{
  "url": "https://openlegislation.uk/eu-regulation-2018-150/annex",
  "json_url": "https://openlegislation.uk/eu-regulation-2018-150/annex.json",
  "legislation": "eur/2018/150",
  "legislation_title": "Commission Implementing Regulation (EU) 2018/150 of 30 January 2018 amending Implementing Regulation (EU) 2016/1240 as regards methods for the analysis and quality evaluation of milk and milk products eligible for public intervention and aid for private storage",
  "legislation_url": "https://openlegislation.uk/eu-regulation-2018-150",
  "path": "annex",
  "kind": "annex",
  "number": null,
  "heading": null,
  "text": "The Annexes to Implementing Regulation (EU) 2016/1240 are amended as follows:\n(1) Annex IV is amended as follows:\n(a) in Part I, point 2, the second sub-paragraph is replaced by the following:\n‘Each sample shall be assessed individually. No resampling or re-evaluation is allowed.’;\n(b) the following Part Ia is inserted:\n‘PART IA\nMethods of analysis of unsalted butter for public intervention\nThe method to be applied shall be approved by the paying agency.\nParameter\nMethod\nFat\nISO 17189 or ISO 3727 part 3\nWater\nISO 3727 part 1\nNon-fat solids\nISO 3727 part 2\nFat acidity\nISO 1740\nPeroxide value\nISO 3976\nNon-milk fat\nISO 17678\nSensory characteristics\nISO 22935 parts 2 and 3 and scoring table hereafter.\nScoring table\nAppearance\nConsistency\nOdour and Flavour\nPoints\nRemarks\nPoints\nRemarks\nPoints\nRemarks\n5\nVery good\nIdeal type\nHighest quality\n(equal dry)\n5\nVery good\nIdeal type\nHighest quality\n(equal spreadable)\n5\nVery good\nIdeal type\nHighest quality\n(absolutely pure finest odour)\n4\nGood\n(no evident defects)\n4\nGood\n(no evident defects)\n4\nGood\n(no evident defects)\n1, 2 or 3\nAny defect\n1, 2 or 3\nAny defect\n1, 2 or 3\nAny defect\n’\n(2) in Annex V the following Part Ia is inserted:\nPART IA\nMethods of analysis of skimmed milk powder for public intervention\nScorched particles' analyses may be conducted systematically. However, such analyses shall always be conducted if no sensory checks are performed.\nThe method to be applied shall be approved by the paying agency (one or both methods).\nThe method to be applied shall be approved by the paying agency.\nSensory checks shall be performed where deemed necessary after risk based analysis approved by the paying agency.\nParameter\nMethod\nProtein\nISO 8968 part 1\nFat\nISO 1736\nWater\nISO 5537\nAcidity\nISO 6091\nLactates\nISO 8069\nPhosphatase test\nISO 11816 part 1\nInsolubility index\nISO 8156\nScorched particles\nADPI\nMicro-organisms\nISO 4833-part 1\nButtermilk\nAppendix I\nRennet whey\nAppendix II and III\nAcid whey\nISO 8069 or On-the-spot inspections\nSensory checks\nISO 22935 part 2 and 3\nAppendix I\nSKIMMED MILK POWDER: QUANTITATIVE DETERMINATION OF PHOSPHATIDYLSERINE AND PHOSPHATIDYLETHANOLAMINE\nMethod: reversed-phase HPLC\n1.\nPURPOSE AND FIELD OF APPLICATION\nThe method describes a procedure for the quantitative determination of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in skimmed milk powder (SMP) and is suitable for detecting buttermilk solids in SMP.\n2.\nDEFINITION\nPS + PE content the mass fraction of substance determined using the procedure here specified. The result is expressed as milligrams of phosphatidylethanolamine dipalmitoyl (PEDP) per 100 g powder.\n3.\nPRINCIPLE OF THE METHOD\nExtraction of aminophospholipids by methanol from reconstituted milk powder. Determination of PS and PE as o-phthaldialdehyde (OPA) derivatives by reversed-phase (RP) HPLC and fluorescence detection. Quantification of PS and PE content in the test sample by reference to a standard sample containing a known amount of PEDP.\n4.\nREAGENTS\nAll reagents shall be of recognised analytical grade. Water shall be distilled or water of at least equivalent purity, unless otherwise specified.\n4.1.\nStandard material: PEDP, at least 99 % pure\nNote: Standard material shall be stored at – 18 °C.\n4.2.\nReagents for standard sample and test sample preparation\n4.2.1.\nHPLC-grade methanol\n4.2.2.\nHPLC-grade chloroform\n4.2.3.\nTryptamine-monohydrochloride\n4.3.\nReagents for o-phthaldialdehyde derivatisation\n4.3.1.\nSodium hydroxide, 12 M water solution\n4.3.2.\nBoric acid, 0,4 M water solution adjusted to pH 10,0 with sodium hydroxide (4.3.1)\n4.3.3.\n2-mercaptoethanol\n4.3.4.\no-phthaldialdehyde (OPA)\n4.4.\nHPLC elution solvents\n4.4.1.\nElution solvents shall be prepared using HPLC-grade reagents.\n4.4.2.\nHPLC-grade water\n4.4.3.\nMethanol of tested fluorimetric purity\n4.4.4.\nTetrahydrofuran\n4.4.5.\nSodium dihydrogen phosphate\n4.4.6.\nSodium acetate\n4.4.7.\nAcetic acid.\n5.\nAPPARATUS\n5.1.\nAnalytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg\n5.2.\nBeakers, 25 and 100 ml capacity\n5.3.\nPipettes, capable of delivering 1 and 10 ml\n5.4.\nMagnetic stirrer\n5.5.\nGraduated pipettes, capable of delivering 0,2, 0,5 and 5 ml\n5.6.\nVolumetric flasks, 10, 50 and 100 ml capacity\n5.7.\nSyringes, 20 and 100 μl capacity\n5.8.\nUltrasonic bath\n5.9.\nCentrifuge, capable of operating at 27 000 × g\n5.10.\nGlass vials, about 5 ml capacity\n5.11.\nGraduated cylinder, 25 ml capacity\n5.12.\npH-meter, accurate to 0,1 pH units\n5.13.\nHPLC equipment\n5.13.1.