Starch (and Sugar) Project

Update

Mary Beth Hall, Ph.D.

Department of Animal Sciences
University of Florida, Gainesville
 Plant Carbohydrates

Cell Cell
Contents Wall

Organic Mono+Oligo- Starches Fructans Pectic Hemicelluloses Cellulose

Acids saccharides Dextrins Substances
Galactans
β -glucans ADF

NDSF NDF
Non-Starch Polysaccharides

NDSC
NFC
University of Florida
Objectives:
To evaluate the ability
of labs to analyze
accurately for starch
and “sugars”, assess
variability among labs,
and seek ways to
improve accuracy.
Sample Enzymatic Starch
Analysis

Gelatinization
Open the
structure
for
hydrolysis Hydrolysis
Must be
complete;
Enzyme End Product
specific Measurement
Released
glucose only
 Glucose to Starch
“ 1 lb starch = 1.1 lb glucose
“ Glucose x 0.9 = starch
CH2OH CH2OH CH2OH CH2O CH2OH CH2OH

O O O O O O

OH OH OH OH OH OH

O O O O O
O
O

OH OH OH OH OH OH

H2O H2O H2O H2O H2O

Can use heat-stable α-amylase (pH ~7) and

amyloglucosidase (pH ~4.5), or amyloglucosidase, alone.

Specificity of enzymes to release Glc only from starch

decides if you are measuring starch, or something else.
Starch Analysis
The basic elements for accuracy:
“ Complete gelatinization
“ Specificity of enzymes
“ Complete hydrolysis to glucose
“ Specific measurement of glucose
“ Minimize interference
Carbohydrates in Feeds
“ Starch is one of many carbohydrates that
contain glucose.

“ Free glucose
CH2OH

“ Sucrose OH
O

“ Lactose OH OH

H
O
OH CH2OH

H2
C
O

“ Fructans
OH

O
O
OH O

H
CH

O
OH
2O

C
H

“ Cellulose

H2
CH OH

O
2 OH O

H
HO
O O
OH

CH
“ and more….
OH
OH

2
OH
OH
OH

O
OH

OH
CH2

O
CH2 O O
CH2 O O
O HO
HO
HO CH2OH
CH2OH
CH2OH OH
OH
OH
Check Test
“ Laboratory participation
¾ Starch analysis: 16 (14 consortium)
¾ Sugar analysis: 9 (7 consortium)
“ Samples: 12
¾ Results reported on an as received basis
 Check Test: Samples
1. Distillers grains
2. 48% soybean meal
’ 3. Isolated potato starch
4. Citrus pulp
5. Corn meal
6. Alfalfa hay
’ 7. Corn starch + glucose
’ 8. Corn starch + fructose
’ 9. Corn starch
’ 10. Corn starch + cellobiose
’ 11. Confectioners’ sugar
12. Corn silage (NFTA)
 Check Test: Purified Samples
Recovery: Measured / Actual
Isolated potato starch: 89.8% starch as received
Corn starch (Sigma Chemical): 90.1% starch as rec’d

Interference: What is actually measured?

Corn starch + glucose (79.4:11.5)
Corn starch + fructose (79.4:11.5)
Corn starch + cellobiose (79.2:11.8)
Confectioners’ sugar (3% starch, 2.3% Glc, 99.8% DM)

Particle size not an issue with these samples.

Check Test: NFC in Feed Samples
Distillers grains
- Fermented, low starch
No gold
48% soybean meal
- Little starch, some sugars standard!
Citrus pulp
- No starch, much sugar + soluble fiber
Corn meal
- Primarily starch
Alfalfa hay
- Some starch, more sugar + soluble fiber
Corn silage (NFTA)
- Mostly starch
 Starch Recovery: Measured/Actual

3. Potato Starch Recovery 9. Corn Starch

1.20

1.01
?
1.05 0.99 1.00 1.03 1.00
0.97 0.99 0.96
1.07 ? 1.01
1.00 0.97 0.98 0.96 1.01 0.98
0.93 0.94 0.92 0.91 0.92 0.90 0.94
Starch Measured / Actual

0.84 0.82
0.80
0.64
0.60
0.53 0.54

0.40

0.20

0.00
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16 L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID Laboratory ID

Potato Starch Recovery Corn Starch Recovery

Average 98.6% SE 1.00% Average 94.4% SE 1.94%
Average 98.1% SE 0.89% Average 93.3% SE 1.76%
 Amyloglucosidase from Aspergillus niger, solution For use in
Total Dietary Fiber Assay, TDF-100A
A9913
Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-
glucosidase, 9032-08-0

