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14 Determination of Lipase and Lipoxygenase Activity (Sardar and Joseph 1992; Shekhar and Reddy 1982)

14 Determination of Lipase and Lipoxygenase Activity (Sardar and Joseph 1992; Shekhar and Reddy 1982)

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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.



Methods for Nutritional Quality Evaluation of Food Materials

2. Squeeze the suspension through four layers of muslin cloth.

3. Centrifuge at 10,000 Â g for 30 min to remove all the debris. Collect the


B. Partial Purification and Assay

1. Adjust the pH of the supernatant to 5.2 with 1 M acetic acid. Centrifuge the

turbid solutin at 10,000 Â g for 30 min.

2. Adjust the pH of the supernatant to 6.8 with 1 M NaOH.

3. Add ammonium sulphate to the solution to 50% saturation.

4. Adjust the pH immediately to 6.8 with 2 N ammonia solution.

5. Centrifuge, collect the precipitate, dissolve in water and dialyze against water.

6. Prepare the substrate by ultrasonically dispersing 50 ml to Tween 20 and 5 ml

double distilled water containing 35 ml linoleic acid (~35 mg).

7. Keep the solution at pH 9.0 by adding 0.2 M NaOH until all the linoleic acid

gets dissolve and the pH remains stable.

8. After adjusting the pH to 6.5–7.0 by adding 0.2 M HCl, 0.1 M phosphate buffer

(pH 6.5–7.0) is added to a total volume of 100 ml.

9. The substrate solution is flushed with and kept under a nitrogen atmosphere.

10. Take 2.0 ml substrate solution; add 100 ml of diluted enzyme extract and 0.9 ml

of 0.1 M phosphate buffer (pH 6.5–7.0).

11. The increase in absorbance at 234 nm is measured for 1 min at 25 C (maximal

DA234 per min).

The activity can be expressed as mmol hydroperoxide formed/min/mg protein

using a molar extinction coefficient of 25,000/M/cm. The specific activity is

expressed in terms of unit/mg protein.


Estimation of Carotenoid Pigments (Ranganna 1986)

The carotenoids are an abundant group of naturally occurring pigments, present in all

green tissues; they are the constituents of the chloroplast and are responsible for most

of the yellow to red colours of flowers and fruits. Carotenoid hydrocarbons are called

carotenes, whereas derivatives containing oxygen functions are the xanthophylls.

They may be acyclic (lycopene) or contain 5–6-membered rings at one or both ends

of the molecule (b –carotene, lutein). Mango, papaya, leafy vegetables, pumpkin,

wheat and durum wheat are rich sources of carotenes. Tomato, watermelon and

apricot contain mainly lycopene, while pigment of red peppers is xanthophylls.

b-carotene is the precursor of vitamin A and is nutritionally important in human

diet. Carbon structure of vitamin A is the same as that of half of – carotene, oxidation

of the latter at the midpoint produces 2 molecules of vitamin A, but oxidation is not

very efficient since 1 unit of carotene is equivalent to 0.6 unit of vitamin A. b-carotene

is unstable in light, particularly light of short wavelength and ultraviolet. It is

also unstable in acidic or alkaline medium and rapidly destroyed when heated in


Estimation of Carotenoid Pigments


the presence of oxygen. Oxidation seems to be the main cause of destruction.

The carotenes must be protected from oxidation and light. They are also sensitive to

auto-oxidation. Yellow colour of the pigment is mainly because of conjugated double

bonds, which are destroyed by light. The methods for the estimation of carotenoid

pigments in fruits, vegetables and wheat are described below.

A. Estimation of Carotenoids in Fruits and Vegetables

(a) Total Carotenoids


Analysis of total carotenoids is based on the extraction of crude pigment mixture in

a lipid solvent and measurement of its optical density at 460 nm. The pigment

content is expressed as b-carotene. The sample is extracted in acetone which

dissolves both the fat and water-soluble pigments. The acetone extract is then

taken in petroleum ether layer. The fat soluble carotenoids pass from acetone to

the petroleum ether leaving all the rest of the pigments in the acetone.

