More Free PowerPoint Templates at SmileTemplates.com Anti-nutritional & Toxic Factors Amino Acid Analysis by Ion-ExchangeChromatography Bleach Test for Tennins in Sorgham Rapid Test for Tennnins in Barley Mycotoxin Analysis Aflatoxin Analysis Analysis of Ochratoxin Analysis of Zearalenone Rapid Mycotoxins test (ELISA) Analysis of DON Analysis of T-2 Toxin
More Free PowerPoint Templates at SmileTemplates.com“Substances which either by themselves orthrough their metabolic products, interferewith food utilization and affect the health andproduction of animals”“Components Of Feed which have toxic effectsand deleterious effects on living cells &tissues are called toxic factors”
More Free PowerPoint Templates at SmileTemplates.como Tennins (Sorgham) Bind with dietary proteino Cyclopropenoid Fatty acids=CFA (cottonseed) Albumindiscolorationo Gossypol (cottonseed) yolk discolorationo Lectins (Sorgham grains) Bind with CHOo Phytates (Sorgham grains) Block absorption ofminerals(Ca, Mg, Fe, Zn, P), Lipid,Proteinso Sponins (Soybean) Bitter, low intakeo Protease inhibitors (Soybean) Amino Acids availableo DON or Deoxynevalenol or Vomitoxin (Barley) Immunityo T-2 Toxin (Corn, Oat) Lesions on beak, egg productiono Cyanogens (Linseed, Sorgham) HCN poisoning
Reagents (Hydrolyasate): 6N HCL: 50 ml of concentrated hydrochloric acid added to 50ml of double distilled water. Nor-leucine standard, 25 mole/ml Sodium citrate buffer, pH=2.2Apparatus: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 tocontain about 30-40 mg of protein. This would beapproximately 60-80 mg of soybean meal samples and 300-350 mg for corn samples when diluting the hydrolysate 100times.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 at110ᵒC for 20 hours. After the tube cools, filter the contentsthrough Whattman No.1 filter paper into a drying tube.
. Wash hydrolysate tube with double distilled water and collect inthe same drying tubeDry the filtrate by evaporating with a rotary evaporator undervacuum, with the water bath temperature at 48ᵒC.Dry the filtrate to a slightly wet residue & Wash residue withdistilled water and dry again.Add 20 ml of citrate buffer and mix well.Take one ml from last step (C-buffer) anddilute to 5 ml with citrate buffer.Filter the diluted sample liquid using0.2 micron Nucleopore membrane filter.Determine amino acids by injecting sampleinto 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-carplayer of sorghum grain, revealing the presence of a black pigmentedtesta layer in the case of tannin sorghums, or its absence in the case ofnon-tannin sorghums.Bleaching reagent5g sodium hydroxide is dissolved in 100 ml of 3.5% sodiumhypochlorite solution (commercial/Household bleach). Reagent can bestored at room temperature in light-proof bottle for up to one month.
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 willcause over bleaching and give false negative results. If in doubt repeatusing less reagent.Incubate beaker at room temperature (20-30 C)for 20minutes, swirling contents of beaker every 5 minutesEmpty contents of beaker into tea strainer, discarding bleachingreagent.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 withanother piece of paper towel
1.Take about of 10 gms of sample (Barley) in bottle2. Add potassium hydroxide and sodium hypochloride solution.3. Close the bottle and shake till crystals dissolve.4. Wait for about 15 minutesTannin sorghum grains are those grains that are black over the entiresurface of the grain, with the exception of the where the germ is whichis somewhat lighter in colour. Non-tannin sorghum grains are thosewhich are either completely white or are brown over part of thesurface of the grain
1-SamplingCollect at least 100 subsamples from the whole lot. For eg. froma truck of 100 bags of maize, collect 100 g maize from each bagto obtain a total sample size of 10 kg.Get about 50 - 100 g subsample from the whole sampleemploying either coning and quartering method2-Toxin extraction (using organic solvents i.e. Acetic acid)3-Clean-up (To remove fat, impurities etc.)4-Identification & Quantification (TLC, HPLC, ELISA etc.)