\nGradient pumping system, capable of operating at 1,0 ml/min at 200 bar\n5.13.2.\nAutosampler with derivatisation capability\n5.13.3.\nColumn heater, capable of maintaining the column at 30 °C ± 1 °C\n5.13.4.\nFluorescence detector, capable of operating at 330 nm excitation wavelength and 440 nm emission wavelength\n5.13.5.\nIntegrator or data processing software capable of peak area measurement\n5.13.6.\nA LiChrospher® — 100 column (250 × 4,6 mm) or an equivalent column packed with octadecylsilane (C 18), 5 μm particle size.\n6.\nSAMPLING\nSampling shall be carried out in accordance with ISO Standard 707.\n7.\nPROCEDURE\n7.1.\nPreparation of the internal standard solution\n7.1.1.\nWeigh 30,0 ± 0,1 mg of tryptamine-monohydrochloride (4.2.3) into a 100 ml volumetric flask (5.6) and make up to the mark with methanol (4.2.1)\n7.1.2.\nPipette 1 ml (5.3) of this solution into a 10 ml volumetric flask (5.6) and make up to the mark with methanol (4.2.1) in order to obtain a 0,15 mM tryptamine concentration\n7.2.\nPreparation of the test sample solution\n7.2.1.\nWeigh 1,000 ± 0,001 g of the SMP sample into a 25 ml beaker (5.2). Add 10 ml of distilled water at 40 °C ± 1 °C by a pipette (5.3) and stir with a magnetic stirrer (5.4) for 30 minutes in order to dissolve any lumps\n7.2.2.\nPipette 0,2 ml (5.5) of the reconstituted milk into a 10 ml volumetric flask (5.6), add 100 μl of the 0,15 mM tryptamine solution (7.1) using a syringe (5.7) and make up to the volume with methanol (4.2.1). Mix carefully by inversion and sonicate (5.8) for 15 min\n7.2.3.\nCentrifuge (5.9) at 27 000 g × g for 10 minutes and collect the supernatant in a glass vial (5.10)\nNote: Test sample solution should be stored at 4 °C until the HPLC analysis is performed.\n7.3.\nPreparation of the external standard solution\n7.3.1.\nWeigh 55,4 mg PEDP (4.1) into a 50 ml volumetric flask (5.6) and add about 25 ml of chloroform (4.2.2) using a graduated cylinder (5.11). Heat the stoppered flask to 50 °C ± 1 °C and mix carefully till the PEDP dissolves. Cool the flask to 20 °C, make up to the volume with methanol (4.2.1) and mix by inversion\n7.3.2.\nPipette 1 ml (5.3) of this solution into a 100 ml volumetric flask (5.6) and make up to the volume with methanol (4.2.1). Pipette 1 ml (5.3) of this solution into a 10 ml volumetric flask (5.6), add 100 μl (5.7) of 0,15 mM tryptamine solution (7.1) and make up to the volume with methanol (4.2.1). Mix by inversion\nNote: Reference sample solution should be stored at 4 °C until the HPLC analysis is performed.\n7.4.\nPreparation of the derivatising reagent\nWeigh 25,0 ± 0,1 mg of OPA (4.3.4) into a 10 ml volumetric flask (5.6), add 0,5 ml (5.5) of methanol (4.2.1) and mix carefully to dissolve the OPA. Make up to the mark with boric acid solution (4.3.2) and add 20 μl of 2-mercaptoethanol (4.3.3) by syringe (5.7).\nNote: The derivatising reagent should be stored at 4 °C in a brown glass vial and is stable for one week.\n7.5.\nDetermination by HPLC\n7.5.1.\nElution solvents (4.4)\nSolvent A: Solution of 0,3 mM sodium dihydrogen phosphate and 3 mM sodium acetate solution (adjusted to pH 6,5 ± 0,1 with acetic acid): methanol: tetrahydrofuran = 558:440:2 (v/v/v)\nSolvent B: methanol\n7.5.2.\nSuggested eluting gradient:\nTime (min)\nSolvent A (%)\nSolvent B (%)\nFlow rate (ml/min)\nInitial\n40\n60\n0\n0,1\n40\n60\n0,1\n5,0\n40\n60\n0,1\n6,0\n40\n60\n1,0\n6,5\n40\n60\n1,0\n9,0\n36\n64\n1,0\n10,0\n20\n80\n1,0\n11,5\n16\n84\n1,0\n12,0\n16\n84\n1,0\n16,0\n10\n90\n1,0\n19,0\n0\n100\n1,0\n20,0\n0\n100\n1,0\n21,0\n40\n60\n1,0\n29,0\n40\n60\n1,0\n30,0\n40\n60\n0\nNote: The eluting gradient may require slight modification in order to achieve the resolution shown in figure 1.\nColumn temperature: 30 °C.\n7.5.3.\nInjection volume: 50 μl derivatising reagent and 50 μl sample solution\n7.5.4.\nColumn equilibration\nStarting up the system on a daily basis, flush the column with 100 % solvent B for 15 minutes, then set at A:B = 40:60 and equilibrate at 1 ml/min for 15 minutes. Perform a blank run by injecting methanol (4.2.1).\nNote: Before long-term storage flush the column with methanol: chloroform = 80:20 (v/v) for 30 minutes.\n7.5.5.\nDetermine the PS + PE content in the test sample\n7.5.6.\nPerform the sequence of the chromatographic analyses keeping constant the run-to-run time in order to obtain constant retention times. Inject the external standard solution (7.3) every 5-10 test sample solutions in order to calculate the response factor\nNote: The column shall be cleaned by flushing with 100 % solvent B (7.5.1) for at least 30 minutes every 20-25 runs.\n7.6.\nIntegration mode\n7.6.1.\nPEDP peak\nPEDP is eluted as a single peak. Determine the peak area by valley-to- valley integration.\n7.6.2.\nTryptamine peak\nTryptamine is eluted as a single peak (Figure 1). Determine the peak area by valley-to-valley integration.\n7.6.3.\nPS and PE peaks groups\nUnder the described conditions (Figure 1), PS elutes as two main partially unresolved peaks preceded by a minor peak. PE elutes as three main partially unresolved peaks. Determine the whole area of each peak cluster setting the baseline as reported in Figure 1.\n8.