Chemistry. 25, 372 – 378.
Francesch, M., Perez-Vendrell, A., Esteve-Garcia, E. and Brufau, J. 1995. Enzyme supplementation of a
barley and sun flower-based diet on laying hen performance. Journal of Applied Poultry Research. 4, 32 – 40.
Friesen, O. D., Guenter, W., Rotter, B. A. and Marquardt, R. R. 1991. Effect of enzyme supplementation on
the nutritive value of rye grain (Secale cereale) for the young broiler chick. Poultry Science. 70, 2501 - 2508.
Fry, S. C. 1988. Wall polymers: chemical characterisation. In The growing plant cell wall: chemical and
metabolic analysis. pp. 102 – 187. Ed. Fry, S. C. Longman scientific & technical publication, New York.
George, R., McFarlane, D. and Nulsen, B. 1997. Salinity Threatens the Viability of Agriculture and
Ecosystems in Western Australia. Journal of Hydrology. 5, 6 - 21.
114 Gerendai, D., K.E.L. Sherif, and T. Gippert, 1997. The effect of Kemzyme and Phylacell enzyme preparation
on the utilization of broiler feeds containing sunflower meal. Proceedings of the Australian Poultry Science Symposium. 9, 211 – 214.
Giger-Reverdin, S. 2000. Characterisation of feedstuffs for ruminants using some physical parameters.
Animal Feed Science and Technology. 86, 53 – 69. Gladstones, J. S. 1970. Lupins as crop plants. Field Crop Abstract, 23, 123 - 148. Gladstones, J. S. 1974. Lupins in Western Australia. Department of Agriculture, Western Australia, Bulletin
No. 3834. Gladstones, J. S. 1982. Breeding the first modern crop lupins. Journal of Agriculture of Western Australia.
23, 70 - 76. Glencross, B. D., Carter, C. G., Duijster, N., Evans, D. R., Dods, K., McCafferty. P., Hawkins, W. E., Maas,
R. and Sipsas, S. 2004. A comparison of the digestibility of a range of lupin and soybean protein products when fed to either Atlantic salmon (Salmo salar) or rainbow trout (Oncorhynchus mykiss). Aquaculture. 237, 333 - 346.
Goh, B. and Gohl, I. 1977. The effect of viscous substances on the transit time of barley digesta in rats.
Journal of the Science of Food and Agriculture. 28, 911 – 915. Gohl, B., Alden, S., Elwinger, K. Thomke, S. 1978. Influence of beta-glucanase on feeding value of barley for
poultry and moisture content of excreta. British Poultry Science. 19, 41 - 47. Gorecki R J. Piotrowicz Cieslak A. Obendorf R L. 1997. Soluble sugars and flatulence-producing
oligosaccharides in maturing yellow lupin (Lupinus luteus) seeds. Seed Science and Research. 7, 185 – 193.
Graham, H. & Balnave, D. 1995, Dietary enzymes for increasing energy availability. In Biotechnology in
Animal Feeds and Animal Feeding. pp. 295 - 309. Ed. Wallace, J. R. and Chesson, A. VCH publication.
Grammer, J. C., McGinnis, J. and Pubols, M. H. 1982. The effect of a pectic enzyme on the growth-
depressing and rachetogenic properties of rye for chicks. Poultry Science. 61, 1891 – 1896. Grammer, J. C., Kirstein, D., McGinnis, J. and Pubols, M. H. 1980. The effects of pectic enzyme fractions on
chicks fed rye, wheat, pectin, and guar. Abstract. Poultry Science. 59, 1614. Grant, G. T. Morris, E. R., Rees. D. A., Smith, P. J. C. and Thom, D. 1973. Biological interactions between
polysaccharides and divalent cation: The egg-box model. FEBS Letters. 32, 195 – 198. Greve, L. C., Labavitch, J. M. and Hungate, R. E. 1984. α-Arabinofuranosidase from Ruminococcus albus 8:
purification and possible role in hydrolysis of alfalfa cell wall. Applied and Environmental Microbiology. 47, 1135 - 1140.
Griffiths, A. J. and Kennedy, J. F. 1988. Biotechnology of polysaccharides. In Carbohydrate Chemistry. Ed.
Kennedy, J. F. Chapter 14. pp. 597 – 635. Clarendon Press, Oxford. Gross, K. C. 1982. A rapid and sensitive spectrophotometric method for assaying polygalacturonase using 2-
cyanoacetamide. Hortscience. 17, 933 – 934. Guevara, M. L. M., Morel, P. C. H., Coles, G. D., Pluske, J. R., Monro, J. A. and Thomas, D. V. 1999.
Additive effects of β-glucan and heat treatment on nutrient digestibility and AME content in broiler chickens. Proceedings of the Australian Poultry Science Symposium. 11, 173.
115 Gupta, J. 1962. A possible enzymatic cause of viscid ruminal contents- its relationship to legume bloat.
American Journal of Veterinary Research. 23, 128 – 133. Gupta, J. and Nichols, R. E. 1962. A possible enzymatic cause of viscid ruminal contents-its relationship to
legume bloat. American Journal of Veterinary Research. 23, 128 - 133. Hagerman, A. E. and Austin, P. J. 1986. Continuous spectrophotometric assay for plant pectin methyl
esterase. Journal of Agriculture and Food Chemistry. 34, 440 – 444. Harris, D. J. and Jago, J. 1985. Report on chemical composition of kernels and hulls of sweet lupin seed from
Western Australia. Government Chemical Laboratory, Perth, Western Australia. Harris, P. J. and Smith, B. G. 2006. Plant cell walls and cell-wall polysaccharides: structures, properties and
uses in food products. International Journal of Food Science and Technology. 41, 129–143. Head, M. J. 1959. Bloat in cattle. Nature. 183, 757 - 757. Heinz, T. and Poppe, S. 1975a. Effect of thermal and mechanical treatment of feedstuffs on the true digestibility of amino
acids. 1. True digestibility of amino acids in soya oil meals after different heat treatments. Arch. Tierern. 25, 353 – 358.
Heinz, T. and Poppe, S. 1975b. Effect of heat and mechanical treatment of feedingstuffs on the true digestibility of the
amino acids. 3. Effect of pelleting cereal meals on the true digestibility of the amino acids. Arch. Tierern. 25, 589 – 592.
Hesselman, K., Elwinger, K., Nilsson, M. and Thomke, S. 1982. The effect of β-glucanase supplementation,
stage of ripeness and storage of barley diets fed to broiler chicks. Poultry Science. 16, 1 - 8. Hill, G. D. 1977. The composition and nutritive value of lupin seed. Nutrition Abstracts and Reviews. Part B.
47, 511 – 529. Huber, D. J. 1983. Polyuronide degradation and hemicellulose modifications in ripening tomato fruit. Journal
of the American Society for Horticultural Science. 108, 405 – 409. Hughes, R. J., Ross, G. M. and Hargreave G. 2004. Feed enzymes improve the nutritive value of faba beans.
Proceedings of the Australian Poultry Science Symposium. 16, 88. Hughes, R. J., Choct, M., Kocher, A. and van Barneveld, R. J. 2000. Effect of food enzymes on AME and
composition of digesta from broiler chickens fed on diets containing non-starch polysaccharides isolated from lupin kernel. British Poultry Science. 41, 318.
