It is been an endeavor to constantly update the technicalinputs to our esteemed customers in VETCARE. This

effort is an extension of the programme

This manual covers the details of analysis of manyingredients used in the feed along with the Mycotoxin

Estimation and Toxin binding estimation. Also find theinteresting Enzyme analysis in this manual

VETCARE

IS - 40, KHB Industrial AreaYelahanka New TownBangalore - 560 064, INDIAPh. : 8460060, 8462055/56 Fax: 8461240E-mail : btan@giasbg01.vsnl.net.inWeb : www.vetcareindia.com

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

CONTENTS

Sl.No.

Title Page 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-Mycotoxinanalysis

34 - 36

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 agentsin 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

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. 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

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 PoultryScience Association (IB),

Department of Poultry ScienceUniversity of Agricultural Sciences

Hebbal, Bangalore - 560 024, INDIA

Preface

The Department of Poultry Science, University of AgriculturalSciences, Bangalore, has been the pioneer in the field of MycotoxinResearch in the Country. Several breakthroughs have been achieved here incounteracting Mycotoxins in Animal and Poultry feeds. Further Research ison in the direction to achieve total solution to the problem of mycotoxins infoods and feeds, sooner or later.

Mycotoxins are always of concern, both in tropical and temperateclimates. Mycotoxins in feed remain to be a potential threat to the health,productivity and livability of poultry and livestock and are also of seriouspublic health concern. Such a situation requires that there be adequatelaboratory facilities everywhere to identify and evaluate the problemprecisely and take necessary steps for good.

While technology for Mycotoxin research is available, much of thedata is relevant to developed countries which make use of sophisticatedinstruments and techniques. And there will always be a lot of hurdles for aperson working on a small scale laboratory to follow those methods.

Having faced such problems initially and developed / adoptedmethods that suit our conditions, we felt that such problems should not bethe excuse. Hence we felt the need of a manual that gives, simple, but indetail, information on the basic laboratory work involved in Mycotoxinresearch. And the fruit of our effort in this direction is this manual.

We trust this manual would be quite useful to the Post Graduatestudents in Animal Sciences and Biological Sciences. Food and FeedAnalysis & Quality Control Laboratories, Feed Manufacturers, ResearchInstitutions, Food Processing Industry, Food Exporters, Private VeterinaryDiagnostic Laboratories etc.

Date : March, 1999 Prof. G. DevegowdaPlace : Bangalore, India Dr. M.V.L.N. Raju

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

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

Table 1. Concentrations of the Mycotoxin standardsrequired (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

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

3,160CF = (normal value : 0.95 - 1.05)

E

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 andabsorptivity 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

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,compounded feed)

3 kg

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

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.)

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.

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 originRf =

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

(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.

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.

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.

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

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

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

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 :

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

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 DAflatoxin content (ppb) = x 1000

T x W

Where, S = Standard which compares with the sample influorescent intensity

C = Concentration of standard (1 µg / ml)

D = Dilution factor in ml

T = Sample which compares with standard in fluorescentintensity

75 x 100W = 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

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 orequivalent)

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

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

S x Y x VOchratoxin A µg / kg =

Z x W

Where, S = Volume in µl of ochratoxin A standard spotcomparable to Z µl of sample spot

Z = Volume in µl of sample spot comparable to S µl ofochratoxin 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 50150

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)

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

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

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)

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 VT - 2 µg / kg =

Z x W

Where, S = Volume in µl of T - 2 standard spot comparable toZ µl of sample spot

Z = Volume in µl of sample spot comparable to S µl ofT - 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 10250 300 20

References

Romer, T.R., Boling, T.M. and Mc Donald, J.L. 1978. Gas liquidchromatographic determination of T-2 toxin and diacetoxyscirpenol incorn and mixed feeds. JAOAC. 61 : 801 - 807.

Rukmini, C. and Bhat, R.V. 1978. Occurrence of T-2 toxin in Fusariuminfested sorghum from India. J. Agric. Food Chem. 26 : 647-649.

