Enzymes AACC Method 22-15

Page 1 of 4

Measurement of Diastatic Activity of Flour or Semolina

Final approval April 13, 1961; Reapproval November 3, 1999

Objective
Processing characteristics of flour are influenced by increased levels of
hydrolytic enzymes, primarily α-amylase. This method estimates diastatic
activity in flour or semolina by measuring the amount of maltose produced
through hydrolysis of starch. Maltose can be measured by ferricyanide procedure
with or without titration with thiosulfate.

Apparatus
1. Constant-temperature bath regulated at 30 ± 0.1°.
2. Filter paper, Whatman No. 4 or equivalent.
3. Microburet, 10-ml capacity.
4. Erlenmeyer flask, 125-ml.

Reagents
1. Buffer solution. Dissolve 3 ml glacial acetic acid and 4.1 g anhydrous
sodium acetate and make to 1 liter with water. The pH of this solution is 4.6–4.8.
2. H2SO4, 3.68N (±0.05). Dilute 10 ml concentrated H2SO4 (specific gravity
1.84) to 100 ml and adjust concentration if necessary.
3. Sodium tungstate solution, 12%. Dissolve 12.0 g Na2WO4·2H2O and dilute
to 100 ml.
4. Alkaline ferricyanide reagent, 0.1N. Dissolve 33 g pure dry K3Fe(CN)6 and
44 g anhydrous Na2CO3 and dilute to 1 liter. To standardize, add to 10 ml of this
solution 25 ml acetic acid-salt solution (reagent 5) and 1 ml soluble starch-KI
solution (reagent 6) and titrate with 0.1N thiosulfate. Exactly 10 ml should be
required to discharge blue color completely.
5. Acetic acid-salt solution. Dissolve completely 70 g KCl and 40 g
ZnSO4·7H2O in 750 ml water; add slowly 200 ml glacial acetic acid, and dilute
to 1 liter with water.
6. Soluble starch-KI solution. Suspend 2 g soluble starch in a small quantity of
cold water and pour slowly into boiling water with constant stirring. Cool thor-
oughly (or resulting mixture will be dark-colored), add 50 g KI, dilute to 100 ml,
and add 1 drop saturated NaOH solution.
7. Thiosulfate solution, 0.1N. Prepare and standardize as directed in Method
70-75.

Procedure
1. Introduce 5 g flour (14% moisture basis) and 1 tsp ignited quartz sand into
100- or 125-ml Erlenmeyer flask, and mix by rotating flask. Add 46 ml buffer
solution at 30°, and again mix by rotating flask until all flour is in suspension.
(Flask and all ingredients should be individually brought to 30° before being
mixed together.)
Enzymes AACC Method 22-15
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Measurement of Diastatic Activity of Flour or Semolina

(continued)

2. Place in constant-temperature bath at 30° and maintain at this temperature

exactly 1 hr, shaking flask by rotation every 15 min.
3. At end of 1 hr, add 2 ml 3.68N H2SO4 and mix thoroughly. Add immedi-
ately 2 ml sodium tungstate solution and again mix thoroughly.
4. Let stand 2 min and filter (Whatman No. 4 or equivalent), discarding first
8–10 drops of filtrate.
5. Mix filtrate, remove 5-ml aliquot. Determine maltose as follows:
6. Pipet 5 ml extract into 125-ml Erlenmeyer flask. Add with pipet exactly 10
ml alkaline ferricyanide (reagent 4), mix, and immerse flask in vigorously boil-
ing water bath so that surface of liquid in flask is 3–4 cm below surface of boil-
ing water. (Delay between filtering of extract and treatment in boiling water bath
should not exceed 15–20 min. Further delay may cause error due to sucrose
hydrolysis in acid solution.)
7. Exactly 20 min after immersion, remove flask from bath and cool under
running water. Add 25 ml acetic acid-salt solution (reagent 5) and 1 ml soluble
starch-KI (reagent 6); mix well by rotation. Titrate with 0.1N thiosulfate to
complete disappearance of blue color. (A 10-ml microburet is recommended for
this titration.)

Calculation
Calculate ml ferricyanide reduced by subtracting ml thiosulfate required from
thiosulfate equivalent of ferricyanide reagent. See Note 2. Report as mg maltose
produced by 10 g flour in 1 hr at 30° by reference to Table I or table of Method
80-60.

