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Study of Amino Acids in DDGS
Y. Zhang, J. V. Simpson and B. A. WrennNational Corn-to-Ethanol Research Center
Edwardsville, IL 62025
Hans SteinUniversity of Illinois Urbana Champaign
Gerald C. ShursonUniversity of Minnesota
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Topics
1. AA testing: snap shots of AOCS collaborative study
2. Variations: crude protein and amino acids
3. CP and AA digestibility
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Part IIntroduction by Amy Johnson (AOCS)
Part II
Data
AA Testing Snap Shots of AOCS Collaborative Study
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Comparison of Testing Methods on Amino Acid Contents in Animal Feed
Joint Meeting of the AAFCO Laboratory Methods and Service Committee
and the AOAC Feed Additive and Contaminants Group of the Ag Community
Wednesday, January 20, 2010Crowne Plaza
Redondo Beach, California
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Instrument and Derivatization Utilized
• Lab 1 - HPLC Post-Column Ninhydrin Derivatization• Lab 2 - HPLC Post-Column Ninhydrin Derivatization• Lab 3 - HPLC Post-Column OPA Derivatization• Lab 4 - HPLC Post-Column Ninhydrin Derivatization• Lab 5 - HPLC Post-Column Ninhydrin Derivatization
Lab 6 - LC/MS/MS Pre-Column Derivatization
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Protocol Used
• Eighteen samples • Hydrolysis procedure is based on the
method of AOAC 994.12. • Some of the samples will be spiked with
Sigma amino acid hydrolysate standard. • Four samples shall be analyzed once per
week for 6 weeks, as part of repeatability assessment.
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Results
• 15 amino acids: alanine, arginine, aspartic acid, glycine, glutamic acid, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine
• Report the final data in µg/mL (wt / vol)
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0
2
4
6
8
10
12
14
16
0 1 2 3 4 5 6
Ala
Arg
Asp
Gly
Glu
His
ILue
Lue
Lys
Phe
Pro
Ser
Thr
Tyr
Val
Precision (RSD, %)(Based on DDGS hydrolysate triplicate)
HPLC-POST LCMSMS-PRE
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6
Pre
cisi
on (R
SD
%)
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70
80
90
100
110
120
130
140
150
160
0 1 2 3 4 5 6
Ala
Arg
Asp
Gly
Glu
His
Ilue
Lue
Lys
Phe
Pro
Ser
Thr
Tyr
Val
% R
ecov
ery
Accuracy (Recovery, %)(Based on Sigma amino acid hydrolysate standard)
HPLC-POST LCMSMS-PRE
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6
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0
20
40
60
80
100
120
0 1 2 3 4 5 6
Ala
Arg
Asp
Gly
Glu
His
ILue
Lue
Lys
Phe
Pro
Ser
Thr
Tyr
Val
Amino Acid Content in DDGS Hydrolysates
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6
HPLC-POST LCMSMS-PRE
Am
ino
acid
con
tent
in D
DG
S h
ydro
lysa
te (μ
g/m
l)
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0
2
4
6
8
10
12
14
16
18
20
0 1 2 3 4 5
Ala
Arg
Asp
Gly
Glu
His
ILue
Lue
Lys
Phe
Pro
Ser
Thr
Tyr
Val
Lab 1 Lab 2 Lab 3 Lab 4 Lab 5
Precision (RSD, %)(DDGS sample, tested in 6-week period)
Pre
cisi
on (R
SD
%)
HPLC-POST
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New Testing Method Development
Tryptophan in DDGS• Microwave, basic hydrolysis• Hydrolyze 12 samples in 2 hours, use
LC/MS/MS for quantitation.• Recovery of 85% for an NIST standard • Look into the recoveries for other AAs
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Summary I
• HPLC-POS is a strong method• Best performance of HPLC-POS:
RSD < 2%Recovery between 95 and 105%
• There is need to develop a faster and more comprehensive hydrolysis method for AA testing
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Data SourcesNCERC: 12 plants (mainly from IL, IN, WI), 76 DDGS samples
(http://www.value-added.org/renewableEnergy/ethanol/ddgs/)
MN: 49 plants (mainly from MN, IA, IL, etc.), 49 DDGS sampleshttp://www.ddgs.umn.edu/profiles/us_profile_comparison_march_
2009.pdf)
Variations:
Crude Protein and Amino Acids
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Variations between Plants
37 (6%)1.1 (6%)1.1 (6%)Thr47 (4%)1.4 (7%)1.4 (4%)Val
46 (4%)1.4 (8%)1.4 (5%)Phe20 (7%)0.6 (8%)0.6 (9%)Met31 (9%)1.0 (13%)0.9 (9%)Lys
116 (4%)3.6 (7%)3.4 (5%)Leu35 (4%)1.2 (7%)1.0 (4%)Ile26 (9%)0.8 (6%)0.8 (10%)His44 (5%)1.4 (9%)1.3 (7%)Arg
30.8 (5%)29.3 (4%)CP
NCERC(AA/ CP, g/kg)
MN(%, w/w dry)
NCERC(%, w/w dry)
In DDGS
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Variations: Between vs. Within Plants(based on NCERC data)
2% (2 – 4 %)4%CP
8% (4 - 12%)6%Thr4% (2 - 6%)5%Phe8% (6 -10%)9%Met
4% (2 - 5%)4%Val
10% (5 - 14%)9%Lys4% (1 - 6%)5%Leu4% (1 - 6%)4%Ile
11% (3 – 19 %)10%His4% (2 – 6 %)7%Arg
CV of within
CV of between
CV range
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Variations within Plants, CP(from two earlier data sources)
Within-plant coefficient of variance (%)
2 4 6 8
NCERC
40 plants
2 – 4 DDGS from each plant
0
20
40
60
80
100
Perc
etna
ge o
f Sam
ples
in
that
Ran
ge MN
10 plants
12 DDGS from each plant
0
10
20
30
40
50
Per
cent
age
of S
ampl
es in
that
Ran
ge
5 10
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AA in DDGS / AA in Corn(Corn data from Feedstuff, 2010)
Val
Thr
Phe
Met
Lys
Leu
Ile
His
Arg
3.84.03.7
4.03.73.7
3.73.63.7
3.53.4
4.03.93.7
3.63.43.4
4.13.73.6
3.43.63.1
3.52.93.2
MNNCERC Pilot Plant Trial
NCERC(website)
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Summary II
• CP: most of the CVs are lower than 5% between and within plants.