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) orAcetic 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 orequivalent)

iv) Developing tank / chamber

v) UV viewing cabinet

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

Dissolve residue in 10 ml of hexane and transfer quantitatively to aseparating 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)

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 VZearalenone µg / kg =

Z x W

Where, S = Volume in µl of Zearalenone standard spotcomparable to Z µl of sample spot

Z = Volume in µl of sample spot comparable to S µl ofZearalenone standard

Y = Concentration of Zearalenone standard (1 µg / ml)

V = Volume (µl) of the dissolved residue before spotting

W= Effective weight of the sample

5050 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

oxalic acid in 100 ml methanol)

Solvents: 1. Acetonitrile2. Iso octane3. Chloroform4. 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 for1 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 waterand 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 thethree extracts

Add 25 ml of 5% NaHC03 to the extract. Shake for 1 minute and drainoff 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 6NHCl 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.

Drain the lower portion, repeat above step with another 50 ml portion ofCHCl3

Collect the CHCl3 extracts and evaporate to near dryness

Add 5 ml CHCl3 to the dried extract and filter through fluorocarbon filterinto 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 dryovernight

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 theintensitites

Calculate the citrinin content of sample using the following formula

Citrinin, g/kg = S x Y x VX x W

Where, S = l standard equal to unknownY = Concentration of standard (g/ml)V = Dilution of sample extract (l)X = l of sample spottedW= gms of sample represented by final extract

= 25 x 100 = 12.376 g202

7. Rapid TLC Method of Multi-Mycotoxin Analysis(Modified Tapia Method)

Reagents : i) 4 % KCl (4 g KCl in 100 ml di. water)

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

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 50C

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 110C

View the spots, identify and quantify the toxins as done with

individual toxins

50Effective weight of the sample = 25 x = 12.255 g

102

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

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

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 - ET BC - EC

Percent toxin adsorption = ------------ x 100 - ------------- x100

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

10. In vitro evaluation of Mycotoxin binding agentsin 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)

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 - ET BC - EC

Percent toxin adsorption = ------------ x 100 - ------------- x 100BT 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

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) 244739Fax : (040) 244794

Orders can also be placed at the following distribution centres

New Delhi : Tel (011) 6899826 / 6897830Fax (011) 6899827

Mumbai : Tel (022) 6325344 / 6325345Fax (022) 6268686

Bangalore : Tel (080) 3316659Fax (080) 3440570

AFLATOXIN BINDING ANALYSIS

Aim - To check the binding capacity of binder with aflatoxin B1 standardand release at different pH (3.2, 6.0,6.5) .

Equipment - U.V. chamber

Chemicals - ChloroformTrisodium citrateHydrochloric acidAcetoneAflatoxin 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 sodiumcitrate 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 andevaporate 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 for30 minutes at 40oC.

4. After 30 minutes centrifuge it. Take out the supernatant in othertest tube and use the sediment for release studies of aflatoxin.

5. To the above supernatant add 400 l of chloroform to extractthe toxin from supernatant.

6. Spot it on the TLC plate and compare the colour intensity withstandard aflatoxin B1. This will give the level of toxin which isnot bound with the binder. The difference between the loadedtoxin and unbound toxin gives the toxin bound with the binder.

7. Use the sediment obtained in step IV for release studies.

8. Add 1ml of buffer (pH 3.2) to the sediment. Vortex and keepfor incubation at 40oC with intermediate shaking. After 30minutes centrifuge separate the supernatant and sediment. Usethe supernatant to find out the toxin released at pH 3.2 byadding 400 l of chloroform. Use the sediment for the furtherrelease study at pH 6.0.

9. Add 1ml of buffer (pH 6.0) to the sediment . Vortex and keepfor incubation at 40oC for 60 minutes with intermediateshaking.

10.After 60 minutes centrifuge it, separate the supernatant andsediment. Use the supernatant to find out the toxin released atpH 6.0 by adding 400 l of chloroform.

11.Add 1ml of buffer (pH 6.5) to the sediment, vortex and keepfor incubation for 30 minutes at 40oC.

