challenges of predicting metabolizable lysine content of...

Challenges of Predicting Metabolizable Lysine Content of

Ingredients

Sarah BoucherWilliam H. Miner Agricultural Research Institute

Pre-Conference Symposium

71st Annual Cornell Nutrition ConferenceEast Syracuse, NY

October 20 – 22, 2009

Presentation

1. Lysine structure

2. Nutritional consequences of heat processing

3. Estimating lysine damage in the RUP fraction of feeds

4. Digestibility of lysine and RUP in blood meal

5. Summary

20 Amino acids

Indispensable 1. Arginine2. Histidine3. Isoleucine4. Leucine5. Lysine6. Methionine7. Phenylalanine8. Threonine9. Tryptophan10. Valine

Dispensable1. Alanine2. Aspartic acid3. Asparagine4. Cysteine5. Glutamic acid6. Glutamine7. Glycine8. Proline9. Serine10. Tyrosine

Amino Acid Structure

C

O

C

OH

R1

+H3N

α-amino group

α

Peptide Bond Formation

CO

COH

R1

+H3N

HO

H

CO

COH

R2

+H3N

C

O

C

H

R1

+H3N CO

COH

R2

N

H

Lysine Structure

CO

COH

CH2

H2N

CH2

CH2

CH2

NH2

Protein metabolism in ruminantsCrude protein Saliva

True protein

Peptides

Aminoacids

Ammonia

NPN

Microbial protein

Urea

Liver

Microbial protein

RUMEN

SMALLINT

Endogenousprotein

Metabolizable protein (absorbed AA)

RUP

Mammarygland

MILK

Aminoacids

RUP

Characteristics of Lysine

Cationic amino acid at physiological pH In direct competition with arginine

for intestinal absorption Absorption stimulated by intracellular

leucine

- S. Bröer 2008. Physiol Rev 88:249-286

Intestinal Lysine Absorption

bo,+y+L

Adapted from: S. Bröer 2008. Physiol Rev 88:249-286

Lysine Structure

Heat Processing Reactions

1. Amino acid racemization

2. Protein cross-linking reactions

3. Maillard reaction

Maillard Reaction

Generally has the greatest impact on nutritional quality of feeds

3 phases:1. Early2. Advanced 3. Final

Simplified Scheme of Maillard Reaction

Adapted from J. Mauron. 1981. Prog. Fd. Nutr. Sci. 5:5-35.

Amadori compound

Melanoidin formation

Reducing sugar + Amino compound

Advanced glycation end products

Challenges with Lysine Analysis

H2N COOHCH

(CH2)4

NH

CH

(HCOH)3

C = O

CH2OHAmadori compound

Boiling 6N HCl

24 h

H2NCH

H2NCH

H2N COOHH2N COOHCH

H2N COOHCH

H2N COOHCH

H2N COOHCH

(CH2)4

COOHCH

(CH2)4

COOHCH

(CH2)4

COOHCH

NH

(CH2)4

COOHCH

NH

(CH2)4

COOHCH

CH

NH

(CH2)4

COOHCH

C = O

CH

NH

(CH2)4

COOHCH

(HCOH)3

C = O

CH

NH

(CH2)4

COOHCH

CH2OH

(HCOH)3

C = O

CH

NH

(CH2)4

COOHCH

Terminology

Bioavailability – proportion of ingested dietary AA that is absorbed in a chemical form that renders these AA potentially suitable for metabolism or protein synthesis

Digestibility – reflects enzymatic hydrolysis of ingested proteins and absorption of AA and peptides from the gastointestinal lumen

Terminology

Basal endogenous AA losses - the minimum quantities of AA inevitably lost by the animal

Standardized digestibility –calculated by subtracting only basal endogenous AA losses from outflow of AA

