LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

VETERINARY ACADEMY
Department of the Food Safety and Quality

Elena Bartkiene

Plant Food analysis methods

Methodical book

Kaunas, 2012
1

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.

CONTENT
I. ENZYMATIC ACTIVITY ANALYSIS
I. 1. Carbohydrate-degrading enzymes: -amylase analysis
I. 2. Carbohydrate-degrading enzymes: -amylase analysis
I. 3. Proteolytic enzymes
I. 4. Cellulase and xylanase activity determination
II. CARBOHYDRATE ANALYSIS
II. 1. Colour reactions to identify the saccharides
II. 2. Determination of Reducing Sugars by Nelson-Somogyi Method
II. 3. Determination of Reducing Sugar by Dinitrosalicylic Acid Method
II. 4. Determination of Glucose by Glucose Oxidase Method
II. 5. Determination of Total Carbohydrate by Anthrone Method
II. 6. Phenol Sulphuric Acid Method for Total Carbohydrate
II. 7. Estimation of Starch by Anthrone Reagent
II. 8. Determination of Amylose
II. 9. Determination of Cellulose
II. 10. Determination of Hemicellulose
II. 11. Determination of Fructose and Inulin
II. 12. Determination of Pectic Substances
II. 12. Determination of Crude Fibre
II. 13. Determination of Pyruvic Acid
II. 14. Determination of Amylose in Flour by a Colorimetric Assay
III. PHYTOESTROGENS ANALYSIS METHODS

4
5
6
6
9
17
17
19
20
21
22
23
24
25
26
26
27
28
31
32
33
35

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

elektrode detector
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
III. 3. The analysis of the dietary fibre components
III. 4. Correlation between NSP constituent sugars of plants and plant lignan
metabolites

37

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

43

47
51

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.

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.
5

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 3,5dinitro-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
6

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.

Test Filtrate
Blank Filtrate
Na2CO3
F-C

Test1
2.00
----5.00
1.00

Test2 Test3 Blank

2.00
2.00
--------- ----2.00
5.00
5.00
5.00
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 Soln
Purified Water
Na2CO3
F-C

Std 1
0.05
1.95
5.00
1.00

Std 2 Std 3 Std 4

0.10 0.20 0.40
1.90 1.80 1.60
5.00 5.00 5.00
1.00
1.00 1.00

Std 5 Std Blank

0.50
0.00
1.50
2.00
5.00
5.00
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.

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- betacellobiosidase; beta-glucosidase; Xylan 1,4-beta-xylosidase.
Description: Typically off-white to tan amorphous powders, or liquids dispersed in foodgrade 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

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 2000ml 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.
10

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

11

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 2000ml 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.
12

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.

13

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 AzoWAX, 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.

14

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 testtubes (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.
15

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
2. Food or Feed A containing Trichoderma sp. xylanase (SA)
3. SA + 70 mU xylanase (in the assay)

0.000
0.859
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.

16

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.

17

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.

18

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.

19

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).
20

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.

21

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.

22

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.

23

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.

24

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.

25

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.

26

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.
27

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.

28

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%.

29

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

30

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.

31

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 )

Weight of the sample

* 100

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.

32

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 alphaamylase and amyloglucosidase.
The amount of glucose produced was then enzymatically determined using a glucose testcombination 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.

33

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.

34

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.

OH

HO
O

HO

HO
O
O
O
b
OCH
a

OH
OH

HO

CH

HO
OH

O
OH

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

35

OH
OCH

HO
OH
HO
OH
HO

O
O
O

OCH

HO

HO

OH
OH

Figure 2. Secoisolariciresinol diglycoside

OH

HO
O

HO

HO
O
O
O
b
OCH

OH

OH

OH
OH

HO

OH
OH

O
OH

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

diethylstilbestrol, d) estriol

36

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 mobile phase, internal standard and
potentials of working electrodes of the coulometric detector, have been chosen.
Selection of the mobile phase. Ten different eluents (E1 - E10) were tested as mobile
phases (Table 1). For their preparation 4 different organic substances: ethanol, methanol,
tetrahydrofuran and acetonitril were used. The optimal retention time (RT) of daidzein and
genistein in the column and the optimal chromatograms obtained from standard solutions
were determined by using the mobile phase E10 (pH 2.6). Also, using this mobile phase, the
isoflavones were eluted isocratically from the column within 11 minutes and a minimum flow
37

rate (0.8 ml/min) was noticed, which contributes to the efficiency of the analysis (less
chemicals are needed).
Table 1. Different eluents which were tested as mobile phases
Eluents
EtOH
5 mmol
H2O
MeOH
THF
ACN
NaAc
ml
E1

6.8
500
500

E2

3.6
450
550

E3

6.8
500

88.24

E4

6.8
500

17.65

E5

6.8
500

10.00

E6

6.8
500

167
E7

6.8
500

300
E8

1.8
500

321
E9
333.3
1.8
500

8.00

E10
333.3

500

8.00

E11
333.3

500

7.8

pH

Flow rate,
ml/min

4.8
4.8
4.5
4.5
4.5
4.5
4.5
4.5
3.0
2.6
3.0

1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
0.8
0.8
0.8

Selection of the internal standard. Various phenolic compounds, such as -estradiol,

bisphenol A, estriol, vanilin, ferulic methylacetate ester, ferulic acetate and vanilin acetate,
were tested as internal standards. The retention time (RT) of these substances are presented in
Table 2.
Finally estriol was chosen for the internal standards due to its electrochemical behaviour
similar to that of daidzein and genistein and its RT in between the analysed isoflavones (8.8
minutes).
Additionally estriol is an endogenic mammalian hormone and does not occur naturally in
plant products.
Table 2. The retention times of internal standards and analyzed substances
Internal standards
RT, min
Analyzed substances
-estradiol
35.3
Daidzein
Bisphenol A
23.4
Genistein
Estriol
8.8
Vanilin
6.8
Ferulic methylacetate ester
6.4
Ferulic acetate
5.9
Vanilin acetate
5.5

RT, min
7.1
10.4

Selection of the potentials of the working electrodes. In oder to get the voltammograms,
the working elektrode of the first cell was set at +200 mV and the potential of the second
elektrode was changed in increments of 50 mV from +150 mV till +500 mV.
The hydrodynamic voltammograms were established by plotting the signals of daidzein,
genistein and estriol agains the potential applied on the second working elektrode (Figure 4).

38

Peak height, nA

1200
1000
800

daidzein
estriol
genistein

600
400
200
0
100 150 200 250 300 350 400 450 500 550

Potential, mV
Figure 4. Hydrodynamic voltammograms of daidzein, genistein and estriol
At potentials lower than +200 mV against electrodes neither the internal standard nor the
isoflavones were oxidized. By increasing the potentials of the second electrode, daidzein and
genistein started to oxidize and at the potential +350 mV also estriol started to oxidize.
The biggest peak areas of all three compounds in the chromatogram were noticed when
potentials ranged from +400 mV till +500 mV.
Considering the hydrodynamic voltammograms, potentials of +350 mV (channel 1) and
+500 mV (channel 2) were chosen for the detection of daidzein, genistein and estriol.
Determination of calibration curves and detection limits. Under chosen chromatographic
conditions a linear correlation between the signals in channel 2 and the concentration of
daidzein and genistein in the working standard solutions was found.
The calibration curves for analyzed compounds were calculated with the linear regression
equation and the correlation coefficients (R), which represent the precission of the
coulometric detector to analyse the substances, were determined.
In all cases R was higher than 0.99 (when daidzein concentration is in the range from 6.5
to 262 g/l, R = 0.99 5 and when genistein from 7. to 2 4 g/l, R = 0.9973). The detection
limits were 0.5 pg daidzein and 1.0 pg genistein.
This shows that a coulometric dual elektrode detector can be used for the separation of
daidzein and genistein and the determination of their quantities.
Method for the determination of the phytoestrogens: matairesinol, daidzein, genistein
and formononetin. The application of the above method for the determination of daidzein,
genistein was extended for other phytoestrogens, such as matairesinol and formononetin,
determination. For this, different internal standards and mobile phases have been analyzed.
Selection of mobile phase. By using the mobile phases E1 - E10, it was not possible to
obtain good isocratically separation of analyzed substances. Therefore a new mobile phase
E11, in which in compare with E10 the concentration of tetrahydrofuran was decreased and
the pH was increased till 3.0 (Table 1), was tested. These changes in E11 allowed to extend
the RT of analysed phytoestrogens and separate without interference these compounds in one
chromatogram.
The selection of the internal standard. In addition to the phenolic compounds described
above, cumestrol and biochanin were used in the experiment. The determined RT of internal
standards and analyzed substances are presented in Table 3.
Estriol was again chosen as internal standard due to its eluation behavior between the
analy ed compounds in the empty part of the chromatogram.
39

