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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

VETERINARY ACADEMY

Department of the Food Safety and Quality

Elena Bartkiene

Plant Food analysis methods

Methodical book

Kaunas, 2012

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This methodical book Plant Food analysis methods is devoted for food safety and food technology students and other interested in plant food analysis methods. This educational tool

information can be used for training purposes.

Reviewers: assoc. prof. Loreta Basinskiene (Kaunas University of Technology,

Department of Food Technology), dr. Aldona Baltusnikiene (Lithuanian University of Health

Sciences, Veterinary Academy, Department of Biochemistry).

Methodical book approved by Lithuanian University of Health Sciences, Veterinary

Academy, Veterinary Faculty Council meeting.

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CONTENT

I. ENZYMATIC ACTIVITY ANALYSIS

4

I. 1. Carbohydrate-degrading enzymes: -amylase analysis 5

I. 2. Carbohydrate-degrading enzymes: -amylase analysis 6

I. 3. Proteolytic enzymes 6

I. 4. Cellulase and xylanase activity determination

9

II. CARBOHYDRATE ANALYSIS

17

II. 1. Colour reactions to identify the saccharides 17

II. 2. Determination of Reducing Sugars by Nelson-Somogyi Method 19

II. 3. Determination of Reducing Sugar by Dinitrosalicylic Acid Method 20

II. 4. Determination of Glucose by Glucose Oxidase Method 21

II. 5. Determination of Total Carbohydrate by Anthrone Method 22

II. 6. Phenol Sulphuric Acid Method for Total Carbohydrate 23

II. 7. Estimation of Starch by Anthrone Reagent 24

II. 8. Determination of Amylose 25

II. 9. Determination of Cellulose 26

II. 10. Determination of Hemicellulose 26

II. 11. Determination of Fructose and Inulin 27

II. 12. Determination of Pectic Substances 28

II. 12. Determination of Crude Fibre 31

II. 13. Determination of Pyruvic Acid 32

II. 14. Determination of Amylose in Flour by a Colorimetric Assay

33

III. PHYTOESTROGENS ANALYSIS METHODS

35

III. 1. Phytoestrogens analysis in soy beans by HPLC with coulometric dual

elektrode detector

37

III. 2. Investigation of metabolism of plant lignans by using in vitro fermentation

with human fecal inoculum and by HPLC with coulometric elektrode array

detector

43

III. 3. The analysis of the dietary fibre components 47

III. 4. Correlation between NSP constituent sugars of plants and plant lignan

metabolites

51

IV. IMPROVING THE QUALITY OF BREADS WITH WHOLE GRAIN

PRODUCTS DIETARY FIBRE SOURCE

53

IV. 1. Nutritional quality of fermented defatted soya and flaxseed flours and

their effect on texture and sensory characteristics of wheat sourdough

bread

54

References

64

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I. ENZYMATIC ACTIVITY ANALYSIS

Introduction. Cereals are a versatile and reliable source of food. They are easy to store

and may be used to produce a myriad of food products.

Cereals processing thus forms a large and important part of the food production chain. It

also plays a lesser, but no less important role in the non-food sector.

There are two main outlets for grain, animal feed, and human consumption, while a small

amount is required annually for seed.

The definition of quality therefore depends on the requirements of the specific market.

Grain attributes which determine its suitability for a specific market include its chemical,

physical and biological properties. All sectors of the market have a basic requirement for

sound grain free from impurities, insect damage and moulds. Other standards are more market

specific and will vary in importance according to species and end product. For wheat these

may include protein quality and quantity, endosperm texture, flour yield and colour, water

adsorption capacity, -amylase activity and specific weight. The major human and industrial uses of wheat are for breadmaking, biscuit manufacture

and distilling.

Specific weight, a measure of the bulk density of grain, is widely used as a wheat quality

indicator. High specific weight grain results in better flour extraction within a specific variety,

but is not always consistent across different varieties. Also important in ensuring that the

plant remains free of pests and disease and is supplied with adequate nutrients and water.

Hard wheat is required for inclusion in breadmaking and a soft texture is required for

biscuit making.

Wheat flour is used for breadmaking as a result of the viscoelastic properties of the dough

when water is added.

The dough may be classed as either strong or weak, depending on the quantity and quality

of the grain proteins, which in turn influences gluten strength. For breadmaking, gluten must

be strong enough to retain the carbon dioxide generated during fermentation, allowing the

bread to rise.

Protein quality is strongly influenced by genotype, although husbandry and environmental

factors can also play an important role. Low grain sulphur will result in low concentrations of

the sulphur-containing amino acids, cystine and methionine, and may result in poor loaf

volume. Protein quality has also been shown to fall as a result of late fungicide sprays that

prolong the grain filling period.

During seed germination endosperm starch is converted into soluble glucose and maltose

to support the developing embryo. This is brought about by enzymatic activity, especially the

enzyme -amylase, present within the grain and activated during the germination process. Some -amylase activity is needed to release sugars and aid fermentation during the

breadmaking process.

Excessive -amylase levels result in the formation of a darkened loaf crust as a result of sugar caramelization and a sticky crumb structure which can cause problems during slicing.

Grain -amylase levels reach their lowest levels during ripening, thereafter increasing significantly. This suggests an optimum date for harvesting, but one which is difficult to

predict and achieve in practice. Nevertheless, it is good practice to harvest crops destined for

the breadmaking market early to avoid the effects of wet weather.

The major components of wheat all contribute to baking quality and are thus targets for

genetic improvement of baking quality. Proteins are essential for the visco-elastic properties

of wheat doughs. Starch and cell wall polysaccharides (e.g., pentosans) also influence baking

quality. The breakdown of starch by amylases is a key process in baking. The pentosans of the

cell wall also have a significant influence on loaf quality.

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I. 1. Carbohydrate-degrading enzymes: -amylase analysis

This enzyme hydrolyzed -(1-4)D-glucosidic linkages from starch oligosaccharides. Analysis of -amylase can be by viscometric, colorimetric, dyed-substrate, turbidometric, gel diffusion, or reducing sugars assay.

Colorimetric methods have been widely used in the past are still used for measuring -amylase in wheat and barley.

-amylase activity is expressed as a function of alpha-amylase concentration and of the velocity constant for the hydrolytic degradation of limit dextrin.

In breadmaking, some -amylase is needed to sustain the production of sugars required for proper fermentation and consequent gas production unless adequate fermentable sugars are

included in the breadmaking formula. In addition, -amylases are unique in modifying starch and its functional properties.

Millers prefer to have a low level of endogenous -amylase and to add a malt or fungal -amylase source to the appropriate level. Fungal amylase is less thermostable than either malt

or bacterial amylases, and bacterial amylase is the most stable of the three. Thus, although a

bacterial source may be added occasionally, tolerances must be more closely watched.

The amount of -amylase that can be tolerated depends on the breadmaking process.

Analysis

Preparation of Chemicals

A soluble potato starch solution prepared to give 20 mg starch/mL.

The starch solution diluted 1: 1 with a 0.04M phosphate buffer at pH 5.9.

The working iodine reagent prepared fresh by diluting 1.0 mL of stock solution (500

mg iodine and 5.0 g potassium iodide/100 mL water) 100 times.

Bacillus amyloliquefaciens -amylase and Taka-therm L-170 -amylase of Bacillus ficheniformis diluted and used for the enzyme assay.

All the chemicals used - reagent grades.

Assay Procedure

Five milliliters of substrate solution added to a test tube and maintained for 10 min at

an incubation temperature in a water bath.

Enzyme (0.5 mL) added to the substrate solution and incubated under the test conditions.

The digest added to 5 mL stopping reagent (0.M HCI).

After mixing, 0.5 mL of this mixture added to 5.0 mL working iodine solution.

The intensity of blue color measured in a colorimeter using a red filter.

The instrument is set to zero with an iodine blank containing neither enzyme nor

substrate. The activity of the enzyme is calculated from the formula:

Activity (unit/mL) = D [(Ro-R)/Ro] X 100

Where:

Ro - the absorbance of the substrate-iodine complex in the absence of enzyme;

R - the absorbance of the digest;

D is the dilution factor of the enzyme.

The enzyme solution diluted when necessary so that the ratio (Ro - R)/Ro was between

0.2 and 0.7.

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I. 2. Carbohydrate-degrading enzymes: -amylase analysis

This enzyme hydrolyzed -(1-4)D-glucosidic bonds in amylose and amylopectin components in a stepwise fashion from the nonreducing end. Linear dextrin chains containing

an even number of D-glucose units produce maltose as the sole product. If the dextrin chain

contains an odd number of D-glucose units, some glucose is also produced. The action of the

enzyme stops in the region of -(1-6)D-glucosidic linkages. As a result, branched starch molecules such as a amylopectin produce a -limit dextrin in addition to maltose.

Upon germination of wheat, the levels of -amylase increase to several times their original level. This is a result of the release of bound enzyme, mediated by proteolytic or

disulfide reductase, rather than de novo synthesis of new enzyme.

Analysis

Assay of -amylase is difficult because of the complicating influence of -amylase. In sound wheat, -amylase activity is minimal and the many methods involving measurement of reducing sugars from a starch substrate may be employed.

In some cases, a separate determination may be made of the amount of saccharifying

activity due to -amylase, which is substrated from the total activity. Commonly used methods for measuring reducing sugars include reduction with 3,5-

dinitro-salicylic acid or neocuproine.

A number of attempts have been made to preferentially inactive the -amylase by some method such as acid treatment at pH 3.4.

In most cases, results have not been entirely satisfactory because such treatments are not

entirely selective.

In many cases, knowing the combined action of - and -amylases may be sufficient. For example, in breadmaking, it is necessery to know that both enzymes are present and

in the correct proportions to ensure that sufficient sugars are produced during the fermentation

period.

-amylase is more heat labile than -amylase, and a heat treatment at 70C for 15 min is routinely used to purify -amylase by eliminating -amylase.

