SOP No : QS-LS 15Effective Date : 01/08/2010Rev No. 00 Revision Date : 01/08/2012Doc. Status : Controlled Standard Operating Procedure (SOP)

Title: Feed Chemistry at Laboratory Services

1. PURPOSETo test proximate analysis of raw material finished feed, sales feed and incoming samples to monitor and ensure high quality raw material and feed quality is done at Laboratory Services department as per end user department requirements.

2. SCOPEProximate analysis (Moisture, crude protein, crude fat, crude ash, calcium, phosphorus, crude fibre, soluble protein, nitrogen, physical test, particle size, water durability) of raw material (as per SOP MMPD-FM-03), finished feed & sales feed (as per SOP FM-010) and incoming samples to monitor and ensure high quality raw material and feed quality is done.3. DEFINITIONS The wordings in this SOP are defined as follows3.1. Analyte: The chemical substance or physical property being measured in a sample.3.2. Stock Standard Solution - A concentrated solution containing one analyte prepared in the laboratory using an assayed reference compound or purchased from a reputable commercial source.3.3. Kjeldahl Method: Kjeldahl digestion in analytical chemistry is a method for the quantitative determination of nitrogen in chemical substances. 3.4. Soxhlet Method: The method described by Soxhlet in 1879 is the most commonly used example of a semi-continuous method applied to extraction of lipids from foods.3.5. Parameter: A variable, measurable property whose value is a determinant of the characteristics of a system.4. REFFERENCES.NoName of analysisMethod of AnalysisReferences

4.1Moisture Oven Drying methodAOAC Official method 930.15AOAC Official methods of analysis 17th Edn (Chapter 4, p 2, Clause 4.1.06)

4.2Crude Protein Kjeldahl method- FOSS automated analyzer AOAC Official method 976.05AOAC Official methods of analysis 17th Edn. (Chapter 4, p 23, Clause 4.2.05)

4.3Crude Fat Soxhlet extraction methodAOAC Official Method 920.39AOAC Official methods of analysis 17th Edn.(2000) (Chapter 4, p 33, Clause 4.5.01)

4.4Crude Ash Furnace method AOAC Official method 942.05AOAC Official methods of analysis 17th Edn. (2000) (Chapter 4, p 5, Clause 4.1.10)

4.5Crude Fiber Acid & Base digestion method AOAC Official method 962.09 AOAC Official methods of analysis 17th Edn.(2000) (Chapter 4, p 34, Clause 4.6.01)

4.6Nitrogen Free extract By difference methodFood Energy - methods of analysis and conversion factors. Food and Agriculture Association of the United Nations,Feeds and Feeding, Handbook for the Student and Stockman", 21st Ed., by Frank B. Morrison, The Morrison Publishing Co., Ithaca, NY, 1950.

4.7Free Fatty Acid Neutral Alcohol solvent method AOCS Official Method Ca 5a-40

4.8Acid Value Neutral Alcohol solvent method AOCS Official Method Ca 5a-40

4.9Acetone Insolubility Acetone solvent method

4.10Calcium Gravimetric methodAOAC Official method 927.02AOAC Official methods of analysis 17th Edn.(2000) (Chapter 4, p 40, Clause 4.8.03)

4.11Magnesium Complexometric method / spectrophotometricAOAC Official method 917.04AOAC Official methods of analysis 17 Edn(2000)(Chapter 4, p 43, Clause 4.8.12)

4.12Salt as NaCl Argentometric AOAC Official method 943.01AOAC Official methods of analysis 17th Edn. (2000) (Chapter 4, p 41, Clause 4.8.05)

4.13TVBN Kjeldhls MethodOfficial Journals of European Commission

4.14Urease Activities Indicator method American Soybean Association

4.15Sieve test Mechanical Sieving Official Method of Analysis of the Association of Analytical Chemist;17th Ed.2000

4.16Bulk Density By Weight Official Method of Analysis of the Association of Analytical Chemist;17th Ed.2000

4.17Peroxide Value Iodometric methodAOAC official method 965.33AOCS official method Cd 8-53

4.18Rancidity Iodometric methodAOAC official method 965.33AOCS official method Cd 8-53

RESPONSIBILITY4.1. Office of the Managing Director Has the sole responsibility of authorizing the preparation of this Standard Operating Procedure, the approval of this SOP and subsequent revision and amendments.4.2. Office of the Manager, Laboratory Services is responsible for preparation of this SOP, revise / review based on the needs of NPC and its implementation.4.3. Office of the General Manager, Quality & Sustainability is responsible for identifying and recommending the needs of NPC core business units; review the SOP & recommends to the Office of the General Manager for any amendments.4.4. Office of the Head, Quality System Department- is responsible for the inspection of the procedures, applications for the above said operations and shall coordinate closely with the Central Laboratory on various issues related to monitoring of this SOP, may from time to time give recommendations and suggestions for the improvement of this SOP.4.5. Technical Committee - Following people shall form the technical committee who will review and recommend for any amendment to the General Manager of NPC. 4.5.1. Manager- Laboratory Services4.5.2. Head Quality System4.5.3. Head Corporate Systems & Certification, Q&S4.5.4. Head Feed Mill4.5.5. Residential Nutritionist6. PROCEDURE 6.1.Estimation of moisture

a. EquipmentHot air oven Instant moisture analyzer

b. Procedure

Regulate air oven to 135 + 2 C. Using low, covered glass dishes weigh about 10 15 g sample into each dish and shake until the contents are evenly distributed. With covers removed, place dishes and covers in hot air oven as quickly as possible and dry samples for 2 hrs. Place covers on dishes and transfer to desiccators and cool. Take the weight. Calculate loss in weight as % moisture.

Alternatively use instant moisture analyzer calibrated against samples tested in air oven at 135 + 2 C.

