Protein/amino acids are among the most expensive nutrients to deliver in poultry nutrition The digestibility of protein in poultry is typically incomplete by the terminal ileum Undigested protein that leaves the ileum is from both exogenous (diet) and endogenous (bird) sources Understanding the digestion of dietary proteins and the recovery of endogenous proteins is important and can provide a basis for the use of exogenous proteases

1. Meeting the challenge of providing cost
effective and efficacious protein nutritioneffective and efficacious protein nutrition
Aaron Cowieson
Principal Scientist, DSM
Professor of Poultry Nutrition, University of Sydneyy , y y y

2. Presentation Overview
• Introduction and key concepts in protein digestion
• Endogenous and exogenous sources of protein in the intestine
• Factors that influence protein/amino acid digestionFactors that influence protein/amino acid digestion
• Optimising the use of exogenous protease and the importance of
benchmarking raw material quality
• Conclusions
Page 1

3. Introduction
• Protein/amino acids are among the most expensive nutrients to deliver in
poultry nutritionp y
• The digestibility of protein in poultry is typically incomplete by the
terminal ileum
• Undigested protein that leaves the ileum is from both exogenous (diet)
and endogenous (bird) sources
• Understanding the digestion of dietary proteins and the recovery of
endogenous proteins is important and can provide a basis for the use of
exogenous proteases
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4. Key Concepts - background
• Substantial input of endogenous protein into the lumen during digestion
E d g t i t f ll d b th t i l il ( ti t• Endogenous proteins are not fully recovered by the terminal ileum (estimates
around 80-90%, Souffrant et al., 1993)
• Endogenous proteolysis requires co-operative effort from several peptidases
• Most (80%) amino acids are recovered from the lumen as di- and tri-peptides,
not as free amino acids (Ganapathy et al., 1994)
• Cytostolic peptidases have limited capacity to hydrolyse tetrapeptides (Sterchi
& Woodley, 1980)
• Dietary protein is generally well recovered and amino acids that exit the
intestine are largely of endogenous origin
• Ileal measurements are more meaningful (microbial synth/metab.)
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5. Low (1980)
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4

6. Endogenous loss (Moughan &
Rutherfurd 2012)Rutherfurd, 2012)
• Sources of endogenous loss
P ti d t iPancreatic and gastric enzymes
Mucin
Bile
Acids
BALANCE OF
SECRETION
Bicarbonate
Intestinal cells
(Microbial protein)
Saliva
SECRETION
AND
ABSORPTION!
Saliva
• ‘Loss’ defined when an endogenous secretion
leaves the ileum (amino acid cost to the animal)( )
where there will be no further reasorption
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7. Amino acid profile of endogenous proteins
12
• mean amino acid profile of 8 sources of endogenous protein
8
10
12
acid
4
6
8
ofaminoa
Mean = 5.3%
0
2
4
%
0
Asp
Thr
Ser
G
lu
Pro
G
ly
Ala
Val
Ile
Leu
Tyr
Phe
H
is
Lys
Arg
C
ys
M
et
• amino acids of most significance, overall, are ser, gly, leu, pro, val, thr, as
Page
g , , , g y, , p , , ,
• of least significance are met and his

8. Energy associated with amino acids
• All amino acids have associated energy – ranges from 2891kcal/kg
for aspartic acid to 6739kcal/kg for phenylalaninep g p y
• The energetic consequence of the ingestion of an antinutrient
will depend on the profile of amino acid response (AA profile of
lost protein) & synthesis energy requirements
6500
7000
)
lost protein) & synthesis energy requirements
4500
5000
5500
6000
6500
aminoacid)
Mean = 4954kcal/kg
3000
3500
4000
4500
kcal/kgofa
Page
2000
2500
Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe His Lys Arg Cys Met
GE(k