Hughes, R. J., Kocher, A. and Choct, M. 1998. Nutritive value of lupins for broilers. Proceedings of the
Australian Poultry Science Symposium. 10, 140 – 143. Hughes, B., Choct, M., Kocher, A. and van Barneveld, R. 1997. Do lupins need to be de-hulled for use in
broiler chicks? PPPI Research and extension highlights 1996. SARDI research publications, South Australia.http://www.sardi.sa.gov.au/livestck/pigspoul/highligh/reshigh6.htm
Igbasan, F. A. and Guenter, W. 1997. The influence of micronization, dehulling, and enzyme supplementation
on the nutritional value of peas for laying hens. Poultry Science. 76, 331 - 337. Igbasan, F. A., Guenter, W. and Slominski, B, A. 1997. The effect of pectinase and alpha-galactosidase
supplementation on the nutritive value of peas for broiler chickens. Canadian Journal of Animal Science. 77, 537 – 539.
Igbasan, F. A. and Guenter, W. 1996. The evaluation and enhancement of the nutritive value of yellow-,
green- and brown-seeded pea cultivars for unpelleted diets given to broiler chickens. Animal Feed Science and Technology. 63, 9 – 24.
116 Ikeda, K. and Kusano, T. 1983. Invitro inhibition of digestive enzymes by indigestible polysaccharides.
Cereal Chemistry. 60, 260 – 263. Inborr, J. 1990. Practical application of feed enzymes. Feed Compounder. 10, 41 – 49. Irish, G. G., Barbour G. W., Classen, H. L., Tyler, R. T. and Bedford, M. R. 1995. Removal of the alpha-
galactosides of sucrose from soybean meal using either ethanol extraction or exogenous alpha-galactosidase and broiler performance. Poultry Science 74, 1484 – 1494.
Irish, G. G. and Balnave, D. 1993. Non-starch polysaccharides and broiler performance on diets containing
soybean meal as the sole protein concentrate. Australian Journal of Agricultural Research. 44,1483–1499.
James, W. P. T., Branch, W. J. and Southgate, D. A. T. 1978. Calcium binding by dietary fibre. Lancet. 80,
638 – 639. Jansen, E. F. and MacDonnell, L. R. 1945a. Influence of methoxyl content of pectic substances on the action
of polygalacturonase. Archives of Biochemistry and Biophysics. 8, 97 – 112. Jansen, E. F. and MacDonnell, L. R. 1945b. Simultaneous action of polygalacturonase and pectin esterase on
pectin. Archives of Biochemistry and Biophysics. 8, 113 – 118. Janssen, W. M. M. A. and Carre, B. 1985. Influence of fibre on digestibility of poultry feeds. 71 – 86. In
Recent Advances in Animal Nutrition. Haresign, W. and Cole, D. J. A. Ed. Butterworths, London, UK.
Jarvis, M. C. 1984. Structures and properties of pectin gels in plant cell walls. Plant, Cell and Environment. 7,
153 – 164. Johnson, Jr., J. C., Miller, J. D., and Bedell, D. M. 1986. Tifwhite-78 lupin seed as a feedstuff for cattle.
Journal of Dairy Science. 6, 142 - 147. Joseleau, J. P., Comtat, J. and Ruel, K. 1992. Chemical structure of xylans and their interaction in the plant
cell walls. In xylans and xylanases. Progress in Biotechnology. Vol. 7, 1 - 15. [ed.] J. Visser. Elsevier science publishers. Wageningen. The Netherlands.
Judd, P. A. and Truswell, A. S. 1985. The hypocholesterolaemic effects of pectins in rats. British Journal of
Nutrition. 53, 409 – 425. Juskiewicz, J. and Zdunczyk, Z. 1997. The utilization of protein and mineral components of diets containing
white lupin or casein supplemented with lupin hulls, manganese and sodium phytate. Journal of Animal Feed Science. 6, 413 – 422.
Karunajeewa, H. and Bartlett, B. E. 1985. The effects of replacing soybean meal in broiler starter diets with
white lupine lupinus-albus cultivar hamburg seed meal of high manganese content. Nutrition Reports International. 31, 53 – 58.
Keller, T. and Jeroch, H. 1997. Effect of different microbial carbohydrases in its combination of preparation
and application rate on contents of N-corrected metabolizable energy of dried peas (Pisum sativum) on broiler chicks Archiv Fur Geflugelkunde. 61, 131 - 134.
Kertesz, Z. I. 1951. Pectic enzymes. In The pectic substances. pp 337 - 358. Interscience publishers Inc. N.Y. Kertesz, Z. I. and McColloch, R. J. 1950. Enzymes acting on pectic substances. Advances in Carbohydrate
Chemistry. 5, 79 – 102. Kertesz, Z. I. 1940. Pectic enzymes. V. The fate of pectins in the animal body. Journal of Nutrition. 20, 289 –
296.
117 Kester, H. C. M., Benen, J. A. E., Visser, J., Warren, M. E., Orlando, R. and C. Bergmann. 2000. Tandem
mass spectrometric analysis of Aspergillus niger pectin methylesterase: mode of action on fully methylesterified oligogalacturonates. Biochemical Journal. 346, 469 – 474.
Kester, H. C. M. and Visser, J. 1990. Purification and characterization of polygalacturonases produced by the
hyphal fungus Aspergillus niger. Biotechnology and Applied Biochemistry. 12,150 – 160. Kim, C. S. and Madhusudhan, K. T. 1988. Hemagglutinating and trypsin inhibitor activities of lupine seed
(Lupinus-angustifolius). Journal of Food Science. 53, 1234 – 1235. Kim, D.W., Kim, D.G., Lee, D.Y., Kim, K.E. and Kim C.W. 2000. Physicochemical characterization of
pectin extracted from Cheju mandarin (Citrus unshiu) peels with citric acid. Food Science and Biotechnology. 9, 95 – 98.
Kim, J. B. and Carpita, N. C. 1992. Changes in esterification of the uronic acid groups of cell wall
polysaccharides during elongation of maize coleoptiles. Plant Physiology. 98, 646 – 653. Kim, J. C., Mullan, B. P., Simmins P. H. and Pluske J. R. 2003. Variation in the chemical composition of
wheats grown in Western Australia as influenced by variety, growing region, season, and post-harvest storage. Australian Journal of Agricultural Research. 54, 541-550.
Kiosseoglou, A., Doxastakis, G., Alevisopoulos. S. and Kasapis. S. 1999. Physical characterization of
thermally induced networks of lupin protein isolates prepared by isoelectric precipitation and dialysis. International Journal of Food Science and Technology. 34, 253-63.
Kirstein, D. and D. McGinnis, J. 1979. Effect of Irgazyme 100 on the feeding value of wheat for broiler
chicks. Poultry Science. 58, 1073 – 1074. Knee, M., Fielding, A. H., Archer, S. A. and Laborda, F. 1975. Enzymatic analysis of cell wall structure in
apple fruit cortical tissue. Phytochemistry. 14, 2213 – 2222. Kocher, A., Choct, M., Morrisroe, L. and Broz, J. 2001. Effect of enzyme supplementation on the replacement
value of canola meal for soybean meal in broiler diets. Australian Journal of Agricultural Research. 52, 447 – 452.
