LABORATORY MANUAL ON QUALITY CONTROL OF ANIMAL FEEDS by Dr. G. DEVEGOWDA, PROFESSOR & HEAD, DEPARTMENT OF POULTRY SCIENCE UNIVERSITY OF AGRI. SCIENCES HEBBAL, BANGALORE

1. It is been an endeavor to constantly update the technical
inputs to our esteemed customers in VETCARE. This
effort is an extension of the programme
This manual covers the details of analysis of many
ingredients used in the feed along with the Mycotoxin
Estimation and Toxin binding estimation. Also find the
interesting Enzyme analysis in this manual

VETCARE
IS - 40, KHB Industrial Area
Yelahanka New Town
Bangalore - 560 064, INDIA
Ph. : 8460060, 8462055/56 Fax: 8461240
E-mail : btan@giasbg01.vsnl.net.in
Web : www.vetcareindia.com

2. LABORATORY MANUAL ON
QUALITY CONTROL OF ANIMAL FEEDS
Dr. G. DEVEGOWDA
M.V.Sc, Ph.D (U.S.A)
PROFESSOR & HEAD
DEPARTMENT OF POULTRY SCIENCE
UNIVERSITY OF AGRI. SCIENCES
HEBBAL, BANGALORE - 560 024
1999

3. CONTENTS
Sl. Title Page No.
No.
I. MYCOTOXIN QUANTIFICATION
1. Mycotoxin Standards 1-5
2. Mycotoxin Analysis 6 - 16
3. Rapid TLC method of Aflatoxin analysis 17 - 19
4. Analysis of Ochratoxin 20 - 23
5. Analysis of T-2 toxin 24 - 28
6. Analysis of Zearalenone 29 - 33
7. Rapid TLC method of Multi-Mycotoxin 34 - 36
analysis
8. Rapid Mycotoxin Test / "ELISA Test" 37 - 39
9. In vitro evaluation of Mycotoxin binding agents 40 - 41
10. In vitro evaluation of Mycotoxin binding agents
in contaminated feeds 42 - 43
11. List of Suppliers of Mycotoxin standards 44
12. Toxin Binding analysis of Mycotoxins (VETCARE)
13. Preparation of Acid hydrolysate of seed samples for
amino acid analysis using ion exchange chromatography.
14. Preparation of seed samples for methiomine and
cystine analysis using performic acid oxidation procedure.
15. Estimation of Sodium and potassium by flame photometry

4. PREPARATION OF ACID HYDROLYSATE OF FEED SAMPLES
FOR AMINOACID
ACID ANALYSIS BY ION-EXCHANGE CHROMATOGRAPHY
Reagents:
1. 6 N HCL: 50 ml of concentrated hydrochloric acid added to 50 ml of
double distilled water.
2. DL-norleucine standard, 25 umole/ml
3. Sodium citrate buffer, pH2.2
Procedure:
1. Grind sample finely (grind to pass a 40 mesh sieve).
2. Weigh the hydrolysate tube.
3. Weigh the sample into the hydrolusate tube, so as to
contain about 30-40 mg
of protein. This would be approximately 60-80 mg of
soybean meal samples and 300-350 mg for corn samples
when diluting the hydrolysate 100 times.
4. Add 6 ml of 6N HCL and 0.6 ml of norleucine internal
standard. Mix well.

5. 5. Charge the tube with nitrogen gas and place in an oven at
1100C for 20
hours.
6. After the tube cools, filter the contents through Whatman
No.1 filter paper
into a drying tube. Wash hydrolysate tube with double
distilled water and collect in the same drying tube.
7. Dry the filtrate by evaporating with a rotary evaporator
under vacuum, with
the water bath temperature at 480C. Dry the filtrate to a
slightly wet residue. Wash residue with distilled water and
dry again.
8. Add 20 ml of citrate buffer and mix well.
9. Take one ml from step 8 and dilute to 5 ml with citrate
buffer.
10. Filter the diluted sample liquid using 0.2 micron
Nucleopore membrane filter.
11. Detemine amino acids by injecting sample into Dionex D-
300 Amino acid analyzer.
ACKNOWLEDGEMENT

6. I would like to put on record my sincere gratitude to
Dr. Malathi, V., Dr. Manju, N.C. and Dr. Manoj, K.B., for their
significant contribution in bringing up this manual so beautifully. My
thanks are also due to Dr. S. Abdul Rahman, Director of Instructions,
Veterinary College, and the Staff and Students of the Department of
Poultry Science, University of Agricultural Sciences, Bangalore, India
for their encouragement and support.
I also thank Dr. Ravikiran, D., Ph.D. Scholar of this Department
for his critical editing of this manual
( G. DEVEGOWDA )
Professor and Head
Vice President, World's Poultry
Science Association (IB),
Department of Poultry Science
University of Agricultural Sciences
Hebbal, Bangalore - 560 024, INDIA

7. Preface
The Department of Poultry Science, University of Agricultural
Sciences, Bangalore, has been the pioneer in the field of Mycotoxin
Research in the Country. Several breakthroughs have been achieved here in
counteracting Mycotoxins in Animal and Poultry feeds. Further Research is
on in the direction to achieve total solution to the problem of mycotoxins in
foods and feeds, sooner or later.
Mycotoxins are always of concern, both in tropical and temperate
climates. Mycotoxins in feed remain to be a potential threat to the health,
productivity and livability of poultry and livestock and are also of serious
public health concern. Such a situation requires that there be adequate
laboratory facilities everywhere to identify and evaluate the problem
precisely and take necessary steps for good.
While technology for Mycotoxin research is available, much of the
data is relevant to developed countries which make use of sophisticated
instruments and techniques. And there will always be a lot of hurdles for a
person working on a small scale laboratory to follow those methods.
Having faced such problems initially and developed / adopted
methods that suit our conditions, we felt that such problems should not be
the excuse. Hence we felt the need of a manual that gives, simple, but in
detail, information on the basic laboratory work involved in Mycotoxin
research. And the fruit of our effort in this direction is this manual.
We trust this manual would be quite useful to the Post Graduate
students in Animal Sciences and Biological Sciences. Food and Feed
Analysis & Quality Control Laboratories, Feed Manufacturers, Research
Institutions, Food Processing Industry, Food Exporters, Private Veterinary
Diagnostic Laboratories etc.
Date : March, 1999 Prof. G. Devegowda
Place : Bangalore, India Dr. M.V.L.N. Raju

8. CHAPTER I
MYCOTOXIN QUANTIFICATION
1. Mycotoxin standards
Pure mycotoxins of known concentration are used in mycotoxin
assays for either fluorescence intensity comparison or calibration.
Reconstitution of Mycotoxin standards
Mycotoxin standards are often supplied in crystalline form in sealed
glass vials. They need to be suitably dissolved in appropriate solvents for
preparation of stock and working solutions of desired final concentration.
Procedure
 Carefully remove the metallic seal from the central injecting area of
the cap of the vial and inject about 1 ml of appropriate solvent into the
vial
 Shake the vial gently to dissolve the mycotoxin in the solvent
 Recover the dissolved mycotoxin using the same syringe and transfer
into a volumetric flask
 Inject again 1 ml of solvent into the vial, shake gently, recover and
transfer to volumetric flask. Repeat 5-6 times for complete recovery
of Mycotoxin

9.  Make up the volume of the dissolved toxin to get the desired
concentration in the stock solution
 Tightly stopper the volumetric flask and store in refrigerator in an
opaque container at 4°C
 For preparation of working standards and those used for
spectrophotometric purity evaluation, dilute the stock standard using
the suitable solvent to get the desired mycotoxin concentration

10. Table 1. Concentrations of the Mycotoxin standards
required (µg/ml)
Stock UV TLC Solvent
Aflatoxins 100 10 1 Benzene : Acetonitrile (98 : 2)
Ochratoxin A 25 25 2 Benzene : Acetic acid (99 : 1)
T-2 toxin 5000 100 50 Ethyl acetate
Zearalenone 100 10 50 Benzene
Citrinin 40 20 40 Chloroform
DON 500 20 20 Ethyl acetate : Methanol (19 : 1)
Sterigmatocystin 100 100 100 Benzene

11. Checking the purity / concentration
 The standards thus prepared are required to be checked periodically
for assessing any possible alteration in their concentration during
storage
 Prepare 0.4 mM potassium dichromate solution by dissolving 125 mg
potassium dichromate in 1 litre 0.018 N Sulphuric acid
(1 ml H2SO4 in 2 litre distilled water)
 Prepare 0.2 mM and 0.1 mM solutions of potassium dichromate by
making two successive dilutions of 0.4 mM solution with
0.018 N Sulphuric acid
 Read absorbance of these 3 solutions at 350 nm using 0.018 N H2SO4
as blank
Abs x 1000
 Calculate (E) of each solution =
mM
 Calculate the average of the three solutions (0.4, 0.2 and 0.1 mM)
 Calculate the correction factor (CF) for the instrument
3,160
CF = (normal value : 0.95 - 1.05)
E

12.  Read the absorbance of the mycotoxin standard at wave length of
maximum absorbance
Abs x Mol wt x CF x 1000
Concn. (µg/ml) =
E
Molecular weight, wave length of maximum absorbance and
absorptivity of some Mycotoxins
Mol. wt. Max. abs. (nm) Absorptivity (E)
Aflatoxin B1 312 353 19,800
Ochratoxin A 403 333 5,550
Zearalenone 318 316 6,020
Citrinin 259 322 16,100
Sterigmatocystin 324 325 15,200

13. 2. Mycotoxin Analysis
A. Sampling
 Collect the samples at the following quantities for ensuring
meaningful representation of the whole lot of feed / feedstuff
Min. sample size
Small particle type (milk, vegetable oils) 500 g
Intermediate particle type (ground meals, flours, 3 kg
compounded feed)
Small grains (wheat, rice, sorghum, ragi, barley etc.) 5 kg
Intermediate grains (maize, cotton seed / cake) 10 kg
Large grains (groundnuts / cake) 20 kg
 Collect at least 100 subsamples from the whole lot. For eg. from a
truck of 100 bags of maize, collect 100 g maize from each bag to
obtain a total sample size of 10 kg
 Get about 50 - 100 g subsample from the whole sample employing
either coning and quartering method (in a series of steps) or using
sample divider
 The subsample thus collected can be directly subjected for analysis

14. B. Outline of Mycotoxin analysis
Sampling
Toxin extraction
(using organic solvents)
Clean-up
(To remove fat, impurities etc.)
Work up
Identification & Quantification
(TLC, HPLC, ELISA etc.)