• AA, variations between and within plants: Arg., Ile, Leu, Phe, Val, CV < 6% His., Lys, Met., Thr., CV between 6% and 19%
• The data set are representative of DDGS produced in the past 1 - 2 years
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CP and AA digestibility
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Amino acids / LysineAnimal Science Ideal Ratio
158
123
156
69
100
386
117
87
147DDGS
76
55
105
59
100
124
69
36
105Poultry
68
65
95
60
100
111
60
32
Swine
ValThrPheMetLysLeuIleHisArg
AA / Lysine
175
121
175
75
100
417
121
104
167Corn
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CP and AA Digestibility of DDGS in Swine Diet
SID (%)(standardized ileal digestbility)
=AID (%)
(apparent ileal digestbility)
+Basal IAAend /AAdiet (%)
(basal ileal endogenous loss)
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SID of DDGS in Swine Diet
7175 (6%)73 (8%)CP
7777 (4%)75 (6%)Val7471 (5%)71 (7%)Thr8383 (3%)81 (5%)Phe
8484 (3%)82 (5%)Met6864 (9%)62 (12%)Lys8585 (3%)84 (5%)Leu7877 (5%)75 (6%)Ile8280 (4%)78 (6%)His7784 (6%)81 (7%)Arg
One Corn Data(Stein et al, JAS, 2006)
Mean of 14 DDGS(Shurson website, 2009)
Mean of 33 DDGS(Stein et al, JAFC, 2008)
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Lysine and Furosine
• Maillard reaction: binding of lysine to reducing sugars produces unreactive lysine
• The unreactive lysine is not bioavailable for pigs, but cannot be detected by the usual testing method for total lysine, which involves acid hydrolysis of DDGS.
• Furosine, a lysine derivative, is produced during DDGS acid hydrolysis, and has been confirmed by food industry to be a quantitative indicator for unreactivelysine (unreactive lysine = 1.25 furosine)
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Testing Furosine in DDGS by LC/MS/MS
• System Suitability – trace compound in aqueous acidic hydrolysate
• Calibration Linearity - r2 = 0.999 for 5 external standards
• Accuracy - recovery between 95% and 105% for synthetic standard spiked in DDGS matrix
• Precision - triplicates, within batch (3%), between batches (5%)
• Limit of Quantitation (LOD) – 50 ppm in DDGS
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Furosine in DDGS
Furosine / Lysine (%)
Furosine(%, w/w as-received)
N = 20(06/2009)
20.01Minimum
300.24Maximum
370.20Maximum
20.02Minimum
Furosine / Lysine (%)
Furosine(%, w/w as-received)
N = 55(12/2008)
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Furosine / Lysine in DDGS
0
10
20
30
40
50
60
70
9 18 27 36Perc
enta
ge o
f Sam
ples
in
that
Ran
ge
0
Furosine / Lysine in DDGS (%)
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Furosine / Lysine in Intermediates from Corn to DDGS Production
0
2
4
6
8
10
12
14
16
corn ferm wet cake syrup DDGS1 DDGS2 DDGS 3
Sample Type
Furo
sine
/Lys
ine
(%)
Higher outlet T than the other DDGS samples
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Animal Trial Study(2009, U. of I)
• 20 DDGS sources • SID of lysine: 63.3 ± 8.1%.
SID of lysine vs. reactive lysine, r2 = 0.90 vs. CP, r2 = 0.02vs. color, r2 = 0.04
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Any DDGS sample from a standard dry-grind plant in the US, likely (with 68% confidence level)
0.9 – 1.21.3 – 1.5Val0.6 – 0.91.0 – 1.2Thr
1.0 – 1.21.2 – 1.5Phe0.4 – 0.60.5 – 0.7Met
0.4 – 0.7 (need furosine test to
confirm)
0.8 – 1.0Lys2.5 – 3.23.1 – 3.7Leu0.7 – 0.90.9 – 1.1Ile0.5 – 0.70.7 – 0.9His0.9 – 1.21.2 - 1.4Arg
18.9 – 24.028.1 – 30.5CP
Digestible Level(%, w/w dry)
Content in DDGS(%, w/w dry)
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Acknowledgement
AOCSIllinois Corn Marking Board