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9 Protein Fractionation in Cereals (Landry and Moureaux 1970)

9 Protein Fractionation in Cereals (Landry and Moureaux 1970)

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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.



Methods for Nutritional Quality Evaluation of Food Materials


1. It is advisable to check for the completeness of extraction of different protein

fractions. It can be done easily by monitoring absorbance of the last extraction at

280 nm. If considerable absorbance is observed, continue the extraction further

with the same solvent.

2. If there is any delay in the estimation of N, then few drops of toluene should be

added to each fraction.

3. Reagents for fractions I and II are stable, while for fractions III, IV and V are

unstable, and these should be prepared not more than 1 week before use.


Estimation of Crude Fibre (AOAC 1965)


Crude fibre consists mainly of cellulose and lignin (97%) plus some minerals. It can

be estimated by treatment of the sample first with acid and subsequently with alkali.

Oxidative hydrolytic degradation of the native cellulose and lignin occur. The

residue obtained after final filtration is weighed incinerated, cooled and weighed

again. The loss in weight gives the crude fibre content.

Equipment and Glassware









Extracting apparatus

Hot plate


Muffle furnace

Tall spoutless beakers, 600 ml


Filtering device

Filtering cloth (muslin cloth)


1. Sulfuric acid solution (0.255 Ỉ 0.005 N) containing 1.25 g H2SO4 per 100 ml.

2. Sodium hydroxide solution. (0.313 Ỉ 0.005 N) containing 1.25 g NaOH per

100 ml, free or nearly so, form Na2CO3. Strength of acid and alkali solutions

must be accurately checked.

3. Methyl alcohol, 95%.

4. Petroleum ether or diethyl ether.


1. Extract 2 g of ground dry matter with ethyl ether or petroleum ether to remove

fat. If material contains less than 1% fat, the extraction may be omitted.


Estimation of Dietary Fibre


2. Add 200 m boiling H2SO4 solution, immediately connect digestion flask to

condenser and heat. (Contents of flask must come to boiling within 1 min and

boiling must continue briskly for 30 min. Keep rotating the flask).

3. After 30 min remove flask, filter immediately through muslin cloth and wash

with boiling water until washings are no longer acidic. Test with BaCl2 solution.

4. Boil with 200 ml of NaOH solution. Connect flask with reflux condenser and

boil for 30 min. Rotate flask frequently until sample is thoroughly wetted.

5. After 30 min remove flask and filter. If filtering cloth is used, thoroughly wash

residue with boiling water and transfer to crucible.

6. For material difficult to filter, after 30 min boiling, filter through funnel using,

vacuum and wash with hot 10% K2SO4 solution (K2SO4 solution may be added

during filtering whenever filtration becomes difficult).

7. Return residue to digestion flask, thoroughly washing all residues from cloth

with hot K2SO4 solution. Filter into crucible.

8. After thorough washing with boiling water, wash with 15 ml alcohol. Dry

crucible and contents at 130 C to constant weight.

9. Cool the crucible in a dessicator and weigh.

10. Ignite contents of crucible in muffle furnace until carbonaceous matter is

consumed (approximately 20 min).

11. Cool in dessicator and weigh.

12. Report loss in weights crude fibre.


Crude fibre % ¼


Loss in weight


Weight of sample

Estimation of Dietary Fibre (Vansoest 1963)


Dietary fibre is composed mainly of cellulose, hemicellulose and pectin. It is broken

down into formic, acetic and galacturonic acid in large intestine. These hydrolytic

products are poorly absorbed by the intestinal wall and rapidly broken down via

intense and metabolic activity and transferred to intestinal microflora. Hemicellulose is determined by subtracting the value of acid detergent fibre (ADF) from

neutral detergent fibre (NDF). NDF is made up of cellulose, silica and lignin.