A1602
Amyloglucosidase from Aspergillus niger
Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-
Amyloglucosidase
glucosidase, 9032-08-0, MFCD00081350
Amyloglucosidase from Aspergillus niger, Aqueous glucose products available from
solution
A3042
Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α- Sigma/Fluka.
glucosidase, 9032-08-0, MFCD00081350
Amyloglucosidase from Aspergillus niger, BioChemika
>50 units/mg
10114
Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-
glucosidase, 9032-08-0, MFCD00081350
Amyloglucosidase from Aspergillus niger, BioChemika
lyophilized powder ~70 units/mg
10115
Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-
glucosidase, 9032-08-0, MFCD00081350
Amyloglucosidase from Aspergillus niger, BioChemika powder
Color white ~120 units/mg
10113
Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-
glucosidase, 9032-08-0, MFCD00081350
Amyloglucosidase from Aspergillus niger, Isoelectric focusing
marker pI 3.6
A2910
Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-
glucosidase, 9032-08-0, MFCD00081350
Amyloglucosidase from Aspergillus niger, Lyophilized powder
30-60 units/mg protein (biuret)
A7420
Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-
glucosidase, 9032-08-0, MFCD00081350
Amyloglucosidase from Aspergillus niger, min. 300 unit/ml
A7095 Glucoamylase, 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-
glucosidase, 9032-08-0, MFCD00081350
Amyloglucosidase from Candida tsukubaensis, Ammonium
sulfate suspension 50-150 units/mg protein
A2330
1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-glucosidase,
Glucoamylase, 9032-08-0
Amyloglucosidase from Rhizopus sp., min. 5,000 units/g solid
A7255 1,4-α-D-Glucan glucohydrolase, Exo-1,4-α-glucosidase,
Glucoamylase, 9032-08-0, MFCD00081350
Product No. Description

A7255 Amyloglucosidase from Rhizopus sp.

min. 5,000 units/g solid
Register or Login for pricing and availability
Identifiers
Synonyms 1,4-α-D-Glucan glucohydrolase
Exo-1,4-α-glucosidase
Glucoamylase
CAS Number 9032-08-0
Enzyme 3.2.1.3
Commission (EC)
Number
EG/EC Number 2328772
MDL number MFCD00081350

Description
Analysis Note Using soluble starch, almost theoretical yields of glucose are obtained.
Unit Definition One unit will liberate 1.0 mg of glucose from starch in 3 min at pH 4.5 at 55°C.

Properties
contains diatomaceous earth, starch and sugar as balance

Storage temp. 2-8°C

 Starch Recovery: Glucose Interference
7. Corn Starch + Glucose
1.20
1.20 1.13
1.09 1.07 1.01
1.00 0.99 0.98 0.97
1.00 0.96 0.96
0.91 0.91
Starch Measured / Actual

0.80

0.61
0.60

0.40

0.20

0.00
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Need to correct for background free glucose.

 Starch Recovery: Fructose Interference
8. Corn Starch + Fructose
1.20
1.05 1.04 0.98
1.00 0.98 0.93 0.93 0.93 0.94 0.94 0.91 0.93
0.92 0.87
Starch Measured / Actual

0.80

0.63
0.60

0.40

0.20

0.00
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Average 94.2% SE 1.20%

 Starch Recovery: Cellobiose Interference
10. Corn Starch + Cellobiose
1.20
1.09
1.00 0.99 1.03 0.97 1.02 0.99
1.00 0.94 0.93 0.94
0.91 0.93 0.89
Starch Measured / Actual

0.80
0.65
0.60

0.40

0.20

0.00
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Average 96.3% SE 1.33%

 Starch Recovery: Conf. Sugar Interference
11. Confectioners' Sugar Sucrose

H
O
CH2OH

H2
1.20

C
O
OH

O
O
OH O

H
1.00

O
OH

C
Starch Measured / Actual

H2
O
H
0.80

0.60

0.40 Glucose
+
0.20
Fructose
5.20 1.82 3.22 2.25 1.53 6.12 8.60 1.00 1.50 8.03 2.00 3.72 0.84 0.47
0.00
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Average 322% SE 7.87%

What to do?
“ Change enzymes
¾ No glucose or starch
¾ No non-amylase activity
“ Reagent blanks
“ Sample blanks
“ Correction factor?
 Starch Analyses of Citrus Pulp
4. Citrus pulp
20
Starch % of as-received sample

15

10 8.57
6.89 7.19
5.67 5.65
5.03
5 4.30
3.64
1.8 1.44 1.44 1.20
0.00 0.51
0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Average 4.10% SE 0.73%

 Starch Analysis of Distillers Grains
1. Distillers grains
20
Starch % of as-received sample

15

10
8.52

4.95 5.40 5.30

5 3.9 3.82 3.88
3.35
2.67
1.34 1.80 1.40
1.07
0.47
0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Average 3.41% SE 0.58%

 Starch Analysis of Corn Meal
5. Corn meal

100
Starch % of as-received sample

80
69.53
65.86 66.76
61.2 63.15 62.70 63.30 62.97
61.60 62.17 62.20
60 54.37 56.89
48.54

40

20

0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Average 61.5% SE 1.41%

 Starch Analysis of Corn Silage
12. Corn silage
40
Starch % of as-received sample

29.32 30.21 29.70 30.15

30 28.00 28.23
26.1

21.01
19.40
20

10

0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Average 26.9% SE 1.34%

“ What error is acceptable?
(Right answer for the right reason.)