12.After 30 minutes centrifuge, separate the sediment andsupernatant. Find out the toxin released in the supernatant atpH 6.5 by adding 400 l of chloroform.

13.Spot on TLC plate and compare the colour intensity withstandard.

Calculations :A x 400 l x Std concentration---------------------------------------B x Total reaction volume

A = l of standard comparible with l of sampleB = l of sample comparible with l of standard

1st supernatant - Unbound toxin with binderloaded toxin - unbound toxin = bound toxin

2nd supernatant - Release at pH 3.2

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 betoxin retained with binder.

AFLATOXIN ANALYSIS IN FEEDSTUFFS

Reagents

Solvents

StandardApparatus

Procedure

0.2 M NaOH (dissolve 8 g NaOH in water and make upvolume to 1liter)0.41 m Ferric chloride (dissolve 66.5 g anhydrous FeCl3 inwater 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 10gKcl in water and make up volume to 1liter)

Aqueous acetone (85 acetone : 15 water)ChloroformChloroform : acetoneAflatoxin B1 1g/ml in Benzene:acetonitrile(98:2)

Wrist action / horizontal shakerHot water (steam) bathTLC plates (precoated silicagel plates or equivalent)Developing tank /chamberUV viewing chamber

1. Add 250 ml of aqueous acetone to 50 g of sample in a glass stopperedconical 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.

4. Add 170 ml of 0.2 M NaOH to 30ml of ferric chloride solution. Add thismixture to the conical flask for 30 seconds and filter.

5. Collect 250ml of filtrate and transfer it to a separating funnel containing250 ml of 0.03% H2SO4.

6. Add 10ml of chloroform to the contents of separating funnel and shakevigorously.

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 repeatthe above steps

10.Gently swirl the separating funnel containing the Chloroform extract andpotassium 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 Waterbath

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 equilibratedChamber

16.Air dry the plate , view under long Wave UV light (365nm) and comparethe intensity of the blue fluorescence spots of sample, with that ofstandard spots and identify the spots, matching with each other.

17.Calculate the aflatoxin B1 content in the following way

S x Y x VAflatoxin B1 g/kg = ----------------Z x W

Where, S = Volume in l of aflatoxin B1 standard spot comparableto Z l of sample spot

Z = Volume in l of sample spot comparable to S l ofaflatoxin B1 standard

Y = Concentration of aflatoxin B1 standard (1g/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:

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.

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

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.

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.

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 mlvolumetric 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

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

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.

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.

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.

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.

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.

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.

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

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:

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.

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

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.

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

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

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

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.

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.

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 = H m + 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.

Calculations:

H = t W - 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 andpreferably .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).

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.

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.

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.

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 concnetratedsulfuric acid in 90 ml. of distilled water.

2. Add 50 ml. of the sulphuric acid solution and slightly swirlsolution so all the meat material is thoroughly wet but thesolution does not come up far on side of the flask.

3. Insert one end of the filter paper strip into the split in thecork so it hangs freely as seen in the figure.

4. Moisten the strip of filter paper with the lead a cetatesolution. Do not have it wet enough so as to drip, for if thelead acetate comes in direct contact with the sulphuric acidsolution the test will be spoiled.

5. Tightly insert the cork and attached filter paper into theflask.

6. Let stand in a warm room for 16 hours.

7. If the sample is badly decomposed, the test paper willdarken quickly.

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.

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.

Appendix 1

Atomic Weights of some Elements

Name Symbol Atomic

weight

Calcium

Carbon

Chlorine

Chromium

Cobalt

Copper

Flourine

Iodine

Iron

Magnesium

Manganese

Molybdenum

Nitrogen

Oxygen

Phosphorus

Potassium

Selenium

Sodium

Sulphur

Zinc

Ca

C

Cl

Cr

Co

Cu

F

I

Fe

Mg

Mn

Mo

N

O

P

K

Se

Na

S

Zn

40.08

12.01

35.45

52.00

58.93

63.55

19.00

126.90

55.85

24.30

54.94

95.94

14.00

16.00

30.97

39.10

78.96

22.99

32.06

65.37

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 acidicColour in basic Solution/mediumsolution/medium

Methyl orange Red/orange/pinkYellow

Methyl red RedYellow

Phenolophthalein ColourlessPink

Methyl red-bromocresol

greed mixture PinkishGreen

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 IndicatorSolution taken acid / alkali of choicein burette

Weak Weak Methyl orangeAlkali

Weak Strong Mixture of methylAcid red and bromocresol

green

Strong Weak Methyl red/MethylAcid orange

Strong Strong Methyl orange/Alkali phenolphtahalein

Weak Strong phenolphtahaleinAlkali

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.