Equipment and Glassware

1. Neutral detergent solution

A. Dissolve 18.61 g disodium ethylene diaminotetracetate (EDTA) dehydrate,

6.81 g Na2B4O7Á10H2O (Sodium borate decahydrate) in 500 ml of water.

Heat till it dissolves.

B. Add 30 g sodium lauryl sulphate and 10 ml of 2-ethoxyethanol.



Methods for Nutritional Quality Evaluation of Food Materials

C. Dissolve 4.56 g of disodium hydrogen phosphate anhydrous in 100 ml of

distilled water, heat it if needed.

Mix solution A, B and C. Check the pH (6.9–7.1). Make the volume to 1 l.

2. Acid detergent solution: Dissolve 20 g cetyl trimethyl ammonium bromide

(CTAB) in 1 l of 1 N H2SO4.

3. Acetone


1. Weigh 500 mg of defatted finely ground sample (100 mesh) and transfer it to 1 l

long without spout.

2. Add 100 ml neutral detergent solution for the determination of neutral detergent

fibre or acid detergent solution for the determination of acid detergent fibre.

3. Reflux for 1 h at 60 C.

4. Filter through previously weighed sintered crucible attached to vacuum.

Washing of the residue with acetone is done after complete filtration.

5. Dry at 110 C for 24 h or till constant weight is obtained.


ADF or NDF % ¼

Loss in weight after drying


Weight of Sample

Hemicellulose ¼ NDF À ADF:


Estimation of Oil Content in Oilseeds (AOAC 1965)

Oil is an important ingredient of food. Estimation of oil in oilseed is generally

carried out using the following two methods –

(a) Soxhlet method

(b) Cold percolation method

(a) Soxhlet Method


The principle is based on the extraction of oil using non-polar solvents viz., ether,

hexane, petroleum ether (40–60 C), chloroform:methanol (2:1). It involves

repeated extraction of oil. The solvent is then distilled off completely. The oil is

dried, weighed and the % of oil is calculated.

Equipment and Glassware

1. Soxhlet extractor assembly.

2. Absorbent cotton.


Estimation of Oil Content in Oilseeds


3. Whatman filter paper/Thimble.

4. Volumetric flasks, 100 ml.

5. Distillation unit, hot plate/heating mentle.


Hexane or diethyl ether/petroleum ether (40–60 C).


1. Fold a piece of filter paper and make into a sample packet in such a way to hold

the seed meal (2.5 g) depending on oil content.

2. Place the sample packet or the thimble (alternatively the sample can be put in a

thimble also) into extract of soxhlet apparatus, after placing some cotton at the

bottom. A piece of cotton is placed at the top to evenly distribute the solvent as it

drops on the sample during extraction.

3. Add organic solvent, two and a half times the capacity of the extractor and

extract oil for a period of 6 h, or for a longer period till the solvent in the

extractor becomes colourless.

4. Put off the heaters and allow cooling. Flash evaporate the solvent or distill. Keep

it in oven at 70 C for 10 min. Cool at room temperature.

5. Weigh the flask after removing moisture. Repeat heating until constant weight is



% oil in the sample =

Weight of oil (g) Â 100


Weight of sample (g)

(b) Cold Percolation Method (Kartha and Sethi 1957)


The extraction of oil from the sample involves the use of non-polar solvents viz.

hexane, petroleum ether (40–60 C) which percolates through a column containing a

mixture of sample powder and anhydrous sodium sulphate. The oil gets eluted by

the solvent and is collected in a conical flask.

Equipment and Glassware






Glass percolator.

Volumetric flask.

Distillation unit.

Cotton wool.

Hot plate/heating mentle/water bath.



Methods for Nutritional Quality Evaluation of Food Materials


1. Anhydrous sodium sulphate

2. CCl4 or hexane or petroleum ether (40–60 C) or solvent ether.


1. Grind 1–2 g of seed material to a fine powder with the help of 20–30 g of

anhydrous sodium sulphate and mix well.

