CELEX: 31992D0608
Language: en
Date: 1992-11-14 00:00:00
Title: 92/608/EEC: Council Decision of 14 November 1992 laying down methods for the analysis and testing of heat- treated milk for direct human consumption

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31992D0608

92/608/EEC: Council Decision of 14 November 1992 laying down methods for the analysis and testing of heat- treated milk for direct human consumption  

Official Journal L 407 , 31/12/1992 P. 0029 - 0046 Finnish special edition: Chapter 3 Volume 47 P. 0164  Swedish special edition: Chapter 3 Volume 47 P. 0164 

COUNCIL DECISION of 14  November 1992 laying down methods for the analysis and testing of heat-treated milk for direct  human consumption (92/608/EEC)THE COUNCIL OF THE EUROPEAN COMMUNITIES, Having regard to the Treaty establishing the European Economic Community, Having regard to Council Directive 85/397/EEC of 5 August 1985 on health and animal-health problems  affecting intra-Community trade in heat-treated milk (1) and in particular Article 11 (6) thereof, Having regard to the proposal from the Commission, Whereas, pursuant to Article 11 (6) of Directive 85/397/EEC, the Council is to lay down the  detailed arrangements for the checks provided for in Article 11 (2); whereas the purpose of such  checks, to be carried out by establishments under the supervision and responsibility of the  official department and with periodic inspection by the official department, is to ensure  compliance with the requirements of Directive 85/397/EEC and in particular those laid down in  Article 3 (A) (3) thereof; Whereas, the laying-down of procedures for checks includes the determination of the methods to be  employed for their implementation; Whereas methods have to be prescribed for determining the total solids content, the fat content,  the solids-non-fat content, the total nitrogen content, the protein content and the density of  heat-treated milk intended for direct human consumption; Whereas, for technical reasons, the initial requirement is to lay down reference methods for  analysis and testing to ensure that the standards prescribed in Article 3 (A) (3) of Directive  85/397/EEC are met; whereas it is particularly important to make a study of the conditions under  which routine methods for analysis and testing are used; whereas, pending the outcome of this  study, it is for the competent authorities to see that appropriate routine methods are used with a  view to ensuring that the said standards are met; Whereas the determination of the aforementioned methods includes in particular the determination of  analytical procedures and the fixing of criteria of reliability in order to ensure uniform  interpretation of the results, HAS ADOPTED THIS DECISION: Article 1The methods for analysing and testing heat-treated milk shall be the  following: - determination of total solids content, - determination of fat content, - determination of total non-fat solids content, - determination of total nitrogen content, - determination of protein content, - determination of specific mass. Article 2The implementation of the reference methods for analysis and testing, the determination  of the criteria of reliability and the collection of samples must be carried out according to the  rules set out in Annex I. Article 3The methods referred to in Article 1 are set out in Annex II. Article 4This Decision is addressed to the Member States. Done at Brussels, 14 November 1992. For the CouncilThe PresidentJ. GUMMER(1) OJ N° L 226, 24. 8. 1985, p. 13, as  last amended by Directive 89/662/EEC (OJ N° L 395, 30. 12. 1989, p. 13).  ANNEX I I. GENERAL PROVISIONS 1. INTRODUCTIONGeneral provisions with respect to  reagents, equipment, expression of results, precision and test reports are described. Competent  authorities of Member States and enforcement laboratories charged with the sampling and testing of  milk must respect these provisions. 2. REAGENTS2.1. Water2.1.1. Wherever mention is made to water for solution, dilution or rinsing  purposes, distilled water, deionized water or demineralized water of at least equivalent purity,  shall be used unless otherwise specified. 2.1.2. Wherever reference is made to 'solution` or 'dilution` without further indication, 'solution  in water` or 'dilution with water` is meant. 2.2. ChemicalsAll chemicals used shall be of recognized analytical quality unless otherwise  specified. 3. EQUIPMENT3.1. Lists of equipmentThe lists of equipment given in the different reference  methods contain only those items with a specialized use and items to a particular specification. 3.2. Analytical balance'Analytical balance` means a balance capable of weighing at 0,1 mg. 4. EXPRESSION OF RESULTS4.1. ResultsUnless otherwise specified, the results stated in the test  report (6) shall be the mean arithmetic value obtained from two tests which satisfy the  repeatability-criterion (5.1.1.) stated for that method. If the repeatability-criterion is not  satisfied, the test must be repeated, if possible, or the result declared invalid. 4.2. Calculation of percentageExcept when otherwise specified, the result shall be calculated as a  percentage by mass of the sample. 5. PRECISION CRITERIA: REPEATABILITY AND REPRODUCIBILITY5.1. The precision criteria given in each  method is defined as follows: 5.1.1. Repeatability (r) is the value below which the absolute difference between two single test  results obtained with the same procedure on identical test material, under the same conditions  (same operator, same apparatus, same laboratory, and a short interval of time) lies. 5.1.2. Reproducibility (R) is the value below which the absolute difference between two single test  results obtained with the same procedure on identical test material, under different conditions  (different operators, different apparatus, different laboratories and/or different time) lies. 5.1.3. Unless otherwise specified for each individual method the values for the repeatability- and  reproducibility-criteria of each procedure represent the 95  % confidence level intervals as  defined by ISO 5725: 2'ed. 1986. 5.1.4. The necessary collaborative trials and studies should be planned and conducted in accordance  with international guidelines. 