Kocher, A. 2000. Enzymatic degradation of non-starch polysaccharides in vegetable proteins in poulty diets.
PhD Thesis. University of New England, Armidale, Australia. Kocher, A. Choct, M., Hughes, R. J. and Broz, J. 2000a. Effect of food enzymes on utilisation of lupin
carbohydrates by broilers. British Poultry Science. 41, 75-82. Kocher, A., Choct, M., Porter, M. D. and Broz, J. 2000b. The effects of enzyme addition to broiler diets
containing high concentrations of canola or sunflower meal. Poultry Science. 79, 1767 – 1774. Kocher, A., Hughes, R. J. and Choct, M. 1999. Lupin oligosaccharides: nutrients or anti-nutrients?.
Proceedings of the Australian Poultry Science Symposium. 11, 120 – 123. Kollar A. 1998. Characterization of an endopolygalacturonase produced by the apple scab fungus, Venturia
inaequalis. Mycological Research. 102, 313 – 319. Konovalov, A. I., Mironov, V. F., Sosnina, N. A., Karaseva, A. N., Karlin, V. V., Minzanova, S. T., Lapin, A.
A., Takunov, I. S. and Kononov, A. S. 1999. Polysaccharides from lupin plants: isolation and structure determination. Lupin- An Ancient crop for the new millennium. Proceeding of the 9th International Lupin Conference. Klink, Muritz, Germany. pp. 151.
Krause, J.-P., Bagger, C. and Schwenke, K. D. Rheological properties of modified lupin proteins. 2001.
Nahrung. 6, 412 – 415.
118 Kravtchenko, T. P., Penci, M., Voragen, A. G. J. and W. Pilnik. 1993. Enzymatic and chemical degradation of
some industrial pectins. Carbohydrate Polymers. 20, 195 – 205. Lacassange, L. 1983. The limits of lupin seed incorporation in broiler diets (L. albus var. Kalina). Prospective
for peas and lupins as protein crops. World Crops. 8, 365 – 371. Langhout, D. J., Schutte, J. B., de Jong, Sloetjes, H., Verstegen, M. W. A. and Tamminga, S. 2000. Effect of
viscosity on digestion of nutrients in conventional and germ-free chicks. British Journal of Nutrition. 83, 533 - 540.
Langhout, D. J., Schutte, J. B., Van Leeuwen, P., Wiebenga, J. and Tamminga, S. 1999. Effect of dietary
high- and low-methylated citrus pectin on the activity of the ileal microflora and morphology of the small intestinal wall of broiler chicks. British Poultry Science. 40, 340 - 347.
Langhout, D. J. and Schutte, J. B. 1996. Nutritional implications of pectins in chicks in relation to
esterification and origin of pectins. Poultry Science. 75, 1236 - 1242. Larbier, M. and Leclercq, B. 1994. Processing of diets and nutritional consequences. In Nutrition and feeding
of poultry. INRA, France. Translated by Wiseman, J. Nottingham University Press, UK. Lazaro, R., Garcia, M., Aranibar, M. J. and Mateos, G. G. 2003. Effect of enzyme addition to wheat-, barley-
and rye-based diets on nutrient digestibility and performance of laying hens. British Poultry Science. 44, 256 – 265.
Lee, B. D. and Campbell, L. D. 1983. Influence of rye and dietary salt level on water and sodium metabolism
in intact and colostomized roosters. Poultry Science. 62, 472 – 479. Lee, S. F. and Forsberg. W. 1987. Purification and characterisation of an α-L-arabinofuranosid-ase from
Clostridium acetobutylicum ATCC 824. Applied and Environmental Microbiology. 53, 644 - 650. Leeson, S. and Summer, J. D. 1997. Ingredient evaluation and diet formulation. In Commercial Poultry
Nutrition. pp. 10 – 111. University Book publication, Ontario, Canada. Leeson, S. and Atteh, J. O. 1995. Response of broiler chicks to dietary full-fat soybeans extruded at different
temperatures prior to or after grinding. Animal Feed Science and Technology. 57, 239 - 245. Leske, K. L., Jevne, C. J., and Coon, C. N. 1993. Extraction methods for removing soybean alpha-
galactosides and improving true metabolizable energy for poultry. Animal Feed Science and Technology. 41, 73 – 78.
Liebert, F. and Wecke, C. 1998. Comparative studies on different treatments of the raw materials cotton seed,
wheat bran and peas as well as the feeding value of compound broiler feeds with high contents of these components. (Abstract). Die Muhle und Mischf. 135, 336 – 340.
Liebert F. 1995. Influence of different energy supply on nutrient- and energy utilization of broilers.
[Abstract]. Archiv Fuer Geflueg. 59, 269 – 273. Liners, F. and van Cutsom, P. 1992. Distribution of pectic polysaccharides throughout walls of suspension-
cultured carrot cells. An immunocytochemical study. Protoplasma. 170, 10 – 21. Liu, Y. K. and Luh, B. S. 1980. Quantitative aspects of pectic acid hydrolysis by endo-polygalacturonase
from Rhizopus arrhizus. Journal of Food Science. 45, 601 – 604. Longstaff, M. and McNab J. M. 1989. Digestion of fiber polysaccharides of pea (Pisum sativum) hulls, carrot
and cabbage by adult cockerels. British Journal of Nutrition. 62, 563 – 577.
119 Longstaff, M. A., Knox, A. and McNab, J .M. 1988. Digestibility of pentose sugars and uronic acids and their
effect on chick weight gain and cecal size. British Poultry Science. 29, 379 – 394. Longstaff, M. and Mcnab, J. M. 1986. Influence of site and variety on starch hemicellulose and cellulose
composition of wheats and their digestibilities by adult cockerels. British Poultry Science. 27, 435 – 449.
Luh, B. S. and Phaff, H. J. 1954a. Properties of yeast polygalacturonase. Archives of Biochemistry and
Biophysics. 48, 23 – 37. Luh, B. S. and Phaff, H. J. 1954b. End products and mechanism of hydrolysis of pectin and pectic acid by
yeast polygalacturonase (YPG). Archives of Biochemistry and Biophysics. 51, 102 – 113. MacAuliffe, T., Pietraszk, A. and McGinnis, J. 1976a. The effect of adding penicillin to diets with different
levels of D3 on the performance of turkey poults. Poultry Science. 55, 183 - 187. MacAuliffe, T., Pietraszk, A. and McGinnis, J. 1976b. Variable rachitogenic effects of grain and alleviation
by excretion or supplementation with vit. D3, fat and antibiotics. Poultry Science. 55, 2142 - 2147. Maga, J. A. 1978. Cis-trans fatty acids rations as influenced by product and temperature of extrusion cooking.
Lebensmittel Wissenschaft und Technologie. 11, 183 – 184. Manning, L., Chadd, S. A. and Baines, R. N. 2007. Water consumption in broiler chicken: a welfare indicator.