Notes
1. The foregoing directions are applicable to all ordinary flours where values
for mg maltose produced from 10 g flour in 1 hr will not exceed 600. If the solu-
tion in the flask is colorless after treatment in a boiling water bath and gives no
blue color after addition of the starch-KI solution, an excess of reducing sugar is
present. Repeat the determination, using a smaller aliquot of extract; i.e., 1, 2, or
3 ml instead of 5 ml. In such cases, dilute to 5 ml and multiply mg maltose found
by an appropriate factor (5, 5/2, or 5/3 according to whether 1-, 2-, or 3-ml
aliquot is taken).
2. A blank determination to indicate maltose or reducing sugar originally
present in flour is unnecessary with normal sound flours. The quantity of reduc-
ing sugars originally present as such in flour milled from sound wheat is so small
and so constant that it may be neglected for all practical purposes. Should a
blank determination be desired, however, proceed as follows:
Enzymes AACC Method 22-15
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Measurement of Diastatic Activity of Flour or Semolina

(continued)

Combine 5 ml ethyl alcohol, 95% by volume; 50 ml acid buffer solution

(dissolve 3 ml glacial acetic acid, 4.1 g anhydrous sodium acetate, and 4.5 ml
H2SO4, specific gravity 1.84, and dilute to 1 liter with water); and 2 ml sodium

TABLE I
Thiosulfate-Maltose (Diastatic Activity) Conversion
0.1N Maltose per 0.1N Maltose per 0.1N Maltose per
Thiosulfate 10 g Flour Thiosulfate 10 g Flour Thiosulfate 10 g Flour
(ml) (mg) (ml) (mg) (ml) (mg)
0.10 618 3.60 360 7.10 145
0.20 608 3.70 353 7.20 140
0.30 598 3.80 347 7.30 135
0.40 588 3.90 341 7.40 130
0.50 578 4.00 334 7.50 126
0.60 568 4.10 328 7.60 121
0.70 558 4.20 322 7.70 116
0.80 550 4.30 315 7.80 111
0.90 542 4.40 308 7.90 106
1.00 534 4.50 302 8.00 101
1.10 527 4.60 295 8.10 96
1.20 519 4.70 288 8.20 90
1.30 512 4.80 282 8.30 85
1.40 505 4.90 276 8.40 80
1.50 499 5.00 270 8.50 76
1.60 492 5.10 264 8.60 71
1.70 485 5.20 257 8.70 65
1.80 478 5.30 251 8.80 60
1.90 472 5.40 244 8.90 56
2.00 465 5.50 237 9.00 51
2.10 458 5.60 231 9.10 46
2.20 451 5.70 225 9.20 41
2.30 445 5.80 218 9.30 36
2.40 438 5.90 213 9.40 31
2.50 431 6.00 207 9.50 25
2.60 425 6.10 201 9.60 20
2.70 418 6.20 195 9.70 15
2.80 412 6.30 188 9.80 10
2.90 406 6.40 182 9.90 5
3.00 398 6.50 176
3.10 392 6.60 171
3.20 385 6.70 166
3.30 379 6.80 161
3.40 373 6.90 156
3.50 367 7.00 151
Enzymes AACC Method 22-15
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Measurement of Diastatic Activity of Flour or Semolina

(continued)

tungstate solution (reagent 3). To 5 ml of this mixture (used in place of 5 ml

flour extract) add 10 ml ferricyanide solution (reagent 4), and proceed as in
determination of maltose, above. It should require 10 ml thiosulfate to discharge a
blue starch-iodine color. If titration (“thiosulfate equivalent”) falls within 10 (±0.05)
ml, reagents need not be discarded, but an appropriate correction should be made
in maltose calculations.

References
1. AOAC International. 1995. Official Methods of Analysis of AOAC International, 16th ed.
Method 932.04. The Association, Arlington, VA.
2. Blish, M. J., and Sandstedt, R. M. 1933. An improved method for the estimation of flour
diastatic value. Cereal Chem. 10:189.
3. Coleman, D. A., Snider, S. R., and Dixon, H. B. 1934. The diastatic activity of whole wheat and
some other cereal grains as determined by the Blish-Sandstedt method. Cereal Chem. 11:523.
4. Davis, C. F., and Worley, D. F. 1934. Correlation between diastatic activity and gassing power
in commercial flours. Cereal Chem. 11:536.
5. Hagedorn, H. C., and Jensen, B. N. 1923. Zur Mikrobestimmung des Blutzuckers mittels Ferri-
cyanid. Biochem. Z. 135:46.
6. Malloch, J. G. 1929. Studies on the resistance of wheat starch to diastatic action. Can. J. Res.
1:111.
7. Markley, M. C., and Bailey, C. H. 1934. Factors affecting the diastatic activity of wheat flour.
Cereal Chem. 11:515.
8. Sandstedt, R. M. 1937. The adaptation of the ferricyanide maltose method to high diastatic
flours. Cereal Chem. 14:603.