I. 3. Proteolytic enzymes

It is becoming increasingly clear that wheat contains a large number of proteolytic

enzymes, each with its unique properties and specificities.

Analysis

To standardize a procedure for the enzymatic assay of Protease using Casein as a

substrate at Sigma-Aldrich St. Louis.

One unit will hydrolyze casein to produce color equivalent to 1.0 mole of tyrosine per minute at pH 7.5 at 37C. Color per Folin & Ciocalteaus reagent.

urified ater - purified water from a deioni ing system with a resistivity c m-1, or equivalent.

Protease Casein + H2O Amino Acids

Analysis conditions: T = 37C, pH=7.5, A660 nm Light Path = 1cm

Method: Spectrophotometric Stop Rate Assay

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Reagents:

1. 50 mM Potassium Phosphate buffer, pH 7.5 at 37C (Buffer)

Prepare 11.4 mg/mL in purified water using Potassium Phosphate, Dibasic, Trihydrate,

Sigma-Aldrich Product Number P5504. Adjust to pH 7.5 at 37C with 1 N HCl.

2. 0.65% (w/v) Casein Solution (Casein)

3. Prepare 6.5 mg/mL in reagent 7.3.1 (Buffer) using Casein, Sigma-Aldrich Product

Number C7078.

4. Heat gently with stirring to 80-85C for approximately 10 minutes until a homogeneous

dispersion is achieved. Do not boil.

5. Adjust the pH to 7.5 at 37C, if necessary, with 0.1 N NaOH or 0.1 N HCl.

6. 110 mM Trichloroacetic Acid Reagent (TCA). Prepare 1:55 dilution of Trichloroacetic

Acid, 6.1N, approximately 100% (w/v), Sigma-Aldrich Product Number T0699, with purified

water.

7. 0.5 Folin & Ciocalteus henol Reagent (F-C) Prepare a 1:4 dilution of 2 M Folin & Ciocalteus henol Reagent, Sigma-Aldrich Product Number F9252, with purified water.

8. 500 mM Sodium Carbonate Solution (Na2CO3)

Prepare 53 mg/mL in purified water using Sodium Carbonate, Anhydrous, Sigma-Aldrich

Product Number S2127.

9. 10 mM Sodium Acetate Buffer with 5 mM Calcium Acetate, pH 7.5 at 37C (Enzyme

Diluent)

Prepare 1.4 mg/mL Sodium Acetate, Trihydrate, Sigma-Aldrich Product Number S8625,

and 0.8 mg/mL Calcium Acetate, Sigma-Aldrich Product Number C1000, in purified water.

Adjust the pH to 7.5 at 37C with 0.1 M Acetic Acid or 0.1 N NaOH.

10. 1.1 mM L-Tyrosine Standard (Std Soln)

Prepare 0.2 mg/mL L-Tyrosine, Free Base, Sigma-Aldrich Product Number T3754, in

purified water. Heat gently (do not boil) until tyrosine dissolves. Cool to room temperature.

11. Protease Enzyme Solution Immediately before use, prepare a solution containing 0.1 0.2 units/mL of Protease in cold (Enzyme Diluent). For samples where little or no protease

detection is expected, prepare sample at 10 mg solid/mL in cold (Enzyme Diluent).

Assay Procedure

Pipette the following into suitable vials (in milliliters):

Test1 Test2 Test3 Blank

Casein 5.00 5.00 5.00 5.00

Let the vials equilibrate in a suitably thermostated water bath at 37C for about 5 minutes,

then add:

Enzyme Solution 1.00 0.70 0.50 -----

Mix by swirling and incubate at 37C for exactly 10 minutes. Then add:

TCA 5.00 5.00 5.00 5.00

Enzyme Solution ----- 0.30 0.50 1.00

Mix by swirling and incubate at 37C for about 30 minutes.

Filter each solution using a 0.45 m syringe filter and use the filtrate in secound step.

Pipette the following reagent into 4 dram vials (in milliliters): for more consistent results,

add F-C immediately following the addition of Na2CO3.

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Test1 Test2 Test3 Blank

Test Filtrate 2.00 2.00 2.00 -----

Blank Filtrate ----- ----- ----- 2.00

Na2CO3 5.00 5.00 5.00 5.00

F-C 1.00 1.00 1.00 1.00

Prepare a standard curve by pipetting the following reagents into suitable vials (in

milliliters).

For impurity samples, low standards may be added as needed:

For more consistent results, add F-C immediately following the addition of Na2CO3.

Std 1 Std 2 Std 3 Std 4 Std 5 Std Blank

Std Soln 0.05 0.10 0.20 0.40 0.50 0.00

Purified Water 1.95 1.90 1.80 1.60 1.50 2.00

Na2CO3 5.00 5.00 5.00 5.00 5.00 5.00

F-C 1.00 1.00 1.00 1.00 1.00 1.00

Mix by swirling and incubate Blanks, Standards, and Tests at 37C for 30 minutes.

Remove the vials and allow to cool to room temperature.

Filter each Blank, Standard, and Test using a 0.45 m syringe filter into suitable cuvettes. Record the A660 nm of each Test, Standard, and Blank solution.

Calculations

Units/mL en yme = (mole Tyrosine equivalents released) (11) (1) (10) (2)

where:

11 = Total volume of assay in milliliters

2 = Volume (in milliliters) used in Colorimetric Determination

1 = volume of enzyme used for assay

10 = time (in minutes) of assay

Units/mg solid = Units/mL enzyme

mg solid/ml enzyme

Units/mg protein= Units/mL enzyme

mg protein/ml enzyme

Final assay concentrations

In a 6.00 ml reaction mix, the final concentrations are 42 mM potassium phosphate,

0.54% (w/v) casein, 1.7 mM sodium acetate, 0.8 mM calcium acetate, and 0.1-0.2 unit

protease.

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I. 4. Cellulase and xylanase activity determination

Enzymes are widely used as technological aids in several processes of food technology.

In recent years, the baking industry has focused its attention on the replacement of several

chemical compounds by enzymes, since they are clean label compounds. Different enzymes

are currently added to the breadmaking process for improving dough handling, fresh bread

quality, and also the shelf life. Complex polysaccharides of cereal grain cell wals,

predominantly cellulose and hemicellulose (arabinoglucuronoxylans), are new regarded as

potential sources of mono- and oligosaccharides.

Cellulases and hemicellulases are used in order to improve the quality of bread. Their

addition to the breadmaking process lead to an increase in the bread volume and better crumb

porosity in whole wheat bread. As the consequence of the hydrolytic action of

hemicellulases/pentosanases/xylanases, some free sugars such pentoses and hexoses can be

released and may be used by the microorganisms.

Cellulase from Penicillium Funiculosum determination procedure

Sources: Produced by the controlled fermentation of non-toxicogenic and non-pathogenic

strains of Penicillium funiculosum and isolated from the growth medium.

Active principles: Cellulase (endo-1,4-beta-glucanase); Endo-1,3(4)-beta-glucanase;

Endo-1,4- beta-xylanase.

Systematic names and numbers: 1,4-(1,3; 1,4)- beta-D-Glucan-4-glucanohydrolase; 1,3-

(1,3; 1,4)- beta-D-Glucan-3(4)-glucanohydrolase; 1,4- beta-D-xylan xylohydrolase.

Reactions catalyzed: Hydrolyzes 1,4-beta-glucan linkages in polysaccharides such as

cellulose, yielding beta-dextrins.

Secondary enzyme activities: alpha-N-Arabinofuranosidase; Cellulose 1,4- beta-

cellobiosidase; beta-glucosidase; Xylan 1,4-beta-xylosidase.

Description: Typically off-white to tan amorphous powders, or liquids dispersed in food-

grade carriers or diluents; soluble in water; practically insoluble in ethanol and ether.

Functional uses: Enzyme preparation. Used in the preparation of breadmaking, fruit

juices, wine, beer and vegetable oils.

General specifications: Must conform to the General Specifications for Enzyme

Preparations Used in Food Processing.

Identification:

Cellulase activity: The sample shows cellulase activity

Xylanase activity: The sample shows xylanase activity

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TESTS

Cellulase activity

Principle

The assay is based on the ability of the enzyme to hydrolyze carboxymethyl cellulose

(CMC) to reducing sugars.

The reaction products are determined photometrically at 540 nm by measuring the

resulting increase in reducing groups using 3,5-dinitrosalicylic acid.

One cellulase unit is defined as the amount of enzyme that liberates reducing sugar at the

rate of mol/min under the conditions of the assay.

Apparatus

Spectrophotometer set at 540 nm.

Water-bath set at 40.0 0.1

Reagents

1. CMC substrate solution (1.0%): Accurately weigh 0.500 g of CMC (SIGMA C5678-7

or equivalent) and sprinkle on to warm 40 ml of water in a beaker.

Place beaker on a hot-plate equipped with a magnetic stirrer, apply heat and stir

vigorously.

When the liquid has started to boil, cover the beaker with a watch glass, turn off the hot

plate and continue stirring until the solution is cool.

Quantitatively transfer the solution into a 50 ml volumetric flask, add 5 ml acetate buffer,

adjust the pH to 5.0 and make up to volume.

2. 3,5-Dinitrosalicylic acid (DNS) solution: Accurately weigh 10 g of DNS into a 2000-

ml beaker. Add 16 g of sodium hydroxide pellets, 300 g of potassium sodium (+)-tartrate and

500 ml of water. Place the beaker on a heater/stirrer and warm gently, whilst stirring, to

dissolve.

Cool to ambient temperature and transfer the contents of the beaker into a 1000-ml

volumetric flask. Rinse the beaker with water, add to volumetric flask and make up to volume

with water. Store the solution at ambient temperature for up to 10 weeks.

It is possible that DNS reagent get overheated during the preparation making the solution

quite dark. The maximum absorbance at 540 nm for a blank (without glucose standard)

measured against water shall not be more than 0.050 absorbance units.