World Poultry Science Journal. 63, 63 - 71. Manunza, B., Deiana, S., Pintore, M. and Gessa, C. 1998. Interaction of Ca++ and Na+ ions with
polygalacturonate chains: A molecular dynamics study. Glycoconjugate Journal. 15, 297 – 300. Manunza, B., Deiana, S., Pintore, M. and Gessa, C. 1997. A molecular dynamics investigation on the
occurrence of helices in polygalacturonic acid. Journal of Molecular Structure. 419, 169 – 172. Matsuura, Y. and Hatanaka, C. 1990. Matrix polysaccharides involved in adhesion of the cell walls of kidney
bean cotyledon tissues. Agricultural and Biological Chemistry. 54, 3013 – 3014. Mavrakis, C., Doxastakis, G. and Kiosseoglou, V. 2003. Large deformation properties of gels and model
comminuted meat products containing lupin protein. Journal of Food Science. 68, 1371 – 1376. May C. D. 1990. Industrial pectins: Sources, production and applications. Carbohydrate Polymers. 12, 79 –
99. McCready, R. M. 1970. Pectin. In Methods in Food Analysis: physical, chemical and instrumental methods of
analysis. pp. 575 – 599. Ed. Joslyn, M. A. 2nd Ed. Academic Press. New York. McCready, R. M. and McComb, E. A. 1954a. Extraction and determination of total pectic materials in fruits.
Analytical Chemistry. 12, 1986 – 1988. McCready, R. M. and McComb, E. A. 1954b. Pectic constituents in ripe and unripe fruit. Food Research. 19,
530 – 535. McCready, R. M. and Seegmiller, C. G. 1954. Action of pectic enzymes on oligogalacturonic acids and some
of their derivatives. Archives of Biochemistry and Biophysics Biophys. 50, 440 – 450. McCready, R. M. and McComb, E. A. 1953. Enzymes. Course of action of polygalacturonase on
polygalacturonic acids. Journal of Agriculture and Food Chemistry. 1, 1165 – 1168. McNab, J. M. and Shannon, D. W. F. 1974. The nutritive value of barley, maize, oats and wheat for poultry.
British Poultry Science. 15, 561 – 567.
120 McNab, J. M. and Shannon, D. W. F. 1975. The nutritive value of triticale and rye for the laying hen. British
Poultry Science. 16, 9 – 15. McNeil, M., Darvill, A. G., Fry, S. C. and Albersheim, P. 1984. Structure and function of the primary cell
walls of plants. Annual Review of Biochemistry. 53, 625 – 663. Mercedes, M., Ridout, C. L., Price, K. R. and Fenwick, G. R. 1993. The saponin content and composition of
sweet and bitter lupin seed. Journal of the Science of Food and Agriculture. 63, 47-52. Moran, E. T. and McGinnis, J. 1968. Growth of chicks and turkey poults fed western barley and corn- based
rations: Effect of autoclaving on supplemental enzyme requirement and asymmetry of antibiotic response between grains. Poultry Science. 47, 152 – 158.
Moran, T. H. and McHugh, P. R. 1982. Cholecystokinin suppresses food intake by inhibiting gastric
emptying. American Journal of Physiology. 242, R491 – R497. Morris, E. R., Powell, D. A., Gidley, M. J. and Rees D. A. 1982. Conformations and interactions of pectins. I.
Polymorphism between gel and solid states of calcium polygalacturonate. Journal of Molecular Biology. 155, 507 – 516.
NHMRC, 1991. Food Standard Code. Australian Government Publishing Service, Canberra, Australia. pp.
B1-4 – B1-5. Nichols, R. E., Dillon, R. D., Penn, K., Bryant, J. and Schreiber, J. 1957. Effects of various surface active
agents on the surface tension and other properties of paunch fluid. Veterinary Medicine. 52, 285. Nichols, R. E. and Deese, D. C. 1966. Bloat producing capacity and pectin methyl esterase activity of alfalfa
stands of various moisture levels. American Journal of Veterinary Research. 27, 623 – 627. Nissinen V. 1994. Enzyme and processing: the effects and interaction of enzyme and hydrothermal
pretreatments and their contribution to feeding value. International Milling Flour and Feed. 21-22. Northcote, D. H. 1958. The cell walls of higher plants; their composition, structure and growth. Biological
Review. 33, 53 – 103. Oi, S. and Satomura, Y. 1965. Increase in viscosity and gel formation of fruit juice by purified pectinesterase.
Agricultural and Biological Chemistry. 29, 936 – 942. Olver, M. D. & Jonker, A. 1997. Effect of sweet, bitter and soaked micronised bitter lupins on broiler
performance. British Poultry Science. 38, 203 – 208. Patel, M. B. and McGinnis, J. 1985. The effect of autoclaving and enzyme supplementation of guar
cyamopsis-tetragonoloba meal on the performance of chicks and laying hens. Poultry Science. 64, 1148 – 1156.
Patel, M. B., McGinnis, J. and Publos, M. H. 1981. Effect of dietary cereal grain, citrus pectin, and guar gum
on liver fat in laying and hens chicks. Poul. Sci. 60, 631 – 636. Patel, M. B. and McGinnis, J. 1980. Effect of gamma irradiating rye or supplementing a rye-containing layer
diet with penicillin or pectic enzymes on egg production. Poultry Science. 59, 10, 2287 – 2289. Pearson, G. and Carr, J. R. 1977. Lupin-seed meal (L. angustifolius var. Uniwhite) as a protein supplement to
barley based diets for growing pigs. Animal Feed Science and Technology. 1, 631 - 638. Penn, K. E., Deese, D. C. and Nichols, R. E. 1966. A factor in ruminal contents that inhibits the gelling of
pectin by pectin methyl esterase--its relationship to legume bloat. American Journal of Veterinary Research. 27, 369 – 372.
121 Perez-Escamilla, R. Vohra, P. and Klasing, K. 1988. Lupins (Lupinus albus var. Ultra) as a replacement for
soya bean meal in diets for growing chickens and turkeys poults. Nutrition Reports International. 38, 583 – 593.
Petersen, C. F. and Sauter, E. A. 1968. Enzyme sources and their value in barley rations for chick growth and
egg production. Poultry Science. 47, 1219 – 1224. Petterson, D. S., Sipsas, S. and Mackintosh, J. B. 1997. The chemical composition and nutritive value of
Australian pulses. 2nd edition. Grain Research and Development Corporation, Canberra. Petterson, D. S. and Mackintosh, J. B. 1994. The chemical composition and nutritive value of Australian grain
legumes. Grain Research and Development Cooperation (GRDC) Canberra. pp. 10 – 3. Pettersson, D. and Aman, P. 1988. Effects of enzyme supplementation of diet based on wheat, rye and triticale
on their productive value for broiler chickens. Animal Feed Science and Technology. 20, 313 – 324. Pettersson, D. and Aman, P. 1989. Enzyme supplementation of a poultry diet containing rye and wheat.
British Journal of Nutrition. 62, 139 - 149. PGH (Premium Grain Handlers, Western Australia) 2007. Information on nutrient composition of barley.
http://www.pgh.com.au/products.php Pilnik, W., Rombouts, F. M. and Voragen, A. G. J. 1973. On the classification of pectin developments:
activity of pectin deplomerases on glycol esters of pectate as new classification criterion. Chem. Microbiol. Technol. 2, 122 - 128.