2. Plug the percolator by putting cotton wool at the bottom of it. Put approximately

2 g of sodium sulphate (anhydrous) over the plug.

3. Pack the powdered material slowly inside the percolator carefully. Ensure that

whole of the material is packed properly, pack little cotton over the material and

cover with a layer of anhydrous sodium sulphate.

4. Add 10 ml solvent in the mortar and pestle to ensure complete removal of the

material and transfer it into the percolator.

5. Keep a flask below the percolator and fill the percolator with the solvent.

6. Let the solvent tickle down under gravity. The solvent will flow down after

extracting oil from the material.

7. Add more solvent, to fill the percolator and allow it to pass through the column.

Repeat this thrice.

8. Flash evaporate or distill off the solvent using heating mentle/water bath. When

5 ml of solvent is left in the flask (round bottom in case of flash evaporator/

distillation), remove it and transfer it into a weighed conical flask and keep in the

oven till the solvent is almost completely evaporated. Weigh to constant weight

after keeping in dessicator for a few minutes.


Weight of flask + Glass bead ẳ X g

Weight of flask ỵ Glass bead ỵ oil ẳ Y g

Weight of oil ẳ YX g

% oil in the sample ẳ


YXị 100


weight of the material (g)

Estimation of Fatty Acids by Gas–Liquid

Chromatography (GLC) Morrison and Smith (1964);

Sharma et al. (1981)

Analysis of complex fatty acid mixtures can be carried out by GLC. In this

procedure the fatty acids are first converted into a volatile form, usually their

methyl esters. The esters of fatty acids are identified by comparing with a set of

standard fatty acid esters and quantified by the method of triangulation.


Estimation of Fatty Acids by Gas–Liquid Chromatography (GLC)



1. Sodium methoxide (0.5 N): Dissolve sodium methoxide in dried methanol

BF3 – methanol reagent.

2. Hexane/Petroleum ether 40–60 C (Spectroscopic grade).

3. Sodium sulphate (anhydrous)

All solvents should be anhydrous.

GLC Conditions

• Column – 10% DEGS (diethylene glycol succinate) in chromosorb P or W

(60–80 mesh).

• Detector – Flame ionization detector (FID).

• Carrier gas – Nitrogen/Argon with a flow rate of 40–50 ml/min. hydrogen

(0.5 kg/cm2) and air (0.8 kg/cm2) are also used in this detector as a fuel.

• Column temperature – 170–200 C.

• Detector temperature – 230 C.

• Injector port temperature – 230 C.

• Recorder speed – 1 cm/min.

Temperature programming: Temperature programming is resorted to when the

mixture to be analysed contains components of widely varying chain lengths. Elution

temperature rather than retention time forms this basis of identification. There is an

approximate linear relationship chain. However, there are practical problems with

temperature programmed GLC. Programming alters the flow rate of carrier gas

through the columns and the detector. Polar columns baseline drift becomes critical

and must be compensated by dual column operation or by an electronic baseline

corrector. Temperature programming shortens the life of polyester columns.

Generally, the injector and detector temperature are kept 50 C above column



1. Grind 0.1 g of oilseed with 5 ml of 0.5 N sodium methoxide in instalment of 1 ml

each in a pestle-mortar. Transfer the contents to an air-tight screw capped 15 ml

vial. Wash the pestle-mortar twice with 1 ml of 0.5 N sodium methoxide and

transfer into vial. Take 20 mg oil (1–2 drops of oil) with 5 ml of 0.5 N sodium

methoxide in screw capped vial.

2. Keep the vial in a boiling water bath for 10–15 min.

3. Cool to room temperature, add 1–2 drops BF3 – methanol reagent and again heat

for 5 min.

4. Cool and add 1–2 ml of hexane, shake and wait till hexane layer separates out.

Take the appropriate amount (2–3 ml) of ethyl ester formed from hexane layer

and inject GLC.