Equipments and Glassware






Separatory funnel

Pestle and mortar

Analytical balance


Conical flasks







Petroleum ether

MgO (magnesium oxide)/alumina neutral (AI2O3)

Sodium sulphate (anhydrous)


1. Weigh 1–2 g of fresh sample and grind it with acetone using acid and alkali

washed sand in a mortar with pestle.

2. Pour the extract in a 250-ml conical flask.

3. Continue the extraction of the residue with acetone till residue is colourless (3 times).

4. Pool all the extracts in conical flask and transfer it into a separatory funnel.

5. Add 10–15 ml of petroleum ether in separatory funnel and shake thoroughly.

6. The yellow pigment is then transferred into the petroleum ether by diluting the

acetone with water containing 5% sodium sulphate.

7. Keep on adding petroleum ether until all colour gets transferred into the petroleum ether layer.

8. Make up the volume with petroleum ether and measure the intensity of the

colour at 460 nm. The results are expressed in terms of b-carotene as mg/100 g

of the material.



Methods for Nutritional Quality Evaluation of Food Materials

(b) b-carotene

The b-carotene is usually estimated by Chromatographic separation.


A. Weigh samples according to their expected b-carotene content (Spinach 1 g,

carrot 2 g, peas 10 g and cauliflower 20 g).

B. Grind weighed sample with pestle and mortar with acetone till all the yellow

colour is extracted and the material becomes white.

C. Transfer the extract to a separatory funnel; add petroleum ether to the acetone

extract till all the colour taken up by the petroleum ether layer becomes


D. Vacuum distill the extract, till the volume is reduced to about 5 ml.

E. Load this carotene extract on to a column of alumina (10 Â 1 cm) which has

previously been activated by keeping in oven at 50–60 C for 48 h. the column

also contains 3% anhydrous sodium sulphate.

F. Elute b-carotene with petroleum ether containing 3% acetone.

G. Note the volume of the eluate and measure the absorbance at 460 nm.

Alternatively, the column can be filled with supercell powder and magnesium

oxide (MgO) in the ration 3:1 up to 10 cm in length and absorbent is pressed 2–3

times with plunger to ensure a tight column. Then place 1 cm of anhydrous Na2SO4

layer over top of the column. Wet the column with 25–50 ml of petroleum ether by

leaving the last ml of petroleum ether above Na2SO4 layer. A 5–10-ml sample is

then loaded.


b - carotene (mg/100 g) ¼

3:1206  A460  Vol. made up or dilution  100


Weight of sample  1; 000

B. Estimation of Carotenoids in Wheat (AOAC 1962)

(a) b-Carotene

Yellow colour in durum wheats imparts attractive appearance to the pasta product

and therefore, majority of the pasta consumers prefer the yellow pigment.

Xanthophylls and specially b-carotene contributes to the colour production in the

semolina. High lipoxygenase activity has been described to be responsible for an

appreciable loss to b-carotene. Linoleic acid acts as a source of substrate for the

enzyme to form hydroperoxides which ultimately oxidize the pigment. b-carotene

acts as a preservative. Durum wheat endosperm contains twice the concentration of

b-carotene than that of T.aestivum. Since b-carotene is highly susceptible to

oxidation, precaution has to be taken for its proper determination.


Estimation of Carotenoid Pigments


Method 1

Equipment and Glassware






Erlenmeyer flask

Volumetric flasks, 50, 100 and 250 ml

Whatman No. 1 filter paper


1. Water-saturated n-butanol (WSB): Prepare a solution n-butanol and water in a

proportion of 6:2 (v/v) and shake vigorously. Use the clear upper layer after

separation of the phases

2. Diethyl ether

3. Synthetic b-carotene, crystalline


1. Preparation of extract: Weigh 10 g of sample and disperse it in 50 ml of watersaturated n-butanol to give a homogenous suspension. Shake gently and allow it

to stand overnight (16 h) at room temperature under the dark. Shake and filter

completely through the filter paper (Whatman No. 1) into a 100-ml volumetric


2. Standard solution of b-carotene: In a 100-ml volumetric flask, weigh 25 mg of

b-carotene. Dissolve it in diethyl ether and make up to the mark with diethyl

ether, 20 ml of this homogenous solution ( ¼ 5 mg b-carotene) is pipetted into a

250-ml volumetric flask. Make up to the mark with water-saturated n-butanol.

