Feed Testing Manual By Dr Devegowda

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

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Feed Testing Manual By Dr Devegowda

  1. 1. It is been an endeavor to constantly update the technical inputs to our esteemed customers in VETCARE. This effort is an extension of the programme This manual covers the details of analysis of many ingredients used in the feed along with the Mycotoxin Estimation and Toxin binding estimation. Also find the interesting Enzyme analysis in this manual  VETCARE IS - 40, KHB Industrial Area Yelahanka New Town Bangalore - 560 064, INDIA Ph. : 8460060, 8462055/56 Fax: 8461240 E-mail : btan@giasbg01.vsnl.net.in Web : www.vetcareindia.com
  2. 2. LABORATORY MANUAL ON QUALITY CONTROL OF ANIMAL FEEDS Dr. G. DEVEGOWDA M.V.Sc, Ph.D (U.S.A) PROFESSOR & HEAD DEPARTMENT OF POULTRY SCIENCE UNIVERSITY OF AGRI. SCIENCES HEBBAL, BANGALORE - 560 024 1999
  3. 3. CONTENTS Sl. Title Page No. No. I. MYCOTOXIN QUANTIFICATION 1. Mycotoxin Standards 1-5 2. Mycotoxin Analysis 6 - 16 3. Rapid TLC method of Aflatoxin analysis 17 - 19 4. Analysis of Ochratoxin 20 - 23 5. Analysis of T-2 toxin 24 - 28 6. Analysis of Zearalenone 29 - 33 7. Rapid TLC method of Multi-Mycotoxin 34 - 36 analysis 8. Rapid Mycotoxin Test / "ELISA Test" 37 - 39 9. In vitro evaluation of Mycotoxin binding agents 40 - 41 10. In vitro evaluation of Mycotoxin binding agents in contaminated feeds 42 - 43 11. List of Suppliers of Mycotoxin standards 44 12. Toxin Binding analysis of Mycotoxins (VETCARE) 13. Preparation of Acid hydrolysate of seed samples for amino acid analysis using ion exchange chromatography. 14. Preparation of seed samples for methiomine and cystine analysis using performic acid oxidation procedure. 15. Estimation of Sodium and potassium by flame photometry
  4. 4. PREPARATION OF ACID HYDROLYSATE OF FEED SAMPLES FOR AMINOACID ACID ANALYSIS BY ION-EXCHANGE CHROMATOGRAPHY Reagents: 1. 6 N HCL: 50 ml of concentrated hydrochloric acid added to 50 ml of double distilled water. 2. DL-norleucine standard, 25 umole/ml 3. Sodium citrate buffer, pH2.2 Procedure: 1. Grind sample finely (grind to pass a 40 mesh sieve). 2. Weigh the hydrolysate tube. 3. Weigh the sample into the hydrolusate tube, so as to contain about 30-40 mg of protein. This would be approximately 60-80 mg of soybean meal samples and 300-350 mg for corn samples when diluting the hydrolysate 100 times. 4. Add 6 ml of 6N HCL and 0.6 ml of norleucine internal standard. Mix well.
  5. 5. 5. Charge the tube with nitrogen gas and place in an oven at 1100C for 20 hours. 6. After the tube cools, filter the contents through Whatman No.1 filter paper into a drying tube. Wash hydrolysate tube with double distilled water and collect in the same drying tube. 7. Dry the filtrate by evaporating with a rotary evaporator under vacuum, with the water bath temperature at 480C. Dry the filtrate to a slightly wet residue. Wash residue with distilled water and dry again. 8. Add 20 ml of citrate buffer and mix well. 9. Take one ml from step 8 and dilute to 5 ml with citrate buffer. 10. Filter the diluted sample liquid using 0.2 micron Nucleopore membrane filter. 11. Detemine amino acids by injecting sample into Dionex D- 300 Amino acid analyzer. ACKNOWLEDGEMENT
  6. 6. I would like to put on record my sincere gratitude to Dr. Malathi, V., Dr. Manju, N.C. and Dr. Manoj, K.B., for their significant contribution in bringing up this manual so beautifully. My thanks are also due to Dr. S. Abdul Rahman, Director of Instructions, Veterinary College, and the Staff and Students of the Department of Poultry Science, University of Agricultural Sciences, Bangalore, India for their encouragement and support. I also thank Dr. Ravikiran, D., Ph.D. Scholar of this Department for his critical editing of this manual ( G. DEVEGOWDA ) Professor and Head Vice President, World's Poultry Science Association (IB), Department of Poultry Science University of Agricultural Sciences Hebbal, Bangalore - 560 024, INDIA
  7. 7. Preface The Department of Poultry Science, University of Agricultural Sciences, Bangalore, has been the pioneer in the field of Mycotoxin Research in the Country. Several breakthroughs have been achieved here in counteracting Mycotoxins in Animal and Poultry feeds. Further Research is on in the direction to achieve total solution to the problem of mycotoxins in foods and feeds, sooner or later. Mycotoxins are always of concern, both in tropical and temperate climates. Mycotoxins in feed remain to be a potential threat to the health, productivity and livability of poultry and livestock and are also of serious public health concern. Such a situation requires that there be adequate laboratory facilities everywhere to identify and evaluate the problem precisely and take necessary steps for good. While technology for Mycotoxin research is available, much of the data is relevant to developed countries which make use of sophisticated instruments and techniques. And there will always be a lot of hurdles for a person working on a small scale laboratory to follow those methods. Having faced such problems initially and developed / adopted methods that suit our conditions, we felt that such problems should not be the excuse. Hence we felt the need of a manual that gives, simple, but in detail, information on the basic laboratory work involved in Mycotoxin research. And the fruit of our effort in this direction is this manual. We trust this manual would be quite useful to the Post Graduate students in Animal Sciences and Biological Sciences. Food and Feed Analysis & Quality Control Laboratories, Feed Manufacturers, Research Institutions, Food Processing Industry, Food Exporters, Private Veterinary Diagnostic Laboratories etc. Date : March, 1999 Prof. G. Devegowda Place : Bangalore, India Dr. M.V.L.N. Raju
  8. 8. CHAPTER I MYCOTOXIN QUANTIFICATION 1. Mycotoxin standards Pure mycotoxins of known concentration are used in mycotoxin assays for either fluorescence intensity comparison or calibration. Reconstitution of Mycotoxin standards Mycotoxin standards are often supplied in crystalline form in sealed glass vials. They need to be suitably dissolved in appropriate solvents for preparation of stock and working solutions of desired final concentration. Procedure  Carefully remove the metallic seal from the central injecting area of the cap of the vial and inject about 1 ml of appropriate solvent into the vial  Shake the vial gently to dissolve the mycotoxin in the solvent  Recover the dissolved mycotoxin using the same syringe and transfer into a volumetric flask  Inject again 1 ml of solvent into the vial, shake gently, recover and transfer to volumetric flask. Repeat 5-6 times for complete recovery of Mycotoxin
  9. 9.  Make up the volume of the dissolved toxin to get the desired concentration in the stock solution  Tightly stopper the volumetric flask and store in refrigerator in an opaque container at 4°C  For preparation of working standards and those used for spectrophotometric purity evaluation, dilute the stock standard using the suitable solvent to get the desired mycotoxin concentration
  10. 10. Table 1. Concentrations of the Mycotoxin standards required (µg/ml) Stock UV TLC Solvent Aflatoxins 100 10 1 Benzene : Acetonitrile (98 : 2) Ochratoxin A 25 25 2 Benzene : Acetic acid (99 : 1) T-2 toxin 5000 100 50 Ethyl acetate Zearalenone 100 10 50 Benzene Citrinin 40 20 40 Chloroform DON 500 20 20 Ethyl acetate : Methanol (19 : 1) Sterigmatocystin 100 100 100 Benzene
  11. 11. Checking the purity / concentration  The standards thus prepared are required to be checked periodically for assessing any possible alteration in their concentration during storage  Prepare 0.4 mM potassium dichromate solution by dissolving 125 mg potassium dichromate in 1 litre 0.018 N Sulphuric acid (1 ml H2SO4 in 2 litre distilled water)  Prepare 0.2 mM and 0.1 mM solutions of potassium dichromate by making two successive dilutions of 0.4 mM solution with 0.018 N Sulphuric acid  Read absorbance of these 3 solutions at 350 nm using 0.018 N H2SO4 as blank Abs x 1000  Calculate (E) of each solution = mM  Calculate the average of the three solutions (0.4, 0.2 and 0.1 mM)  Calculate the correction factor (CF) for the instrument 3,160 CF = (normal value : 0.95 - 1.05) E
  12. 12.  Read the absorbance of the mycotoxin standard at wave length of maximum absorbance Abs x Mol wt x CF x 1000 Concn. (µg/ml) = E Molecular weight, wave length of maximum absorbance and absorptivity of some Mycotoxins Mol. wt. Max. abs. (nm) Absorptivity (E) Aflatoxin B1 312 353 19,800 Ochratoxin A 403 333 5,550 Zearalenone 318 316 6,020 Citrinin 259 322 16,100 Sterigmatocystin 324 325 15,200
  13. 13. 2. Mycotoxin Analysis A. Sampling  Collect the samples at the following quantities for ensuring meaningful representation of the whole lot of feed / feedstuff Min. sample size Small particle type (milk, vegetable oils) 500 g Intermediate particle type (ground meals, flours, 3 kg compounded feed) Small grains (wheat, rice, sorghum, ragi, barley etc.) 5 kg Intermediate grains (maize, cotton seed / cake) 10 kg Large grains (groundnuts / cake) 20 kg  Collect at least 100 subsamples from the whole lot. For eg. from a truck of 100 bags of maize, collect 100 g maize from each bag to obtain a total sample size of 10 kg  Get about 50 - 100 g subsample from the whole sample employing either coning and quartering method (in a series of steps) or using sample divider  The subsample thus collected can be directly subjected for analysis
  14. 14. B. Outline of Mycotoxin analysis Sampling Toxin extraction (using organic solvents) Clean-up (To remove fat, impurities etc.) Work up Identification & Quantification (TLC, HPLC, ELISA etc.)