6.2.Estimation of crude protein

a. Reagentsb. Conc. Sulphuric acidi. Kjeltabs or equivalent (3.5 g K2SO4 + 0.4 CuSO4)ii. Alkali40% sodium hydroxide (LR) solution. Use 400 g NaOH per liter of solution. Commercially available in concentrations up to 50%. Do not use concentrations above 40% as this will lead to crystal formation impairing the function of the pumps.

iii. Receiver solution Boric acid 1% with bromocresol green / methyl red indicator solution. Dissolve 100 g boric acid in 10-lit de-ionized water, followed by addition of 100 ml of bromocresol green solution (100 mg in 100 ml methanol) and 70 ml of methyl red solution (100 mg in100 ml of methanol). In order to obtain accurate results the receiver solution is adjusted[footnoteRef:1] so that a small (0.05-0.15ml) positive blank is obtained when running a blank sample. [1: Adjustment of the boric acid solution is made by the following procedure:Transfer 25 ml boric acid solution to a receiver flask and add 100 ml of distilled water. If the solution in the flask is still red, titrate with 0.1 M sodium hydroxide solution until a neutral gray color is obtained. Calculate the amount of sodium hydroxide solution necessary to adjust the boric acid solution in the 10 lit flask with the formulaMl 1.0 M alkali = ml titrant x 40Add the calculated amount of 1.0 M alkali solution to the boric acid solution. Mix. For boric acid 1%, addition of 30 ml 0.1 M NaOH usually gives a good adjustment.To check proceed as follows using 25 ml of the boric acid solution; Run a blank. If the value of this blank is high (0.5 ml of 0.2 M HCl) the boric acid is incorrectly adjusted. This might create irregular blanks. For correction add HCl directly into the boric acid tank, mix it carefully and repeat until a reading of 0.05-0.15 ml HCl is obtained. If a positive blank is not achieved, add further small quantities of 1M NaOH and repeat the check until a satisfactory value is achieved. The addition of alkali is to achieve a positive blank value. This should, however, be kept between 0.05 0.15 ml titrant, to obtain good repeatability when testing blanks.]

iv. Titrant solution0.2 M standard HCl. Standardization is done using the following method

Weigh approximately 10 g of anhydrous sodium carbonate (Na2CO3). Use mortar to make fine powder. Dry it for 1 hr at 265 C or 2 hrs at 200 C. After cooling in a desiccator, transfer the sodium carbonate to a beaker with a tight lid. Store it in a desiccator.

Dissolve 0.1 g methyl red in 100 ml ethanol.

Weigh approximately 0.4 g of the standard substance, using an analytical balance; note the weight (W1). Transfer the sodium carbonate to a receiver flask and add 40 ml of H20 (distilled or de-ionized). Add 10 drops of the indicator solution. Titrate to pink. Note the amount in ml used (A1). Boil this solution for a few minutes. The solution will turn green. Cool rapidly to room temperature under running water. Continue the titration until the next pink color change occurs. Note also this volume (A2). Boil the solution for a few minutes. Cool rapidly to room temperature under running water. Continue the titration until the next pink color occurs. Note also this volume (A3).The color change of this official procedure (AOAC 936.15) may be difficult to see, therefore a pH meter to a mixed indicator (e.g. 0.1 g methyl red and 0.1 g bromocresol green in 100 ml ethanol) will make it much easier to perform.

Calculation18.870 x W1Molarity (M)= ----------------(A1 + A2 +A3)

b. ApparatusDigester DS 20 with exhaust manipulator and Kjeltec 2300 Analyzer

c. Procedure

i. Sample preparationAnalytical sample from the laboratory sample of the bulk product may involve one or more treatments such as:

Physical treatments: shaking, stirring, mortaring, riffling, coning and quartering

Mechanical treatments: grinding, blending, homogenizing or milling. As a recommendation the particle size should be equal or less than 1 mm.

ii. Sample size

Actual weight of the sample required is dependant on Nitrogen content and homogeneity.

Homogeneous samples (excluding water) 0.1 1.0 gNon-homogeneous samples1.0 3.0 g or moreWater samples (dependant on the N content)1.0 100 ml

Using a titrant concentration of 0.2 N the analytical sample should ideally contain 10 100 mg N and the appropriate sample weight can be calculated as follows.

1000Minimum weight in mg = ------- XWhere x = approximate % N anticipated

iii. Digestion

During digestion the nitrogen or protein in the sample is converted to ammonium sulphate according to the formula Salt K2SO4Protein + H2SO4 -------------(NH2)2 SO4 Catalyst

Prepare a representative sample[footnoteRef:2] and weigh based on the nitrogen content to 0.1 mg accuracy into a digestion tube. Add one kjeltab Cu 3.5 (alternatively 3.5 g K2SO4 and 0.4 g CuSO4 x 5 H2O). Carefully add 10 ml of conc. sulphuric acid and gently shake to wet the sample with the acid. [2: For samples containing high fat or carbohydrate levels use 15 ml H2SO4.]

Place the samples in the preheated (420 C) digester with the exhaust manifold on the top and heat shields in place. The water aspirator must operate at full flow the first five minutes of the digestion. This is done to evacuate moisture. After five minutes it is essential to decrease the aspirating effect to a minimum until the acid fumes are just contained within the exhaust head to prevent acid losses.