The determination of the calibration curves. A linear correlation was determined

between the peak areas and the concentration of matairesinol (14.2 - 42 g/l), daid ein ( 4.6
- 46 g/l), genistein ( 3.6 - 36 g/l) and formononetin (24 - 240 g/l) in the working
standard solutions. For the analyzed substances a linear regression equation of calibration
curves and correlation coefficients were calculated: for matairesinol R=0.9827; daidzein
R=0.9994; genistein R=0.9721 and formononetin R=0.9386.
In all cases R was higher than 0.93, so the coulometric dual elektrode detector could be
used for the separation of matairesinol, daidzein, genistein and formononetin and as well for
the determination of their quantities.
Table 3. The retention times of internal standards and analyzed substances
Internal standards
RT, min
Analyzed
RT, min
substances
-estradiol
36.4
Matairesinol
9.5
Cumestrol
35.0
Daidzein
11.5
Bisphenol A
25.1
Genistein
15.1
Estriol
13.5
Formononetin
20.6
Vanilin
9.4
Vanilin acetate
8.5
Ferulic acetate
8.2
Ferulic methyl acetate ester
7.9
Biochanin
7.4
Daidzein and genistein in soy beans. To apply the developed HPLC method for the
qualitative and quantitative analysis of analyzed phytoestrogens in soy beans, two methods of
sample preparation described in literature were tested.
The result of the investigations (Figure 5) showed that the levels of phytoestrogens were
lower by using only sample extraction in compare with the combination of sample extraction
and acid hydrolysis: 7.3 - 10.6% less daidzein and 11.9 - 37.9% less genistein. Therefore the
method for sample preparation by using simultaneously extraction and acid hydrolysis was
chosen for phytoestrogens analysis.
During these procedures phytoestrogen conjugates are converted into their respective
aglycons. The results showed that using the proposed method, relative high concentrations of
phytoestrogen aglycons were formed. Therefore low sample amounts can be used for analysis,
which cause a decreased disturbance in the chromatograms of the sample matrix.
Additionally, by using this method of sample preparation the quantities of phytoestrogens
can be determined in soy beans without defatting and a cleaning up procedure.
The analysis of phytoestrogens showed that the quantities of maitairesinol and
formononetin in tested soy beans were less then the detection limits for these substances, and
under chosen chromatographic conditions the peaks of maiteresinol and formononetein could
not be seen in the chromatograms. Therefore by using the developed method, matairesinol,
daidzein, genistein and formononetin can not be quantified in one chromatogram.
It was shown that the recovery of daidzein and genistein determined with standard
solutions and the standard addition method both depends on the matrix of the food and that
isoflavones behave very similar to the internal standard. By analyzing three different soy bean
samples (each three times) a recovery of 81.9 7.4. for estriol (internal standard) was
found. This value was adopted as recovery for daidzein and genistein. It could be mentioned
that the recovery for these substances determined by using the developed method in compare
with other HPLC methods is reasonably high.

40

Daidzein

g/kg
1.5

extraction

extraction and
hydrolysis

0.5
0
1

Genistein

g/kg
2.5

extraction

2
1.5
1

extraction and
hydrolysis

0.5
0
1

Figure 5. The quantities of daidzein and genistein determined by using different methods
of sample preparation
The results on the precision of HPLC analysis are given in Table 4.
The repeatability was calculated from the three independent single test results, obtained
on the same test material using the same equipment. It was observed that in the concentration
range from 1.54 to 1.94 g daidzein/kg, and 2.04 to 2.40 g genistein/kg, the coefficients of
variation of repeatability are less than 12% and 11%, respectively.
Therefore the HPLC method with coulometric dual electrode detector is enough precise
and could be used for the simple and rapid determination of daidzein and genistein in soy
beans.
The following advantages of the developed method should be mentioned:
- Relative low quantities of chemicals used, because the flow rate of the mobile phase
[ethanol/tetrahydrofuran/buffer solution (sodium acetate/ trichloroacetic acid), 396/9/595,
v/v/v, pH 2.6] for substances separation on the column is low, only 0.8 ml/min.
- Daidzein and genistein were isocritically eluted from the column within 11 minutes with
the rapid chromatographic analysis.
- Simple identification and quantification of daidzein and genistein, because using chosen
potentials of working electrodes: +350 mV (channel 1) and +500 mV (channel 2) only
analyzed substances are oxidized at the second working electrode.

41

- The method is sensitive and precise for the qualitative and quantitative determination of
daidzein and genistein in one chromatogram. The recovery for these substances is 81.9
7.4, and the coefficient of variation of repeatability is less than 12%.
Table 4. Statistical results
Daidzein

Genistein

Parameters
Average mean,
g/kg
Repeatability
standard deviation
sr, g/kg
Coefficient of
variation of
repeatability, %

Samples
1

1.79

1.54

1.94

2.38

2.04

2.40

0.10

0.17

0.12

0.11

0.22

0.18

5.59

11.04

6.19

4.62

10.78

7.50

The influence of technological parameters on the content of phytoestrogens in soy

beans. The results of the investigation of daidzein and genistein content in different genotypes
of soy beans, untreated and heated to 100oC and 280oC, are summarized in Table 5.
Results showed that the content of daidzein in soy beans ranged from 1.36 g/kg to 2.10
g/kg. The content of genistein were slightly higher from 1.83 g/kg till 2.63 g/kg. It should
be mentioned that the content of tested phytoestrogens in soy beans of various genotypes
grown in Lithuania are slightly higher than those mentioned in literature. According to the
investigation it can be assumed that the genotype did not influence the content of daidzein and
genistein in soy beans.
The investigation of the thermostability of daidzein and genistein is also important
because heat treatment of soy beans is performed to eliminate enzyme inhibitors and various
soy products are therefore processed at higher temperatures. Therefore three sorts of soy
beans, untreated, heated at 100oC for 30 minutes and at 280oC for 5 minutes were analyzed to
investigate the influence of temperature on the content of phytoestrogens. Results showed that
heat treatment did not alter the daidzein and genistein content in soy beans. The deviations
between the values at different temperatures could be due to the inhomogeneity of the
powdered soy beans caused by milling. Therefore, these compounds are not decomposed in
food prepared by higher temperatures. These results also correspond to literature mentioned
that isoflavones are thermostabile phytoestrogens.

Table 5. The influence of temperature on the contents of daidzein and genistein in soy
beans
Samples of soy beans Time and temperature
Daidzein, g/kg
Genistein, g/kg
of heat treatment
Sample 1
Untreated (25oC)
1.790.10
2.380.11
1.930.19
2.630.34
100oC, 30 min
o
1.720.10
2.460.14
280 C, 5 min
Sample 2

Range

1.72-1.93

2.38-2.63

Untreated (25oC)
100oC, 30 min
280oC, 5 min
Range

1.540.17
1.360.10
1.420.25
1.36-1.54

2.040.22
1.830.05
1.970.37
1.83-2.04

42

Table 5 continue. The influence of temperature on the contents of daidzein and genistein
in soy beans
Samples of soy beans Time and temperature
Daidzein, g/kg
Genistein, g/kg
of heat treatment
Sample 3
Untreated (25oC)
1.940.12
2.400.18
100oC, 30 min
1.800.13
1.900.08
280oC, 5 min
2.100.16
2.400.19
Range
1.80-2.10
1.90-2.40

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
A carbonate-phosphate buffer solution with trace elements was held in an anaerobic
chamber for 2 days prior to fermentation. Feces were collected from three healthy human
volunteers, who suffered no digestive disease, and had not received antibiotics for at least 3
month. Freshly passed feces were immediately taken in an anaerobic chamber, pooled, and
homogenized with an equal weight of culture medium using a Waring blender. The slurry was
diluted to 16.7% (v/v) with the culture medium, filtered through a 1 mm sieve, and used
immediately as inoculums.
0.1 g of a analyzed food sample was weighed into 50 ml glass vials, and 10 ml of the
inoculum was added and stored in a 30C anaerobic chamber. The vials were sealed with
rubber stoppers and shaken in a 37C water bath for 24 h. The fermentation was stopped by
plunging the vials into iced water, after which the vial contents were freeze-dried. Dublicate
incubations were carried out for each sample. Also, dublicate blanks, containing only culture
medium and inoculum, were incubated for 0 and 24 h.
Quantitative analysis of END and ENL by HPLC with coulometric elektrode array
detector. The HPLC system consisted of a pump model 580 (ESA, Chelmsford, USA) and an
automatic injector model 540 (ESA, Chelmsford, USA). An intersil ODS-3 (GL Science Inc.,
Japan) column (3.0150 mm, 3.3 m, 9LI 500 10) in combination with precolumn Quick
Releate C18 (Upchurch Scientific Inc., WA) and for detection Coulochem Electrode Array
Detector (ESA, Chelmsford, USA) equipped with a eight electrode cell were used.
The freeze-dried incubated samples were weighed (approximately 20 mg) and 500 l of
water and 10 l of 6 M HCl was added. The samples were extracted twice with 5 ml of
diethyl ether. The extracts were combined and evaporated to dryness under N2 flow. The
samples were dissolved in 500 l MeOH and subsequent diluted with the mobile phase.
The mobile phase consisted of 20% solution B (50 mM NaAc, pH 5/MeOH/ACN,
40/40/20, v/v/v) and 80 solution A (50 mM NaAc, pH 5/MeOH, 80/20, v/v).
Prepared sample extracts or standard solutions (END: 7.0 to 350 g/l, ENL: 10.834 to
541.7 g/l) were injected, the compounds were separated (flow rate of 1.2 ml/min) on the
reversed phase column and were detected at +180 mV (channel 1) till +720 mV (channel 8).
The END and ENL were quantified using calibration curves and by evaluation of their
quantities determined in a blank sample. The precision of the method was determined using
END and ENL standard solutions. Duplicate samples were analyzed for each incubation
sample and the standard deviation was calculated.
The quantities of END and ENL produced in 24 h fecal blanks were subtracted from the
results obtained from the incubations carried out with the food samples.