Pilnik, W. and Voragen, A. G. J. 1970. Pectic substances and other uronides. In The Biochemistry of fruits
and their products. Vol. 1. pp. 53 – 87. Ed. Hulme, A. C. Academic Press, New York. Pipa, F. and Frank, G. 1989. High-pressure conditioning with annular gap expander. A new way of feed
processing. Advances in Feed Technology. 2, 22 - 30. Plavnik, I. and Sklan, D. 1995. Nutritional effects of expansion and short time extrusion on feeds for broilers.
Animal Feed Science and Technology. 55, 247 – 251. Polizeli, M. L. T. M., Jorge, J. A. and Terenzi, H. 1991. Pectinase production by Neurospora crassa:
purification and biochemical characterisation of extracellular polygalacturonase activity. Journal of General Microbiology. 137, 1815 – 1823.
Powell, D. A., Morris, E. R., Gidley, M. J. and Rees D. A. 1982. Conformations and interactions of pectins.
II. Influences of residue sequence on chain association in calcium pectate gels. Journal of Molecular Biology. 155, 517 – 531.
Pressey, R. and Avants, J. K. 1971. Effect of substrate size on the activity of tomato polygalacturonase. J.
Food Sci. 36, 486 – 489. Pressey, R. and Avants, J. K. 1982. Solubilization of cell walls by tomato polygalacturonases: effects of
pectinesterase. Journal of Food Biochemistry. 6, 57 – 74. Proctor, A. and Peng, L. C. 1989. Pectin transition during blueberry fruit development and ripening. Journal
of Food Science. 54, 385 – 387. Qureshi, A. A. 1993. Lupins as an alternative source of protein. Misset World Poultry. 9, 10 - 13. Rackis, J. J. 1975. Oligosaccharides of food legumes: alpha-galactosidase activity and flatus problem. In
Physiological effects of food carbohydrates. pp. 207 – 222. Ed. Allen, J. and Heilge, Journal of American Chemistry Society. Washington, D. C.
122 Rassmussen, P. B. and Petterson, D. 1997. A multi component carbohydrase improves the production
performances of broilers fed diets containing soybean meal in mixtures or soybean meal and canola meal as plant protein source. Proceedings of the Australian Poultry Science Symposium. 9, 157 – 161.
Ravindran, V., Cabahug, S., Ravindran, G., Selle, P. H. and Bryden, W. L. 1999a. Influence of microbial
phytase on apparent ileal amino acid digestibility of feedstuffs for broilers. Poultry Science. 78, 677 - 706.
Ravindran, V. P. H. Selle and W. L. Bryden. 1999b. Effects of phytase supplementation, individually and in
combination with glycanase on the nutritive value of wheat and barley. Poultry Science. 78, 1588 - 1595.
Ravindran, V., Bryden, W. L. and Kornegay, E. T. 1995. Phytates: occurrence, bioavailability and
implications in poultry nutrition. Poultry and Avian Biological Review. 6, 125 - 143. Rebole, A., Rodriguez, M. L., Alzueta, C., Ortiz, L. T. and Trevino, J. 1999. A short note on effect of enzyme
supplement on the nutritive value of broiler chick diets containing maize, soya bean meal and full-fat sunflower seed. Animal Feed Science and Technology. 78, 153 – 158.
Rees, D. A. and Wight, A. W. 1969. Molecular cohesion in plant cell walls. Methylation analysis of pectic
polysaccharides from the cotyledons of white mustard. Biochemical Journal. 115, 431 – 439. Reichert, R. D. 1981. Quantitative isolation and estimation of cell wall materials from dehulled pea flour and
concentrates. Cereal Chemistry. 54, 266 – 270. Reid, W. W. 1952. The pectic enzymes of the fungus Byssochlamys fulva. Biochemical Journal. 50, 289 –
292. Reid, W. W. 1950. The enzymatic degradation of pectin and other polysaccharides. 1. Introduction and a
preliminary study on the degradation of the polysaccharides of fruits by the enzymes produced by micro-fungi (moulds). Journal of the Science of Food and Agriculture. 1, 234 – 240.
Rexova-Benkova, B, L. R., Marcokva, M., Luknar, O. and Kohn, R. 1977. Pectic enzymes. Collection of
Czechoslovak Chemical Communications. 42, 3204 – 3213. Rexova-Benkova, B, L. R. and Markovic, O. 1976. Pectic enzymes. Advances in Carbohydrate Chemistry
and Biochemistry. 33, 323 – 385. Ricke, S. C. Aar, P. J. van Der. Fahey, G. C., Jr. Berger, L. L. 1982. Influence of dietary fibers on
performance and fermentation characteristics of gut contents from growing chicks. Poultry Science. 61, 1335 – 1343.
Ritz, C. W., Hulet, M. R., Self, B. B. and Denbow, D. M. 1995. Effect of protein level and enzyme
supplementation upon the growth and rate of digesta passage of male turkeys. Poultry Science. 74, 1323 - 1328.
Robertson, J. A. and Eastwood, M. A. 1981a. An examination of factors which may affect the water holding
capacity of dietary fibre. British Journal of Nutrition. 45, 83 – 88. Robertson, J. A. and Eastwood, M. A. 1981b. An investigation of factors which may affect the water-holding
capacity of dietary fibre. Journal of the Science of Food and Agriculture. 32, 819 – 825. Rolin, C. and de Vries, J. 1990. Pectin. In Food gels. Chapter 10. Ed. Harris, P. pp. 401 – 434. Elsevier
Applied Science Publication, London.
123 Rombouts, F. M. and Thibault, J.-F. 1986. Sugar beet pectins: chemical structure and gelation through
oxidative coupling. In Chemistry and function of pectins. pp. 49 – 60. Ed. Fishman, M. L. and Joseph, J. J. American Chemical Society (ACS) symposium, series 310. Washington.
Rombouts, F. M. and Pilnik, W. 1980. Pectic enzymes. In Economic microbiology. 5, 277 – 282. Ed. Rose, A.
H. Academic Press. London. Ruiz, R. G., Price, K. R., Rose, M. E., Arthur, A. E., Petterson, D. S. and Fenwick, R. 1995. The effect of
cultivar and environment on saponin content of Australian sweet lupin seed. Journal of the Science of Food and Agriculture. 69, 347 - 351.
Saini, H. S. and Gladstones, J. S. 1986. Variability in the total and component galactosyl sucrose
oligosaccharides of lupinus species. Australian Journal of Agricultural Research. 37, 157 – 166. Sajjaanantakul, T. and Pitifer, L. A. 1991. Pectinesterase. In The Chemistry and Technology of Pectins. Ed.
Walter, R. H. pp. 135 – 164, Academic Press, San Diego, CA. Sakai, T., Sakamoto, T., Hallaert, J. and Vandamme, E. J. 1993. Pectin, pectinase and protopectinase:
production, properties and applications. In Advances in applied microbiology. Ed. Neidleman, S. and Laskin, A. I. 39, 213 – 294. Academic Press, New York.