6. TEST REPORTThe test report shall specify the method of analysis used as well as the results  obtained. In addition, it shall give any details of procedure used not specified in the method of  analysis or which are optional, as well as any other circumstances that may have influenced the  results obtained. The test report shall give all the information necessary for the complete  identification of the sample. II. SAMPLING OF HEAT-TREATED MILK 1. SCOPE AND FIELD OF APPLICATIONThis procedure specifies the  reference method of sampling, transport and storage of samples of heat treated milk. 2. GENERALSampling of heat-treated milk in tanks etc., shall be carried out by a skilled operator  who has had suitable training before undertaking the sampling of milk. If they consider it appropriate, the competent authorities or testing laboratory shall instruct  sampling personnel in sampling techniques to ensure that the sample is representative of, and in  conformity with, the entire batch. If they consider it appropriate, the competent authorities or testing laboratory shall instruct  sampling personnel on marking the sample to ensure the unambiguous identity of the sample. 3. SAMPLING EQUIPMENT3.1. GeneralSampling equipment shall be made of stainless steel, or other  suitable material of adequate strength and of a construction suitable for the intended purpose  (mixing, sampling etc.). Plungers and agitators for mixing liquids in containers shall have a  sufficient area to produce adequate mixing of the product, but without causing the development of a  rancid flavour. Dippers must have a solid handle of sufficient length to enable a sample at any  depth of the container to be obtained. The capacity of the dipper shall be not less than 50 ml. Sample containers and closures should be of glass, suitable metals or plastics. The materials of which sampling equipment (including containers and closures) is constructed must  not cause any change in the sample which could affect the results of the examinations. All surfaces  of sampling equipment and sample containers shall be clean and dry, smooth and free from crevices,  and corners shall be rounded. 4. SAMPLING TECHNIQUE4.1. GeneralIrrespective of the tests to be performed, the milk shall be  thoroughly mixed prior to sampling, by either manual or mechanical means. The sample shall be taken immediately after mixing while the milk is still agitated. The volume of the sample shall be adequate to the testing requirements. The capacity of the sample  containers used shall be such that they are filled almost completely by the sample, thus allowing  proper mixing of the contents before testing, but avoiding churning during transport. 4.2. Manual sampling4.2.1. Sampling a divided bulkWhere the quantity of milk to be sampled is in  more than one container, take a representative quantity from each container and note the quantity  of milk to which each sample relates. Unless the samples from each container are to be tested  individually, mix portions of these representative quantities in amounts which are proportional to  the quantity in the container from which each sample was taken. Take sample(s) from these bulked  proportionate amounts after mixing. 4.2.2. Sampling from large vessels - Storage, rail and road tanks4.2.2.1. Mix the milk by an  appropriate procedure, before sampling. To mix the contents of large vessels or of storage, rail or road tanks, the use of mechanical  agitation is advised (4.2.2.2.). The extent of mixing shall be appropriate to the period of time over which the milk has been at  rest. The efficiency of the procedure of mixing applied in any particular circumstances shall be  demonstrated as being adequate for the purposes of the analysis envisaged; the criterion of mixing  efficiency particularly influences the similarity between analytical results from samples taken  either from different parts of the consignments, or from the outlet of the tank at intervals during  discharge. A procedure of mixing milk (untreated milk or whole milk) shall be considered efficient  if the difference in fat content between two samples, taken under these conditions, is less than  0,1  %. In a large vessel with a bottom discharge outlet there may be, at the discharge point, a small  quantity of milk which is not representative of the whole contents even after mixing. Therefore  samples should preferably be taken through a manhole. If samples are taken from the discharge  outlet, run off sufficient milk to ensure that the samples are representative of the whole. 4.2.2.2. Mixing of the contents of large vessels or of storage, rail or road tanks can be carried  out: - by a mechanical agitator built into the tanks and driven by an electric motor; - by a propellor or agitator driven by an electric motor and placed on the manhole with the  agitator suspended in the milk; - in the case of rail or road tankers by recirculation of the milk through the transfer hose  attached to the tanker unloading pumps and inserted through the manhole; - by clean filtered compressed air. In this case, minimal air pressure and volume should be used to  prevent the development of rancid flavour. 4.3. Sampling of heat-treated milk for direct consumption in retail-packingsSamples of  heat-treated milk for direct consumption in retail packages are to be the complete sealed package.  If possible, the samples must be taken from the packaging machine or cold room in the treatment  establishment as soon as possible after processing (for pasteurized milk on the same day as  processing). The samples are taken from each type of heat-treated milk (pasteurized, UHT-treated and sterilized)  in numbers corresponding to the examinations which will be made and in accordance with instructions  laid down by the testing laboratory or other competent authority. 5. IDENTIFICATIONS OF THE SAMPLEThe sample shall be marked with an identification code so that it  can be readily identified using instructions given by the testing laboratory or competent  authority. 6. PRESERVATION, TRANSPORT AND STORAGE OF SAMPLESIn accordance with the competent national  authority, instructions concerning the conditions of preservation (chemical, temperature),  transport, storage and time between sampling and analysis of milk shall be prepared by the testing  laboratory according to the type of milk and the procedure of analysis to be used. In the instruction the following points shall be included: - During transport and storage, precautions shall be taken to prevent exposure to contaminating  odours and to direct sunlight. If the container used for samples is transparent, it shall be stored  in a dark place.  