Ruminant Digestive Tract

Estimating Intestinal AA Digestibility In Vivo

Avian Digestive Tract

Standardized Digestibility –SBM Products

0

10

20

30

40

50

60

70

80

90

100

RUP-Lysine RUP-Total AA

SE = 2.9 SE = 1.7

Stan

dard

ized

dig

esitb

ility

, %

HSPHSBMSP2SP3SBM2SBM3

a,b,c Means within a series with different superscripts differ, P < 0.01

c

b

a a a a

c

b

a a a a

Standardized Digestibility -DDGS

0

10

20

30

40

50

60

70

80

90

100

RUP-Lysine RUP-Total AA

SE = 2.93 SE = 1.28

Sta

ndar

dize

d di

gest

ibilit

y, %

HDDGSDDGS1DDGS2DDGS3DDGS4


c

b

a a,b a,b

b

a a a a

Standardized Digestibility –Fish meals

0

10

20

30

40

50

60

70

80

90

100

RUPLysine RUP-Total AA

SE = 1.88 SE = 1.44

Stan

dard

ized

dig

estib

ility

, %

ANVYCFSHMNHN1MNHN2PLCK


a

b

a a a a

b

a a a

Terminology

Blocked lysine – lysine molecules in which the ε-amino group is bound to another compound

Reactive lysine – lysine molecules in which the ε-amino group is notbound to another compound

Estimating Blocked RUP-Lys

H2N COOHCH

(CH2)4

NH

CH

(HCOH)3

C = O

CH2OHfructosyllysine

Boiling 6N HCl

24 h

H2NCH

H2NCH

H2N COOHH2N COOHCH

H2N COOHCH

H2N COOHCH

H2N COOHCH

(CH2)4

COOHCH

(CH2)4

COOHCH

(CH2)4

COOHCH

NH

(CH2)4

COOHCH

NH

(CH2)4

COOHCH

CH

NH

(CH2)4

COOHCH

C = O

CH

NH

(CH2)4

COOHCH

(HCOH)3

C = O

CH

NH

(CH2)4

COOHCH

CH2OH

(HCOH)3

C = O

CH

NH

(CH2)4

COOHCH

Estimating Reactive RUP-Lys

NH2

CH2

CH2

CH2

CH2

C COOHH2N

H

Lysine

CNH

O

H2N

CH3

H

C

H

COOHC

H

H2N COOH

H

CH2N COOH

H

CH2

CH2N COOH

H

CH2

CH2

CH2N COOH

H

CH2

CH2

CH2

CH2N COOH

H

CH2

CH2

CH2

CH2

CH2N COOH

H

NH2

CH2

CH2

CH2

CH2

CH2N COOH

H

NHCH2

CH2

CH2

CH2

CH2N COOH

H

NH2

CH2

CH2

CH2

CH2

CH2N COOH

H

NH2

CH2

CH2

CH2

CH2

CH2N COOH

H

C NH2

NH

Homoarginine

Modified TSP Gargallo et al. (2006)

5 g of rumen undegraded resiude weighed into polyester bags in duplicate Incubated in a pepsin/HCl

solution 1 h, 38°C Incubated in a pancreatin

solution 24 h, 38°C

Bag residues analyzed for AAGargallo et al. (2006) J. Anim. Sci. 84:2163-2167.