1.0

2.0

3.0

4.0

4.14.24.34.44.54.64.74.84.9

5.0

5.15.25.35.45.55.6

TITLE

DETERMINATION OF VETCARE SFCase ACTIVITY USING 3, 5- DINITROSALICYLIC ACID

PRINCIPLE

The assay is based on the production of reducing sugar from a solutionof Sun Flower Cake. The reducing sugar is then reacted with 3, 5-Dinitrosalicylic acid (DNS). The colour change produced isproportional to the amount of reducing sugar released which inturn isproportional to the activity of the SFCase present in the sample. Theoptical density is read at 540 nm & converted into milligrams ofglucose produced using a standard curve.

UNIT DEFINITION

One Vetcare unit is defined as the amount of enzyme required toliberate 0.5 mg of reducing sugar from 200 mg of Standard SunflowerCake substrate (6.2) in a total reaction mixture of 4 ml at 40C in 2hours at pH - 4.8. This corresponds to the release of 0.125 mgreducing sugar per ml of the reaction mixture.

REAGENTS

Citric acid monohydrateGlacial Acetic acidSunflower cake3, 5 Dinitrosalicylic acid (AR)Glucose (AR)Potassium Sodium Tartarate Tetrahydrate (AR)Sodium HydroxidePhenolSodium metabisulphite

EQUIPMENT

Water bath set at 40 1 CTimerUV-VIS Double beam Spectrophotometer - Shimadzu 1601Boiling water bathCooling water bathCentrifuge

6.0

6.1

6.2

6.3

PREPARATION OF REAGENTS AND SUBSTRATE

Citrate buffer - 0.05M , pH - 4.8

Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. AddNaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and checkpH. 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.

Standard Sunflower Cake substrate preparation

Take 100 gms of SFC and grind it in a mixie to a fine powder. Mix the abovepowder with 1 litre of distilled water. Filter the above solution through muslincloth. Squeeze out all the water from SFC. Repeat the above procedure bymixing the residue with 500 ml distilled water. Autoclave the washed SFC at15 psi for 20 minutes. Dry the SFC at 105C overnight. Grind the dry SFC to afine powder. Seive the powder through a Nylobolt filter cloth. Collect theresidue as substrate.

DNS SOLUTION

a) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litreflask.

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 ofNa2S2O5.

j) Store this final solution (i) in a amber bottle.

7.0 PROCEDURE FOR STANDARD GRAPH

Prepare a stock solution by dissolving 300 mg of glucose with citratebuffer pH 4.8 in a 100 ml volumetric flask. This stock glucose is stable for3 months.

Prepare working dilutions from stock according to table below.

Standardglucose

soln (ml)

Volume ofbuffer (ml)

mg of glucosein tube (ml)

DNS(ml)

Water(ml)

0.0

0.1

0.2

0.4

0.6

0.8

1.0

1.5

1.4

1.3

1.1

0.9

0.7

0.5

0.0

0.3

0.6

1.2

1.8

2.4

3.0

3.0

3.0

3.0

3.0

3.0

3.0

3.0

Boil

for

5

minu-

tes

15

15

15

15

15

15

15

8.0 ASSAY PROCEDURE

a) Take 1 gm of Standard Sunflower cake powder in 100 ml Erlenmeyerflask.

b) Prepare the enzyme dilution of 10 to 15 mg/ml and make up to volumewith 0.05M citrate buffer of pH 4.8.

c) Make the reaction volume to 20 ml with 0.05M citrate buffer, pH 4.8 byusing 1 ml of the enzyme dilution.

d) Incubate this suspended mixture for 2 hours in a shaking waterbathmoving at 140 rpm at a constant temperature of 40C.

