Meeting the challenge of providing cost effective and efficacious protein nutritioneffective and efficacious protein nutrition

Aaron Cowieson Principal Scientist, DSMProfessor of Poultry Nutrition, University of Sydneyy , y y y

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

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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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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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Low (1980)

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Endogenous loss (Moughan & Rutherfurd 2012)Rutherfurd, 2012)

• Sources of endogenous lossP ti d t iPancreatic and gastric enzymesMucinBileAcids

BALANCE OF SECRETION

BicarbonateIntestinal 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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Amino acid profile of endogenous proteins

12

• mean amino acid profile of 8 sources of endogenous protein

8

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Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr

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• 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

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

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lost protein) & synthesis energy requirements

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Mean = 4954kcal/kg

3000350040004500

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20002500

Asp Thr Ser Glu Pro Gly Ala Val Ile Leu Tyr Phe His Lys Arg Cys Met

GE

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Adaptation to new diets – Corring (1980)

• GIT physiology is fluid and adapts readily to changing diet composition.

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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 gmicroflora

• 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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Li & Sauer (1994)

• Effect of added fat (canola oil) on amino acid digestibility in piglets

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Fat removal may compromise di tibilit f AAdigestibility of AA

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C

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Cowieson et al., 2010: 2% fat removed (PC to NC)

Page

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d21 d42

Bye et al (2013)Bye et al. (2013)

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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 influence grouping based on their ability to influence protein solubility

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Hofmeister Series

• Effect of ions on protein solubilityp y

CO32- > SO4

2- > HPO42- > 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)

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from Hess & van der Vegt, 2009)

Huang et al. (2005) British Poultry Science

• Wheat/Canola – overall a decrease in AA digestibility d14-42C /S ll i i AA di tibilit d14 42

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• Corn/Soy – overall an increase in AA digestibility d14-42

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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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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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 3511618

inal

• Pattern of response is correlated with the AA profile of intestinal mucin (Fig below)

R² = 0.351

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id p

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muc

in (%

)• We need to be able to predict

control digestibility to better predict Protease enzyme effect

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Change in amino acid digestibility with protease (%)

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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Performance Model: key terms that influence effect of proteaseinfluence effect of proteaseSUBJECTIVE1. Relative performance of control birds (index Ross standard)

OBJECTIVE1. Diet CP, %2. Diet AME, kcal/kg, g3. dLys, %4. Limestone inclusion, % 5. AME:dLys ratio6 CP:dThr ratio6. CP:dThr ratio7. AME:dSAA ratio8. dLys:dThr ratio

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Models – Grower/FinisherLOWER 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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Application in the future

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Application in the future

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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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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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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 proteins Endogenous 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

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• In the future the value of Protease enzyme may extend beyond feed cost savings to offer performance enhancement

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