43

The mammalian lignan production of enterodiol and enterolactone from various

plant foods using in vitro fermentation.
The quantative results of enterodiol (END) and enterolactone (ENL) produced during the
24 h fecal incubation of lignans of berries, vegetable and cereal products are summarized in
Tables 6, 7 and 8.
Berries. The mammalian lignan production from various berries ranged from 7.8 to 382.8
nmol/g, for END from 0 to 327.2 nmol/g and for ENL from 7.8 to 55.6 nmol/g (Table 6).
By comparing different kind of berries on a wet (as-is) basis, the cloudberry (382.8
nmol/g), raspberry (227.9 nmol/g), and strawberry (150.8 nmol/g) were the highest lignans
producers. The lignan production from other berries, such as lingonberry, blueberry and
cranberry was 12 to 18 times lower than that from the cloudberry.
The buckthorn and crowberry did not appear to be good producers, because the total
lignan quantity was lower than 10 nmol/g.
It has been noticed that of the higher quantities of total lignans (in cloudberry, raspberry,
strawberry), more END was produced than ENL and the ratios of END to ENL ranged from
6:1 to 5:1. In berries in which lower quantities of total lignans were found, the levels of ENL
were higher than END.
The reason for it could be the particularities of mammalian lignan precursors in different
berries and END oxidation to the more stable ENL.
It can be affirmed that by longer fermentation (48 h or 72 h) this ratio of END to ENL
could be different, so the total lignan amount could better characterize the mammalian lignans
production.
By knowing that matairesinol can be transformed by the bacterial enzymes directly to
ENL through reactions involving hydrolysis, dehydroxylation and demethylation, the higher
levels of ENL rather than END in a majority of the berries suggest that matairesinol may be
more abundant present.

Table 6. Mammalian lignan production from different berries

Analyzed berries
Cloudberry
Raspberry
Strawberry
Cranberry
Blueberry
Lingonberry
Chokeberry
Blackcurrant
Crowberry
Buckthorn
Minimum
Average
Maximum

Enterodiol, nmol/g
Wet basis
327.2
191.9
124.5
3.2
6.3
0
1.8
5.0
0
0
0
66.0113.9
327.2

Enterolactone, nmol/g

Dry basis Wet basis

1817.5112
55.6
1066.0 42
36.0
803.3 4.6
26.3
23.2 2.2
30.4
34.9 1.8
26.8
0
21.6
12.6 2.2
14.2
33.3 1.3
8.9
0
8.6
0
7.8
0
7.8
379.1636.9 23.615.0
1817.5
55.6

Total lignans, nmol/g

Dry basis Wet basis Dry basis

308.7 36
382.8
2126.2
199.6 17
227.9
1265.6
169.4 0.6
150.8
972.7
217.2 3
33.6
240.4
148.7 3.5
33.1
183.6
154.5 0.5
21.6
154.5
101.7 4.1
16.0
114.3
59.6 1.3
13.9
92.9
47.8 0.4
8.6
47.8
45.9 1.8
7.8
45.9
45.9
7.8
45.9
145.384.4 89.6126.6 524.4725.1
308.7
382.8
2126.2

44

Vegetables. The quantities of mammalian lignans produced from different vegetables

ranged from 10.5 to 91.2 nmol/g, END from 5.8 to 45.7 nmol/g and ENL from 4.7 to 45.5
nmol/g (Table 7).
Table 7. Mammalian lignan production from different vegetables
Analyzed
Enterodiol, nmol/g Enterolactone, nmol/g Total lignans, nmol/g
vegetables
Wet basis Dry basis Wet basis Dry basis Wet basis Dry basis
Potato
45.7
190.513.9
45.5
189.7 13.5
91.2
380.2
Garlic
42.9
214.4 2.2
23.3
116.5 1.5
66.2
330.9
Zucchini
33.6
239.9 32.9
26.8
191.5 35.4
60.4
431.4
Broccoli
24.9
177.6 1.1
17.4
124.0 0.3
42.3
301.6
Red paprika
15.3
153.4 3
20.0
199.8 4.1
35.3
353.2
Red cabbage
10.1
111.6 2.3
22.1
245.4 1.2
32.2
357.0
16.0
114.4 0.2
16.0
114.6 0.2
32.0
229.0
Pumpkin
Onion
19.2
137.3 0.3
10.2
72.9 0.2
29.4
210.2
Carrot
12.7
106.1 0.8
13.8
115.0 0.4
26.5
221.1
Rape
5.8
55.1 4.9
4.7
44.4 2.7
10.5
99.5
Minimum.
5.8
55.1
4.7
44.4
10.5
99.5
22.614.2 150.056.3 20.011.1 141.462.8 42.623.5 291.499.7
Average
Maximum
45.7
239.9
45.5
245.4
91.2
380.2

Considering individual vegetables on a wet basis, potatoes produced the highest quantity
of total lignans (91.2 nmol/g). Garlic (66.2 nmol/g), zucchini (60.4 nmol/g) and broccoli (42.3
nmol/g) were the other good producers of lignans in this food product group.
Rape in compare with other vegetables was the lowest lignan producer (10.5 nmol/g).
In most of the vegetables (although the END to ENL ratio was close to 1) slightly more
END was produced than ENL with the exception of red cabbage where the level of ENL was
higher than the level of END.
Considering this, the larger quantities of END rather than ENL produced from vegetables
may be a reflection of their higher concentration of the other lignan precursor
secoisolariciresinol coupled with a low rate of conversion of END to ENL.
Cereal products. From cereal products the production of mammalian lignans ranged from
78.3 to 321.9 nmol/g depending on the product type, END from 8.7 to 149.3 nmol/g and
ENL from 64.4 to 278.3 nmol/g (Table 9).
Cereal brans were the highest producers of mammalian lignans (294.1 - 321.9 nmol/g).
The lignan production from bran was 2.2 - 2.3 times higher than that from whole flour of the
same kind of cereals.
It is known that dietary fibre of cereals with their associated substances (as
phytoestrogens) are distributed in the outer layer of grains.
The higher quantities of dietary fibre were found in the aleurone and pericarp layers of
grains, and during milling end up in the bran fraction.

45

Table 8. Mammalian lignan production from different cereal products

Analyzed cereal
Total lignans, nmol/g
Enterodiol, nmol/g
Enterolactone, nmol/g
products
Wet basis Dry basis Wet basis Dry basis Wet basis
Dry basis
Rye bran
34.6
40.2 0.2
278.3
323.6 1.1
312.9
363.8
Wheat bran
141.3 164.3 2.6
180.6
210.0 0.1
321.9
374.3
Oat bran
149.3 173.7 3.3
171.4
198.9 0.8
320.7
372.6
Barley bran
121.5 141.2 6.7
172.6
200.7 4.8
294.1
341.9
Whole wheat
119.3 138.7 5.7
111.3
129.4 1.5
230.6
268.1
flour
Whole rye flour
66.1
76.8 5.1
76.8
89.2 4.9
142.9
166.0
Oat whole flour
65.9
76.6 3.5
71.8
83.5 1.2
137.7
160.1
Barley whole
62.5
72.7 4.2
64.4
74.9 3.6
126.9
147.6
flour
Wheat flour
8.7
10.1 0.2
69.6
80.9 1.1
78.3
91.0
Minimum
8.7
10.1
64.4
74.9
78.3
91.0
85.4749.3 99.3757.3 132.9872.9 154.5784.8 218.497.6 253.93113.5
Average
Maximum
149.3
164.3
278.3
323.6
321.9
374.3
Flaxseed
4350.8 5059.1 41 6082.4
7072.6 41 10433.2
12131.7
From most of analyzed cereal products slightly more ENL was produced than END, and
with rye bran and wheat flour the ratios of ENL to END were even 8:1. It seems that in this
food product group matairesinol is a quantitatively more important precursor than
secoisolariciresinol.
Comparison of enterodiol and enterolactone production from different products. The
results of investigation showed that the amount of mammalian lignans produced from
flaxseed (10433.2 nmol/g) was of another magnitude than in other food product groups (Table
8), where vegetables (42.6 nmol/g) and berries (89.6 nmol/g) produced less than cereal
products (321.9 nmol/g). A larger amount of END (5059.1 nmol/g) rather than ENL (7072.6
nmol/g) produced from flaxseed was found. Similar results of mammalian lignans production
from flaxseed were noticed in literature.
On a wet basis, the total lignan production from flaxseed was 27 times higher than that
from the berries, 32 times higher than that from the cereal products and 114 times higher than
from the vegetables. Cloudberry, raspberry, strawberry, wheat bran, oat bran, rye bran, barley
bran, whole wheat flour, rye and whole rye flour were the other good producers of lignans,
but still there magnitudes were lower than flaxseed.
Considering the ratio of END to ENL, it can be suggested that in most of the cereal
products, and berries (except cloudberries, raspberries, and strawberries) matairesinol is one
of the most important mammalian lignan precursor. Vice versa in vegetables, other lignan
precursors such as secoisolariciresinol may be more abundant present.
It can be noticed that berries and vegetables did not appear to be good producers because
of their high moisture content (up to 96%) in compare with cereal products (about 14%).
However, when the data are expressed on a dry matter basis, eliminating the moisture effect,
in most of the berries and vegetables the highest amount of these substances could be
determined. On a dry matter basis, the total amount of lignans produced from such berries as
cloudberry (2126.2 nmol/g), raspberry (1265.6 nmol/g), strawberry (972.7 nmol/g) and such
vegetables as zucchini (431.4 nmol/g), potato (380.2 nmol/g), red cabbage (357.0 nmol/g) and
red paprika (353.2 n mol/g) were higher or similar than that from cereal brans (341.9 374.3
nmol/g).

