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30 Estimation of Lignin (Goering and Van Soest 1975)

30 Estimation of Lignin (Goering and Van Soest 1975)

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Methods for Nutritional Quality Evaluation of Food Materials


Round bottom flask

Refluxing Set

Muffle Furnace


C. Acid Detergent Fibre (ADF)

1. Place 1 g of powdered sample in a round bottom flask and 100 ml of acid

detergent solution. Heat to boil in 5–10 min. Reduce heat to avoid foaming as

boiling begins. Reflux for 1 h after the onset of boiling. Adjust boiling to slow,

even level.

2. Remove container, swirl and filter the contents through a pre-weighed sintered

glass crucible (G-2) by suction and wash with hot water twice.

3. Wash with acetone breaks up the lumps. Repeat acetone washing until the filtrate

is colourless.

4. Dry at 100 C for overnight.

5. Weigh after cooling in a desiccator.

6. Express ADF content in percentage i.e. W/S x 100, where W is the weight of the

fibre and S is the weight of the sample.

D. Determination of Acid Detergent Lignin (ADL)

1. Transfer ADF to a 100-ml beaker with 25–50 ml of 72% sulphuric acid. Add 1 g

asbestos. Allow it to stand for 3 h with intermittent stirring with a glass rod.

2. Dilute the acid with distilled water and filter with pre-weighed Whatman No. 1

filter paper. Wash the glass rod and the residue several times to get rid of the


3. Dry the filter paper at 100 C and weigh after cooling in a desiccator.

4. Transfer the filter paper to a pre-weighed silica crucible and ash the filter paper

with the content in a muffle furnace at 550 C for about 3 h.

5. Cool the crucible in a desiccator and weigh. Calculate the ash content.

6. For blank, take 1 g asbestos, add 72% H2SO4 and follow the steps 2–5.

• For preparing pre-weighed filter paper, see the note under pectic substance.

NDF ẳ Hemicellulose + Cellulose + Lignin + Minerals

ADF ¼ Cellulose + Lignin + Minerals

Hemicellulose ¼ NDF – ADF

Cellulose ¼ ADF – Residue after extraction with 72% H2SO4

Lignin ẳ Residue after extraction with 72% H2SO4 ash

Wet materials can also be used for lignin estimation, provided the wet sample

is equivalent to 1 g of dry material.

• If the weight loss of asbestos blank on ashing is below 0.002 g/g, discontinue

the determination of blank.


Estimation of Capsaicin in Chillies



Weight of 72% H2SO4

% ADL =


washed fibre ðTest À Asbestos blankÞ À Ash ðTest À Asbestos blankÞ Â 100


Weight of sample

Estimation of Capsaicin in Chillies

Capsaicin is a protoalkaloid which is responsible for the pungency of chillies.

The quality of chilli fruits, extracts or oleoresins is determined by the capsaicin


(i) Colorimetric Method


The phenolic group in capsaicin reduces phosphomolybdic acid to lower acids

of molybdenum. The resulting component is blue in colour and is read at 650 nm.

The colour intensity is directly proportional to the concentration of capsaicin.


1. 0.4% sodium hydroxide

2. 3% Phosphomolybdic Acid

3. Dry acetone (Add about 25 g anhydrous sodium sulphate to 500 ml acetone of

analytical grade at least 1 day before use).

4. Stock Standard Capsaicin Solution: Dissolve exactly 50 ml of 0.4% sodium

hydroxide solution (1,000 mg/ml).

5. Working Standard: Dilute 10 ml of the stock standard to 50 ml with 0.4% sodium

hydroxide solution (200 mg/ml).


• Weigh 0.5 g dry chilli powder into a glass-stoppered test tube or volumetric


• Pipette out 10 ml dry acetone into the flask and shake it for 3 h in a mechanical

shaker. Let the contents settle down or centrifuge (10,000 rpm for 10 min).

Pipette out 1 ml of clear supernatant into a test tube and evaporate to dryness in a

hot water bath. Dissolve the residue in 5 ml of 0.4% sodium hydroxide solution.

• Add 3 ml of 3% phosphomolybdic acid. Shake the contents and let stand for 1 h.

Filter the solution quickly into centrifuge tubes to remove any floating debris.

Centrifuge at about 5,000 rpm for 10–15 min. Transfer the clear blue coloured

solution directly into the cuvette and read the absorbance at 650 nm.



Methods for Nutritional Quality Evaluation of Food Materials

• Run a reagent blank along with the test samples.

• Prepare a standard graph using 0–200 mg capsaicin simultaneously i.e. pipette

out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard solution and proceed as above.

(ii) Spectrophotometric Method


Capsaicin is extracted from red pepper (chillies) with ethyl acetate and made to

react with ethyl acetate solution of vanadium oxychloride. Then it is read at 720 nm.

This method is sensitive and is useful to measure small quantities (less than 0.05%).


1. Vanadium oxychloride (0.5%) in ethyl acetate.

2. Pure capsaicin (0.01%) in ethyl acetate (10 mg in 100 ml)


1. Grind the sample well to pass through No. 40 sieve.

2. Place 2 g sample in a 100-ml volumetric flask.

3. Let it stand for 24 h to extract (otherwise reflux the contents for 2.5 h) and then

make up to volume. Dilute 1 ml of extract to 5 ml with ethyl acetate. Add 0.5 ml

vanadium oxychloride solution (just before reading) and shake.

4. Read at 720 nm in a spectrophotometer.

5. Subtract the absorbance value.

6. Prepare a standard curve using 0.5, 1.0, 1.5, 2.0 and 2.5 ml of standard capsaicin

solution containing 50, 100, 150, 200 and 250 mg capsaicin, respectively.


% capsaicin ¼


mg capsaicin

100 100 mg capsaicin





1; 000 Â 1; 000




Estimation of Glucosinolates Content in Oil Seeds

(Brezezinski and Mendelewski 1984)

Rapeseed mustard meal obtained after oil extraction is a valuable component

of animal feedstuff. However, its utilization is limited as its feeding causes undesirable physiological effects due to the presence of glucosinolates (GSL’s).

The glucosinolate content in defatted meal ranges from 7 to 10%. There is need

to screen varieties containing low glucosinates produced by breeding programmes.


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.

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30 Estimation of Lignin (Goering and Van Soest 1975)

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