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37 Determination of Phytin Phosphorus (Wheeler and Ferrel 1971)

37 Determination of Phytin Phosphorus (Wheeler and Ferrel 1971)

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316



13



OH



I



II



O



OH



P



OH



O



O



P

HO



Methods for Nutritional Quality Evaluation of Food Materials



HO

HO O



OH

HO



O

P



O



O



OH



H H H

H H H



HO



OH

O



O

P

O



OH

HO



O

P



OH

OH



HO

HO

HO



P



HO



P



O



HO



HO OH



O



P



O



P



HHH

HHH



O



O

O



P

O



OH



P



O



O



HO

HO



OH

OH

OH



OH

OH



O



Fig. 13.3 Inositol hexaphate (phytic acid)



suggested as factors responsible for anti-nutritional activity. The phosphorus in

phytic acid is not nutritionally available to the monogastric animals. Phytic acid

also interferes with calcium and iron absorption. Hence, estimation of phytic acid in

food grains becomes essential especially in cereals.

Principle

The phytate is extracted with trichloroacetic acid and precipitated as ferric salt.

The iron content of the precipitate is determined colorimetrically and the phytate

phosphorus content calculated from this value assuming a constant 4 Fe: 6P

molecular ratio in the precipitate (Fig. 13.3).

Materials

1.

2.

3.

4.

5.

6.

7.



3% Trichloroacetic Acid

3% Sodium Sulphate in 3% TCA

1.5 N NaOH

3.2 N HNO3

FeCl3 Solution (Dissolve 583 mg FeCl3 in 100 ml of 3% TCA).

1.5 M Potassium thiocynate (KSCN)(Dissolve 29.15 g in 200 ml water)

Standard Fe(NO3) solution



Procedure

1. Weigh a finely ground (40 mesh) sample estimated to contain 5–30 mg phytate

P into a 125-ml Erlenmeyer flask.

2. Extract in 500 ml, 3% of TCA for 30 min with mechanical shaking or with

occasional swirling by hand for 45 min.

3. Centrifuge the suspension and transfer a 10-ml aliquot of the supernatant to a

40 ml conical centrifuge tube.

4. Add 4 ml of FeCl3 solution to the aliquot by blowing rapidly from the pipette.



13.38



Estimation of Trypsin Inhibitor in Legumes



317



5. Heat the contents in a boiling water bath for 45 min. If the supernatant is not

clear after 30 min, add one or two drops of 3% sodium sulphate in 3%TCA and

continue heating.

6. Centrifuge (10–15 min) and carefully decant the clear supernatant.

7. Wash the precipitate twice by dispersing well in 20–25 ml 3% TCA, heat in

boiling water for 5–10 min and centrifuge.

8. Repeat washing with water.

9. Disperse the precipitate in a few ml of water and add 3 ml 1.5 N NaOH with

mixing.

10. Bring volume to approximately 30 ml with water and heat in boiling water for

30 min.

11. Filter hot (quantitatively) through a moderately retentive paper Whatman No.2.

12. Wash the precipitate with 60–70 ml hot water and discard the filtrate.

13. Dissolve the precipitate from paper with 40 ml hot 3.2 N HNO3 into a 100-ml

volumetric flask.

14. Wash paper with several portions of water, collecting the washings in the same

flask.

15. Cool flask and contents to room temperature and dilute to volume with water.

16. Transfer a 5-ml aliquot to another 100 ml volumetric flask and dilute to

approximately 70 ml.

17. Add 20 ml of 1.5 M KSCN dilute to volume, and read colour immediately

(within 1 min) at 480 nm.

18. Run a reagent blank with each set of samples.

Standard

Weigh accurately 433 mg Fe(NO3)3 and dissolve in 100 ml distilled water in a

volumetric flask. Dilute 2.5 ml of this stock standard and make up to 250 ml in

a volumetric flask. Pipette out 2.5, 5, 10, 15 and 20 ml of this working standard

into a series of 100 ml volumetric flasks and proceed from step 16.

Calculation

Find out the mg iron present in the test from the standard curve, and calculate the

phytate P as per the equation.

Phytate P mg/100 g sample ¼



13.38



mg Fe  15

:

Weight of sample (g)



Estimation of Trypsin Inhibitor in Legumes

(Kakade et al. 1974)



The food legumes are rich sources of proteins and their importance has been very

well recognized in human nutrition, particularly in the countries where cereals form

the staple diet of the people. Though grain legumes are rich in proteins, historically



318



13



Methods for Nutritional Quality Evaluation of Food Materials



they are also known to possess a wide variety of chemical substance which

interferes with the nutritive value when ingested. Among such anti-nutritional

factors, enzyme inhibitors, the substances which have the ability to inhibit

the activity of the enzymes, are present in an appreciable quantity in the grains.