Principle:It is the cheapest and most commonly used method. It makes useof heterogenous equilibrium established during the flow of asolvent(mobile phase) through a fixed phase (stationary phase)to separate ≥2 components from materials carried by solvent(differential migration).Apparatus: ChromatogramTLC plateMicropippete/MicrocapTLC Scanner
Spotting the extract:o Place 5 - 20 µl of sample as a small circular spot (< 5 mm), 1 - 2cm from the end of the TLC plate. Micropipette may be used forthe 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 tanko Keep the plate at a slight angle with the spots little above theupper level of the solvent. Due to capillary action, solvent movesupward on the plate.o Allow the solvent to travel at least about 8-10 cms
Detection:o Air dry the developed plate and view in a UV cabinet undereither longwave (365 nm) or short wave (254 nm) range toidentify the fluorescing mycotoxins. In case of mycotoxinswhich do not fluoresce, spray the plate with suitable reagent (like50 % aqueous H2SO4 , Triflouro,etc). to develop fluorescence. Resolving front value (Rf):o Each mycotoxin has its characteristic color of fluorescence underUV light and a constant Rf value in a particular developingsolvent(See Table ).o Rf value is computed using the formula;
Confirmation:o The presence of mycotoxin can be confirmed either by sprayingthe plate with suitable reagents (like 50 % aqueous H2SO4, Triflouro, Acetic Acid etc). Detection by Scanner:o The fluorescence intensity of sample and standard spots can bemeasured by using TLC Scanner / fluorodensitometer to avoidpossible human errors in comparison
This is an extension of TLC method. The sample spots on thedeveloped TLC plate are scraped out along with the sorbent(silica gel) and extracted with methanol for 3 minutes. Theextract is filtered and the absorbance of the filtrate is measuredin a spectrophotometer at 363 nm.This is an improvised version of TLC, where sample applicationand detection of fluorescence intensity are fully automated andcarried 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 thismethod.
Reagents:o 0.2 M NaOH (dissolve 8 g NaOH in water and make up volumeto 1 lit)o 0.41 M Ferric Chloride (dissolve 66.5 g anhydrous FeCl3 inwater 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 inwater and make up volume to 1lit)Solvents:o Acetoneo Chloroformo Developing solvent==Chloroform : Acetone : Water (88 : 12 : 1)
Standard:o Aflatoxin B1 1 µg/ml in Benzene : Acetonitrile (98:2)Procedure:o Take 25 g sample in a conical flask, add 100 ml distilled waterand blend for 2 minuteso Add 150 ml acetone and blend again for 2 minuteso Filter through Whattman no.1 filter paper and transfer 75 ml offiltrate to a conical flask containing 3 g cupric carbonateo Prepare ferric gel by adding 85 ml of 0.2 M NaOH to 15 ml of0.41 M FeCl3o Add this mixture to the flask containing extract and cupriccarbonate
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 thecontents slowlyCollect the chloroform layer into a 100 ml beakerAdd again 10 ml of chloroform to the separating funnel andrepeat the above step. Combine both the chloroform extractsTake 100 ml potassium wash solution in a separate separatingfunnelAdd the chloroform extract to the second separating funnel andmix it slowly
Collect the chloroform layer through anhydrous sodium sulfatebed drop by drop to remove moistureDry the chloroform extract in an oven at 50 CDissolve the dried residue in 0.2 ml chloroform and spot on TLCplat along with the standardCompare the flourescence intensities of the sample and standardspots and identify the ones matching with each other2 ml of final chloroform extract is placed in the column (20 cmlength, 6 mm internal diameter with tapering end 2 mm) andeluted with chloroform : acetone (9 : 1). Aflatoxin,if present istrapped as a band above florisil layer which can be viewed underlong wave UV light as a blue fluorescent band
Principle :o Antibody coated column is used to trap the mycotoxin. Thistrapped toxin is then eluted using approximate solvent andquantified in fluorometer.Equipments :o Immuno affinity columno Affinity column stand with syringeo Cuvetteo Calibrated Fluorometero Blendero Fluted filter paperReagent :oTest developero2. Methonol : water(80 : 20 by volume)o3. Mycotoxin wash buffer
Calibrated FluorometerImmuno affinity columnQuartz 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 vesselAflatoxin, Ochratoxin : 10 mlZearalenone : 1 mlDilute with purified water and mixAflatoxin, Ochratoxin : 40 mlZearalenone : 49 mlFilterRemove top cap and attach the syringe (cut 1/8 inch bottom ofcolumn )
Pass filtered diluted extract at the rate of 1-2 drops/secondAflatoxin : 2 mlOchratoxin, Zearalenone : 10mlPass water at the rate of 1-2 drops/secondAflatoxin, Zearalenone : 5 mlOchratoxin : First 10 ml Mycotoxin wash buffer,Later 10 ml distilled waterElute toxin in glass cuvetteAflatoxin, Zearalenone : Pass 1 ml HPLC grade methanolOchratoxin : Pass 1.5 ml Ochratoxin eluting solution.Add 1 ml of developer to the cuvette and mix wellRead in calibrated fluorometer
Measured by ELISAPoultry are the most tolerant of livestock species toDON. Some studies show that feeding 20-50 ppmDON has no effect on production.