Continue digestion until all samples are clear with a blue / green solution. This will normally be after 30 60 minutes depending on the sample type. Remove the rack of he tubes with exhaust still in place and put in the stand to cool for 10 20 mins. iv. Distillation

All samples after digestion will form ammonium sulphate ((NH4)2SO4). The distillation principle is to convert ammonium (NH4) into ammonia (NH3) by using alkali (NaOH) and thereafter steam distil it into a receiver flask containing boric acid and titrate with standard acid solution using colorimetric end point detection.Place the digestion tube in the distillation unit and close the safety door[footnoteRef:3]. Dispense 50 ml of 40% NaOH in to the tube[footnoteRef:4]. Then the distillation cycle is controlled automatically. [3: Dilution water is automatically added on the Kjeltec 2300 if the option is selected.] [4: In general 4 times the amount of the acid = the amount of alkali to use with the concentration of 40%. When 20 ml concentrated H2SO4 is used during digestion the amount of alkali used (40%) should be 20 x 4 = 80 ml of 40% NaOH. It is very important that alkali is present in excess; otherwise hydrogen sulphide (H2S) will be formed. Hydrogen sulphide is a strongly acidic gas that will make the indicator turn red and no result will be achieved.]

v.Blank determination

Full chemical blanks should be run before each batch of analyses to compensate for any contribution from the reagents used. Blanks should be treated identically to samples to be meaningful. vi.Chemical check Use ammonium sulphate (NH4)2SO4 > 99.5%Molecular weight = 132.14 g/mol.Dry ammonium sulphate for 4 hrs at 102 C, and store in a desiccator.% Nitrogen in ammonium sulphate (99.5%) = 21.09Weigh 0.15 g Ammonium Sulphate into a tubeAdd 75 ml distilled water and 50 ml 40% NaOH and distil

(ml blank) x N x 1.401% Nitrogen = ------------------------------ g sample

N = Normality of titrant to 4 places of decimal. Actual % Nitrogen% Recovery = ------------------------ x 10021.09

Use ammonium iron (II) sulphate (NH4)2 Fe(SO4)2 x 6 H2O

Molecular weight = 392.14 g/mol% Nitrogen in ammonium iron (II) sulphate = 7.145Weight 0.5 g ammonium iron (II) sulphate into a tube.Add about 75 ml distilled water, and 50 ml 40% NaOH. (ml blank) x N x 1.401% Nitrogen = ---------------------------------- g sample N = Normality of titrant to 4 places of decimal. Actual % Nitrogen% Recovery = ------------------------ x 100 7.145

Please note that the above calculations must be adjusted if other purity levels of ammonium salts are used. For a full check of your chemistry a substance like glycine can be digested and used as a test sample.d.Calculation of results

The most common ways of reporting Kjeldhal results are as % Nitrogen, % protein, mg N/liter (ppm), g N/liter and mg N/100 ml. The calculations are as follows:

(T B) x N x 14.007 x 100%N = ----------------------------------Weight of the sample in mg% Protein = % N x F

(T B) x N x 14.007 x 1000mg N / liter =-----------------------------------Volume of the sample in ml

(T B) x N x 14.007g N/liter = ------------------------------------- Volume of the sample in ml

(T B) x N x 14.007 x 100mg N/100 ml = -------------------------------------- Volume of the sample in ml

WhereT = Titration volume for the sample (ml)B = Titration volume for blank (ml)N = Normality of the acid to 4 places of decimal.F = Conversion factor for Nitrogen to protein e.g. 6.25, 5.7, 6.38 depending on sample.

6.3. Estimation of crude fat

a. Regents Hexane, petroleum ether or any suitable reagent.b. EquipmentSoxtec Avanti 2055 systemc. Sample preparationSamples prepared using physical treatments such as shaking, stirring, mortaring, riffling, coning and quartering or mechanical treatments such as grinding, blending, homogenizing, or milling is placed into extraction thimble to allow for maximum solvent contact. Recommended particle size is 20 25%0.5 1 g + 0.1 mg

e. Instrument settings Control unit / service unit is set at temperatures to get an even boiling of the solvent with a reflux rate of about 3 5 drops / sec. This setting varies with the solvent used and the type of extraction cups used (whether glass or aluminum). If too high temperature is used the boiling of the solvent can become too vigorous which for some samples increases the risk for foaming. The risk of oxidation of the extractable matter during the evaporation phase is also increased.f. ProcedurePress the mains button (switch lamp should light up). Set temperature to achieve a reflux of 3 5 drops of solvent per second. (See application sub note to achieve desired reflux of solvent). Select the proper program and check time settings for boiling / rinsing / evaporation / pre-drying on the control unit. Open the cold-water tap for the reflux condensers. With cooling water at approximately 15 C the flow should be adjusted to 2 l/min to prevent solvent evaporation from the condensers. Prepare the thimbles and attach them to adapters. Prepare and weigh sample into thimbles with a precision of + 0.1 mg . Use the thimble support with the balance. Move the thimbles to the thimble stand. Use thimble handler. Put a thin layer of de-fatted cotton on the top of the sample and place the thimbles into thimble stand. Use the thimble handler and move the thimbles to the thimble supports. Insert the thimbles in to the extraction unit. Attach them to magnets. Remove the thimble supports.

Use the cup holder to insert the pre-dried extraction cups, tared with 5 10 glass pellets (5 6 mm) to prevent shock boiling, loaded with the solvent to be used. See ASN for recommended volume. The buzzer signal will inform you when the temperature on the hot plate is sufficient for lowering the thimbles with the handle. Boil the thimbles immersed according to boiling time in the ASN. Make sure that the condenser valves are open. Move the thimbles to rinsing position. Rinse according to rinsing time in the ASN. Also note that the condenser valves are open. After rinsing, close the condenser valves by turning a quarter turn. Move the handle to recovery position. When almost all the remaining solvent is collected, press the air button. The last traces of solvent will now be collected in the condenser /solvent collection vessel. Remove the cups. Dry the cups at 103 C for 120 150 mins. Cool the cups in the desiccator and weigh them. g. CalculationW3 W2Crude fat % = ------------- x 100 W1 WhereW1 = Sample weight (g)W2= Extraction cup weight (g) W3= Extraction cup + residue weight (g)6.4.Estimation of crude asha. EquipmentMuffle furnaceb. ProcedureWeigh 3g sample into silica crucible and place in temperature controlled furnace preheated to 600 C. Hold at this temperature for 2 hrs. Transfer crucible directly to desiccator, cool and weigh immediately. Calculate the % of crude ash. Weight of the ash% Crude Ash= --------------------------- X 100Weight of the sample