ANEX II I. DETERMINATION OF TOTAL SOLIDS CONTENT1. SCOPE AND FIELD OF APPLICATIONThis  procedure specifies the reference method for the determination of the total solids content of  milk. 2. DEFINITIONTotal solids content: The mass remaining after completion of the specified drying  procedure and expressed as a percentage by mass. 3. PRINCIPLEEvaporation of the water from a test portion at a temperature of 102±2 oC in a drying  oven. 4. APPARATUS AND GLASSWAREUsual laboratory equipment and, in particular: 4.1. Analytical balance4.2. Desiccator, provided with an efficient desiccant (for example freshly  dried silica gel with a water content indicator). 4.3. Drying oven, ventilated, thermostatically controlled at 102±2 oC throughout the total working  space. 4.4. Flat-bottom dishes, of height 20 to 25 mm, diameter 50 to 75 mm, and of appropriate material  provided with well-fitting, readily removable lids. 4.5. Boiling water bath4.6. Homogenizer5. PREPARATION OF THE TEST SAMPLEBring the sample of milk  to a temperature of 20 to 25 oC. Thoroughly mix to ensure a homogeneous distribution of the fat  throughout the sample. Avoid agitating so vigorously as to cause frothing of the milk or churning  of the fat. If it is found difficult to disperse the cream layer, warm slowly to between 25 and 40  oC and with careful mixing, incorporate any cream adhering to the container. Cool the sample  quickly to 20-25 oC. If desired, a homogenizer may be used to assist the dispersion of the fat. Correct results cannot be expected if the sample contains separated liquid fat or visible separate  irregularly shaped white particles adhering to the walls of the container. 6. PROCEDURE6.1. Preparation of the dishHeat a dish (4.4.) with its lid placed next to it in the  oven (4.3.), controlled at 102 ± 2 oC, for at least 30 minutes. Place the lid on the dish and  immediately transfer to the desiccator (4.2.), allow to cool to room temperature (i.e. for at least  30 minutes) and weigh to the nearest 0,1 mg. 6.2. Test portionImmediately weigh, to the nearest 0,1 mg, 3 to 5 g of the prepared test sample  (5.) into the prepared dish (6.1.). 6.3. Determination6.3.1. Pre-dry the dish for 30 minutes by heating it on the boiling water bath  (4.5). 6.3.2. Heat the dish, with its lid alongside, in the oven (4.3), controlled at 102±2 oC, for two  hours. Place the lid on the dish and remove from the oven. 6.3.3. Allow to cool in the desiccator (4.2.) to room temperature (i.e. for at least 30 minutes)  and weigh to the nearest 0,1 mg. 6.3.4. Heat the dish again, with its lid alongside, in the oven for one hour. Place the lid on the  dish and remove from the oven. Allow to cool for approximately 30 minutes in the desiccator and  weigh to the nearest 0,1 mg. 6.3.5. Repeat the operations described in 6.3.4. until the difference in mass between two  consecutive weighings does not exceed 0,5 mg. Record the lowest mass. 7. EXPRESSION OF RESULTS7.1. Calculation and formulaCalculate the total solids content as a  parcentage by mass from:  WT =m2     m0m1     m0 × 100whereWT =   the total solids content in g per 100 g, m0 =   the mass, in grams, of the dish and lid (see 6.1.), m1 =   the mass, in grams, of the dish, lid and test portion (see 6.2.), m2 =   the mass, in grams, of the dish, lid and dried test portion (see 6.3.5.). Round the value obtained to the nearest 0,01  % (percentage by mass). 7.2. Precision7.2.1. Repeatability (r): 0,10 g of total solids per 100 g of product. 7.2.2. Reproducibility (R): 0,20 g of total solids per 100 g of product. II. DETERMINATION OF FAT CONTENT 1. SCOPE AND FIELD OF APPLICATIONThis procedure specifies the  reference method for the determination of the fat content of raw milk and of whole milk, partly  skimmed milk and skimmed milk. 2. DEFINITIONFat content of milk: all the material as determined by the specified method. It is  expressed as a percentage by mass. 3. PRINCIPLEAn ammoniacal ethanolic solution of a test portion is extracted with diethyl ether and  light petroleum, the solvents removed by distillation or evaporation, and the mass of the extracted  material soluble in light petroleum, is determined. (The procedure is usually known as the  Roese-Gottlieb method). 4. REAGENTSAll reagents shall be of recognized analytical quality and shall leave no appreciable  residue when taken through a blank test. To test the quality of the reagents, carry out a determination as specified in 6.3. Use an empty  flask, beaker or metal dish (5.8.) for weighing, prepared as specified in 6.4. as a tare (to allow  correction for the effects on the weighing result, caused by changes in the atmospheric  conditions). If the residue, corrected for the apparent change of the mass of the tare is larger  than 2,5 mg, determine the residue or the solvents separately by evaporating 100 ml diethyl ether  (4.4.) and 100 ml light petroleum (4.5.) respectively. Also use a tare for the weighing. When the  residue is larger than 2,5 mg, cleanse the solvent by means of distillation or replace the  solvent. 4.1. Ammonia solution, containing approximately 25  % (m/m) of NH3. A more concentrated ammonia  solution may also be used (see 6.5.1. and A.1.5.1.). 4.2. Ethanol, at least 94  % (v/v). Ethanol denatured by methanol may be used provided it is  certain that the results of the determination are not affected. 4.3. Congo red or Cresol red solutionDissolve 1 g of Congo red or Cresol red in water and dilute  to 100 ml. Note: The use of this solution, which allows the interface between the solvent and aqueous layers  to be seen more clearly, is optional (see 6.5.2.). Other aqueous colour solutions may be used  provided that they do not affect the result of the determination. 4.4. Diethylether, free from peroxides not containing more than 2 mg/kg of antioxidants, and  meeting the requirements of the blank test (6.3.). 4.5. Light petroleum, having any boiling range between 30 and 60 oC. 4.6 Mixed solvent, prepared shortly before use by mixing equal volumes of diethyl ether (4.4.) and  light petroleum (4.5.). 5. APPARATUS AND GLASSWAREWarning: Since the determination involves the use of volatile flammable  solvents, any electrical apparatus employed shall comply with legislation relating to the use of  such solvents. Usual laboratory equipment and, in particular: 5.1. Analytical balance5.2. Centrifuge, in which the fat-extraction flasks or tubes (5.6.) can be  spun at a rotational frequency of 500 to 600 rev min 1 to produce a gravitational field of 80 to 90  g at the outer end of the flasks or tubes. Note:  The use of a centrifuge is optional (6.5.5.). 