  15. 15. C. Different methods of Mycotoxin analysis C. 1. Thin layer chromatography (TLC)  Principle It is the cheapest and most commonly used method. It makes use of heterogenous equilibrium established during the flow of a solvent (mobile phase) through a fixed phase (stationary phase) to separate ≥ 2 components from materials carried by solvent (differential migration).  Spotting the extract Place between 5 - 20 µl of sample extract / standard as a small circular spot (< 5 mm), 1 - 2 cm from the end of the TLC plate. Micropipette / microcaps may be used for the purpose. Leave at least 1 cm gap between two adjacent spots.  Developing the plate Place about 50 - 100 ml of mobile phase (solvent) in a tank and keep the plate at a slight angle with the spots little above the upper level of the solvent. Due to capillary action, solvent moves upward on the plate. Allow the solvent to travel at least about 8-10 cms.  Detection Air dry the developed plate and view in a UV cabinet under either longwave (365 nm) or short wave (254 nm) range to identify the fluorescing mycotoxins. In case of mycotoxins which do not fluoresce, spray the plate with suitable reagent to develop fluorescence.
  16. 16.  Resolving front value (Rf) Each mycotoxin has its characteristic color of fluorescence under UV light and a constant Rf value in a particular developing solvent (Table 3). Rf value is computed using the formula, Distance travelled by sample spot from the origin Rf = Distance travelled by solvent front from the origin  Confirmation The presence of mycotoxin can be confirmed either by spraying the plate with suitable reagents (like 50 % aqueous H2SO4, Triflouro Acetic Acid etc.) or placing an internal standard right over the top of the sample spot (superimposing).  Detection by Scanner The fluorescence intensity of sample and standard spots can be measured by using TLC Scanner / fluorodensitometer to avoid possible human errors in comparison. Table 3. TLC characteristics of mycotoxins Toxin Rf * Color Color (UV) after
  17. 17. (UV) spray * * Aflatoxin B1 0.31 Blue Pink Aflatoxin B2 0.26 Blue Pink Aflatoxin G1 0.23 Green Blue Aflatoxin G2 0.17 Green Blue Ochratoxin A 0.55 Green Blue T-2 toxin 0.36 Yellow Blue Zearalenone 0.78 Blue Yellow DAS 0.33 Yellow Variable Sterigmatocystin 0.85 Red-brown Yellow *TEF : Toluene : ethyl acetate : formic acid ( 6:3:1 ) * *P - anisaldehyde C. 2. Spectrophotometry This is an extension of TLC method. The sample spots on the developed TLC plate are scraped out alongwith the sorbent (silica gel) and extracted with methanol for 3 minutes. The extract is filtered and the absorbance of the filtrate is measured in a spectrophotometer at 363 nm. Reference : Nabney and Nesbitt. 1965. Analyst 90 : 155-160.
  18. 18. C. 3. High Performance Thin Layer Chromatography (HPTLC) This is an improvised version of TLC, where sample application and detection of fluorescence intensity are fully automated and carried out by using automated sample applicator (like Linomat IV of Camag, Switzerland) and densitometer, respectively. Mycotoxin levels less than 0.1 ppb can be detected by this method. C. 4. Minicolumn method A glass column of 20 cm length, 6 mm internal diameter with tapering end (2 mm) is packed serially from the bottom with glass wool, calcium or sodium sulphate (8-10 mm), florisil (8-10 mm), silica gel (18-20 mm), neutral aluminia (8-10 mm), calcium or sodium sulphate (8-10 mm) and a cap of glass wool. 2 ml of final chloroform extract (in case of aflatoxin) is placed in the column and eluted with chloroform : acetone (9 : 1). Aflatoxin, if present is trapped as a band above florisil layer which can be viewed under long wave UV light as a blue fluorescent band. This method can be used as a qualitative test for rapid identification of mycotoxin. C. 5. Immuno assays These assays are developed on the basic principle of Antigen - Antibody reaction. Antibodies are highly specific to the Mycotoxin - Protein conjugate (Hapten) used. Hence the results will be highly specific.
  19. 19. Commonly employed immuno assays  Radio immuno assay (RIA) Standard mycotoxin, labelled onto a radioactive compound like Tritium is used. Mycotoxin levels as low as 2-5 ppb can be detected. The disadvantages of this method include high cost, difficulty in labelling, radio active waste disposal problem and risk of handling.  Enzyme linked immuno sorbent assay (ELISA) It has received great attention in recent times and has been the most popular and widely practiced immuno assay method. ELISA is rapid, more sensitive, highly specific and simple to operate. It does not require any extensive extraction or clean- up. Commercial ELISA kits Various companies have been marketing commercial kits which basically work on ELISA principle. These have gained wider acceptance as considerable amount of time is saved on antibody production. Sample is extracted with methanol : water (60 : 40) or acetonitrile : water (50 :50) and the extract is directly subjected to analysis.
  20. 20. Elisa tests are good for quick identification of mycotoxins in feed samples, various tests are developed based on Antigen - Antibody principle. Some companies which produce ELISA kits are : 1. Neogen Corp, 620, Lesher place, Lansing, Michigan 48912, U.S.A. 2. Vicam, 313, Pleasant St., Watertown, Massachusetts - 02172, U.S.A. C. 6. High performance liquid Chromatography (HPLC) It is highly sensitive and can detect upto 5 x 10-6 ppb level of mycotoxin. Stainless steel columns (< 18) of 15 cm length and 4 mm internal diameter, packed with silica gel (particle size - 5 microns) are used. Sample is first extracted with suitable solvent (generally 60 % aqeous methanol) and the extract is cleaned - up. This purified extract (20 µl) is injected into the column and the eluent (generally a mixture of methanol, water and acetonitrile) is passed at a flow rate of 0.75 ml / min and at a pressure of 3000 psi. The eluted toxins coming out of the column are detected and quantified by fluorimeter. The columns may be either normal phase (polar stationary phase) or reverse phase (polar mobile phase) type. The latter type is most commonly used. C. 7. Bio - assays
  21. 21. Mostly are useful as confirmatory tests. Toxin extract is injected as a single dose into stomach (day-old duckling bioassay, guinea pig bioassay), fertile eggs (chick embryo bioassay) or into skin of rabbits (skin bioassay). Presence of toxin is confirmed by noticing pathological changes or mortality. Safety precautions in mycotoxin analysis  Carryout the mycotoxin analysis in a separate work area in the laboratory  Cover the bench top with non absorbent material  Solvents used are highly inflammable. So avoid using electric stoves, bunsen burners etc.  Do not stock the solvents in larger quantities  Wear protective clothing, gloves and mask to minimise the risk of inhalation / contact with hazardous mycotoxins  Some of the solvents (like benzene, chloroform) are toxic. Avoid direct skin contact with them  Any spillage should be immediately mopped-up with cotton. Such cotton should be incinerated  After completing the work, decontaminate the area with 4 % sodium hypochlorite solution
  22. 22.  Decontaminate the glassware by soaking for atleast 2 hours in 1 % sodium hypochlorite solution  Spray the TLC plate with reagent only in a fume cup-board / spray cabinet  At the UV cabinet, always view the TLC plate only through the UV filter  Avoid eating, drinking and smoking in the laboratory  Keep the lab well ventilated using exhaust fans
  23. 23. 3. Rapid TLC method of Aflatoxin analysis (Modified Romer's method) Reagents : i) 0.2 M NaOH (dissolve 8 g NaOH in water and make up volume to 1 lit) ii) 0.41 M Ferric Chloride (dissolve 66.5 g anhydrous FeCl3 in water and make up volume to 1 lit) iii) 0.03 % H2SO4 (0.3 ml conc. H2SO4 + 999.7 ml water) iv) Potassium wash solution (dissolve 1.12 g KOH and 10 g KCl in water and make up volume to 1 lit) Solvents : i) Acetone ii) Chloroform iii) Developing solvent Chloroform : Acetone : Water (88 : 12 : 1) Standard : Aflatoxin B1 - 1 µg/ml in Benzene : Acetonitrile (98:2) Procedure :
  24. 24.  Take 25 g sample in a conical flask, add 100 ml distilled water and blend for 2 minutes  Add 150 ml acetone and blend again for 2 minutes  Filter through Whatman no. 1 filter paper and transfer 75 ml of filtrate to a conical flask containing 3 g cupric carbonate  Prepare ferric gel by adding 85 ml of 0.2 M NaOH to 15 ml of 0.41 M FeCl3. Add this mixture to the flask containing extract and cupric carbonate  Mix the contents slowly by swirling movements  Filter through Whatman no. 1 filter paper  Take 100 ml of filtrate in a 250 ml separating funnel  Add 100 ml of 0.03 % H2SO4 and 10 ml of chloroform. Mix the contents slowly  Collect the chloroform layer into a 100 ml beaker  Add again 10 ml of chloroform to the separating funnel and repeat the above step. Combine both the chloroform extracts  Take 100 ml potassium wash solution in a separate separating funnel  Add the chloroform extract to the second separating funnel and mix it slowly  Collect the chloroform layer through anhydrous sodium sulfate bed drop by drop to remove moisture  Dry the chloroform extract in an oven at 50°C  Dissolve the dried residue in 0.2 ml chloroform and spot on TLC plate along with the standard
  25. 25.  Compare the flourescence intensities of the sample and standard spots and identify the ones matching with each other  Calculate the aflatoxin content in the following way S x C x D Aflatoxin content (ppb) = x 1000 T x W Where, S = Standard which compares with the sample in fluorescent intensity C = Concentration of standard (1 µg / ml) D = Dilution factor in ml T = Sample which compares with standard in fluorescent intensity 75 x 100 W = Effective weight 25 x = 4.286 g 250 x 175 4. Analysis of Ochratoxin (By thin layer chromatography) Reagents : Sodium bicarbonate and diatomaceous earth mixture : Add 25 ml of 5 % aqueous NaHCO3 to 50 g
  26. 26. diatomaceous earth (Celite 545), mix well and store in tightly closed container Solvents : i) Chloroform ii) Hexane iii) Acetic acid : benzene ( 2 : 98 ) iv) Acetic acid : benzene (1 : 99 ) Standard : Ochratoxin A 2 µg / ml in acetic acid : benzene (1 : 99) Apparatus : i) Wrist action / horizontal shaker ii) Hot water (steam) bath iii) TLC plates (precoated silica gel plates or equivalent) iv) Developing tank / chamber v) UV viewing cabinet Procedure :  Take 25 g of sample in a 250 ml glass stoppered conical flask, add 12.5 ml water and mix  Add 125 ml chloroform and shake for 1 hour  Filter through Whatman No.1 filter paper and collect the filtrate  Plug the bottom of a glass column (2 cm x 30 cm) with glass wool, put 6 g of NaHCO3 - Celite mixture and tamp firmly with a glass rod