\nCALCULATION AND EXPRESSION OF RESULTS\nPS and PE content in the test sample shall be calculated as follows:\nC = 55,36 × ((A2)/(A1)) × ((T1)/(T2))\nwhere:\nC PS or PE content (mg/100 g powder) in the test sample\nA 1 PEDP peak area of the standard sample solution (7.3)\nA 2 PS or PE peak area of the test sample solution (7.2)\nT 1 Tryptamine peak area of the standard sample solution (7.3)\nT 2 Tryptamine peak area of the test sample solution (7.2).\n9.\nACCURACY OF THE METHOD\nNote: The values for repeatability were calculated according to the IDF International Standard (*).\n9.1.\nRepeatability\nThe relative standard deviation of the repeatability, which expresses the variability of independent analytical results obtained by the same operator using the same apparatus under the same conditions on the same test sample and in a short interval of time, should not exceed 2 % relative. If two determinations are obtained under these conditions, the relative difference between the two results should not be greater than 6 % of the arithmetic mean of the results.\n9.2.\nReproducibility\nIf two determinations are obtained by operators in different laboratories using different apparatus under different conditions for the analysis on the same test sample, the relative difference between the two results should not be greater than 11 % of the arithmetic mean of the results.\n10.\nREFERENCES\n10.1.\nResmini P., Pellegrino L., Hogenboom J.A., Sadini V., Rampilli M., ‘Detection of buttermilk solids in skimmilk powder by HPLC quantification of aminophospholipids’. Sci. Tecn. Latt.-Cas., 39,395 (1988).\nFigure 1\nHPLC pattern of OPA-derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in methanol extract of reconstituted skim-milk powder. Integration mode for the peaks of PS, PE and tryptamine (internal standard) is reported\nAppendix II\nDETECTION OF RENNET WHEY IN SKIMMED MILK POWDER FOR PUBLIC STORAGE BY DETERMINATION OF CASEINOMACROPEPTIDES HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)\n1.\nSCOPE AND FIELD OF APPLICATION\nThis method allows detection of rennet whey in skimmed milk powder intended for public storage by determination of the caseinomacropeptides.\n2.\nREFERENCE\nInternational Standard ISO 707 - Milk and Milk Products - Guidance on sampling.\n3.\nDEFINITION\nThe content of rennet whey solids is defined as the percentage by mass as determined by the caseinomacropeptide content by the procedure described.\n4.\nPRINCIPLE\nReconstitution of the skimmed milk powder, removal of fat and proteins with trichloroacetic acid, followed by centrifugation or filtration;\nDetermination of the quantity of caseinomacropeptides (CMP) in the supernatant by high-performance liquid chromatography (HPLC);\nEvaluation of the result obtained for the samples by reference to standard samples consisting of skimmed milk powder with or without the addition of a known percentage of whey powder.\n5.\nREAGENTS\nAll reagents shall be of recognised analytical grade. The water used shall be distilled water or water of at least equivalent purity.\n5.1.\nTrichloroacetic acid solution\nDissolve 240 g of trichloroacetic acid (CCl3COOH) in water and make up to 1 000 ml. The solution should be clear and colourless.\n5.2.\nEluent solution, pH 6,0\nDissolve 1,74 g of dipotassium hydrogen phosphate (K2HPO4), 12,37 g of potassium dihydrogen phosphate (KH2PO4) and 21,41 g of sodium sulphate (Na2SO4) in about 700 ml of water. Adjust, if necessary, to pH 6,0, using a solution of phosphoric acid or potassium hydroxide.\nMake up to 1 000 ml with water and homogenise.\nNote: The composition of the eluent can be updated to comply with the certificate of the standards or the recommendations of the manufacturer of the column packing material.\nFilter the eluent solution, prior to use, through a membrane filter with a 0,45 μm pore diameter.\n5.3.\nFlushing solvent\nMix one volume acetonitrile (CH3CN) with nine volumes water. Filter the mixture prior to use through a membrane filter with a 0,45 μm pore diameter.\nNote: Any other flushing solvent with a bactericidal effect which does not impair the columns' resolution efficiency may be used.\n5.4.\nStandard samples\n5.4.1.\nSkimmed milk powder meeting the requirements of this Regulation (i.e. [0])\n5.4.2.\nThe same skimmed milk powder adulterated with 5 % (m/m) rennet-type whey powder of standard composition (i.e. [5])\n6.\nAPPARATUS\n6.1.\nAnalytical balance\n6.2.\nOptional centrifuge capable of attaining a centrifugal force of 2 200 g, fitted with stoppered or capped centrifuge tubes of about 50 ml capacity\n6.3.\nMechanical shaker\n6.4.\nMagnetic stirrer\n6.5.\nGlass funnels, diameter about 7 cm\n6.6.\nFilter papers, medium filtration, diameter about 12,5 cm\n6.7.\nGlass filtration equipment with 0,45 μm pore diameter membrane filter\n6.8.\nGraduated pipettes allowing delivery of 10 ml (ISO 648, Class A, or ISO/R 835) or a dispensing system capable of delivering 10,0 ml in two minutes\n6.9.\nDispensing system capable of delivering 20,0 ml water at ca. 50 °C\n6.10.\nThermostatic water bath, set at 25 ± 0,5 °C\n6.11.