5. Measure the retention time and identify fatty acids by comparing with the

retention time of standard. The area of each peak should be calculated by



Methods for Nutritional Quality Evaluation of Food Materials

measuring peak height and width at half height (Triangulation method).

Calculate the percentage fatty acids.

Peak area ¼ Peak height  width at half height:


Determination of Lipase and Lipoxygenase Activity

(Sardar and Joseph 1992; Shekhar and Reddy 1982)

The storage and other environmental parameters influence the nutritional status of

food grains especially; the oilseeds and/or oil bearing materials. The two enzymes,

namely; “Lipase” and “Lipoxygenase” are of special significance as both together

contribute to the deterioration of fats and development of off flavours in soybean

and brown rice during storage. The enzymes liase (Triacylglycerol acylhydrolase

EC hydrolyses triacylglycerols to release free fatty acids and glycerol which

makes the food stuffs more susceptible to oxidative rancidity generally caused by

lipoxygenase. Lipoxygenase (Linoleate:oxygen oxidoreductase, EC is

one of the primary catalysts of oxidation. It is implicated with heme proteins and

specially catalyzes the oxidation of methylene interrupted unsaturated fatty acids

such as linoleic, linolenic and arachidonic acid to their respective peroxides. In view

of the involvement of lipase and lipoxygenase in deterioration of oil/fat, the methods

of the determination of activity of these two enzymes are described below:

A. Lipase Activity


The quantity of fatty acids released in unit time is measured by titrating it against a

standard alkali solution. Lipase activity can be expressed either in terms of 0.05 N

NaOH used to neutralize free fatty acids librated from 1 g material or as micromoles

of fatty acids released per minute.

Equipment and Glassware

1. Water bath with thermostat

2. Sonicator

3. Centrifuge







Olive oil

Polyvinyl alcohol

Tris-Cl buffer (50 mM, pH 8.0)

Tris-Cl buffer (50 mM, pH 8.3)

Sodium hydroxide (0.05 N)


Determination of Lipase and Lipoxygenase Activity


6. Acetone:Ethanol (1:1 v/v)

7. Phenolphthalein indicator

Preparation of Substrate

Take 100 ml of olive oil, add 2.5 g of polyvinyl alcohol, sonicate it, till an emulsion

is formed.


1. Grind 2 g of defatted sample thoroughly using acid washed sand, with 20 ml of

50 mM Tris-Cl buffer (pH 8.0) centrifuge at 20,000 Â g for 30 min.

2. Collect the supernatant, which is used as crude intracellular lipase extract.

3. Prepare the reaction mixture as follows:

(i) 2.5 ml of emulsified substrate

(ii) 2 ml of 50 mM Tris-Cl buffer (pH 8.3)

(iii) 1 ml of enzyme extract incubate at 37 C for 30 min

4. Stop the reaction after 30 min by adding 10 ml of acetone:ethanol (1:1) mixture.

5. Titrate the librated fatty acid with 0.05 N NaOH, using phenolphthalein indicator.

6. Express the result in terms of ml of 0.05 N NaOH used to neutralize free fatty

acids librated from 1 g material.

Calculate in terms of units as one unit of lipase activity is defined as the amount

of enzyme that liberate 1 macro mole of fatty acids/min/ml at 37 C.

1 Unit ¼ m moles of fatty acids released/min.

B. Lipoxygenase Activity


The enzyme extract (crude or purified) is allowed to react with the substrate

(Linoleic acid). The rate of maximal A234 nm per minute between 1 and 3 min

interval is used for calculating the specific activity.

Equipments and Glassware







Refrigerated centrifuge

Centrifuge tubes

Muslin cloth

Volumetric flask (50 ml)

Assay of Lipoxygenase in Rice Sample


A. Preparation of Enzyme Extract

1. Suspend 1 g of defatted rice flour in 4 ml of 0.1 M sodium phosphate buffer

(pH 6.8) at 3–5 C for 45 min.

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