5.3. Distillation or evaporation apparatus, to permit the solvents and ethanol to be distilled from  the flasks or to be evaporated from beakers and dishes (sec 6.5.12. and 6.5.15.) at a temperature  not exceeding 100 oC. 5.4. Oven, electrically heated, with ventilation port(s) fully open, capable of being controlled at  a temperature of 102±2 oC throughout the working space. The oven shall be fitted with a suitable  thermometer. 5.5. Water bath, capable of being maintained at a temperature of 35 40 oC. 5.6. Mojonnier-type fat extraction flasksNote: It is also possible to use fat-extraction tubes  with siphon or wash-bottle fittings, but the procedure is then different and is specified in the  Appendix. The flasks (or tubes) shall be provided with ground-glass or good quality bark corks or other  stoppers unaffected by the reagents used. Rark corks shall be extracted with the diethyl ether  (4.4.) kept in water at 60 oC or more for at least 15 minutes, and shall then be allowed to cool in  water so that they are saturated when used. 5.7. Rack, to hold the fat-extraction flasks (or tubes) (see 5.6.). 5.8. Wash bottle, suitable for use with the mixed solvent (4.6.). A plastic wash bottle shall not  be used. 5.9. Fat-collecting vessels, for example boiling flasks (flat-bottom), or Erlenmeyer flasks of  capacity 125-250 ml or metal dishes. If metal dishes are used, they shall preferably be of  stainless steel, shall be flat-bottomed, preferably with a spout, and shall have a diameter of 80  to 100 mm and a height of approximately 50 mm. 5.10. Boiling aids, fat-free, of non-porous porcelain or silicon carbide or glass beads (optional  in the case of metal dishes). 5.11. Measuring cylinders, of capacities 5 and 25 ml. 5.12. Pipettes, graduated, of capacity 10 ml. 5.13. Tongs, made of metal, suitable for holding flasks, beakers or dishes. 6. PROCEDURENote: The alternative procedure using fat-extraction tubes with siphon or wash-bottle  fittings (see the note to 5.6.) is described in the Appendix. 6.1. Preparations of the test sampleAdjust the temperature of the laboratory sample to  approximately 35-40 oC for 15 minutes, by means of a water bath is necessary. Mix the sample  thoroughly, but gently, by repeatedly inverting the sample bottle without causing frothing or  churning, and cool quickly to approximately 20 oC. 6.2. Test portionMix the test sample (6.1.) by gently inverting the bottle three or four times and  immediately weigh, to the nearest 1 mg, 10 to 11 g of the test sample, directly or by difference,  into one of the extraction flasks (5.6.). The test portion shall be delivered as completely as possible into the lower (small) bulb of the  extraction flasks. 6.3. Blank testCarry out the blank test simultaneously with the determination using the same  procedure and same reagents, but replacing the test portion by 10 to 11 ml of water. The change in apparent mass of the fat collecting vessel, corrected for apparent change in mass of  the control vessel, should not be greater than 2,5 mg. 6.4. Preparation of fat-collecting vesselDry a vessel (5.9.) together with a few boiling aids  (5.10.) to promote gentle boiling during the subsequent removal of solvent in the oven (5.4.) for  one hour. Allow the vessel to cool (not in a desiccater but protected from dust) to the temperature  of the weighing room (for glass vessels allow at least one hour, for metal dishes allow at least 30  minutes). Taking care to avoid temperature variations, use tongs to place the vessel on the balance  and weigh to the nearest 0,1 mg. 6.5. Determination6.5.1. Add 2 ml of the ammonia solution (4.1.) or an equivalent volume of a more  concentrated ammonia solution and mix thoroughly with the test portion in the small bulb of the  flask. After the addition of the ammonia, carry out the determination without delay. 6.5.2. Add 10 ml of the ethanol (4.2.) and mix gently but thoroughly by allowing the contents of  the flask to flow backwards and forwards between the two bulbs; avoid bringing the liquid too near  to the neck of the flask. If desired, add two drops of the Congo red or Cresol red solution  (4.3.). 6.5.3. Add 25 ml of diethyl ether (4.4.), close the flask with a cork saturated with water or with  a stopper wetted with water (see 5.6.), and shake the flask vigorously, but not excessively (in  order to avoid the formation of persistant emulsions), for one minute with the flask in a  horizontal position and the small bulb extending upwards. Periodically allow the liquid in the  large bulb to run into the small bulb. If necessary, cool the flask in running water, then  carefully remove the cork or stopper and rinse it and the neck of the flask with a little of the  mixed solvent (4.6.) using the wash bottle (5.8.) so that the rinsings run into the flask. 6.5.4. Add 25 ml of light petroleum (4.5.), close the flask with the rewetted cork or stopper  (rewet by dipping in water), and shake the flask gently for 30 seconds as described in 6.5.3. 6.5.5. Centrifuge the closed flask for one to five minutes at a rotational frequency of 500 to 600  rev min 1 (5.2.). If a centrifuge is not available (see note to 5.2.) allow the closed flask to  stand in the rack (5.7.) for at least 30 minutes until the supernatant layer is clear and  distinctly separated from the aqueous layer. If necessary, cool the flask in running water. 6.5.6. Carefully remove the cork or stopper and rinse it and the inside of the neck of the flask  with a little of the mixed solvent (4.6.) so that the rinsings run into the flask. If the interface is below the bottom of the neck of the flask, raise it slightly above this level  by gently adding water down the side of the flask to facilitate decantation of solvent. 6.5.7. Holding the extraction flask by the small bulb, carefully decant as much as possible of the  supernatant layer into the prepared fat-collecting vessel (6.4.) containing a few boiling aids  (5.10.) in the case of flasks (optional with metal dishes), avoiding decantation of any of the  aqueous layer. 6.5.8. Rinse the outside of the neck of the extraction flask with a little of the mixed solvent  (4.6.), collecting the rinsings in the fat-collecting vessel and taking care that the mixed solvent  does not spread over the outside of the extraction flask. If desired, the solvent or part of the solvent may be removed from the vessel by distillation or  evaporation as described in 6.5.12. 