Take 25 ml of this solution and place in a 100-ml volumetric flask and make

up with water-saturated n-butanol. This standard solution has the following

concentration. 1 ml ¼ 0.005 mg ¼ 5 mg b-carotene

3. Preparation of calibration curve: Prepare suitable dilutions of the standard

solution with water-saturated n-butanol in calibrated 10 ml volumetric flasks

(e.g. from 0.5 to 3 ml of standard solution in 10 ml). Measure the absorbance, of

each dilution and establish a calibration curve (b-carotene in 10 ml of solution as

a function of absorbance).

4. Determination of b-carotene content: Measure the absorbance of the clear filtrate

at 440 nm. Unfiltered water-saturated n-butanol may be used as blank. Evaluation of the contents is based on a b-carotene calibration curve (related to the

b-carotene content in a 10 mL solution).


The yellow pigment, Yp, expressed as milligrams of b-carotene in 100 g dry matter, is

Yp =



100 À H



Methods for Nutritional Quality Evaluation of Food Materials


a ¼ b-carotene content of a 10-ml extract equivalent to 2 g of the test sample, in mg.

H ¼ moisture content of the test sample, expressed as percentage by mass.

Method 2

This method can be used for screening a large number of samples for their

b-carotene content.

Glassware and Reagents

Same as described previously.


Preparation of extract

1. Weigh 8 g semolina or flour into 150 ml glass-stoppered Erlenmeyer flask and add

40 ml water-saturated n-butanol. Shake contents for 1 min and let it stand for 16 h.

Shake and filter through Whatman No. 1 filter paper into 50 ml volumetric flask.

2. Measure transmission of extract in colorimeter at 440 nm employing standard

containing reagent water-saturated n-butanol. To obtain the values for carotene

content (ppm) the transmission reading of unknown sample is put in the equation.

b À carotene (ppm) = 0:174 ỵ 16:57 L;

where, L ẳ Transmission (apparent density).

As described earlier yellow pigment is essentially a preferable feature of durum

wheats. Range of b-carotene is generally 4–8 ppm but durums with less than 5 ppm

of b-carotene are not acceptable in the international market. Some countries offer

little preference to yellow pigment.


Estimation of Lycopene

Lycopene is responsible for the red colour of tomato, fleshy part of water melon,

fruits and vegetables. It is a carotene with the formula C40H56. Colour of tomato due

to lycopene has a great role in consumer acceptability.


The carotenoids in the sample are extracted in acetone and then taken up in

petroleum ether. Lycopene has absorption maxima at 473 and 503 nm. One mole

of lycopene when dissolved in 1 l light petroleum (40–60 C) and measured in a

spectrophotometer at 503 nm in 1 cm light path gives an absorbance of 17.2 Â 104.

Therefore, a concentration 3.1206 mg lycopene/mL gives unit absorbance.


Estimation of Chlorophylls








Petroleum ether 40–60 (AR)

Anhydrous sodium sulphate

5% Sodium sulphate


1. Take sample of 3–4 tomato fruits and pulp it well to a smooth consistency in a

waring blender.

2. Weigh 5–10 g of this pulp.

3. Extract the pulp repeatedly with acetone using pestle and mortar or a waring

blender until the residue is colourless.

4. Pool the acetone extracts and transfer to a separating funnel containing about

20 mL petroleum ether and mix gently.

5. Add about 20 mL of 5% sodium sulphate solution and shake the separating

funnel gently. Volume of petroleum ether might be reduced during these

processes because of its evaporation. Therefore, add 20 ml more of petroleum

ether to the separating funnel for clear separation of two layers. Most of the

colour will be noticed in the upper petroleum ether layer.

6. Separate the two phases and re-extract the lower aqueous phase with additional

20 ml petroleum ether until the aqueous phase is colourless.

7. Pool the petroleum ether extracts and wash once with a little distilled water.

8. Pour the washed petroleum ether extract containing carotenoids into a brown

bottle containing about 10 g anhydrous sodium sulphate. Keep it aside for

30 min or longer.