15. C. Different methods of Mycotoxin analysis
C. 1. Thin layer chromatography (TLC)
 Principle
It is the cheapest and most commonly used method. It makes use of
heterogenous equilibrium established during the flow of a solvent
(mobile phase) through a fixed phase (stationary phase) to separate ≥
2 components from materials carried by solvent (differential
migration).
 Spotting the extract
Place between 5 - 20 µl of sample extract / standard as a small circular
spot (< 5 mm), 1 - 2 cm from the end of the TLC plate. Micropipette /
microcaps may be used for the purpose. Leave at least 1 cm gap
between two adjacent spots.
 Developing the plate
Place about 50 - 100 ml of mobile phase (solvent) in a tank and keep
the plate at a slight angle with the spots little above the upper level of
the solvent. Due to capillary action, solvent moves upward on the
plate. Allow the solvent to travel at least about 8-10 cms.
 Detection
Air dry the developed plate and view in a UV cabinet under either
longwave (365 nm) or short wave (254 nm) range to identify the
fluorescing mycotoxins. In case of mycotoxins which do not
fluoresce, spray the plate with suitable reagent to develop
fluorescence.

16.  Resolving front value (Rf)
Each mycotoxin has its characteristic color of fluorescence under UV
light and a constant Rf value in a particular developing solvent
(Table 3). Rf value is computed using the formula,
Distance travelled by sample spot from the origin
Rf =
Distance travelled by solvent front from the origin
 Confirmation
The presence of mycotoxin can be confirmed either by spraying the
plate with suitable reagents (like 50 % aqueous H2SO4, Triflouro
Acetic Acid etc.) or placing an internal standard right over the top of
the sample spot (superimposing).
 Detection by Scanner
The fluorescence intensity of sample and standard spots can be
measured by using TLC Scanner / fluorodensitometer to avoid
possible human errors in comparison.
Table 3. TLC characteristics of mycotoxins
Toxin Rf * Color Color (UV) after

17. (UV) spray * *
Aflatoxin B1 0.31 Blue Pink
Aflatoxin B2 0.26 Blue Pink
Aflatoxin G1 0.23 Green Blue
Aflatoxin G2 0.17 Green Blue
Ochratoxin A 0.55 Green Blue
T-2 toxin 0.36 Yellow Blue
Zearalenone 0.78 Blue Yellow
DAS 0.33 Yellow Variable
Sterigmatocystin 0.85 Red-brown Yellow
*TEF : Toluene : ethyl acetate : formic acid ( 6:3:1 )
* *P - anisaldehyde
C. 2. Spectrophotometry
This is an extension of TLC method. The sample spots on the
developed TLC plate are scraped out alongwith the sorbent (silica gel)
and extracted with methanol for 3 minutes. The extract is filtered and
the absorbance of the filtrate is measured in a spectrophotometer
at 363 nm.
Reference :
Nabney and Nesbitt. 1965. Analyst 90 : 155-160.

18. C. 3. High Performance Thin Layer Chromatography (HPTLC)
This is an improvised version of TLC, where sample
application and detection of fluorescence intensity are fully automated
and carried out by using automated sample applicator (like Linomat
IV of Camag, Switzerland) and densitometer, respectively.
Mycotoxin levels less than 0.1 ppb can be detected by this method.
C. 4. Minicolumn method
A glass column of 20 cm length, 6 mm internal diameter with
tapering end (2 mm) is packed serially from the bottom with glass
wool, calcium or sodium sulphate (8-10 mm), florisil (8-10 mm),
silica gel (18-20 mm), neutral aluminia (8-10 mm), calcium or
sodium sulphate (8-10 mm) and a cap of glass wool.
2 ml of final chloroform extract (in case of aflatoxin) is placed
in the column and eluted with chloroform : acetone (9 : 1). Aflatoxin,
if present is trapped as a band above florisil layer which can be
viewed under long wave UV light as a blue fluorescent band. This
method can be used as a qualitative test for rapid identification of
mycotoxin.
C. 5. Immuno assays
These assays are developed on the basic principle of
Antigen - Antibody reaction. Antibodies are highly specific to the
Mycotoxin - Protein conjugate (Hapten) used. Hence the results will
be highly specific.

19. Commonly employed immuno assays
 Radio immuno assay (RIA)
Standard mycotoxin, labelled onto a radioactive compound like
Tritium is used. Mycotoxin levels as low as 2-5 ppb can be
detected. The disadvantages of this method include high cost,
difficulty in labelling, radio active waste disposal problem and
risk of handling.
 Enzyme linked immuno sorbent assay (ELISA)
It has received great attention in recent times and has been the
most popular and widely practiced immuno assay method.
ELISA is rapid, more sensitive, highly specific and simple to
operate. It does not require any extensive extraction or clean-
up.
Commercial ELISA kits
Various companies have been marketing commercial kits
which basically work on ELISA principle. These have gained
wider acceptance as considerable amount of time is saved on
antibody production. Sample is extracted with methanol : water
(60 : 40) or acetonitrile : water (50 :50) and the extract is
directly subjected to analysis.

20. Elisa tests are good for quick identification of
mycotoxins in feed samples, various tests are developed based
on Antigen - Antibody principle. Some companies which
produce ELISA kits are :
1. Neogen Corp,
620, Lesher place,
Lansing, Michigan 48912, U.S.A.
2. Vicam,
313, Pleasant St.,
Watertown, Massachusetts - 02172, U.S.A.
C. 6. High performance liquid Chromatography (HPLC)
It is highly sensitive and can detect upto 5 x 10-6 ppb level of
mycotoxin. Stainless steel columns (< 18) of 15 cm length and 4 mm
internal diameter, packed with silica gel (particle size - 5 microns) are
used. Sample is first extracted with suitable solvent (generally 60 %
aqeous methanol) and the extract is cleaned - up.
This purified extract (20 µl) is injected into the column and the
eluent (generally a mixture of methanol, water and acetonitrile) is
passed at a flow rate of 0.75 ml / min and at a pressure of 3000 psi.
The eluted toxins coming out of the column are detected and
quantified by fluorimeter.
The columns may be either normal phase (polar stationary
phase) or reverse phase (polar mobile phase) type. The latter type is
most commonly used.
C. 7. Bio - assays

21. Mostly are useful as confirmatory tests. Toxin extract is
injected as a single dose into stomach (day-old duckling bioassay,
guinea pig bioassay), fertile eggs (chick embryo bioassay) or into skin
of rabbits (skin bioassay). Presence of toxin is confirmed by noticing
pathological changes or mortality.
Safety precautions in mycotoxin analysis
 Carryout the mycotoxin analysis in a separate work area in the
laboratory
 Cover the bench top with non absorbent material
 Solvents used are highly inflammable. So avoid using electric stoves,
bunsen burners etc.
 Do not stock the solvents in larger quantities
 Wear protective clothing, gloves and mask to minimise the risk of
inhalation / contact with hazardous mycotoxins
 Some of the solvents (like benzene, chloroform) are toxic. Avoid
direct skin contact with them
 Any spillage should be immediately mopped-up with cotton. Such
cotton should be incinerated
 After completing the work, decontaminate the area with 4 % sodium
hypochlorite solution

22.  Decontaminate the glassware by soaking for atleast 2 hours in 1 %
sodium hypochlorite solution
 Spray the TLC plate with reagent only in a fume cup-board / spray
cabinet
 At the UV cabinet, always view the TLC plate only through the UV
filter
 Avoid eating, drinking and smoking in the laboratory
 Keep the lab well ventilated using exhaust fans

23. 3. Rapid TLC method of Aflatoxin analysis
(Modified Romer's method)
Reagents : i) 0.2 M NaOH (dissolve 8 g NaOH in water and
make up volume to 1 lit)
ii) 0.41 M Ferric Chloride (dissolve 66.5 g anhydrous
FeCl3 in water and make up volume to 1 lit)
iii) 0.03 % H2SO4 (0.3 ml conc. H2SO4 + 999.7 ml
water)
iv) Potassium wash solution (dissolve 1.12 g KOH
and 10 g KCl in water and make up volume to 1
lit)
Solvents : i) Acetone
ii) Chloroform
iii) Developing solvent
Chloroform : Acetone : Water (88 : 12 : 1)
Standard : Aflatoxin B1 - 1 µg/ml in Benzene : Acetonitrile (98:2)
Procedure :

24.  Take 25 g sample in a conical flask, add 100 ml distilled water and
blend for 2 minutes
 Add 150 ml acetone and blend again for 2 minutes
 Filter through Whatman no. 1 filter paper and transfer 75 ml of filtrate
to a conical flask containing 3 g cupric carbonate
 Prepare ferric gel by adding 85 ml of 0.2 M NaOH to 15 ml of 0.41 M
FeCl3. Add this mixture to the flask containing extract and cupric
carbonate
 Mix the contents slowly by swirling movements
 Filter through Whatman no. 1 filter paper
 Take 100 ml of filtrate in a 250 ml separating funnel
 Add 100 ml of 0.03 % H2SO4 and 10 ml of chloroform. Mix the
contents slowly
 Collect the chloroform layer into a 100 ml beaker
 Add again 10 ml of chloroform to the separating funnel and repeat the
above step. Combine both the chloroform extracts
 Take 100 ml potassium wash solution in a separate separating funnel
 Add the chloroform extract to the second separating funnel and mix it
slowly
 Collect the chloroform layer through anhydrous sodium sulfate bed
drop by drop to remove moisture
 Dry the chloroform extract in an oven at 50°C
 Dissolve the dried residue in 0.2 ml chloroform and spot on TLC plate
along with the standard

25.  Compare the flourescence intensities of the sample and standard spots
and identify the ones matching with each other
 Calculate the aflatoxin content in the following way
S x C x D
Aflatoxin content (ppb) = x 1000
T x W
Where, S = Standard which compares with the sample in
fluorescent intensity
C = Concentration of standard (1 µg / ml)
D = Dilution factor in ml
T = Sample which compares with standard in fluorescent
intensity
75 x 100
W = Effective weight 25 x = 4.286 g
250 x 175
4. Analysis of Ochratoxin
(By thin layer chromatography)
Reagents : Sodium bicarbonate and diatomaceous earth mixture :
Add 25 ml of 5 % aqueous NaHCO3 to 50 g