6.5.9. Add 5 ml of the ethanol (4.2.) to the contents of the extraction flask, using the ethanol to  rinse the inside of the neck of the flask and mix as decribed in 6.5.2. 6.5.10. Carry out a second extraction by repeating the operations described in 6.5.3. to 6.5.8.  inclusive, but using only 15 ml of diethyl ether (4.4.) and 15 ml of light petroleum (4.5.); use  the ether to rinse the inside of the neck of the extraction flask. If necessary, raise the  interface to the middle of the stem of the flask to enable the final decantation of solvent to be  as complete as possible. 6.5.11. Carry out a third extraction by further repeating the operations described in 6.5.3. to  6.5.8. inclusive, but using only 15 ml of diethyl ether (4.4.) and 15 ml of light petroleum (4.5.);  use the ether to rinse the inside of the neck of the extraction flask. If necessary, raise the  interface to the middle of the neck of the flask to enable the final decantation of solvent to be  as complete as possible. The third extraction can be omitted for skimmed milk. 6.5.12. Remove the solvents (including ethanol) as completely as possible from the flask by  distillation, or from the beaker or dish by evaporation (5.3.), rinsing the inside of the neck of  the flask with a little of the mixed solvent (4.6.) before commencing the distillation. 6.5.13. Heat the fat-collecting vessel (with the flask placed on its side to allow solvent vapour  to escape) for one hour in the oven (5.4.). Remove the fat-collecting vessel from the oven, allow  to cool (not in a desiccator, but protected from dust) to the temperature of the weighing room (for  glass vessels allow at least one hour, for metal dishes allow at least 30 minutes) and weigh to the  nearest 0,1 mg. Do not wipe the vessel immediately before weighing. Place the vessel on the balance using tongs and  avoid, in particular, temperature variations. 6.5.14. Repeat the operations described in 6.5.13. until the mass of the fat-collecting vessel  decreases by 0,5 mg or less, or increases, between two successive weighings. Record the minimum  mass observed as the mass of the fat-collecting vessel and extracted matter. 6.5.15. Add 25 ml of light petroleum to the fat-collecting vessel in order to verify whether or not  the extracted matter is wholly soluble. Warm gently and swirl the solvent until all the fat is  dissolved. If the extracted matter is wholly soluble in the light petroleum, take the mass of fat as the  difference between the final mass of the vessel containing the extracted matter (6.5.14.) and its  initial mass (6.4.). 6.5.16. If the extracted matter is not wholly soluble in the light petroleum, or in case of doubt,  extract the fat completely from the vessel by repeatedly washing with warm light petroleum. Allow any trace of insoluble material to settle and carefully decant the light petroleum without  removing any insoluble material. Repeat this operation three more times, using the light petroleum  to rinse the inside of the neck of the vessel. Finally, rinse the outside of the top of the vessel with mixed solvent so that the solvent does not  spread over the outside of the vessel. Remove light petroleum vapour from the vessel by heating the  vessel for one hour in the oven, allow to cool and weigh, as described in 6.5.13. and 6.5.14. Take the mass of fat as the difference between the mass determined in 6.5.14. and this final mass. 7. EXPRESSION OF RESULTS7.1. Calculation and formulaCalculate the fat content as a percentage by  mass by:  F =(m1     m2)     (m3     m4)m0 × 100whereF  =   the fat content, m0 =   the mass, in grams, of the test portion (6.2.), m1 =   the mass, in grams, of the fat-collecting vessel and extracted matter determined in  6.5.14., m2 =   the mass, in grams, of the prepared fat-collecting vessel or, in the case of undissolved  material, of the fat-collecting vessel and insoluble residue determined in 6.5.16., m3 =   the mass, in grams, of the fat-collecting vessel used in the blank test (6.3.) and any  extracted matter determined in 6.5.14., m4 =   the mass, in grams, of the prepared fat-collecting vessel (see 6.4.) used in the blank test  (6.3.), or in the case of undissolved material, of the fat-collecting vessel and insoluble residue  determined in 6.5.16. Report the result to the nearest 0,01  %. 7.2. Precision7.2.1. Repeatability (r): - for whole milk and partly skimmed milk: 0,02 g of fat per 100 g of product, - for skimmed milk: 0,01 g of fat per 100 g of product. 7.2.2. Reproducibility (R): - for whole milk: 0,04 g of fat per 100 g of product, - for partly skimmed milk: 0,03 g of fat per 100 g of product, - for skimmed milk: 0,025 g of fat per 100 g of product. III. DETERMINATION OF TOTAL NON-FAT SOLIDS1. SCOPE AND FIELD OF APPLICATIONThis procedure  specifies the reference method for the determination of the content of total non-fat solids in  heat-treated milk. 2. DEFINITION AND CALCULATIONThe total non-fat solids content must be expressed as a percentage by  mass. The content of non-fat solids is: The content of total solids (see Section I) minus the content of fat (see Section II). IV. DETERMINATION OF TOTAL NITROGEN CONTENT1. SCOPE AND FIELD OF APPLICATIONThis procedure  specifies the reference method for the determination of the total nitrogen content of raw milk and  of whole milk, partly skimmed milk and skimmed milk. 2. DEFINITIONThe total nitrogen content of milk: the nitrogen content, expressed in per cent by  mass, as determined by the specified Kjeldahl method. 3. PRINCIPLEA weighed quantity of the milk sample is digested with concentrated sulphuric acid and  potassium sulphate and copper (II) sulphate as catalyst, in order to convert the nitrogen of the  organic compounds into ammonium sulphate. The ammonia is released by the addition of sodium  hydroxide solution and then distilled and absorbed in a boric acid solution. This is titrated with  an acid solution. 4. REAGENTS4.1. Potassium sulphate (K2SO4). 4.2. Copper sulphate solution. Dissolve 5,0 g of copper (II) sulphate pentahydrate (CuSO4,    5H2O)  in water and dilute to 100 ml (at 20 oC) in a volumetric flask. 4.3. Sulphuric acid, at least 98,0  % (m/m) H2SO4. 