Reagents:1. Urease enzyme solution2. Standard Urea Solutions (0, 0.5, 1, 1.5,..........5%)3. Phenol red indicator (0.1%)OrCresol 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 spotplate.Add 2-3 drops of phenol red indicator and then add 2-3 drops ofurease solution.
Stand for 3-5 minutesIf Urea presents solution will become red- purple in contrast tothe yellow colour of indicatorColour can be compared with the colour developed in standardsolution of varying levels of urea.white porcelein spot plate Whatman #41
Reagents :o 30% ammonium sulphate (dissolve 30 g (NH4)2SO4 in waterand make up volume to 100 ml)o Celite 545o Potassium wash solution (dissolve 1.12 g KOH and 10 g KCl inwater and make up volume to 1 lit)o Sodium sulphateo Silica gelo Methanol : H2SO4 ( 1 : 1 v/v )
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 ofextract 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 intoanother separating funnelRepeat the extraction with another 10 ml of chloroform
More Free PowerPoint Templates at SmileTemplates.com Combine both the extracts and add 100 ml ofpotassium wash solution Swirl gently for 30 seconds and let layersseparate Drain the lower chloroform layer through abed of Sodium sulphate (in a funnel) to dryand collect 10 ml of clear filtrate Column Preparation :o Plug the bottom of a glass column ( 2x30cm )with glass wool and add 5 g anhydroussodium sulphate. Fill the column to half levelwith chloroform and add 10 g silica gel.
More Free PowerPoint Templates at SmileTemplates.como Wash sides of column with chloroform andstir to eliminate air bubbles.o Drain off chloroform leaving about 7 cmabove the upper level of silica gel. Add 15 ganhydrous sodium sulphate withoutdisturbing the silica gel.o Drain off chloroform to the upper level ofsodium sulphate Wash the column serially with 50 ml ofdiethyl ether and 10 ml of chloroform anddiscard the washings
More Free PowerPoint Templates at SmileTemplates.com Mix 10 ml of sample extract with 30 ml ofhexane and add to the column and slowlydrain until solvent is about 1 cm aboveSodium sulphate Add in succession 30 ml benzene and 40 mlacetone : benzene (5: 95) and discard boththe washings Elute T-2 with diethyl ether until 30 ml ofeluate is collected and evaporate the eluate
More Free PowerPoint Templates at SmileTemplates.com Dissolve the residue in 0.5-1.0 ml diethylether. Spot on TLC along with the standard(5-20 µL or any other suitable range) anddevelop the plate in toluene : ethyl acetate :formic acid (6 : 3 : 1) Air dry the plate and spray withmethanol:H2SO4 (1:1) Dry at 110 C for 10 minutes Observe blue fluorescence under long waveUV light (365 nm) Compare the intensities of the blue fluorescent spotsof the sample with those of standard and identify theones matching each other
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 AgricultureUniversity, 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