Sakai, S. and Ozaki, Y. 1988. Protopectin solubilizing enzyme that does not catalyze the degradation of
polygalacturonic acid. Agricultural and Biological Chemistry. 52, 1091 – 1093. Salih, M. E., Classen, H. L. and Campbell, G. L. 1991. Response of chickens fed on hulless barley to dietary
beta-glucanase at different ages. Animal Feed Science and Technology. 33, 139 – 149. Savory, C. J. and M. J. Gentle, 1980. Intravenous injection of cholecystokinin and caerulin suppress food
intake in fowls. Experientia Basel. 36, 1191 – 1197. Schaus, E. E., de Lumen, B. O., Chow, F. I., Reyes, P. and Omaye, S. T. 1985. Bioavailability of vitamin E in
rats fed graded levels of pectin. Journal of Nutrition. 115, 263 – 270. Schejter, A. and Marcus, L. 1988. Isozymes of pectinesterase and polygalacturonase from Botrytis cinerea
Pers. Methods in Enzymology. 161, 366 – 367. Scott, T. A., Kampen, R., and Silversides, F. G. 1999. The effect of phosphorus, phytase enzyme, and calcium
on the performance of layers fed corn-based diets. Poultry Science. 78, 1742 – 1749. Scott, T. A., Swift, M. L. and Bedford, M. R. 1997. The influence of feed milling, enzyme supplementation,
and nutrient regimen on broiler chick performance. Journal of Applied Poultry Research. 6, 391 – 398.
Selle, P. H., Ravindran, V., Caldwell, R. A. and Bryden, W. L. 2000. Phytate and phytase: consequences for
protein utilization. Nutrition Research Reviews. 13, 255 - 278. Selvendran, R. R. 1985. Development in the chemistry and biochemistry of pectic and hemicellulosic
polymers. Journal of Cell Science. Supplement. 2, 51 - 88. Sethu, K. M. P., Prabha, T. N. and Tharanathan, R. N. 1996. Post-harvest biochemical changes associated
with the softening phenomenon in Capsicum annum fruits. Phytochemistry. 42, 961 – 966. Sherif, K. E., Gerendai, D. and Gippert, T. 1997a. Complete substitution of sunflower meal for soybean meal
with or without enzyme supplementation in broiler rations. Archiv fur Geflugelkunde. 61, 8–14. Sherif, K.E.L., Gerendai, D. and Gippert, T. 1997b. Substitution of soyabean meal by sunflower meal with or
without enzyme supplementation in broiler diets. Proceedings of the Australian Poultry Science Symposium. 9, 195 – 198.
124 Sipsas, S. 1994. Australian sweet lupin (L. angustifolius): Variations in anti-nutritional factors. Proceedings
of the 1st Australian Lupin Technical Symposium. Perth, Western Australia. 308 – 309. Slominski, B. A., Campbell, L. D. and Guenter, W. 1994. Oligosaccharides in canola meal and their effect on
non-starch polysaccharide digestibility and true metabolizable energy in poultry. Poultry Science. 73, 156 – 162.
Slominski, B. A. and Campbell, L. D. 1990. Non-starch polysaccharides of low glucosinolate rapeseed
(canola) meal: quantification, digestibility in poultry and potential benefit of dietary enzyme supplementation. Journal of the Science of Food and Agriculture. 53, 175 - 184.
Smetana, P. and Morris, R. H. 1972. Lupin seed meal in broiler rations. Proceedings of the Australasian
Poultry Science Convention. pp. 209 – 217. Auckland, New Zealand. Smith, C. G. 2003. The Australian lupin collection - Annual report. Plant Industries, The Department of
Agriculture and Food Western Australia. Standing Committee on Agriculture (SCA) - Poultry subcommittee. 1987. Feeding standards for Australian
livestock, Poultry. CSIRO, Melbourne. Australia. Steenfeldt, S., Gonzalez, E. and Bach Knudsen, K. E. 2003. Effects of inclusion with blue lupins (Lupinus
angustifolius) in broiler diets and enzyme supplementation on production performance, digestibility and dietary AME content. Animal Feed Science and Technology. 110, 185–200.
Taverner, M. R., Curic, D. M. and Rayner, C. M. 1983. A composition of the extend and site of energy and
protein digestion of wheat, lupin and meat-and-bone meal by pigs. Journal of the Science of Food and Agriculture. 34, 122 – 128.
Taverner, M. R. 1975. Sweet lupin seed meal as a protein source for growing pigs. Animal Production. 20,
413 - 419. Themmen, A. P. N., Tucker, G. A. and Grierson, D. 1982. Degradation of isolated tomato cell walls by purified
polygalacturonase in vitro. Plant Physiology. 69, 122 - 124. Thibault, J. F., Renard, C. M. G. C., Axelos, M. A. V., Roger, P. and Crepeau, M. J. 1993. Studies of the
length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydrate Research. 238, 271 – 286.
Thibault, J. F. and Mercier, C. 1978. Aspergillus niger endopolygalacturonase: 2. Characterization and some
properties. Journal of Food Biochemistry. 2, 379 – 393. Tomoda M. and Kitamura, M. 1967.The polysaccharide from Lupinus luteus seed. II. Purification and
properties of two polysaccharides. Chemical and Pharmaceutical Bulletin. 15, 1021 – 1024. Um, J. S. and Paik, I. K. 1999. Effects of microbial phytase supplementation on egg production, eggshell
quality, and mineral retention of laying hens fed different levels of phosphorus. Poultry Science. 78, 75-79.
Underwood, E. J. and Gladstones, J. S. 1979. Subterranean clover and other legumes. In: G.H. Burvill, Editor,
Agriculture in Western Australia 1829–1979, University of Western Australia Press, Perth, Western Australia. pp. 139–156.
van Alebeek, G. J.,W. M., Zabotina, O., Beldman, G., Schols, H. A. and Voragen, A. G. J. 2000.
Esterification and glycosidation of oligogalacturonides: examination of the reaction products using MALDI-TOF MS and HPAEC. Carbohydrate Polymers. 43, 39 – 46.
125 van Buren, J. P. 1991. Function of pectin in plant tissue structure and firmness. In The chemistry and
technology of pectin. pp. 1 – 22. Ed. Walter, R. H. Academic Press publication, San Diego, CA. van der Klis, J. D. 1993. Effect of endoxylanase on the nutritive value of different wheat varieties for broilers.
Journal of the Science of Food and Agriculture. 63, 113 – 114. van der Klis, J. D. van der and Voorst, A. van der. 1993. The effect of carboxymethyl cellulose (soluble
polysaccharide) on the on rate of marker excretion from the gastrointestinal tract of broilers. Poultry Science. 72, 503 - 512.
van Kampen, M. 1983. Effect of water : Food intake ratios in laying hens on food metabolisable energy.
British Poultry Science. 24, 169 – 172. Veldman, A., Veen, W. A. G., Barug, D. and van Paridon, P. A. 1993. Effect of α-galactosides and α-
galactosidase in feed on ileal piglet digestive physiology. Journal of Animal Physiology and Animal Nutrition. 69, 57 – 65.
Verma, S. V. S. and McNab, J. M. 1982. Guar meal in diets for broiler chickens. Poult. Sci. 23, 95 – 105. Vest, L. 1996. Influence of expanders on broiler performance. Poultry Digest. 55, 18 – 24. Vohra, P. and Kratzer, F. S. 1964. Growth inhibitory effect of certain polysaccharides for chickens. Poultry
Science. 43, 1164 – 1170. Vukic-Vranjes, M. and Wenk, C. 1995. The influence of extruded vs. untreated barley in the feed, with and
without dietary enzyme supplement on broiler performance. Animal Feed Science and Technology. 54, 21 - 32.