9. Decant the petroleum ether extract into a 100-ml volumetric flask through a

funnel containing cotton wool. Wash sodium sulphate slurry with petroleum

ether until it is colourless and transfer the washings to the volumetric flask.

10. Make up the volume and measure the absorbance in a spectrophotometer at

503 nm using petroleum ether as blank.


Absorbance (1 unit) ¼ 3.1206 mg lycopene/ml.

mg lycopenein 100 g sample =


31:206 Â Absorbance


Wt. of sample (g)

Estimation of Chlorophylls

The chlorophylls are the essential pigments for photosynthesis and are present in

chloroplasts as green pigments in all photosynthetic plant tissues. They are bound

loosely to proteins but are readily extracted in organic solvents such as acetone or ether.



Methods for Nutritional Quality Evaluation of Food Materials

Chemically, each chlorophyll molecule contains a porphyrin (tetrapyrole) head with

a chelated magnesium atom at the centre and a long-chain hydrocarbon (phytol) side

chain attached through a carboxylic acid group. There are at least five types of

chlorophylls in plants. Chlorophylls a and b occur in higher plants, ferns and mosses.

Chlorophylls c, d and e are only found in algae and in certain bacteria.


Chlorophyll is extracted in 80% acetone and are read at 663 and 645 nm using

spectrophotometer. Using the absorption coefficients, the amount of chlorophyll is



80% Acetone (prechilled).


1. Weigh 1 g of finely cut leaf or fruit tissue of sample into a clean mortar.

2. Grind the tissue to a fine pulp with the addition of 20 ml of 80% acetone.

3. Centrifuge at 5,000 rpm for 5 min and transfer the supernatant to a 100-ml

volumetric flask.

4. Grind the residue with 20 ml of 80% acetone, centrifuge and transfer the

supernatant to the same volumetric flask.

5. Repeat this procedure until the residue is colourless. Wash the mortar and pestle

thoroughly with 80% acetone and collect the clear washings in the volumetric


6. Make up the volume to 100 ml with 80% acetone.

7. Read the absorbance of the solution at 645, 663 and 652 nm against the solvent

(80% acetone) blank.

• The amount to tissue taken for extraction may be varied. Accordingly amount

of 80% acetone used may be altered so that the final extract has volume based

on 10 mg plant material extracted in 1 ml of acetone.


Calculate the amount of chlorophyll present in the extract mg chlorophyll per g

tissue using the following equations:


• mg chlorphyll a/g tissue ẳ 12:7A663 ị 2:69A645 ị

1; 000 w


mg chlorophyll b/g tissue ẳ 22:9A645 ị 4:68A663 ị

1; 000 w


mg total chlorophyll/g tissue ẳ 20:2A645 ị 8:02A663 ị


1; 000 w



A ¼ absorbance at specific wavelengths,

V ¼ final volume of chlorophyll extract in 80% acetone

W ¼ fresh weight of tissue extracted.



Estimation of Thiamine


Estimation of Curcumin

Turmeric rhizome contains a number of pigments. It is used as a natural dye

in food industries and in cosmetic and pharmaceutical products as an antimicrobial principle. The powder contains a large number of aromatic compounds;

curcumin is the major compound responsible for the characteristic colour in

Indian recipes. In pure form it is an orange yellow crystalline powder, soluble

in alcohol and glacial acetic acid. Curcumin content is used as measure of

turmeric quality.


Curcumin is quantitatively extracted by refluxing the material in alcohol and is

estimated spectrometrically at 425 nm.


1. Absolute alcohol

2. Stoppered flask and air condenser


1. Dissolve 0.2–0.5 g of weighed, moisture-free turmeric powder in 250 ml of

absolute ethanol.

2. Reflux the contents in the flask fitted with an air condenser over a heating mantle

for 3–5 h, compensate alcohol loss if any due to evaporation by adding alcohol

freshly into the flask.

3. Cool and decant the extract into a volumetric flask and make up the volume.

4. Dilute a suitable aliquot (1–2 ml) to 10 ml with absolute alcohol. Measure the

intensity of yellow colour at 425 nm using spectrophotometer.