6.5.Estimation of acid insoluble ash

a. Equipment Muffle furnace

b.ProcedureMoisten the contents with 5-10 ml of conc. HCl, after ashing as described in the procedure for estimation of crude ash. Boil for 2 min, evaporate to dryness and heat on steam bath for 3 hrs to render SiO2insoluble. Moisten residue with 5 ml conc. HCl, boil for 2 min, add about 50 ml distilled water. Heat on a water bath for 5 min, filter through Whatman filter paper No. 1 and wash thoroughly with distilled water. Then ignite at 500 550 C, cool and weigh to calculate acid insoluble ash % by difference

Weight of acid insoluble ashAcid insoluble ash % = -------------------------------------- X 100 Weight of the sample6.6.Estimation of crude fibre

a. ReagentsSulphuric acid: 1.25 % (12.5 g of H2SO4 diluted to 1 lt and mixed)Sodium hydroxide (NaOH) or potassium hydroxide (KOH) 1.25% (12.5 g of NaOH or KOH dissolved in deionized water and diluted to 1 lit.n-OctanolAcetoneb. EquipmentMuffle furnaceHot air ovenc. ProcedureGrind the sample to pass through 1 mm screen in a grinder. Weigh and transfer 2 g (Wo) of defatted sample in to a beaker. Add 200 ml preheated 1.2% sulphuric acid and a few drops of octanol to prevent foaming. And heat to boiling. Adjust heat and boil for 30 mins. Filter through a 200 micron cloath and wash with hot de-ionized water three times. Transfer again to the same beaker and add 200 ml hot 1.25 % NaOH or KOH solution and boil for 30 min. Filter and then wash with de-ionized hot water three times and then finally with acetone (25 ml each) three times. Collect the residue, dry it in hot air oven at 100 C over night or at 130 C for 2 hrs. Cool and weigh (W1) the material. Ash the sample in the crucibles at 550 C for 2 hrs. Cool the crucibles to room temperature and weigh again (W2). Calculate the fibre content from the formula W1 W2Crude fibre % = -------------- x 100 Wo

6.7. Estimation of water durability

1Objective:To assess the water durability of shrimp feed

2Parameters of the judgment

Swelling:Indicates the degree of water absorption. The higher the water absorption by the pellets the higher the risk for disintegration

Cracks:Cracks are the result of the pelletizing process. They are fine as hairs and are visible under the microscope or when put into water. Cracks are the entrance for penetration of water into pellets.

Disintegration:Due to absorption of water, the particles of the pellets fall off. Falling off particles from the pellet is the beginning of the disintegration

3Assay

Drop ~5 g feed sample, just enough to form one layer on the bottom of a 250 ml glass beaker filled with water. This is stirred automatically and continuously so as to simulate the pond water movement during aeration.

4Procedure of judgment

Rating (judgment) is done at the end of 1 hour and 2 hours

Rating of the water durability is done by applying 1 to 10 points of fractions thereof, preferably by three persons

Feed has to be observed carefully after putting into water for any quick changes with reference to swelling, cracks and disintegration, especially at the end of the intervals given above. Testing may be stopped if the samples get a failure rating as per judgment chart given below.

5Judgment chart

Chart for the judgment of the water durability of shrimp feed

Swelling:Cracks 1 (in pellets)DisintegrationPoints ( 1 to 10 )

Little to normalNoneNone10

NormalLess than 1/5 th None9

Normal1/5 th to 1/3 rdVery less8.5

StandardNormal1/3 rd to 1/2~ 15% 28

Normal1/2 to 2/3 rd~ 20 %7.5

Normal to full2/3 rd to 3/4 th ~ 25%7

Normal to full> 3/4 th~ 50%6

FullTotal~ 75%5

FullTotalNearly total ( ~90%)4

Full Total Total 3

Full (within 5 min)-Total (within 5 min)2

Full (immediate) -Total (immediate)1

1Means number of pellets actually developed cracks but have not fallen apart

2Visual estimation of fallen off particles in comparison to the volume of the feed present

6.8.Estimation of calcium

a. Reagents

i) Ammonium oxalate solution: Boil 45g Ammonium oxalate in 1L of distilled water, cool and filter.

ii) 0.1N Potassium permanganate (Std. KMnO4 soln.): Weigh accurately about 3.2g of KMnO4 crystals (AR Grade) into a beaker. Add cold water and stir thoroughly with a glass rod. Break up the crystals with glass rod and dissolve maximum crystals. Decant the solution through a plug of glass wool supported in a funnel. Leave the undisclosed residue in the beaker and dissolve this in fresh water. Continue this until all the crystals dissolve. Dilute the solution to 1L, shake well and keep for 24 hrs in an amber coloured bottle with stopper.

Standardization of KMnO4 solution: Weigh accurately about 0.3g Sodium Oxalate (Na2C2O4) AR (preheated for 2 hrs at 1050C) into a 500ml beaker, add 250ml distilled water and 10ml conc.H2SO4. Warm on a hot plate up to 700C and titrate against KMnO4 while hot till a pink colour persists.

Normality of KMnO4 = W x 1000/V x 67

Where W = Weight in g of Na2C2O4 takenV= Titre volume of KMnO4

iii) Acetic acid Glacial

iv) Phenol Red indicator solution: 0.1g dissolved in 30ml 0.01N NaOH solution and diluted to 250 ml with distilled water.

b.ProcedureAsh 1g of feed in a crucible as per the standard procedure for determination of %Crude Ash. Cool and transfer quantitatively all the contents in the crucible into a beaker. Rinse the crucible with a few drops distilled water followed by a few drops of (1+1) HCl Soln and these washings also transferred to the same beaker. Add 10ml (1+1) HCl Soln, cover the beaker with a watch glass and heat just below boiling point on a hot plate for 20 minutes. Filter through Whatman 541 9cm filter paper in to a 250ml volumetric flask. Wash the filter paper well with distilled water and make up the volume to 250ml.