Vukic-Vranjes, M., Pfirter, H. P. and Wenk, C. 1995. Influence of processing on dietary enzyme effect and
nutritive value of diets for laying hens. Canadian Journal of Animal Science. 75, 453 – 460. Vukic-Vranjes, M., Pfuter, H. P. and Wenk, C. 1994. Influence of processing treatment and type of cereal on
the effect of dietary enzymes in broiler diets. Animal Feed Science and Technology. 46, 261 – 270. Wagner, D. D. and Thomas, O. P. 1978. Influence of diets containing rye or pectin on the intestinal flora of
chicks. Poultry Science. 57, 971 – 975. Wagner, D. D. and Thomas, O. P. 1977a. A rye type depression of chicks fed pectin. Poultry Science. 56, 615
– 619. Wagner, D. D. and Thomas, O. P. 1977b. Influence of rye and pectin on the intestinal flora of chicks. Poultry
Science. 56, 1766 – 1767. Wallner, S. J. and Bloom, H. L., Characteristics of tomato cell wall degradation in vitro. 1977. Implications
for the study of fruit-softening enzymes. Plant Physiology. 60, 207 – 210. Watkins, B. A., Manning, B. and Al Athari, A. K. 1988. The effects of lupinus-albus cultivar ultra on broiler
performance. Nutrition Reports International. 38, 173 – 182. Watkins, B. A. and Mirosh, W. 1987. White lupin as a protein source for layers. Poultry Science. 66, 1798 –
1806. Watkins, J. B. 1964. Changes in the pectic substances of stored Elberta peaches. Queensland Journal of
Agricultural Science. 21, 47 – 58. White, W., Bird, H. R., Sunde, M. L., Prentice, N., Burger, W. C. and Marlett, J. A. 1981. The viscosity
interaction of barley beta-glucan with Trichoderma viride cellulase in the chick intestine. Poultry Science. 60, 1043 - 1048.
126 Wiggins, H. S. 1984. Nutritional value of sugars and related compounds undigested in the small gut.
Proceedings of the Nutrition Society. 43, 69 – 75. Wikipedia, 2008. Cell-wall structure and pectin content. http://en.wikipedia.org/wiki/Cell_wall Wilkie, K. C. B. 1985. New perspectives on non-cellulosic cell-wall polysaccharides (hemicelluloses and
pectic substances) of new lands. In Society for Experimental Biology. Ed. Brett, C. T. and Hillman, J. R. Seminar series. 28, 1 – 37.
Willats, W. G. T., McCartney, L., Mackie, W. and Knox , J. P. 2001. Pectin: cell biology and prospects for
functional analysis. Plant Molecular Biology. 47, 9 – 27. Williams, R. B. 1996. The ratio of the water and food consumption of chickens and its significance in the
chemotherapy of coccidiosis. Veterinary Research Communications. 20, 437 - 447. Wittert, G. A., Fraser, R. and Morley, H. E. 1997. The endocrine system of the gastrointestinal tract. In
Endocrinology; basic and clinical principles. pp. 325 – 348. Ed. Conn, P. M. and Melmed, S. Uhmana Press, Totowa, New Jersey.
Wong, K. K. Y. and Saddler, J. N. 1992. Trichodem xylanases, their properties and application. Critical
Reviews in Biotechnology. 12, 413 - 435. Wood, T. M. and McCrea, S. I. 1979. Synergism between enzymes involved in the solubilisation of native
cellulose. In: Hydrolysis of cellulose, mechanism of enzymatic and acid catalysis. Advances in Chemistry Series, 181, 181 - 229.
Wood, P. J. and Siddiqui, I. R. 1971. Determination of methanol and its application to measurement of pectin
ester content and pectin methyl esterase activity. Analytical Biochemistry. 39, 418 – 428. Worth, H. G. J. 1967. The chemistry and biochemistry of pectic substances. Chemical Reviews. 67, 465 – 473. Yamaguchi, F., Shimizu, N. and Hatanaka C. 1994. Preparation and physiological effect of low-molecular
weight pectin. Bioscience, biotechnology and biochemistry. 58, 679 – 682. Yule, M. A. and Fuller, M. F. 1992. The utilization of orally administered D-xylose, L-arabinose and D-
galacturonic acid in the pig. International Journal of Food Science and Nutrition. 43, 31 – 40. Yule, W. J. and McBridge, R. L. 1976. Lupin and rapeseed meals in poultry diets: effects on broilers
performance and sensory evaluation of carcasses. British Poultry Science. 17, 230 - 239. Zitko, V. and Bishop, C. T. 1966. Structure of a galacturonan from sunflower pectic acid. Canadian Journal
of Chemistry. 44, 1275 – 1282.
127
Appendix Additional information on the nutrient composition and content of cell wall pectin of
legumes, with more emphasis on lathyrus being a new legume produced in Australia, were
used as control treatments in Chapter 7.
What is lathyrus?
Lathyrus is a legume crop grown mainly in southern Australia and was developed for fine-
textured, neutral to alkaline soils where narrow-leaf lupins are poorly adapted. The seed is
of brown colour and about half the size of a field pea with an angular, blocky appearance.
Lathyrus is tolerant to waterlogging and herbicides, well adapted to low rainfall and harsh
environments, and more resistant to diseases such as black spot than field peas. The Centre
for Legumes in Mediterranean Agriculture (CLIMA) at UWA has commercially released a
cultivar, Lathyrus cicera cv Chalus to have low levels of the neurotoxin, 3-(-N-oxalyl)-L-
2,3-diamino propionic acid, a substance which causes paralysis (lathyrism) in the lower
limbs of monogastrics. This cultivar now appears safe for pig and poultry (Mullan et al,
1999,2006; Trezona et al, 2000, Hanbury & Hughes, 2003).
Uses of lathyrus.
Australia produces about 300,000 tonnes of lathyrus annually, worth $54 million. The
animal industry is the main user in Australia. Lathyrus are used as a multipurpose crop as a
feed grain, fodder, hay and green manure for cattle and sheep (White et al, 2002; Hanbury
& Siddique, 1998).
There has been an increasing interest in using lathyrus as a source of protein and energy for
pigs (Mullan et al, 2006, 2000, 1999; Trezona et al, 2000) but little interest for poultry
(Hanbury & Hughes, 2003). However, it may prove a cheap and good-quality source of
protein and energy for poultry particularly if feed enzymes improve its nutritive value.
Botanical name of lathyrus
The botanical name of lathyrus is Lathyrus cicera and Lathyrus sativus (Petterson &
Mackintosh, 1997). Lathyrus is known under a number of names such as grass pea,
128
chickling vetch, khesari and Indian pea of species L. sativus, and as dwarf chickling, flat
pod pea, red vetchling and chickling pea of species L. cicera. The latter, L. cicera is the
main species grown in southern Australia.
Nutrient composition of lathyrus
Lathyrus appears to have a better nutrient profile than lupins and may prove an excellent
feed for poultry. There are four reasons we wish to investigate lathyrus for broilers and
layers.
1. Lathyrus contains much more starch than lupins (42 vs. 1%) and comparable amounts
of protein (28 vs. 32%). The metabolisable energy of lathyrus is 34% higher than that
of lupins (13.4 vs. 10.0MJ/kg) for broilers (Hanbury et al, 2000).
