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A. Principle
Samples are hydrated and starch is hydrolyzed to maltodextrins with thermostable -amylase at 95100C. Temperature
and pH are adjusted, and maltosaccharides are quantitatively
hydrolyzed to glucose with highly purified amyloglucosidase.
Glucose is determined with high-purity glucose oxidaseperoxidase reagent. Samples containing high-amylose starches or
resistant starch are pretreated with dimethyl sulfoxide (at 95C)
before treatment with -amylase. Starch content is calculated
and reported as percentage of sample on as is basis.
B. Apparatus
(a) Grinding mill.Centrifugal, equipped with 12-tooth
rotor and 0.5 mm sieve, or similar device. Alternatively, cyclone mill can be used for small samples.
(b) Bench centrifuge.Capable of holding 16 120 mm
glass test tubes, with rating of ca 1000 g.
(c) Water bath.Capable of maintaining 50 0.1C.
(d) Boiling water bath.Capable of boiling (e.g., appropriate deep-fat fryer filled with H2O) at 95100C.
(e) Vortex mixer.
(f) pH meter.
(g) Stop-clock timer (digital).
(h) Top-loading balance.
(i) Analytical balance.
(j) Laboratory oven.With forced-convection; capable of
maintaining 103 1C; used for determining dry weight of
sample.
(k) Spectrophotometer. Capable of operating at 510 nm.
(l) Pipets.Capable of delivering 100 and 200 L; with
disposable tips. Alternatively, motorized hand-held dispenser
can be used.
(m) Positive-displacement pipetter.Equipped with
5.0 mL tips; capable of accurately delivering 100 and 200 L;
and 50 mL tips, capable of delivering 3.0 mL.
(n) Dispenser.500 mL capacity set to deliver 3.0 mL.
Used for glucose oxidaseperoxidaseaminoantipyrine buffer
mixture.
(o) Glass test tubes.16 120 mm, 17 mL capacity, suitable for centrifugation at ca 1000 g; and 18 150 mm.
(p) Test tube racks.48 place, capable of holding 16
120 mm and 18 150 mm tubes.
(q) Thermometer.Capable of reading 103 1C.
(r) Filter paper.Fast, ashless.
C. Reagents
(a) 3-(N-Morpholino)propanesulfonic acid (MOPS) buffer.pH 7.0. Contains 50 mM MOPS, 5 mM calcium chloride,
and 0.02% sodium azide. In 1 L volumetric flask dissolve
11.55 g MOPS in 900 mL H2O and adjust pH to 7.0 with 1M
HCl (ca 17 mL). Add 0.74 g calcium chloride dihydrate and
0.2 g sodium azide. Dilute to volume with H2O. Buffer is stable
at room temperature.
(b) Thermostable
-amylase
solution.10 mL;
3000 U/mL. Dilute 1 mL -amylase solution (in 50% glycerol)
to 30 mL with MOPS buffer, (a). Thermostable -amylase so-
G. Calculations
Calculate total starch content (%, on as is basis) in test sample as follows:
Total starch, % = A F 1000
=A
100 162
1
1000 W 180
F
90
W
Negative Controls
The negative controls used to demonstrate assay specificity
were -cellulose, barley -glucan, and wheat arabinoxylan.
These were assayed by the standard assay procedure E(a) with
incubation with -amylase and amyloglucosidase. In another
control, the incubation contained buffers and enzymes only
(i.e., no sample was added). Portions from each of these controls were incubated with glucose oxidaseperoxidase buffer
mixture, and the absorbance was read against the reagent blank
(glucose oxidaseperoxidase buffer plus distilled water) D(c).
For all control solutions, the color formed was negligible, that
is,<0.001 absorbance unit.
Ref.: J. AOAC Int. 80, 571579(1997)
Results and Discussion
The precision for both assay procedures [E(a) and E(b)] was
calculated according to AOAC guidelines (9). Total starch values
determined by each collaborator by the non-DMSO, (E(a), and
DMSO, E(b), procedures as a percentage on a dry-weight basis,
are shown in Tables 1 and 2. Means, repeatability values (r), repeatability relative standard deviations (RSDr), reproducibility
values (R), reproducibility relative standard deviations (RSDR),
HORRAT values, and ranges for test results on each sample also
are given. Results requested but not supplied by the collaborator
are identified by a dash. The Cochran test identified as outliers the
results for the following samples: high-amylose maize starch (collaborator 12, non-DMSO procedure, and collaborator 19, DMSO
procedure), white wheat flour (collaborator 32), oat bran (collaborator 5), spaghetti (collaborator 19), and wheat starch (collaborator 5, DMSO procedure). The Grubbs test identified as outliers
the results of collaborator 18 for green peas.