  27. 27.  Add 50 ml of chloroform extract to the column and elute until meniscus reaches top of the NaHCO3 - Celite column  Wash the column with 70 ml hexane followed by 70 ml chloroform and discard washings  Elute Ochratoxin with 100 ml acetic acid : benzene ( 2 : 98)  Collect the eluate and evaporate on steam bath  Dissolve the residue in 5 - 10 ml chloroform, transfer to a small vial ( 10 ml capacity) and evaporate on steam bath  Dissolve the residue in 0.5 ml acetic acid : benzene (1 : 99) by vigorous shaking  Spot on TLC plate along with the standard (5, 10, 15 and 20 µ1 or in any other suitable range)  Develop the plate using toluene : ethyl acetate : formic acid ( 5 : 4 : 1) in an unequilibrated chamber  Air dry the plate, view under long wave UV light (365 nm) and compare the intensity of greenish blue fluorescent spots of the sample with that of standard spots and identify the spot, matching each other  Calculate the Ochratoxin A content using the formula SxYxV Ochratoxin A µg / kg = ZxW
  28. 28. Where, S = Volume in µl of ochratoxin A standard spot comparable to Z µl of sample spot Z = Volume in µl of sample spot comparable to S µl of ochratoxin A standard Y = Concentration of ochratoxin A standard (2 µg / ml) V = Volume (µl) of the dissolved residue before spotting W= Effective weight of the sample 25 x 50 150 Confirmation Expose the developed plate to NH3 fumes. Greenish blue fluorescence of Ochratoxin will turn to bright blue. Reference AOAC. 1995. Official methods of analysis. 16th ed. Assoc. Off. Anal. Chem., Washington, D.C. 5. Analysis of T-2 toxin (By thin layer chromatography) Reagents : i) 30 % ammonium sulphate (dissolve 30 g (NH4)2 SO4 in water and make up volume to 100 ml) ii) Celite 545 iii) Potassium wash solution (dissolve 1.12 g KOH and 10 g KCl in water and make up volume to 1 lit)
  29. 29. iv) Sodium sulphate v) Silica gel vi) Methanol : H2SO4 ( 1 : 1 v/v ) Solvents : i) Methanol : water (1 : 1 v/v) ii) Chloroform iii) Diethyl ether iv) Hexane v) Benzene vi) Acetone : Benzene (5 : 95 v/v) vii) Developing solvent mixture - Toluene : ethyl acetate formic acid (6 : 3: 1 v/v) Standard : T-2 toxin 50 µg / ml in Benzene or diethyl ether Apparatus : i) Wrist action / horizontal shaker ii) TLC plates (precoated silica gel plates or equivalent) iii) Developing tank / chamber iv) UV viewing cabinet Procedure :  Take 50 g of sample in a glass stoppered conical flask
  30. 30.  Add 250 ml of methanol : water (1 : 1) and shake for 1 hour  Filter using whatman No.1 filter paper and collect 60 ml of extract into a beaker  Add 240 ml 30 % (NH4)2 SO4 and stir vigorously for 1 minute  Add 20 g of celite and stir for 1 minute  Filter and collect 200 ml of filtrate  Transfer filtrate to a separating funnel  Add 10 ml of chloroform and shake vigorously for 1 minute  Allow the layers to separate and collect the bottom layer into another separating funnel  Repeat the extraction with another 10 ml of chloroform  Combine both the extracts and add 100 ml of potassium wash solution  Swirl gently for 30 seconds and let layers separate  Drain the lower chloroform layer through a bed of Sodium sulphate (in a funnel) to dry and collect 10 ml of clear filtrate  Column Preparation : Plug the bottom of a glass column ( 2 cm x 30 cm ) with glass wool and add 5 g anhydrous sodium sulphate. Fill
  31. 31. the column to half level with chloroform and add 10 g silica gel. Wash sides of column with chloroform and stir to eliminate air bubbles. Drain off chloroform leaving about 7 cm above the upper level of silica gel. Add 15 g anhydrous sodium sulphate without disturbing the silica gel. Drain off chloroform to the upper level of sodium sulphate  Wash the column serially with 50 ml of diethyl ether and 10 ml of chloroform and discard the washings  Mix 10 ml of sample extract with 30 ml of hexane and add to the column and slowly drain until solvent is about 1 cm above Sodium sulphate  Add in succession 30 ml benzene and 40 ml acetone : benzene (5: 95) and discard both the washings  Elute T-2 with diethyl ether until 30 ml of eluate is collected and evaporate the eluate  Dissolve the residue in 0.5-1.0 ml diethyl ether. Spot on TLC along with the standard (5-20 µ1 or any other suitable range) and develop the plate in toluene : ethyl acetate : formic acid (6 : 3 : 1)  Air dry the plate and spray with methanol : H2SO4 (1 : 1)  Dry at 110°C for 10 minutes and observe blue fluorescence under long wave UV light (365 nm)
  32. 32.  Compare the intensities of the blue fluorescent spots of the sample with those of standard and identify the ones matching each other  Calculate the T-2 content of sample using the following formula S x Y x V T - 2 µg / kg = Z x W Where, S = Volume in µl of T - 2 standard spot comparable to Z µl of sample spot Z = Volume in µl of sample spot comparable to S µl of T - 2 standard Y = Concentration of T - 2 standard (50 µg / ml) V = Volume (µl) of the dissolved residue before spotting W= Effective weight of the sample 50 x 60 x 200 x 10 250 300 20 References Romer, T.R., Boling, T.M. and Mc Donald, J.L. 1978. Gas liquid chromatographic determination of T-2 toxin and diacetoxyscirpenol in corn and mixed feeds. JAOAC. 61 : 801 - 807. Rukmini, C. and Bhat, R.V. 1978. Occurrence of T-2 toxin in Fusarium infested sorghum from India. J. Agric. Food Chem. 26 : 647-649.
  33. 33. 6. Analysis of Zearalenone (By thin layer chromatography) Reagents : i) Aluminium chloride solution (dissolve 20g AlCl3 6H2O in 100 ml methanol) ii) Celite 545 Solvents : i) Chloroform : water mixture ( 10 : 1 v/v) ii) Hexane iii) Chloroform iv) Diethyl ether v) Benzene vi) Acetone : benzene ( 5 : 95 v/v) vii) Acetonitrile viii) Developing solvent - Methanol : Chloroform (5 : 95 v/v) or Acetic acid : Benzene (5 : 95 v/v) Standard : Zearalenone 50 µg / ml in Benzene Apparatus : i) Wrist action / horizontal shaker ii) Hot water (steam) bath iii) TLC plates (precoated silica gel plates or equivalent) iv) Developing tank / chamber v) UV viewing cabinet
  34. 34. Procedure :  Take 50 g of sample in a glass stoppered conical flask and add 300 ml of chloroform : water (10 : 1) and 25 g of celite  Shake for 1 hour and filter using Whatman No. 1 filter paper  Column Preparation : Plug the bottom of a glass column (2 cm x 30 cm) with glass wool and add 5 g anhydrous sodium sulphate. Fill the column about half full with chloroform and add 10 g silica gel. Wash sides of column with chloroform and stir to eliminate air bubbles. Drain off chloroform leaving about 7 cm above the upper level of silica gel. Add 15 g anhydrous sodium sulphate without disturbing the silica gel. Drain off chloroform to the upper level of sodium sulphate  Transfer 50 ml of sample extract together with 150 ml hexane into the silica gel column  Drain until the solvent reaches top of the column and discard the washings  Wash the column serially with 150 ml of diethyl ether and 150 ml of benzene and discard both the washings  Elute Zearalenone with 250 ml of acetone : benzene ( 5 : 95 )  Add few silica chips to the eluate and evaporate on steam bath, preferably under gentle stream of N2
  35. 35.  Dissolve residue in 10 ml of hexane and transfer quantitatively to a separating funnel  Repeat the above step for 3 times using 10 ml of hexane each time  Finally rinse the residue with 10 ml of acetonitrile and add to the hexane washes present in the separating funnel. Shake well and let phases separate  Collect the lower acetonitrile phase into a 100 ml beaker  Add another 5 ml acetonitrile to the hexane washes present in the separating funnel and repeat the above step. Combine both the acetonitrile fractions.  Evaporate the combined acetonitrile fractions on steam bath ( under stream of N2)  Transfer the residue to a small vial (about 10 ml capacity) using about 5 - 10 ml of chloroform and evaporate as in the previous step  Add about 0.5 ml benzene to the residue and shake vigorously  Spot on TLC plate (5, 10, 15, 20 µl or other suitable volumes) along with the standard and develop the plate in methanol : chloroform (5 : 95) or acetic acid : benzene (5 : 95)  Air dry the plate and spray the spots with aluminium chloride solution, heat at 130° C for 5 min and examine under longwave UV light (365 nm)
  36. 36.  Compare the intensities of the blue fluorescent spots of sample with those of the standard and identify the ones, matching with each other. Calculate the Zearalenone content of the sample in the following way S x Y x V Zearalenone µg / kg = Z x W Where, S = Volume in µl of Zearalenone standard spot comparable to Z µl of sample spot Z = Volume in µl of sample spot comparable to S µl of Zearalenone standard Y = Concentration of Zearalenone standard (1 µg / ml) V = Volume (µl) of the dissolved residue before spotting W= Effective weight of the sample 50 50 x 300 References : AOAC, 1995. Official methods of analysis. 16th ed. Assoc. Off. Anal. Chem. Washington, D.C. ANALYSIS OF CITRININ Reagents: 1. 4% KCL (4 gms KCL in 100 ml distilled water) 2. 20% H2SO4 (20 ml concentrated H2SO4 + 5% NaHC03 ( 5 gms of NaHC03 in 100 ml DW) 3. 6 N HCl (185.4 ml of HCl in 1 lt distilled water) 4. 10% Oxalic acid in methanol (W/V) (10 gm
  37. 37. oxalic acid in 100 ml methanol) Solvents: 1. Acetonitrile 2. Iso octane 3. Chloroform 4. Chloroform – methanol – hexane (64:1:35) Standards: 1. Citrinin : 10 g/ml in methanol Procedure:  Take 25 gms of sample, add 180 ml acetonitile, 20 ml 4% KCl, 2 ml 20% H2SO4 and shake for 15 minutes  Filter and collect 100 ml of the filtrate  Transfer filtrate to separating funnel and add 50ml iso octane. Shake for 1 minute.  Collect the lower layer, add 50 ml iso octane and repeat the above step.  Collect the lower layer into another separating funnel, add 25 ml water and extract with 50 ml chloroform  Drain the chloroform layer into a separating funnel  Extract again with two 10 ml portion of chloroform and combine all the three extracts  Add 25 ml of 5% NaHC03 to the extract. Shake for 1 minute and drain off the lower portion.  Re-extract with two 25 ml 5% NaHC03 portion and repeat the above step.  Combine the extracted portion in a 600 ml beaker and acidify it with 6N HCl to pH 1-2, transfer it to separating funnel  Rinse the beaker with 50 ml CHCl3, and transfer it to seperating funne, swirl for 30 seconds.
