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32.2.05A
AOAC Official Method 996.11
Starch (Total) in Cereal Products
Amyloglucosidase–a-Amylase Method
First Action 1996
AOAC–AACC Method
(Applicable to determination of total starch in cereal products.)
See Table 996.11 for the results of the interlaboratory study supporting the acceptance of the method.
Caution: See Appendix B, safety notes. Glucose oxidaseperoxidase-aminoantipyrine buffer mixture, MOPS,
and acetate buffers contain sodium azide. Avoid contact with skin and eyes. In case of contact, immediately
flush contact surfaces with plenty of water. Disposal of
these reagents into sinks with copper or lead plumbing
should be followed immediately with large quantities
of water to prevent potential explosive hazards.
Dimethyl sulfoxide is a skin irritant and should be used
with caution.
A. Principle
Test samples are hydrated and starch is hydrolyzed to
maltodextrins with thermostable α-amylase at 95–100°C. Temperature and pH are adjusted and maltosaccharides are quantitatively hydrolyzed to glucose with highly purified amyloglucosidase. Glucose
is determined with high purity glucose oxidase–peroxidase reagent.
Products containing high-amylose starches or resistant starch are
pre-treated with dimethyl sulfoxide at 95°C before treatment with
α-amylase. Starch content is reported on “as is” basis.
B. Apparatus
(a) Grinding mill.—Centrifugal, with 12-tooth rotor and 0.5 mm
sieve, or similar device. Alternatively, cyclone mill can be used for
small quantities.
Table 996.11
(b) Bench centrifuge.—Holding 16 × 120 mm glass test tubes,
with rating of ca 1000 × g.
(c) Water bath.—Maintaining 50 ± 0.1°C.
(d) Boiling water bath.—Boiling H2O at 95–100°C (e.g., fryer
filled with H2O).
(e) Vortex mixer.
(f) pH Meter.
(g) Stopclock timer (digital).
(h) Top-loading balance.
(i) Analytical balance.
(j) Laboratory oven.—With forced-convection; maintaining
103 ± 1°C; used for determining dry weight of test sample.
(k) Spectrophotometer.—Operating at 510 nm.
(l) Pipets.—Delivering 100 and 200 µL; with disposable tips. Alternatively, motorized hand-held dispenser can be used.
(m) Positive displacement pipetter.—With 5.0 mL tips accurately delivering 100 and 200 µL; and 50 mL tips delivering 3.0 mL.
(n) Dispenser.—500 mL, to deliver 3.0 mL. Used for glucose
oxidase–peroxidase–aminoantipyrine buffer mixture.
(o) Glass test tubes.—16 × 120 mm, 17 mL, for centrifugation at
ca 1000 × g; and 18 × 150 mm.
(p) Test tube racks.—48 place, holding 16 × 120 and
18 × 150 mm tubes.
(q) Thermometer.—Reading 103 ± 1°C.
(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 CaCl2.2H2O and 0.2 g sodium azide. Dilute to volume
with H2O. Buffer is stable at room temperature.
(b) Thermostable a-amylase solution.—10 mL; 3000 U/mL. Dilute 1 mL α-amylase solution (in 50% glycerol) to 30 mL with
MOPS buffer, (a). Thermostable a-amylase solution is stable up to
Interlaboratory study results for determination of total starch in processed cereal products by amyloglucosidase–
a-amylase method
Mean total
starcha, %
Moisture, %
No. of labs
sra
sRa
RSDr, %
RSDR, %
rb
Rc
Chicken feed pellets
44.9
11.4
32
1.4
2.1
3.1
4.7
3.9
5.9
White bread
60.9
10.6
32
1.6
3.0
2.6
4.9
4.5
8.4
Green pea
38.5
12.4
31
1.3
1.9
3.4
4.9
3.6
5.3
High amylose maize starch
74.8
13.4
26
2.2
3.6
2.9
4.8
6.2
White wheat flour
68.0
12.8
31
2.0
2.9
2.9
4.3
5.6
8.1
Wheat starch
85.3
12.2
26
2.8
3.3
3.3
3.9
7.8
9.2
Oat bran
38.5
8.8
31
1.5
1.9
3.9
4.9
4.2
5.3
Spaghetti
67.5
11.8
31
2.6
3.2
3.9
4.7
7.3
9.0
High amylose-maize starch
(DMSO method)
84.2
13.4
31
1.8
2.4
2.1
2.9
5.0
6.7
Wheat starch (DMSO method)
84.7
12.2
31
2.6
3.9
3.1
4.6
7.3
10.9
Sample
a
Calculated on “as is” basis.