9. Adaptation to new diets – Corring (1980)
• GIT physiology is fluid and adapts readily to changing diet composition.
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10. Recovery of endogenous protein
• Whilst much (perhaps 85%) of the endogenous protein is recovered and
retained, some is lost and either excreted or modified by the hind gut, y g
microflora
• Some endogenous protein sources are more readily recovered than others
• Hydrophobic and/or refractory proteins are poorly recovered• Hydrophobic and/or refractory proteins are poorly recovered
• Approximately 70% of endogenous protein is secreted distally from the
stomach/gizzard and so does not readily undergo gastric digestion (Fuller &
Reeds 1998)Reeds, 1998)
• Glycosylated domains of mucin (rich in Ser, Thr, Pro) are poorly recovered
(Forstner & Forstner, 1994)
C i h bi d i h f d i d/ d (i• Can we assist the bird with recovery of endogenous protein and/or reduce (in
an appropriate way) endogenous secretion?
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11. Li & Sauer (1994)
• Effect of added fat (canola oil) on amino acid digestibility in piglets
Page
10

12. Fat removal may compromise
di tibilit f AAdigestibility of AA
4
C
2
3
mPCtoNC
-1
0
1
Thr
S
er
Ile
C
ys
A
sp
V
al
G
ly
Lys
H
is
P
ro
Leu
A
rg
A
la
Tyr
G
lu
P
he
M
et
E
A
N
estibilityfro
-3
-2
T
S
C
A
V
G
L
P
L
A
A
T
G
P
M
M
E
inilealdige
6
-5
-4
%change
Cowieson et al., 2010: 2% fat removed (PC to NC)
Page
-6
d21 d42

13. Bye et al (2013)Bye et al. (2013)
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14. Franz Hofmeister
• Prof. Franz Hofmeister (1850-1922)
• Born in Prague, 1850
Pharmace tical chemistr• Pharmaceutical chemistry
• Proposer of the ‘Hofmeister Series’ ionic
grouping based on their ability to influencegrouping based on their ability to influence
protein solubility
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15. Hofmeister Series
• Effect of ions on protein solubilityp y
CO3
2- > SO4
2- > HPO4
2- > OH- > F- > HCOO- > CH3COO- > Cl- > Br-
> NO - > I- > SCN- > ClO -> NO3 > I > SCN > ClO4
• Fig. 1 Representation of Hofmeister anions with
increasing chaotropic potency from left to right (adapted
f L idi 2002 Zh & C 2006)from Leontidis, 2002; Zhang & Cremer, 2006).
Cs+ > Rb+ > NH4
+ > K+ > Na+ > Li+ > Mg+ > Sr2+ > Ca2+
4 g
• Fig. 2 Representation of Hofmeister cations with
increasing chaotropic potency from left to right (adapted
from Hess & van der Vegt 2009)
Page
from Hess & van der Vegt, 2009)

16. Huang et al. (2005) British Poultry Science
• Wheat/Canola – overall a decrease in AA digestibility d14-42
C /S ll i i AA di tibilit d14 42
Page 15
• Corn/Soy – overall an increase in AA digestibility d14-42

