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32 Estimation of Glucosinolates Content in Oil Seeds (Brezezinski and Mendelewski 1984)

32 Estimation of Glucosinolates Content in Oil Seeds (Brezezinski and Mendelewski 1984)

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Estimation of Glucosinolates Content in Oil Seeds



Sinigrin (a glucosinolate) on reaction with sulphuric acid is first hydrolyzed to

thioglucose which then dehydrates to thiofurfural derivative. This furfural reacts

with thymol to a coloured compound whose absorbance is measured at 505 nm

against the sample blank.

Equipment and Glassware







Fat extractor apparatus

Micro ion-exchange columns ; 100 Â 18

Water bath for boiling.



1. Potassium sulphate solution (0.3 M): Weight 26.1 g potassium sulphate into

500 ml volumetric flask and make up the volume with water.

2. Petroleum ether (40–60 C boiling range)

3. Sulphuric acid solution (80%)

4. Thymol reagent: Weigh 1 g thymol into a 100-ml volumetric flask and make up

the volume with ethanol.

5. Lead acetate solution: Weigh 22.75 g lead acetate trihydrate, dissolve and

dilute to 100 ml with water.

6. Pyridine/acetic acid buffer (0.5 M): Add 30 ml glacial acetic acid and 40 ml

pyridine to 930 ml water, mix and store in refrigerator.

7. Sodium hydroxide (0.5 N): Weigh 20 g NaOH and dissolve. Dilute it to

1,000 ml with water.

8. DEAE: Sephadex A-25

9. Formic acid (30%)

10. Allyl glucosinolate stock solution (5 mmoles/ml): Weigh out 519.5 mg sinigrin

into 250 ml volumetric flask and make up the volume with water.

11. Allyl glucosinolate standard solution (0.3 mmoles/ml): Dilute 3 ml of the stock

(5 mmoles/ml) sinigrin with 0.3 M K2SO4.

Preparation of oil free meals: Weigh about 4 g raw mustard seed and extract

with petroleum ether in a soxhlet extractor. Dry the extracted meals and store in

screw-cap vials.

Extraction of Glucosinolates

1. Weigh 100 mg meal into a test tube.

2. Add 9 ml cold water and place the mixture for 15 min in a boiling water bath.

3. Cool and add 1 ml of lead acetate solution and allow the mixture to stand for

15 min.

4. Centrifuge the mixture (4 min, 1,400 Â g).

5. Use the supernatant for purification on DEAE-Sephadex A-25 column.



Methods for Nutritional Quality Evaluation of Food Materials

Preparation of DEAE-Sephadex A-25 Pyridine Acetate Column

1. Stir suspension of sephadex in an excess of 0.5 M pyridine/acetic acid buffer

such that the settled volume of the resin is one half of the total.

2. Fill half of the column with water and add 1 ml of sephadex suspension.

3. After setting of the column with water and insert the sieve.

4. Allow it to sink on the bed.

5. Wash the column twice with water and drain the water between each wash.

Isolation and Measurement of Glucosinolates

1. Add 3 ml of meal extract to the prepared columns without disturbing the surface.

2. Wash the column with 2 Â 2 ml water, 2 Â 2 ml 30% formic acid and 2 Â 2 ml


3. Place clean graduated tubes or vials beneath the column.

4. Elute the column with 2 Â 5 ml of 0.3 M potassium sulphate and dilute to 10 ml


5. Take 1 ml aliquot in the glass tubes with screw caps.

6. Add 7 ml of 80% sulphuric acid and 1 ml of 1% thymol solution to each tube.

7. Thoroughly mix the contents and place at 100 C for 60 min.

8. Immediately cool the tubes under tap water and mix again.

9. Measure the absorbance at 505 nm against 0.3 M potassium sulphate blanks.

Calculation of Total Glucosinolate Content

1. Calculate the micromolar concentration of glucosinolates from the absorbance

reading of the micromolar extinction coefficient of the sinigrin standard solution.

2. Prepare a set of (4 or 5) blanks and sinigrin standard with each batch of samples.

3. Heat them and read on the spectrophotometer.

4. Prepare the blank by mixing 1 ml 0.3 M potassium sulphate, 7 ml 80% sulphuric

acid and 1 ml of 1% thymol solution.

5. Prepare sinigrin standard by taking 1 ml sinigrin standard (0.3 mmoles/ml), add

to it 7 ml 80% sulphuric acid and 1 ml of 1% thymol solution.