b
r = 2.8 × sr.
c
R = 2.8× sR.
© 2000 AOAC INTERNATIONAL
3 years when frozen. (Note: One unit [U] of α-amylase activity is
amount of enzyme required to release 1 µmole p-nitrophenol from
“end-blocked” p-nitrophenyl maltoheptaoside in presence of saturating levels of α-glucosidase and amyloglucosidase [i.e., Ceralpha
α-amylase assay reagent] at 40°C and pH 6.0.) Thermostable α-amylase solution should be free of detectable levels of free glucose.
(c) Amyloglucosidase solution.—10 mL; 200 U/mL. Use directly
without dilution. Solution is viscous; for dispensing, use positive
displacement dispenser. Amyloglucosidase solution is stable up to
3 years at 4°C. (Note: One unit [U] of enzyme activity is amount of
enzyme required to release 1 µmole p-nitrophenol from
p-nitrophenyl β-maltoside in the presence of saturating levels of
β-glucosidase [i.e., amyloglucosidase assay reagent] at 40°C and
pH 4.5.) Amyloglucosidase solution should be free of detectable
levels of free glucose.
(d) Glucose oxidase–peroxidase–aminoantipyrine buffer mixture.—Mixture of glucose oxidase, 12000 U/L; peroxidase,
650 U/L; and 4-aminoantipyrine, 0.4 mM.
Prepare buffer concentrate by dissolving 13.6 g KH2PO4, 4.2 g
NaOH, and 3.0 g 4-hydroxybenzoic acid in 90 mL distilled H2O. Adjust to pH 7.4 with either 2M HCl (16.7 mL HCl/100 mL) or
2M NaOH (8.0 g NaOH/100 mL). Dilute solution to 100 mL, add
0.4 g sodium azide, and mix until dissolved. Buffer concentrate is
stable up to 3 years at 4°C.
To prepare glucose oxidase–peroxidase–aminoantipyrine buffer
mixture, dilute 50 mL buffer concentrate to 1.0 L. Use part of diluted
buffer to dissolve the entire contents of vial containing freeze-dried
glucose oxidase–peroxidase mixture. Transfer contents of vial to 1 L
volumetric flask containing diluted buffer. Reagent is stable
2–3 months at 4°C and 2–3 years at –20°C. Color formed with glucose is stable several hours. (Note: Glucose oxidase must not be contaminated with β- and/or α-glucosidase and chromogen color
complex must be stable at least 60 min.)
Check color formation and stability of glucose
oxidase–peroxidase–aminoantipyrine buffer mixture by incubating
(in duplicate) 3.0 mL glucose oxidase-peroxidase-aminoantipyrine
buffer mixture with glucose standard (100 µg dried crystalline glucose in 0.2 mL 0.2% sodium benzoate solution). After 15, 20, 30,
and 60 min incubation, read absorbance, A, of solution at 510 nm.
Maximum color formation should be achieved within 20 min, and
color should be stable at least 60 min at 50°C.
(e) Aqueous ethanol.—About 80% (v/v). Dilute 80 mL 95% ethanol (laboratory grade) to 95 mL with H2O.