46

How much the different foods contribute to the total lignan production in humans depends
on their level of use in the diet. Flaxseed produced the highest quantity of mammalians
lignans (that shows also this investigation), but currently its dietary contribution is low
because it is not widely used for food since it contains a high concentration of toxic
cyanoglycosides. In the Western type of diet whole grain foods, fruits, berries and vegetables
due to their higher level of intake are the main sources of lignans.
In Lithuania cereal products are the most important sources of mammalian lignan
precursors. It needs to pay attention to increase in the diet the intake of whole grain foods,
especialy rye products. Among grains, rye has a relatively high content of important dietary
fiber components combined with other bioactive compounds, such us plant lignans, which are
termostable and are not lost during baking.
Although fresh berries and vegetables, on the average, produce lower quantities of
mammalian lignans, among them there are some with high mammalian lignan producing
capability, such as cloudberry, raspberry and strawberry, potato and zucchini, which could
contribute to reduce the risk of cancer.
No data were found in literature as of the ability of berries to produce mammalian lignans.
Although the data on lignan production of cereals and vegetables differ slightly with the data
available from literature tendencies seem to be the same with this findings. The reason could
be that intestinal bioconversion of plant lignans to mammalian lignans is subject to large
interindividual variation, probably due to differences in bacteria microflora and also the use of
antibiotics.
Concerning the latter antibiotics we recall that our human volunteers in the experiment did
not received antibiotics for at least 3 month. It is probably that the diet-host interactions are
largely determined by genetic variation between individuals, and also by dietary differences
with respect to the content and type of non-digestible food components which provide
substrates for the gut microflora. Therefore, much research is still needed to understand plant
lignan metabolism and absorbtion in humans, to verify their role and mechanisms in desease
prevention and to create recommendations for their intake.

III. 3. The analysis of the dietary fibre components

The analysis of the dietary fibre components: NDF (neutral detergent fibre), ADF (acid
detergent fibre) and ADL (acid detergent lignin) was carried out by the Van Soest and Wine
detergent method. The NDF fraction contains hemicelluloses, cellulose, cutin and lignin, the
ADF cellulose, cutin and lignin, and the ADL cutin and lignin.
The hemicelluloses and cellulose contents were calculated as difference between NDF
and ADF fractions, and ADF and ADL fractions, respectively. Using this method lignin could
be determined together with cutin by means of the quantity of the ADL fraction.
Before extraction of the dietary fibre components, starch was removed enzymatically with
-amylase and amyloglucosidase. For NDF analysis, starch-free material was extracted with a
neutral detergent, and the residue was filtered and weighed after washing, drying and ashing.
By sample extraction with acid detergent hemicelluloses were removed and the ADF fraction
was precipitated. The quantities of lignin and cutin were determined by extraction of the ADF
fraction with sulphuric acid.
-glucan was determined enzymatically according to the McCleary and Cood method,
using a Megazyme kit (BBG 3/96, Ireland). Samples were suspended in sodium phosphate
buffer solution (pH 6.5) and digested sequentially with lichenase and -glucosidase enzymes.
The -glucan was hydroly ed to -glucan-oligosaccharides by lichenase and oligosaccharides
were hydroly ed to glucose by -glucosidase. By adding glucose-oxidaze-peroxidase reagent
chinoidic colors are formed. Light absorption of the colored solution is measured by a
spectrophotometer at 5 0 nm, which values correlate with the -glucan quantity.
47

The constituent sugars of NSP were measured according to the modified H. N. Englyst
and J.H. Cummings method by GLC.
In the modified method, starch including resistant starch that is resistant to gelatinization
in boiling water, was dispersed with dimethyl sulphoxide and removed from the sample
matrixes enzymatically (with -amylase and amyloglucosidase). NSP was precipitated with
ethanol and then hydrolyzed with sulfuric acid for 2 h to monosaccharides. The constituent
sugars of NSP: arabinose, xylose, mannose, glucose and galactose were determined by GLC.
Alditol acetates prepared by using N-methylimidazole to catalyze the acetylation were used
for GLC determination of released monosaccharides. The total NSP were expressed as sum of
constituent sugars. GLC was performed by using a gas chromatograph of Shimadzu GC 17
AAF FID with a flame ionization detector (FID) and an analytical column RTX 50 (30 m0.25
mm, 0.25 m) and an automatic sample injector AOC-17. The column was maintained at 230
C and the injector and detector were kept at 300 C. The carrier gas (helium) flow rate was
1.67 ml/min (30 cm3/min). The contents of monosaccharides were determined by using a
compatible computer provided with CLASSGC10 Chemstation software.
Complex analysis of dietary fibre of cereals grown in Lithuania. The quantities of
hemicelluloses, cellulose, lignin + cutin also -glucan determined in Lithuanian wheat, rye,
barley and oat are presented in Figure 6.
40

35
30
lignin + cutin

25

-glucan

20

cellulose

15

hemicelluloses

10
5
0
rye

wheat

barley

oat

Figure 6. Dietary fibre fractions of cereal

The highest quantities of total dietary fibre and most of its fractions were found in oats. In
this type of cereals the hemicellulose and cellulose content was, respectively, 1.3 and 2.7
times higher than in barley, 1.6 and 5.5 times higher than in rye and 1.8 and 5.1 higher than in
wheat. The lignin and cutin content together in oats was from 1.8 to 2.4 times higher in
compare with other cereals.
Among the tested cereals, barley was specific for the highest content of -glucan (5.1
0.4 % dm), which was 1.4 times higher than in oats, 2.8 times higher than in rye and 6.4 times
higher than in wheat. In compare with rye and wheat the content of hemicellulose (11.2 0.7
% dm) and cellulose (4.9 0.8 % dm) in barley were also about 1.4 and 2 times higher,
respectively.
In rye only the content of -glucan (1.8 0.3 % dm) was higher than in wheat (0.8 0.3
% dm). The quantities of hemicellulose, cellulose and lignin in rye and wheat were
equivalent.
48

By comparing different genotypes of cereals it was noticed that the variation in

composition of dietary fibre fractions depends not only on the kind of cereals but also on the
genotype.
No significant relationships have been determined between the amounts of mammalian
lignans produced from cereal products and the amounts of total dietary fibre and their
fractions. Obviously, the lignan production more depends on dietary fibre specific
composition and structure, which varies between different kind of cereals, than on the amount
of dietary fibre.
Non-starch polysaccharides and their constituent sugars in plant products. The
results of the investigation of non-starch polysaccharides (NSP) of different berries,
vegetables and cereal products, flaxseed and soy beans are presented in Figures 7, 8, 9.
Berries. For berries, the total NSP content ranged from 1.08 to 2.88 g/100 g. The main
constituent sugar was glucose, which content varied from 0.67 to 1.99 g/100 g. The contents
of other constituent sugars were substantially lower: xylose (0.11 - 0.63 g/100 g), arabinose
(0.08 - 0.30 g/100 g), galactose (0.11 - 0.24 g/100 g) and mannose (0.04 - 0.14 g/100 g). The
following proportion of NSP components in berries was found: 49.1 - 69.1 % glucose, 7.3 37.3 % xylose, 4.7 - 16.2 % arabinose, 5.9 - 11.1 % galactose and 2.4 - 5.6 % mannose
(Figure 7).
%
100
Arabinose

80

Xylose

60

Mannose
40

Glucose

20

Galactose

0
1

Figure 7. The proportion of NSP constituent sugars in berries (1- crowberry, 2raspberry, 3- blueberry, 4- lingonberry, 5- strawberry)
The comparison of different berries showed, that the highest total NSP content was in
crowberry (2.88 g/100 g) and raspberry (2.71 g/100 g). The highest glucose content was in
crowberry and raspberry, arabinose in lingonberry and crowberry, mannose and galactose
also in crowberry, xylose in blueberry and raspberry.
Vegetables. For the analyzed vegetables, the total NSP content varied from 0.29 to 3.39
g/100 g. As in berries, the dominant constituent sugar was glucose (0.11 - 1.87 g/100 g), the
lowest level was of xylose (0.03 - 0.20 g/100 g). The proportion of NSP components in
vegetables was the following: 23.9 - 55.2 % glucose, 13.3 - 53.5 % galactose, 4.0 - 24.8 %
mannose, 4.1 - 27.4 % arabinose and 2.7 - 19.6 % xylose (Figure 8).
The highest NSP content was determined in garlic (3.40 g/100 g), and the lowest in red
paprika (0.28 g/100 g). Among the analyzed vegetables, garlic had the highest amounts of
glucose (1.87 g/100 g) and mannose (0.84 g/100 g), broccoli the highest amount of xylose
(1.41 g/100 g) and arabinose (2.30 g/100 g), and onion the highest amount of galactose
(3.78 g/100 g).