David S. Jackson, Department of Food Science and Technology, University of Nebraska, Lincoln, NE
E. Rabe, Federal Centre for Cereal, Potato and Lipid Research, Detmold, Germany
Naresh Patel, Campden and Chorleywood Food Research
Association, Chipping Campden, United Kingdom
Philip C. Williams, Canadian Grain Commission, Grain Research Laboratory, Winnipeg, Manitoba, Canada
Janette Gelroth, American Institute of Baking, Manhattan, KS
Mary Ellen Camire, University of Maine, ME
Ravindra N. Chibbar, National Research Council, Saskatoon, Canada
Mark Ingelin, Kansas State University, Department of Grain
Science, Manhattan, KS
Claudia Niemann, Technische Universitt Berlin, Institut
fr Lebensmitteltechnologie, Berlin, Germany
Linda A. Grant, U.S. Department of Agriculture, Biosciences Research Laboratory, Agricultural Research Services,
Fargo, ND
David M. Peterson, U.S. Department of Agriculture-ARS,
Cereal Crops Research Unit, Madison, WI
Harold Corke, University of Hong Kong, Department of
Botany, Hong Kong
Peter Sanders, NIKO, Groningen, The Netherlands
Jane Muir, Deakin University, School of Nutrition and Public
Health, Victoria, Australia
Mingan Choct, CSIRO Division of Human Nutrition,
OHalloran Hill, Australia
Jenny Schmidt, GFW Ingredients Limited, Tamworth, Australia
Richard Walker, Bread Research Institute of Australia, Inc.,
North Ryde, Australia
A.B. Blakeney, NSW Agriculture, Yanco Agriculture Institute,
Yanco, Australia
Sue Logue, The University of Adelaide, Waite Campus,
Glen Osmond, Australia
Allen Tarr, Western Australian Department of Agriculture,
South Perth, Australia
Ian Batey, CSIRO Grain Quality Research Laboratory,
North Ryde, Australia
John Dynes, Defiance Milling Company, Toowoomba, Australia
Teresa Miklaszewicz, Bunge Albury Mills, Albury, Australia
Joe Panozzo, Victorian Institute for Dryland Agriculture,
Horsham, Australia
Betty W. Li, Agricultural Research Service, U.S. Department
of Agriculture, Beltsville, MD
Petrea Hofer, Plant and Soil Science Department, Montana
State University, Bozeman, MT
Elizabeth Arndt, Con Agra, Omaha, NE
Michelle Thomas, Weston Cereals Laboratories, Australia
Rochelle Depalo, Arnotts Biscuits Ltd., Homebush, Australia
References
(1) McCleary, B.V., Solah, V., & Gibson, T.S. (1994) J. Cereal
Sci. 20, 5158
(2) McCleary, B.V., Gibson, T.S., Solah, V., & Mugford, D.C.
(1994) Cereal Chem. 71, 501505
(10) Peeler, J.T., Horwitz, W., & Albert, R. (1989) J. Assoc. Off.
Anal. Chem. 72, 784806
(11) McCleary, B.V., & Sheehan, H. (1987) J. Cereal Sci. 6,
237251
(12) McCleary, B.V., Bouhet, F., & Driguez, H. (1991) Biotechnol. Tech. 5, 255258
(13) Horwitz, W., Albert, R., Deutsch, M.J., & Thompson, J.N.
(1990) J. Assoc. Off. Anal. Chem. 73, 661680
(14) Horwitz, W., Albert, R., Deutsch, M.J., & Thompson, J.N.
(1992) J. Assoc. Off. Anal. Chem. 75, 227239
(15) Anon. (1987) Starch 39, 414416
Table 996.11. Method performance for determination of total starch in processed cereal products by amyloglucosidase
-amylase method
Sample
Chicken feed pellets
White bread
Green pea
High-amylose maize starch
White wheat flour
Wheat starch
Oat bran
Spaghetti
High-amylose maize starch (DMSO method)
Wheat starch (DMSO method)
a
b
c
Calculated on as is basis.
r = 2.8 sr.
R = 2.8 sR.
Moisture, %
No. of labs
a a
sr
44.9
60.9
38.5
74.8
68.0
85.3
38.5
67.5
84.2
84.7
11.4
10.6
12.4
13.4
12.8
12.2
18.8
11.8
13.4
12.2
32
32
31
26
31
26
31
31
31
31
1.4
1.6
1.3
2.2
2.0
2.8
1.5
2.6
1.8
2.6
sRa
RSDr, %
RSDR, %
bb
c c
2.1
3.0
1.9
3.6
2.9
3.3
1.9
3.2
2.4
3.9
3.1
2.6
3.4
2.9
2.9
3.3
3.9
3.9
2.1
3.1
4.7
4.9
4.9
4.8
4.3
3.9
4.9
4.7
2.9
4.6
3.9
4.5
3.6
6.2
5.6
7.8
4.2
7.3
5.0
7.3
5.9
8.4
5.3
10.11
8.1
9.2
5.3
9.0
6.7
10.91
Table 1. Results for determination of total starch in processed cereal products and plant materials (part 1)
Chicken feed pellets
White bread
Laboratory No.