(f) Sodium acetate buffer.—(1) 200 mM, pH 4.5.—Pipet 11.8 mL
glacial acetic acid (1.05 g/mL) to 900 mL H2O. Adjust pH to 4.5 with
1M NaOH solution (ca 60 mL is required). Add 0.2 g sodium azide
and dilute to 1 L with H2O. (Caution: Sodium azide should not be
added until pH is adjusted. Acidification of sodium azide releases
poisonous hydrazoic acid.) Buffer is stable 12 months at room temperature. (2) 50 mM, pH 4.5.—Dilute 200 mM acetate buffer 1 + 3
with H2O.
(g) Dimethyl sulfoxide (DMSO).—Laboratory grade.
(h) Glucose standard stock solution.—1 mg/mL. Before preparing solution, dry powdered crystalline glucose (purity ≥97%) 16 h at
60°C under vacuum. Dissolve 0.1 g dried glucose, weighed to nearest mg, in 100 mL distilled water.
(i) Corn starch.—Containing known content of starch (e.g., ca
98% dry weight).
Items (a)–(c), (h), and (i) are supplied in Total Starch Assay kit
available from Megazyme International Ireland Ltd, Bray Business
Park, Bray, Co. Wicklow, Ireland.
D. Preparation of Test Samples, Standards, and Reagent Blank
(a) Test sample.—Grind ca 50 g laboratory sample in grinding
mill to pass 0.5 mm sieve. Transfer all material into wide-mouthed
plastic jar and mix well by shaking and inversion.
(b) D-Glucose standard working solutions.—50 and 100 µg. Add
50 and 100 µL D-glucose standard stock solution, C(h), to separate
test tubes, and adjust volume in each tube to 100 µL with H2O. Prepare solutions immediately before use.
(c) Reagent blank.—Transfer 0.1 mL H2O into test tubes and proceed with total starch determination using standard assay procedure
starting from step E(a)(7).
E. Determination of Total Starch
(a) Standard procedure α-amylase/amyloglucosidase
(AA/AMG).—Run D-glucose working standard solutions (in quadruplicate), reagent blank (in duplicate), and corn starch with each
set of tests. Use reagent blank to zero spectrophotometer. (1) Accurately weigh 90–100 mg ground test portion directly into glass
test tube. Tap tube gently on laboratory bench to ensure that all particles drop to bottom of tube. (Note: When analyzing cereal products containing high levels of glucose [processed cereal products
{e.g., breakfast cereals} and all products of unknown or uncertain
composition {i.e., products containing free glucose or
maltodextrins}], pre-extract 90–100 mg of weighed, ground test
sample 2× with 10 mL 80% aqueous ethanol, C(e), at ca 80°C over
10 min/extraction. Centrifuge slurry at 1000 × g and discard
supernatant. Use sediment for analysis.) (2) Add 0.2 mL 80%
aqueous ethanol to tube and stir on Vortex mixer to ensure that test
portion is wet. Add 3.0 mL thermostable α-amylase, C(b), and mix
contents of tube on Vortex mixer to ensure complete dispersion.
(3) Immediately place tube in boiling water bath for 2 min, remove
from water bath, and mix vigorously on Vortex mixer. Return tube
to boiling water bath for additional 3 min and then mix contents
vigorously on Vortex mixer. (Note: Some solids will adhere to side
of test tube; however, this will not affect analysis since tube contents are treated with enzyme in this step.) (4) Place tubes in water
bath set at 50°C and let equilibrate 5 min. Add 4.0 mL 200 mM sodium acetate buffer, C(f)(1), and 0.1 mL amyloglucosidase solution, C(c), and vigorously mix contents on Vortex mixer. Cap tube
with marble and incubate 30 min at 50°C. (5) Quantitatively transfer the entire contents of test tube to 100 mL volumetric flask. Use
water wash bottle to rinse tube contents thoroughly. Dilute to 100
mL with H2O. (Note: If product contains <10% starch, adjust volume to 10.0 mL [instead of 100 mL]. Make appropriate adjustments
to calculations.) Thoroughly mix contents of flask. Centrifuge
aliquot of suspension 10 min at 1000 × g. Alternatively, filter
aliquot through filter paper. (6) Carefully and accurately transfer 0.1
mL aliquot of each supernatant (or filtrate) to bottoms of separate
test tubes; use 2 tubes/supernatant (or filtrate). (7) Add 3.0 mL glucose oxidase–peroxidase–aminoantipyrine buffer mixture, C(d), to
each tube (reaction solutions from test portion and corn starch, re© 2000 AOAC INTERNATIONAL
agent blank, and D-glucose standard working solutions), and incubate 20 min at 50°C. (8) Measure and record absorbance, A, of each
test solution at 510 nm against reagent blank. Average A values for
each test and use in Calculations, G.