  38. 38.  Drain the lower portion, repeat above step with another 50 ml portion of CHCl3  Collect the CHCl3 extracts and evaporate to near dryness  Add 5 ml CHCl3 to the dried extract and filter through fluorocarbon filter into a 25 ml beaker  Rinse the beaker twice with small amounts of CHCl3, filter and evaporate  Dip the TLC plate in 10% oxalic acid solution for 2 min and air dry overnight  Spot the sample and standard in 5-20 l range  Develop plate in chloroform-methanol-hexane (64:1:35) for 45 minutes. Air dry and observe under long wave UV light for comparing the intensitites  Calculate the citrinin content of sample using the following formula Citrinin, g/kg = S x Y x V X x W Where, S = l standard equal to unknown Y = Concentration of standard (g/ml) V = Dilution of sample extract (l) X = l of sample spotted W= gms of sample represented by final extract = 25 x 100 = 12.376 g 202 7. Rapid TLC Method of Multi-Mycotoxin Analysis (Modified Tapia Method) Reagents : i) 4 % KCl (4 g KCl in 100 ml di. water)
  39. 39. ii) 5 N HCl (405.9 ml Conc. HCl in 1 l di. water) iii) Na2So4 ( anhydrous ) iv) 20 % KOH (20 g KOH in 100 ml di. water) v) 20 % H2SO4 in ethanol (20 ml Conc. H2SO4 + 80 ml ethanol) Solvents : i) Acetonitrile ii) Hexane iii) Chloroform iv) Chloroform : Acetone : Water (88 : 12 : 1) v) Toluene : Ethyl acetate : Formic acid (5 : 4 : 1) Standards : Aflatoxin B1 1 µg/ml in Acetonitrile: Benzene (2 : 98) Ochratoxin A 2 µg/ml in Acetonitrile: Benzene (2: 98) Zearalenone 50 µg / ml in Benzene T-2 toxin 50 µg / ml in Ethyl acetate Sterigmatocystin Citrinin Oosporein Procedure :  Take 25 g sample, add 85 ml acetonitrile, 15 ml 4 % KCl and 2 ml 5 N HCl and blend at high speed for 3 minutes  Filter through Whatman no. 1 filter paper
  40. 40.  Transfer 50 ml filtrate into a 250 ml separating funnel  Add 50 ml water, followed by 50 ml hexane and shake well  Collect the lower layer, add 50 ml hexane again and repeat the above step  Collect the lower layer into another separating funnel and extract with two 10 ml portions of chloroform  Drain the chloroform layer through anhydrous Sodium sulphate and evaporate in oven at 50C  Dissolve the residue in 0.2 ml chloroform and spot on TLC plate along with the standards  Develop plate in chloroform : acetone : water (88 : 12 : 1) in one direction and toluene : ethyl acetate : formic acid (5 : 4 : 1) in the second direction  Spray zone of spots, corresponding to sterigmatocystin with 20 % aqueous KOH  Spray zone of spots, corresponding to T-2 toxin with 20 % H2So4 in ethanol and heat at 110C  View the spots, identify and quantify the toxins as done with individual toxins 50 Effective weight of the sample = 25 x = 12.255 g
  41. 41. 102
  42. 42. 8. Rapid Mycotoxin Test / "ELISA Test" Principle : Antibody coated column is used to trap the mycotoxin. This trapped toxin is then eluted using approximate solvent and quantified in fluorometer. Equipments : 1. Immuno affinity column 2. Affinity column stand with syringe 3. Cuvette 4. Calibrated Fluorometer 5. Blender 6. Fluted filter paper Reagent : 1. Test developer 2. Methonol : water (80 : 20 by volume) 3. Mycotoxin wash buffer Procedure :  50 gms of sample + 5 gms of NaCl + 100 ml of methanol water (80 : 20) Note : NaCl is not added in case of Ochra Test  Blend at high speed and filter  Pipette filtered extract into clean vessel Aflatoxin, Ochratoxin : 10 ml
  43. 43. Zearalenone : 1 ml  Dilute with purified water and mix Aflatoxin, Ochratoxin : 40 ml Zearalenone : 49 ml  Filter  Remove top cap and attach the syringe (cut 1/8 inch bottom of column )  Pass filtered diluted extract at the rate of 1-2 drops/second Aflatoxin : 2 ml Ochratoxin, Zearalenone : 10ml  Pass water at the rate of 1-2 drops/second Aflatoxin, Zearalenone : 5 ml Ochratoxin : First 10 ml Mycotoxin wash buffer, Later 10 ml distilled water Courtesy : VICAM  Elute toxin in glass cuvette Aflatoxin, Zearalenone : Pass 1 ml HPLC grade methanol Ochratoxin : Pass 1.5 ml Ochratoxin eluting soln.  Add 1 ml of developer to the cuvette and mix well  Read in calibrated fluorometer 9. In vitro evaluation of Mycotoxin binding agents
  44. 44. Objective : To evaluate the mycotoxin binding efficacy of binding agents (adsorbants) in percentage under simulated GI tract conditions of chicken. Methodology : 1. Take 2 sets of triplicate samples of 25 g each of compounded broiler / layer feed in 250 ml Erlenmeyer flasks 2. Add known quantity of mycotoxin to the feed in all the flasks 3. Add the binding agent to the feed in one set of flasks (treated) and leave the feed in the remaining set of flasks untreated (control) 4. Add 100 ml buffer solution of either 4.5 or 6.5 pH to all the flasks and mix the contents thoroughly for 30 minutes using a wrist action / horizontal shaker 5. Incubate the flasks at 37°C for 3 hours 6. Filter the contents and dry the residue at 35-45°C for 2 hours 7. Analyse the dried residue for the unbound toxin content as per the standard procedure (AOAC, 1995) 8. Calculate the percentage adsorption by subtracting the percent difference in toxin content in the control flasks from that of the treated flasks BT - E T BC - E C
  45. 45. Percent toxin adsorption = ------------ x 100 - ------------- x 100 BT BC Where, BT = Toxin content in the treated flasks at the beginning ET = Toxin content in the treated flasks at the end BC = Toxin content in the control flasks at the beginning EC = Toxin content in the control flasks at the end Note :  Buffer composition - For every one litre 4.5 pH : 273 ml 0.1 M citric acid, 227 ml 0.2 M Di Sodium hydrogen phosphate (Na2HPO4) and 500 ml distilled water 6.5 pH : 145 ml 0.1 M citric acid, 335 ml 0.2 M Di Sodium hydrogen phosphate (Na2HPO4) and 500 ml distilled water
  46. 46. 10. In vitro evaluation of Mycotoxin binding agents in contaminated feeds Objective : To evaluate the mycotoxin binding efficacy of binding agents (adsorbants) in percentage under simulated GI tract conditions of chicken in contaminated feeds. Methodology : 1. Analyse the contaminated feed for mycotoxin 2. Take 2 sets of triplicate samples of 25 g each of contaminated feed in 250 ml Erlenmeyer flasks 3. Add the binding agent to the feed in one set of flasks (treated) and leave the feed in the remaining set of flasks untreated (control) 4. Add 100 ml buffer solution of either 4.5 or 6.5 pH to all the flasks and mix the contents thoroughly for 30 minutes using a wrist action / horizontal shaker 5. Incubate the flasks at 37°C for 3 hours 6. Filter the contents and dry the residue at 35-45°C for 2 hours 7. Analyse the dried residue for the unbound toxin content as per the standard procedure (AOAC, 1995)
  47. 47. 8. Calculate the percentage adsorption by substracting the percent difference in toxin content in the control flasks from that of the treated flasks in the following way - BT - E T BC - EC Percent toxin adsorption = ------------ x 100 - ------------- x 100 BT BC Where BT = Toxin content in the treated flasks at the beginning ET = Toxin content in the treated flasks at the end BC = Toxin content in the control flasks at the beginning EC = Toxin content in the control flasks at the end
  48. 48. 11. List of suppliers of Mycotoxin standards : Mycotoxin standards are supplied in pure crystalline form by several companies. They can be obtained from Sigma Chemical Co., P.O. Box 14508, St. Louis, Missouri 63178 - 9916, USA. Ph : (314) 771 - 5750 (314) 771 - 5757 Internet : http://www. sigma/ sial. com Sigma - Aldrich Corpn., Plot no. 70, Road no. 9, Jubilee Hills, Hyderabad - 500 033. Ph : (040) 244739 Fax : (040) 244794 Orders can also be placed at the following distribution centres New Delhi : Tel (011) 6899826 / 6897830 Fax (011) 6899827 Mumbai : Tel (022) 6325344 / 6325345 Fax (022) 6268686 Bangalore : Tel (080) 3316659 Fax (080) 3440570
  49. 49. AFLATOXIN BINDING ANALYSIS Aim - To check the binding capacity of binder with aflatoxin B1 standard and release at different pH (3.2, 6.0,6.5) . Equipment - U.V. chamber Chemicals - Chloroform Trisodium citrate Hydrochloric acid Acetone Aflatoxin B1 (Sigma standard) TLC Plates (Merck) Preparation of buffer - Sodium citrate - 0.588gms in 100 ml (0.1 M) Hydrochloric acid - 0.782ml in 100 ml Add the hydrochloric acid solution to sodium citrate solution to get the pH 3.2, 6.0, 6.5. Assay - 1. Take 400 g level of std aflatoxin B1 in a test tube and evaporate to dryness. 2. Add different levels of binders to the above test tubes. 3. Add 1ml of 3.2 pH buffer , vortex it and keep for incubation for 30 minutes at 40oC. 4. After 30 minutes centrifuge it. Take out the supernatant in other test tube and use the sediment for release studies of aflatoxin. 5. To the above supernatant add 400 l of chloroform to extract the toxin from supernatant. 6. Spot it on the TLC plate and compare the colour intensity with standard aflatoxin B1. This will give the level of toxin which is not bound with the binder. The difference between the loaded toxin and unbound toxin gives the toxin bound with the binder.