3. DNS-lactose solution: Dissolve lactose monohydrate with water to obtain 0.120 g/l

solution. Mix l50 ml of DNS solution and 50 ml of Lactose solution. Use freshly prepared

mixture.

4. Samples preparation: Dissolve known quantity of sample in distilled water. Make serial

dilutions to get a working solution in the absorbance range of 0.150 - 0.400.

5. Glucose standard solution: Accurately weigh 0.5g of anhydrous glucose and make up

to volume in a 100 ml volumetric flask. Dilute the solution with water to get 5, 10 and 15

moles/l of glucose.

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Procedure

Measurement of enzyme activity

Add 1 ml of substrate solution (pre-warmed to 40.0 0.1 for 5 min) to an equal volume

of sample solution also pre-warmed to 40.0 0.1.

Mix the resulting solution thoroughly and transfer to a water-bath maintained at 40.0

0.1 . After 10 minutes (reaction step) remove the test tube from the water bath, and add 4 ml

of DNS-Lactose solution and mix to stop the enzymatic reaction.

Cover tubes and place in a boiling water bath for 15 min. and then cooled to room

temperature with a cooling water bath. Remove insoluble substances by centrifugation (3000

rpm, 10 min).

Determine the absorbance at 540 nm against water blank. Prepare a reaction blank in a

similar manner but without a reaction step.

Prepare a reagent blank omitting substrate and read absorbance against water.

Standard curve

Prepare the glucose standard curve by adding 1 ml glucose standard solution (5, 10 and

5 moles/l) instead of CMC substrate solution in the procedure described above. Draw the standard curve in a coordinate system using glucose concentration (mol/l) as

the abscissa and absorbance as the ordinate.

The standard curve is a straight line passing through the origin and linear regression can

therefore be applied.

Calculate the glucose concentration in the sample from the standard curve and calculate

the enzyme activity as follows.

Calculation

Calculate the sample enzyme activity (U/g) by reading the equivalent glucose

concentration on the standard curve for the sample and the reaction blank and inserting them

in the following formula:

Where:

CG : Reading from the standard curve for sample en yme, mol/l

CRB : Reading from the standard curve for reagent blank, mol/l

D : Dilution factor of the sample

W : Weight of sample taken, g

10 : Incubation time, min

V : Volume of sample solution taken, 1 ml

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Xylanase activity (A)

Principle

This assay is based on the enzymatic hydrolysis of sodium arabinoxylan.

The resulting reducing sugar is allowed to react with 3,5-dinitrosalicylic acid and is

determined photometrically at 540 nm.

One xylanase unit is defined as that quantity of enzyme that liberates reducing sugar at a

rate of 1 mol /min under the conditions of the assay.

Apparatus

Spectrophotometer set at 540 nm.

Water bath set at 40.0 0.1

Reagents and solutions

1. Xylan substrate solution (1.0%): Accurately weigh 1.0 g xylan (dry base, from oat

spelts; such as SIGMA X-0627), transfer to a beaker with 60 ml of 0.2 M acetate buffer (pH

4.5).

Stir for 30 min and incubate at 60 for l hr with gradually stirring and check pH (4.50

0.05). Transfer the solution into a 100 ml volumetric flask and make up to volume with water.

2. 3,5-Dinitrosalicylic acid (DNS) solution: Accurately weigh 10 g of DNS into a 2000-

ml beaker. Add 16 g of sodium hydroxide pellets, 300 g of potassium sodium (+)-tartrate and

500 ml of water.

Place the beaker on a heater/stirrer and warm gently, whilst stirring, to dissolve. Cool to

ambient temperature and transfer the contents of the beaker into a 1000-ml volumetric flask.

Rinse the beaker with water, add rinsings to the volumetric flask and make up to volume

with water.

Store the solution at ambient temperature for up to 10 weeks. It is possible that DNS

reagent get overheated during the preparation making the solution quite dark.

The maximum absorbance at 540 nm for a blank (without xylose standard) measured

against water shall not be more than 0.050 absorbance units.

3. DNS-lactose solution: Dissolve lactose monohydrate with water to obtain 0.120 g/l

solution. Mix l50 ml of DNS solution and 50 ml of Lactose solution. Use freshly prepared

mixture.

4. Samples preparation: Dissolve known quantity of sample in distilled water. Make serial

dilutions to get a working solution in the absorbance range of 0.150 - 0.400.

5. Xylose standard dilutions: Accurately weigh 0.5g of anhydrous xylose with distilled

water and make up to 100 ml in a volumetric flask. Dilute with water to get working standard

solutions containing 250, 500 and 750 moles/l of xylose.

Procedure

Measurement of enzyme activity

Add 0.1 ml of sample solution to 1.9 ml of substrate solution pre-warmed to 40.0 0.1

for 5 min.

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Mix the resulting solution thoroughly and transfer to a water-bath maintained at 40

0.1. After 10 minutes (reaction step) remove the test tube from the water bath, and add 4 ml

of DNS-Lactose solution and mix to stop the enzymatic reaction.

Cover tubes and place in a boiling water bath for 15 min. and then cooled to room

temperature with a cooling water bath. Remove insoluble substances by a centrifuge (3000

rpm, 10 min).

Determine the absorbance at 540 nm against water blank.

Prepare a reagent blank in a similar manner but without a reaction step.

Standard curve

Prepare the xylose standard curve by adding 0.1 ml xylose standard solution (250, 500

and 750 moles/l) instead off xylan substrate solution in the procedure described above. Draw the standard curve in a coordinate system using glucose concentration (mol/l) as

the abscissa and absorbance as the ordinate. The standard curve is a straight line passing

through the origin and linear regression can therefore be applied.

Calculate the xylose concentration in the sample from the standard curve and calculate the

enzyme activity as follows.

Calculation

Calculate the sample enzyme activity (U/g) by reading the equivalent xylose

concentration on the standard curve for the sample and the reaction blank and inserting them

in the following formula:

Where:

Cx : Reading from the standard curve for sample enzyme, mol/l

CRB: Reading from the standard curve for reagent blank, mol/l

D : Dilution factor of the sample

W : Weight of sample taken, g

10 : Incubation time, min

V : Volume of sample taken, 0.1 ml

Xylanase activity in food and feed samples (B)

Arabinoxylan is the major endosperm cell-wall polysaccharide of wheat and rye and is

found in significant proportions in most cereal solutions and slurries of high viscosity, and in

nutrition it reduces the rate of nutrient absorption from the gut.

endo-b-D-Xylanase (xylanase) is added to feeds to catalyse depolymerisation of this

polysaccharide.

It can be demonstrated that endo-cleavage by xylanase of just one bond per thousand in

the arabinoxylan backbone can significantly remove viscosity properties.

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Of the carbohydrase enzymes used as feed supplements, one of the most difficult to

measure has been xylanase. These problems are attributed to several factors, including the low

levels of enzyme added to the food and feed, inactivation of enzyme during pelleting, binding

of the enzyme to feed components and the presence of specific xylanase inhibitors.

The only biochemical methods which are sufficiently sensitive, specific and robust to

measure xylanase in feeds are viscometric assays and those employing dyed xylan or

arabinoxylan polysaccharides.

Viscometric assays are tedious, whereas assays employing dyed xylan substrates are

rapid, reproducible and simple to perform. We recommend the use of either Xylazyme AX

tablets or Azo-Wheat Arabinoxylan (Azo-WAX).

Xylazyme AX based assays are about 5-fold more sensitive than assays employing Azo-

WAX, however, this latter substrate does have sufficient sensitivity in most applications, and

results are slightly more reproducible than with Xylazyme AX.

It is generally accepted that xylanase enzymes which are best suited to feed applications

have optimal activity at pH 6.0. Consequently, these enzymes are generally assayed at this pH

in 100 mM sodium phosphate buffer. In recovery experiments, however, we found that

sodium phosphate buffer extracts only a small proportion (< 20%) of the amount of enzyme

added to the feed.

Thus a wide range of alternative extractants and extraction conditions have been

evaluated. For feeds containing Trichoderma sp. xylanases, the best and most consistent

results have been obtained using 100 mM acetic acid or 100 mM sodium acetate buffer (pH

4.7) at room temperature.

Optimal extraction of Humicola sp. xylanases was achieved with a buffer containing 100

mM MES buffer (pH 6.0) and 1 % w/v sodium dodecyl sulphate (SDS).

KIT components:

Kits containing the required reagents to measure xylanase in foods and feeds are available

from Megazyme.

These kits contain:

1. Xylazyme AX test tablets (200 tablets).

2. A. niger control xylanase (~ 295 mU/mL at 40C and pH 4.7) in 50 % (v/v) glycerol

(activity stated on vial).

3. T. longibrachiatum control xylanase (~ 386 mU/mL at 40C and pH 6.0) in 50% (v/v)

glycerol (activity stated on vial).

Extraction Buffers: (not enclosed):

(A) Acetic acid (0.1 M)

Add 5.8 mL of glacial acetic acid (1.05 g/mL) to 900 ml of distilled water and adjust the

volume to 1 litre. Stable at room temperature for > 12 months.

(B) MES buffer (100 mM) plus SDS (1 % w/v)

Add 19.5 g of MES free acid (Sigma M-8250) to 800 mL of distilled water and dissolve.

Adjust the pH to 6.0 with 1 M sodium hydroxide.

Add 10 g of sodium lauryl sulphate (SDS; Sigma L-4509) and dissolve.

Adjust the volume to 1 litre and add 0.2 g of sodium azide and dissolve.

Stable at room temperature for > 12 months.

15

Equipment (Recommended):

1. Glass test tubes (round bottomed; 16 x 100 mm and 16 x 120 mm)

2. Micro-pipettors eg: Gilson Pipetman 200 L and 500 L.

3. Positive displacement pipettor eg: Eppendorf Multipette - with 5.0 mL Combitip [to

dispense 0.2 mL aliquots of xylanase control in 50% (v/v) glycerol].