6. Read the blanks before and after the sample batch.

Calculate the micromolar extinction (absorption) coefficient (K) from the

absorption reading of the standard and blank as follows:


Mean absorbance of standardsị Mean absorbance of blanksị

0:3 ðthe micromolar concentration of the standardÞ

Calculate the micromolar concentration of glucosinolates in the samples on a dry

weight basis as follows:

mM/g dry basis ¼

Corrected absorbance  Dilution Factor  100


K Â Wt. of the sample (g) Â ð100 À % moisture of the sample)


Determination of Polyphenols in Pulse Grains



Determination of Polyphenols in Pulse Grains

(Swain and Hills 1959)

Polyphenols are tannin-like compounds and are termed as anti-nutritional

constituents of pulse grains and some other food grains. Polyphenol combine with

the proteins present in grains thereby adversely affecting their digestibility to a

considerable extent. Although, polyphenols are nutritionally undesirable to a

human being, however, they are useful to plants as they are involved in the defence

mechanism of plants conferring them disease resistance.


Polyphenols reduce phosphotungstomolybdic acid in alkaline solution and produce

a highly coloured blue solution, the intensity of which is proportional to the amount

of polyphenols present in grain sample. The intensity is measured at 725 nm.


1. Folin-Denis reagent: Dissolve 100 g sodium tungstate and 20 g

phosphomolybdic acid in 750 ml distilled water in a suitable flask and add

50 ml phosphoric acid. Reflux the mixture for 2 h and make up to 1 l with

water. Protect the reagent from exposure to light.

2. Sodium carbonate solution: Dissolve 350 g sodium carbonate in 1 l of water at

70–80 C. Filter through glasswool after allowing it to stand overnight.

3. Standard tannic acid solution: Dissolve 100 mg tannic acid in 100 ml of distilled


4. Working standard solution: Dilute 5 ml of the stock solution to 100 ml with

distilled water. One ml contains 50 mg tannic acid.


1. Take 500 mg of powdered sample and add 10 ml methanol containing 1 ml of

1% HCl and keep for 24 h at room temperature with occasional shaking or reflux

for 1 h and cool.

2. Then centrifuge the contents at 2,000 rpm for 20 min of filter and transfer the

supernatant into 25 ml volumetric flask.

3. Make up the volume up to mark by 80% methanol, take 1 ml of this solution into

another 25 ml volumetric flask and add 5 ml distilled water followed by the

addition of 1 ml Folin-Denis reagent and keep it for 30 min.

4. After this add 2 ml of sodium carbonate solution. Make up the volume up to

25 ml with the help of distilled water.

5. After this keep the whole contents for 1 h to develop blue colour.

6. Read the colour intensity at 725 nm.

7. Run a blank using distilled water in place of sample.

8. Prepare a standard graph by using 0–100 mg tannic acid.



Methods for Nutritional Quality Evaluation of Food Materials


Calculate the polyphenol content of the samples as tannic acid equivalents from the

standard graph.

Modified method for tannins (A.O.A.C 1995a, b)


1. Weigh 1 g of the powdered material 4 times and transfer to 500 ml conical flasks.

Add 150 ml water to each flask.

2. Add to three flasks 10, 15 and 20 ml of standard tannic acid solution, respectively.

3. Heat all the flasks gently and boil for 30 min. Then centrifuge at 2,000 rpm for

20 min.

4. Collect supernatant in 250 ml flasks and make up the volume.

5. Transfer 10 ml of the supernatant extract in 100 ml flasks and add 75 ml of water,

2.5 ml of Folin-Denis reagent and add 5 ml of sodium carbonate solution and

make up the volume.

6. After 30 min take the reading at 740 nm.

7. Prepare a graph by plotting O.D. vs. tannic acid concentration. The value

wherever it cuts X-axis was taken as new origin. The difference in O.D. in first

and second origin is taken as a measure of the content of tannins in the samples.


Estimation of Aldehydes in Food Stuffs

The occurrence of aldehydes in food stuffs is highly undesirable. These compounds

are usually formed via the process of auto-oxidation of oil/fats or oxidation of

alcohols. In order to maintain the quality standards of food items, it is important to

know the contents of these undesirable molecules whose regular intake might create

serious health problem to the consumers.


Aldehydes possess a unique property of reacting with hydroxylamine hydrochloride. The liberated acid when reacts with alcoholic KOH, gives yellow colour. From

the weight of material taken, volume of alkali used and the factor corresponding to a

particular aldehyde, its percentage in food can be calculated with the help of

formula derived for this purpose.

Equipment and Glassware

1. Tubes for weighing the sample

2. Volumetric flasks (100 ml)


1. Benzene.

2. Ethanol (60% v/v)

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32 Estimation of Glucosinolates Content in Oil Seeds (Brezezinski and Mendelewski 1984)

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