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 - 10minutes.

g) Similarly prepare the enzyme blank by stopping the reaction immediatelyafter addition of enzyme solution.

9.0

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. Add0.5 ml of supernatent. Add 3ml DNS. boil for 5 minutes in a boilingwater 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 theamount of sugar released with the help of Standard Graph.

n) Reading should be between 0.01 to 0.1.

CALCULATIONS

(1) Total Sugar produced = Sugar produced in 0.5 ml ofsupernatent X 100.

Total sugar released in the reaction mixture X 1000(2) VSU/g = ---------------------------------------------------------------

0.5 X Amount of Enzyme used in the reactionmixture

1.0

2.0

3.0

4.0

5.0

AIM

DETERMINATION OF CELLULASE (FILTER PAPER UNITS) INTHE GIVEN SAMPLE USING 3, 5 - DINITRO SALICYLIC ACID

PRINCIPLE

The assay is based on the production of reducing sugar from a filterpaper (Whatman no.1). The reducing sugar is then reacted with 3, 5-Dinitrosalicylic acid (DNS). The colour developed is proportional tothe amount of reducing sugar released which inturn is proportional tothe activity of the cellulase present in the sample. The colourdeveloped is read at 540 nm & converted into milligrams of glucoseproduced using a standard curve.

UNIT DEFINITION

One filter unit is defined as the amount of enzyme required to liberate1 mole of reducing sugar per min from 50 mg of filter paper(Whatman no.1) at 50C at pH - 4.8.

REAGENTS

Citric acid monohydrateSodium Hydroxide (AR)3, 5 Dinitrosalicylic acid (AR)D (+) Glucose (AR)Potassium Sodium Tartarate Tetrahydrate (AR)PhenolSodium metabisulphiteWhatman no.1 filter paper

EQUIPMENT

Water bath set at 40 1 CTimerVisible range Spectrophotometer set at 540 nmBoiling water bathCooling water bathCentrifuge

6.0

a)

b)

c)

7.0

a)

PREPARATION OF REAGENTS AND SUBSTRATE

Citrate buffer - 0.05M , pH - 4.8

Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. AddNaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and checkpH. This is 1M citrate buffer. Prepare 0.05 M buffer solution as a workingsolution. Adjust the pH to 4.8 with Acetic acid or NaOH.

DNS SOLUTION

1) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litreflask.

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) anddissolve 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.

Substrate - Whatman no.1 filter paper - 50mg (1 cm x 6 cm)

PROCEDURE FOR STANDARD GRAPH

Prepare a stock standard glucose solution by dissolving 300 mg of glucose with0.05 M citrate buffer (pH 4.8) in a 100 ml volumetric flask. This stock glucoseis stable for 3 months.

b) Prepare working dilutions from stock according to table below.

Standardglucose

soln (ml)

Volume ofbuffer (ml)

mg of glucosein tube (ml)

DNS(ml)

Water(ml)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.5

1.4

1.3

1.2

1.1

1.0

0.9

0.8

0.7

0.6

0.5

--

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3.0

3.0

3.0

3.0

3.0

3.0

3.0

3.0

3.0

3.0

3.0

3.0

Boil

for

5

minu-

tes

15

15

15

15

15

15

15

15

15

15

15

8.0

a)

b)

c)

d)

e)

f)

g)

ASSAY PROCEDURE

Prepare different dilutions of enzymes.

Take 1ml of citrate buffer in different test tubes. To each tube add 0.5ml ofdifferent enzyme dilutions. Incubate at 50C for 5 minutes.

To the above test tubes, add 50mg of Whatman no.1 filter paper. Vortex it.

Incubate the tubes in water bath at 50C for 1 hour.

After 1 hour, cool the tubes under flowing tap water.

Add 3ml DNS. Boil for 5 minutes in a boiling water bath. Cool and add15ml of distilled water.