(b) Modified procedure (DMSO/AA/AMG).—For products containing enzyme-resistant starch. (1) Accurately weigh 90–100 mg
ground test portions directly into glass test tube. Tap tube gently on
laboratory bench to ensure that all particles drop to bottom of tube.
(Note: When analyzing cereal products containing high levels of
glucose, pre-extract 90–100 mg weighed, ground test sample 2×
with 10 mL aqueous ethanol, C(e), at ca 80°C over 10 min/extraction. Centrifuge slurry at 1000 × g and discard supernatant. Use sediment for analysis.) (2) Add 0.2 mL 80% aqueous ethanol to tube and
stir on Vortex mixer to ensure that test portion is wet. (3) Immediately add 2 mL DMSO solution, C(g), and stir tube on Vortex mixer.
Place tube in vigorously boiling water bath and remove after 5 min.
Add 3.0 mL thermostable α-amylase solution, C(b), and mix contents on Vortex mixer to ensure complete dispersion. (4) Immediately proceed according to standard procedure (AA/AMG) starting
from step E(a)(3).
(2) Centrifuge reaction solution 10 min at 1000 × g and carefully
pour supernatant into 100 mL volumetric flask.
(3) Resuspend pellet in 5 mL 50 mM sodium acetate buffer,
C(f)(2), by vigorous stirring on Vortex mixer. Add additional 5 mL
50 mM sodium acetate buffer, mix, and centrifuge 10 min at
1000 × g.
(4) Combine supernatant with that from step (2), and dilute to
volume. Analyze for starch starting with step E(a)(6).
(5) To pellet from (3) add 2 mL DMSO and analyze for starch by
modified procedure E(b) starting from step E(b)(3) [proceed to procedure E(a) as stated].
(6) At step E(a)(5), adjust volume to 10 mL (instead of 100 mL).
(7) Proceed with standard procedure, E(a), starting from step
E(a)(6). Make appropriate adjustments to calculations.
G. Calculations
Calculate total starch content (percent, on as is basis) in test sample as follows:
Total starch, % = A × F ×1000 ×
=A×
F. Determination of Enzyme-Resistant Starch (Optional)
(Note: This part of method was not validated in collaborative study.)
Level of enzyme-resistant starch in products depends on nature of
original starch (e.g., high amylose) and on processing conditions.
This may vary from 0.1–30% total starch in product. Determine
level of enzyme resistant starch as follows:
(1) Analyze product according to standard procedure,
E(a)(1)–(4).
1
100 162
×
×
1000 W
180
F
× 90
W
where A = absorbance of reaction solutions read against reagent
blank; F = factor to convert absorbance values to µg glucose =
100 µg glucose/absorbance value for 100 µg glucose; 1000 = volume correction, i.e., 0.1 mL taken from 100 mL; 1/1000 = conversion from µg to mg; 100/W = conversion to 100 mg test portion;
162/180 = factor to convert from free glucose, as determined, to
anhydroglucose, as occurs in starch.
Reference: J. AOAC Int. 80, 571(1997).
Revised: March 1998
© 2000 AOAC INTERNATIONAL