2. Unlike lupins, lathyrus does not require dehulling and can be ground with its hulls
giving savings in processing costs of about $65/tonne.
3. Lathyrus is better source of total lysine (7 vs. 5g/16g nitrogen) and, depending on its
availability, it may be superior to lupins in providing lysine to birds.
4. Lathyrus contains less cell walls than lupins (25 vs. 35%) and hence less indigestible
materials.
However, lathyrus, like soybean, contains high levels of anti-nutritional factors (ANF).
Chymotrypsin and trypsin inhibitors are the most likely ANFs and are well known to
depress the digestion of protein, thereby reducing feed utilisation and growth. Such ANFs
might be destroyed with heat such as cooking, autoclaving and micronisation. A full
chemical composition and proximate analysis is given for lathyrus and lupins in Table 1.
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Table 1. Chemical composition of Lathyrus cicera, Lupinus angustifolius, Vicia faba and Pisum sativum.
Nutrient
Lathyrus
Lupins
Faba bean
Field pea
Moisture (%) 10.6 10.6 10.3 9.8 Fat (%) 0.7 4.1 1.4 1.7 Protein (%) 26.8 32.2 23.7 21.0 Ash (%) 3.1 3.6 3.8 3.3 NDF (%) 24.5 35.7 20.2 14.6 ADF (%) 10.7 23.6 13.1 8.1 Starch (%) 42 1 40.0 45.3 Lignin (%) 0.2 0.8 2.4 1.1 Minerals P (%) 0.33 0.46 0.41 0.34 K (%) 0.91 1.32 0.96 0.91 Na (%) 0.07 0.02 0.01 0.01 Ca (%) 0.25 0.23 0.12 0.07 Mg (%) 0.13 0.19 0.10 0.12 S (%) 0.17 0.26 0.13 0.18 Fe (mg/kg) 156 41 77 53 Mn (mg/kg 11 23 30 14 Zn (mg/kg) 20 32 28 30 Cu (mg/kg) 9 9 10.3 4.8 Essential amino acids (g/16g N) Cystine 1.23 1.56 1.37 1.49 Methionine 0.82 0.56 0.78 0.85 Threonine 3.68 3.79 3.54 3.35 Valine 4.57 4.24 4.30 4.29 Isoleucine 4.01 4.46 3.80 3.89 Leucine 7.36 7.47 7.27 6.54 Phenylalanine 4.46 4.46 4.12 4.17 Lysine 7.13 5.35 6.29 6.81 Histidine 2.45 2.90 2.54 2.37 Arginine 8.81 12.26 9.64 10.0 Anti-nutritional factors Tannins (total) (%) 1.08 0.32 0.75 0.25 Tannin, catechin (%) 0.51 0.36 nm 0.06 Trypsin inhibitor activity (g/kg) 2.07 0.13 0.39 1.01 Oligosaccharides (%) 4.12 4.57 2.93 3.69 Chymotrypsin inhibitor activity (g/kg) 3.46 0.09 0.40 1.60 Phytic acid (%) 0.91 0.56 nm 0.48 ODAP (%) 0.11 0.00 nm nm Alkaloids (%) nm 0.02 nm nm
Results are expressed on a DM basis. NDF, neutral detergent fibre; ADF, acid detergent fibre; ODAP, 3-(-N-oxalyl)-l-2,3-diamino propionic acid; nm, not measured. Data obtained from Hanbury et al, (2000) and Petterson & Mackintosh (1994).
130
Pectin in lathyrus
Cell walls of legumes are more complex than those of cereals because there are more side
branches and a higher proportion of pectin (Table 2). The pectin content of lupins is high at
7.9% but we know of no data for lathyrus. One way to estimate pectin content is by
difference. For example, if lathyrus contains 25% cell walls, approximately 13%
hemicellulose and 7% cellulose, there should be about 4 –5% (25 – 13 – 7 ≈ 4) pectic
substances. Another measure of pectin is the water-holding capacity. We have recently
determined the water-holding capacity for lathyrus and found values of 4.5g water/g seed
which are similar to lupins and field peas. This would suggest that lathyrus has a similar
content of pectin to lupins. Whatever the real value we believe that lathyrus is likely to
contain significant amounts of pectin.
Table 2. Pectin content of feedstuffs
Cereal % Legume % Wheat 1.2 Soybean 7.0 Barley 0.5 Lupins 7.9
Oat 0.6 Field pea 4.5 Corn 0.2 Broad bean 5.3
Data obtained from McNab & Shannon (1974), Brillouet & Carre (1983), Ali et al, (2000) and Leeson & Summer (1997). References Hanbury, C. D., White, C. L., Mullan, B. P., and Siddique, K. H. M. 2000. A review of the potential of
Lathyrus sativus L. and L. cicera L. grain for use as animal feed. Anim.. Feed Sci. Technol. 87, 1 – 27. McNab, J. M. and Shannon, D. W. F. 1974. The nutritive value of barley, maize, oats and wheat for poultry.
Brit. Poul. Sci. 15, 561 – 567. Leeson, S. and Summer, J. D. 1997. Ingredient evaluation and diet formulation. In Commercial Poultry
Nutrition. 10 – 111. University Book publication, Ontario, Canada. Brillouet, J.-M. and Carre, B. 1983. Composition of cell walls from cotyledons of Pisum sativum, Vicia faba
and Glycine max. Phytochemistry. 22, 841 – 847. Mullan, B. P., Hanbury, C. D., Hooper, J. A, Nicholls, R. R., Hagan, C. R. and Siddique, K. H. M. 1999.
Lathyrus (Lathyrus cicera cv. Chalus): a potential new ingredient in pig grower diets. In: Rec. Adv. Anim. Aust. 12, 12A.
Mullan, B. P., Pluske, J. R., Trezona, M., D. J. Harris, Allen, J. G., Siddique, K. H. M., Hanbury, C. D. 2009. Chemical composition and standardised ileal digestible amino acid contents of Lathyrus (Lathyrus cicera) as an ingredient in pig diets. Anim. Feed Sci. Technol. 150, 139 – 143.
Trezona, M., Mullan, B. P., Pluske, J. R., Hanbury, C. D., Siddique, K. H. M. 2000. Evaluation of Lathyrus (Lathyrus cicera) as an ingredient in diets for weaner pigs. Proc. Nutr. Soc. Aust. 24, 119.
White, C. L., Hanbury, C. D., P. Young, N. Phillips, S. C. Wiese, J. B. Milton, R. H. Davidson, K. H. M. Siddique and D. Harris. 2002. The nutritional value of Lathyrus cicera and L. angustifolius grain for sheep. Anim. Feed Sci. Technol. 99, 24 – 64.
Hanbury, C. D. and Siddique, K. H. M. 1998. Growing dwarf chickling (Lathyrus cicera). Farmnote, Agriculture Western Australia. No. F02498.
Hanbury, C. D. and Hughes, R. J. 2003. New grain legume for layers. Evaluation of Lathyrus cicera as a feed ingredient for layers. A report for the Australian Egg Corporation Limited. UWA-61A.but little interest for poultry (Hughes & Hanbury, 2003).