26. diatomaceous earth (Celite 545), mix well and store in
tightly closed container
Solvents : i) Chloroform
ii) Hexane
iii) Acetic acid : benzene ( 2 : 98 )
iv) Acetic acid : benzene (1 : 99 )
Standard : Ochratoxin A 2 µg / ml in acetic acid : benzene (1 : 99)
Apparatus : i) Wrist action / horizontal shaker
ii) Hot water (steam) bath
iii) TLC plates (precoated silica gel plates or
equivalent)
iv) Developing tank / chamber
v) UV viewing cabinet
Procedure :
 Take 25 g of sample in a 250 ml glass stoppered conical flask, add
12.5 ml water and mix
 Add 125 ml chloroform and shake for 1 hour
 Filter through Whatman No.1 filter paper and collect the filtrate
 Plug the bottom of a glass column (2 cm x 30 cm) with glass wool,
put 6 g of NaHCO3 - Celite mixture and tamp firmly with a glass rod

27.  Add 50 ml of chloroform extract to the column and elute until
meniscus reaches top of the NaHCO3 - Celite column
 Wash the column with 70 ml hexane followed by 70 ml chloroform
and discard washings
 Elute Ochratoxin with 100 ml acetic acid : benzene ( 2 : 98)
 Collect the eluate and evaporate on steam bath
 Dissolve the residue in 5 - 10 ml chloroform, transfer to a small vial
( 10 ml capacity) and evaporate on steam bath
 Dissolve the residue in 0.5 ml acetic acid : benzene (1 : 99) by
vigorous shaking
 Spot on TLC plate along with the standard (5, 10, 15 and 20 µ1 or in
any other suitable range)
 Develop the plate using toluene : ethyl acetate : formic acid ( 5 : 4 :
1) in an unequilibrated chamber
 Air dry the plate, view under long wave UV light (365 nm) and
compare the intensity of greenish blue fluorescent spots of the sample
with that of standard spots and identify the spot, matching each other
 Calculate the Ochratoxin A content using the formula
SxYxV
Ochratoxin A µg / kg =
ZxW

28. Where, S = Volume in µl of ochratoxin A standard spot
comparable to Z µl of sample spot
Z = Volume in µl of sample spot comparable to S µl of
ochratoxin A standard
Y = Concentration of ochratoxin A standard (2 µg / ml)
V = Volume (µl) of the dissolved residue before spotting
W= Effective weight of the sample
25 x 50
150
Confirmation
Expose the developed plate to NH3 fumes. Greenish blue fluorescence
of Ochratoxin will turn to bright blue.
Reference
AOAC. 1995. Official methods of analysis. 16th ed. Assoc. Off. Anal.
Chem., Washington, D.C.
5. Analysis of T-2 toxin
(By thin layer chromatography)
Reagents : i) 30 % ammonium sulphate (dissolve 30 g (NH4)2
SO4 in water and make up volume to 100 ml)
ii) Celite 545
iii) Potassium wash solution (dissolve 1.12 g KOH
and 10 g KCl in water and make up volume to 1 lit)

29. iv) Sodium sulphate
v) Silica gel
vi) Methanol : H2SO4 ( 1 : 1 v/v )
Solvents : i) Methanol : water (1 : 1 v/v)
ii) Chloroform
iii) Diethyl ether
iv) Hexane
v) Benzene
vi) Acetone : Benzene (5 : 95 v/v)
vii) Developing solvent mixture - Toluene : ethyl
acetate formic acid (6 : 3: 1 v/v)
Standard : T-2 toxin 50 µg / ml in Benzene or diethyl ether
Apparatus : i) Wrist action / horizontal shaker
ii) TLC plates (precoated silica gel plates
or equivalent)
iii) Developing tank / chamber
iv) UV viewing cabinet
Procedure :
 Take 50 g of sample in a glass stoppered conical flask

30.  Add 250 ml of methanol : water (1 : 1) and shake for 1 hour
 Filter using whatman No.1 filter paper and collect 60 ml of extract
into a beaker
 Add 240 ml 30 % (NH4)2 SO4 and stir vigorously for 1 minute
 Add 20 g of celite and stir for 1 minute
 Filter and collect 200 ml of filtrate
 Transfer filtrate to a separating funnel
 Add 10 ml of chloroform and shake vigorously for 1 minute
 Allow the layers to separate and collect the bottom layer into another
separating funnel
 Repeat the extraction with another 10 ml of chloroform
 Combine both the extracts and add 100 ml of potassium wash solution
 Swirl gently for 30 seconds and let layers separate
 Drain the lower chloroform layer through a bed of Sodium sulphate
(in a funnel) to dry and collect 10 ml of clear filtrate
 Column Preparation : Plug the bottom of a glass column ( 2 cm x
30 cm ) with glass wool and add 5 g anhydrous sodium sulphate. Fill

31. the column to half level with chloroform and add 10 g silica gel.
Wash sides of column with chloroform and stir to eliminate air
bubbles. Drain off chloroform leaving about 7 cm above the upper
level of silica gel. Add 15 g anhydrous sodium sulphate without
disturbing the silica gel. Drain off chloroform to the upper level of
sodium sulphate
 Wash the column serially with 50 ml of diethyl ether and 10 ml of
chloroform and discard the washings
 Mix 10 ml of sample extract with 30 ml of hexane and add to the
column and slowly drain until solvent is about 1 cm above Sodium
sulphate
 Add in succession 30 ml benzene and 40 ml acetone : benzene (5: 95)
and discard both the washings
 Elute T-2 with diethyl ether until 30 ml of eluate is collected and
evaporate the eluate
 Dissolve the residue in 0.5-1.0 ml diethyl ether. Spot on TLC along
with the standard (5-20 µ1 or any other suitable range) and develop
the plate in toluene : ethyl acetate : formic acid (6 : 3 : 1)
 Air dry the plate and spray with methanol : H2SO4 (1 : 1)
 Dry at 110°C for 10 minutes and observe blue fluorescence under
long wave UV light (365 nm)

32.  Compare the intensities of the blue fluorescent spots of the sample
with those of standard and identify the ones matching each other
 Calculate the T-2 content of sample using the following formula
S x Y x V
T - 2 µg / kg =
Z x W
Where, S = Volume in µl of T - 2 standard spot comparable to
Z µl of sample spot
Z = Volume in µl of sample spot comparable to S µl of
T - 2 standard
Y = Concentration of T - 2 standard (50 µg / ml)
V = Volume (µl) of the dissolved residue before spotting
W= Effective weight of the sample
50 x 60 x 200 x 10
250 300 20
References
Romer, T.R., Boling, T.M. and Mc Donald, J.L. 1978. Gas liquid
chromatographic determination of T-2 toxin and diacetoxyscirpenol in
corn and mixed feeds. JAOAC. 61 : 801 - 807.
Rukmini, C. and Bhat, R.V. 1978. Occurrence of T-2 toxin in Fusarium
infested sorghum from India. J. Agric. Food Chem. 26 : 647-649.

33. 6. Analysis of Zearalenone
(By thin layer chromatography)
Reagents : i) Aluminium chloride solution (dissolve 20g
AlCl3 6H2O in 100 ml methanol)
ii) Celite 545
Solvents : i) Chloroform : water mixture ( 10 : 1 v/v)
ii) Hexane
iii) Chloroform
iv) Diethyl ether
v) Benzene
vi) Acetone : benzene ( 5 : 95 v/v)
vii) Acetonitrile
viii) Developing solvent -
Methanol : Chloroform (5 : 95 v/v) or
Acetic acid : Benzene (5 : 95 v/v)
Standard : Zearalenone 50 µg / ml in Benzene
Apparatus : i) Wrist action / horizontal shaker
ii) Hot water (steam) bath
iii) TLC plates (precoated silica gel plates or
equivalent)
iv) Developing tank / chamber
v) UV viewing cabinet

34. Procedure :
 Take 50 g of sample in a glass stoppered conical flask and add 300 ml
of chloroform : water (10 : 1) and 25 g of celite
 Shake for 1 hour and filter using Whatman No. 1 filter paper
 Column Preparation : Plug the bottom of a glass column
(2 cm x 30 cm) with glass wool and add 5 g anhydrous sodium
sulphate. Fill the column about half full with chloroform and add 10 g
silica gel. Wash sides of column with chloroform and stir to
eliminate air bubbles. Drain off chloroform leaving about 7 cm above
the upper level of silica gel. Add 15 g anhydrous sodium sulphate
without disturbing the silica gel. Drain off chloroform to the upper
level of sodium sulphate
 Transfer 50 ml of sample extract together with 150 ml hexane into the
silica gel column
 Drain until the solvent reaches top of the column and discard the
washings
 Wash the column serially with 150 ml of diethyl ether and 150 ml of
benzene and discard both the washings
 Elute Zearalenone with 250 ml of acetone : benzene ( 5 : 95 )
 Add few silica chips to the eluate and evaporate on steam bath,
preferably under gentle stream of N2

35.  Dissolve residue in 10 ml of hexane and transfer quantitatively to a
separating funnel
 Repeat the above step for 3 times using 10 ml of hexane each time
 Finally rinse the residue with 10 ml of acetonitrile and add to the
hexane washes present in the separating funnel. Shake well and let
phases separate
 Collect the lower acetonitrile phase into a 100 ml beaker
 Add another 5 ml acetonitrile to the hexane washes present in the
separating funnel and repeat the above step. Combine both the
acetonitrile fractions.
 Evaporate the combined acetonitrile fractions on steam bath ( under
stream of N2)
 Transfer the residue to a small vial (about 10 ml capacity) using about
5 - 10 ml of chloroform and evaporate as in the previous step
 Add about 0.5 ml benzene to the residue and shake vigorously
 Spot on TLC plate (5, 10, 15, 20 µl or other suitable volumes) along
with the standard and develop the plate in methanol : chloroform
(5 : 95) or acetic acid : benzene (5 : 95)
 Air dry the plate and spray the spots with aluminium chloride
solution, heat at 130° C for 5 min and examine under longwave UV
light (365 nm)

36.  Compare the intensities of the blue fluorescent spots of sample with
those of the standard and identify the ones, matching with each other.
Calculate the Zearalenone content of the sample in the following way
S x Y x V
Zearalenone µg / kg =
Z x W
Where, S = Volume in µl of Zearalenone standard spot
comparable to Z µl of sample spot
Z = Volume in µl of sample spot comparable to S µl of
Zearalenone standard
Y = Concentration of Zearalenone standard (1 µg / ml)
V = Volume (µl) of the dissolved residue before spotting
W= Effective weight of the sample
50
50 x
300
References :
AOAC, 1995. Official methods of analysis. 16th ed. Assoc. Off. Anal.
Chem. Washington, D.C.
ANALYSIS OF CITRININ
Reagents: 1. 4% KCL (4 gms KCL in 100 ml distilled water)
2. 20% H2SO4 (20 ml concentrated H2SO4 + 5%
NaHC03 ( 5 gms of NaHC03 in 100 ml DW)
3. 6 N HCl (185.4 ml of HCl in 1 lt distilled
water)
4. 10% Oxalic acid in methanol (W/V) (10 gm

37. oxalic acid in 100 ml methanol)
Solvents: 1. Acetonitrile
2. Iso octane
3. Chloroform
4. Chloroform – methanol – hexane (64:1:35)
Standards: 1. Citrinin : 10 g/ml in methanol
Procedure:
 Take 25 gms of sample, add 180 ml acetonitile, 20 ml 4% KCl, 2 ml 20%
H2SO4 and shake for 15 minutes
 Filter and collect 100 ml of the filtrate
 Transfer filtrate to separating funnel and add 50ml iso octane. Shake for
1 minute.
 Collect the lower layer, add 50 ml iso octane and repeat the above step.
 Collect the lower layer into another separating funnel, add 25 ml water
and extract with 50 ml chloroform
 Drain the chloroform layer into a separating funnel
 Extract again with two 10 ml portion of chloroform and combine all the
three extracts
 Add 25 ml of 5% NaHC03 to the extract. Shake for 1 minute and drain
off the lower portion.
 Re-extract with two 25 ml 5% NaHC03 portion and repeat the above step.
 Combine the extracted portion in a 600 ml beaker and acidify it with 6N
HCl to pH 1-2, transfer it to separating funnel
 Rinse the beaker with 50 ml CHCl3, and transfer it to seperating funne,
swirl for 30 seconds.