The protease inhibitors which are proteinaceous in nature are the inhibitors of the

enzyme – trypsin, chymotrypsin, papain, elastase, carboxypeptidase A and B,

pepsin etc.

Principle

The trypsin inhibitor activity is measured indirectly by inhibiting the activity of

trypsin. A synthetic substrate (BApNA) is subjected to hydrolysis by trypsin to

produce yellow coloured p-nitroanilide. The degree of inhibition by the extract of

the yellow colour production is measured at 410 nm.

Reagents

1. 30% Glacial acetic acid (v/v)

2. Trypsin: Dissolve 6.25 mg lyophilized trypsin and make up to 25 ml with

0.001 M HCl. Dilute 2 ml of this solution to 25 ml for assay.

3. Substrate: Benzoyl-DL-Arginine-paranitroanilide (BApNA):Completely dissolve

40 mg BAPNA in 0.5 ml of dimethyl sulphoxide and then make up to 100 ml with

Tris-HCl buffer pH 8.2.

4. Tris-HCl Buffer (pH 8.2): Weigh 6.05 g Tris (hydroxymethyl aminomethane)

and 2.94 g CaCl2ÁH2O, dissolve in 90 ml water, adjust to pH 8.2 with dilute HCl

and make up to 1,000 ml with distilled water.

5. Trypsin inhibitor source: Extract 0.5 g sample in 25 ml water by grinding in

prechilled mortar and pestle. Extract the ground sample in a refrigerator for

2–3 h with occasional shaking for complete extraction of TI. Centrifuge the

homogenate at 12,000 rpm for 20 min at 4–6 C. Dilute 1 ml of the supernatant to

10 ml with distilled water and use as TI source.

Procedure

1. Pipette out 0–1 ml of extract in duplicate sets of test tubes, one to serve as

endogenous (E) and the other test (T).

2. Make up the volume to 2 ml with buffer in the endogenous set.

3. Make up the volume to 1 ml in the test set.

4. Add 1 ml of trypsin solution (20 mg) to each tube in the test set. Pipette out into

a separate test tube 1 ml of buffer and 1 ml of trypsin solution for standard (S).

5. Incubate all the tubes in a water bath at 37 C.

6. After a few minutes, add 2.5 ml of substrate (1 mg BApNA) to each tube.

7. Allow the reaction to proceed for 10–60 min at 37 C.

8. Stop the reaction by adding 0.5 ml of 30% glacial acetic acid.

9. Read the absorbance at 410 nm in a spectrophotometer.

10. Determine the protein content in the extract by Lowry’s method.



13.39



Estimation of Cyanogens



319



Calculation

Find out T and S absorbance. Plot the absorbance against the volume of extract.

Determine the aliquot size of the extract required to inhibit 50% of the trypsin

activity (S/2). That aliquot size is considered to be one unit of trypsin inhibitor.

One unit of activity corresponds to that amount of trypsin inhibitor in mg protein

which gives 50% inhibition of enzyme activity under experimental conditions.

The trypsin inhibitor activity is expressed as trypsin inhibitor units (TIU) per

gram sample or per mg protein. (The dilutions of trypsin inhibitor source are

made in such a way that 0.5 ml produces 50% inhibition).



13.39



Estimation of Cyanogens



Cyanogens are cyanogenic glucosides and are widespread in the plant kingdom in

trace amounts. These glucosides release hydrogen cyanide by an endogenous

enzymatic reaction. Relatively high concentration is found in certain grasses, root

crops and fruit kernels. Young sorghum plants contain high amounts of cyanogens

which could result in adverse effects on the livestock.

Principle

Hydrocyanic acid which is evolved from the sample forms a red coloured compound with sodium picrate and the intensity of developed colour is measured at

625 nm.

Materials

1. Chloroform.

2. Whatman No.1 filter Paper: Cut filter paper into strips 10–12 cm long and 0.5 cm

wide, and saturate them with alkaline picrate solution.

3. Alkaline picrate solution: Dissolve 25 g sodium carbonate and 5 g picric acid in

1 l of water.

4. Standard hydrogen cyanide solution: Dissolve 0.241 g of KCN/litre of water.

This gives a solution containing 100 mg hydrogen cyanide/ml.

Procedure

1. Homogenize 1 g of the sample in 25 ml water with 3–4 drops of chloroform.

2. Place the homogenate in 500 ml conical flask.

3. Place the saturated filter paper in the hanging position with the help of a cork

stopper inside the conical flask.