49

100

80
Arabinose
Xylose

60

Mannose
40

Glucose
Galactose

20
0
1

Figure 8. The proportion of NSP constituent sugars in vegetables (1- garlic, 2

broccoli, 3- onion, 4- potato, 5 zucchini, 6 - red paprika )
Cereal products. The NSP content of cereal products ranged from 3.10 to 15.38 g/100 g.
The proportion of NSP components in cereal products was the following: 29.1 - 68.3 %
glucose, 12.1 - 43.1 % xylose, 12.4 - 22.4 % arabinose, 1.9 - 20.3 % galactose and 3.6 - 7.1 %
mannose (Figure 9).
100

80
Arabinose
Xylose

60

Mannose
40

Glucose
Galactose

20
0
1

Figure 9. The proportion of NSP constituent sugars in cereal products (1- rye bran, 2whole barley flour, 3- whole oat flour, 4- wheat flour)
The highest total NSP content was in rye bran (15.38 g/100 g), also the highest
arabinose and xylose content (3.45 g/100 g and 6.63 g/100 g, respectively). The reason could
be that during cereal processing the outer layers of the grains, containing the highest amount
of dietary fibre, are passed to the rye bran fraction.
Therefore, the lower amounts of NSP are passed to the flour, especially with low ash
content. For example, wheat flour contained the lowest amount of total NSP (3.10 g/100 g)
and specific proportion of constituent sugars: the highest amount of galactose and the lowest
amounts of other monosaccharides. Compared to wheat flour, whole grain flour (whole barley

50

and oat flour) produced using simple milling schemes contained higher amounts of NSP, also
xylose, glucose and arabinose.
Comparison of NSP composition of various plant foods. For this, additional
investigation of flaxseed and soy beans which according to literature have the highest amount
of phytoestrogens, was carried out.
Compared with other analyzed plant foods, flaxseed and soy beans next to rye bran
contain the highest amount of NSP. Also, galactose content was the highest in these products,
especially in soy beans. The amount of this monosaccharide in soy beans and flaxseed was,
respectively, 19.3 and 7.8 times higher - than in berries, 8.8 and 3.5 than in vegetables and
7.4 and 3.0 than in cereal products. Mannose content in flaxseed (0.15 g/100 g) was lower
than in cereal products and garlic. Vice versa, soy beans as garlic contained the most of this
monosaccharide (0.68 g/100 g).
Among analyzed plant food groups, the total NSP contain in cereal products was about 6.4
times higher than in vegetables and 4.1 times higher than in berries, however by comparing
these results on a dry matter basis the differences are insignificant.
Considering the NSP composition of different plant foods groups, it can be suggested that
the main NSP constituent sugars in berries are glucose and xylose, in vegetables glucose,
galactose and mannose, and in cereal products glucose, xylose, and arabinose. This confirms
that in berries higher quantities of cellulose are present, in vegetables cellulose, galactans
and mannans, and in cereal products cellulose, -glucan and arabinoxylans. The analysis of
NS constituent sugars of flaxseed and soy beans shows that cellulose, -glucan,
arabinoxylans and galactans are main NSP components in flaxseed, and cellulose, galactans
and arabinans in soy beans.
Analysis of different types of sugars also showed that there are differences between
investigated plant food groups. In soy beans, berries and vegetables the amount of hexoses
were respectively 2.9; 3.0 and 5.8 times higher than that of pentoses, therefore in cereal
products and flaxseed the amounts of hexoses and pentoses were almost similar (content of
hexoses compared to pentoses was only 1.2 - 1.3 times higher). These variations could have
influence on the amount of produced mammalian lignans and their precursors in different
plant products.

III. 4. Correlation between NSP constituent sugars of plants and plant lignan
metabolites
By analyzing the NSP composition of selected plant foods and mammalian lignans
produced during their fermentation it was noticed that some correlations existed between
quantities of total NSP, their constituent sugars and mammalian lignans (Table 9).
For berries, an intermediate correlation was found between the total NSP and ENL values,
and a loose correlation between total NSP and the total amount of mammalian lignans.
The studies showed that among the NSP components, the biggest influence on the amount
of lignans has hexoses, especially glucose.
An intermediate correlation was found between the glucose and ENL values and a loose
correlation between the glucose and total amount of mammalian lignans.
On the contrary to ENL, no correlations have been found between NSP, their constituent
sugars and the less stable END.

51

Table 9. Squared correlation coefficient (R2) between total NSP, their constituent sugars
and mammalian lignans
MammaNSP constituent sugars
lian
ara
xyl
man
glu
gal
pen
hex
Total
lignans
berries
END
0.0451 0.1296 0.0009
0.2247 0.0106 0.1762 0.1713 0.2435
ENL
0.0037 0.1691 0.1126
0.6339 0.0558 0.3510 0.5535 0.7264
Total
0.0422 0.1347 0.0024
0.2488 0.0070 0.1891 0.1922 0.2711
vegetables
END
0.0025 0.0350 0.0053
0.4294 0.1729 0.0010 0.2602 0.3615
ENL
0.0285 0.1963 0.0018
0.0318 0.0009 0.0743 0.0110 0.0036
Total
0.0037 0.1101 0.0586
0.2009 0.0583 0.0259 0.0670 0.1278
cereal products
END
0.0052 0.0193 0.0143
0.7425 0.9382 0.0139 0.6444 0.0191
ENL
0.0836 0.0850 0.9841 0.1537 0.8687
0.9710 0.9887 0.6797
Total
0.2709 0.0021 0.9671 0.3724 0.9780
0.9772 0.9610 0.7640
ara arabinose, xyl xylose, man mannose, glu glucose, gal galactose, hex hexose,
pen - pentose
For vegetables, higher correlations between NSP and END were found. The intermediate
correlations were found between the total NSP, glucose and END values. It can be suggested
that hexoses, especially glucose are more important for lignan structure and content in
vegetables and their bioconversion to mammalian lignans.
For cereal products, close correlations were found between the total NSP and the total
amount of mammalian lignans, and also ENL, between pentoses and the total amount of
mammalian lignans, and also ENL, between arabinose, xylose and ENL, between galactose
and END values.
The results showed that there is a correlation between the particularities of fermented food
matrixes and the production of mammalian lignans. Considering the correlations between
glucose and ENL found in berries and between glucose and END found in vegetables, it can
be assumed that in these plant materials lignans are present in a conjugated form as
glucosides. While in cereal products, in which close correlations were found between hexoses
and END as well as between pentoses and ENL, lignans could occur as different glycosides
e.g. not only conjugated with glucose but also with other NSP constituent sugars as xylose,
arabinose. This indicates that pentoses are closely related to the quantities of plant lignans in
cereal products.

52

IV. IMPROVING THE QUALITY OF BREADS WITH WHOLE GRAIN

PRODUCTS DIETARY FIBRE SOURCE

There is a growing amount of evidence that high-fibre diets, especially those

containing cereal fibre, have definite health benefits in reducing the risks of developing
chronic diseases such as diabetes, cancer, coronary heart disease. A high fibre intake is
also positively related to control of obesity and physical gastrointestinal tract disorders. As
the life expectancy of the population is growing in Western countries, health -related issues,
such as functional foods and individual well-being will certainly also be very appealing
topics for the majority of the population in future decades.
Consumers will be interested in buying functional cereal products that will promote
their health and help them avoid becoming overweight. High-fibre cereal products will be
thus asked for and undoubtedly bought. However, as always with food items, the major
criteria for consumer acceptability of cereal products are good flavour and texture. In other
words, consumers expect functional cereal products such as high-fibre breads to have at
least equally good quality attributes as standard wheat bread.
Traditionally, introducing fibre into baked products in such amounts that health
benefits can be expected has meant an unavoidable deterioration of quality, in terms of
both the flavour and texture of cereal products.
However, due to recent studies of the role of fibre in baking, there are now several
alternatives for bakers that overcome problems related to high-fibre baking. Fibre
ingredients with improved technological and flavour properties have been developed
during the last decade.
Also, by employing different process and recipe modifications in high-fibre baking, it
is possible to produce high-quality, high-fibre cereal products with definite health benefits
and consumer acceptability.
Interest in dietary fibre (DF) originates from observations that populations consuming
diets high in DF have reduced incidence of chronic diseases that are common in Western
countries with low-fibre diets. High consumption of DF has been associated with a lower
risk of cardiovascular disease, diabetes, hypertension, obesity, and gastrointestinal
disorders. The physiological mechanisms underlying the health protective properties of DF
have been intensively studied, and a range of functions throughout the gastrointestinal tract
have been elucidated. A large part of the research has concentrated on the effects of soluble
viscous fibre to lower cholesterol or attenuated glucose responses, as reviewed for oat // glucan.
The current definitions of DF allow dietary constituents other than plant cell wall
polymers to be classified as fibre provided that they are shown to have beneficial
physiological functions. Usually only one beneficial effect is needed to fulfill the
definition, but the criteria for substantiation of the effect are not defined. Improved bowel
function (e.g. altered faecal frequency and quality of faeces), fermentability by colon
microbiota and attenuated blood glucose and cholesterol levels are the physiological effects
most commonly associated with increased use of DF.
Dietary fibre can also be classified as cereal based fibre and non-cereal based fibre.
This categorisation is often used in the baking industry. Cereal based fibre includes, for
example, wholegrain flour, resistant starch and bran (made of wheat, oat, rye, barley or
corn). Oat and barley bran are classified as soluble dietary fibre and resistant starch, wheat
and rye bran as insoluble fibre.
Non-cereal based fibres include inulin, psyllium, pea hulls, pea cell wall fibres and
fibres from various fruit and vegetables origins, e.g. sugar beet, citrus fruits, algae, rice and
asparagus. Inulin and psyllium are soluble fibres whereas pea hulls, pea cell walls and