“ Gelatinization method
“ Hydrolysis
“ Types of blanks?
“ What are the challenges that
starch analysis poses to a lab?
“ NIR?
 Mono- & Oligosaccharides:“Sugars” (LMW)
CH2OH

H
CH2OH

2O

HO

CH
CH
O O Sucrose

2
O
O

H
OH OH

H
O
OH

O
OH OH O

O
O
O
OH OH

CH
C
H2
Glucose

2
O

O
O
H

H
O
H
CH
2 OH
HOCH2 O OH CH OH

O
CH2OH

O
O
OH O

H
2
HO HO OH
O O HOH C
CH2OH OH
2

OH OH OH
OH
Fructose Raffinose
OH Lactose
 Measurement Units
“ As is
“ Total sugars as invert – monosaccharide basis
“ Acid hydrolyzed – monosaccharide basis
CH2OH CH2OH

O O
OH OH
OH
Glucose: 1 lb = 1 lb monosacch.
OH OH OH
OH OH

CH2OH
O Sucrose: 1 lb = 1.05 lb monosacch
H

CH2OH
2O

+H2O OH
H

O
C

OH OH
O

OH
HOCH2 O OH
O

OH O OH
H

H
O

OH
HO
C
H2

CH2OH
O
H

OH
CH
2 OH
CH OH
2 OH O
HO
OH
O O
OH
Lactose?
OH
OH
 Sugar Analysis of Starch + Glucose
7. Starch + glucose
40
Sugar % of as-received sample

29.5 30.03
30

20 18.96

14.60 14.3 15.08 15.2

13.08
10.20 10.55
10

0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Actual: TSAI 11.5%, As is 11.5%

 Sugar Analysis of Starch + Fructose
8. Starch + fructose
20
Sugar % of as-received sample

15
13.10
12.5 12.40
11.20 11.26
9.58
10

5.18
5
2.29 2.71

0
0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Actual: TSAI 11.5%, As is 11.5%

 Sugar Analysis of Starch + Cellobiose
10. Starch + cellobiose
20
Sugar % of as-received sample

15
13.38

10.80
10.3 9.88
9.70
10 9.13
8.40

5
2.35

0.1 0.27
0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Actual: TSAI 12.4%, As is 11.8%

 Sugar Analysis of Confec. Sugar
11. Confectioners sugar
101.30 103.24
108 98.85 100.00
100 96.6
90.60 92.44
90.14
Sugar % of as-received sample

80

60
48.15

40

20

0
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L13 L14 L15 L16
Laboratory ID

Actual: TSAI 101.5%, As is 96.8%

“ What units and methods
to work with for sugars?
“ Which extractants?
“ NIR
Enzyme Specificity
“ Hydrolyze only α-(1Æ4) & α-(1Æ6) glucan

“Amylase “Amyloglucosidase
– Endoenzyme – Exoenzyme
– Neutral pH – pH 4.5
– Heat-stable – 60°C
– Breaks starch into pieces – Releases glucose

“ Measure glucose released from starch

“ Glucose oxidase-peroxidase (Karkalas, 1985);
YSI system with glucose oxidase membrane.
Interfering Substances

“ Increase or decrease starch assay values

“ Free glucose
“ Glucose released from non-starch CHO
“ Tannins?
“ Remove low molecular weight CHO
“ Assure specificity of enzymes***
“ Account for free glucose
“ Measure only glucose
 Starch Analysis Recommendations

•O
•OH
•CH•2•OH
“ Run the complete assay from start to finish ASAP

•O
•OH
•O
“ Use an analysis that is specific for glucose

•OH
•CH•2•OH
•O
•OH
“ Use glucose as the standard for the assay

•O

•OH
•O
•CH•2•O

•OH
“ Check enzyme specificity on sucrose

•O

•OH
•CH•2•OH
•O
“ Include a starch control & reagent blank in each run

•OH
•O

•OH
•CH•2•OH
•O
“ Remove interfering substances

•OH
•O
“ Account for free glucose

•OH
•CH•2•OH
•O
C
H H2 O

•OH
OH O

•O
O
C
H H2 O
OH O OH O

CH2O CH2O CH2O OH CH O CH2O CH2O

2
H O H O H O O H O H O
OH OH OH OH OH OH
O O O O O O
O

University of Florida
OH OH OH OH OH OH