11
12
13
14
15
16
17
18
19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Moisture, %
d
Number of labs
e
Outliers
Average, %
f
sr
g
RSDr
h
r
i
sR
j
RSDR
k
R
l
HORRAT
Range
49.75
54.94
50.54
52
53.69
46.25
48.39
50.54
51.66
48.05
51.89
45.23
56.51
49.97
54.26
49.07
49.75
49.86
55.39
49.75
51.10
51.21
49.97
52.57
49.63
51.78
50.54
51.21
53.69
49.52
52.23
54.37
49.86
53.81
47.60
50.65
49.29
45.35
50.31
49.63
52.23
47.60
49.07
45.46
51.10
51.89
50.31
49.63
48.84
49.97
55.27
50.65
49.75
48.73
52.23
50.54
49.63
54.03
50.87
51.10
50.08
51.10
51.78
49.75
66.59
70.51
69.28
71.52
63.46
62.67
67.15
68.38
68.49
67.49
64.91
60.44
77.11
70.84
78.79
64.35
65.71
69.17
71.29
67.61
67.71
72.64
68.38
70.96
66.37
69.95
67.49
67.15
62.56
70.51
68.94
73.87
66.03
70.73
65.36
71.52
62.67
63.01
64.02
68.72
69.05
67.15
67.15
63.46
69.28
70.06
70.96
62.90
67.82
69.39
69.50
67.71
66.03
69.17
68.38
70.51
66.59
72.86
67.82
67.38
64.47
67.26
67.82
71.85
a
b
c
d
e
f
g
h
i
j
k
l
11.35
32
0
50.7
1.6
3.1
4.4
2.4
4.6
6.6
2.1
45.455.3
10.65
32
0
68.1
1.8
2.7
5.2
3.4
5
9.5
2.4
62.074.9
Green pea
C
42.69
43.26
46.00
46.00
42.35
42.58
44.98
44.75
40.41
40.75
36.64
42.12
44.86
43.84
44.29
44.52
45.09
43.72
42.58
43.95
44.18
43.38
40.87
44.29
42.24
44.06
45.21
46.00
43.49
40.87
44.18
41.89
43.04
43.72
c
c
26.48c
23.52c
46.92
47.95
44.63
43.38
43.38
42.47
45.55
46.58
48.63
45.55
44.29
44.86
43.95
42.58
46.23
45.78
44.29
44.41
43.95
43.15
49.89
43.84
43.49
39.61
43.26
44.41
44.75
47.03
12.4
31
c c
1
44
1.5
3.4
4.2
2.1
4.8
6
2.1
39.447.4
Maize starch
D
81.02
81.25
76.98
80.55
a a
78.94
79.86
90.36
87.94
88.98
88.40
80.78
83.21
b
b
87.25b
72.48b
86.90
90.83
90.83
88.86
89.90
86.56
83.67
86.67
86.79
85.05
88.52
90.48
85.29
87.71
83.44
81.13
96.83
86.67
86.90
88.75
89.32
89.21
83.21
83.90
90.71
91.06
88.52
88.98
85.98
86.56
75.59
85.29
88.29
84.25
86.21
89.32
88.63
90.36
13.35
26
b b
1
86.9
2.5
2.9
6.9
4.9
5.7
13.8
2.8
78.7100.7
76.95
76.72
81.31
79.47
76.83
80.16
82.91
78.10
77.52
72.36
69.50
73.74
76.26
75.92
77.75
77.41
79.24
81.77
76.61
76.49
78.78
75.23
74.43
69.84
83.37
88.19
79.70
80.50
80.85
75.34
72.82
74.54
75.92
77.87
79.47
79.47
80.96
75.46
77.64
76.49
75.92
73.39
87.16
78.21
78.67
79.24
80.16
78.78
76.83
77.18
81.88
79.13
78.33
77.52
78.21
78.33
79.36
78.67
76.61
76.72
79.82
79.01
c
b
83.37c
69.15b
12.8
31
b b
1
78
2.2
2.9
6.3
3.3
4.2
9.2
2
71.685.8
Table 2. Results for determination of total starch in processed cereal products and plant materials (part 2)
Wheat starch
Laboratory No.