53

different fibres of plant origin are mostly classified as insoluble. Fibre contents of some
common fibre sources are presented in Table 10.
Table 10. Fibre contents of some common fibre sources
Sources of fibre
Total dietary fibre
References
content (%)
Rice bran

20

(Lebesi and Tzia, 2009)

Oat bran

15.2

(Lebesi and Tzia, 2009)

Corn bran

51.05

(Gull et al., 2009)

Rye bran

43

(Kamal-Eldin et al., 2009)

Wheat bran

39.9-53.1

(Kamal-Eldin et al., 2009)

Inulin

92.1

(Collar etal., 2006)

Sugar beet fibre

73.0

(Collar et al, 2006)

Pea hull fibre

80.0

(Collar et al., 2006)

Pea cell wall fibre

35.0

(Collar et al., 2006)

Hull less barley

272

(Jacobs et al., 2008)

Apple pomace

78.2-89.8

(Figuerola et al., 2005)

Orange peel

64.3

(Figuerola et al., 2005)

Grapefruit peel

44.4-62.6

(Figuerola et al., 2005)

Lemon peel

60.1-68.3

(Figuerola et al., 2005)

Asparagus by-products

62-77

(Fuentes-Alventosa et al, 2009)

IV. 1. Nutritional quality of fermented defatted soya and flaxseed flours and
their effect on texture and sensory characteristics of wheat sourdough
bread

Improving the nutritional value of bread with whole grains or other seeds has become
more popular due to documented positive health effects. As plant foods have a number of
biologically active substances that may prevent chronic diseases, it may be appropriate for
that reason to select plant crops with more nutritional valuable components for bread
production. The main sources of biologically active compounds are seeds (flax, sesame),
beans, whole grain cereals, berries, nuts, vegetables, fruits and beverages.
Soya is extremely versatile and is used for new product development, such as breakfast
cereals and snacks, meat analogues and bakery products. Flaxseeds are increasingly being
incorporated into a variety of food products, such as bread, breakfast cereals or used as a
supplement.
Plant products rich in bioactive compounds such as soya products have an unattractive
taste and are generally consumed in limited amounts. Another problem of soya is that wide
application in food processes is unattractive because of its poor digestibility due to the

54

presence of a number of antinutritional factors trypsin inhibitor, agglutinin, phytic acid and
non-digestible oligosaccharides.
However, fermentation can play an important role in producing attractive flavours and
improve the digestibility and bioavailability of nutrients as well.
Numerous fermented soya products appear to have a beneficial health effect and have
therefore been developed. Tempeh, a fermented soya product made from whole soya beans,
has been associated with a better memory related to the fact that it contains high levels of the
vitamin folate, which is known to reduce the risk of dementia.
Fermented soya beverages possess several additional desirable nutritional properties, such
as lowering cholesterol and saturated fat levels, and increasing isoflavone bioavailability.
Flaxseed products enriched with 0.71.4% supplemental fat in the diet of cows can
slightly improve the nutritive value of milk fat for better human health. However, there is a
lack of knowledge about the effect of supplementation of fermented soya and flaxseed flours
on nutritional value and quality of wheat bread. Over the last few years, there has been an
explosion of basic and applied research on antimicrobial peptides produced by lactic acid
bacteria (LAB) and exhibiting bactericidal and/or bacteriostaticmodes of action against
closely related species and even food-borne pathogens. The highly promising results of these
studies underline the potential application of selected bacteriocinogenic LAB strains in the
food industry as starter cultures, co-cultures or bio-protective cultures, to improve food
quality and safety.
On the other hand, fermented foods especially protein-rich foods, e.g. fermented
vegetables and legumes, contain biogenic amines (BAs) that might be significant to food
safety and human health. BAs are formed through the decarboxylation of specific free amino
acids by exogenous decarboxylases released from the microbial population associated with
the raw material.
BAs are of concern in relation to food spoilage, food safety and food intolerance, and
their content in foods should be as low as possible. The most frequent foodborne intoxications
and intolerances, caused by BAs, involve histamine and tyramine. Compared with foods of
animal origin (e.g.milk and meat products), relatively little information is available on the
levels of BAs in vegetable foods and in novel and convenience foods. The objective of this
study was to compare the functional properties of soya and flaxseed flours in different
sourdough fermentation processes for wheat bread production, and to determine the criteria of
fermented products that add to the nutritional and sensory values of the bread. Another
essential point of study was the development of different drying processes for preservation of
plant sourdoughs intended for centralized production.
Materials and methods
Plant materials and microorganisms
Partially defatted flaxseed (Linum usitatissimum L.) and soya (Glycine max L.) flours
were purchased at a pharmacy network in Kaunas (Lithuania) and used as the starting material
for sourdough production.
Wheat flour (type 550D) was obtained from Kauno Grudai Ltd mill (Kaunas, Lithuania).
Characteristics of plant flour and wheat flour used in bread making are reported in Table 11.
Homofermentative Pediococcus acidilactici KTU05-7 (collected from Kaunas University
of Technology, Kaunas, Lithuania) previously isolated from spontaneous rye sourdough
(Digaitiene et al. 2005) was used for fermentation of plant materials. LAB strain was stored at
-80C in MRS broth (CM0359, Oxoid Ltd, Hampshire, UK), supplemented with 20% (v/v)
glycerol. P. acidilactici was cultured at 358C for 72 h in MRS broth with the addition of 39
mmol/l fructose and 20mmol/l maltose. Enumeration of LAB was carried out by plating the
diluted samples onto MRS agar at 30C for 48 h.

55

Table 11. Characteristics of plant flour and wheat flour used in bread making
Wheat
Soya
Flaxseed
Property
flour
flour
flour
Moisture

14.2

8.2

9.8

Protein* (g/100 g d.m.)

13.5

49.2

40.3

Total carbohydrate
(g/100 g d.m.)
Fibre (g/100 g d.m.)

61.8

26.3

26.4

9.8

4.6

11.4

Fat (g/100 g d.m.)

6.1

9.2

Ash (g/100 g d.m.)

0.7

5.6

2.9

Falling number (s)

350

Gluten (g/100 g d.m.)

31.6

Notes: Data are the mean SD of three analyses. d.m., dry matter; * Nitrogen factor 5.7 for
wheat flour and 6.25 for soya and flaxseed flours.
Preparation of fermented plant products
Plant flour (100 g), tap water (165 g) and bacterial suspension (5 g), containing an
average of 10.2 log10 CFU (colony-forming units)/g of P. acidilactici, were used to prepare
270 g of sourdough (moisture content 65%). Incubation was carried out at 35C, till a
constant acidity was reached followed by pH measurement. Spontaneous sourdough (without
bacterial inoculum) was also produced by naturally fermented defatted soya or flaxseed flour
at 30C for 72 h. To obtain the dried fermented product, prepared sourdough was freeze-dried
at -40C in a LD85 freeze-dryer (Millrock Technology, Inc., Kingston, NY, USA) or dried at
45C in a lab spray dryer SD-1500 (Triowin Lab Technology Co. Ltd, Shanghai, China).
Microbiological analysis
The vitality of LAB after thermal treatment was evaluated by comparing the cell numbers
between non-treated, freeze-dried and spray-dried sourdough samples. Ten grams of sample
were homogenized with 90 ml of saline (0.9%). The suspension was diluted, and the 10-4 and
10-8 solutions were plated onto MRS agar. The plates were incubated under anaerobic
conditions at 30C for 72 h. After incubation, the LAB cell number was expressed as a log10
of CFU/g.
Bread making
Experimental bread making was carried out according to the traditional procedure used
for bread making in Lithuania. The wheat flour (2 kg) was substituted by (i) soya or flaxseed
sourdough; (ii) soya or flaxseed freeze-dried sourdough and (iii) defatted untreated soya or
flaxseed flour (Table 12). Water content was calculated with reference to the moisture content
of the raw materials and the required moisture content of the end-product (45%). The dough
was then mixed for 8min and fermented at 28C for 30 min. Then the dough shaping and
proofing at 35C temperature for 40 minwas performed. Dough balls (each of 450 g) were
baked in a MIWE condo deck oven (MIWE Michael WenzGmbH, Arnstein, Germany) at
230C for 25 min.