\nHPLC equipment, consisting of:\n6.11.1. Pump\n6.11.2. Injector, hand or automatic, with a 15 to 30 μl capacity\n6.11.3. Two TSK 2 000-SW columns in series (length 30 cm, internal diameter 0,75 cm) or equivalent columns (e.g. single TSK 2 000-SWxl, single Agilent Technologies Zorbax GF 250) and a precolumn (3 cm × 0,3 cm) packed with I 125 or material of equivalent effectiveness\n6.11.4. Thermostatic column oven, set at 35 ± 1 °C\n6.11.5. Variable wavelength UV detector, permitting measurements at 205 nm with a sensitivity of 0,008 Å\n6.11.6. Integrator capable of valley-to-valley integration\nNote: Working with columns kept at room temperature is possible, but their power of resolution is slightly lower. In that case, the temperature should vary by less than ± 5 °C in any one range of analyses.\n7.\nSAMPLING\n7.1.\nSamples shall be taken in accordance with the procedure laid down in International Standard ISO 707. However, Member States may use another method of sampling provided that it complies with the principles of the abovementioned standard\n7.2.\nStore the sample in conditions which preclude any deterioration or change in composition\n8.\nPROCEDURE\n8.1.\nPreparation of the test sample\nTransfer the milk powder into a container with a capacity of about twice the volume of the powder, fitted with an airtight lid. Close the container immediately. Mix the milk powder well by means of repeated inversion of the container.\n8.2.\nTest portion\nWeight 2,000 ± 0,001 g of test sample into a centrifuge tube (6.2) or a suitable stoppered flask (50 ml).\n8.3.\nRemoval of fat and proteins\n8.3.1.\nAdd 20,0 ml of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes using a mechanical shaker (6.3). Place the tube into the water bath (6.10) and allow to equilibrate to 25 °C\n8.3.2.\nAdd 10,0 ml of the trichloroacetic acid solution (5.1) of ca. 25 °C in two minutes, while stirring vigorously with the aid of the magnetic stirrer (6.4). Place the tube in a water bath (6.10) and leave for 60 minutes\n8.3.3.\nCentrifuge (6.2) for 10 minutes at 2 200 g, or filter through paper (6.6), discarding the first 5 ml of filtrate\n8.4.\nChromatographic determination\n8.4.1.\nInject 15 to 30 μl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus (6.11) operating at a flow rate of 1,0 ml of eluent solution (5.2) per minute\nNote 1. Another flow rate may be used, dependent of the internal diameter of the columns used or the instructions of the manufacturer of the column.\nNote 2. Rinse the columns with water during each interruption. Never leave the eluent solution in them (5.2).\nPrior to any interruption of more than 24 hours, rinse the columns with water then wash them with solution (5.3) for at least three hours at a flow rate of 0,2 ml per minute.\n8.4.2.\nThe results of chromatographic analysis of the test sample [E] are obtained in the form of chromatogram in which each peak is identified by its retention time RT as follows:\nPeak II:\nThe second peak of the chromatogram having an RT of about 12,5 minutes.\nPeak III:\nThe third peak of the chromatogram, corresponding to the CMP, having an RT of 15,5 minutes.\nThe choice of the column(s) can affect the retention times of the individual peaks considerably.\nThe integrator (6.11.6) automatically calculates the area A of each peak:\nAII:\narea of peak II,\nAIII:\narea of peak III,\nIt is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order to detect any abnormalities due either to malfunctioning of the apparatus or the columns, or to the origin and nature of the sample analysed.\nIf in doubt, repeat the analysis.\n8.5.\nCalibration\n8.5.1.\nApply exactly the procedure described from point 8.2 to point 8.4.2 to the standard samples (5.4)\nUse freshly prepared solutions, because CMP degrade in an 8 % trichloroacetic environment. The loss is estimated at 0,2 % per hour at 30 °C.\n8.5.2.\nPrior to chromatographic determination of the samples, condition the columns by repeatedly injecting the standard sample (5.4.2) in solution (8.5.1) until the area and retention time of the peak corresponding to the CMP are constant\n8.5.3.\nDetermine the response factors R by injecting the same volume of filtrates (8.5.1) as used for the samples\n9.\nEXPRESSION OF RESULTS\n9.1.\nMethod of calculation and formulae\n9.1.1.\nCalculation of the response factors R:\nPeak II:\nRII = 100/(AII[0])\nwhere:\nRII the response factors of peaks II,\nAII [0] the areas of peaks II of the standard sample [0] obtained in 8.5.3.\nPeak III:\nRIII = W/(AIII[5] – AIII[0])\nwhere:\nRIII the response factor of peak III,\nAIII [0] and AIII [5] the areas of peak III in standard samples [0] and [5] respectively obtained in 8.5.3,\nW the quantity of whey in standard sample [5], i.e. 5.\n9.1.2.\nCalculation of the relative area of the peaks in the sample [E]\nSII[E] = RII × AII[E]\nSIII[E] = RIII × AIII[E]\nSIV[E] = RIV × AIV[E]\nwhere:\nSII [E], SIII [E], SIV [E] the relative areas of peaks II, III and IV respectively in the sample [E],\nAII [E], AIII [E] the areas of peaks II and III respectively in the sample [E] obtained in 8.4.2,\nRII, RIII the response factors calculated in 9.1.1.\n9.1.3.\nCalculation of the relative retention time of peak III in sample [E]:\nRRTIII[E] = (RTIII[E])/(RTIII[5])\nwhere:\nRRTIII [E] the relative retention time of peak III in sample [E],\nRTIII [E] the retention time of peak III in sample [E] obtained in 8.4.2,\nRTIII [5] the retention time of peak III in control sample [5] obtained in 8.5.3.\n9.1.4.