38.  Drain the lower portion, repeat above step with another 50 ml portion of
CHCl3
 Collect the CHCl3 extracts and evaporate to near dryness
 Add 5 ml CHCl3 to the dried extract and filter through fluorocarbon filter
into a 25 ml beaker
 Rinse the beaker twice with small amounts of CHCl3, filter and evaporate
 Dip the TLC plate in 10% oxalic acid solution for 2 min and air dry
overnight
 Spot the sample and standard in 5-20 l range
 Develop plate in chloroform-methanol-hexane (64:1:35) for 45 minutes.
Air dry and observe under long wave UV light for comparing the
intensitites
 Calculate the citrinin content of sample using the following formula
Citrinin, g/kg = S x Y x V
X x W
Where, S = l standard equal to unknown
Y = Concentration of standard (g/ml)
V = Dilution of sample extract (l)
X = l of sample spotted
W= gms of sample represented by final extract
= 25 x 100 = 12.376 g
202
7. Rapid TLC Method of Multi-Mycotoxin Analysis
(Modified Tapia Method)
Reagents : i) 4 % KCl (4 g KCl in 100 ml di. water)

39. ii) 5 N HCl (405.9 ml Conc. HCl in 1 l di. water)
iii) Na2So4 ( anhydrous )
iv) 20 % KOH (20 g KOH in 100 ml di. water)
v) 20 % H2SO4 in ethanol (20 ml Conc. H2SO4
+ 80 ml ethanol)
Solvents : i) Acetonitrile
ii) Hexane
iii) Chloroform
iv) Chloroform : Acetone : Water (88 : 12 : 1)
v) Toluene : Ethyl acetate : Formic acid (5 : 4 : 1)
Standards : Aflatoxin B1 1 µg/ml in Acetonitrile: Benzene (2 : 98)
Ochratoxin A 2 µg/ml in Acetonitrile: Benzene (2: 98)
Zearalenone 50 µg / ml in Benzene
T-2 toxin 50 µg / ml in Ethyl acetate
Sterigmatocystin Citrinin Oosporein
Procedure :
 Take 25 g sample, add 85 ml acetonitrile, 15 ml 4 % KCl and 2 ml 5
N HCl and blend at high speed for 3 minutes
 Filter through Whatman no. 1 filter paper

40.  Transfer 50 ml filtrate into a 250 ml separating funnel
 Add 50 ml water, followed by 50 ml hexane and shake well
 Collect the lower layer, add 50 ml hexane again and repeat the above
step
 Collect the lower layer into another separating funnel and extract with
two 10 ml portions of chloroform
 Drain the chloroform layer through anhydrous Sodium sulphate and
evaporate in oven at 50C
 Dissolve the residue in 0.2 ml chloroform and spot on TLC plate
along with the standards
 Develop plate in chloroform : acetone : water (88 : 12 : 1) in one
direction and toluene : ethyl acetate : formic acid (5 : 4 : 1) in the
second direction
 Spray zone of spots, corresponding to sterigmatocystin with 20 %
aqueous KOH
 Spray zone of spots, corresponding to T-2 toxin with 20 % H2So4 in
ethanol and heat at 110C
 View the spots, identify and quantify the toxins as done with
individual toxins
50
Effective weight of the sample = 25 x = 12.255 g

41. 102

42. 8. Rapid Mycotoxin Test / "ELISA Test"
Principle :
Antibody coated column is used to trap the mycotoxin. This trapped
toxin is then eluted using approximate solvent and quantified in fluorometer.
Equipments :
1. Immuno affinity column
2. Affinity column stand with syringe
3. Cuvette
4. Calibrated Fluorometer
5. Blender
6. Fluted filter paper
Reagent :
1. Test developer
2. Methonol : water (80 : 20 by volume)
3. Mycotoxin wash buffer
Procedure :
 50 gms of sample + 5 gms of NaCl + 100 ml of methanol water (80 :
20)
Note : NaCl is not added in case of Ochra Test
 Blend at high speed and filter
 Pipette filtered extract into clean vessel
Aflatoxin, Ochratoxin : 10 ml

43. Zearalenone : 1 ml
 Dilute with purified water and mix
Aflatoxin, Ochratoxin : 40 ml
Zearalenone : 49 ml
 Filter
 Remove top cap and attach the syringe (cut 1/8 inch bottom of column )
 Pass filtered diluted extract at the rate of 1-2 drops/second
Aflatoxin : 2 ml
Ochratoxin, Zearalenone : 10ml
 Pass water at the rate of 1-2 drops/second
Aflatoxin, Zearalenone : 5 ml
Ochratoxin : First 10 ml Mycotoxin wash buffer,
Later 10 ml distilled water
Courtesy : VICAM
 Elute toxin in glass cuvette
Aflatoxin, Zearalenone : Pass 1 ml HPLC grade methanol
Ochratoxin : Pass 1.5 ml Ochratoxin eluting soln.
 Add 1 ml of developer to the cuvette and mix well
 Read in calibrated fluorometer
9. In vitro evaluation of Mycotoxin binding agents

44. Objective :
To evaluate the mycotoxin binding efficacy of binding agents
(adsorbants) in percentage under simulated GI tract conditions of chicken.
Methodology :
1. Take 2 sets of triplicate samples of 25 g each of compounded broiler /
layer feed in 250 ml Erlenmeyer flasks
2. Add known quantity of mycotoxin to the feed in all the flasks
3. Add the binding agent to the feed in one set of flasks (treated) and leave
the feed in the remaining set of flasks untreated (control)
4. Add 100 ml buffer solution of either 4.5 or 6.5 pH to all the flasks and
mix the contents thoroughly for 30 minutes using a wrist action /
horizontal shaker
5. Incubate the flasks at 37°C for 3 hours
6. Filter the contents and dry the residue at 35-45°C for 2 hours
7. Analyse the dried residue for the unbound toxin content as per the
standard procedure (AOAC, 1995)
8. Calculate the percentage adsorption by subtracting the percent
difference in toxin content in the control flasks from that of the treated
flasks
BT - E T BC - E C

45. Percent toxin adsorption = ------------ x 100 - ------------- x
100
BT BC
Where, BT = Toxin content in the treated flasks at the beginning
ET = Toxin content in the treated flasks at the end
BC = Toxin content in the control flasks at the beginning
EC = Toxin content in the control flasks at the end
Note :
 Buffer composition - For every one litre
4.5 pH : 273 ml 0.1 M citric acid, 227 ml 0.2 M Di Sodium
hydrogen phosphate (Na2HPO4) and 500 ml distilled
water
6.5 pH : 145 ml 0.1 M citric acid, 335 ml 0.2 M Di Sodium
hydrogen phosphate (Na2HPO4) and 500 ml distilled
water

46. 10. In vitro evaluation of Mycotoxin binding agents
in contaminated feeds
Objective :
To evaluate the mycotoxin binding efficacy of binding agents
(adsorbants) in percentage under simulated GI tract conditions of chicken in
contaminated feeds.
Methodology :
1. Analyse the contaminated feed for mycotoxin
2. Take 2 sets of triplicate samples of 25 g each of contaminated feed in
250 ml Erlenmeyer flasks
3. Add the binding agent to the feed in one set of flasks (treated) and leave
the feed in the remaining set of flasks untreated (control)
4. Add 100 ml buffer solution of either 4.5 or 6.5 pH to all the flasks and
mix the contents thoroughly for 30 minutes using a wrist action /
horizontal shaker
5. Incubate the flasks at 37°C for 3 hours
6. Filter the contents and dry the residue at 35-45°C for 2 hours
7. Analyse the dried residue for the unbound toxin content as per the
standard procedure (AOAC, 1995)

47. 8. Calculate the percentage adsorption by substracting the percent
difference in toxin content in the control flasks from that of the treated
flasks in the following way -
BT - E T BC - EC
Percent toxin adsorption = ------------ x 100 - ------------- x 100
BT BC
Where BT = Toxin content in the treated flasks at the beginning
ET = Toxin content in the treated flasks at the end
BC = Toxin content in the control flasks at the beginning
EC = Toxin content in the control flasks at the end

48. 11. List of suppliers of Mycotoxin standards :
Mycotoxin standards are supplied in pure crystalline form by several
companies. They can be obtained from
Sigma Chemical Co.,
P.O. Box 14508,
St. Louis,
Missouri 63178 - 9916,
USA.
Ph : (314) 771 - 5750
(314) 771 - 5757
Internet : http://www. sigma/ sial. com
Sigma - Aldrich Corpn.,
Plot no. 70, Road no. 9,
Jubilee Hills,
Hyderabad - 500 033.
Ph : (040) 244739
Fax : (040) 244794
Orders can also be placed at the following distribution centres
New Delhi : Tel (011) 6899826 / 6897830
Fax (011) 6899827
Mumbai : Tel (022) 6325344 / 6325345
Fax (022) 6268686
Bangalore : Tel (080) 3316659
Fax (080) 3440570

49. AFLATOXIN BINDING ANALYSIS
Aim - To check the binding capacity of binder with aflatoxin B1 standard
and release at different pH (3.2, 6.0,6.5) .
Equipment - U.V. chamber
Chemicals - Chloroform
Trisodium citrate
Hydrochloric acid
Acetone
Aflatoxin B1 (Sigma standard)
TLC Plates (Merck)
Preparation of buffer - Sodium citrate - 0.588gms in 100 ml (0.1 M)
Hydrochloric acid - 0.782ml in 100 ml
Add the hydrochloric acid solution to sodium
citrate solution to get the pH 3.2, 6.0, 6.5.
Assay -
1. Take 400 g level of std aflatoxin B1 in a test tube and
evaporate to dryness.
2. Add different levels of binders to the above test tubes.
3. Add 1ml of 3.2 pH buffer , vortex it and keep for incubation for
30 minutes at 40oC.
4. After 30 minutes centrifuge it. Take out the supernatant in other
test tube and use the sediment for release studies of aflatoxin.
5. To the above supernatant add 400 l of chloroform to extract
the toxin from supernatant.
6. Spot it on the TLC plate and compare the colour intensity with
standard aflatoxin B1. This will give the level of toxin which is
not bound with the binder. The difference between the loaded
toxin and unbound toxin gives the toxin bound with the binder.