Pipette 25ml of the sample solution to a 300ml flask, add 50ml distilled water and 5 drops Phenol red indicator solution (Solution turns to yellow colour). Then add drops of (1+1) ammonia solution with stirring till the colour of the solution turns to purplish red (pH =8.5). Bring back the colour to just yellow by adding drops of Acetic acid (Glacial) carefully. Boil the solution on a hot plate. At the same time, boil 25ml of ammonium oxalate solution in another beaker. When both the solutions are started to boil, add ammonium oxalate solution portion by portion into the solution with stirring. Allow to cool and keep overnight for complete precipitation.

Filter the solution through Whatman No: 1 filter paper, wash the precipitate and beaker with hot water until the washings are free from oxalate (non-cloudiness with CaCl2 solution). Take 100ml distilled water in the beaker, add 4ml Conc.H2SO4 into it and heat to boiling. Pour this through the filter paper into a fresh conical flask with punching the filter paper, wash the filter paper thoroughly with hot water and collect all washings in the conical flask. Also put the filter paper into the conical flask and heat the flask to 700C. Then titrate while hot against Std.KMnO4 solution till a pale pink colour persists.

Calculation

% Calcium = N x V x 20/ W

Where N= Normality of Std. KMnO4 V = Titre Volume of Std. KMnO4 W = Weight of feed sample in g taken for ashing

6.9.Estimation of phosphorus (Gravimetric method)

a. Reagents

Concentrated Nitric acid Nitric acid 1:1- Mixture of equal volumes of concentrated nitric acid and water Ammonium molybdate stock solution Take 200g of powdered ammonium molybdate (AR grade) in a stoppered graduated cylinder of 1000 ml capacity .Add to it 800 ml of distilled water and shake well for 25 minutes to dissolve the ammonium molybdate. Add gradually 25 % (m/v) ammonium hydroxide solution till the solution is clear (about 100 to 140 ml of ammonium hydroxide may be required.) Avoid adding excess of ammonia. Make up the volume to one liter, if necessary, filter the solution through a filter paper and stock this solution. Nitric acid solution 2 % Potassium nitrate solution 3% Standard sodium hydroxide 0.1 N Phenolphthalein indicator solution Dissolve 0.1g of phenolphthalein in 100 ml of 60% alcohol.

b. ProcedurePrecipitation Take 10 ml aliquot of the prepared solution (general solution as prepared for cal) In a 150ml beaker. In a dry baker, prepare ammonium molydbdate solution by pouring into it, quickly and simultaneously. 10 ml of conc. nitric acid or take 10 ml of conc. nitric acid solution in the beaker and into thispour quickly 10 ml of ammonium molybdate solution, whirling the beaker during addition. Pour this freshly prepared clear liquid in to the beaker containing the aliquol and stir. But much better, if the above analysis is done fast, so that the temp raised due to nitric acid will be enough and if it is done slow , care should be taken .

Note: The temperature developed in the molydbdate solution is sufficient to precipitate all the phosphorus present in the aliquant. Under no circumstances the phosphomolybdate precipitate should be heated either on a water bath or directly over a burner so as to avoid precipitation of molybdie anhydrate.

Filtration and Washing Allow the precipitate to stand overnight and then filter through whatman filter paper no 42 over an ordinary funnel. As far as possible only the supernatant liquid is passed thru the filter paper, retaining the precipitate in the beaker. When the supernatant liquid is decanted off the precipitate is washed twice with dilute nitric acid and then with potassium nitrate solution until the washings are free from acid. Freedom from acidity may be tested by collecting sufficient filtrate in test tube to which as few drops of phenolphthalein indicator solution and one drop of the standard sodium hydroxide solution are added. If the pink color appears with one drop of the standard alkali, the precipitate is free from acid.

Titration Transfer the precipitate with the filter paper back to the beaker in which precipitation was carried out. Add sufficient quantity of the standard solution hydroxide solution from a burette just to sufficient to dissolve the ppt and then add 5ml in excess. See that no yellow ppt sticks to the filter paper. Note the total volume of the standard sodium hydroxide solution added. Add about 2-3 drops of phenolphthalein indicator solution and filtrate the excess of alkali with the standard nitric acid.

Calculation: phosphorous = 336.75(AN1- BN2) --------------- M x 100 Where,

A - Volume in ml of the standard sodium hydroxide solution used N1- Normality of the standard sodium hydroxide solution B - Volume in ml of the standard nitric acid used into neutralize the excess alkali.N2- Normality of the standard nitric acid. M Mass in gram of the material taken for the test (it means that total weight of sample which is taken for ash)

6.10.Estimation of salt (Chlorine as sodium chloride)a.Reagents Ferric sulphate solution: dissolve 60 g of ferric sulphate [Fe2(SO4)3] in one liter of water.Ammonium hydroxide solution: Prepared by mixing one volume of ammonium hydroxide with 12 volumes of water.Concentrated nitric acid.Ferric sulphate indicator solution: Prepare as 25 % (m/v) solution. Filter and add equal volume of nitric acid.Standard silver nitrate solution: 0.1 N (follow procedure as described later).Standard potassium thiocyanate solution: 0.1 n (follow procedure as described later).b.Procedure

Weigh accurately about 1 g of the dried material and transfer to a 250 ml volumetric flask. Add 50 ml of the ferric sulphate solution with a pipette, gently swirling the flask. Add 100 ml of the ammonium hydroxide solution to bring it to the mark. Swirl the flask enough to ensure through mixing but avoid vigorous shaking. Allow to settle for 10 min. filter through 11 cm Whatman filter paper # 41 or its equivalent. Transfer from the filtrate, an aliquot of 25 ml to a 250 ml beaker. Add 10 ml of nitric acid and 10 ml of the ferric sulphate indicator solution. Then add with constant stirring, known quantity of the standard silver nitrate solution in slight excess. Heat the solution to the boil and cool to room temperature and stir to coagulate the precipitate. Titrate the excess of silver nitrate with the standard potassium thiocyanate solution. The end point is indicated by the first appearance of reddish tint that persists for 15 seconds.