  50. 50. 7. Use the sediment obtained in step IV for release studies. 8. Add 1ml of buffer (pH 3.2) to the sediment. Vortex and keep for incubation at 40oC with intermediate shaking. After 30 minutes centrifuge separate the supernatant and sediment. Use the supernatant to find out the toxin released at pH 3.2 by adding 400 l of chloroform. Use the sediment for the further release study at pH 6.0. 9. Add 1ml of buffer (pH 6.0) to the sediment . Vortex and keep for incubation at 40oC for 60 minutes with intermediate shaking. 10.After 60 minutes centrifuge it, separate the supernatant and sediment. Use the supernatant to find out the toxin released at pH 6.0 by adding 400 l of chloroform. 11.Add 1ml of buffer (pH 6.5) to the sediment, vortex and keep for incubation for 30 minutes at 40oC. 12.After 30 minutes centrifuge, separate the sediment and supernatant. Find out the toxin released in the supernatant at pH 6.5 by adding 400 l of chloroform. 13.Spot on TLC plate and compare the colour intensity with standard. Calculations : A x 400 l x Std concentration --------------------------------------- B x Total reaction volume A = l of standard comparible with l of sample B = l of sample comparible with l of standard 1st supernatant - Unbound toxin with binder loaded toxin - unbound toxin = bound toxin 2nd supernatant - Release at pH 3.2
  51. 51. 3rd supernatant - Release at pH 6.0 4th supernatant - Release at pH 6.5 The difference between the bound toxin and total release toxin will be toxin retained with binder. AFLATOXIN ANALYSIS IN FEEDSTUFFS Reagents 0.2 M NaOH (dissolve 8 g NaOH in water and make up volume to 1liter) 0.41 m Ferric chloride (dissolve 66.5 g anhydrous FeCl3 in water and make up volume to 1 lit) 0.03% H2SO4 (0.3 ml con. H2SO4 + 999.7ml water ) Potassium Wash solution (dissolve 1.12 g KOH and 10g Kcl in water and make up volume to 1liter) Solvents Aqueous acetone (85 acetone : 15 water) Chloroform Chloroform : acetone Aflatoxin B1 1g/ml in Benzene:acetonitrile(98:2) Standard Wrist action / horizontal shaker Apparatus Hot water (steam) bath TLC plates (precoated silicagel plates or equivalent) Developing tank /chamber UV viewing chamber Procedure 1. Add 250 ml of aqueous acetone to 50 g of sample in a glass stoppered conical flask and shake for 1 hour. 2. Filter whatman No.1 filter paper. 3. Add 150 ml of filtrate to a conical flask containing 3g cupric carbonate.
  52. 52. 4. Add 170 ml of 0.2 M NaOH to 30ml of ferric chloride solution. Add this mixture to the conical flask for 30 seconds and filter. 5. Collect 250ml of filtrate and transfer it to a separating funnel containing 250 ml of 0.03% H2SO4. 6. Add 10ml of chloroform to the contents of separating funnel and shake vigorously. 7. Allow the chloroform layer to separate at the bottom of the funnel. 8. Drop the separated Chloroform layer into another separating funnel, Containing 100 ml of potassium Wash solution 9. Add another 10 ml of Chloroform to the first separating funnel and repeat the above steps 10.Gently swirl the separating funnel containing the Chloroform extract and potassium wash solution and allow the layers to separate 11.Drain the Chloroform layer through Na2SO4 and collec 12.Measure 10 ml aliquot of the chloroform extract and evaporate on Water bath 13.Dissolve the dried residue in Chloroform(about 0.3 ml) 14.Spot on TLC plate along with the Standard 15.Develop the plate using Chloroform : Acetone (85: 15) in an equilibrated Chamber 16.Air dry the plate , view under long Wave UV light (365nm) and compare the intensity of the blue fluorescence spots of sample, with that of standard spots and identify the spots, matching with each other. 17.Calculate the aflatoxin B1 content in the following way S x Y x V Aflatoxin B1 g/kg = ---------------- Z x W
  53. 53. Where, S = Volume in l of aflatoxin B1 standard spot comparable to Z l of sample spot Z = Volume in l of sample spot comparable to S l of aflatoxin B1 standard Y = Concentration of aflatoxin B1 standard (1g/ml) V = Volume (l) of the dissolved residue before spotting W = Effective weight of the sample : 50 x 150 x 250 x 10 ------ ----- ---- 250 350 20 PREPARATION OF FEED SAMPLES FOR METHIONINE AND CYSTINE ANALYSIS USING PERFORMICACID OXIDATION PROCEDURE Reagents:
  54. 54. 1. Hydrogen peroxide (H2O2), 30% W/W 2. 88% formic acid 3. 48% hydrobromic acid 4. 6N HCL: 50 ml of concentrated hydrochloric acid added to 50 ml double distilled water. 5. DL-Norleucine standard, 25 umole/ml 6. Sodium citrate buffer, pH 2.2 Procedure: 1. Weigh finely ground sample (ground to pass a 40-mesh sieve) containing 30- 40 mg protein into a 50 ml Erlenmeyer flask. This would be approximately 60-80 mg for soybean meal samples and 300-350 mg for corn samples when diluting the hydrolysate 100 times. 2. Add 6 ml of cold, freshly prepared, performic acid2 to each flask, cover and reactat 00C for 4 hours for amino acids and soluble proteins, or overnight (16 hours) for proteins that do not dissolve in the performic acid mixture.
  55. 55. 3. Add 0.75 ml of 48% hydrobromic acid with swirling of the flask in an ice bath. 4. Remove performic acid and hydrobromic acid with swiling of the flask in an ice bath. 5. Add 0.6 ml DL-norleucine (25 umole/ml) so that after the final dilution the concentraion of norleucine will be 0.15 umole/ml. 6. Transfer contents into hydrolysate tube (1.5 cm internal diameter, 15 cm length) by repeated washings with 6 N HCL so that the final volume is about 10-12 ml. 7. Charge the tubes with nitrogen gas, cover with screw cap and place in an oven at 1100 for 20 hours. 8. After the tube cools, filter the contents through Whatman No. 1 filter paper into a drying tube. Wash hydrolysate tube and filter paper with double distilled water and collect in the same drying tube. 9. Dry the filtrate by evaporating with a rotary evaporator under vacuum, with
  56. 56. the water bath temperature at 480C. Dry the filtrate to a slightly wet residue Wash residue with distilled water and dry again. 10. Add 20 ml of citrate buffer and mix well. 11. Take one ml from step 10 and dilute to 5 ml with citrate buffe, pH 2.2. Mis well. 12. Filter the diluted sample liquid using 0.2 micron Nuclepore3 membrane filter 13. Determine methionine sulfone and cysteic acid by injection sample into Dionex D-300 amino acid analyzer.
  57. 57. ESTIMATION OF SODIUM AND POTASSIUM BY FLAMEPHOTOMETRY PRINCIPLE: Depends on the fact that when a metal is burned in flame, its molecules are energized, emitting a charateristic colour. The intensity of the colour is proportional to the concentration of the element in the solution. Preparation of sample 1. Transfer about 1gm of representative sample into a 100 ml conical flask. 2. Add 10 ml of conc. HNO3 and keep overnight. 3. Place this flask on the hot plate, allow it to boil, until yellow flumes subside cool. 4. Add 5ml of triacid mixture and digest on hot plate until it forms white geletinous semi-liquid mass. 5. Cool the flask and make it to a known volume i.e., 100 ml by double glass distilled glass distilled water.
  58. 58. Preparation of triacid mixture: Take a clean dry 500 ml pyrex beaker, add 100 ml conc. HNO3 gently, and add 40 ml perchloric acid and 10 ml of conc. H2SO4 , carefully. Mix it gently and store it in dry and clear amber coloured bottle. Preparation of standard solution: Stock sodium standard solution : Dissolve 0.2543 g sodium chloride (dry) in 100 ml volumetric flask and make the volume upto the mark (1000 ppm solution). Stock potassium standard solution : Dissolve 0.1910 g potassium chloride (KCL) and dissolve in 100 ml water (1000 ppm). Working standard solution : Sodium Make the visual dilution of stock standard to get 10, 20, 30, ..... 100 ppm solution by taking 1 ml, 2, 3...... and 10 ml respectively in 100 ml volumetric flasks and make the volume
  59. 59. Potassium Take 1,.. 2,.. 3,.. and 1 ml of stock standard solution in 100 ml flasks and make the volume by distilled water (1,2,3. . . . and 10 ppm solution). UREA TEST Scope: Applicable to most feeds. Purpose: Is useful in determining the presence of urea in feeds. Apparatus: Test tubes Reagents: Dissolve 2 grams of dimethylaminobenzaldehyde (DMAB) in a solution of 90 ml. of methyl alcohol (methanol) and 10 ml. of concentrated hydrochloric acid. Procedure: 1. To a teaspoonful of feed in a test tube, add about 3 teaspoonfuls of water and mix
  60. 60. 2. Let settlle and pour the supernatant through a filter into another test tube containing approximately a teaspoonful of DMAB reagent. 3. Any deeping of the yellow color indicates the presence of urea.
  61. 61. QUALITATIVE TEST FOR UREA IN FISH MEAL Reagents: 1. Urease enzyme solution 2. Standard Urea Solutions (0, 0.5, 1, 1.5,..........5%) 3. Phenol red indicator (0.1%) or Cresol red indicator (0.1%) Procedure: 1. Weigh 10 g. tested sample and add 100 ml of distilled water. Mix thoroughly and then filter with whatman No. 41 filter paper. 2. Take 1 ml of tested sample aliquate into white porcelein spot plate. 3. Add 2-3 drops of phenol red indicator and then add 2-3 drops of urease solution. 4. Stand for 3-5 minutes, if Urea presents solution will become red purple in contrast to the yellow colour of indicator.
