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4 Estimation of Methionine (Mc Carthy and Paoille 1959)

4 Estimation of Methionine (Mc Carthy and Paoille 1959)

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Estimation of Starch



1. Weight 2 g of defatted sample into a 250-ml conical flask. To this add 25 ml of

2 N HCl and mix well. Autoclave at 15 lb pressure for 1 h.

2. Add a pinch of activated charcoal to the autoclaved sample. Heat to boiling and

filter when hot. Wash the charcoal with hot water. Also Collect the washings.

3. Neutralize the filtrate with 10 N NaOH to bring the pH to 6.5. Make up the

volume to 100 ml with water.

4. Transfer 50 ml of the hydrolysate in 250 ml conical flask and add 6 ml of 10%

NaOH followed by 0.3 ml of 10% sodium nitroprusside solution. Keep aside the

contents for 10 min with occasional shaking. Add 2 ml of 3% glycine solution

after 10 min. Allow it to stand for 10 min.

5. Add 4 ml of concentrated orthophosphoric acid and shake the contents


6. Read the intensity of colour after 10 min at 520 nm against a blank prepared by

omitting sodium nitroprusside.

Standard Curve

Prepare standard curve by taking different concentrations of methionine (0, 2, 4, 6,

8 and 10 mg) in duplicate and water to make up the total volume to 50 ml. Develop

the colour in the same way as mentioned above. Finally, draw the standard curve.


% methionine ¼


mg methionine from standard curve


Weight of sample

Estimation of Starch (Clegg 1956)


Starch is extracted with perchloric acid after removal of sugars by extracting the

sample flour with hot 70% alcohol. In hot acidic medium starch is hydrolysed to

glucose and dehydrated to hydroxymethyl furfural. This compound when reacts

with anthrone forms green colour.

Equipment and Glassware






Measuring flasks of various capacities (10, 25, 50, 100 ml)

Measuring cylinders (50, 100 ml)




Methods for Nutritional Quality Evaluation of Food Materials


1. Anthrone reagent: Dissolve 200 mg anthrone in 100 ml of ice-cold 95%

sulphuric acid.

2. 70% ethanol.

3. 52% perchloric acid.

4. Standard glucose solution: Dissolve 200 mg glucose in 100 ml distilled water.

Standard (100 mg/ml) – 10 ml of standard stock solution of glucose is diluted to

100 ml with distilled water.


1. Extract oven dried sample flour (100 mg) with hot 70% ethanol. Centrifuge and

retain the residue. Wash the residue with 70% hot ethanol. Dry the residue.

2. To the residue add 5.0 ml of water and 6.5 ml of 52% perchloric acid.

3. Shake the content for 5 min, centrifuge and collect the supernatant.

4. Repeat the extraction using 5 ml fresh perchloric acid for 10 min and centrifuge

to collect the supernatant.

5. Re-extract the pellet using 5 ml fresh perchloric acid and shake for 30 min.

6. Pool the supernatants and make up the volume to 100 ml with water.

7. Take suitable aliquot for glucose estimation. Add 5 ml of anthrone reagent. Heat

on boiling water bath for 10 min.

8. Cool rapidly and read the intensity of colour formed at 620 nm.

9. Find out glucose content using the standard curve. Multiply the value by a factor

0.9 as 0.9 g of starch yields 1 g of glucose on hydrolysis.


Estimation of Amylose (Williams et al. 1970)

Starch is composed of two components namely amylose and amylopectin. Amylose

is a linear and non-branched polymer of glucose, joined together by a 1–4 glucosidic linkages. Amylase when reacts with iodine, which is absorbed within the

helical coils, produces a blue coloured complex. The blue colour is measured


Equipment and Glassware







Centrifuge tubes

Volumetric flask, 100 and 50 ml


1. Stock iodine solution: Dissolve 20 g of KI and 2 of resublimed I2 in minimum

amount of water and make up the volume to 100 ml.

