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33 Determination of Polyphenols in Pulse Grains (Swain and Hills 1959)

33 Determination of Polyphenols in Pulse Grains (Swain and Hills 1959)

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310



13



Methods for Nutritional Quality Evaluation of Food Materials



Calculation

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)

Procedure

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.



13.34



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.

Principle

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)

Reagents

1. Benzene.

2. Ethanol (60% v/v)



13.35



Estimation of ODAP



311



3. Hydroxylamine hydrochloride (0.5 M). Dissolve 3.475 g of pure hydroxylamine

hydrochloride in 95 ml ethanol (60% v/v). Add 10 drops of methyl orange and

adjust, using 0.5 M alcoholic potassium hydroxide to a yellow colour. Make up

the volume to 100 ml with 60% ethanol.

4. Alcoholic potassium hydroxide (0.5 M). Dissolve approximately 2.8 g of KOH

in a few drops of water in a flask and then make up its volume to 100 ml with

alcohol.

Procedure

1. Transfer 5 g of sample to a weighed tube, add ml benzene and 15 ml 0.5 M

hydroxylamine hydrochloride solution.

2. Shake vigorously and titrate the liberated acid with 0.5 M alcoholic potassium

hydroxylamine hydrochloride solution.

Calculation

Percent aldehydes ¼



t  f  1:008  100

;

W



where, t ¼ titre; W ¼ weight of the sample (g)

The factor “f ” are as follows:

Benzaldehyde – 0.053

Cinnamic aldehyde – 0.066

Citral – 0.076

Cuminaldehyde – 0.074

Decyclic aldehyde – 0.078



13.35



Estimation of ODAP (Rao 1978)



Lathyrus sativus L. (Khesari) contains a neurotoxin, b-N-Oxaly-a–bdiaminopropionic acid (ODAP) and prolonged consumption of this pulse causes

neurolathyrism/lower limb paralysis.

Principle

ODAP on hydrolysis with KOH yield a,b-diaminopropionic acid (DAP) which

reacts with o-Phthalaldehyde (OPT) under alkaline condition to give an intense

yellow colour.

Equipments and Glassware

1. Spectrophotometer

2. Centrifuge



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13



Methods for Nutritional Quality Evaluation of Food Materials



Reagents

1. 0.5 M Dipotassium tetraborate buffer (pH 9.9).

2. o-Phthalaldehyde (OPT) reagent: 100 mg of OPT in 1 ml of 95% ethanol

and 0.2 ml of mercaptoethanol are added to 99 ml of potassium borate buffer.

This reagent although freshly made and used, can also be used for as long as

3–4 days.

3. 3 N potassium hydroxide in distilled water.

Procedure

1. Boil 25 mg of finely ground powder of Lathyrus sativus seed in 5 ml of distilled

water for 30 min.

2. Centrifuge at 4,000 rpm for 10 min. Collect the supernatant.

3. Take 0.1 ml of the supernatant in duplicate in test tubes and add 0.2 ml of 3 N

KOH.

4. Keep one set of tubes in boiling water bath for 30 min for hydrolysis, while the

other set is kept at room temperature.

5. Cool the tubes and make the volume to 100 ml with water and add 2.0 ml of

0.5 M Borate buffer (pH 9.9) along with 2 ml of reagent.

6. Keep the tubes at room temperature for 30 min for colour development and read

the absorbance at 420 nm.

7. The difference between the absorbance readings with and without hydrolysis

gives an estimation of DAP.

8. Run a set of control which includes all the reagents except the experimental

material.

9. Make a standard curve using different concentration (10–100 n mol) of DAP.

10. Calculate DAP content using the standard curve.

11. For calculating ODAP content, multiply DAP content by a factor of 1.25

ODAP in sample (% ) = 1:25 Â DAP (% ):



13.36



Assessment of Rancidity of Oil and Fats (A.O.A.C 1984)



Oils and fat undergo changes during storage which result in production of unpleasant taste and odour commonly referred to as “Rancidity”. Similar changes also

occur when oils/fats are subjected to heating processes during cooking. The rancidity caused by air is known as oxidative rancidity. When it is caused by microorganisms, it is known as ketonic rancidity. Generally, oils with high unsaturated

fatty acid content are prone to oxidative rancidity. The situation is caused in

numerous ways involving features such as light, air, high temperature, enzymes,

micro-organisms, metals and presence of free fatty acids but prime among them is

enzyme-lipoxygenase during storage of oilseeds.



13.36



Assessment of Rancidity of Oil and Fats



313



The principle toxic substance occurring in oxidized oils is lipid hydroperoxide, a

primary product of rancid oils. The hydroxyl and carbonyl compounds are

originated only by decomposition of hydroperoxides on prolonged aeration which

make even greater contribution to the toxicity than hydroperoxides; these decomposition products are also injurious to health.

The two important parameters of oxidative type of rancidity are peroxide value

and carbonyl value which are of nutritional significance. The procedures for their

determination have been described below:

(i) Peroxide value

The presence of peroxide oxygen in fat resulting from auto-oxidation is determined

by iodometric method. The peroxide value is expressed as the number of milliequivalents peroxide in 1 kg of fat.

Equipment and Glassware

1. Erlenmeyer flasks (250 ml)

2. Volumetric flasks (100 ml)

3. Pestle and mortar

Reagents

1.

2.

3.

4.



Acetic acid: Chloroform mixture (3:1 v/v)

Saturated KI solution (15% approximately).

Sodium thiosulphate solution (0.1 N)

Soluble starch solution (1%).