  62. 62. Colour can be compared with the colour developed in standard solution of varying levels of urea. UREASE TEST (RAPID METHOD) Occurring in: Soybean meal Type of analysis : Enzymatic 1. Principle The urease enzyme activity of soybean meals is measures qualitatively by the conversion of urea to ammonia in the presence of Phenol red-indicator. 2. Reagents A. Sodium hydroxide, 0.1N B. Sulfuric acid, 0.1N C. Urea-phenol red solution, 1.4 g phenol red, 70 ml 0.1N NaOH, 350 ml H2O -- 210 g reagent grade urea, 3000 ml H2O.
  63. 63. D. Add 25 ml of amber colored phenol red solution. Swirl gently to spread sample evenly in dish. E. Let stand 5 minutes. 3. Method A. Transfer small amount of Phenol red solution to beaker. 25 ml is needed for each test. B. Adjust to amber color with 0.1N HCL. If solution turns yellow, adjust with 0.1N NaOH. C. Place one level tablespoon of well mixed soybean meal into a petri dish. D. Add 25 ml of amber colored phenol red solution. Swirl gently to spread sample evenly in dish. E. Let stand 5 minutes. 4. Scale A. Slightly active: Few scattered red particles B. Moderately active : Surface appears to be approximately 25% covered with red particles.
  64. 64. C. Active : Surface appears to be approximately 50% covered with red particles. D. Very active : Surface appears to be approximately 75% covered with red particles. If more than 75% of the particles are colored red, the urease activity is estimated to be greater than 0.2. E. Overcooked : No visible red color after 5 minutes. Allow sample to set for additional 25 minutes. If still no colored particles, the meal is overcooked. F. UREASE TEST (Association of Oficial Agricultural Methods - U. S. Test) Objective: Urease index is used to evaluate soybean meal quality. The procedure measures only underprocessing and not overprocessing. The method determines the activity of the residual urease in the soybean products under the contitions of the test.
  65. 65. Adequate Values: 0.02 to 0.20 pH. The urease index should not be less than 0.02 or more than 0.20. Materials Tested: Soybean meals, soy flour, and soybean meal feeds except where urea has been added. UREASE ACTIVITY PROCEDURE A. Apparatus: 1. Water bath capable of being maintained at a temperature of 300 + .5o C 2. pH meter equipped with glass and calomel electrodes and with provision for testing 5 ml. of solutions. It should be a precision instrument with a temperature compensator having a sensitivity of + 0.02 pH units or better. Follow manufacturer’s instructions for operation of the instrument and detemination of pH. Calibrate the meter with standard buffers with values at or near the range at which measurements are to be made.
  66. 66. 3. Test tubes, 20 mm. X 150 mm., fitted with rubber stoppers. B. Solutions: 1. Phosphate buffer solution, 0.05M. Dissolve 3.403 g. of monobasic potassium phosphate (KH2PO4, AR grade) in approximately 100 ml. of freshly distilled water. Dissolve 4.355 g. of dibasic potassium phosphate (K2HPO4, AR grade) in approximately 100 ml. of water. Combine the two solutions and make to 1000 ml. If reagents are pure, pH should be at 7.0. If it is not, adjust to 7.0 with a solution of a strong acid or base before using. The useful life of the buffer solution, prepared as described, is less than 90 days. 2. Buffered urea solution. Dissolve 15 g. urea (AR grade) in 500 ml. of the phosphate buffer solution. Add 5 ml. of toluene to serve as a presergvative and to prevent mold formation. Adjust the pH of the urea solution to 7.0 as in B,1. C. Preparation of Sample: 1. Grind the sample as fine as possible without raising the temperature and mix. At least 60% of the sample should
  67. 67. pass a No. 40 U.S. Standard sieve. Soy flour requires no grinding but make certain it is well mixed. D. Procedure: 1. Weigh 0.200 g (+ 0.001 g.) of sample into a test tube and add 10 ml. of the buffered urea solution. Stopper, mix and place in water bath at 300C. Do not invert the tube during the processing of mixing. 2. Prepare a blank by weighing 0.200 g (+ 0.001 G.) sample into a test tube and to this add 10 ml of the phosphate buffer solution. Stopper, mix and place in water bath at 300C. Allow a time interval of 5 minutes between the preparation of the test and the blank portions. Agitate the contents of each tube at 5 minute intervals. 3. Remove the test and blank portions from the water bath after 30 minutes. Transfer the supernatant liquids to a 5.0 ml. beaker, maintaining the 5-minute interval between the test and blank. Determine the pH of the supernatant liquids at exactly 5 minutes after removal from the bath. (See Note 1.) E. Calculations:
  68. 68. 1. The difference between the pH of the test and the pH of the blank is an index of urease activity. F. Note: 1. Care must be exercised to prevent contamination of all glassware or electrodes. Should the pH instrument fail to deliver a prompt and stable reading, investigate. Frequently, the flow the electrolyte through the porous fibers in the calomel electrode may be retarded by a coating of the soluble fraction from soybean. 2. This method is a modification of the procedure of Caskey, C. D. and knapp, F. C., Ind. Eng. Chem., Anal.Ed.16, 640 (1944). DETERMINATION OF TANNINS INTRODUCTION : 1. Tannins (quercitannin acid and gallotannic acid ) are plant toxin.
  69. 69. 2. Plant feed ingredients containing more than 5% tannins when used in poultry feeds retards the growth of birds and reduces egg production. 3. Tannins are determined by using gelatin as a precipitating agent. REAGENTS AND CHEMICALS: 1. Indigo carmine (0.6%) (1.5 g indigo carmine + 100 ml H2O + 12.5 ml H2SO4 - dilute to 250 ml and filter.. 2. Gelatin solution (2.5%) 25 g gelatin+800 ml saturated Nacl - dissolve and make the Vol. to 1000 ml with sat. Nacl). 3. Acid sodium chorode solution (975 ml saturated Nacl + 25 conc. H2SO4 4. Standard Kmno4 (0.1 N) 5. Sodium chloride 6. Kaolin Powder
  70. 70. PROCEDURE : 1. Reflux about 5 g fat-free sample in 400 ml water for 30 min. 2. Transfer the contents to a 500 ml volumetric flask and dilute to 500 ml. 3. Filter through whatman No.1 filter paper and transfer 10 ml filtrate into a 1 litre beaker. 4. Add 25 ml indigo carmine solution and 750 ml water. 5. Titrate against 0.1 N Kmno4. The colour of the solution changes from dark blue to light green to bright yellow which is the end point. 6. Let this reading be (A). 7. At step (3), transfer 100 ml filtrate into a 500 ml vol. flask and add to it 50 ml gelatin solution, 100 ml acid sodium chloride and 10 g kaolin powder. 8. Shake for several min. and allow it to settle ; filter through whatman No.1 filter paper. 9. Transfer 25 ml filtrate into a 1 litre beaker; and 25 ml indigo carmine solution solution and 740 ml water.
  71. 71. 10. Repeat step (5) 11. Let this reading be (B). 12. Calculate the percentage of tannins by using the following formula : % Tannins = 21 x X x 100 W DM Where, `W’ is weight of sample, `X’ is the difference between A and B. RAPID QUALITATIVE TEST FOR TANNIN 1. Take about of 10 gms of jowar in bottle 2. Add potassium hydroxide and sodium hypochloride solution. 3. Close the bottle and shake till crystals dissolve. 4. Wait for about 15 minutes
  72. 72. 5. If the grain is very dark/black it contains tannin or if bleached white/light yellow, it contains no tannin. BOMB CALORIMETRY - PARR ADIABATIC BOMB CALORIMETER WITH AUTOMATIC WATER TEMPERATURE CONTROLLER I. Setting Up 1. Select location free from drafts and rapid temperature changes. 2. Attach cold water line to heater inlet. Flush heater using manual hot and cold water switch on controller to remove air after controller is connected. 3. Connect copper tubings to jacket. Jacket fittings are non-specific. 4. Attach multiple heater plug to controller. 5. Mount thermistors on thermometers so that thermistor end is aligned with center of the bulb. II. Initial Balance
  73. 73. 1. After flushing heater, turn swtich on and allow 10- 15 minutes to warm up. 2. Assemble bomb and place in bucket with 2,000 g. H2O. Place in jacket. 3. Start calorimeter and controller with switch found on controller. 4. Balancing may be done at any temperature. Equality of thermometers is not necessary. 5. Open throttling valves about 1 turn. 6. Add hot or cold water with manual control switch until galvanometer spot swings toward center. 7. Make fine adjustments with throttling valves. They will probably have to be turned down. 8. Galvanometer should not vary more than 10 marks in either direction when balanced. 9. Bring thermometers to true temperature equality using the balance knob. Find true equality by correcting thermometer readings. (Thermometers may read slightly
  74. 74. different after correcting, but it is true temperature which is important.) 10. Lock balance knob and recheck galvanometer balance. 11. Check manual #131 for any additional information. III. Bomb Standardization 1. Weigh 1 gram Benzonic Acid pellet to nearest .1 mg. in cup. 2. Place cup in circular electrode and attach 10 cm. fuse wire to each the electrodes. Touch fuse wire to pellet. 3. Assemble bomb and fill with O2 (30 atmospheres pressure). It may be necessary to wet rubber seal to prevent O2 escape at filling. 4. Place bomb in bucket and bucket in the jacket. Attach contact wire. Fill bucket with2,000 ml. distilled water from volumetric flask. Allow same drainage time for all samples and standards. A drop of acetone on bucket-jacket connections aids in contacts.
  75. 75. 5. Close cover, lower thermometer-thermistor units, and start calorimeter motor. 6. Bring jacket temperature to near bucket temperature using manual control. It is usually best to bring jacket to a few tenths degree C. below the bucket and then allow the automatic control to equalize the temperature. 7. Allow four minutes at equilibrium temperature. 8. Read initial bucket temperature, ignite. Check temperature rise. If too much hot overshoot occurs, turn down valve located on bomb - not throttling vlave. About 1/4 turn open for this valve is sufficient. 9. After exactly 8 minutes, read final temperature. 10. Release pressure from bomb slowly - over a period of 1 minute. 11. Rinse inside of bomb with distilled water and titrate with .0725N Na2CO3 (.001 Kcal./ml.) using methyl orange indicator.