\nExperiments have shown that there is a linear relation between the relative retention time of peak III, i.e. RRTIII [E] and the percentage of whey powder added up to 10 %\nThe RRTIII [E] is < 1,000 when the whey content is > 5 %;\nThe RRTIII [E] is ≥ 1,000 when the whey content is ≤ 5 %.\nThe uncertainty allowed for the values of RRTIII is ± 0,002.\nNormally the value of RRTIII [0] deviates little from 1,034. Depending on the condition of the columns, the value may approach 1,000, but it shall always be greater.\n9.2.\nCalculation of the percentage of rennet whey powder in the sample:\nW = SIII[E] – [1, 3 + (SIII[0] – 0,9)]\nwhere:\nW the percentage m/m of rennet whey in the sample [E];\nSIII [E] the relative area of peak III of test sample [E] obtained as in 9.1.2;\n1,3 represents the relative average area of peak III expressed in grams of rennet whey per 100 g determined in non-adulterated skimmed milk powder of various origins. This figure was obtained experimentally;\nSIII [0] represents the relative area of peak III which is equal to RIII × AIII [0]. These values are obtained in 9.1.1 and 8.5.3 respectively;\n(SIII [0] – 0,9) represents the correction to be made to the relative average area 1,3 when SIII [0] is not equal to 0,9. Experimentally the relative average area of peak III of the control sample [0] is 0,9.\n9.3.\nAccuracy of the procedure\n9.3.1.\nRepeatability\nThe difference between the results of two determinations carried out simultaneously or in rapid succession by the same analyst using the same apparatus on identical test material shall not exceed 0,2 % m/m.\n9.3.2.\nReproducibility\nThe difference between two single and independent results, obtained in two different laboratories on identical test material shall not exceed 0,4 % m/m.\n9.4.\nInterpretation\n9.4.1.\nAssume the absence of whey if the relative area of peak III, SIII [E] expressed in grams of rennet whey per 100 g of the product is ≤ 2,0 + (SIII[0] – 0,9)\nwhere\n2,0\nis the maximum value allowed for the relative area of peak III taking into account the relative average area of peak III, i.e. 1,3, the uncertainty due to variations in the composition of skimmed milk powder and the reproducibility of the method (9.3.2),\n(SIII [0] – 0,9)\nis the correction to be made when the area SIII [0] is different from 0,9 (see point 9.2)\n9.4.2.\nIf the relative area of peak III, SIII [E] is > 2,0 + (SIII[0] – 0,9) and the relative area of peak II, SII [E] ≤ 160, determine the rennet whey content as indicated in point 9.2.\n9.4.3.\nIf the relative area of peak III, SIII [E] is > 2,0 + (SIII[0] – 0,9) and the relative area of peak II, SII [E] ≤ 160, determine the total protein content (P %); then examine graphs 1 and 2.\n9.4.3.1.\nThe data obtained after analysis of samples of unadulterated skimmed milk powders with a high total protein content have been assembled in graphs 1 and 2.\nThe continuous line represents the linear regression, the coefficients of which are calculated by the least squares method.\nThe dashed straight line fixes the upper limit of the relative area of peak III with a probability of not being exceeded in 90 % of cases.\nThe equations for the dashed straight lines of graphs 1 and 2 are:\nSIII = 0,376 P % – 10,7\n(graph 1),\nSIII = 0,0123 SII [E] + 0,93\n(graph 2),\nrespectively where:\nSIII is the relative area of peak III calculated either according to total protein content or according to the relative area of peak SII [E],\nP % is the total protein content expressed as a percentage, by weight,\nSII [E] is the relative area of sample calculated in point 9.1.2.\nThese equations are equivalent to the figure of 1,3 mentioned in point 9.2.\nThe discrepancy (T1 and T2) between the relative area SIII [E] found and the relative area SIII is given by means of the following: T1 = SIII[E] – [(0,376 P% – 10,7) + (SIII[0] – 0,9)]T2 = SIII[E] – [(0,0123 SII[E] + 0,93) + (SIII[0] – 0,9)]\n9.4.3.2.\nIf T1 and/or T2\nare zero or less, the presence of rennet whey cannot be determined.\nIf T1 and T2\nexceed zero, rennet whey is present.\nThe rennet whey content is calculated according to the following formula: W = T2 + 0,91\nwhere:\n0,91 is the distance on the vertical axis between the continuous and dotted straight lines.\nAppendix III\nDETERMINING RENNET WHEY SOLIDS IN SKIMMED MILK POWDER\n1.\nPURPOSE: DETECTING THE ADDITION OF RENNET WHEY SOLIDS TO SKIMMED MILK POWDER\n2.\nREFERENCES: INTERNATIONAL STANDARD ISO 707\n3.\nDEFINITION\nThe content of rennet whey solids is defined as the percentage by mass as determined by caseinomacropeptide content by the procedure described.\n4.\nPRINCIPLE\nSamples are analysed for caseinomacropeptide A by a reversed-phase high-performance liquid chromatography procedure (HPLC procedure). Evaluation of the result is obtained by reference to standard samples consisting of skimmed milk powder with and without a known percentage of whey powder. Results higher than 1 % (m/m) show that rennet whey solids are present.\n5.\nREAGENTS\nAll reagents shall be of recognised analytical grade. The water used shall be distilled water or water of at least equivalent purity. Acetonitrile should be of spectroscopic or HPLC quality.\n5.1.\nTrichloroacetic acid solution\nDissolve 240 g of trichloroacetic acid (CCl3COOH) in water and make up to 1 000 ml. The solution should be clear and colourless.