Lysine Estimates – Soy-productsMethod

FurosineHomo-

arginine MTSP Rooster

Sample

Blocked RUP-Lys,

%

Reactive RUP-Lys,

%

RUP-Lys digestibility,

%


%

Heated SP - 36.5 66.4 37.8

SP1 - 81.5 92.7 89.5

SP2 0.1 77.4 94.1 84.9

Heated SBM - 43.3 68.4 55.0

SBM1 0.7 85.2 97.7 90.1

SBM2 1.0 85.1 98.8 89.6

Lysine Estimates – DDGS

Method

FurosineHomo-


Sample

Blocked RUP-Lys,

%

Reactive RUP-Lys,

%


%


%

Heated DDGS - 31.5 32.4 10.3

DDGS2 26.0 70.3 79.2 63.0

DDGS3 7.6 76.8 92.1 79.5

DDGS4 15.3 73.7 86.7 75.8

DDGS5 21.0 71.3 89.1 72.7

Lysine Estimates – Fish meal

Method

FurosineHomo-


Sample

Blocked RUP-Lys,

%

Reactive RUP-Lys,

%


%


%

ANVY 0.4 84.9 95.9 87.5

CFSH - 71.3 66.9 63.2

MNHN1 0.2 79.5 95.4 88.6

MNHN2 - 79.1 92.6 84.3

PLCK - 89.4 92.0 89.9

Blood mealMethod

Homo-arginine

MTSP1 MTSP2 Rooster

Sample

Reactive RUP-Lys,

%


%


%


%

Heated bovine BM 45.3 56.7 62.0 93.2

Bovine BM1 67.0 71.3 81.7 93.9

Bovine BM2 73.0 71.2 76.7 85.8

Heated porcine BM 86.8 17.2 19.5 83.9

Porcine BM1 71.3 93.4 80.4 92.0

Porcine BM2 93.1 - 94.7 95.3

Ohio State Modifications ofthe Three-Step Procedure

1. Foundation for the procedure is the Calsamiglia and Stern (1995) Minnesota three-step procedure

2. Modifications minimized inter –assay variation to 5%

3. Validated by Nostfsger and St-Pierre (2003, J. Dairy Sci. 86:958-969) lactational performance trial

4. Patent pending procedure is intellectual property of Venture Milling

Blood Meal RUP digestibility

Blood meal RUP digestibility

0102030405060708090

100110120

0 10 20 30 40 50 60 70 80 90 100 110 120

RUP (%CP)

RUP

diges

tibilit

y %

Data courtesy of Venture Milling. Ring dried blood meal data generated from non-published Ohio State University research using a Modified Minnesota 3-Way analytical procedure.

n = 403

Average RUP digestibility, % = 64.6; SE = 23.1

Blood Meal RUP-Lysine Digestibility

Blood meal RUP lysine digestibility vs RUP digestibility

0102030405060708090

100

0 10 20 30 40 50 60 70 80 90 100

RUP digestibility

RUP

Lys

dig

estib

ility

Data courtesy of Venture Milling. Ring dried blood meal data generated from non-published Ohio State University research using a Modified Minnesota 3-Way analytical procedure.

n = 403

Average RUP- Lys digestibility, % = 56.0; SE = 27.1

NRC Crude Protein Fractions

Feed Crude Protein

Fraction CUndegraded

Fraction BRest of CP

Fraction ANPN

NRC, 2001

RUP digestibility, NRC (2001)

RUP digestibility values are the approximate mean values reported using the mobile bag technique and the three step procedure

Assumes digestibility of individual AA in RUP is the same as RUP

CNCPS Crude Protein Fractions

TOTAL

BORATEBUFFER

NEUTRALDETERGENT

ACIDDETERGENT

SOLA

B1

INSOL

B2B3

C

SOLA

B1B2

INSOL

B3

C

SOLA

B1B2B3

INSOL

C

RUP digestibility, CNCPS v.6.1

Absorption of escaped feed protein is calculated by multiplying each protein fraction by its respective intestinal digestibility A – 100% B1 – 100% B2 – 100% B3 – 80% C – 0%

Assumes digestibility of individual AA in RUP is the same as RUP

Ingredient % of DMCorn silage 36.8Grass silage 16.1Alfalfa hay 5.8Corn grain 10.7Soybean hulls 5.0Soybean meal 2.1DDGS 10.7Soybean meal, expellers 2.3Blood meal 1.3Rumen protected Met 0.05Urea 0.25Inert fat 1.1Vitamins/minerals 2.5

Example diet

Balanced in NRC (2001)

Animal:

~ 85 lbs milk

DMI = 53 lbs

NRC (2001) Library

DDGS Blood mealring-dried

RUP, % CP* 50 78

RUP digestibility, % 80 80

Lys, % CP 2.7 8.9

*At DMI = 4.0% of BW and DMI = 50% forage

RUP-Lys digestibility

Scenario 1: DDGS RUP-Lys digestibility = 63% Blood meal RUP-Lys digestibility = 56%

Scenario 2: DDGS RUP-Lys digestibility = 80% Blood meal RUP-Lys digestibility = 90%

Example

NRC (2001) Scenario 1 Scenario 2

Lys, % of MP 6.22 5.99 6.30

Met, % of MP 2.07 2.07 2.07

Lys: Met 3.0:1 2.89:1 3.04:1

Summary

Heat processing of feeds can damage lysine

Intestinal digestibility of lysine in the RUP fraction of heated feeds is less than digestibility of total RUP

Determining digestible or available lysine content of feeds can improve predictions of MP-Lys supply

Acknowledgements

Special thanks to Venture Milling and Normand St-Pierre

Financial support for some of the research presented was provided by: Adisseo West Central

Special thanks to research collaborators Carl Parsons Pam Utterback Hans Stein Sergio Calsamiglia Marshall Stern Carsten Pedersen Lawrence Novtony &

Deon Simon at SDSU

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