17. Importance of benchmarking
• Enzymes act on substrates – substrate type and concentration is clearly
important e.g. phytate, fibre, refractory proteins and starch
• Enzymes can degrade antinutrients such as trypsin inhibitors and phytate
• INHERENT DIGESTIBILITY OF FOCAL NUTRIENTS is absolutely central to the
magnitude and consistency of enzyme effects (Cowieson, 2010)
– Xylanase, protease and phytase all follow this rule
• So, how do we integrate these thoughts in order to optimise the use of
enzymes in our diets?
• Meta-analysis of large databases to show key leverage terms that promote
enzyme efficacy
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18. Methodology
• Digestibility meta-analysis included 804 datapoints from 25 independent
experiments
• Performance meta-analysis included 673 datapoints from 63 independent
experiments
• Data were generated from experiments run between 2006 and 2013
• Most trials were conducted in EU, US and Brazil
• Models were constructed using the statistical software ‘R’
– trials nested in region and Alkaline Protease treatment nested in trial
– compared with the appropriate controlp pp p
• Predictors were assessed based on degree of statistical significance and
biological relevance
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19. Digestibility
• Mean response to Alkaline Protease
was around 4% ranging from 5.6%
for Thr to 2.7% for Glu
• AME was significantly increased by
49 Kcal/kg and fat dig by 1%
• Inherent digestibility in the control• Inherent digestibility in the control
diet explained around 47% of the
variance in response (Fig above)
P f i l d
y = 3.319x - 6.239
R² 0 351
16
18
inal
• Pattern of response is correlated
with the AA profile of intestinal
mucin (Fig below)
R² = 0.351
6
8
10
12
14
6
idprofileofintesti
mucin(%)
• We need to be able to predict
control digestibility to better
predict Protease enzyme effect
0
2
4
6
2.5 3 3.5 4 4.5 5 5.5 6
Aminoaci
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Change in amino acid digestibility with protease (%)

20. Performance Modelling
• Considered 93 separate leverage terms
• Significance set at P < 0.05
Non significant terms re introduced once a• Non-significant terms re-introduced once a
beta-model was in place to confirm lack of
importance
• Heat mapping used to check for co-linearity
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21. Performance Model: key terms that
influence effect of proteaseinfluence effect of protease
SUBJECTIVE
1. Relative performance of control birds (index Ross standard)
OBJECTIVE
1. Diet CP, %
2. Diet AME, kcal/kg, g
3. dLys, %
4. Limestone inclusion, %
5. AME:dLys ratio
6 CP:dThr ratio6. CP:dThr ratio
7. AME:dSAA ratio
8. dLys:dThr ratio
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22. Models – Grower/Finisher
LOWER VALUE OF PROTEASE
HIGH VALUE OF PROTEASE:
- Low CP
- High AME
- Poor bird performanceLOWER VALUE OF PROTEASE:
- High CP
- Low AME
- Good bird performance
- High limestone
- Inappropriate AA balance:
Poor bird performance
- Low limestone
- Appropriate AA balance:
- HIGH dLys:dSAA
- LOW dLys:dThr
- HIGH CP:dThrInappropriate AA balance:
- LOW dLys:dSAA
- HIGH dLys:dThr
- LOW CP:dThr
- HIGH AME:dSAA
- LOW AME:dSAA
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23. Application in the future
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24. Application in the future
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25. Where do the performance effects of
Protease come from?Protease come from?
• Mechanisms responsible for the ‘extra-proteinaceous’ effects of Protease
enzyme may include:
– Gut health
– Mucosal integrity
– Tight junction integrityTight junction integrity
– Collagen structure
– Mucin and enzyme flow
– Litter quality
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26. Implications
• Alkaline Protease is currently widely used to reduce feed cost and does so
very successfully
• DSM are currently working on further enhancement of the application of
Protease enzyme to deliver additional value through:Protease enzyme to deliver additional value through:
– Possible further feed cost savings linked to raw material quality
– Improved performance of birds via diet balance (meta-analysis)
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27. Conclusions
• Protein digestion in poultry (and other animals) is a complex process of
hydrolysis of incoming proteins, absorption, further processing and the
concurrent secretion and recovery of endogenous protein
• Endogenous proteins are often less well recovered that exogenous proteinsEndogenous proteins are often less well recovered that exogenous proteins
and Alkaline Protease may assist the animals with digestion of both
fractions
Th h P t tl d li b t ti l l th h th• Though Protease enzyme currently delivers substantial value through the
CP/AA matrices and/or DIF values and the focus of use is feed cost saving
there may be additional advantages in performance in the future
• Work is ongoing to further explore the mechanisms responsible for the
effect of Protease enzyme on gut health, litter quality, performance etc.
In the future the value of Protease enzyme may extend beyond feed cost
Page
• In the future the value of Protease enzyme may extend beyond feed cost
savings to offer performance enhancement
26