4. Adjustable volume dispenser set at 5.0 mL (to dispense Trizma base solution)

5. Top-pan balance correct to 0.01 g

6. Spectrophotometer set at 590 nm

7. Vortex mixer (e.g. IKAYellowline Test Tube Shaker TTS2).

8. Whatman No. 1 (9 cm) filter circles and filter funnels.

Extraction and assay of xylanase in food and feed samples:

Trichoderma sp. Xylanases:

Extraction:

1. Mill feed samples (approx. 50 g) to pass a 0.5 mm screen and mix thoroughly.

2. Weigh 0.5 g ( 0.01 g) of each sample in quadruplicate into glass test-tubes (16 x 120 mm).

3. Add 5 mL of 0.1 M acetic acid to each sample and stir on a vortex mixer. Add 0.2 mL of

distilled to two of these tubes with stirring.

To the other two tubes add 0.2 mL of control xylanase solution (approx. 60-80 mU/0.2 mL;

see vial label) with vigorous and immediate stirring on a vortex mixer.

4. Incubate the slurries at room temperature and stir occasionally over the following 20 min.

5. Centrifuge the tubes at 1,500 g for 10 min in a bench centrifuge and use the supernatant

directly in the assays. Assays should be initiated within 30 min of obtaining these extracts to

minimise loss of enzyme activity in the extracts.

Assay:

1. Accurately transfer 0.5 mL aliquots of supernatant solutions (in duplicate) to glass test-

tubes (16 x 100 mm) at room temperature.

2. Add a Xylazyme AX tablet (without stirring) to each tube and immediately place the tubes

in a water bath set at 50 0.1C and incubate for exactly 30 minutes.

3. After exactly 30 minutes, add 5 mL of Trizma Base solution (pH ~ 9), stir vigorously on a

vortex mixer and store at room temperature for 5 minutes.

NOTE

1. This treatment terminates the reaction.

2. The tubes must be stored at room temperature and not at 50oC, as the substrate is not stable under alkaline

conditions at elevated temperatures (ie: absorbance values will increase due to substrate breakdown).

4. Stir the tubes on a vortex mixer and filter the slurry through a Whatman No. 1 (9 cm) filter

paper.

5. Measure the absorbance of the filtrates at 590 nm against a Reaction Blank.

Prepare the Reaction Blank by adding Trizma Base solution (5 mL) to the feed extract

(0.5 mL), followed by a Xylazyme AX tablet.

Stir the slurry and store at room temperature for 5 min before filtration through Whatman

No. 1 filter paper.

A single Reaction Blank is required for each feed sample.

16

Calculation of activity:

The level of xylanase in the flour sample is calculated as follows:

Activity in feed sample (0.5 g) = Added activity x SA

TA - SA

Where:

Added activity = the amount of xylanase added to the feed sample slurry at the time of assay

eg: 70 mU in the control xylanase solution (0.2 mL).

SA = the reaction absorbance obtained for extracts of the food and feed sample to which no

control xylanase was added.

TA = the total absorbance i.e. the absorbance of extracts of the food and feed sample to which

the control xylanase was added.

Example calculation:

Sample Abs/30 min. incubation

1. Food or Feed A 0.000

2. Food or Feed A containing Trichoderma sp. xylanase (SA) 0.859

3. SA + 70 mU xylanase (in the assay) 1.344

Activity in food or feed sample (0.5g) = Added activity x SA

TA - SA

where:

SA = absorbance of extract of the sample [assayed by the standard format (eg: 0.859)]

TA = total absorbance; i.e. the absorbance of extracts of the sample to which the additional

xylanase (0.2 ml of 350 mU/ml) was added (eg. Abs = 1.299)

Thus:

Activity in food or feed (U/0.5g)

= 70/1000 Units x 0.859/(1.299 - 0.859)

= 0.07 x 0.859/0.440 = 0.137 U/0.5 grams

= 0.137 x 2000 = 274 U/Kg or 274,000 Units/ton

NOTE:

Through the equation, the activity calculated is at 40C and the pH at which the particular enzyme was

standardised e.g. A. niger xylanase at pH 4.7 and T. longibrachiatum xylanase at pH 6.0.

17

II. CARBOHYDRATE ANALYSIS

Carbohydrates are widely prevalent in the plant kingdom, comprising the mono-, di-,

oligo-, and polysaccharides. The common monosaccharides are glucose, fructose, galactose,

ribose etc. The disaccharides, i.e., the combination of two monosaccharides include sucrose,

lactose and maltose.

Starch and cellulose are polysaccharides consisting of many monosaccharide residues.

Cellulose is the most abundant organic compound on this planet since it forms part of the cell

wall in plants.

Aldehydes (CHO) and ketones (=CO) are active groups in carbohydrates. Carbohydrates contain many hydroxyl groups as well. The number of hydroxyl groups varies with the

number of carbon atoms. Monosaccharides contain the free aldehyde or ketone group. Some

disaccharides have the free aldehyde group (maltose) and some do not have the free ones

(sucrose). The polysaccharides, starch and cellulose, are polymers of monosaccharides linked

through the active groups.

The chemical properties of saccharides vary depending upon the number of hydroxyl

groups and the presence or absence of CHO / =CO groups. These variations are the basis in the development of colour reactions to identify the saccharides.

II. 1. Colour reactions to identify the saccharides

Reagents:

Iodine solution: Add a few crystals of iodine to 2% potassium iodide solution till the

colour becomes deep yellow.

Fehlings reagent A: Dissolve 34.65 g copper sulphate in distilled water and make up to 500 mL.

Fehlings reagent B: Dissolve 125 g potassium hydroxide and 173 g Rochelle salt (potassium sodium tartrate) in distilled water and make up to 500 mL.

Benedicts qualitative reagent: Dissolve 173 g sodium citrate and 100 g sodium carbonate in about 500 mL water. Heat to dissolve the salts and filter, if necessary. Dissolve 17.3 g

copper sulphate in about 100 mL water and add it to the above solution with stirring and make

up the volume to 1 L with water.

Barfoeds reagent: Dissolve 24 g copper acetate in 450 mL boiling water. Immediately add 25 mL of 8.5% lactic acid to the hot solution. Mix well, Cool and dilute to 500 mL.

Seliwanoffs reagent: Dissolve 0.05 g resorcinol in 100 mL dilute (1:2) hydrochloric acid. Bials reagent: Dissolve 1.5 g orcinol in 500 mL of concentrated HCl and add 20 to 30

drops of 10% ferric chloride.

The reactions of carbohydrates:

1. Molischs Test Add two drops of olischs reagent (5% -naphthol in alcohol) to about 2 mL of test

solution and mix well.

Incline the tube and add about 1 mL of concentrated sulphuric acid along the sides of the

tube.

Observe the colour at the junction of the two liquids.

Observation: A red-cum-violet ring appears at the junction of the two liquids.

18

Remarks: The colour formed is due to the reaction of alpha-naphthol with furfural and/or

its derivatives formed by the dehydration of sugars by concentrated sulphuric acid. All

carbohydrates react positively with this reagent.

2. Iodine Test

Add a few drops of iodine solution to about 1 mL of the test solution.

Observation: Appearance of deep blue colour.

Remarks: This indicates the presence of starch in the solution. The blue colour is due to

the formation of starch-iodine complex.

3. Fehlings Test To mL of Fehlings solution A, add mL of Fehlings solution B and a few drops of

the test solution. Boil for a few minutes.

Observation: Formation of yellow or brownish-red precipitate.

Remarks: The blue alkaline cupric hydroxide present in Fehlings solution, when heated in the presence of reducing sugars, gets reduced to yellow or red cuprous oxide and it gets

precipitated. Hence, formation of the coloured precipitate indicates the presence of reducing

sugars in the test solution.

4. Benedicts Test To 2 mL of Benedicts reagent add five drops of the test solution. Boil for five minutes in

a water bath. Cool the solution.

Observation: Formation of red, yellow or green colour/precipitate.

Remarks: As in Fehlings test, the reducing sugars because of having potentially free aldehyde or keto group reduce cupric hydroxide in alkaline solution to red coloured cuprous

oxide. Depending on the sugar concentration yellow to green colour is developed.

5. Barfoeds Test To 1 mL of the test solution add about 2 mL of Barfoeds reagent. Boil it for one minute and allow to stand for a few minutes.

Observation: Formation of brick-red precipitate.

Remarks: Only monosaccharides answer this test. Since Barfoeds reagent is weakly acidic, it is reduced only by monosaccharides.

6. Seliwanoffs Test To 2 mL of Seliwanoffs reagent add two drops of test solution and heat the mixture to

just boiling.

Observation: Appearance of deep red colour.

Remarks: In concentrated HCl, ketoses undergo dehydration to yield furfural derivatives

more rapidly than do aldoses. These derivatives form complexes with resorcinol to yield deep

red colour.

It is a timed colour reaction specific for ketoses.

7. Bials Test To 5 mL of Bials reagent add 23 mL of solution and warm gently. When bubbles rise to

the surface cool under the tap.

Observation: Appearance of green colour or precipitate.

Remarks: It is specific for pentoses. They get converted to furfural. In the presence of

ferric ion orcinol and furfural condense to yield a coloured product.

19

8. Test for non-reducing sugarssuch as sucrose:

(a) Do Benedicts test with the test solution. (b) Add 5 drops of concentrated HCl to 5 mL of test solution in another test tube. Heat for

five minutes on a boiling water bath. Add 10% sodium hydroxide solution to give a slightly

alkaline solution (test with red litmus paper). Now perform Benedicts test with this hydrolysed solution.

Observation: No characteristic colour formation. Appearance of red or yellow colour.

Remarks: Indicates the absence of reducing sugars in the given solution. Indicates the

formation of reducing sugars from non-reducing sugars after hydrolysis with acid.

9. Mucic Acid Test

Add a few drops of conc. HNO3 to the concentrated test solution or substance directly and

evaporate it over a boiling water bath till the acid fumes are expelled. Add a few drops of

water and leave it overnight.