Procedure

A. Standardization of sodium thiosulphate (Hypo) solution

1. Mix 10 ml of 0.1 N K2Cr2O7 and 5 ml of 1 N HCl and 10 ml of saturated KI in a

100-ml volumetric flask. Titrate against sodium thiosulphate solution.

2. When brownish yellow colour is formed, add 2–3 drops of 1% starch solution.

On addition of starch, blue colour is formed.

3. Titrate it further till the disappearance of blue colour and the solution turns light

green. Note down the titre value. The normality of Hypo is calculated by using

the equation:

N1V1 (for Hypo) ¼ N2V2 (for K2Cr2O7)

B. Procedure

1. Weigh 1.0 g oil/fat in 250 ml Erlenmeyer flask. Add 30 ml of acetic

acid–chloroform mixture. Dissolve the fat completely by using wrist action

shaker for 5–10 min.

2. Add 0.5 ml of saturated KI and allow to stand for 1 min with occasional shaking

and then add 30 ml water.

3. Titrate against standardized Hypo solution with vigorous shaking until yellow

colour disappears.



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Methods for Nutritional Quality Evaluation of Food Materials



4. Add 2–3 drops of 1% starch and continue titration to release all the iodine from

chloroform or until blue colour disappears.

Calculation

(i) Weight of fat ¼ W g.

(ii) Volume of Hypo solution ¼ V ml

(iii) Normality of Hypo solution ¼ N

Peroxide value ¼



V Â N Â 1; 000

:

W



(ii) Carbonyl value

When fat is made to react with TCA and 2,4-dinitrophenyl hydrazine, corresponding

hydrozones are formed which on treatment with KOH give rise to development of

colour, the intensity of which can be measured spectrophotometrically. Carbonyl

value is expressed in terms of number of micromoles per gram of oil/fat.

Equipment and Glassware

1. Spectrophotometer

2. Water bath.

3. Volumetric flask (50 ml)

Reagents

1. Carbonyl-free benzene: Analytical reagent grade benzene is usually sufficiently

carbonyl-free as received, but if the blank has an absorbancy greater than 0.35

against water at 43 nm, then the benzene should be purified as follows: To 1 l of

benzene add 5 g of 2, 4-dinitrophenyl hydrazine and 1 g of trichloroacetic acid;

reflux for 1 h and then distill through a short column.

2. Trichloroacetic acid solution (4.3%): Dissolve 43 g of trichloroacetic acid in

carboxyl-free benzene and make to 1 l with water.

3. 2,4 Dinitrophenyl hydrazine solution (0.05% in water): Dissolve 0.5 g

2,4-dinitrophenyl hydrazine twice re-crystallized from carboxyl-free methanol

(which can be prepared in same manner as carbonyl-free ethanol).

4. Potassium hydroxide solution (4%): Dissolve 4 g of potassium hydroxide in

100 ml of absolute carbonyl-free ethanol with the aid of gentle heating and

shaking. Filter the solution through fine glasswool using suction.

5. Carbonyl-free ethanol: To 1 l of ethyl alcohol add 5–10 g of aluminium granules

and 8–10 g KOH and reflux the mixture for 1 h. Distill it, discard the first 50 ml

of distillate, and stop the distillation before the last 50 ml has distilled.

Procedure

1. Weigh 1 g fat in 50 ml volumetric flask, add 5 ml of benzene (The solution of

fat should not have more than 250 Â 10À6 molar carbonyls). Shake till the fat

is dissolved.



13.37



Determination of Phytin Phosphorus



315



2. Add 3.0 ml of 4.3% TCA and 5 ml of 0.05% 2,4-dinitrophenyl hydrazine

solutions.

3. Stopper the flask and heat it in a water bath at 60 C for 30 min. Cool to room

temperature. The solution is stable for several hours.

4. Develop the colour by adding 10 ml of 4% KOH solution. Dilute the solution

with carbonyl-free absolute ethanol. Mix well.

5. After exactly 10 min. Read the absorbance at 430 and 460 nm against a blank

prepared exactly in the same manner, substituting 5 ml of carbonyl-free benzene

for the sample solution.

Calculation

When the measurements are made in 1 cm cuvettes with Beckman DU spectrophotometer, the analysis can be calculated using the following equations.

Unsaturated ¼



3:861 A460 À 3:012 A430

0:854



Saturated ¼ 3:861A460 À 2:170 Unsaturated:

Important Points

1. In preparation of samples for analysis, fats and oils are easily dissolved in

benzene and 5 ml aliquots used. Solid foods are ground in a mortar or a mill.

Samples are weighed out into glass-stoppered centrifuge bottles, benzene is

added, the bottle is stoppered, shaken and centrifuged. Five ml aliquots of

these extracts are then taken for analysis.

2. Solutions and extracts of fat for this test should be protected from undue

exposure to light and air before use to prevent further oxidation of fat and

deterioration of existing carbonyl compounds.

3. Potassium hydroxide solution should be prepared fresh daily.

4. Using the present method of analysis the 2,4-dinitrophenyl hydrozones of

saturated aldehydes exhibited maximum absorption at 432 nm, and the aM is

16,670, while for this derivative of crotonaldehyde, an a,b-unsaturated aldehyde, maximum absorption is at 150 nm and aM is 28,100. The most suitable

wavelengths for this determination therefore, are 430 and 460 nm.



13.37



Determination of Phytin Phosphorus

(Wheeler and Ferrel 1971)



Phytic acid (1, 2, 3, 4, 5, 6,-hexakis dihydrogen phosphate myoinositol) is a

common storage form of phosphorus in seeds and is also considered as an antinutritional factor. The complexing of phytic acid with nutritionally essential

elements and the possibility of interference with proteolytic digestion have been



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.



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