50. 7. Use the sediment obtained in step IV for release studies.
8. Add 1ml of buffer (pH 3.2) to the sediment. Vortex and keep
for incubation at 40oC with intermediate shaking. After 30
minutes centrifuge separate the supernatant and sediment. Use
the supernatant to find out the toxin released at pH 3.2 by
adding 400 l of chloroform. Use the sediment for the further
release study at pH 6.0.
9. Add 1ml of buffer (pH 6.0) to the sediment . Vortex and keep
for incubation at 40oC for 60 minutes with intermediate
shaking.
10.After 60 minutes centrifuge it, separate the supernatant and
sediment. Use the supernatant to find out the toxin released at
pH 6.0 by adding 400 l of chloroform.
11.Add 1ml of buffer (pH 6.5) to the sediment, vortex and keep
for incubation for 30 minutes at 40oC.
12.After 30 minutes centrifuge, separate the sediment and
supernatant. Find out the toxin released in the supernatant at
pH 6.5 by adding 400 l of chloroform.
13.Spot on TLC plate and compare the colour intensity with
standard.
Calculations :
A x 400 l x Std concentration
---------------------------------------
B x Total reaction volume
A = l of standard comparible with l of sample
B = l of sample comparible with l of standard
1st supernatant - Unbound toxin with binder
loaded toxin - unbound toxin = bound toxin
2nd supernatant - Release at pH 3.2

51. 3rd supernatant - Release at pH 6.0
4th supernatant - Release at pH 6.5
The difference between the bound toxin and total release toxin will be
toxin retained with binder.
AFLATOXIN ANALYSIS IN FEEDSTUFFS
Reagents 0.2 M NaOH (dissolve 8 g NaOH in water and make up
volume to 1liter)
0.41 m Ferric chloride (dissolve 66.5 g anhydrous FeCl3 in
water and make up volume to 1 lit)
0.03% H2SO4 (0.3 ml con. H2SO4 + 999.7ml water )
Potassium Wash solution (dissolve 1.12 g KOH and 10g
Kcl in water and make up volume to 1liter)
Solvents Aqueous acetone (85 acetone : 15 water)
Chloroform
Chloroform : acetone
Aflatoxin B1 1g/ml in Benzene:acetonitrile(98:2)
Standard Wrist action / horizontal shaker
Apparatus Hot water (steam) bath
TLC plates (precoated silicagel plates or equivalent)
Developing tank /chamber
UV viewing chamber
Procedure
1. Add 250 ml of aqueous acetone to 50 g of sample in a glass stoppered
conical flask and shake for 1 hour.
2. Filter whatman No.1 filter paper.
3. Add 150 ml of filtrate to a conical flask containing 3g cupric carbonate.

52. 4. Add 170 ml of 0.2 M NaOH to 30ml of ferric chloride solution. Add this
mixture to the conical flask for 30 seconds and filter.
5. Collect 250ml of filtrate and transfer it to a separating funnel containing
250 ml of 0.03% H2SO4.
6. Add 10ml of chloroform to the contents of separating funnel and shake
vigorously.
7. Allow the chloroform layer to separate at the bottom of the funnel.
8. Drop the separated Chloroform layer into another separating funnel,
Containing 100 ml of potassium Wash solution
9. Add another 10 ml of Chloroform to the first separating funnel and repeat
the above steps
10.Gently swirl the separating funnel containing the Chloroform extract and
potassium wash solution and allow the layers to separate
11.Drain the Chloroform layer through Na2SO4 and collec
12.Measure 10 ml aliquot of the chloroform extract and evaporate on Water
bath
13.Dissolve the dried residue in Chloroform(about 0.3 ml)
14.Spot on TLC plate along with the Standard
15.Develop the plate using Chloroform : Acetone (85: 15) in an equilibrated
Chamber
16.Air dry the plate , view under long Wave UV light (365nm) and compare
the intensity of the blue fluorescence spots of sample, with that of
standard spots and identify the spots, matching with each other.
17.Calculate the aflatoxin B1 content in the following way
S x Y x V
Aflatoxin B1 g/kg = ----------------
Z x W

53. Where, S = Volume in l of aflatoxin B1 standard spot comparable
to Z l of sample spot
Z = Volume in l of sample spot comparable to S l of
aflatoxin B1 standard
Y = Concentration of aflatoxin B1 standard (1g/ml)
V = Volume (l) of the dissolved residue before spotting
W = Effective weight of the sample :
50 x 150 x 250 x 10
------ ----- ----
250 350 20
PREPARATION OF FEED SAMPLES FOR METHIONINE AND
CYSTINE ANALYSIS USING PERFORMICACID OXIDATION
PROCEDURE
Reagents:

54. 1. Hydrogen peroxide (H2O2), 30% W/W
2. 88% formic acid
3. 48% hydrobromic acid
4. 6N HCL: 50 ml of concentrated hydrochloric acid added to
50 ml double distilled water.
5. DL-Norleucine standard, 25 umole/ml
6. Sodium citrate buffer, pH 2.2
Procedure:
1. Weigh finely ground sample (ground to pass a 40-mesh
sieve) containing 30-
40 mg protein into a 50 ml Erlenmeyer flask. This would be
approximately 60-80 mg for soybean meal samples and
300-350 mg for corn samples when diluting the hydrolysate
100 times.
2. Add 6 ml of cold, freshly prepared, performic acid2 to each
flask, cover and
reactat 00C for 4 hours for amino acids and soluble
proteins, or overnight (16 hours) for proteins that do not
dissolve in the performic acid mixture.

55. 3. Add 0.75 ml of 48% hydrobromic acid with swirling of the
flask in an ice bath.
4. Remove performic acid and hydrobromic acid with swiling of
the flask in an ice bath.
5. Add 0.6 ml DL-norleucine (25 umole/ml) so that after the
final dilution the concentraion of norleucine will be 0.15
umole/ml.
6. Transfer contents into hydrolysate tube (1.5 cm internal
diameter, 15 cm length) by repeated washings with 6 N HCL
so that the final volume is about 10-12 ml.
7. Charge the tubes with nitrogen gas, cover with screw cap
and place in an oven at 1100 for 20 hours.
8. After the tube cools, filter the contents through Whatman
No. 1 filter paper
into a drying tube. Wash hydrolysate tube and filter paper
with double distilled water and collect in the same drying
tube.
9. Dry the filtrate by evaporating with a rotary evaporator
under vacuum, with

56. the water bath temperature at 480C. Dry the filtrate to a
slightly wet residue Wash residue with distilled water and
dry again.
10. Add 20 ml of citrate buffer and mix well.
11. Take one ml from step 10 and dilute to 5 ml with citrate
buffe, pH 2.2. Mis well.
12. Filter the diluted sample liquid using 0.2 micron
Nuclepore3 membrane filter
13. Determine methionine sulfone and cysteic acid by injection
sample into Dionex D-300 amino acid analyzer.

57. ESTIMATION OF SODIUM AND POTASSIUM BY
FLAMEPHOTOMETRY
PRINCIPLE: Depends on the fact that when a metal is
burned in flame, its molecules are energized, emitting a
charateristic colour. The intensity of the colour is proportional
to the concentration of the element in the solution.
Preparation of sample
1. Transfer about 1gm of representative sample into a 100 ml
conical flask.
2. Add 10 ml of conc. HNO3 and keep overnight.
3. Place this flask on the hot plate, allow it to boil, until yellow
flumes subside cool.
4. Add 5ml of triacid mixture and digest on hot plate until it
forms white geletinous semi-liquid mass.
5. Cool the flask and make it to a known volume i.e., 100 ml
by double glass distilled glass distilled water.

58. Preparation of triacid mixture:
Take a clean dry 500 ml pyrex beaker, add 100 ml conc.
HNO3 gently, and add 40 ml perchloric acid and 10 ml of
conc. H2SO4 , carefully. Mix it gently and store it in dry
and clear amber coloured bottle.
Preparation of standard solution:
Stock sodium standard solution :
Dissolve 0.2543 g sodium chloride (dry) in 100 ml
volumetric flask and
make the volume upto the mark (1000 ppm solution).
Stock potassium standard solution :
Dissolve 0.1910 g potassium chloride (KCL) and dissolve
in 100 ml water
(1000 ppm).
Working standard solution :
Sodium
Make the visual dilution of stock standard to get 10, 20,
30, ..... 100 ppm solution by taking 1 ml, 2, 3...... and 10 ml
respectively in 100 ml volumetric flasks and make the volume

59. Potassium
Take 1,.. 2,.. 3,.. and 1 ml of stock standard solution in
100 ml flasks and make the volume by distilled water (1,2,3. .
. . and 10 ppm solution).
UREA TEST
Scope: Applicable to most feeds.
Purpose: Is useful in determining the presence of urea in
feeds.
Apparatus: Test tubes
Reagents: Dissolve 2 grams of
dimethylaminobenzaldehyde (DMAB) in a
solution of 90 ml. of methyl alcohol (methanol) and
10 ml. of concentrated hydrochloric acid.
Procedure:
1. To a teaspoonful of feed in a test tube, add about 3
teaspoonfuls of
water and mix

60. 2. Let settlle and pour the supernatant through a filter
into another
test tube containing approximately a teaspoonful of
DMAB reagent.
3. Any deeping of the yellow color indicates the
presence of urea.

