Special tests for anti-nutritional and toxic factors

in poultry feed

11-ARID-961, 962, 969, 972, 975, 978

Faculty of Veterinary & Animal SciencesPMAS Arid Agriculture University, Rawalpindi

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Anti-nutritional & Toxic Factors Amino Acid Analysis by Ion-Exchange

Chromatography Bleach Test for Tennins in Sorgham Rapid Test for Tennnins in Barley Mycotoxin Analysis Aflatoxin AnalysisAnalysis of OchratoxinAnalysis of Zearalenone Rapid Mycotoxins test (ELISA)Analysis of DON Analysis of T-2 Toxin

Contents:

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“Substances which either by themselves or

through their metabolic products, interfere

with food utilization and affect the health and

production of animals”

“Components Of Feed which have toxic effects

and deleterious effects on living cells &

tissues are called toxic factors”

Anti-Nutritional Factors:

Toxic Factors:

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o Tennins (Sorgham) Bind with dietary protein

o Cyclopropenoid Fatty acids=CFA (cottonseed) Albumin

discoloration

o Gossypol (cottonseed) yolk discoloration

o Lectins (Sorgham grains) Bind with CHO

o Phytates (Sorgham grains) Block absorption of minerals(Ca,

Mg, Fe, Zn, P), Lipid,Proteins

o Sponins (Soybean) Bitter, low intake

o Protease inhibitors (Soybean) Amino Acids available

o DON or Deoxynevalenol or Vomitoxin (Barley) Immunity

o T-2 Toxin (Corn, Oat) Lesions on beak, egg production

o Cyanogens (Linseed, Sorgham) HCN poisoning

Anti-nutritional & Toxic Factors

Reagents (Hydrolyasate):

6N HCL: 50 ml of concentrated hydrochloric acid added to 50

ml of double distilled water.

Nor-leucine standard, 25 mole/ml

Sodium citrate buffer, pH=2.2

Apparatus:

Amino Acid Analysis by Ion-Exchange Chromatography

Rotary Evaporator40 mesh sieve

Grind sample finely (grind to pass a 40 mesh sieve).Weigh the hydrolysate tube.Weigh the sample into the hydrolysate 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.

Add 6 ml of 6N HCL and 0.6 ml of norleucine standard & Mix well.

Charge the tube with nitrogen gas and place in an oven at 110ᵒC for 20 hours. After the tube cools, filter the contents through Whattman No.1 filter paper into a drying tube.

Procedure

. Wash hydrolysate tube with double distilled water and collect in the same drying tube

Dry the filtrate by evaporating with a rotary evaporator under vacuum, with the water bath temperature at 48ᵒC.

Dry the filtrate to a slightly wet residue & Wash residue with distilled water and dry again.

Add 20 ml of citrate buffer and mix well.Take one ml from last step (C-buffer) and

dilute to 5 ml with citrate buffer.Filter the diluted sample liquid using

0.2 micron Nucleopore membrane filter.Determine amino acids by injecting sample

into Amino acid analyzer.

Scope:

Applicable to whole grain sorghumPrinciple:

Sorghum grain is immersed in a sodium hypochlorite solution (bleach) containing alkali. The solution dissolves away the outer peri-carp layer of sorghum grain, revealing the presence of a black pigmented testa layer in the case of tannin sorghums, or its absence in the case of non-tannin sorghums.

Bleaching reagent

5g sodium hydroxide is dissolved in 100 ml of 3.5% sodium hypochlorite solution (commercial/Household bleach). Reagent can be stored at room temperature in light-proof bottle for up to one month.

Bleach Test for Tennins in Sorgham

Apparatus

Glass beakers (50 ml)

Tea strainer

Aluminum foil

Paper towel

One hundred whole, sound sorghum grains are placed in a beakerBleaching reagent is added to just cover the sorghum grainsClose beaker with aluminum foil. Too much bleaching reagent will

cause over bleaching and give false negative results. If in doubt repeat using less reagent.

Incubate beaker at room temperature (20-30°C)for 20 minutes, swirling contents of beaker every 5 minutes

Empty contents of beaker into tea strainer, discarding bleaching reagent.

Rinse sorghum grains in tea strainer with tap waterEmpty contents of tea strainer onto sheet of paper towel. Spread grains out into a single layer and gentle blot them dry with

another piece of paper towel

Procedure

1.Take about of 10 gms of sample (Barley) 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

Tannin sorghum grains are those grains that are black over the entire surface of the grain, with the exception of the where the germ is which is somewhat lighter in colour. Non-tannin sorghum grains are those which are either completely white or are brown over part of the surface of the grain

Rapid Test For Tennins

Results For Both Tennins Tests

Mycotoxin (Myc.) Analysis

1• High Performance Liquid

Chromatography HPLC

2 • Immuno Assay

3 • Mini-column Method4 • High Performance Thin Layer Chromatography HPTLC

5 • Spactrophotometry

6 • Thin Layer Chromatography , TLC

1-Sampling

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

2-Toxin extraction (using organic solvents i.e. Acetic acid)

3-Clean-up (To remove fat, impurities etc.)