2. Iodine reagent: Dilute 10 ml of stock solution 1–100 ml at the time of use.


Estimation of Gluten in Wheat


3. KOH (0.5 N).

4. HCl (0.1 N).


1. Take 20 mg of dry, finely ground material (minimum 60 mesh). Add 10 ml of

0.5 N KOH and stir thoroughly with a magnetic stirrer for 10 min.

2. Make up the volume to 100 ml.

3. Take 10 ml of aliquot into 50 ml volumetric flask and add 5 ml of 0.1 N HCl and

0.5 ml of 12 reagent 2. Make up the volume.

4. Read the colour at 625 nm.

5. Calculate the amount of amylase from standard curve prepared by using standard

solution containing different amounts of amylase (0.2–2 mg) and amylopectin

(0.8–8 mg) keeping the ratio of amylase and amylopectin constant (1:4 w/w).

6. Amylopectin content can be calculated by subtracting the amylase from total

starch content.


Absorbance corresponding to 10 ml of the test solution = y mg

Amylose/g dry wt = y  10  50 mg:


Estimation of Gluten in Wheat (Misra and Gupta 1995)

When water is mixed with wheat flour and the contents are kneaded, a cohesive

mass of dough is formed. This mass on washing removes starch, bran and yields a

visco-elastic gum like material known as gluten. Gluten is mainly composed of

gliadin and glutenin proteins.

Equipment and Glassware







Glass rod

Sieve (100 mesh)


Analytical balance


1. KI solution: Dissolve 200 mg iodine and 2 g KI in water and make up to 100 ml.


1. Take 10 g of wheat sample in a beaker.

2. Add to it 7 ml distilled water and make a dough with the help of glass rod for

around 2 min.



Methods for Nutritional Quality Evaluation of Food Materials

3. After proper mixing, make a small ball of the dough and immerse it in a beaker

containing water.

4. The ball is left for 30 min.

5. Place the beaker under tap water; put a sieve (100 mesh) on it. Now the dough is

washed gently under running water till a chewing gum type cohesive mass

separates out. Bran will settle over the sieve and starch will pass down to the


6. To ensure complete removal of bran, wash the extract gluten with excess of

water by stretching inside the fingers.

7. To check whether whole of the starch is washed out or not, add KI to last extract.

Absence of any violet colour indicates completes removal of starch.

8. Squeeze out the adhered water from the extracted gluten and weigh. This will be the

weight of wet gluten. Dry at 105 C for 6 h and weigh to get the yield of dry gluten.


Estimation of Lysine (Tsai et al. 1972)

Lysine is an essential amino acid generally deficient in cereals. The method

described is for non-pigmented cereals (maize, sorghum etc.). The sample is

hydrolysed with papain or other proteolytic enzyme releasing free amino acids

and low molecular weight peptides. The a-amino group of the free amino acids are

blocked with copper, while the e-amino group of lysine remains free. The

e-dinitropyridyl derivative of lysine is then formed on reaction with 2-chloro-3,

5-dinitropyridine. Excess of 2-chloro-3 5-dinitropyridine is removed with ethyl

acetate and the colour of e-dinitropyridyl derivative is read at 390 nm.

Equipment and Glassware








Centrifuge tubes, 15 ml

Screw-cap glass vials, 30 ml



1. Copper phosphate reagent: The reagent is a suspension of copper phosphate

in borate buffer.

(i) Borate buffer (0.5 M, pH 9.0)

(ii) Copper phosphate suspension:

Solution A: 2.8 g CuCl2Á2H2O dissolved in 100 ml distilled water

Solution B: 13.8 g Na3PO4Á12H2O dissolved in 200 ml distilled water

Pour solution A into B with swirling and centrifuge (300 Â g) for 5 min. to

collect the precipitate. Discard the supernatant. Resuspend the pellet 3 times

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4 Estimation of Methionine (Mc Carthy and Paoille 1959)

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