5.845 (AN1 BN2)Chlorine as sodium chloride percent by mass = ---------------------m

WhereA =Volume in ml of the standard silver nitrate solution used.N1 =Normality of the standard silver nitrate solutionB =Volume in ml of the standard potassium thiocyanate solution used up by the excess silver nitrate solutionN2 =Normality of the standard potassium thiocyanate solutionm =mass in g of the material taken for the test.

6.11.Estimation of magnesiuma.Reagents Buffer solution pH 10: Dissolve 67.5 ammonium chloride in 200 ml distilled water. Add 570 ml ammonium hydroxide and dilute to 1 liter. Potassium hydroxide (KOH) potassium cyanide (KCN) solution: dissolve 280 g KOH and 66 g KCN in 1 liter distilled water. Potassium cyanide solution 2 % : Dissolve 2 g KCN in 100 ml distilled water. Erichrome black T indicator: Mix 1 g indicator with 99 g potassium nitrate. A quantity of 30 to 40 mg is to be used. Magnesium standard solution: 0.25 and 1.0 mg / ml : Dissolve 0.25 and 1.00 g magnesium turnings in HCL (1+10) and dilute each to 1 liter with distilled water. Calcium standard solution 1mg / ml: Dissolve 2.4973 g calcium carbonate, AR grade, previously dried for 2 hrs at 285 C, in HCL (1+10). Dilute to 1 liter with distilled water. Pattons and reeders reagent: 1% indicator mixed with 99%sodium sulphate. Disodium dihydrogen EDTA (Na2H2EDTA) standard solution: 0.4 % : dissolve 4 g Na2H2EDTA in liter distilled water. Standardize against standard Ca and Mg solutions. 0.1%: Prepare as above (i), using 1 g Na2H2EDTA and standardize against 0.25 mg / ml Mg standard solution.b.Standardization: For Calcium: Pipet 10 ml standard Ca solution into 250 ml conical flask and add 10 ml distilled water. Add 10 ml KOH-KCN solution and about 35 g pattons and reeders reagent. Titr with 0.4% EDTA standard solution till end point. Titr 3 aliquots and use average to calculate titr Ca solution = 10 / ml EDTA solution.

For Magnesium: Pipet 10 ml 0.25 and 1.00 mg / ml Mg standard solutions into 250 ml conical flasks and add 150 ml distilled water. Add 7 ml pH 10 buffer, 2 ml 2% KCN solution, and 30 to 40 mg erichrome black T indicator. Titr. with 0.1 and 0.4% EDTA standard solutions respectively, until color changes permanently from wine red to pure blue. Titr 3 aliquots and use average to calculate titer Mg solution = 2.5. ml EDTA solution, or 10 / ml EDTA solution, respectively.c.DeterminationDry sample at 110 C to constant weight and cool to room temperature. Grind to pass # 60 or 80 ASTM sieve and mix thoroughly. Accurately weigh about 0.5 g into 250ml beaker, add 20 ml HCL (1+1), and evaporate to dryness on hot plate. Dissolve residue in 5 ml HCl (1+10), dilute to about 100 ml wit distilled water and digest over water bath for 1 hr. Coll, transfer to 250 ml volumetric flask, dilute to volume, mix and let settle or filter. For Calcium: Pipette 10 ml aliquot into 250 ml conical flask and titer. As in II a, observing end point thru solution and away from light. % Ca = (titer EDTA standard solution for Ca x ml EDTA standard solution x 2 / g sample).

For Magnesium (for samples containing 4 % Mg) : For Ca + Mg, pipet 10 ml aliquot into 250 ml conical flask and titr. with 0.4% EDTA solution as in II b. % Mg = (Titer EDTA standard solution for Mg ) x [(ml EDTA standard solution in Ca + Mg titrn) - (ml EDTA standard solution in Ca titrn)] x 2 / g sample.

For Magnesium (for samples containing 2 4% Mg): Pipette 10 ml aliquot (0.5 1.0 mg Mg) into 250 ml conical flask and add exact volume of 0.4% EDTA solution required for Ca determination. Titrate with 0.1% EDTA solution as in II b. % Mg = (Titer EDTA standard solution for Mg) x ml EDTA standard 0.1% solution x 2 / g sample.

6.12.Estimation of urease activity in soybean meala. ReagentsUrea crystalsPhenol red indicator (0.62 % solution in distilled water)b.ProcedureWeigh 0.2 g soybean meal into a test tube, add 0.02 g urea crystals and 2 drops of phenol red indicator. Finally add 2 3 ml distilled water into the test tube. Shake the test tube for 10 seconds and observe for the appearance of pink color in the test tube. Record the time span for color development. c.Identification of the strength of the urease activityIf color appears within 1 minute -Very strongIf color appears between 1 to 5 minutes-strongIf color appears between 5 to 15 minutes-Some activityIf color appears between 15 to 30 minutes -No activityd.ConclusionIf soybean meal sample did not have pink or red color appearance within 10 minutes, means this soybean meal is acceptable for feed production.

6.13.Estimation of Acid Value[footnoteRef:5] [5: mg potassium hydroxide necessary to neutralize acids present in 1 g sample. ]

a. ReagentsSodium hydroxide (NaOH) 0.1 N Ethyl alcohol 95%Add little phenolphthalein and neutralize with alkali to a faint but permanent pink color just before using. Phenolphthalein indicator 1% in 95% ethyl alcohol.Petroleum ether or hexane.b. Preparation of sampleWarm the sample to soften the product (do not heat over 60 C) and then mix thoroughly.c. ProcedureWeigh about 2 g of sample into a 250 ml conical flask. Dissolve in 50 ml of petroleum ether or hexane by mixing gently. Then add 50 ml of neutral alcohol and mix. Add 4 drops of indicator and while mixing titrate with 0.1 N NaOH to the first pink color, which persists for 5 sec. Adding the bulk of the alkali solution rapidly until near the end point, and then slowing down to about 4, then 1 2 drops fairly easily ascertain the end point, at a time. ml. titrant x N x 56.1Acid value (mg KOH / g ) of sample[footnoteRef:6] = ------------------------ [6: Although sodium hydroxide is used in the procedure, results are expressed based on mg KOH per gram. The factor used in calculation is based on the fact that 1.0 ml of 1N NaOH solution is equivalent to 56.1 mg KOH.]