Observation: Formation of crystals.

Remarks: The both end carbon groups are oxidized to carboxylic groups. The resultant

saccharic acid of galactose is called mucic acid which is insoluble in water.

10. Osazone Test

To 0.5 g of phenylhydrazine hydrochloride add 0.1 g of sodium acetate and 10 drops of

glacial acetic acid. To this mixture add 5 mL of test solution and heat on a boiling water bath

for about half an hour. Allow the tube to cool slowly and examine the crystals under a

microscope.

Observation: Glucose, fructose and mannose produce needle-shaped yellow osazone

crystals, whereas lactosazone is mushroomshaped.

Different osazones show crystals of different shapes. Maltose produces flower-shaped

crystals.

Remarks: The ketoses and aldoses react with phenylhydrazine to produce a

phenylhydrazone which in turn reacts with another two molecules of phenylhydrazine to form

the osazone.

NOTES:

1. For osazone test, the reaction mixture should be between pH 5 and 6. Fructose takes 2 min to form the

osazone whereas for glucose it is 5 min. The disaccharides take a longer time to form osazones. Dissacharides

form crystals only on cooling.

2. When a mixture of carbohydrates is present in the test sample, chromatographic methods should be

employed to identify the individual sugars.

II. 2. Determination of Reducing Sugars by Nelson-Somogyi Method

Sugars with reducing property (arising out of the presence of a potential aldehyde or keto

group) are called reducing sugars. Some of the reducing sugars are glucose, galactose, lactose

and maltose. The Nelson-Somogyi method is one of the classical and widely used methods for

the quantitative determination of reducing sugars.

Principle

The reducing sugars when heated with alkaline copper tartrate reduce the copper from the

cupric to cuprous state and thus cuprous oxide is formed. When cuprous oxide is treated with

arsenomolybdic acid, the reduction of molybdic acid to molybdenum blue takes place. The

blue colour developed is compared with a set of standards in a colorimeter at 620 nm.

20

Materials

Alkaline Copper Tartrate

(i) Dissolve 2.5 g anhydrous sodium carbonate, 2 g sodium bicarbonate, 2.5 g potassium

sodium tartrate and 20 g anhydrous sodium sulphate in 80 mL water and make up to 100 mL.

(ii) Dissolve 15 g copper sulphate in a small volume of distilled water. Add one drop of

sulphuric acid and make up to 100 mL.

Mix 4 mL of B and 96 mL of solution A before use.

Arsenomolybdate reagent: Dissolve 2.5 g ammonium molybdate in 45 mL water. Add 2.5

mL sulphuric acid and mix well. Then add 0.3 g disodium hydrogen arsenate dissolved in 25

mL water. Mix well and incubate at 37C for 2448 hours. Standard glucose solution: Stock: 100 mg in 100 mL distilled water.

Working standard: 10 mL of stock diluted to 100 mL with distilled water [100 g/mL].

Procedure

1. Weigh 100 mg of the sample and extract the sugars with hot 80% ethanol twice (5 mL

each time).

2. Collect the supernatant and evaporate it by keeping it on a water bath at 80C.

3. Add 10 mL water and dissolve the sugars.

4. Pipette out aliquots of 0.1 or 0.2 mL to separate test tubes.

5. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard solution into a series of

test tubes.

6. Make up the volume in both sample and standard tubes to 2 mL with distilled water.

7. Pipette out 2 mL distilled water in a separate tube to set a blank.

8. Add 1 mL of alkaline copper tartrate reagent to each tube.

9. Place the tubes in a boiling water for 10 minutes.

10. Cool the tubes and add 1 mL of arsenomolybolic acid reagent to all the tubes.

11. Make up the volume in each tube to 10 mL with water.

12. Read the absorbance of blue colour at 620 nm after 10 min.

13. From the graph drawn, calculate the amount of reducing sugars present in the sample.

Calculation

Absorbance corresponds to 0.1 mL of test = x mg of glucose

10 mL contains = x/0.1 * 10 mg of glucose = % of reducing sugars

II. 3. Determination of Reducing Sugar by Dinitrosalicylic Acid Method

For sugar estimation an alternative to Nelson-Somogyi method is the dinitrosalicylic acid

method-simple, sensitive and adoptable during handling of a large number of samples at a

time.

Materials

Dinitrosalicylic Acid Reagent (DNS Reagent)

Dissolve by stirring 1 g dinitrosalicylic acid, 200 mg crystalline phenol and 50 mg sodium

sulphite in 100 mL 1% NaOH. Store at 4C. Since the reagent deteriorates due to sodium

sulphite, if long storage is required, sodium sulphite may be added at the time of use.

40% Rochelle salt solution (Potassium sodium tartrate).

21

Procedure

1. Follow, steps 1 to 3 as in Nelson-Somogyis method to extract the reducing sugars from the test material.

2. Pipette out 0.5 to 3 mL of the extract in test tubes and equalize the volume to 3 mL

with water in all the tubes.

3. Add 3 mL of DNS reagent.

4. Heat the contents in a boiling water bath for 5 min.

5. When the contents of the tubes are still warm, add 1 mL of 40% Rochelle salt solution.

6. Cool and read the intensity of dark red colour at 510 nm.

7. Run a series of standards using glucose (0500 g) and plot a graph.

Calculation

Calculate the amount of reducing sugars present in the sample using the standard graph.

II. 4. Determination of Glucose by Glucose Oxidase Method

Glucose is a widely distributed simple sugar with an active aldehyde group. Estimation of

glucose by glucose oxidase gives the true glucose concentration eliminating the interference

by other reducing sugars.

Principle

Glucose oxidase catalyses the oxidation of alpha-D-glucose to D-glucono-1, 5 lactone

(gluconic acid) with the formation of hydrogen peroxide. The oxygen liberated from hydrogen

peroxide by peroxidase reacts with the O-dianisidine and oxidises it to a red chromophore

product.

glucose Glucose + O2 H2O2 + Gluconic Acid oxidase

proxidase H2O2 + O-dianisidine Red-coloured product

Materials

Glucose Oxidase Peroxidase Reagent

Dissolve 25 mg O-dianisidine completely in 1 mL of methanol. Add 49 mL of 0.1 M

phosphate buffer (pH 6.5). Then add 5 mg of peroxidase and 5 mg of glucose oxidase to the

above prepared O-dianisidine solution.

Standard: Dissolve 100 mg glucose in 100 mL water. Dilute 10 mL of this stock to 100

mL to obtain the working standard.

22

Procedure

1. To 0.5 mL of deprotinised plant extract (deproteinization is not necessary in samples

with very low protein content) add 0.5 mL distilled water and 1 mL glucose

oxidaseperoxidase reagent.

2. Into a series of test tubes pipette out 0 (blank), 0.2, 0.4, 0.6, 0.8 and 1 mL of working

standard glucose solution and make up the volume to 1.0 mL with distilled water. Then add 1

mL of glucose oxidase-peroxidase reagent.

3. Incubate all the tubes at 35C for 40 minutes.

4. Terminate the reaction by the addition of 2 mL of 6 N-HCl.

5. Read the colour intensity at 540 nm.

Calculation

From the standard graph, calculate the amount of glucose present in the sample

preparation.

II. 5. Determination of Total Carbohydrate by Anthrone Method

Carbohydrates are the important components of storage and structural materials in the

plants.

They exist as free sugars and polysaccharides. The basic units of carbohydrates are the

monosaccharides which cannot be split by hydrolysis into more simpler sugars. The

carbohydrate content can be measured by hydrolysing the polysaccharides into simple sugars

by acid hydrolysis and estimating the resultant monosaccharides.

Principle

Carbohydrates are first hydrolysed into simple sugars using dilute hydrochloric acid. In

hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This compound forms

with anthrone a green coloured product with an absorption maximum at 630 nm.

Materials

2.5 N HCl

Anthrone reagent: Dissolve 200 mg anthrone in 100 mL of ice-cold 95% H2SO4. Prepare

fresh before use.

Standard glucose: StockDissolve 100 mg in 100 mL water. Working standard10 mL of stock diluted to 100 mL with distilled water. Store refrigerated after adding a few drops of

toluene.

Procedure

1. Weigh 100 mg of the sample into a boiling tube.

2. Hydrolyse by keeping it in a boiling water bath for three hours with 5 mL of 2.5 N HCl

and cool to room temperature.

3. Neutralise it with solid sodium carbonate until the effervescence ceases.

4. Make up the volume to 100 mL and centrifuge.

5. Collect the supernatant and take 0.5 and 1 mL aliquots for analysis.

6. Prepare the standards by taking 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard.

0 serves as blank.

23

7. Make up the volume to 1 mL in all the tubes including the sample tubes by adding

distilled water.

8. Then add 4 mL of anthrone reagent.

9. Heat for eight minutes in a boiling water bath.

10. Cool rapidly and read the green to dark green colour at 630 nm.

11. Draw a standard graph by plotting concentration of the standard on the X-axis versus

absorbance on the Y-axis.

12. From the graph calculate the amount of carbohydrate present in the sample tube.

Calculation

Amount of carbohydrate present in 100 mg of the sample

= (mg of glucose / Volume of test sample) * 100

NOTE:

Cool the contents of all the tubes on ice before adding ice-cold anthrone reagent.

II. 6. Phenol Sulphuric Acid Method for Total Carbohydrate

The phenol sulphuric acid method to estimate total carbohydrates is described below.

Principle

In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This forms a

green coloured product with phenol and has absorption maximum at 490 nm.

Materials

Phenol 5%: Redistilled (reagent grade) phenol (50 g) dissolved in water and diluted to

one litre.

Sulphuric acid 96% reagent grade.

Standard glucose: Stock-100 mg in 100 mL of water. Working standard-10 mL of stock

diluted to 100 mL with distilled water.