\n5.2.\nEluents A and B\nEluent A: 150 ml of acetonitrile (CH3CN), 20 ml of isopropanol (CH3CHOHCH3), and 1,00 ml of trifluoroacetic acid (TFA, CF3COOH) are placed in a 1 000 ml volumetric flask. Make up to 1 000 ml with water.\nEluent B: 550 ml of acetonitrile, 20 ml of isopropanol and 1,00 ml of TFA are placed in a 1 000 ml volumetric flask. Make up to 1 000 ml with water. Filter the eluent solution, prior to use, through a membrane filter with a 0,45 μm pore diameter.\n5.3.\nConservation of the column\nAfter the analyses the column is flushed with eluent B (via a gradient) and subsequently flushed with acetonitrile (via a gradient for 30 minutes). The column is stored in acetonitrile.\n5.4.\nStandard samples\n5.4.1.\nSkimmed milk powder meeting the requirements for public storage (i.e. [0]).\n5.4.2.\nThe same skimmed milk powder adulterated with 5 % (m/m) rennet-type whey powder of standard composition (i.e. [5]).\n5.4.3.\nThe same skimmed milk powder adulterated with 50 % (m/m) rennet-type whey powder of standard composition (i.e. [50])\n6.\nAPPARATUS\n6.1.\nAnalytical balance\n6.2.\nOptional centrifuge capable of attaining a centrifugal force of 2 200 g, fitted with stoppered or capped centrifuge tubes of about 50 ml capacity\n6.3.\nMechanical shaker\n6.4.\nMagnetic stirrer\n6.5.\nGlass funnels, diameter about 7 cm\n6.6.\nFilter papers, medium filtration, diameter about 12,5 cm\n6.7.\nGlass filtration equipment with 0,45 μm pore diameter membrane filter\n6.8.\nGraduated pipettes, allowing delivery of 10 ml (ISO 648, Class A, or ISO/R 835), or a dispensing system capable of delivering 10,0 ml in two minutes\n6.9.\nDispensing system capable of delivering 20,0 ml water at ca. 50 °C\n6.10.\nThermostatic water bath, set at 25 ± 0,5 °C\n6.11.\nHPLC equipment, consisting of:\n6.11.1. Binary gradient pumping system\n6.11.2. Injector, hand or automatic, with a 100 μl capacity\n6.11.3. Agilent Technologies Zorbax 300 SB-C3 column (length 25 cm, 0,46 cm internal diameter) or an equivalent wide-pore silica based reversed-phase column\n6.11.4. Thermostatic column oven, set at 35 ± 1 °C\n6.11.5. Variable wavelength UV detector, permitting measurements at 210 nm (if necessary, a higher wavelength up to 220 nm may be used) with a sensitivity of 0,02 Å\n6.11.6. Integrator capable of setting the integration to common baseline or valley-to-valley\nNote: Operation of the column at room temperature is possible, provided that the room temperature does not fluctuate more than 1 °C, otherwise too much variation in the retention time of CMPA takes place.\n7.\nSAMPLING\n7.1.\nSamples shall be taken in accordance with the procedure laid down in International Standard ISO 707. However, Member States may use another method of sampling provided that it complies with the principles of the abovementioned standard\n7.2.\nStore the sample in conditions which preclude any deterioration or change in composition.\n8.\nPROCEDURE\n8.1.\nPreparation of the test sample\nTransfer the milk powder into a container with a capacity of about twice the volume of the powder, fitted with an airtight lid. Close the container immediately. Mix the milk powder well by means of repeated inversion of the container.\n8.2.\nTest portion\nWeigh 2,00 ± 0,001 g of test sample into a centrifuge tube (6.2) or suitable stoppered flask (50 ml).\nNote: In the case of mixtures, weigh such an amount of the test sample that the defatted sample portion corresponds to 2,00 g.\n8.3.\nRemoval of fat and proteins\n8.3.1.\nAdd 20,0 ml of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes using a mechanical shaker (6.3). Place the tube into the water bath (6.10) and allow to equilibrate to 25 °C\n8.3.2.\nAdd 10,0 ml of the trichloroacetic acid solution of ca. 25 °C (5.1) constantly over two minutes, while stirring vigorously with the aid of the magnetic stirrer (6.4). Place the tube in a water bath (6.10) and leave for 60 minutes\n8.3.3.\nCentrifuge (6.2) 2 200 g for 10 minutes, or filter through paper (6.6), discarding the first 5 ml of filtrate\n8.4.\nChromatographic determination\n8.4.1.\nThe reversed-phase HPLC method excludes the possibility false-positive results due to the presence of acid buttermilk powder.\n8.4.2.\nBefore the reversed phase HPLC-analysis is carried out, the gradient conditions should be optimised. A retention time of 26 ± 2 minutes for CMPA is optimal for gradient systems having a dead volume of about 6 ml (volume from the point where the solvents come together to the volume of the injector loop, inclusive). Gradient systems having a lower dead volume (e.g. 2 ml) should use 22 minutes as an optimal retention time\nTake solutions of the standard samples (5.4) without and with 50 % rennet whey.\nInject 100 μl of supernatant or filtrate (8.3.3) into the HPLC apparatus operating at the scouting gradient conditions given in Table 1.\nTable 1\nScouting gradient conditions for optimisation of the chromatography\nTime(min)\nFlow(ml/min)\n% A\n% B\nCurve\nInitial\n1,0\n90\n10\n*\n27\n1,0\n60\n40\nlinear\n32\n1,0\n10\n90\nlinear\n37\n1,0\n10\n90\nlinear\n42\n1,0\n90\n10\nlinear\nComparison of the two chromatograms should reveal the location of the peak of CMPΑ.