  76. 76. 12. Measure fuse wire remaining on dispenser card. Subtract this amount from 23 cal. This will give calories used in ignitioin. Calculation for Standardization W= Hm+a+b t where W = energy equivalent of calorimeter H = Heat of combustion benzoic acid m = mass of benzoic acid pellet a = ml. Na2CO3 used b = calories used in ignition - fuse wire t = difference in true initial and true final bucket temperature Note: W must be calculated for each bomb-bucket combination. IV. Sample Determinations 1. Make good firm pellet using pelleting machine. 2. Weigh to 0.1 mg. and place in bomb as with standard. 3. Perform operations identically with standardization procedure.
  77. 77. Calculations: H= tW - a - b m where H = Heat of combustion of sample t = true temperature difference W = energy equivalent of calorimeter a = ml. Na2CO3 used b = calories used in ignition-fuse wire m = mass of sample in grams. Duplicates should agree within .1 kcal/gram and preferably .05 kcal/gram. TREATED SEED TEST Scope : Applicable to grain suspected of having been treated and to feed containing grain suspected of having been treated. Purpose : To detect corn or other grain containing residues of arasan (tetramethyl thirum disulfide) (TMTD) (THIRAM).
  78. 78. Apparatus : 1. Small flasks: 125 ml. or 250 ml. Erlenmeyer are suitable. 2. Funnels: short stem, approximately 9 cm. diameter. 3. Test tubes. Reagents : 1. Chloroform 2. Cupric choride, analytical reagent. Procedure: 1. Place 10-15 grams of suspect grain or feed in a 250 ml. flask. Add 25 ml. chloroform and shake for 3 minutes. 2. Filter the solution through cotton placed in small funnel into a small. 3. Add a few crystals of cupric chloride to the filtered solution and shake for 3 minutes.
  79. 79. 4. Observe the resulting color. If arasan (TMTD) is present a characteristic amber to brown color will appear. In case the sample is a mixed feed containing alfalfa or some other green plant, the color of the solution after filtration in step 2 may appear yellow-green or green. After addition of the cupric chloride in step 3, the resulting color will be darker, a muddy brown if arasan (TMTD) is present, or a muddy green to yellow-green if no arasan (TMTD) is present in thte sample. A quantitative method for determining residue of thiram or treated seed corn is available from the DuPont Company, Retail Products Section, Grasselli Chemical Department,Wilmington,Delaware.
  80. 80. RANCIDITY TEST Purpose: To detect rancidity of fats in a feedstuff. Apparatus: 100 ml. Erlenmeyer flasks. Reagents: 1. Combine 60 ml. of concentrated glacial acetic acid (CH3COOH) and 40 ml. of chloroform (CHCL3). 2. Saturated potasium iodide (KI) solution. 3. Starch indicator. Procedure: 1. Place 5 grams of the sample in the flask and add 40 ml. of the acetic acid-chloroform mixture. 2. Add 1 ml. of the saturated potassium iodide solution. 3. Agitate thoroughly. Add 50 ml. of water and starch indicator. 4. The development of a blue color indicates rancidity.
  81. 81. DECOMPOSITION TEST (Eber’s Sulfide Test) Scope: Used for animal and marine products. Apparatus: 1. 250 cc. Erlenmeyer flask 2. Cork:Must fit tightly in flask and have a split in bottom. 3. 2 x 1/4 inch filter paper. Reagents: 1. 10% sulphuric acid (H2SO4) solution: 10 ml. of concnetrated sulfuric acid in 90 ml. of distilled water. 2. Add 50 ml. of the sulphuric acid solution and slightly swirl solution so all the meat material is thoroughly wet but the solution does not come up far on side of the flask. 3. Insert one end of the filter paper strip into the split in the cork so it hangs freely as seen in the figure. 4. Moisten the strip of filter paper with the lead a cetate solution. Do not have it wet enough so as to drip, for if the lead acetate comes in direct contact with the sulphuric acid solution the test will be spoiled. 5. Tightly insert the cork and attached filter paper into the flask. 6. Let stand in a warm room for 16 hours. 7. If the sample is badly decomposed, the test paper will darken quickly.
  82. 82. FEED MICROSCOPY A fast, simple and inexpensive method of determining the adulteration and contamination of feed ingredients and compound feeds is important in quality control in feed manufacturing industy. Objectives 1. To identify and to evaluate feed ingredients and foreign materials alsone or in mixture, particularly where the food material is finely ground. 2. Detection and identification of major adulterants, the presence of which may or may not be suspected from consideration of the results of proximate or specific chemical analysis. 3. Recognition and identification of contaminants such as presence of fungal, insect or rodent contamination.
  83. 83. Equipments * Steriomicroscope. * Test sieves with screen of 10, 20 or 30 mesh. * Sharp point forceps. * Petridish. * Beakers and stainless steel spoon. * Mortor and pestle * Chemicals.
  84. 84. Appendix 1 Atomic Weights of some Elements Name Symbol Atomic weight Calcium Ca 40.08 Carbon C 12.01 Chlorine Cl 35.45 Chromium Cr 52.00 Cobalt Co 58.93 Copper Cu 63.55 Flourine F 19.00 Iodine I 126.90 Iron Fe 55.85 Magnesium Mg 24.30 Manganese Mn 54.94 Molybdenum Mo 95.94 Nitrogen N 14.00 Oxygen O 16.00 Phosphorus P 30.97 Potassium K 39.10 Selenium Se 78.96 Sodium Na 22.99 Sulphur S 32.06 Zinc Zn 65.37
  85. 85. Colour of some common indicators in solution The colour of some indicators in acid and basic medium is presented in the following table: Name of indicator Colour in acidic Colour in basic Solution/medium solution/medium Methyl orange Red/orange/pink Yellow Methyl red Red Yellow Phenolophthalein Colourless Pink Methyl red-bromocresol greed mixture Pinkish Green
  86. 86. Selection of indicator for use in titration For the determination of neutralization or end point in an acid base titration the choice of indicator has been given in the following table: Nature of Nature of Indicator Solution taken acid / alkali of choice in burette Weak Weak Methyl orange Alkali Weak Strong Mixture of methyl Acid red and bromocresol green Strong Weak Methyl red/Methyl Acid orange Strong Strong Methyl orange/ Alkali phenolphtahalein Weak Strong phenolphtahalein Alkali
  87. 87. Point of Neutralization / End point The point at which an acid is neutralized by an alkali and vice-versa is called point of neutralization or end point. It is determined by the change in the colour of indicator during titration. Titration The process of gradual mixing of a solution of known normality with the help of a burette into a known volume of another solution to complete the reaction as indicated by the change in the colour of indicator is called titration. The volume of the solution of known normality used for neutralization (end point) is known as titre.
  88. 88. 1.0 TITLE DETERMINATION OF VETCARE SFCase ACTIVITY USING 3, 5 - DINITROSALICYLIC ACID 2.0 PRINCIPLE The assay is based on the production of reducing sugar from a solution of Sun Flower Cake. The reducing sugar is then reacted with 3, 5- Dinitrosalicylic acid (DNS). The colour change produced is proportional to the amount of reducing sugar released which inturn is proportional to the activity of the SFCase present in the sample. The optical density is read at 540 nm & converted into milligrams of glucose produced using a standard curve. 3.0 UNIT DEFINITION One Vetcare unit is defined as the amount of enzyme required to liberate 0.5 mg of reducing sugar from 200 mg of Standard Sunflower Cake substrate (6.2) in a total reaction mixture of 4 ml at 40C in 2 hours at pH - 4.8. This corresponds to the release of 0.125 mg reducing sugar per ml of the reaction mixture. 4.0 REAGENTS 4.1 Citric acid monohydrate 4.2 Glacial Acetic acid 4.3 Sunflower cake 4.4 3, 5 Dinitrosalicylic acid (AR) 4.5 Glucose (AR) 4.6 Potassium Sodium Tartarate Tetrahydrate (AR) 4.7 Sodium Hydroxide 4.8 Phenol 4.9 Sodium metabisulphite 5.0 EQUIPMENT 5.1 Water bath set at 40  1 C 5.2 Timer 5.3 UV-VIS Double beam Spectrophotometer - Shimadzu 1601 5.4 Boiling water bath 5.5 Cooling water bath 5.6 Centrifuge
  89. 89. 6.0 PREPARATION OF REAGENTS AND SUBSTRATE 6.1 Citrate buffer - 0.05M , pH - 4.8 Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. Add NaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and check pH. This is 1M citrate buffer. When diluted to 0.05M, pH should be 4.8. Adjust the pH to 4.8 with Acetic acid or NaOH. 6.2 Standard Sunflower Cake substrate preparation Take 100 gms of SFC and grind it in a mixie to a fine powder. Mix the above powder with 1 litre of distilled water. Filter the above solution through muslin cloth. Squeeze out all the water from SFC. Repeat the above procedure by mixing the residue with 500 ml distilled water. Autoclave the washed SFC at 15 psi for 20 minutes. Dry the SFC at 105C overnight. Grind the dry SFC to a fine powder. Seive the powder through a Nylobolt filter cloth. Collect the residue as substrate. 6.3 DNS SOLUTION a) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litre flask. b) Dissolve 24 gms of NaOH in 200 ml of distilled water in a 500 ml flask. c) Dissolve 5 gm of phenol in 80 ml of distilled water in a 200 ml flask. d) Add 20 ml of DNS solution (a) to the phenol solution (c). e) Add 180 ml of NaOH solution (b) slowly to the remainder of the solution (a) and stir until the solution is homogenous. f) Add 200 gm of Sodium potassium tartarate to the resulting solution (e). g) Add 5 gms of NaHSO3 to the solution (d) and dissolve completely. h) Mix the solutions (f) and (g) in a 2 litre flask and make the volume to 1 litre. i) Filter the solution (h) through a bed of absorbent cotton and add 5gms of Na2S2O5. j) Store this final solution (i) in a amber bottle.