Procedure

1. Follow the steps 1 to 4 as given in anthrone method for sample preparation.

2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard into a series of test

tubes.

3. Pipette out 0.1 and 0.2 mL of the sample solution in two separate test tubes. Make up

the volume in each tube to 1 mL with water.

4. Set a blank with 1 mL of water.

5. Add 1 mL of phenol solution to each tube.

6. Add 5 mL of 96% sulphuric acid to each tube and shake well.

7. After 10 min shake the contents in the tubes and place in a water bath at 2530C for 20 min.

8. Read the colour at 490 nm.

9. Calculate the amount of total carbohydrate present in the sample solution using the

standard graph.

24

Calculation

Absorbance corresponds to 0.1 mL of the test = x mg of glucose

100 mL of the sample solution contains = (x / 0.1) * 100 mg of glucose = % of total

carbohydrate present.

II. 7. Estimation of Starch by Anthrone Reagent

Starch is an important polysaccharide. It is the storage form of carbohydrate in plants

abundantly found in roots, tubers, stems, fruits and cereals.

Starch, which is composed of several glucose molecules, is a mixture of two types of

components namely amylose and amylopectin.

Starch is hydrolysed into simple sugars by dilute acids and the quantity of simple sugars

is measured colorimetrically.

Principle

The sample is treated with 80% alcohol to remove sugars and then starch is extracted with

perchloric acid. In hot acidic medium starch is hydrolysed to glucose and dehydrated to

hydroxymethyl furfural.

This compound forms a green coloured product with anthrone.

Materials

Anthrone: Dissolve 200 mg anthrone in 100 mL of ice-cold 95% sulphuric acid.

80% ethanol.

52% perchloric acid.

Standard glucose: Stock-100 mg in 100 mL water. Working standard-10 mL of stock

diluted to 100 mL with water.

Procedure

1. Homogenize 0.1-0.5 g of the sample in hot 80% ethanol to remove sugars. Centrifuge

and retain the residue. Wash the residue repeatedly with hot 80% ethanol till the washings do

not give colour with anthrone reagent. Dry the residue well over a water bath.

2. To the residue add 5.0 mL of water and 6.5 mL of 52% perchloric acid.

3. Extract at 0C for 20 min. Centrifuge and save the supernatant.

4. Repeat the extraction using fresh perchloric acid. Centrifuge and pool the supernatants

and make up to 100 mL.

5. Pipette out 0.1 or 0.2 mL of the supernatant and make up the volume to 1 mL with

water.

6. Prepare the standards by taking 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard and

make up the volume to 1 mL in each tube with water.

7. Add 4 mL of anthrone reagent to each tube.

8. Heat for eight minutes in a boiling water bath.

9. Cool rapidly and read the intensity of green to dark green colour at 630 nm.

25

Calculation

Find out the glucose content in the sample using the standard graph. Multiply the value by

a factor 0.9 to arrive at the starch content.

II. 8. Determination of Amylose

Starch is composed of two components, namely amylose and amylopectin. Amylose is a

linear or non-branched polymer of glucose. The glucose units are joined by -1-4 glucosidic linkages. Amylose exists in coiled form and each coil contains six glucose residues.

Principle

The iodine is adsorbed within the helical coils of amylose to produce a blue-coloured

complex which is measured colorimetrically.

Materials

Distilled ethanol.

1 N NaOH.

0.1% phenolphthalein.

Iodine reagent: Dissolve 1 g iodine and 10 g KI in water and make up to 500 mL.

Standard: Dissolve 100 mg amylose in 10 mL 1 N NaOH; make up to 100 mL with

water.

Procedure

1. Weigh 100 mg of the powdered sample, and add 1 mL of distilled ethanol. Then add 10

mL of 1 N NaOH and leave it overnight.

2. Make up the volume to 100 mL.

3. Take 2.5 mL of the extract, add about 20 mL distilled water and then three drops of

phenolphthalein.

4. Add 0.1 N HCl drop by drop until the pink colour just disappears.

5. Add 1 mL of iodine reagent and make up the volume to 50 mL and read the colour at

590 nm.

6. Take 0.2, 0.4, 0.6, 0.8 and 1 mL of the standard amylose solution and develop the

colour as in the case of sample.

7. Calculate the amount of amylose present in the sample using the standard graph.

8. Dilute 1 mL of iodine reagent to 50 mL with distilled water for a blank.

Calculation

Absorbance corresponds to 2.5 mL of the test solution

= x mg amylose 100 mL contains = x/2.5 * 100 mg amylose = % amylose.

NOTES:

1. The sample suspension may be heated for 10 min in a boiling water-bath instead of overnight dissolution.

2. The amount of amylopectin is obtained by subtracting the amylose content from that of starch.

26

II. 9. Determination of Cellulose

Cellulose, a major structural polysaccharide in plants, is the most abundant organic

compound in nature, and is composed of glucose units joined together in the form of the

repeating units of the disaccharide cellobiose with numerous cross linkages. It is also a major

component in many of the farm wastes.

Principle

Cellulose undergoes acetolysis with acetic/nitric reagent forming acetylated cellodextrins

which get dissolved and hydrolyzed to form glucose molecules on treatment with 67% H2SO4.

This glucose molecule is dehydrated to form hydroxymethyl furfural which forms green

coloured product with anthrone and the colour intensity is measured at 630 nm.

Materials

Acetic/Nitric reagent: Mix 150 mL of 80% acetic acid and 15 mL of concentrated nitric

acid.

Anthrone reagent: Dissolve 200 mg anthrone in 100 mL concentrated sulphuric acid.

Prepare fresh and chill for 2 h before use.

67% sulphuric acid.

Procedure

1. Add 3 mL acetic/nitric reagent to a known amount (0.5 g or 1 g) of the sample in a test

tube and mix in a vortex mixer.

2. Place the tube in a water-bath at 100C for 30 min.

3. Cool and then centrifuge the contents for 1520 min. 4. Discard the supernatant.

5. Wash the residue with distilled water.

6. Add 10 mL of 67% sulphuric acid and allow it to stand for 1 h.

7. Dilute 1 mL of the above solution to 100 mL.

8. To 1 mL of this diluted solution, add 10 mL of anthrone reagent and mix well.

9. Heat the tubes in a boiling water-bath for 10 min.

10. Cool and measure the colour at 630 nm.

11. Set a blank with anthrone reagent and distilled water.

12. Take 100 mg cellulose in a test tube and proceed from Step No. 6 for standard.

Instead of just taking 1 mL of the diluted solution (Step 7) take a series of volumes (say 0.42 mL corresponding to 40200 g of cellulose) and develop the colour.

Calculation

Draw the standard graph and calculate the amount of cellulose in the sample.

II. 10. Determination of Hemicellulose

Hemicelluloses are non-cellulosic, non-pectic cell wall polysaccharides. They are

regarded as being composed of xylans, mannans, glucomannans, galactans and

arabinogalactans.

Hemicelluloses are categori ed under unavailable carbohydrates since they are not split by the digestive enzymes of the human system.

27

Principle

Refluxing the sample material with neutral detergent solution removes the water-solubles

and materials other than the fibrous component. The left out material is weighed after

filtration and expressed as Neutral Detergent Fibre (NDF).

Materials

Neutral Detergent Solution

Weigh 18.61 g disodium ethylenediamine tetraacetate and 6.81 g sodium borate

decahydrate. Transfer to a beaker. Dissolve in about 200 mL of distilled water by heating and

to this, add a solution (about 100200 mL) containing 30 g of sodium lauryl sulphate and 10 mL of 2-ethoxy ethanol. To this add a solution (about 100 mL) containing 4.5 g of disodium

hydrogen phosphate. Make up the volume to one litre and adjust the pH to 7.0.

Decahydronaphthalene.

Sodium sulphite.

Acetone.

Procedure

1. To 1 g of the powdered sample in a refluxing flask add 10 mL of cold neutral detergent

solution.

2. Add 2 mL of decahydronaphthalene and 0.5 g sodium sulphite.

3. Heat to boiling and reflux for 60 min.

4. Filter the contents through sintered glass crucible (G-2) by suction and wash with hot

water.

5. Finally give two washings with acetone.

6. Transfer the residue to a crucible, dry at 100C for 8 h.

7. Cool the crucible in a desiccator and weigh.

Calculation

Hemicellulose = Neutral detergent fibre (NDF) Acid detergent fibre (ADF) NOTE:

See Lignin for determining acid detergent fibre.

II. 11. Determination of Fructose and Inulin

Fructose, a keto-hexose (called as fruit sugar), is usually accompanied by sucrose in fruits

like apple. Honey is a rich source of fructose.

Principle

The hydroxymethyl furfural formed from fructose in acid medium reacts with resorcinol

to give a red colour product.

Materials

Resorcinol reagent: Dissolve 1 g resorcinol and 0.25 g thiourea in 100 mL glacial acetic

acid. This solution is indefinitely stable in the dark.

Dilute HCl: Mix five parts of conc. HCl with one part of distilled water.

28

Standard fructose solution: Dissolve 50 mg of fructose in 50 mL water. Dilute 5 mL of

this stock to 50 mL for a working standard.

Procedure

1. To 2 mL of the solution containing 2080 g of fructose add mL of resorcinol reagent.

2. Then add 7 mL of dilute hydrochloric acid.

3. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard and make up the volume

to 2 mL with water. Add 1 mL of resorcinol reagent and 7 mL of dilute HCl as above.

4. Set a blank along with the working standard.

5. Heat all the tubes in a water-bath at 80C for exactly 10 min.

6. Remove and cool the tubes by immersing in tap water for 5 min.

7. Read the colour at 520 nm within 30 min.

8. Draw the standard graph and calculate the amount of fructose present in the sample

using the standard graph.

Inulin

Inulin is a polymer made of fructose units with -2-1 linkage. It is found in onion, garlic and in many other plant parts.

Sample Extraction

Grind the sample and extract in 80% ethanol for six hours to remove free sugars. Dry the

sample and take 500 mg in a 100 mL conical flask.