\nUsing the formula given below, the initial solvent composition to be used for the normal gradient (see 8.4.3) can be calculated % B = 10 – 2,5 + (13,5 + (RTcmpA – 26) / 6) * 30 / 27 % B = 7,5 + (13,5 + (RTcmpA – 26) / 6) * 1,11\nWhere:\nRTcmpA retention time of CMPΑ in the scouting gradient\n10 the initial % B of the scouting gradient\n2,5 % B at midpoint minus % B at initial in the normal gradient\n13,5 midpoint time of the scouting gradient\n26 required retention time of CMPΑ\n6 ratio of slopes of the scouting and normal gradient\n30 % B at initial minus % B at 27 minutes in the scouting gradient\n27 run-time of the scouting gradient.\n8.4.3.\nTake solutions of the test samples\nInject 100 μl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus operating at a flow rate of 1,0 ml of eluent solution (5.2) per minute.\nThe composition of the eluent of the start of the analysis is obtained from 8.4.2. It is normally close to A:B = 76:24 (5.2). Immediately after the injection a linear gradient is started, which results in a 5 % higher percentage of B after 27 minutes. Subsequently a linear gradient is started, which brings the eluent composition to 90 % B in five minutes. This composition is maintained for five minutes, after which the composition is changed, via a linear gradient in five minutes to the initial composition. Depending on the internal volume of the pumping system, the next injection can be made 15 minutes after reaching the initial conditions.\nNote 1. The retention time of the CMPA should be 26 ± 2 minutes. This can be achieved by varying the initial and end conditions of the first gradient. However, the difference in the % B for the initial and end conditions of the first gradient shall remain 5 % B.\nNote 2. The eluents should be degassed sufficiently and should also remain degassed. This is essential for proper functioning of the gradient pumping system. The standard deviation for the retention time of the CMPA peak should be smaller than 0,1 minutes (n = 10).\nNote 3. Every five samples the reference sample [5] should be injected and used to calculate a new response factor R. (9.1.1).\n8.4.4.\nThe results of the chromatographic analysis of the test sample (E) are obtained in the form of a chromatogram in which the CMPA peak is identified by its retention time of about 26 minutes\nThe integrator (6.11.6) automatically calculates the peak height H of the CMPA peak. The baseline location should be checked in every chromatogram. The analysis or the integration should be repeated if the baseline was incorrectly located.\nNote: If the CMPA peak is sufficiently separated from other peaks valley-to-valley baseline allocation should be used, otherwise use dropping perpendiculars to a common baseline, which should have starting point close to the CMPA peak (thus not at t = 0 min!).Use for the standard and the samples the same type integration type and check in case of common baseline its consistency for the samples and the standard.\nIt is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order to detect any abnormalities due either to malfunctioning of the apparatus or the column, or to the origin and nature of the sample analysed. If in doubt, repeat the analysis.\n8.5.\nCalibration\n8.5.1.\nApply exactly the procedure described from point 8.2 to point 8.4.4 to the standard samples (5.4.1 to 5.4.2). Use freshly prepared solutions, because CMP degrades in an 8 % trichloroacetic acid environment at room temperature. At 4 °C the solution remains stable for 24 hours. In the case of long series of analyses the use of a cooled sample tray in the automatic injector is desirable\nNote: 8.4.2. may be omitted if the % B at initial conditions is known from previous analyses.\nThe chromatogram of the reference sample [5] should be analogous to Figure. 1. In this figure the CMPA peak is preceded by two small peaks. It is essential to obtain a similar separation.\n8.5.2.\nPrior to chromatographic determination of the samples inject 100 μl of the standard sample without rennet whey [0] (5.4.1)\nThe chromatogram should not show a peak at the retention time of the CMPA peak.\n8.5.3.\nDetermine the response factors R by injecting the same volume of filtrate (8.5.1) as used for the samples.\n9.\nEXPRESSION OF RESULTS\n9.1.\nMethod of calculation and formulae\n9.1.1.\nCalculation of the response factor R:\nCMPA peak: R = W/H\nWhere:\nR the response factor of the CMPA peak\nH the height of the CMPA peak\nW the quantity of whey in the standard sample [5].\n9.2.\nCalculation of the percentage of rennet whey powder in the sample\nW(E) = R × H(E)\nWhere:\nW(E) the percentage (m/m) of rennet whey in the sample (E).\nR the response factor of the CMPA peak (9.1.1)\nH(E) the height of the CMPA peak of the sample (E)\nIf W(E) is greater than 1 % and the difference between the retention time and that of the standard sample [5] is smaller than 0,2 minutes then rennet whey solids are present.\n9.3.\nAccuracy of the procedure\n9.3.1.\nRepeatability\nThe difference between the results of two determinations carried out simultaneously or in rapid succession by the same analyst using the same apparatus on identical test material shall not exceed 0,2 % m/m.\n9.3.2.\nReproducibility\nNot determined.\n9.3.3.\nLinearity\nFrom 0 to 16 % of rennet whey a linear relationship should be obtained with a coefficient of correlation > 0,99.