4-Identification & Quantification (TLC, HPLC, ELISA etc.)

Common Steps in All Myc. Ana. Meth.

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).

Apparatus: ChromatogramTLC plateMicropippete/MicrocapTLC Scanner

Thin Layer Chromatography , TLC

Apparatus

UV Cabnet

TLC Scanner

Micropipette

Chromatogram

Spotting the extract:o Place 5 - 20 µl of sample as a small circular spot (< 5 mm), 1 - 2

cm from the end of the TLC plate. Micropipette may be used for the purpose. Leave at least 1 cm gap between two adjacent spots.

Developing the plate:o Place about 50 - 100 ml of mobile phase (solvent) in a tankoKeep 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.

oAllow the solvent to travel at least about 8-10 cms

Procedure

Detection:oAir 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 (like 50 % aqueous H2SO4 , Triflouro,etc). to develop fluorescence.

Resolving front value (Rf):oEach mycotoxin has its characteristic color of fluorescence under

UV light and a constant Rf value in a particular developing solvent(See Table ).

oRf value is computed using the formula;

Cont…

Confirmation:oThe presence of mycotoxin can be confirmed either by spraying

the plate with suitable reagents (like 50 % aqueous H2SO4 , Triflouro, Acetic Acid etc).

Detection by Scanner:oThe fluorescence intensity of sample and standard spots can be

measured by using TLC Scanner / fluorodensitometer to avoid possible human errors in comparison

Cont…

Table For Characteristics of Myc.

This is an extension of TLC method. The sample spots on the developed TLC plate are scraped out along with 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.

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.

Spectrophotometry

High Performance Thin Layer Chromatography

Apparatus

Conical Flask

100ml Beaker

Spectrophotometer Linomat IVBlender

Reagents:o 0.2 M NaOH (dissolve 8 g NaOH in water and make up volume

to 1 lit)o 0.41 M Ferric Chloride (dissolve 66.5 g anhydrous FeCl3 in

water and make up volume to 1 lit)o 0.03 % H2SO4 (0.3 ml conc. H2SO4+ 999.7 m water)o Potassium wash solution (dissolve 1.12 g KOH and 10 g KCl in

water and make up volume to 1lit)Solvents:oAcetoneoChloroformoDeveloping solvent==Chloroform : Acetone : Water (88 : 12 : 1)

Rapid TLC method of Aflatoxin analysis(Modified Romer's method)

Standard:oAflatoxin B1 1 µg/ml in Benzene : Acetonitrile (98:2)Procedure:oTake 25 g sample in a conical flask, add 100 ml distilled water

and blend for 2 minutesoAdd 150 ml acetone and blend again for 2 minuteso Filter through Whattman no.1 filter paper and transfer 75 ml of

filtrate to a conical flask containing 3 g cupric carbonateo Prepare ferric gel by adding 85 ml of 0.2 M NaOH to 15 ml of

0.41 M FeCl3

oAdd this mixture to the flask containing extract and cupric carbonate

Procedure:

Mix the contents slowly by swirling movementsFilter through Whattman no. 1 filter paperTake 100 ml of filtrate in a 250 ml separating funnelAdd 100 ml of 0.03 % H2SO4 and 10 ml of chloroform. Mix the

contents slowlyCollect the chloroform layer into a 100 ml beakerAdd again 10 ml of chloroform to the separating funnel and

repeat the above step. Combine both the chloroform extractsTake 100 ml potassium wash solution in a separate separating

funnelAdd the chloroform extract to the second separating funnel and

mix it slowly

Cont…

Collect the chloroform layer through anhydrous sodium sulfate bed drop by drop to remove moisture

Dry the chloroform extract in an oven at 50°CDissolve the dried residue in 0.2 ml chloroform and spot on

TLC plat along with the standardCompare the flourescence intensities of the sample and standard

spots and identify the ones matching with each other

2 ml of final chloroform extract is placed in the column (20 cm length, 6 mm internal diameter with tapering end 2 mm) 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

Cont…

Mini-column Method

Enzyme-Linked Immuno-Sorbant Assay ELISA

RadioImmuno Assay IRA

Immunoaffinity Column Assay ICA

Aflatoxin analysis Faster Tests

Principle :oAntibody coated column is used to trap the mycotoxin. This

trapped toxin is then eluted using approximate solvent and quantified in fluorometer.