Reagent blank : Instead of taking 0.5ml enzyme dilution, take 0.5ml citratebuffer.

h) Enzyme blank : Take 1ml of citrate buffer and incubate at 50C 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.

Enzymedilution

(ml)

Buffer(ml)

DNS(ml)

Enzymedilution

(ml)

Distilledwater(ml)

EnzymeSample

EnzymeBlank

ReagentBlank

0.5

--

--

1.0

1.0

1.5

Incubatefor

1 hourat

50C

3.0

3.0

3.0

--

0.5

--

Boil for5

minutes

15

15

15

i)

j)

9.0

Read enzyme sample and enzyme blank tubes against reagent blank at540nm.

Subtract the enzyme blank reading from enzyme sample reading. Find outthe amount of sugar released with the help of Standard graph.

CALCULATIONS

mg of glucose released X dilution factor X 1000FPU/gm = ------------------------------------------------------------

180 X 60 X 0.5

1.0

2.0

3.0

4.0

5.0

AIM

DETERMINATION OF XYLANASE (HEMICELLULASE)ACTIVITY USING 3, 5 - DINITRO SALICYLIC ACID

PRINCIPLE

The assay is based on the production of reducing sugar from a solutionof Xylan. The reducing sugar then reacts with 3, 5-Dinitro salicylicacid (DNS). The colour change produced is proportional to theamount of reducing sugar (expressed as Xylose) released which inturnis proportional to the activity of the Xylanase present in the sample.The optical density is read at 540 nm and converted into micromolesof Xylose produced using a standard curve.

UNIT DEFINITION

One unit is the amount of enzyme required to produce 1 micromole ofreducing sugar (as Xylose) per minute at pH - 4.8 at 40C.

REAGENTS

Citric acid monohydrateGlacial Acetic acidOat spelt xylan (Sigma)3, 5 Dinitrosalicylic acid (AR)D (+) - Xylose (AR)Potassium Sodium Tartarate Tetrahydrate (AR)Sodium Hydroxide (Analytical grade)

EQUIPMENT

Water bath set at 40 1 CTimerVisible range Spectrophotometer set at 540 nmBoiling water bathCooling water bathGrade A GlasswareWhirl mixerGlass or automatic pipettesCentrifuge

6.0

a)

b)

c)

PREPARATION OF REAGENTS AND SUBSTRATE

0.05 M Citrate buffer (pH - 4.8)

Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. AddNaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and checkpH. This is 1M stock citrate buffer. Prepare 0.05 M buffer solution as aworking solution from the stock solution. Adjust the pH to 4.8 with Acetic acidor NaOH.

XYLAN SUBSTRATE

Take 2g of Xylan in the mortor pestle and grind it with 1-2 beads of SodiumHydroxide and then slowly add citrate buffer to it till the volume reachesapproximately 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 withcitrate buffer. Store in refrigerator at 2-8C. This solution must be prepareddaily.

DNS SOLUTION

1) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litreflask.

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) anddissolve 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.

7.0

a)

b)

c)

PROCEDURE FOR STANDARD GRAPH

Dry xylose to constant weight at 105C. Label the container and store indesiccator.

Prepare a stock solution by dissolving 225.2 mg (15 micromoles of xylosedry weight) in approximately 80ml of 0.05 M citrate buffer. Transfer to a100ml volumetric flask and make to mark with 0.05 M buffer. This stockxylose is stable for 3 months in refrigerator.

Prepare working dilutions from stock according to table below (ormultiples of same).

d)

Substrate(2% Xylan)

(ml)

StandardXylose soln

(ml)

Soln 6.3Buffer

(ml)

Micromoles ofXylose in the r x n

mixture

1.8

1.8

1.8

1.8

1.8

1.8

0.0

0.066

0.10

0.133

0.167

0.20

0.2

0.134

0.1

0.067

0.033

0.0

0.0

1.0

1.5

2.0

2.5

3.0

e)

f)

g)

The graph must be prepared after every 2 months.

Into a series of test tubes, pipette in duplicate Xylan substrate, Std. Xylosesolution and buffer according to table give.