Weight of the sample

Where N = Exact normality of the standard NaOH solution6.14. Estimation of Acetone insolubility

a.Reagents: Acetone

b.Procedure

Weigh 5 g lecithin sample accurate to 0.001 g into the beaker. Weigh filter. When convinced, by means of thermometer, that the acetone is at 0 C, add about 20 ml thereof to the lecithin and start to knead with glass rod. Allow the solids to settle for a short period and filter off the oil-containing acetone through the filter into the conical flask. Repeat the kneading step at least 5 times using in each case 20 ml acetone at 0 C. The initially greasy tacky material must finally have disintegrated to a fine powder. Transfer as much as possible of the kneaded lecithin to the filter and wash with cold acetone at 0 C until a drop of the filtrate evaporates on a glass plate without leaving a residue. Then dry the filter together with the remainder of the kneaded lecithin for 30 min in a hot air oven at 105 C. when the material is dried, cool it in a desiccator, weigh it.

Acetone Insolubility % = [(F-E) x 100] W

Where F = Final weight of filter with sample in g

E = Empty weight of filter in g

W= Original sample weight in g

6.15.Determination of pepsin digestibility[footnoteRef:7] (Filtration method) [7: Defatted sample is digested for hrs in the presence of warm acid solution of pepsin under constant agitation. Insoluble residue is isolated by filtering, washed, dried and weighed to determine % residue. Residue is analyzed for protein.]

a.ReagentsPepsin solution - % pepsin (activity 1:10,000) in 0.075N HCl, freshly prepared. Do not use pepsin NF or pepsin of activity other than 1:10,000. Prepare just before use by diluting 6.1 ml HCl to 1 liter and heating to 42 45 C. Add pepsin and stir gently until dissolved. Do not heat pepsin solution on hot plate or over heat.b.EquipmentAgitator continuous, slow speed (15 rpm), end-over-end type, to operate inside incubator at 45 + 2 C and carry 250 ml screw cap prescription bottles, or equivalent.

Filtering device Buchner funnel or equivalentGlass fiber filter paper, for indigestible residues.45 angle settling rack

c.ProcedureGrind sample to pass through # 20 ASTM sieve. Weigh accurately 1.000 g of material into the thimble and extract with hexane as in procedure for determination of crude fat. Transfer the defatted sample to 250 ml sample bottle. Add 150 ml freshly prepared 0.2% pepsin HCl solution and heat to 42 to 45 C. Be sure sample is completely wetted by pepsin solution. Stopper bottle, clamp in agitator, and incubate with constant agitation for 16 hrs at 45 + 2 C remove bottles from agitator. Place in 45 angle settling rack and loosen the caps. Let residue settle for 15 min or more.

Place weighed filter paper in funnel. Filter the sample and quantitatively transfer residue on to filter washing properly with hot water. If filtration rate is slow it may be accelerated by adding acetone coupled with suction. Add 15 ml acetone into the bottle. Hold thumb over bottleneck and shake vigorously. Release pressure, replace thumb over bottleneck and shake bottle in inverted position over filter. Repeat rinse two or three times till the bottle is free from residue. Dry the residue in the filter paper in a hot air oven as in the procedure of determination of moisture. Calculate the % indigestible residue. Determine indigestible protein by transferring filter-containing residue directly to Kjeldahl flask and determine crude protein by Kjeldahl method as described previously.Make blank determination on 1 sheet of glass filter and subtract from each sample determination if necessary.Calculate % of protein based on original sample weight. Result represents % indigestible protein in sample.

% Indigestible crude protein in sample% Protein indigestible = -------------------------------------------- x100 % Crude protein

% Pepsin digestibility = 100 - % protein indigestible.

6.17. Estimation of wet gluten index in wheat flour

Weigh 20 g wheat flour in to a bowl and add 12 ml tap water, mix to form a firm paste by thumb (only). Let the dough stand still for 5 min. Wash the dough in the bowl several times (app. 20 min) till the water is clear. Each washing has to be passed through 100 ASTM mesh to prevent gluten pieces from escaping. Gluten pieces, if any, found to be sticking to the mesh, are to be collected back.

Test the completeness of washing by squeezing out water from the gluten ball, which should not form a ring or thick starch cloud in a beaker full of water. Remove excess water adhering to the gluten ball by rolling over a cotton cloth and weigh it.

Weight of wet glutenWet gluten index % = --------------------------- X 100Weight of wheat flour

6.18.Estimation of wet gluten index in wheat gluten

Take 10 g sample of wheat gluten, add 40 ml tap water and then soak it for 15 min in a bowl. Then the contents are transferred into a filter paper in a funnel to let the excess water filter out.

Wet gluten content in the filter paper is made into a ball and thoroughly washed over a mesh (100 ASTM) for 20 30 min to remove excess of starch. Pure wet gluten ball is then made free of adhering water over a cotton cloth and weighed for calculating the percent gluten index.Weight of the wet glutenWet gluten index % =-------------------------------- X 100Weight of the dry gluten

6.19.Estimation of dry matter retention

a.EquipmentSmaller pore size meshAquariumAeratorHot air oven

b.Procedure Take 20 g pelletized feed in to a mesh of smeller pores than feed and place it in slowly circulating fresh water in an aquarium. Water can be circulated with the help of aerator. After 2 hrs, collect the sample and dry it in hot air oven at 135 C till it is fully dried. Weigh dried feed pellets and calculate the dry matter % retained in the mesh.6.20.Abrasion test

a.Equipment : Slowly rotating mechanized tumbler b.Procedure :

Place 500g pelletized feed in to the tumbler and rotate at slow speed ( about 30 to 50 rpm) for15 min. Collect the sample and sieve it through smaller pore size mesh .The dust and fines generated the period of tumbling are calculated as percent by weight .