4.4. Sodium hydroxide solution, 47  % (m/m) 704 g NaOH/l (20 oC). Note: A less concentrated sodium hydroxide solution may be used for example: 40  % (m/m) 572 g/l,  20 oC; or 30  % (m/m) 399 g/l, 20 oC. 4.5. Boric acid solution. Dissolve 40 g of boric acid (H3BO3) in one litre of hot water, allow to  cool, and store in a borosilicate glass bottle. 4.6. Indicator solution. Dissolve 0,01 g methyl red, 0,02 g bromothymol blue and 0,06 g bromocresol  green in 100 ml of ethanol. Store the solution in a brown closed bottle, in a cool, dark place. 4.7. Volumetric solutionc (1/2 H2SO4) or c (HCl) = 0,1 mol/l standardized to the nearest 0,0001  mol/l. 4.8. Nitrogen-free sucrose. 4.9. Ammonium salt, pure, such as ammonium oxalate (NH4)2C2O4,  H2O or ammonium sulfate (NH4)2SO4. 4.10. Tryptophan (C11H12N2O2), phenacetin (C10H7CH2CONH2) or lysine mono- or  di-hydrochloride(C6H14N2O2   7  HCl or C6H14N2O2   7  2HCl). Note: The purity of reagents in 4.9. and 4.10. should be of higher quality than 'analytical grade`.  If available, certified ammonium salt solution (4.9.) should be used. 5. APPARATUS AND GLASSWAREUsual laboratory equipment and, in particular: 5.1. Kjeldahl flasks of capacity 500 ml. 5.2. Suitable boiling aids, for example, glass beads of diameter approximately 5 mm, Hengar  granules, pumice. 5.3 Burette or automatic pipette, to deliver 1,0 ml. 5.4. Graduated measuring cylinders, glass, of capacities 50, 100 and 250 ml. 5.5. Digestion apparatus in an inclined position (approximately 45o), with electric heaters or gas  burners that do not heat the flasks above the level of their contents, with a fume extraction  system. 5.6. Distillation apparatus, made of borosilicate glass, to which a Kjeldahl flask (5.1.) can be  fitted, consisting of an efficient splash-head connected to an efficient condenser with straight  inner tube and an outlet tube attached to its lower end; the connecting tubing and stopper (s)  shall be close-fitting and preferably of neoprene rubber. 5.7. Pipette or automatic pipette, to deliver 0,10 ml. 5.8. Conical flasks, of capacity 500 ml, graduated at 200 ml. 5.9. Burette of capacity 50 ml, gratuated in 0,1 ml, maximum error ±0,05 ml. 5.10. Magnifying lens, for reading the burette (5.9.). 5.11. pH meter5.12. Automatic burette. 6. PROCEDURE6.1. To the Kjeldahl flask (5.1.) add boiling aid (5.2.) (eg. three glass beads), 15 g  of potassium sulphate (4.1.), 1,0 ml of copper sulphate solution (4.2.), approximately 5 g of milk  sample (weighed to the nearest 0,001 g) and 25 ml of sulphuric acid (4.3.). Use the acid to wash  down any copper sulphate solution, potassium sulphate or milk on the neck of the flask, and gently  mix the contents of the flask. Note: Because organic matter consumes sulphuric acid during boiling, use 30 ml of H2SO4 (4.3.),  instead of 25 ml for digestion, if the milk contains more than 5,0  % (m/m) of fat. This should  also be done in the blank test. 6.2. Heat each Kjeldahl flask on the digestion apparatus (5.5.), very gently at first so that all  the black froth stays within the bulb. When the initial frothing has ceased and copious white  vapour appears, boil vigorously (acid vapour will condense half-way up the neck of the flask) until  no black particles remain and until the digest becomes clear pale blue-green in colour. Then boil  gently for at least 1,5 hours. Note the following requirements: (a) The time for the digest to become clear should not be more than one hour, and the total  digestion time shall not be less than 2,5 hours. If more than one hour is necessary to achieve  clearing, the total digestion time shall be increased accordingly. (b) The added potassium sulphate promotes the digestion as it raises the boiling temperature of the  mixture. If the residual volume of H2SO4 is less than approximately 15 ml at the end of the  digestion time, nitrogen may have been lost because of excessive heating. If heating by gas, heat  the flask on a plate of heat-insulating material, provided with a circular opening of such a  diameter that the free flame only touches the part of the flask that is below the surface of the  liquid contents (5.5.). (c) If black particles enter the neck of the flask and are not all washed down into the bulb by the  acid refluxing during the inital stages of the vigorous boiling period (this may be facilitated by  rotating the flask) allow the flask to cool sufficiently and carefully wash with the minimum of  water. Then continue the digestion as described above. 6.3. When the Kjeldahl flasks are cool, add 300 ml of water (see note) to each so as to wash  carefully down the neck of the flask, and mix the contents thoroughly ensuring that the crystals  which have separated out are dissolved. Add some boiling aid (5.2.) to ensure uniform boiling. Then  to each flask, add 70 ml of sodium hydroxide solution (4.4.) (see note) by gently pouring the  solution down the inclined neck of the flask to form a bottom layer in the bulb; do not wet the top  of the neck with the sodium hydroxide solution. Note: It is necessary that the combined volume of water and sodium hydroxide solution total 370 ml  to enable approximately 150 ml of distillate to be collected just before irregular boiling  ('bumping`) ensues (6.4.). Thus, if a larger equivalent volume of a sodium hydroxide solution which  is less concentrated than 47  % (m/m) is added, the volume of water added shall be reduced  accordingly. For example, if 85 ml of 40 % (m/m) or 125 ml of 30  % (m/m) sodium hydroxide solution  are to be added, the volume of water added shall be 285 ml or 245 ml respectively. 6.4. Immediately connect each Kjeldahl flask to a distillation apparatus (5.6.). Ensure that the  tip of the condenser outlet-tube is immersed in 50 ml of boric acid solution (4.5.) together with  0,20 ml (5 6 drops) of indicator solution (4.6.) all contained in a conical flask (5.8.). Swirl the  contents of each Kjeldahl flask to mix thoroughly and boil, but gently at first to prevent  excessive frothing. When 100 to 125 ml of distillate have been collected, lower each conical flask  until the tip of the condenser outlet-tube is approximately 40 mm above the 200 ml mark. Continue  each distillation until irregular boiling ('bumping`) starts and then immediately stop the heating.  