Place in a 40 1C water bath for 5 minutes. Add 3ml of DNS reagent intoeach tube. Cover tubes and place all tubes together into a boiling water bathfor 5 minutes exactly. Cool the tubes under flowing tap water. Add 15ml ofdistilled water to each test tube. Take the absorbance against reagent blank(no xylose). Plot the O.D versus Xylose concentration.

8.0

a)

b)

c)

d)

e)

f)

g)

9.0

ASSAY PROCEDURE

For each sample to be analysed, pipette 1.8ml of xylan substrate into a rowof 4 test tubes, 3 for the test and one for the enzyme blank. Incubate for 2-3minutes at 40 1C.

Add 0.2ml of enzyme dilution to each tube of the triplicate test tubes andincubate for 5 minutes at 40C. Prepare the enzyme blanks by adding 3ml ofDNS to the 4th tube, followed by 0.2ml of enzyme dilution.

Prepare a reagent blank using 0.2ml buffer (Soln. 7.3) in place of enzymedilution.

Stop the reactor by adding 3ml of DNS reagent to each tube at the same timeintervals. Cover tubes and place in a boiling water bath for exactly 5minutes.

Cool the tubes under flowing tap water. Add 15ml distilled water.

Determine the O.D. at 540nm of each sample, using the reagent blank to zerothe spectrophotometer. Read all enzyme blanks and enzyme samples againstreagent blank.

Subtract the enzyme blank from enzyme sample.

CALCULATIONS

The micromoles of Xylose produce may be read directly from the standardgraph or determined using factor derived from the regression of the use.

MICROMOLES OF XYLOSE x DILUTION FACTORIU/G = ----------------------------------------------------------------------

TIME x WEIGHT OF SAMPLE USED

PHYTASE ACTIVITY

Principle

Unit of activity

Assay conditions

Equipment

Reagents

Phytase acts on phytate to release inorganicphosphate. The determination of released inorganicphosphate is based on the colour formed by thereduction of a phosphomolybdate complex.

One phytase unit is the amount of enzyme whichliberates, under standard conditions, 1 umole ofinorganic phosphate from sodium phytate in oneminute.

Substrate Sodium phytatepH 5.0Incubation temperature 37.0

Incubation time 15 mins

Water bath 37 0

Water bath 50 0

SpectrophotometreTest tube mixer

Prepare all the solutions in distilled water.

1. Citrate Buffer- 0.2 M, pH 5.0

Prepare 0.2 M solutions of both sodium citrate and citricacid in water. Adjust the pH of the citric solution to 5.0 with0.2M citric acid.

2. Substrate

Dissolve 1 gm of sodium phytate in about 70 ml citratebuffer. Adjust the pH to 5.0 with 0.2M citric acid and adjustthe volume to 100 ml with citrate buffer. Fresh substratesolution must be prepared daily.

3. 15% TCA solution

4. 10% ascorbic acid solution

5. 2.5% ammonium molybdate solution

6. 1M sulphuric acid- Add 55.6 ml of concentrated H2SO4to about 800 ml of water, with stirring . Allow to cool

and make upto 1000 ml with distilled water.

Reagent C- Mix 3 volumes of 1M sulphuric acid with onevolume of 10%ascorbic acid and mix well. Fresh reagent Cmust be prepared daily.

Samples are diluted in citrate buffer. Weigh the sampleaccurately in a volumetric flask, dissolve in the buffer andfill to the mark. Dilute further if necessary.

Hydrolysis

Pipette 1.0 ml of sample dilution containing 0.02-0.19phytase units in two test tubes. Add 2.0 ml of 15% TCAsolution to one of the tubes (Blanks) and mix. Put the tubeswithout TCA in a water bath at 37 0C and let themequilibtate for 5 mins. Using stop watch start the hydrolysisby adding sequentially at proper intervals 1.0 ml of substrateto each tube and mix. After exactly 15 mins incubation stopthe reaction by adding 2.0 ml of T C A to each tube. Mix andcool to room temperature. Add 1.0 ml substrate to blanktubes and mix. If precipitate occures it must be seperated bycentrifugation .