56

Table 12. Ingredients (%) used for the production of wheat bread, supplemented with
different soya and flaxseed additives
Ingredients
Breads
WF
FS/SS
FSD/SSD
F/S
Wheat flour
100
86.5
95
95
Sourdough

13.5

Dried sourdough

Soya/flaxseed flour

1.5

1.5

1.5

1.5

Bakers yeast

Water

56

50

58

57

Salt

Determination of the quality and sensory evaluation of the breads were performed 24 h
after baking.
Bread quality evaluation
Specific loaf volume of the bread was determined by measuring the volume of rapeseed
displaced by the bread (AACC method 10.05, 2003) and known weight of loaf, being
expressed for 100 g of product. Bread crumb porosity was determined by the determination of
the total volume of pores of a known volume of crumb, knowing its mass and density. To
obtain an average of porosity, bread was cross-sectioned, removing the crust and crumb
shaped in three cylinders, from three different areas, which were subjected to the
measurement method. Bread before crumb texture analysis was immediately sliced (thickness
6mm), and crumb hardness was fixed as a maximum compression force (60% compression,
10-mm diameter plunger, compression rate 2 mm/s) using the Stevens-LFRA Texture
Analyzer (Voland Corp., New York, NY, USA). Sensory analysis of breads was carried out
by 12 panelists according to the ISO 8586-1 method (ISO 1993) for crumb colour, crumb
texture, flavour/taste and overall acceptability using a 150-mm hedonic line scale ranging
from 150 (extremely like) to 0 (extremely dislike) for each sensory characteristic.
Protein determination
Total nitrogen was determined by using the Kjeldahl method (AACC method 4612,
2003) and was multiplied by a factor of 6.25 to determine the protein content in soya and
flaxseed flours. For soluble protein determination, each sample (5 g) was extracted with 20 ml
of distilled water (pH 7) only at room temperature and centrifuged (5000 g, 4C, 20 min). The
sediment was re-extracted with 20ml of 0.5mol/l NaCl. After centrifugation, supernatants
were collected, diluted with water to 50ml and 20ml of solution was used for the protein
content determination.
Determination of in vitro protein digestibility
Determination of protein digestibility was carried out according to Lqari et al. (2002).
Samples containing 62.5mg of protein were suspended in 10ml of water and the pH was
adjusted to 8 with 0.1 mol/l NaOH. An enzymatic solution containing 1.6mg of trypsin (18
Units/mg), 3.1mg of a-chymotrypsin (40 Units/mg) and 1.3mg of protease (15 Units/mg) per
millilitre was added to the protein suspension in a 1:10 (v/v) ratio. The pH of the mixture was
measured exactly after 10 min and the in vitro digestibility calculated as a percentage of
digestible protein (DP) using the equation DP = 210.464 18.103 * pH.

57

Determination of trypsin inhibitor activity

Porcine trypsin inhibitor activities of fermented and untreated defatted soya and flaxseed
flours were determined by assaying trypsin activity in the presence and absence of soya or
flaxseed flour using casein as the substrate for the protease activity determination
BA analysis
Extraction of samples and determination of BAs were carried out according to the
procedures developed by Ben-Gigirey et al. (1999). Perchloric acid (0.4 mol/l, 10 ml)
containing a known amount of 1,7-diaminoheptane used as an internal standard was added to
3 g of sourdough sample, and the mixture was homogenized with Ultra-Turrax (IKA
Labortechnik, Staufen, Germany) and centrifuged (3000 g, 4C, 10 min). The residue was
extracted again with an equal volume of 0.4mol/l perchloric acid. Both supernatants were
combined, and the final volume was adjusted to 30ml with 0.4mol/l perchloric acid. The
extract was filtered through Whatman No. 1 paper. One millilitre of extract or standard
solution was mixed with 200 ml of 2 mol/l sodium hydroxide and 300 ml of saturated sodium
bicarbonate. A 5-(dimethylamino)naphthalene- 1-sulphonyl chloride (dansyl chloride reagent)
(10 mg/ml, 2 ml) prepared in acetone was added to the mixture and incubated at 40C for 45
min. Residual dansyl chloride was removed by the addition of 100 ml of 25% ammonium
hydroxide. After incubation at room temperature for 30 min, the mixture was adjusted to 5ml
with acetonitrile. Finally, the mixture was centrifuged (3000 g, 5min), the supernatant was
filtered through 0.2 mmfilters (Millipore Co., Bedford, MA, USA) and stored at -25C until
HPLC analysis. An Agilent 1200 HPLC (Carlsbad, CA, USA) equipped with diode-array
detector and Chemstation LC software was employed. A Chromolith C18 HPLC column
(100mm x 4.6mm x 4 mm, Merck KGaA/EMD Chemicals, Darmstadt, Germany) was used.
Ammonium acetate (0.1 mol/l) and acetonitrile were used as the mobile phases by a flow rate
of 0.45 ml/min. The sample volume injected was 10ml and the amines were monitored by 254
nm. The detection limits for standard amine solutions were approximately 0.1 mg/kg.
Analyses
Total fibre, fat, ash contents of flours, the falling number and the wet gluten content of
wheat flour were determined using approved AACC methods 32-06, 30-25, 08-16, 56-81B
and 38-12, respectively (AACC 2003). Total carbohydrate was calculated based on the
percentage difference in the other parameters. Total titratable acidity (TTA) was determined
on 10 g of sample homogenized with 90 ml of distilled water and expressed as the amount
(ml) of 0.1M NaOH to get pH 8.2. Moisture content was determined by drying the sample at
105C temperature to constant weight.
Results and discussion
The influence of lactic acid fermentation on soya and flaxseed protein properties. Partially
defatted flaxseed flour was found to have a higher content of soluble protein compared with
defatted soya flour (Table 13), despite the fact that the solubility profile of analysed plant
proteins is comparable.
LAB posseses a complex proteolytic system capable to hydrolyse proteins to peptides and
amino acids, though tested P. acidilactici KTU05-7 strain previously was considered as weak
proteolytic bacteria (between 2.97 0.24 and 45.74 1.31 PU/g). The microbial starter and
the processing conditions (e.g. fermentation temperature and time) were reported to be the
main factors for the proteolytic activity of sourdough with the positive influence on the levels
of amino acids and the accumulation of hydrophobic and basic amino acids.

58

Table 13. Soluble protein (g/100 g d.m.), trypsin inhibitor activity and protein digestibility of
flaxseed and soya flours before and after fermentation with P. acidilactici
Soluble protein*
Trypsin inhibitor
In vitro protein
Flour
activity
digestibility
Untreated Fermented
Untreated
Fermented Untreated Fermented
Flaxs

27.3

31.2

8.4

5.9

64.1

72.7

Soya

19.7

23.0

16.8

11.3

75.3

88.7

eed

Notes: Data are the mean SD of three analyses. d.m., dry matter; * nitrogen factor 6.25 for
soya and flaxseed flours; percentage of protease inhibition; grams of proteins
digested/100 g of protein
The results indicated the increase in protein digestibility by 11.8% and 15.1%, and the
decrease in the trypsin inhibitor activity by 29.8% and 32.7% in fermented products of
flaxseed and soya, respectively, compared with untreated flours (Table 13). If we compare the
nutritional value of flaxseed with soya and concluded that protein utilization of flaxseed was
slightly lower than that of soya. It was reported that the protein digestibility of low-fat soya
flour was 74%, while the digestibility of flaxseed protein varied from 69% to 72.3% of
defatted seeds.
The study confirmed that lactic acid fermentation increased protein digestibility, herewith
creating a higher nutritional value of defatted soya and flaxseed flour which is in agreement
with the results of other authors. Natural flaxseed and soya flours have some nutritional and
functional features to cereal flours. Proteins are highly soluble and characterized by foaming
and emulsifying properties. The amino acid composition of flaxseed proteins is comparable to
that of soya, with both seeds having relatively high levels of aspartic acid, glutamic acid,
leucine and arginine.
BA contents in soya and flaxseed fermented products
BA contents measured in untreated soya flour and flaxseed flour and sourdoughs
fermented with P. acidilactici before and after freeze-drying or spraydrying are shown in
Table 14. Putrescine and spermidine mean levels showed dominance in comparison with
histamine and tyramine mean values in soya flour samples. Untreated flaxseed flour contained
the highest amounts of phenylethylamine and tyramine, and the lowest amounts of
histamine and spermine. Cadaverine and histamine concentrations were found at the same
levels in both kinds of flour.
The highest total BA levels were found in soya and flaxseed spontaneous sourdoughs
(492.7 and 390.2 mg/kg of dry matter (d.m.), respectively), while the soya and flaxseed
sourdoughs fermented with P. acidilactici contained lower amounts by 48% and 59%,
respectively, of amines than those in flours (224.8 and 299.9 mg/kg d.m.). Although the total
content of BAs in soya flour was found lower than that in flaxseed flour, the total level of
BAs after spontaneous fermentation was higher on an average by 20.8% than in flaxseed
fermented products.
Phenylethylamine was found to be the predominant amine in soya and flaxseed
spontaneous sourdough samples (Table 14). The level of this amine was most abundant in P.
acidilactici flaxseed sourdough, whereas in that kind of soya sourdough, putrescine was
predominant. Putrescine concentration was found considerably decreased, reaching a level of
ninefold lower after the spontaneous fermentation of soya flour.
The fermentation of soya flour by P. acidilactici had no significant effect on the formation
of putrescine.