11
12
13
14
15
16
17
18
19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Moisture, %
c
Number of labs
d
Outliers
Average, %
e
sr
f
RSDr
g
r
h
sR
i
RSDR
j
R
k
HORRAT
Range
a
b
c
d
e
f
g
h
i
j
k
95.84
95.84
101.991
100.741
a a
93.45
93.11
96.52
99.03
95.50
100.061
95.38
95.95
89.23
102.451
99.49
110.541
101.991
100.511
98.80
91.51
87.98
95.61
97.21
94.25
96.07
95.84
96.87
96.30
98.80
95.38
92.99
99.60
99.15
96.41
97.89
98.12
96.18
95.95
99.49
99.94
97.44
96.75
96.52
96.98
95.84
96.75
92.31
91.85
101.541
100.971
102.791
97.66
12.25
26
0
97.2
3.2
3.3
9.0
3.7
3.8
10.4
1.9
191.8105.0
Oat bran
Spaghetti
40.79
43.09
37.17
44.63
b
44.85b
45.72
42.11
44.19
42.11
42.65
40.68
39.91
42.76
43.31
42.87
39.91
40.90
44.63
45.07
41.56
46.38
42.11
43.20
43.97
41.34
46.60
44.74
43.09
41.23
41.01
43.75
39.36
41.23
43.97
40.24
44.74
b
23.68b
47.92
40.02
41.78
42.65
40.90
41.45
38.49
42.21
42.43
37.50
39.69
41.67
43.20
42.32
42.76
40.46
45.18
41.67
42.54
40.46
43.75
40.90
41.78
42.11
39.36
40.90
40.57
8.8
31
b b
1
42.2
1.6
3.8
4.5
2.1
5.0
6.0
2.2
38.746.8
Maize starchDMSO
N
76.87
75.62
81.41
78.57
77.78
75.28
80.73
80.61
78.57
67.46
71.88
74.38
79.48
76.64
78.46
78.23
81.63
81.41
76.30
77.10
69.61
79.93
74.38
72.45
76.76
63.49
79.02
75.62
72.79
73.02
71.20
73.70
76.30
77.89
73.24
73.36
b
b
83.45b
17.69b
77.44
77.44
76.08
73.81
77.78
85.71
77.10
78.46
78.80
78.12
76.98
77.10
80.39
79.82
77.21
78.12
77.21
77.55
77.21
76.42
70.86
71.32
80.84
77.55
80.39
76.87
11.8
31
b b
1
76.6
3.0
3.9
8.4
3.7
4.8
10.3
2.3
70.181.8
94.86
93.59
98.33
99.48
101.671
99.60
101.671 102.141
96.94
89.44
100.061 101.441
96.02
100.291
97.40
97.63
99.37
97.63
96.13
95.21
89.32
93.94
94.40
102.371
98.67
100.081
97.17
97.29
100.981
99.02
93.25
95.44
98.56
99.37
97.17
97.86
b
10.39b b103.29b
100.751
98.56
94.06
95.44
100.521
94.40
97.17
97.52
96.36
98.79
95.79
96.48
97.29
97.52
94.75
94.86
94.40
94.86
95.44
94.86
97.63
97.40
94.75
96.71
99.02
95.33
13.35
31
b b
1
97.2
2.0
2.1
5.7
2.8
2.9
7.8
1.4
191.6101.9
Wheat starchDMSO
H
94.47
94.25
99.03
97.55
97.44
96.18
89.23
100.401
b
b
2.96b
78.52b
104.841 103.251
97.55
93.79
96.18
96.98
98.46
92.42
94.36
95.61
86.72
85.36
89.12
95.84
97.66
98.12
98.80
99.03
84.33
94.36
94.70
94.93
100.171 103.251
98.12
98.12
101.421 105.981
100.971
99.26
94.59
96.41
103.131
95.61
96.30
95.04
97.44
98.69
92.99
92.42
99.60
99.03
96.07
95.61
94.59
94.70
99.49
97.55
94.25
91.85
98.12
88.66
105.071
99.37
12.25
31
b b
1
96.5
3.0
3.1
8.4
4.4
4.6
12.4
2.3
186.0104
Table 3. Comparison of results obtained from interlaboratory evaluation of analytical procedures for the
measurement of total starch contents of a range of samples
Sample
White wheat flour
Method
a a
A
B
A
B
A
B
A
B
A
B
A
B
A
B
c c
C
b b
White bread
Chicken feed
Oat bran
Green peas
Spaghetti
High amylose maize starch
a
b
c
Starch content, %
(dry wt)
No. of
outliers
RSDr
RSDR
80.0
78.0
69.8
68.1
47.5
50.7
45.6
42.2
44.2
44.0
75.1
76.6
98.2
86.3
97.2
1
1
2
0
0
0
0
1
0
1
1
1
1
1
1
2.6
2.9
1.5
2.7
5.7
3.1
7.3
3.8
3.6
3.4
6.2
3.9
1.5
2.9
2.1
5.4
4.2
4.1
5
10.91
4.6
9.4
5
5.7
4.8
11.31
4.8
4.9
5.7
2.9