6.21.ESTIMATION OF COARSENESSa.Equipment : Test sieves ASTM-40, 60 and 80

b.Procedure

10-20 g of sample is weighed and then sieved through any of the sieves in order to find out the quality of coarse material. Depending on the type of material, particular sieve has to be used. Quantity of coarse material retained over the sieve is weighed and calculated as percent.

6.22.Preparation of standard potassium permanganate solution Dissolve3.2g of potassium permanganate (KMnO4) in one litter distilled water to prepare 0.1N solution. Boil the solution for 1 hr. Protect from dust and let stand overnight. Standardization: For 0.1N solution, transfer 0.3g dried (1hr at 150c) Sodium oxalate (Na2C2O4) to 500ml beaker. Add 250ml sulphuric acid (5+95) , previously boiled 10-15 min and then cooled to 27-30C . Stir until sodium oxalates dissolved. Add 39-40ml KMnO4 solutions. If too concentrated, discard and begin again, adding few ml less of KMnO4 solution. Heat to 55-60c and complete titrn by adding KMnO4 solution until faint pink persists 30sec. Add last 0.5 1ml drop wise, letting each drop decolorize before adding next. Determine excess of KMnO4 solution required to turn soln pink by matching with color obtained by adding KMn O4 soln to same vol, of boiled and cooled dil. sulphuric acid at 55- 60c. This correction is usually 0.03 0.05ml. From net vol, KMnO4, calculated normality.Normality= g Sodium oxalate X 1000 / ml KMnO4 X66.9996.23.Standardization of 0.1N nitric acid solution

Titrate dilute nitric acid solution against 20 ml standard NaOH solution using phenolphthalein indicator. Note down the volume of dilute nitric acid required to neutralize 20 ml standard NaOH. Determine the normality of the nitric acid using the formula: N2 =N1 V1 / V2

N1= Normality of standard NaOH

V1= Volume standard NaOH used

N2= Normality of standard nitric acid to be obtained

V2 = Volume of nitric acid used.

6.24.Preparation of standard silver nitrate solution

a.Preparation of standard solution

Dissolve slightly more than theoretical weight of silver nitrate (AgNO3) in distilled water and dilute to volume. Thoroughly clean glassware, avoid contact with dust and keep prepared solution in amber glass stopper bottles away from light.

b. ReagentsPotassium chloride Dry KCl at 110c and then heat at about 500 C to constant weight Potassium chromate solution 5% solution of K2Cr2O4 in distilled water. c.Standardization

Accurately weigh enough KCl to yield titration of about 40ml (approximately 0.03g for 0.01Nsolution) and titrate with AgNO3 solution until first perceptible pale red brown appears. From titration volume subtract ml of the AgNO3 solution required to produce end point color in 75 ml distilled water containing 1ml K2CrO4 solution. From net volume AgNO3, calculate normality. Normality = g KCl x 1000/ml AgNO3 x 74.555.

6.25. Preparation of standard potassium thiocyanate solution

Preparation of about 0.1N solution using 9.718 g potassium thiocyanate (KSCN) / L.

Standardization:

Accurately weigh enough purified AgNO3 to give titration of about 40ml (about 0.7g for 0.1N solution) and transfer with distilled water through glass funnel to 250ml conical flask. Dissolve in about 75ml distilled water and add 5ml HNO3 (1+1 ) and 2ml Fe alum ( Ammonium Iron 111 sulphate or Ferric salt ) solution .Titrate with thiocyanate solution until titrated solution is reddish brown .which remains after shaking vigorously 1 min. Record burette reading and set the flask aside 5min ,shaking occasionally and maintaining end point color by addition of thiocyanate solution as required .Then add additional thiocyanate solution , If necessary , to produce permanent end point color, matching with color of reference solution . From total volume thiocyanate solution used in titration, subtract volume, contained in the reference solution. Normality = g AgNO3 X 1000/ml titer X 169.87

6.26. Determination of the concentration of volatile nitrogenous bases (TVB-N)

10g 0.1g of sample is homogenized/blended with 90mL of Perchloricacid (PCA) 6% and filtered by 150mm filter (Whatman No. 1) The extract thereby obtained can be kept for at least seven days at a temperature between approximately 2 6 0C 10mL of the extract is estimated in automated 2300 Kjeltec distillation unit (Foss Tecator AB). Silicone antifoaming agent is added if foaming starts. Blank test is carried out with 10mL aqueous solution (6.0%) of PCA. 10 ml of filtrates (6.1 clause ) taken in to digestion tube and set the Kjeltec analyzer as mg N/100 gm. Enter the weight of sample ( filtrated solution ) taken for test. TVB- N value is expressed in mg N/100g sample. Duplicate analysis is required for each analysis. After one week, the same filtrate is rechecked for TVB-N Value

6.27. The feed analysis is being carried out by laboratory services according to feed mill requirements.

6.28. Apart from the chemical analysis physical examination of feed sample will be done by visual observation.

7.0 DOCUMENTATION

7.1 Annexure - I Raw materials Analysis report Format - (RMI-05)7.2 Annexure II Sales feed analysis Test Report Format (LS-FA-01)7.3 Annexure - III Finished feed analysis report Format FAN-05

All the document formats quoted here are given in Annexure I - III

8.0 REVISION HISTORY

Issue No. DateRevision No. & DateAuthor(s)Change ReferenceReason for Change

Issue 1, 01.08.2010----

9.0 APPROVALS

Prepared by:

Reviewed by:

Process Control, LSRecommended by:

Department Manager, Laboratory Service Verified for Endorsement:

Department Head, Quality Systems

Endorsed by:

General Manager Quality & Sustainability Approved by:

General Manager Quality & Sustainability