4. Incubate the mixture at room temperature (20 C) for 20–24 h.

5. The sodium picrate present in the filter paper is reduced to reddish compound in

proportion to amount of hydrocyanic acid evolved.



320



13



Methods for Nutritional Quality Evaluation of Food Materials



6. Elute the colour by placing the paper in a clean test tube containing 10 ml

distilled water and compare it with standards at 625 nm.

7. Preparation of the Standard Curve: Place 5 ml of the alkaline picrate solution

and 5 ml of the potassium cyanide solution in a test tube. Heat for 5 min in

boiling water. Deliver the following volumes from the above KCN alkaline

picrate solution of six test tubes: 0.1, 0.2, 0.4, 0.6, 0.8 and 1 ml. Bring the

volume of each test tube to 10 ml by adding distilled water. Press rubber stopper

to the tubes and keep them in a cool place. Measure the absorbance at 625 nm.

The amount of hydrogen cyanide in the above tubes is equal to 5, 10, 20, 30, 40

and 50 mg, respectively. Prepare a blank with 10 ml distilled water (as KCN is

poisonous, therefore use automatic pipette for the transfer of cyanide solution).

Calculation

Calculate the hydrogen cyanide content of the sample from the standard graph.



13.40



Estimation of Indoleactic Acid

(Knegt and Bruinsma 1973)



Indole-3-acetic acid (IAA) is an important hormone involved in plant growth and

development.

Principle

IAA is reacted with trifluoroacetic acid and acetic anhydride to convert it into

indole-a-pyrone which is measured fluorimetrically.

Materials

1. Methanol (redistilled)

2. Trifluoroacetic Acid-Acetic. Anhydride Reagent: Mix equal volume of each

liquid, pre-cool to 0 C. Freshly prepare and store on ice until use.

3. Spectrophotofluorimeter: Measure primary filter at 440 nm and secondary filter

at 490 nm (for low concentration measurement).

Extraction of IAA

Put 5 g of plant material in liquid nitrogen and grind to a fine powder using a pestle

and mortar. Continue grinding with 10 ml methanol (redistilled) to a fine suspension. Filter the homogenate through a G4 glass filter under suction into a 100-ml

flask. Extract the material on the filter twice by adding 10 ml methanol and then

once with 5 ml. Evaporate the filtrate in a rotary evaporator at 30 C to an aqueous

residue. To the aqueous residue add 10 ml of cold 0.5 M K2HPO4 solution so that

pH reaches to about 8.5. Transfer to a suitable separating funnel and shake with

10 ml light petroleum ether. Repeat this step again and then shake with 10 ml



13.41



Estimation of Ethylene



321



diethyl ether each time the lipid fraction is discarded. Adjust the aqueous layer to

pH 3 by adding about 3 ml of 2.8 M phosphoric acid. Extract IAA with 10 ml

diethyl ether.

The 10 ml diethyl ether is then extracted with 10 ml cold 50 mM, K2HPO4

solution. The pH of the solution is adjusted to 3 with phosphoric acid (0.28 M) and

the IAA is passed into a final 10 ml diethyl ether. The ether is then evaporated in a

few minutes under reduced pressure. Dissolve the residue in a known volume (5 ml)

of cold redistilled methanol.

Procedure

1. Pipette out 1 ml of the above methanolic extract each in four different test tubes.

2. To each tube add 1 ml of methanol containing 0, 10, 20 or30 ng of IAA,

respectively.

3. Dry the contents in each tube completely under reduced pressure and cool

to 0 C.

4. To each flask add 0.2 ml ice-cold trifluoroacetic acid-acetic anhydride reagent

and mix.

5. Place the tubes on ice for exactly 15 min to ensure the complete conversion of

IAA into indole-a-pyrone. Stop the reaction adding 3 ml water.

6. A blank may be prepared occasionally by adding first 3 ml water to one of four

aliquots and 0.2 ml reagent after 15 min.

7. Take the readings in a spectrophotofluorimeter with excitation at 440 nm and

emission at 490 nm for low concentration samples.

8. Calculate the amount in unknown sample.



13.41



Estimation of Ethylene



Ethylene, the ripening hormone is usually estimated for different physiological

studies.

Principle

The ethylene evolved is measured in a gas chromatography based on the adsorption

principle on activated silica gel or poropak.

Materials

1.

2.

3.

4.



Conical Flasks with facility to seal with rubber gaskets

Air-tight syringes

Ethylene gas

GLC



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