Equipments :o Immuno affinity columnoAffinity column stand with syringeoCuvetteoCalibrated FluorometeroBlendero Fluted filter paper

Rapid Mycotoxin Test / "ELISA Test"

Reagent :oTest developero2. Methonol : water (80 : 20 by volume)o3. Mycotoxin wash buffer

Apparatus

Calibrated Fluorometer

Immuno affinity column

Quartz CuvetteFluted filter paperAffinity column stand with syringe

50 gms of sample + 5 gms of NaCl + 100 ml of methanol water (80 :20)

Note : NaCl is not added in case of Ochratoxin TestBlend at high speed and filterPipette filtered extract into clean vessel

Aflatoxin, Ochratoxin : 10 ml

Zearalenone : 1 mlDilute with purified water and mix

Aflatoxin, Ochratoxin : 40 ml

Zearalenone : 49 mlFilterRemove top cap and attach the syringe (cut 1/8 inch bottom of

column )

Procedure:

Pass filtered diluted extract at the rate of 1-2 drops/second

Aflatoxin : 2 ml

Ochratoxin, Zearalenone : 10mlPass water at the rate of 1-2 drops/second

Aflatoxin, Zearalenone : 5 ml

Ochratoxin : First 10 ml Mycotoxin wash buffer,Later 10 ml distilled waterElute toxin in glass cuvette

Aflatoxin, Zearalenone : Pass 1 ml HPLC grade methanol

Ochratoxin : Pass 1.5 ml Ochratoxin eluting solution.Add 1 ml of developer to the cuvette and mix wellRead in calibrated fluorometer

Cont…

Measured by ELISA

Poultry are the most tolerant of livestock species to DON. Some studies show that feeding 20-50 ppm DON has no effect on production.

DON

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:Weigh 10 g. tested sample and add 100 ml of distilled water.

Mix thoroughly and then filter with whatman No. 41 filter paper.Take 1 ml of tested sample aliquate into white porcelein spot

plate.Add 2-3 drops of phenol red indicator and then add 2-3 drops of

urease solution.

TEST FOR UREA IN FISH MEAL

Stand for 3-5 minutesIf 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.

Cont…

white porcelein spot plate Whatman #41

Reagents :o 30% ammonium sulphate (dissolve 30 g (NH4)2SO4 in water

and make up volume to 100 ml)oCelite 545o Potassium wash solution (dissolve 1.12 g KOH and 10 g KCl in

water and make up volume to 1 lit)o Sodium sulphateo Silica geloMethanol : H2SO4 ( 1 : 1 v/v )

Analysis of T-2 Toxin

Solvents : oMethanol : water (1 : 1 v/v)oChloroformoDiethyl etheroHexaneoBenzeneoAcetone : Benzene (5 : 95 v/v)oDeveloping solvent mixture - Toluene : ethylacetate formic acid

(6 : 3: 1 v/v)Standard : oT-2 toxin 50 µg / ml in Benzene or diethyl ether

Cont…

Apparatus

Developing Tank/Chamber

UV cabnet

Pre-coated Silica Gel plate

Horizontal Shaker

Glass Stoppered Conical Flask

Whatman FP #1

Take 50 g of sample in a glass stoppered conical flaskAdd 250 ml of methanol : water (1 : 1) and shake for 1 hourFilter using whatman No.1 filter paper and collect 60 ml of

extract into a beakerAdd 240 ml 30 % (NH4)2SO4 and stir vigorously for 1 minuteAdd 20 g of celite and stir for 1 minuteFilter and collect 200 ml of filtrateTransfer filtrate to a separating funnelAdd 10 ml of chloroform and shake vigorously for 1 minuteAllow the layers to separate and collect the bottom layer into

another separating funnelRepeat the extraction with another 10 ml of chloroform

Procedure

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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 : o Plug the bottom of a glass column ( 2x30cm )

with glass wool and add 5 g anhydrous sodium sulphate. Fill the column to half level with chloroform and add 10 g silica gel.

Cont…

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o Wash sides of column with chloroform and stir to eliminate air bubbles.

o 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.

o 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

Cont…

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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

Cont…

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Dissolve the residue in 0.5-1.0 ml diethyl ether. Spot on TLC along with the standard (5-20 µL 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 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

Cont…

References:

o Commercial Poultry Nutritiono Analysis of Toxins by layer chromatography by R.W. Nicol & R.C. Sinhao Laboratory Manual on Laboratory Control of Animal feed by Dr.

G.Devegowdao Lecture of Dr. Naeem Tahir (PMAS Arid Agriculture University, Rawalpindi)o Simple Sorgham Grain test for tannin by John R.N Tayloro www.foa.como www.hamletprotein.como www.allaboutfeed.neto www.poultryhub.org

ANY QUESTION ?

Thank

you!!!