61. QUALITATIVE TEST FOR UREA IN FISH MEAL
Reagents:
1. Urease enzyme solution
2. Standard Urea Solutions (0, 0.5, 1, 1.5,..........5%)
3. Phenol red indicator (0.1%)
or
Cresol red indicator (0.1%)
Procedure:
1. Weigh 10 g. tested sample and add 100 ml of
distilled water. Mix thoroughly and then filter with whatman
No. 41 filter paper.
2. Take 1 ml of tested sample aliquate into white
porcelein spot plate.
3. Add 2-3 drops of phenol red indicator and then add
2-3 drops of urease solution.
4. Stand for 3-5 minutes, if Urea presents solution will
become red purple in contrast to the yellow colour of indicator.

62. Colour can be compared with the colour developed in
standard solution of varying levels of urea.
UREASE TEST (RAPID METHOD)
Occurring in: Soybean meal
Type of analysis : Enzymatic
1. Principle
The urease enzyme activity of soybean meals is
measures qualitatively by the conversion of urea to
ammonia in the presence of Phenol red-indicator.
2. Reagents
A. Sodium hydroxide, 0.1N
B. Sulfuric acid, 0.1N
C. Urea-phenol red solution, 1.4 g phenol red, 70
ml 0.1N NaOH, 350 ml H2O -- 210 g reagent
grade urea, 3000 ml H2O.

63. D. Add 25 ml of amber colored phenol red
solution. Swirl gently to spread sample evenly in dish.
E. Let stand 5 minutes.
3. Method
A. Transfer small amount of Phenol red solution
to beaker. 25 ml is needed for each test.
B. Adjust to amber color with 0.1N HCL. If
solution turns yellow, adjust with 0.1N NaOH.
C. Place one level tablespoon of well mixed
soybean meal into a petri dish.
D. Add 25 ml of amber colored phenol red
solution. Swirl gently to spread sample evenly
in dish.
E. Let stand 5 minutes.
4. Scale
A. Slightly active: Few scattered red particles
B. Moderately active : Surface appears to be
approximately 25% covered with red particles.

64. C. Active : Surface appears to be approximately
50% covered with red particles.
D. Very active : Surface appears to be
approximately 75% covered with red particles.
If more than 75% of the particles are colored
red, the urease activity is estimated to be
greater than 0.2.
E. Overcooked : No visible red color after 5
minutes. Allow sample to set for additional 25
minutes. If still no colored particles, the meal
is overcooked.
F. UREASE TEST
(Association of Oficial Agricultural Methods - U. S. Test)
Objective:
Urease index is used to evaluate soybean meal quality.
The procedure measures only underprocessing and not
overprocessing. The method determines the activity of the
residual urease in the soybean products under the contitions
of the test.

65. Adequate Values: 0.02 to 0.20 pH.
The urease index should not be less than 0.02 or more
than 0.20.
Materials Tested:
Soybean meals, soy flour, and soybean meal feeds except
where urea has been added.
UREASE ACTIVITY PROCEDURE
A. Apparatus:
1. Water bath capable of being maintained at a
temperature of 300 + .5o C
2. pH meter equipped with glass and calomel
electrodes and with provision for testing 5 ml. of solutions. It
should be a precision instrument with a temperature
compensator having a sensitivity of + 0.02 pH units or better.
Follow manufacturer’s instructions for operation of the
instrument and detemination of pH. Calibrate the meter with
standard buffers with values at or near the range at which
measurements are to be made.

66. 3. Test tubes, 20 mm. X 150 mm., fitted with rubber
stoppers.
B. Solutions:
1. Phosphate buffer solution, 0.05M. Dissolve 3.403 g.
of monobasic potassium phosphate (KH2PO4, AR grade) in
approximately 100 ml. of freshly distilled water. Dissolve 4.355
g. of dibasic potassium phosphate (K2HPO4, AR grade) in
approximately 100 ml. of water. Combine the two solutions
and make to 1000 ml. If reagents are pure, pH should be at
7.0. If it is not, adjust to 7.0 with a solution of a strong acid or
base before using. The useful life of the buffer solution,
prepared as described, is less than 90 days.
2. Buffered urea solution. Dissolve 15 g. urea (AR
grade) in 500 ml. of the phosphate buffer solution. Add 5 ml.
of toluene to serve as a presergvative and to prevent mold
formation. Adjust the pH of the urea solution to 7.0 as in B,1.
C. Preparation of Sample:
1. Grind the sample as fine as possible without raising
the temperature and mix. At least 60% of the sample should

67. pass a No. 40 U.S. Standard sieve. Soy flour requires no
grinding but make certain it is well mixed.
D. Procedure:
1. Weigh 0.200 g (+ 0.001 g.) of sample into a test tube
and add 10 ml. of the buffered urea solution.
Stopper, mix and place in water bath at 300C. Do
not invert the tube during the processing of mixing.
2. Prepare a blank by weighing 0.200 g (+ 0.001 G.)
sample into a test tube and to this add 10 ml of the phosphate
buffer solution. Stopper, mix and place in water bath at 300C.
Allow a time interval of 5 minutes between the preparation of
the test and the blank portions. Agitate the contents of each
tube at 5 minute intervals.
3. Remove the test and blank portions from the water
bath after 30 minutes. Transfer the supernatant liquids to a
5.0 ml. beaker, maintaining the 5-minute interval between the
test and blank. Determine the pH of the supernatant liquids at
exactly 5 minutes after removal from the bath.
(See Note 1.)
E. Calculations:

68. 1. The difference between the pH of the test and the
pH of the blank is an index of urease activity.
F. Note:
1. Care must be exercised to prevent contamination of
all glassware or electrodes. Should the pH instrument fail to
deliver a prompt and stable reading, investigate. Frequently,
the flow the electrolyte through the porous fibers in the
calomel electrode may be retarded by a coating of the soluble
fraction from soybean.
2. This method is a modification of the procedure of
Caskey, C. D. and knapp, F. C., Ind. Eng. Chem., Anal.Ed.16,
640 (1944).
DETERMINATION OF TANNINS
INTRODUCTION :
1. Tannins (quercitannin acid and gallotannic acid ) are
plant toxin.

69. 2. Plant feed ingredients containing more than 5% tannins
when used in poultry feeds retards the growth of birds and
reduces egg production.
3. Tannins are determined by using gelatin as a
precipitating agent.
REAGENTS AND CHEMICALS:
1. Indigo carmine (0.6%)
(1.5 g indigo carmine + 100 ml H2O + 12.5 ml H2SO4 -
dilute to 250 ml and filter..
2. Gelatin solution (2.5%) 25 g gelatin+800 ml saturated
Nacl - dissolve and make the Vol. to 1000 ml with sat. Nacl).
3. Acid sodium chorode solution (975 ml saturated Nacl
+ 25 conc. H2SO4
4. Standard Kmno4 (0.1 N)
5. Sodium chloride
6. Kaolin Powder

70. PROCEDURE :
1. Reflux about 5 g fat-free sample in 400 ml water for 30
min.
2. Transfer the contents to a 500 ml volumetric flask and
dilute to 500 ml.
3. Filter through whatman No.1 filter paper and transfer 10
ml filtrate into a 1 litre beaker.
4. Add 25 ml indigo carmine solution and 750 ml water.
5. Titrate against 0.1 N Kmno4. The colour of the solution
changes from dark blue to light green to bright yellow which is
the end point.
6. Let this reading be (A).
7. At step (3), transfer 100 ml filtrate into a 500 ml vol. flask
and add to it 50 ml gelatin solution, 100 ml acid sodium
chloride and 10 g kaolin powder.
8. Shake for several min. and allow it to settle ; filter
through whatman No.1 filter paper.
9. Transfer 25 ml filtrate into a 1 litre beaker; and 25 ml
indigo carmine solution solution and 740 ml water.

71. 10. Repeat step (5)
11. Let this reading be (B).
12. Calculate the percentage of tannins by using the
following formula :
% Tannins = 21 x X x 100
W DM
Where, `W’ is weight of sample,
`X’ is the difference between A and B.
RAPID QUALITATIVE TEST FOR TANNIN
1. Take about of 10 gms of jowar in bottle
2. Add potassium hydroxide and sodium hypochloride
solution.
3. Close the bottle and shake till crystals dissolve.
4. Wait for about 15 minutes

72. 5. If the grain is very dark/black it contains tannin or if
bleached white/light yellow, it contains no tannin.
BOMB CALORIMETRY - PARR ADIABATIC BOMB
CALORIMETER WITH AUTOMATIC WATER TEMPERATURE
CONTROLLER
I. Setting Up
1. Select location free from drafts and rapid
temperature changes.
2. Attach cold water line to heater inlet. Flush heater
using manual hot and cold water switch on controller to
remove air after controller is connected.
3. Connect copper tubings to jacket. Jacket fittings are
non-specific.
4. Attach multiple heater plug to controller.
5. Mount thermistors on thermometers so that
thermistor end is aligned with center of the bulb.
II. Initial Balance

73. 1. After flushing heater, turn swtich on and allow 10-
15 minutes to warm up.
2. Assemble bomb and place in bucket with 2,000 g.
H2O. Place in jacket.
3. Start calorimeter and controller with switch found
on controller.
4. Balancing may be done at any temperature.
Equality of thermometers is not necessary.
5. Open throttling valves about 1 turn.
6. Add hot or cold water with manual control switch
until galvanometer spot swings toward center.
7. Make fine adjustments with throttling valves. They
will probably have to be turned down.
8. Galvanometer should not vary more than 10 marks
in either direction when balanced.
9. Bring thermometers to true temperature equality
using the balance knob. Find true equality by correcting
thermometer readings. (Thermometers may read slightly

74. different after correcting, but it is true temperature which is
important.)
10. Lock balance knob and recheck galvanometer
balance.
11. Check manual #131 for any additional information.
III. Bomb Standardization
1. Weigh 1 gram Benzonic Acid pellet to nearest .1 mg.
in cup.
2. Place cup in circular electrode and attach 10 cm.
fuse wire to each the electrodes. Touch fuse wire to pellet.
3. Assemble bomb and fill with O2 (30 atmospheres
pressure). It may be necessary to wet rubber seal to prevent O2
escape at filling.
4. Place bomb in bucket and bucket in the jacket.
Attach contact wire. Fill bucket with2,000 ml. distilled water
from volumetric flask. Allow same drainage time for all
samples and standards. A drop of acetone on bucket-jacket
connections aids in contacts.

75. 5. Close cover, lower thermometer-thermistor units,
and start calorimeter motor.
6. Bring jacket temperature to near bucket
temperature using manual control. It is usually best to bring
jacket to a few tenths degree C. below the bucket and then
allow the automatic control to equalize the temperature.
7. Allow four minutes at equilibrium temperature.
8. Read initial bucket temperature, ignite. Check
temperature rise. If too much hot overshoot occurs, turn down
valve located on bomb - not throttling vlave. About 1/4 turn
open for this valve is sufficient.
9. After exactly 8 minutes, read final temperature.
10. Release pressure from bomb slowly - over a period
of 1 minute.
11. Rinse inside of bomb with distilled water and titrate
with .0725N Na2CO3 (.001 Kcal./ml.) using methyl orange
indicator.