Disconnect each Kjeldahl flask and rinse the tip of each condenser outlet-tube with a little water,  collecting the rinsings in the conical flask. Note the following requirements: (a) The distillation rate shall be such that approximately 150 ml of distillate are collected when  irregular boiling ('bumping`) starts, the volume of the contents of each conical flask will then be  approximately 200 ml. (b) The efficiency of each condenser should be such that the temperature of the contents of each  conical flask does not exceed 25 oC during the distillation. 6.5 Titrate each distillate with standard volumetric solution (4.7.) until the pH is 4,6±0,1, using  a pH meter and if desired an automatic burette. Addition of an indicator helps to check whether the  titration is proceeding correctly. Take each burette reading to the nearest 0.01 ml with the aid of  a magnifying lens (5.10.) avoiding errors of parallax. The titrating may be carried out with the indicator only. Titrate until the colour of the  distillate corresponds to that of a solution recently prepared from 150 ml of water to which has  been added 50 ml of the boric acid solution and 0,20 ml of the indicator contained in a conical  flask (5.8). 6.6. Carry out a blank test according to 6.1. to 6.5. inclusive, taking 5 ml of distilled water  together with about 0,1 g of sucrose (4.8.) through the procedure instead of the milk sample. Note: The titration of the blank distillate will require only a very small volume of the standard  volumetric solution (4.7.). 6.7. Regularly check the accuracy of the procedure by using two recovery trials following the  procedure according to 6.1. to 6.5. inclusive. 6.7.1. Check that no loss of nitrogen occurs as a result of excessive heat or mechanical leaks  during distillation, by using a test portion of 0,15 g of ammonium oxalate or sulphate (4.9.)  weighed to the nearest 0,001 g together with 0,1 g of sucrose (4.8.). The percentage of nitrogen recovered shall be between 99,0 and 100,0  %. Lower or higher results will indicate failures in the procedure and/or inaccurate concentration of  the standard solution (4.7.). 6.7.2. Check that the digestion procedure is sufficient to release all the protein nitrogen by  using a test portion of 0,20 g of pure tryptophan, 0,35 g of phenacetin or 0,20 g of lysine  hydrochloride (4.10.). All weighings should be to the nearest 0,001 g. At least 98-99  % of the  nitrogen should be recovered. 7. SAFETY PRECAUTIONSWhen working with concentrated sulphuric acid and sodium hydroxide and when  handling Kjeldahl flasks, always wear a laboratory coat, safety goggles and acid resistant gloves. During distillation, never leave Kjeldahl flasks unattended. Because of potential danger, stop  distillation immediately if flask contents 'bump` too vigorously. If the power goes off for more  than two to three minutes, lower the collecting flask so that the distillation tip is out of the  liquid. 8. EXPRESSION OF RESULTS8.1. Calculation and formula: Calculate the nitrogen content (WN), expressed in grams of nitrogen per 100 g of product by:  WN =1,40 (V     VO) cmwhere: WN =   the nitrogen content. V =   the volume in millilitres of the standard volumetric solution of acid used in the  determination. VO =   the volume in millilitres of the standard volumetric solution of acid used in the blank  test. c =   the concentration, expressed as moles per litre of the acid standard volumetric solution  (4.7.). m =   the mass in grams of the test portion. Round off the result to the nearest 0,001 g per 100 g. 8.2. Precision8.2.1. Repeatability (r): 0,007 g per 100 g. 8.2.2. Reproducibility (R): 0,015 g per 100 g. 9. MODIFIED PROCEDURES9.1. Use a block digestion apparatus fitted with cylindrical flasks, instead  of the digestion apparatus and the Kjeldahl flasks described in 5.5. and 5.1. In this case to  identify potential trouble, each spot has to be checked individually (6.7.). 9.2. Use of steam distillation instead of direct heating of the flasks (6.4.). When the apparatus  does not allow the use of distilled water, care should be taken that the water does not contain  acid or alkaline volatiles. 9.3. A test portion of 1 g of milk (semi-macro Kjeldahl) can be used instead of 5 g (6.1.)  provided: - the amounts of the reagents used for mineralization (6.1.): H2SO4, CuSO4   7  5 H2O, K2SO4, are  reduced to the same ratio (1/5). - the total digestion time (6.2.) is reduced to 75 minutes. - the amount of sodium hydroxide solution (6.3.) is reduced to the same ratio (1/5). - an acid standard solution (4.7.) of lower concentration (0,02 to 0,03 mol/l) has to be used. Note: Using one or more of these options is acceptable only if the repeatability value (8.2.1.) and  the two accuracy tests results (6.7.) are in accordance with the requirements given in this  method. V. DETERMINATION OF PROTEIN CONTENT 1. SCOPE AND FIELD OF APPLICATIONThis procedure specifies  the reference method for the determination of the content of protein in heat treated milk (Article  3 A3 in Directive 85/397/EEC). 2. DEFINITIONProtein content: The value obtained by multiplying the total nitrogen content,  expressed as a percentage by mass, determined in accordance with the method described in Section  IV(3) by an appropriate factor (3.). 3. CALCULATIONProtein content of milk as percentage by mass = 6,38 x total N content of milk %. VI. DETERMINATION OF SPECIFIC MASS 1. SCOPE AND FIELD OF APPLICATIONThis procedure specifies the  reference method for the determination of the specific mass at 20 oC of raw milk and of whole milk,  partly skimmed milk and skimmed milk. 2. DEFINITIONThe specific mass of the milk is the ratio of the mass of a certain volume of milk at  20 oC to that of the mass of the same volume of water at 20 oC. 3. PRINCIPLEThe specific mass at 20 oC is determined by a hydrometer. 4. APPARATUS AND GLASSWAREUsual laboratory equipment and, in particular: 4.1. HydrometerThe specific gravity hydrometer is an instrument consisting of a glass float,  which, at its lower end, is wide and heavy. There is a cylindrically shaped glass rod attached to  the upper and of the float and coaxially orientated to it; the upper end of the rod is closed. The glass float contains the load (lead, mercury, etc.) intended to adjust the hydrometer mass. The  rod includes a graduated scale from 1,025 to 1,035 g/ml. The hydrometer should he checked by the pycnometric method, using a pycnometer of a capacity of  approximately 100 ml equipped with a precision thermometer. 