59

Table 14. BA contents (mg/kg d.m.) in untreated soya and flaxseed flours before and after
fermentation
Spontaneous
Flour
LAB fermentation
fermentation
BA
Soya
Flaxseed
Soya
Flaxseed
Soya
Flaxseed
Phenylethylamine

37.8

141.9

277.2

186.2

19.2

63.3

Putrescine

63.8

18.6

7.2

39.4

66.4

11.3

Cadaverine

34.3

34.5

92.9

29.8

8.8

18.3

Histamine

5.4

77

18.7

44.7

3.8

4.0

Tyramine

7.1

82.4

26.8

39.6

4.6

19.3

Spermidine

60.3

10.4

56.1

24.1

11.0

4.9

Spermine

16.1

4.4

13.8

26.4

2.8

2.0

Total

224.8

299.9

492.7

390.2

116.6

123.1

Notes: Data are the mean SD. Means within a row with different letters are significantly
different ( p 0.05)
Lactic acid fermentation decreased cadaverine concentration by 74% and 47% in P.
acidilactici sourdoughs of soya and flaxseed, respectively.
Spontaneous fermentation lowered by 13.6% cadaverine level in flaxseed, but increased
amine concentration by 63% in soya.
The concentration of histamine showed a rising trend during the spontaneous
fermentation, reaching levels of more than 3.5- and 6-fold higher in soya and flaxseed
sourdoughs, respectively, than those in flours, in contrast to fermentation using P. acidilactici
(concentrations of tested amines were found by 29.6% and 48% lower, respectively) (Table
14).
Spontaneous fermentation decreased on an average by 52% tyramine concentration in
flaxseed. However, after spontaneous fermentation of soya, an opposite trend was observed.
In spontaneous fermentation of soya, sourdough occurred more by 73.5% tyramine.
The lactic fermentation lowered levels of amine by 35% and 76.5% in soya and flaxseed,
respectively.
The significant decrease (on an average by 82%) in concentrations of spermidine and
spermine was observed in soya samples and by 53% and 54.5%, respectively, in flaxseed
samples after lactic fermentation.
Spontaneous fermentation slightly lowered (by 7% and 14%, respectively) both amine
levels in soya, and increased by 56.8% and 83%, respectively, the spermidine and spermine
contents in flaxseed samples (Table 14).
Foods that have been prepared by a spontaneous fermentative process, or have been
exposed to microbial contamination during ageing or storage are likely to contain amines.
Tyramine is formed at the beginning of the fermentation through a microbial
contamination. The simultaneous formation of phenylethylamine, putrescine and cadaverine is
typical for such a phenomenon in the fermentation.
However, not all amines are of equal toxicity, consequently the levels of histamine and
tyramine are of concern. Tyramine is usually the major amine found in fermented products.
Histamine and tyramine intake should be in the range of 50100 and 100800 mg/kg,
respectively; over 1080 mg/kg tyramine becomes toxic. Histamine intake higher than 100 mg
may cause slight or intermediate poisoning.
60

Putrescine, spermine, spermidine and cadaverine have no adverse health effect, but they
may react with nitrite to form carcinogenic nitrosamines and also can be proposed as
indicators of spoilage. Overall, the histamine and tyramine concentrations of all soya and
flaxseed fermented products were within acceptable limits, and could be considered as safe
for consumption and herewith could be used as supplements in bread making.
The levels of BAs usually increase in fermented products due to decarboxylase activity of
the LAB used as starter culture. Low acid conditions, such as those occurring during
fermentation, favour the decarboxylation of amino acids.
Homofermentative P. acidilactici KTU05-7 previously isolated from spontaneous rye
sourdough has been characterized as a pediocin Ac05-7 producing strain with weak
proteolytic activity (from 2.97 0.24 to 45.74 1.31 PU/g). Results of our study confirm that
the above-mentioned strain does not belong to the group of LAB producing BAs.
Also the antimicrobial compounds produced by P. acidilactici can reduce microbial
contamination as compared with spontaneous fermentation, may inhibit the growth of moulds
and prolong microbiological spoilage, ensuring the stability of the fermented plant products.
The amine concentrations of all analysed soya and flaxseed fermented products were far
below those levels causing a health risk.
The influence of soya and flaxseed flours on fermentation process and wheat bread
quality
Special attention has been paid to the changes of the acidity on fermented products of
soya and flaxseed and to the effect of the treatment based on the combination of
fermentation/freeze-drying or fermentation/spray-drying on cell count of LAB. The values of
pH decreased from 4.72 0.02 and 5.12 0.01 at the start of the fermentation to 4.16 0.02
and 4.64 0.04 in soya and flaxseed sourdough, respectively. The TTA was 17.8 0.1 and
13.8 0.3 after fermentation at 35C temperature of flaxseed and soya flour, respectively.
Organic acids, such as lactic and acetic, produced during sourdough fermentation, have the
capacity to lower the postprandial blood glucose and insulin responses in humans. Biological
acidification such as sourdough fermentation is more effective than chemical acidification in
decreasing the rate of starch hydrolysis and glycaemic index.
Results of microbiological analysis show that freeze-drying did not adversely affect the
viability of sourdough microflora. A significant difference was not found between colony
counts of non-treated and dried sourdough samples of soya (8.51 0.22 and 8.65 0.24 log10
CFU/g, respectively) as well as flaxseed (9.57 0.16 and 9.37 0.16 log10 CFU/g,
respectively). Contrary, the spray-drying significantly affected LAB that responded to
lowering of bacteria colony by an average of 11.6%.
Therefore, freezedrying could be a more appropriate and applicable means to ensure the
long-term stability of sourdoughs.
In this case, the centralized production of dried sourdoughs would be much simpler,
because it does not require special storage conditions, and no need for ordinary observation
and upgrading.
Substitution of wheat flour by both kinds of plant flours affected the texture of bread
(Table 15).
When plant sourdoughs were added, these features markedly improved texture, even if
they remained slightly lower than those of the control sample [wheat flour (WF) bread]. The
values of specific volume were higher by 8.9%, 16.1% and 19.0% for flaxseed flour (F),
flaxseed sourdough (FS) and flaxseed sourdough freeze-dried (FSD) breads, respectively,
than those made with soya products (soya flour, S; SD and soya sourdough freeze-dried,
SSD).

61

Table 15. Crumb texture and sensory analysis of wheat breads with different soya and
flaxseed additives
Breads
WF

FS

SS

FSD

SSD

Texture analysis
Specific
volume (cm3
100-1 g-1)
Crumb
hardness
(TAU)

386

301

274

351

276

382

298

48

46

51

42

66

25

40

Sensory analysis
Pore
uniformity
Crumb
colour
Elasticity

26

31

31

103

49

60

40

32

18

14

96

72

15

114

26

34

113

84

26

40

Additive
taste
Sweetness

17

28

82

145

14

53

44

18

10

18

Acid taste

17

21

40

74

28

50

Acceptability

120

42

18

85

70

Notes: Data are the mean SD. Means within a row with different superscript letters are
significantly different ( p 0.05). Breads: F, wheat flour; SS, soya sourdough; FS,
flaxseed sourdough; SSD, soya sourdough freeze-dried; FSD, flaxseed sourdough freezedried; S, soya flour; F, flaxseed flour. TAU, texture analyzer units

Generally, all wheatflaxseed sourdough breads had a higher specific volume, the greater
uniformity of crumb pores and lowest crumb hardness than soya sourdough (SS) breads, and
breads substituted with defatted soya and flaxseed flours (Table 15). All the texture features
pointed out that FSD bread was softer and more similar to the common wheat bread.
The use of plant sourdoughs caused an intense colouring of the crumb and caused an
increase in the elasticity and loss of the crispness of breads. The incorporation of soya and
flaxseed flour modified the crumb colour of the breads from creamy white to slight brown.
The highest intensity of crumb colour had breads with flaxseed (96 6) or soya (72 8)
sourdough, whereas the breads with freeze-dried flaxseed and soya sourdoughs as well as
flours had a much lighter colour (Table 15).
An additive taste (145 6) was intensively expressed in the bread with SS (Table 15).
The breads prepared with soya flour (sample S) had the highest values of sweetness (44 2).
The lowest intensity of overall taste and the lowest sweet taste have been noticed in all
wheatflaxseed bread samples.
The crumb texture analysis showed that the highest moisture content and greater
uniformity of crumb pores were for FS bread (103 7). Soya and flaxseed flour addition at
5% level significantly decreased crumb pore uniformity (31 4). Study showed an increase in
the elasticity and a loss of crispness of breads with a high concentration of flaxseed meal. The
highest crumb elasticity values were determined for FS bread (113 8), while the lowest were
for FSD (26 2) and F (26 4) breads (Table 15).

62

Generally, like in objective evaluation of bread quality, the higher positive effect of
fermented flaxseed products has been manifested during the sensory rating of the bread. The
incorporation of fermented soya products adverse affected the overall acceptability related to
their specific taste and odour.

Conclusions
Investigation of such nutritional points as protein digestibility and BA content after lactic
acid fermentation in soya and flaxseed sourdoughs for bread making was the main goal of this
study. However, plant flours other than wheat flour were chosen with the aim of replacing
wheat flour without altering and possibly to improve the nutritional, texture and sensory
features of baked goods.
The lactic acid fermentation of soya and flaxseed flours significantly improved protein
extraction and increased protein digestibility. Fermentation with P. acidilactici did not affect
the concentrations of certain BAs such as tyramine, histamine and putrescine in soya and
flaxseed sourdoughs. The levels of total BAs do not present a health risk for consumers
because of their relatively low levels in the plant fermented products. Thus, the use of
fermented soya and flaxseed as food ingredients is from a nutritional point of view
advantageous and creates safe products for bread enrichment with a low fat and BA contents
and a high protein utilization.
Fermented and fermented/freeze-dried plant additives resulted in a better bread quality
compared with bread of untreated flours. Flaxseed generally provides a positive effect on the
quality and texture of bread and does not disimprove sensory properties of it. The evaluation
of experimental results suggest that the production of wheat bread enriched with fermented
flaxseed and possibly with fermented soya could improve the nutritional properties of bread.
The application of freeze-drying could be appropriate as the treatment for preservation of the
plant fermented products intended for centralized production. Specific properties of soya and
flaxseed proteins, such as the ability to absorb higher amounts of water and the use up to 5%
of plant flours may not alter the costeffectiveness of the production such kind of bread.

63

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