Work out the curcumin content using the formula

Curcumin content g/100 g ¼

0:0025  A425  volume made up  dilution factor  100


0:42 Â weight of the sample (g) Â 1; 000

since 0.42 absorbance at 425 nm ¼ 0.0025 g curcumin.


Estimation of Thiamine

Thiamine is one of the vitamins of B complex group whose deficiency is associated

with disease known as beriberi. This vitamin usually occurs in outer layers of grains

including cereals. Therefore, unpolished rice and foods made of whole wheat are



Methods for Nutritional Quality Evaluation of Food Materials


























Thiamine hydrochloride
















Fig. 13.1 Formation of thiochrome during estimation of thiamine

good sources of this vitamin. Thiamine is water-soluble, and overcooking may

leach out or destroy thiamine originally present in the food sources. Among a

number of chemical and micro-biological methods available for the estimation of

thiamine, fluorimetric method is easier and popular (Fig. 13.1).


Alkaline potassium ferricyanide oxidizes thiamine to thiochrome which is a fluorescent compound. The thiochrome is extracted in isobutyl alcohol and measured in

a fluorimeter.








15% NaOH

1% Potassium ferricyanide

Isobutyl alcohol

Anhydrous sodium sulphate

0.1 N H2SO4

Standard thiamine hydrochloride stock solution: Dissolve 50 mg thiamine

hydrochloride in 500 ml of about 0.1 N sulphuric acid containing 25% alcohol

(100 mg/ml). Store this solution in a brown bottle in a refrigerator.

7. Working Standard Solution: Just before the experiment, dilute 5 ml of the

stock solution to 100 ml with 0.1 N sulphuric acid and again dilute 5 ml of

the second solution to 100 ml with 0.1 N H2SO4 (0.25 mg/ml) and use.


Extraction of Thiamine

1. Weigh accurately 5 g finely ground sample into a 250-ml conical flask in


2. Slowly add 10 mL 0.1 N H2SO4 without shaking; stopper the flask and let it

stand overnight.


Estimation of Thiamine


3. On next day shake the contents vigorously and filter through Whatman No.1

filter paper, discarding first 10–15 ml of filtrate.


1. Pipette out 10 ml of the extract in duplicate into 100 mL separating funnels.

2. Pipette out 10 ml of working standard (in 4–5 replicates).

3. Add 3 ml of 15% NaOH into each separating funnel immediately followed by

four drops (0.2 ml) of ferricyanide solution.

4. Shake gently for exactly 30 s.

5. Add 15 ml of isobutanol rapidly from a quick delivery burette or a measuring


6. Stopper immediately, shake vigorously for 60 s and allow the layers to


7. Drain off the bottom layer carefully and discard it.

8. Add 1–2 g of sodium sulphate directly into the separating funnel, stopper and

swirl gently to clarify the extract. If the extract is not clear, add a little more

Na2SO4 and clarify.

9. Collect the clear extract from the top using a Pasteur pipette into a clean dry test


10. Prepare a set of sample blanks by pipetting out 10 mL of the extract and follow

the above procedure excluding addition of ferricyanide.

11. Prepare a blank for the standard (in duplicate) separately.

12. Select suitable primary (366 nm) and secondary filters as per the make of the


13. Set fluorimeter by initially adjusting the standard blank to 0 reading and

standard to 100. Then read the sample blank and sample. Since the light

intensity sometimes changes progressively,5–6 readings should be recorded.

Since the standard has to be read a number of times during the measurement

it is convenient to oxidize the standard in 4 or 5 replicates and to combine all

the oxidized extracts in one conical flask. Portions of this may be read at a time

and discarded.


mg thiaminecontent in 100 g sample ¼


a ¼ reading of standard ¼ 100

a1 ¼ reading of standard bland ¼ 0

x ¼ reading of standard sample and

x1 ¼ reading of standard sample blank

0:25 Â 10 ðx À x1 Þ Â 100 10



a À a1



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14 Determination of Lipase and Lipoxygenase Activity (Sardar and Joseph 1992; Shekhar and Reddy 1982)

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