  90. 90. 7.0 PROCEDURE FOR STANDARD GRAPH Prepare a stock solution by dissolving 300 mg of glucose with citrate buffer pH 4.8 in a 100 ml volumetric flask. This stock glucose is stable for 3 months. Prepare working dilutions from stock according to table below. Standard Volume of mg of glucose DNS Water glucose buffer (ml) in tube (ml) (ml) (ml) soln (ml) 0.0 1.5 0.0 3.0 15 Boil 0.1 1.4 0.3 3.0 15 for 0.2 1.3 0.6 3.0 15 5 0.4 1.1 1.2 3.0 15 minu- 0.6 0.9 1.8 3.0 15 tes 0.8 0.7 2.4 3.0 15 1.0 0.5 3.0 3.0 15 8.0 ASSAY PROCEDURE a) Take 1 gm of Standard Sunflower cake powder in 100 ml Erlenmeyer flask. b) Prepare the enzyme dilution of 10 to 15 mg/ml and make up to volume with 0.05M citrate buffer of pH 4.8. c) Make the reaction volume to 20 ml with 0.05M citrate buffer, pH 4.8 by using 1 ml of the enzyme dilution. d) Incubate this suspended mixture for 2 hours in a shaking waterbath moving at 140 rpm at a constant temperature of 40C. e) After 2 hours incubation, take the suspension out of the water bath. f) Stop the reaction by keeping the flask in a boiling water bath for 5 - 10 minutes. g) Similarly prepare the enzyme blank by stopping the reaction immediately after addition of enzyme solution.
  91. 91. h) Centrifuge the suspension at 10,000 rpm for 10 minutes. i) Use the supernatent for estimating the reducing sugar. j) Estimation of reducing sugar : Take 1 ml of buffer in a test tube. Add 0.5 ml of supernatent. Add 3ml DNS. boil for 5 minutes in a boiling water bath. Cool it. Add 15 ml of distilled water. k) Reagent blank : Instead of taking 0.5 ml of enzyme treated supernatent, take 0.5 ml of distilled water. l) Read all the tubes against reagent blank at 540 nm. m) Substract the enzyme blank reading from Sample reading. Find out the amount of sugar released with the help of Standard Graph. n) Reading should be between 0.01 to 0.1. 9.0 CALCULATIONS (1) Total Sugar produced = Sugar produced in 0.5 ml of supernatent X 100. Total sugar released in the reaction mixture X 1000 (2) VSU/g = --------------------------------------------------------------- 0.5 X Amount of Enzyme used in the reaction mixture
  92. 92. 1.0 AIM DETERMINATION OF CELLULASE (FILTER PAPER UNITS) IN THE GIVEN SAMPLE USING 3, 5 - DINITRO SALICYLIC ACID 2.0 PRINCIPLE The assay is based on the production of reducing sugar from a filter paper (Whatman no.1). The reducing sugar is then reacted with 3, 5- Dinitrosalicylic acid (DNS). The colour developed is proportional to the amount of reducing sugar released which inturn is proportional to the activity of the cellulase present in the sample. The colour developed is read at 540 nm & converted into milligrams of glucose produced using a standard curve. 3.0 UNIT DEFINITION One filter unit is defined as the amount of enzyme required to liberate 1 mole of reducing sugar per min from 50 mg of filter paper (Whatman no.1) at 50C at pH - 4.8. 4.0 REAGENTS Citric acid monohydrate Sodium Hydroxide (AR) 3, 5 Dinitrosalicylic acid (AR) D (+) Glucose (AR) Potassium Sodium Tartarate Tetrahydrate (AR) Phenol Sodium metabisulphite Whatman no.1 filter paper 5.0 EQUIPMENT Water bath set at 40  1 C Timer Visible range Spectrophotometer set at 540 nm Boiling water bath Cooling water bath Centrifuge
  93. 93. 6.0 PREPARATION OF REAGENTS AND SUBSTRATE a) Citrate buffer - 0.05M , pH - 4.8 Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. Add NaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and check pH. This is 1M citrate buffer. Prepare 0.05 M buffer solution as a working solution. Adjust the pH to 4.8 with Acetic acid or NaOH. b) DNS SOLUTION 1) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litre flask. 2) Dissolve 24 gms of NaOH in 200 ml of distilled water in a 500 ml flask. 3) Dissolve 5 gm of phenol in 80 ml of distilled water in a 200 ml flask. 4) Add 20 ml of DNS solution (1) to the phenol solution (3). 5) Add 180 ml of NaOH solution (2) slowly to the remainder of the solution (1) and stir until the solution is homogenous. 6) Add 200 gm of Sodium potassium tartarate to the resulting solution (5). 7) Add 5 gms of Sodium metatrisulphite (NaHSO3) to the solution (4) and dissolve completely. 8) Mix the solutions (6) and (7) and make the volume to 1 litre. 9) Filter the solution through absorbent cotton and add 5gms of Na2S2O5. 10)Store this final solution in a dark bottle. c) Substrate - Whatman no.1 filter paper - 50mg (1 cm x 6 cm) 7.0 PROCEDURE FOR STANDARD GRAPH a) Prepare a stock standard glucose solution by dissolving 300 mg of glucose with 0.05 M citrate buffer (pH 4.8) in a 100 ml volumetric flask. This stock glucose is stable for 3 months.
  94. 94. b) Prepare working dilutions from stock according to table below. Standard Volume of mg of glucose DNS Water glucose buffer (ml) in tube (ml) (ml) (ml) soln (ml) 0.0 1.5 -- 3.0 15 0.1 1.4 0.3 3.0 15 0.2 1.3 0.6 3.0 Boil 15 0.3 1.2 0.9 3.0 for 15 0.4 1.1 1.2 3.0 5 15 0.5 1.0 1.5 3.0 minu- 15 0.6 0.9 1.8 3.0 tes 15 0.7 0.8 2.1 3.0 15 0.8 0.7 2.4 3.0 15 0.9 0.6 2.7 3.0 15 1.0 0.5 3.0 3.0 15 8.0 ASSAY PROCEDURE a) Prepare different dilutions of enzymes. b) Take 1ml of citrate buffer in different test tubes. To each tube add 0.5ml of different enzyme dilutions. Incubate at 50C for 5 minutes. c) To the above test tubes, add 50mg of Whatman no.1 filter paper. Vortex it. d) Incubate the tubes in water bath at 50C for 1 hour. e) After 1 hour, cool the tubes under flowing tap water. f) Add 3ml DNS. Boil for 5 minutes in a boiling water bath. Cool and add 15ml of distilled water. g) Reagent blank : Instead of taking 0.5ml enzyme dilution, take 0.5ml citrate buffer.
  95. 95. h) Enzyme blank : Take 1ml of citrate buffer and incubate at 50C for 1 hour. Add 3ml DNS followed by 0.5ml of enzyme dilution and boil for 5 minutes, cool and make up the volume to 15ml. Enzyme Buffer DNS Enzyme Distilled dilution (ml) (ml) dilution water (ml) (ml) (ml) Enzyme 0.5 1.0 3.0 -- 15 Sample Incubate Boil for for 5 Enzyme -- 1.0 1 hour 3.0 0.5 minutes 15 Blank at 50C Reagent -- 1.5 3.0 -- 15 Blank i) Read enzyme sample and enzyme blank tubes against reagent blank at 540nm. j) Subtract the enzyme blank reading from enzyme sample reading. Find out the amount of sugar released with the help of Standard graph. 9.0 CALCULATIONS mg of glucose released X dilution factor X 1000 FPU/gm = ------------------------------------------------------------ 180 X 60 X 0.5
  96. 96. 1.0 AIM DETERMINATION OF XYLANASE (HEMICELLULASE) ACTIVITY USING 3, 5 - DINITRO SALICYLIC ACID 2.0 PRINCIPLE The assay is based on the production of reducing sugar from a solution of Xylan. The reducing sugar then reacts with 3, 5-Dinitro salicylic acid (DNS). The colour change produced is proportional to the amount of reducing sugar (expressed as Xylose) released which inturn is proportional to the activity of the Xylanase present in the sample. The optical density is read at 540 nm and converted into micromoles of Xylose produced using a standard curve. UNIT DEFINITION 3.0 One unit is the amount of enzyme required to produce 1 micromole of reducing sugar (as Xylose) per minute at pH - 4.8 at 40C. REAGENTS 4.0 Citric acid monohydrate Glacial Acetic acid Oat spelt xylan (Sigma) 3, 5 Dinitrosalicylic acid (AR) D (+) - Xylose (AR) Potassium Sodium Tartarate Tetrahydrate (AR) Sodium Hydroxide (Analytical grade) EQUIPMENT 5.0 Water bath set at 40  1 C Timer Visible range Spectrophotometer set at 540 nm Boiling water bath Cooling water bath Grade A Glassware Whirl mixer Glass or automatic pipettes Centrifuge
  97. 97. 6.0 PREPARATION OF REAGENTS AND SUBSTRATE a) 0.05 M Citrate buffer (pH - 4.8) Dissolve 210 gms of citric acid monohydrate in 750 ml distilled water. Add NaOH pellets to it until the pH reaches to 4.3. Dilute it to 1000 ml and check pH. This is 1M stock citrate buffer. Prepare 0.05 M buffer solution as a working solution from the stock solution. Adjust the pH to 4.8 with Acetic acid or NaOH. b) XYLAN SUBSTRATE Take 2g of Xylan in the mortor pestle and grind it with 1-2 beads of Sodium Hydroxide and then slowly add citrate buffer to it till the volume reaches approximately 80 ml. Adjust solution pH to 4.8  0.05 with Glacial acetic acid. Transfer the solution to 100ml volumetric flask and make up to the mark with citrate buffer. Store in refrigerator at 2-8C. This solution must be prepared daily. c) DNS SOLUTION 1) Dissolve 8 gms of DNS monohydrate in 500 ml of distilled water in a 1 litre flask. 2) Dissolve 24 gms of NaOH in 200 ml of distilled water in a 500 ml flask. 3) Dissolve 5 gm of phenol in 80 ml of distilled water in a 200 ml flask. 4) Add 20 ml of DNS solution (1) to the phenol solution (3). 5) Add 180 ml of NaOH solution (2) slowly to the remainder of the solution (1) and stir until the solution is homogenous. 6) Add 200 gm of Sodium potassium tartarate to the resulting solution (5). 7) Add 5 gms of Sodium metatrisulphite (NaHSO3) to the solution (4) and dissolve completely. 8) Mix the solutions (6) and (7) and make the volume to 1 litre. 9) Filter the solution through absorbent cotton and add 5gms of Na2S2O5. 10)Store this final solution in a dark bottle.

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