76. 12. Measure fuse wire remaining on dispenser card. Subtract
this amount from 23 cal. This will give calories used in
ignitioin.
Calculation for Standardization
W= Hm+a+b
t
where W = energy equivalent of calorimeter
H = Heat of combustion benzoic acid
m = mass of benzoic acid pellet
a = ml. Na2CO3 used
b = calories used in ignition - fuse wire
t = difference in true initial and true final
bucket temperature
Note: W must be calculated for each bomb-bucket
combination.
IV. Sample Determinations
1. Make good firm pellet using pelleting machine.
2. Weigh to 0.1 mg. and place in bomb as with
standard.
3. Perform operations identically with standardization
procedure.

77. Calculations:
H= tW - a - b
m
where H = Heat of combustion of sample
t = true temperature difference
W = energy equivalent of calorimeter
a = ml. Na2CO3 used
b = calories used in ignition-fuse wire
m = mass of sample in grams.
Duplicates should agree within .1 kcal/gram and
preferably .05
kcal/gram.
TREATED SEED TEST
Scope : Applicable to grain suspected of having been
treated and to feed containing grain suspected of having been
treated.
Purpose : To detect corn or other grain containing
residues of arasan (tetramethyl thirum disulfide) (TMTD)
(THIRAM).

78. Apparatus :
1. Small flasks: 125 ml. or 250 ml. Erlenmeyer
are suitable.
2. Funnels: short stem, approximately 9 cm.
diameter.
3. Test tubes.
Reagents :
1. Chloroform
2. Cupric choride, analytical reagent.
Procedure:
1. Place 10-15 grams of suspect grain or feed in a
250 ml. flask. Add 25 ml. chloroform and shake for 3 minutes.
2. Filter the solution through cotton placed in
small funnel into a small.
3. Add a few crystals of cupric chloride to the
filtered solution and shake for 3 minutes.

79. 4. Observe the resulting color. If arasan (TMTD)
is present a characteristic amber to brown color will appear. In
case the sample is a mixed feed containing alfalfa or some
other green plant, the color of the solution after filtration in
step 2 may appear yellow-green or green. After addition of the
cupric chloride in step 3, the resulting color will be darker, a
muddy brown if arasan (TMTD) is present, or a muddy green
to yellow-green if no arasan (TMTD) is present in thte sample.
A quantitative method for determining residue of thiram or
treated seed corn is available from the DuPont Company,
Retail Products Section, Grasselli Chemical
Department,Wilmington,Delaware.

80. RANCIDITY TEST
Purpose: To detect rancidity of fats in a feedstuff.
Apparatus: 100 ml. Erlenmeyer flasks.
Reagents:
1. Combine 60 ml. of concentrated glacial acetic acid
(CH3COOH) and 40 ml. of chloroform (CHCL3).
2. Saturated potasium iodide (KI) solution.
3. Starch indicator.
Procedure:
1. Place 5 grams of the sample in the flask and add 40 ml.
of the acetic acid-chloroform mixture.
2. Add 1 ml. of the saturated potassium iodide solution.
3. Agitate thoroughly. Add 50 ml. of water and starch
indicator.
4. The development of a blue color indicates rancidity.

81. DECOMPOSITION TEST (Eber’s Sulfide Test)
Scope: Used for animal and marine products.
Apparatus:
1. 250 cc. Erlenmeyer flask
2. Cork:Must fit tightly in flask and have a split in bottom.
3. 2 x 1/4 inch filter paper.
Reagents:
1. 10% sulphuric acid (H2SO4) solution: 10 ml. of concnetrated
sulfuric acid in 90 ml. of distilled water.
2. Add 50 ml. of the sulphuric acid solution and slightly swirl
solution so all the meat material is thoroughly wet but the
solution does not come up far on side of the flask.
3. Insert one end of the filter paper strip into the split in the
cork so it hangs freely as seen in the figure.
4. Moisten the strip of filter paper with the lead a cetate
solution. Do not have it wet enough so as to drip, for if the
lead acetate comes in direct contact with the sulphuric acid
solution the test will be spoiled.
5. Tightly insert the cork and attached filter paper into the
flask.
6. Let stand in a warm room for 16 hours.
7. If the sample is badly decomposed, the test paper will
darken quickly.

82. FEED MICROSCOPY
A fast, simple and inexpensive method of determining the
adulteration and contamination of feed ingredients and
compound feeds is important in quality control in feed
manufacturing industy.
Objectives
1. To identify and to evaluate feed ingredients and foreign
materials alsone or
in mixture, particularly where the food material is finely
ground.
2. Detection and identification of major adulterants, the
presence of which
may or may not be suspected from consideration of the
results of proximate or specific chemical analysis.
3. Recognition and identification of contaminants such as
presence of fungal,
insect or rodent contamination.

83. Equipments
* Steriomicroscope.
* Test sieves with screen of 10, 20 or 30 mesh.
* Sharp point forceps.
* Petridish.
* Beakers and stainless steel spoon.
* Mortor and pestle
* Chemicals.

84. Appendix 1
Atomic Weights of some Elements
Name Symbol Atomic
weight
Calcium Ca 40.08
Carbon C 12.01
Chlorine Cl 35.45
Chromium Cr 52.00
Cobalt Co 58.93
Copper Cu 63.55
Flourine F 19.00
Iodine I 126.90
Iron Fe 55.85
Magnesium Mg 24.30
Manganese Mn 54.94
Molybdenum Mo 95.94
Nitrogen N 14.00
Oxygen O 16.00
Phosphorus P 30.97
Potassium K 39.10
Selenium Se 78.96
Sodium Na 22.99
Sulphur S 32.06
Zinc Zn 65.37

85. Colour of some common indicators in solution
The colour of some indicators in acid and basic medium
is presented in the following table:
Name of indicator Colour in acidic
Colour in basic Solution/medium
solution/medium
Methyl orange Red/orange/pink
Yellow
Methyl red Red
Yellow
Phenolophthalein Colourless
Pink
Methyl red-bromocresol
greed mixture Pinkish
Green

86. Selection of indicator for use in titration
For the determination of neutralization or end point in an
acid base titration the choice of indicator has been given in the
following table:
Nature of Nature of Indicator
Solution taken acid / alkali of choice
in burette
Weak Weak Methyl orange
Alkali
Weak Strong Mixture of methyl
Acid red and bromocresol
green
Strong Weak Methyl red/Methyl
Acid orange
Strong Strong Methyl orange/
Alkali phenolphtahalein
Weak Strong phenolphtahalein
Alkali

87. Point of Neutralization / End point
The point at which an acid is neutralized by an alkali and
vice-versa is called point of neutralization or end point. It is
determined by the change in the colour of indicator during
titration.
Titration
The process of gradual mixing of a solution of known
normality with the help of a burette into a known volume of
another solution to complete the reaction as indicated by the
change in the colour of indicator is called titration. The volume
of the solution of known normality used for neutralization
(end point) is known as titre.

88. 1.0 TITLE
DETERMINATION OF VETCARE SFCase ACTIVITY USING 3, 5
- DINITROSALICYLIC ACID
2.0 PRINCIPLE
The assay is based on the production of reducing sugar from a solution
of Sun Flower Cake. The reducing sugar is then reacted with 3, 5-
Dinitrosalicylic acid (DNS). The colour change produced is
proportional to the amount of reducing sugar released which inturn is
proportional to the activity of the SFCase present in the sample. The
optical density is read at 540 nm & converted into milligrams of
glucose produced using a standard curve.
3.0 UNIT DEFINITION
One Vetcare unit is defined as the amount of enzyme required to
liberate 0.5 mg of reducing sugar from 200 mg of Standard Sunflower
Cake substrate (6.2) in a total reaction mixture of 4 ml at 40C in 2
hours at pH - 4.8. This corresponds to the release of 0.125 mg
reducing sugar per ml of the reaction mixture.
4.0 REAGENTS
4.1 Citric acid monohydrate
4.2 Glacial Acetic acid
4.3 Sunflower cake
4.4 3, 5 Dinitrosalicylic acid (AR)
4.5 Glucose (AR)
4.6 Potassium Sodium Tartarate Tetrahydrate (AR)
4.7 Sodium Hydroxide
4.8 Phenol
4.9 Sodium metabisulphite
5.0 EQUIPMENT
5.1 Water bath set at 40  1 C
5.2 Timer
5.3 UV-VIS Double beam Spectrophotometer - Shimadzu 1601
5.4 Boiling water bath
5.5 Cooling water bath
5.6 Centrifuge

89. 6.0 PREPARATION OF REAGENTS AND SUBSTRATE
6.1 Citrate buffer - 0.05M , pH - 4.8
Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. Add
NaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and check
pH. This is 1M citrate buffer. When diluted to 0.05M, pH should be 4.8.
Adjust the pH to 4.8 with Acetic acid or NaOH.
6.2 Standard Sunflower Cake substrate preparation
Take 100 gms of SFC and grind it in a mixie to a fine powder. Mix the above
powder with 1 litre of distilled water. Filter the above solution through muslin
cloth. Squeeze out all the water from SFC. Repeat the above procedure by
mixing the residue with 500 ml distilled water. Autoclave the washed SFC at
15 psi for 20 minutes. Dry the SFC at 105C overnight. Grind the dry SFC to a
fine powder. Seive the powder through a Nylobolt filter cloth. Collect the
residue as substrate.
6.3 DNS SOLUTION
a) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litre
flask.
b) Dissolve 24 gms of NaOH in 200 ml of distilled water in a 500 ml flask.
c) Dissolve 5 gm of phenol in 80 ml of distilled water in a 200 ml flask.
d) Add 20 ml of DNS solution (a) to the phenol solution (c).
e) Add 180 ml of NaOH solution (b) slowly to the remainder of the solution (a)
and stir until the solution is homogenous.
f) Add 200 gm of Sodium potassium tartarate to the resulting solution (e).
g) Add 5 gms of NaHSO3 to the solution (d) and dissolve completely.
h) Mix the solutions (f) and (g) in a 2 litre flask and make the volume to 1 litre.
i) Filter the solution (h) through a bed of absorbent cotton and add 5gms of
Na2S2O5.
j) Store this final solution (i) in a amber bottle.

