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7 Estimation of Gluten in Wheat (Misra and Gupta 1995)

7 Estimation of Gluten in Wheat (Misra and Gupta 1995)

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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


Estimation of Lysine


in 15 ml of borate buffer and centrifuge after each suspension. After the third

washing, resuspend the pellet in 80 ml of borate buffer. The reagent can be

used for 2 weeks.

2. Pyridine reagent: Just before use, prepare a 3% solution of 2-chloro

3,5 dinitroypyridine in methanol.

3. Papain solution: Papain is dissolved in 0.1 N sodium acetate buffer (pH 7.0) to a

final concentration of 4 mg/ml. Prepare the enzyme solution daily. Filter the

solution, if required.

4. Sodium carbonate buffer: 0.05 M, pH 9.0.

5. Amino acid mixture: (in milligrams): alanine, 3; arginine, 50; aspartic acid, 60;

cystine, 20; glutamic acid, 300; glycine, 40; histidine, 30; isoleucine, 30;

leucine, 50; threonine, 30; tyrosine, 30 and valine, 40. Dissolve 100 mg of this

mixture in 10 ml of carbonate buffer (0.05 M, pH 9.0).

6. HCl (1.2 N) solution.

7. Ethyl acetate.

Standard Curve

Prepare a standard curve in a range of 0–200 mg lysine/ml. Dissolve 62.5 mg of pure

lysine mono-hydrochloride in 20 ml of carbonate buffer (0.05 M, pH 9.0). Prepare

the solution by dilution with carbonate buffer to get the following concentrations 0,

250, 500, 750 and 1,000 of lysine (mg/ml). From each of these solution take 1 ml

and add 4 ml of papain (5 mg/ml) solution. Now the respective concentration of

lysine will be 0, 50, 100, 150 and 200 mg/ml. Pipette out 1 ml of each solution into

centrifuge tube, add 0.5 ml of copper phosphate suspension. Follow the same

procedure as described below for the sample.


1. Weigh 100 mg defatted finely group sample into a glass vial. Add 5 ml of papain

solution. Cap the vials and mix well so that the sample is totally wet. Incubate at

65 C in the oven overnight. Include a blank (5 ml of papain solution without any

sample) with every group of samples.

2. Remove the sample from the oven, shake well, cool to room temperature

centrifuge and decant the clear digest.

3. From the supernatant fraction transfer 1 ml into a 15-ml centrifuge tube and add

0.5 ml of carbonate buffer and 0.5 ml of copper phosphate suspension.

4. Shake the mixture for 5 min in a vortex mixer and centrifuge to precipitate the

excess copper phosphate.

5. Pipette out 1 ml of the supernatant into a 30-m test tube and add 0.1 ml of the

pyridine reagent and mix well. Cap the tubes with rubber/velvet corks and shake

occasionally for 2 h at room temperature.

6. Add 5 ml of 1.2 N HCl and mix well by vortexing.

7. Add 5 ml of ethyl acetate, invert capped tubes at least 10 times, then remove

the top layer (ethyl acetate) using a syringe adapted with a polyethylene tube.

This step should be repeated 3 times.



Methods for Nutritional Quality Evaluation of Food Materials

8. Determine the absorbance of the aqueous phase in a colorimeter/spectrophotometer at 390 nm. Relate absorbance to the corresponding lysine value on the

standard curve.


Plot absorbance reading against mg standard. Read off mg of sample from curve.

% Lysine in sample =

mg lysine from standard curve


mg sample


Lysine content (g per 16 g N) =


mg lysine from standard curve  0:16


% in the sample

Protein Fractionation in Cereals

(Landry and Moureaux 1970)

Seed proteins have been classified into five major groups based on their solubility

(Mendel and Osbome). These include: water-soluble albumins, salt soluble

globulins, alcohol soluble prolamins, alkali soluble gluteline and residue (insoluble) proteins. Protein fractionation procedure as given by Landry and Moureaux

(1970) separates 5 fractions namely: albumins + globulins, prolamin, prolamin-like,

glutelins-like and true glutelin which are obtained by following the scheme

described herein. This method is good for maize, sorghum and barley. However,

it has been found that the extractability of prolamin is greater for barley when 50%

isopropanol with 2% mercaptoethanol is used instead of 70% isopropanol

containing 0.6% mercaptoethanol.

Although solubility is one of the classic criteria for separating seed proteins, it is

not an ideal classification as the solubility is critically dependent on a number of

conditions like the exact nature of the solvent, the ratio of solvent to sample, the

hydration state of sample, the temperature of the solvent, fineness of the sample, etc

(Table 13.1).

Equipment and Glassware









Kjeldahl digestion and distillation set

pH meter

Conical flask

Centrifuge tubes


Protein Fractionation in Cereals

Table 13.1 Landry-Moureaux fractionation sequence


Time of

Fraction Solvent

temperature agitation (min)



NaCl, 0.5 M

4 C







Isopropanol, 70%

20 C






Isopropanol, 70%

20 C

+ 2-ME, 0.6% (v/v)


Borate buffer, 0.05 M

20 C


pH 10 + 2-ME, 0.6%(v/v) 20 C




Borate buffer pH 10


+ 2-ME, 0.6% (v/v)


+ SDS, 0.5% (v/v)



Protein fraction

Albumins and globulins






As specified in the scheme.


1. Take 2 g defatted finely ground sample (100 mesh) in a stoppered conical flask

or centrifuge tube.

2. Add 20 ml 0.5 M NaCl solution and shake for 60 min at 4 C.

3. Centrifuge at 6,000 Â g for 5 min and collect the supernatant.

4. Re-extract the residue by following the same extraction procedure twice except

that the shaking should be done for 30 min each time.

5. Then extract the residue twice by using distilled water in the same manner.

6. Collect all the supernatants and make up volume to 100 ml with saline. This

represents fraction I which contains albumins and globulins.

7. Extract prolamin, prolamin-like, glutelin-like and glutelin from the residue

obtained in step 6 at 20 C following the same procedure but using solvents

and shaking time specified in scheme given earlier. The residue obtained at the

last extraction should also be analysed and represented as residue protein.

8. To obtain albumins and globulins from fraction I, add equal volume of 10%

TCA and allow it to remain cold for 30 min then centrifuge at 6,000 Â g for

15 min at 4 C. The precipitate obtained represents albumins + globulins, while

the supernatant will contain non-protein nitrogen (free amino acids and


9. Estimate nitrogen in all the fractions after digestions and distillation following

micro-Kjeldahl method.

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