4.2. Cylinders (glass or stainless steel). Minimum dimensions should be: - internal diameter approximately 35 mm- internal height approximately 225 mm. 4.3. Water bath regulated at 20±0,1 oC. 4.4. Water bath regulated at 40±2 oC. 4.5. Thermometer, graduated to 0,5 oC. 5. PROCEDURE5.1. Mix the sample by inversion to disperse the fat and place in the water bath  (4.4.). Allow the sample to reach a temperature of 40 oC and maintain at that temperature for five  minutes. Mix thoroughly by careful inversion to ensure that the fat is homogeneously distributed.  Cool to 20 oC in the second water bath (4.3). 5.2. Mix the sample thoroughly by careful inversion to avoid inclusion of air. Pour the milk into  the cylinder (4.2.), held inclined so as to avoid the formation of foam or of bubbles. Use  sufficient milk sample to ensure that some will overflow from the cylinder when the hydrometer  (4.1.) is placed in it. Carefully lower the hydrometer into the milk and allow to float freely when  it reaches its balance position. The cylinder is to be placed vertical. The hydrometer must be  positioned in the middle of the liquid column and should not touch the sides of the cylinder. 5.3. When the hydrometer reaches equilibrium read the graduation at the top of the meniscus. 5.4. Immediately after taking the hydrometer reading introduce the thermometer (4.5.) into the  sample and read the temperature with an accuracy of 0,5 oC. The temperature must not differ more  than ±2 oC from ±20 oC. 6. TEMPERATURE CORRECTION6.1. If the temperature of the milk sample is not exactly 20 oC when the  measurement of its specific mass is made, then the result obtained must be corrected adding to the  determined specific mass 0,0002 for each degree Celcius above 20 oC, or subtracting 0,0002 for  every degree Celsius below 20 oC. This correction is only valid if the temperature of the milk  sample differs by not more than 5 oC from 20 oC. 7. EXPRESSION OF RESULTSThe method of calculation and formula specific mass of the sample is  expressed in g/ml of skimmed milk at 20 oC according to the following formula:  1 000   7  mv   MG   7  mv=0,92 mv (1 000     MG)1 000 MG   7  mv0,92920     MG   7   mvwheremv  =   the specific mass of the sample read on the hydrometer (5.4) in g/lMG = the fat  content of the sample in g/l0,92 = the density of fat. 8. PRECISION8.1. Repeatability (r): 0,0003 g/ml. 8.2. Reproducibility (R): 0,0015 g/ml. Appendix (to Annex II)ALTERNATIVE PROCEDURE USING FAT-EXTRACTION TUBES WITH SIPHON OR  WASH-BOTTLE FITTINGS A.1. PROCEDUREA.1.1. Preparation of the test sampleSee 6.1. A.1.2. Test portionProceed as specified in 6.2. but using the fat-extraction tubes (see 5.6.). The test portion shall be delivered as completely as possible at the bottom of the extraction  tube. A.1.3. Blank testSee 6.3. A.1.4. Preparation of fat-collecting vesselSee 6.4. A.1.5. DeterminationA.1.5.1. Add 2 ml of the ammonia solution (4.1.), or an equivalent volume of a  more concentrated ammonia solution, and mix thoroughly with the pretreated test portion at the  bottom of the tube. After the addition of the ammonia, carry out the determination without delay. A.1.5.2. Add 10 ml of the ethanol (4.2.) and mix gently but thoroughly at the bottom of the tube.  If desired, add two drops of the Congo red or Cresol red solution (4.3.). A.1.5.3. Add 25 ml of diethyl ether (4.4.), close the tube with a cork saturated with water or with  a stopper wetted with water (5.6.), and shake the tube vigorously, but not excessively (in order to  avoid the formation of persistent emulsions), with repeated inversions for one minute. If  necessary, cool the tube in running water, then carefully remove the cork or stopper and rinse it  and the neck of the tube with a little of the mixed solvent (4.6.) using the wash bottle (5.8.) so  that the rinsings run into the tube. A.1.5.4. Add 25 ml of light petroleum (4.5.), close the tube with the rewetted cork or stopper  (rewet by dipping in water), and shake the tube gently for 30 seconds as described in A.1.5.3. A.1.5.5. Centrifuge the closed tube for one to five minutes at a rotational frequency of 500 to 600  rev min-1 (5.2.). If the centrifuge is not available (see note to 5.2.), allow the closed tube to  stand in the rack (5.7.) for at least 30 minutes until the supernatant layer is clear and distincly  separated from the aqueous layer. If necessary, cool the tube in running water. A.1.5.6. Carefully remove the cork or stopper and rinse it and the neck of the tube with a little  of the mixed solvent so that the rinsings run into the tube. A.1.5.7. Insert a siphon fitting or a wash-bottle fitting into the tube and push down the long  inner limb of the fitting until the inlet is approximately 3 mm above the interface between the  layers. The inner limb of the fitting shall be parallel to the axis of the extraction tube. Carefully transfer the supernatant layer out of the tube into the prepared fat-collecting vessel  (6.4.) containing a few boiling aids (5.10.) in the case of flasks (optional with metal dishes),  avoiding the transfer of any of the aqueous layer. Rinse the outlet of the fitting with a little of  the mixed solvent, collecting the rinsings in the fat-collecting vessel. A.1.5.8. Loosen the fitting from the neck of the tube, slightly raise the fitting and rinse the  lower part of its long inner limb with a little of the mixed solvent. Lower and re-insert the  fitting and transfer the rinsings to the fat-collecting vessel. Rinse the outlet of the fitting with a little of the mixed solvent, collecting the rinsings in the  vessel. If desired, the solvent or part of the solvent may be removed from the vessel by  distillation or evaporation as described in 6.5.12. A.1.5.9. Again loosen the fitting from the neck, slightly raise the fitting and add 5 ml of the  ethanol to the contents of the tube, using the ethanol to rinse the long inner limb of the fitting;  mix as described in A.1.5.2. A.1.5.10. Carry out the second extraction by repeating the operations described in A.1.5.3. to  A.1.5.8., but using only 15 ml of diethyl ether (4.4.) and 15 ml of light petroleum (4.5.). Use the  ether to rinse the long inner limb of the fitting during the removal of the fitting from the tube  after the previous extraction. A.1.5.11. Carry out a third extraction by again repeating the operations described in A.1.5.3. to  A.1.5.8. using 15 ml of the diethyl ether and 15 ml of the light petroleum and rinsing the long  inner limb of the fitting as described in A.15.10. The third extraction can be omitted for skimmed milk. A.1.5.12. Proceed as described in 6.5.12. to 6.5.16.