90. 7.0 PROCEDURE FOR STANDARD GRAPH
Prepare a stock solution by dissolving 300 mg of glucose with citrate
buffer pH 4.8 in a 100 ml volumetric flask. This stock glucose is stable for
3 months.
Prepare working dilutions from stock according to table below.
Standard Volume of mg of glucose DNS Water
glucose buffer (ml) in tube (ml) (ml) (ml)
soln (ml)
0.0 1.5 0.0 3.0 15
Boil
0.1 1.4 0.3 3.0 15
for
0.2 1.3 0.6 3.0 15
5
0.4 1.1 1.2 3.0 15
minu-
0.6 0.9 1.8 3.0 15
tes
0.8 0.7 2.4 3.0 15
1.0 0.5 3.0 3.0 15
8.0 ASSAY PROCEDURE
a) Take 1 gm of Standard Sunflower cake powder in 100 ml Erlenmeyer
flask.
b) Prepare the enzyme dilution of 10 to 15 mg/ml and make up to volume
with 0.05M citrate buffer of pH 4.8.
c) Make the reaction volume to 20 ml with 0.05M citrate buffer, pH 4.8 by
using 1 ml of the enzyme dilution.
d) Incubate this suspended mixture for 2 hours in a shaking waterbath
moving at 140 rpm at a constant temperature of 40C.
e) After 2 hours incubation, take the suspension out of the water bath.
f) Stop the reaction by keeping the flask in a boiling water bath for 5 - 10
minutes.
g) Similarly prepare the enzyme blank by stopping the reaction immediately
after addition of enzyme solution.

91. h) Centrifuge the suspension at 10,000 rpm for 10 minutes.
i) Use the supernatent for estimating the reducing sugar.
j) Estimation of reducing sugar : Take 1 ml of buffer in a test tube. Add
0.5 ml of supernatent. Add 3ml DNS. boil for 5 minutes in a boiling
water bath. Cool it. Add 15 ml of distilled water.
k) Reagent blank : Instead of taking 0.5 ml of enzyme treated supernatent,
take 0.5 ml of distilled water.
l) Read all the tubes against reagent blank at 540 nm.
m) Substract the enzyme blank reading from Sample reading. Find out the
amount of sugar released with the help of Standard Graph.
n) Reading should be between 0.01 to 0.1.
9.0
CALCULATIONS
(1) Total Sugar produced = Sugar produced in 0.5 ml of
supernatent X 100.
Total sugar released in the reaction mixture X 1000
(2) VSU/g = ---------------------------------------------------------------
0.5 X Amount of Enzyme used in the reaction
mixture

92. 1.0 AIM
DETERMINATION OF CELLULASE (FILTER PAPER UNITS) IN
THE GIVEN SAMPLE USING 3, 5 - DINITRO SALICYLIC ACID
2.0 PRINCIPLE
The assay is based on the production of reducing sugar from a filter
paper (Whatman no.1). The reducing sugar is then reacted with 3, 5-
Dinitrosalicylic acid (DNS). The colour developed is proportional to
the amount of reducing sugar released which inturn is proportional to
the activity of the cellulase present in the sample. The colour
developed is read at 540 nm & converted into milligrams of glucose
produced using a standard curve.
3.0 UNIT DEFINITION
One filter unit is defined as the amount of enzyme required to liberate
1 mole of reducing sugar per min from 50 mg of filter paper
(Whatman no.1) at 50C at pH - 4.8.
4.0 REAGENTS
Citric acid monohydrate
Sodium Hydroxide (AR)
3, 5 Dinitrosalicylic acid (AR)
D (+) Glucose (AR)
Potassium Sodium Tartarate Tetrahydrate (AR)
Phenol
Sodium metabisulphite
Whatman no.1 filter paper
5.0 EQUIPMENT
Water bath set at 40  1 C
Timer
Visible range Spectrophotometer set at 540 nm
Boiling water bath
Cooling water bath
Centrifuge

93. 6.0 PREPARATION OF REAGENTS AND SUBSTRATE
a) Citrate buffer - 0.05M , pH - 4.8
Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. Add
NaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and check
pH. This is 1M citrate buffer. Prepare 0.05 M buffer solution as a working
solution. Adjust the pH to 4.8 with Acetic acid or NaOH.
b) DNS SOLUTION
1) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litre
flask.
2) Dissolve 24 gms of NaOH in 200 ml of distilled water in a 500 ml flask.
3) Dissolve 5 gm of phenol in 80 ml of distilled water in a 200 ml flask.
4) Add 20 ml of DNS solution (1) to the phenol solution (3).
5) Add 180 ml of NaOH solution (2) slowly to the remainder of the solution (1)
and stir until the solution is homogenous.
6) Add 200 gm of Sodium potassium tartarate to the resulting solution (5).
7) Add 5 gms of Sodium metatrisulphite (NaHSO3) to the solution (4) and
dissolve completely.
8) Mix the solutions (6) and (7) and make the volume to 1 litre.
9) Filter the solution through absorbent cotton and add 5gms of Na2S2O5.
10)Store this final solution in a dark bottle.
c) Substrate - Whatman no.1 filter paper - 50mg (1 cm x 6 cm)
7.0 PROCEDURE FOR STANDARD GRAPH
a) Prepare a stock standard glucose solution by dissolving 300 mg of glucose with
0.05 M citrate buffer (pH 4.8) in a 100 ml volumetric flask. This stock glucose
is stable for 3 months.

94. b) Prepare working dilutions from stock according to table below.
Standard Volume of mg of glucose DNS Water
glucose buffer (ml) in tube (ml) (ml) (ml)
soln (ml)
0.0 1.5 -- 3.0 15
0.1 1.4 0.3 3.0 15
0.2 1.3 0.6 3.0 Boil 15
0.3 1.2 0.9 3.0 for 15
0.4 1.1 1.2 3.0 5 15
0.5 1.0 1.5 3.0 minu- 15
0.6 0.9 1.8 3.0 tes 15
0.7 0.8 2.1 3.0 15
0.8 0.7 2.4 3.0 15
0.9 0.6 2.7 3.0 15
1.0 0.5 3.0 3.0 15
8.0 ASSAY PROCEDURE
a) Prepare different dilutions of enzymes.
b) Take 1ml of citrate buffer in different test tubes. To each tube add 0.5ml of
different enzyme dilutions. Incubate at 50C for 5 minutes.
c) To the above test tubes, add 50mg of Whatman no.1 filter paper. Vortex it.
d) Incubate the tubes in water bath at 50C for 1 hour.
e) After 1 hour, cool the tubes under flowing tap water.
f) Add 3ml DNS. Boil for 5 minutes in a boiling water bath. Cool and add
15ml of distilled water.
g) Reagent blank : Instead of taking 0.5ml enzyme dilution, take 0.5ml citrate
buffer.

95. h) Enzyme blank : Take 1ml of citrate buffer and incubate at 50C for 1 hour.
Add 3ml DNS followed by 0.5ml of enzyme dilution and boil for 5 minutes,
cool and make up the volume to 15ml.
Enzyme Buffer DNS Enzyme Distilled
dilution (ml) (ml) dilution water
(ml) (ml) (ml)
Enzyme 0.5 1.0 3.0 -- 15
Sample Incubate Boil for
for 5
Enzyme -- 1.0 1 hour 3.0 0.5 minutes 15
Blank at
50C
Reagent -- 1.5 3.0 -- 15
Blank
i) Read enzyme sample and enzyme blank tubes against reagent blank at
540nm.
j) Subtract the enzyme blank reading from enzyme sample reading. Find out
the amount of sugar released with the help of Standard graph.
9.0 CALCULATIONS
mg of glucose released X dilution factor X 1000
FPU/gm = ------------------------------------------------------------
180 X 60 X 0.5

96. 1.0 AIM
DETERMINATION OF XYLANASE (HEMICELLULASE)
ACTIVITY USING 3, 5 - DINITRO SALICYLIC ACID
2.0 PRINCIPLE
The assay is based on the production of reducing sugar from a solution
of Xylan. The reducing sugar then reacts with 3, 5-Dinitro salicylic
acid (DNS). The colour change produced is proportional to the
amount of reducing sugar (expressed as Xylose) released which inturn
is proportional to the activity of the Xylanase present in the sample.
The optical density is read at 540 nm and converted into micromoles
of Xylose produced using a standard curve.
UNIT DEFINITION
3.0
One unit is the amount of enzyme required to produce 1 micromole of
reducing sugar (as Xylose) per minute at pH - 4.8 at 40C.
REAGENTS
4.0 Citric acid monohydrate
Glacial Acetic acid
Oat spelt xylan (Sigma)
3, 5 Dinitrosalicylic acid (AR)
D (+) - Xylose (AR)
Potassium Sodium Tartarate Tetrahydrate (AR)
Sodium Hydroxide (Analytical grade)
EQUIPMENT
5.0 Water bath set at 40  1 C
Timer
Visible range Spectrophotometer set at 540 nm
Boiling water bath
Cooling water bath
Grade A Glassware
Whirl mixer
Glass or automatic pipettes
Centrifuge

97. 6.0 PREPARATION OF REAGENTS AND SUBSTRATE
a) 0.05 M Citrate buffer (pH - 4.8)
Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. Add
NaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and check
pH. This is 1M stock citrate buffer. Prepare 0.05 M buffer solution as a
working solution from the stock solution. Adjust the pH to 4.8 with Acetic acid
or NaOH.
b) XYLAN SUBSTRATE
Take 2g of Xylan in the mortor pestle and grind it with 1-2 beads of Sodium
Hydroxide and then slowly add citrate buffer to it till the volume reaches
approximately 80 ml. Adjust solution pH to 4.8  0.05 with Glacial acetic acid.
Transfer the solution to 100ml volumetric flask and make up to the mark with
citrate buffer. Store in refrigerator at 2-8C. This solution must be prepared
daily.
c) DNS SOLUTION
1) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litre
flask.
2) Dissolve 24 gms of NaOH in 200 ml of distilled water in a 500 ml flask.
3) Dissolve 5 gm of phenol in 80 ml of distilled water in a 200 ml flask.
4) Add 20 ml of DNS solution (1) to the phenol solution (3).
5) Add 180 ml of NaOH solution (2) slowly to the remainder of the solution (1)
and stir until the solution is homogenous.
6) Add 200 gm of Sodium potassium tartarate to the resulting solution (5).
7) Add 5 gms of Sodium metatrisulphite (NaHSO3) to the solution (4) and
dissolve completely.
8) Mix the solutions (6) and (7) and make the volume to 1 litre.
9) Filter the solution through absorbent cotton and add 5gms of Na2S2O5.
10)Store this final solution in a dark bottle.