Add 20 mL of water and heat it in a water bath at 90C for 10 min.

Collect the extract and then add 70 mL of water.

Replace the flask for another 30 min with occasional shaking to dissolve the fructosan,

then remove and cool it at room temperature.

Combine the extracts and filter the solution if it is not clear and make up to 100 mL in a

standard flask.

To estimate the inulin content in the extract follow the procedure given for fructose

estimation. The amount of inulin is expressed in terms of fructose concentration.

II. 12. Determination of Pectic Substances

Pectic substances abundantly exist in the middle lamella of the plant cells. There are three

types of pectic substances-pectic acids, pectin and protopectin. Pectic acid is an unbranched

molecule made up of about 100 units of D-galacturonic acid residues.

The monomers are linked through 1-4 linkages. Pectin is an extensively esterified pectic

acid. Several carboxyl groups exist as methyl esters.

Pectic acid is water soluble whereas pectin forms a colloidal solution.

Protopectin is a larger molecule than pectic acid and pectin.

During ripening of fruits, conversion of protopectin into pectic acid and pectin takes

place.

The pectins in fruits vary in their methoxyl content and in jellying power.

Two methods are described below for the estimation of pectin: one gravimetric and the

other, colorimetric.

29

A. Gravimetric Method

Principle

Pectin is extracted from plant material and saponified. It is precipitated as calcium pectate

by the addition of calcium chloride to an acid solution. After thoroughly washing to eliminate

chloride ions, the precipitate is dried and weighed.

MATERIALS

1 N Acetic acid (Dilute 30 mL of glacial acetic acid to 500 mL with water).

1 N Calcium chloride solution: Dissolve 27.5 g anhydrous CaCl2 in water and dilute to

500 mL.

1% Silver nitrate: Dissolve 1 g AgNO3 in 100 mL water.

0.01 N HCl

0.05 N HCl

0.3 N HCl

Procedure

1. Weigh 50 g of blended sample into a 1 L beaker and add 300 mL 0.01 N HCl. Boil for

30 min and filter under suction. Wash the residue with hot water and collect the filtrate.

2. To the residue add 100 mL 0.05 N HCl, boil for 20 min filter, wash and collect the

filtrate.

3. To the residue now add 100 mL 0.3 N HCl, boil for 10 min, filter, wash and collect the

filtrate.

4. Pool the filtrates. Cool and make to volume (500 mL).

5. Pipette out 100200 mL aliquots into 1 L beakers. 6. Add 250 mL water and neutralize the acid with 1 N NaOH using phenolphthalein

indicator. Add an excess of 10 mL of 1 N NaOH with constant stirring and allow it to stand

overnight.

7. Add 50 mL 1 N acetic acid and after 5 min, add 25 mL 1 N calcium chloride solution

with stirring. Allow it to stand for 1 h.

8. Boil for 1 to 2 min.

9. Filter through a pre-weighed Whatman No. 1 filter paper (see note 1).

10. Wash the precipitate with almost boiling water until the filtrate is free from chloride.

11. Test the filtrate with silver nitrate for chloride.

12. Transfer the filter paper with the calcium pectate, dry overnight at 100C in a

weighing dish, cool in a desiccator and weigh.

Calculation

The pectin content is expressed as % calcium pectate

% calcium pectate = Wt. of calcium pectate 500 100

mL of filtrate taken Wt. of smaple for estimation

NOTES:

The filter paper for Step No. 9 should be prepared as described below:

1. Wet the filter paper in hot water, dry in oven at 102C for 2 h. Cool in a desiccator and weigh in a

covered dish.

2. The theoretical yield of calcium pectate from pure galacturonic anhydride is 110.6%.

30

B. Colorimetric Method

Principle

Galacturonic acid is reacted with carbazole in the presence of H2SO4 and the colour

developed is measured at 520 nm.

Materials

60% Ethyl alcohol (Mix 500 mL 95% alcohol and 300 mL water).

95% Ethyl alcohol.

Purified ethyl alcohol (Reflux 1 L of 95% ethyl alcohol with 4 g zinc dust and 2 mL conc.

H2SO4 for 15 h and distill in all glass distillation apparatus. Redistill with 4 g zinc dust

and 4 g KOH).

1 N and 0.05 N Sodium hydroxide.

H2SO4 (Analytical grade).

0.1% Carbazole reagent: Weigh 100 mg recrystallized carbazole, dissolve and dilute to

100 mL with purified alcohol.

Procedure

1. Weigh 100 mg pectin (see notes section for the preparation of pectin) and dissolve in

100 mL of 0.05 N NaOH.

2. Allow it to stand for 30 min to deesterify the pectin.

3. Take 2 mL of this solution and make up to 100 mL with water.

4. Pipette out 2 mL of deesterified pectin solution and add 1 mL carbazole reagent. A

white precipitate will be formed.

5. Add 12 mL conc. H2SO4 with constant stirring.

6. Close the tubes with rubber stopper and allow to stand for 10 min to develop the

colour.

7. To set a blank add 1 mL of purified ethyl alcohol in the place of carbazole reagent.

8. Read the colour at 525 nm against blank, exactly 15 min after the addition of acid.

Standard

Weigh 120.5 mg galacturonic acid monohydrate (from a sample vacuum dried for 5 h at

30C) and transfer to a 1 L volumetric flask. Add 10 mL 0.05 N NaOH and dilute to volume

with water.

After mixing, allow it to stand overnight. Dilute 10, 20, 40, 50, 60 and 80 mL of this

standard solution to 100 mL with water.

Take 2 mL of these solutions for colour developing and proceed as in the case of the

sample. Draw a standard curve-the absorbance versus concentration.

Calculation

Read the concentration of the anhydrogalacturonic acid corresponding to the reading of

the sample, and calculate as follows:

% anhydrogalacturonic acid = g of anhydrogalacturonic acid in the aliquot Dilution 00 mL taken for estimation Wt. of pectin sample 1,000,000

31

NOTES:

1. Carbazole is recrystallized from toluene.

2. An alternate procedure adopted for colour development is as follows: Take 12 mL of conc.

H2SO4 in a test tube, cool in an ice-bath, and add 2 mL of the deesterified pectin solution and again cool.

Heat the contents in a boiling water-bath for 10 min, cool to 20C and add 1 mL of 0.15% carbazole reagent in

purified ethyl alcohol.

Allow it to stand for 25 5 min at room temperature to develop the colour.

Read the absorbance at 520 nm. Standards should also be treated similarly.

C. Extraction and Purification of Pectin

1. Blend the fresh sample. If the material is dry grind.

2. Transfer 100 g macerated sample (10 g dry tissue) to a pre-weighed 1 L beaker

containing 400 mL water.

3. Add 1.2 g freshly ground sodium hexametaphosphate and adjust to pH 4.5.

4. Heat with stirring at 9095C for 1 h. Check the pH in every 15 min and maintain at pH 4.5 with citric acid or NaOH. Replace water lost by evaporation at intervals. However, do

not add water at the last 20 min.

5. Add 4 g filter aid and 4 g ground paper pulp. Filter rapidly through a fast filter paper

coated with 3 g moistened fast filter aid.

6. Collect at least 200 mL of the filtrate in a preweighed container. Cool as rapidly as

possible. Now, note the weight of the filtrate.

7. If the filtrate contains less than 0.2% pectin, concentrate the filtrate under vacuum to

attain this concentration.

8. To three volumes of ethanol, isopropanol or acetone containing 0.5 N HCl, pour the

cooled, weighed filtrate. The slurry should be at pH 0.71. Stir for 30 min. 9. Centrifuge or filter. Wash the precipitate with the same solvent containing HCl. Then,

wash repeatedly with 70% alcohol or acetone until the precipitate is essentially chloridefree or

the pH is above 4.

10. Dehydrate the precipitate further in 400 mL acetone. Dry overnight in vacuo with a

slow stream of dry air passing through the oven.

11. Weigh the precipitate and use this pectin for analysis.

12. The dried pectin should be free from ammonia for which a small sample of the pectin

is heated with 1 mL of 0.1 N NaOH and ammoniacal odour can be noticed or tested with a

moistened litmus paper. If ammonium ions are present wash with acidified 6% alcohol,

followed by neutral alcohol to remove the acid and dry.

II. 12. Determination of Crude Fibre

Crude fibre consists largely of cellulose and lignin (97%) plus some mineral matter. It

represents only 6080% of the cellulose and 46% of the lignin. The crude fibre content is commonly used as a measure of the nutritive value of poultry

and livestock feeds and also in the analysis of various foods and food products to detect

adulteration, quality and quantity.

Principle

During the acid and subsequent alkali treatment, oxidative hydrolytic degradation of the

native cellulose and considerable degradation of lignin occur. The residue obtained after final

filtration is weighed, incinerated, cooled and weighed again. The loss in weight gives the

crude fibre content.

32

Materials

Sulphuric acid solution (0.255 0.005 N): 1.25 g concentrated sulphuric acid diluted to

100 mL (concentration must be checked by titration).

Sodium hydroxide solution (0.313 0.005 N): 1.25 g sodium hydroxide in 100 mL

distilled water (concentration must be checked by titration with standard acid).

Procedure

1. Extract 2 g of ground material with ether or petroleum ether to remove fat (Initial

boiling temperature 3538C and final temperature 52C). If fat content is below 1%, extraction may be omitted.

2. After extraction with ether boil 2 g of dried material with 200 mL of sulphuric acid for

30 min with bumping chips.

3. Filter through muslin and wash with boiling water until washings are no longer acidic.

4. Boil with 200 mL of sodium hydroxide solution for 30 min.

5. Filter through muslin cloth again and wash with 25 mL of boiling 1.25% H2SO4, three

50 mL portions of water and 25 mL alcohol.

