SOP: Determination of Starch by the Enzymic Method

GIFSQ — Global Institute for Food Safety & Quality

Principle

The method is suitable for products containing natural starch. Sugars are removed by leaching with alcohol. If much fat and proteinaceous material is present, the sample is treated with hot ethanolic-KOH and washed with 80% ethanol. The starch in the residue is gelatinized and incubated with amyloglucosidase enzyme at pH 4.5, which converts the starch to glucose; the glucose is measured enzymically using glucose oxidase.

Apparatus required

• 250-ml centrifuge bottles
• Boiling water bath
• Watch glasses
• Aluminium foil
• Incubator (55–58°C)
• 100-ml volumetric flasks
• Glass-stoppered test tubes
• Spectrophotometer (absorbance at 526 nm)

Reagents

a. 8 N H₂SO₄.

b. 0.2 N acetic acid: dilute 12 ml of glacial acetic acid to 1000 ml.

c. 0.2 N sodium acetate: make up 27.2 g of sodium acetate trihydrate (CH₃COONa·3H₂O) to 1000 ml.

d. Enzyme solution: dissolve 0.5 g of amyloglucosidase in 50 ml of pH 4.5 buffer prepared by mixing 20 ml of 0.2 N sodium acetate and 30 ml of 0.2 N acetic acid; prepare the solution just before use; allow the insoluble material to settle.

e. Glucose oxidase-peroxidase-chromogen reagent: mix 45 ml of 0.2 N sodium acetate and 5 ml of 0.2 N acetic acid, and make up to 100 ml with water (pH 5.5); weigh 20 mg of purified crystalline o-dianisidine dihydrochloride into a tube and dissolve in 2 ml of water and 1 ml of ethanol; into another tube weigh 2 mg of horseradish peroxidase and dissolve in 5 ml of pH 5.5 buffer, then add 0.3 ml of glucose oxidase prepared from Aspergillus niger, Type V, containing 1000 units/ml; transfer the solution in the two tubes to a 100-ml volumetric flask, make up to volume with pH 5.5 buffer, and store in an amber coloured glass-stoppered bottle; prepare the reagent just before use.

f. Glucose standards: weigh 100 mg of glucose, dissolve in water and make up to 1000 ml; pipette 10, 20, 40 and 60 ml aliquots into 100-ml volumetric flasks and make up to volume to get standard solutions having 10, 20, 40 and 60 µg of glucose per ml respectively.

Sample preparation — removal of sugars

Render the sample free of sugars by one of the following:

A. Take a known weight of the sample containing 0.1–0.2 g of starch into a 250-ml centrifuge bottle; add 150 ml of 80% ethanol; cover with a watch glass and heat on a boiling water bath for 1 hr; centrifuge for 10 min and decant the supernatant; add 50 ml of 80% ethanol, centrifuge again for 10 min, and decant the supernatant. (The alcohol from the supernatant solutions may be evaporated and the residue used for sugar estimation.)

B. If the sample contains much fat and protein: to the residue add 150 ml of hot ethanolic solution; cover with a watch glass and heat on a boiling water bath with occasional stirring for 1 hr; centrifuge for 10 min and discard the supernatant; to the residue add 50 ml of hot 80% ethanol, centrifuge and discard the supernatant.

Procedure — Gelatinization and enzyme digestion

To the residue, either from A or from B, add 30 ml of water and heat on a boiling water bath for 1 hr to remove traces of alcohol

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Cover the mouth of the centrifuge bottle with aluminium foil and continue heating for 2 hr to gelatinize the starch

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Remove the bottle and cool to 40°C; add 20 ml of 0.2 N sodium acetate and then 30 ml of 0.2 N acetic acid (pH 4.5); mix

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Add 2.0 ml of the freshly prepared amyloglucosidase enzyme reagent and mix; add 2 drops of toluene

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Cover the bottle with aluminium foil and incubate overnight (16 hr) at 55–58°C

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Cool the contents; make up to 100 ml with water in a volumetric flask; mix and allow to settle

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Dilute 2.0 ml of the clear supernatant to 100 ml with water

Procedure — Colour development

Take glass-stoppered test tubes: to one tube (blank) add 1.0 ml of water; to four tubes add 1.0 ml each of the 10, 20, 40 and 60 µg/ml glucose standards; to the sample tube add 1.0 ml of the sample digest

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Add 2.0 ml of the glucose oxidase-peroxidase-chromogen reagent to each tube; stopper, mix and incubate at 37°C for 1 hr

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Add 5.0 ml of 8 N H₂SO₄ to each tube; mix and cool to room temperature

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Measure the colour at 526 nm, using distilled water to set the instrument to 100% transmission

Calculation

Correct the absorbances of the standards for the blank value and draw a calibration curve of glucose concentration (µg/ml) versus corrected absorbance. Correct the absorbance of the sample for the blank value, find the glucose concentration from the calibration curve, and calculate as follows:

% Starch = (µg of glucose/ml × 100 × 100 × 100 × 0.9) ÷ (1 × 2 × wt. of sample × 1.0 × 100,000)

Where,

• µg of glucose/ml = glucose concentration read from the calibration curve
• wt. of sample = weight of the sample taken (g)

Precautions

• Prepare the enzyme solution and the glucose oxidase-peroxidase-chromogen reagent just before use.
• Incubate the starch digestion for the full 16 hr at 55–58°C.
• Add the 2 drops of toluene before covering and incubating the bottle.
• Cool to room temperature before measuring the colour at 526 nm.

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

McRae, J.C., Planta (Berlin), 96, 101 (1971) — cited in the book.

Osborne, D.R. & Voogt, P., The Analysis of Nutrients in Foods, Academic Press, London, 1978, p. 139 — cited in the book.