6. Remove the residue and transfer to ashing dish (preweighed dish W1).

7. Dry the residue for 2 h at 130 2C. Cool the dish in a desiccator and weigh (W2).

8. Ignite for 30 min at 600 15C.

9. Cool in a desiccator and reweigh (W3).

Calculation

% crude fibre in ground sample = Loss in weight on ignition (W2- W1 ) - (W3 W1 ) * 100

Weight of the sample

II. 13. Determination of Pyruvic Acid

Pyruvic acid or pyruvate is an important metabolic intermediate. It is greatly produced in

the terminal step of glycolysis and funnels to TCA cycle for further oxidation for releasing the

chemical energy. It can be determined following the procedure given below:

Principle

The DNPH (2,4-dinitrophenyl hydrazine) reacts with pyruvate after the addition NaOH

giving a brown colored hydrazone product which can be estimated colorimetrically at 510 nm.

Materials

Phosphate buffer pH 9.4

A: 0.2 M solution of monobasic sodium phosphate NaH2PO4H2O (27.8 g in 1000 mL).

B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HPO4*7H2O in 1 L or 17.7

g of Na2HPO4*12H2O in 1 L).

19 mL of A and 81 mL of B, diluted to a total of 200 mL

Store in refrigerator.

33

Pyruvate, Standard

Dissolve 22 mg sodium pyruvate in 100 mL water in a standard flask.

2, 4-Dinitrophenyl hydrazine (DNPH)

Dissolve 19.8 mg of DNPH in 10 mL of conc. HCl and make to 100 mL with water.

Store it in an amber bottle at room temperature.

Sodium hydroxide 0.8 N

Dissolve 16 g sodium hydroxide in one litre water.

Plant extract

Grind 6 g of plant material in 15 mL of phosphate buffer. Centrifuge at 25.000 g for 15

min. Use the supernatant as plant extract.

Procedure

1. Pipette out 50 L, 75 L, 00 L, 50 L, 200 L of pyruvate standard solution and 0.5 mL, 1.0 mL, 1.5 mL, and 2.0 mL of sample extract into test tubes and make up the volume

to 2.0 mL with phosphate buffer (pH 7.4).

2. Set a blank with no pyruvate solution.

3. Add 0.5 mL of DNPH solution to each tube.

4. Incubate at 37C for 2030 min. 5. Add 5 mL of NaOH solution to each tube, mix well and incubate for 10 min at room

temperature.

6. Record the absorbance at 610 nm.

Calculation

Draw the standard graph and calculate the amount of pyruvic acid present in the sample

using the graph.

II. 14. Determination of Amylose in Flour by a Colorimetric Assay

Starch content determination

The enzymatic method for the determination of starch was essentially that of Holm et a1.

Starch was completely hydrolysed to glucose by the successive action of thermostable alpha-

amylase and amyloglucosidase.

The amount of glucose produced was then enzymatically determined using a glucose test-

combination kit from Boehringer Mannheim, Germany.

Amylose determination

A l00 mg test portion was weighed into a 30ml teflon centrifuge tube with screw cap

(nr3114-030 from Nalge Company, Rochester, NY, USA) and 10.0ml 90% (v/v) DMSO

solution were added. The tube was immediately capped, vigorously mixed on a vortex mixer

and placed in a water bath at 80C.

Starch was extracted during 60min, under constant stirring.

Every 15min, the tube was additionally mixed on the vortex mixer. The tube was then

cooled to room temperature under constant stirring, centrifuged for 15min at 2900g, and the

supernatant transferred into a 50ml teflon centrifuge tube with screw cap.

34

The residue was washed with 5.0ml 90% (v/v) DMSO solution, centrifuged for 15min at

2900g, and the supernatant collected with the first fraction into the 50ml centrifuge tube.

Starch was precipitated by the addition of 22.5m1 ethanol, the tube centrifuged for 15min at

2900g, and the supernatant cautiously discarded, to avoid any loss of sample material.

The residue was washed with 20ml ethanol, the tube centrifuged for 15min at 2900g, and

the supernatant cautiously discarded. This operation was repeated with 20ml acetone.

Finally the residue was dried under a gentle stream of nitrogen.

Briefly, starch was redissolved in urea-dimethylsulfoxide and the resulting solution

defatted with ethanol.

An aliquot of the lipid-free solution was then reacted with iodine and the absorbance of

the blue-coloured amylose-iodine complex measured.

The iodine-binding capacity of starch, expressed as Blue value, was calculated according

to the following formula:

Blue ValueT = (ml + m2) * A * 10

m3 * ml * (c/100) * 1000

where:

ml = mass of the test portion (g);

m2 = mass of the UDMSO solution (g);

m3 =mass of the starch-UDMSO solution aliquot (8);

A = absorbance at 635nm, measured at temperature T,

c = starch content (Yo).

The correction factors proposed by Morrison and Laignelet were used to convert the Blue

Value measured at temperature T into Blue Value at 20C and the amylose content was

calculated using the suggested regression equation.

35

III. PHYTOESTROGENS ANALYSIS METHODS

Phytoestrogens are plant-derived xenoestrogens functioning as the primary female sex

hormone (estrogen) not generated within the endocrine system but consumed by eating

phytoestrogenic plants. Also called "dietary estrogens", they are a diverse group of naturally

occurring nonsteroidal plant compounds (Figure 1 and 2) that, because of their structural

similarity with estradiol (17--estradiol) or other synthetic or mammals estrogens (Figure 3), have the ability to cause estrogenic or/and antiestrogenic effects.

It has been proposed that plants use the phytoestrogens as part of their natural defence

against the overpopulation of the herbivore animals by controlling the male fertility.

The similarities, at molecular level, of estrogens and phytoestrogens allow them to mildly

mimic and sometimes act as antagonists of estrogen. Phytoestrogens were first observed in

1926, but it was unknown if they could have any effect in human or animal metabolism.

In the 1940s it was noticed for the first time that red clover (a phytoestrogens-rich plant)

pastures had effects on the fecundity of grazing sheep. Researchers are exploring

the nutritionalrole of these substances in the regulation of cholesterol, and the maintenance of

proper bone density post-menopause. Evidence is accruing that phytoestrogens may have

protective action against diverse health disorders, such as prostate, breast, bowel, and

other cancers, cardiovascular disease, brain function disorders and osteoporosis, though there

is no evidence to support their use in alleviating the symptoms of menopause.

Phytoestrogens cannot be considered as nutrients, given that the lack of these in diet does

not produce any characteristic deficiency syndrome, nor do they participate in any essential

biological function.

Figure 1. a) matairesinol, b) cumestrol, c) genistein, d) zearalenone

OCH 3

HO

O

O

OH

O

O

O

OH

OH

HO

c

b

HO

HO

O

O

OHa

HO

OH

O

O CH 3

d

36

O O

OH

HO OH

HO

OH

OCH 3

OH

OCH 3

O

O

HO

HO

OH

HO

Figure 2. Secoisolariciresinol diglycoside

OCH 3

HO

O

O

OH

O

O

O

OH

OH

HO

c

b

HO

HO

O

O

OHa

d

OH

OH

OH

OH

Figure 3. Mammals and sinthetic estrogens: a) estrone, b) 17-estradiol, c) diethylstilbestrol, d) estriol

37

III. 1. Phytoestrogens analysis in soy beans by HPLC with coulometric dual

elektrode detector

The HPLC system consisted of a pump model 5200A (ESA, Chelmsford, USA) and a

Rheodyne 7125 injector (Cotati, C.A., USA) adapted with a 5l sample loop. A Lichrospher

60 RP Select B (Merck, Darmstadt, Germany) column (2504 mm, 5 m) in combination

with a precolumn (104 mm, same material) and for detection a Coulochem II (ESA, Chelmsford, USA) equipped with a dual electrode cell, model 5010 (ESA, Chelmsford, USA)

were used. Data acquisition and evaluation were carried out with an IBM PC/AT compatible

computer provided with Bischoff (Leonberg, Germany) Mc Dacq software.

Sample preparation of soy beans. Two types of sample preparation for phytoestrogens

analysis were performed: 1) simultaneously extraction and acid hydrolysis, and 2) only

extraction.

From 10 g of milled soy beans a 50 mg sample was taken. To each sample 35 ml ethanol,

5 ml of 10 M HCl and 1 ml internal standard (125 mg estriol) were added. The prepared

mixture in the first case was refluxed for two hours and cooled afterwards to room

temperature, and in the second case the mixture was just kept by room temerature for two

hours. The pH was adjusted to 3 with 6 M NaOH and the solution was filled up to 50 ml with

ethanol. After that 1 ml of the solution was diluted with mobile phase [ethanol/THF/buffer

solution (sodium acetate), 396/9/595, v/v/v, pH 2.6 was adjusted by adding glacial acetic

acid] in a flask to 10 ml.

Qualitative and quantitative analysis of daidzein and genistein. 5 l of the diluted and

filtered sample extracts or standard solutions (daid ein: 6.5 to 262 g/l, genistein: 7. to 2 4 g/l, the concentration of estriol was held constant at 250 g/l) were injected; the substances were separated (flow rate: 0.8 ml/min) on the reversed phase column and were detected at

+350 mV (channel 1) and +500 mV (channel 2).

Daidzein and genistein were identified by measuring the retention time (RT) and the

hydrodynamic voltammograms of the substances in samples and standard solutions and were

quantified using the calibration curves. The detection limits for daidzein and genistein were

determined using diluted standard solutions. Triplicate samples were analyzed and the

standard deviation was calculated. The recovery was obtained by adding estriol as internal

standard.

Preparation of the methodology and results

The use of HPLC with coulometric detector for the determination of phytoestrogens in

soy beans. Two methods for the analysis of phytoestrogens by HPLC with coulometric dual

electrode detection have been developed: first for the determination of daidzein and genistein and second for a wider spectrum of phytoestrogens, such as daidzein, genistein, matairesinol, and formononetin.

Method for the determination of daidzein and genistein. To determine daidzein and

genistein, optimal chromatographic conditions, such as a