\n9.4.\nInterpretation\nThe 1 % limit includes the uncertainty due to reproducibility.\nFigure 1\nNi—4.6 standard\n(*)\nInternational IDF Standard 135B/1991. Milk and milk products. Precision characteristics of analytical methods. Outline of collaborative study procedure.\n(3) the following Annexes are added:\n‘ANNEX VI\nMethods of analysis of butter under private storage\nThe method to be applied shall be approved by the paying agency.\nParameter\nMethod\nFat\nISO 17189 or ISO 3727 part 3\nWater\nISO 3727 part 1\nNon Fat Solids (excluding salt)\nISO 3727 part 2\nSalt\nISO 15648\nANNEX VII\nMethods of analysis of skimmed milk powder under private storage\nParameter\nMethod\nFat\nISO 1736\nProtein\nISO 8968 part 1\nWater\nISO 5537\nANNEX VIII\nMethods of analysis of cheeses under private storage\n1.\nThe method of analysis laid down in the Appendix shall be used to ensure that cheese made exclusively from ewe's milk, goat's milk or buffalo milk or from a mixture of ewe's milk, goat's milk and buffalo milk does not contain cow's milk casein.\nCow's milk casein is considered to be present if the cow's milk casein content of the analysed sample is equal to or higher than the content of the reference sample containing 1 % cow's milk as laid down in the Appendix.\n2.\nMethods for detecting cow's milk casein in cheeses referred to in paragraph 1 may be used provided that:\n(a) the detection limit is maximum 0,5 % and\n(b) there are no false-positive results and\n(c) cow's milk casein is detectable with the required sensitivity even after long ripening periods, as may occur in usual commercial conditions.\nIf any of the above mentioned requirements is not met, the methods laid down in the Appendix shall be used.\nAppendix\nMETHOD FOR THE DETECTION OF COW'S MILK AND CASEINATE IN CHEESES FROM EWE'S MILK, GOAT'S MILK OR BUFFALO MILK OR MIXTURES OF EWE'S MILK, GOAT'S MILK AND BUFFALO MILK\n1.\nSCOPE\nDetection of cow's milk and caseinate in cheeses made from ewe's milk, goat's milk, buffalo milk or mixtures of ewe's, goat's and buffalo milk by isoelectric focusing of γ-caseins after plasminolysis.\n2.\nFIELD OF APPLICATION\nThe method is suitable for sensitive and specific detection of native and heat-treated cow's milk and caseinate in fresh and ripened cheeses made from ewe's milk, goat's milk, buffalo milk or mixtures of ewe's, goat's and buffalo milk. It is not suitable for the detection of milk and cheese adulteration by heat-treated bovine whey protein concentrates.\n3.\nPRINCIPLE OF THE METHOD\n3.1.\nIsolation of caseins from cheese and the reference standards\n3.2.\nDissolving of the isolated caseins and submitting to plasmin (EC.3.4.21.7) cleavage\n3.3.\nIsoelectric focusing of plasmin-treated caseins in the presence of urea and staining of proteins\n3.4.\nEvaluation of stained γ3 and γ2 -casein patterns (evidence of cow's milk) by comparison of the pattern obtained from the sample with those obtained in the same gel from the reference standards containing 0 % and 1 % cow's milk.\n4.\nREAGENTS\nUnless otherwise indicated, analytical grade chemicals shall be used. Water shall be double-distilled or of equivalent purity.\nNote: The following details apply to laboratory prepared polyacrylamide gels containing urea, of dimensions 265 × 125 × 0,25 mm. Where other sizes and types of gel are used, the separation conditions may have to be adjusted.\nIsoelectric focusing\n4.1.\nReagents for production of the urea containing polyacrylamide gels\n4.1.1.\nStock gel solution\nDissolve:\n4,85 g acrylamide\n0,15 g N, N′-methylene-bis-acrylamide (BIS)\n48,05 g urea\n15,00 g glycerol (87 % w/w),\nin water and make up to 100 ml and store in a brown glass bottle in the refrigerator.\nNote: A commercially available pre-blended acrylamide/BIS solution may be used in preference to the quoted fixed weights of the neurotoxic acrylamides. Where such a solution contains 30 % w/v acrylamide and 0,8 % w/v BIS, a volume of 16,2 ml shall be used for the formulation instead of the fixed weights. The shelf life of the stock solution is a maximum of 10 days; if its conductivity is more than 5 μS, de-ionize by stirring with 2 g Amberlite MB-3 for 30 minutes, then filter through a 0,45 μm membrane.\n4.1.2.\nGel solution\nPrepare a gel solution by mixing additives and ampholytes (*) with the stock gel solution (see 4.1.1).\n9,0 ml stock solution\n24 mg β-alanine\n500 μl ampholyte pH 3,5-9,5\n250 μl ampholyte pH 5-7\n250 μl ampholyte pH 6-8\nMix the gel solution and de-gas for two to three minutes in an ultrasonic bath or in vacuum.\nNote: Prepare the gel solution immediately prior to pouring it (see 6.2).\n4.1.3.\nCatalyst solutions\n4.1.3.1.\nN, N, N′ N′ — tetramethylethylenediamine (Temed)\n4.1.3.2.\n40 % w/v ammonium persulphate (PER):\nDissolve 800 mg PER in water and make up to 2 ml.\nNote: Always use freshly prepared PER solution.\n4.2.\nContact fluid\nKerosene or liquid paraffin\n4.3.\nAnode solution\nDissolve 5,77 g phosphoric acid (85 % w/w) in water and dilute to 100 ml.\n4.4.\nCathode solution\nDissolve 2,00 g sodium hydroxide in water and dilute to 100 ml with water.\nSample preparation\n4.5.\nReagents for protein isolation\n4.5.1.\nDilute acetic acid (25,0 ml of glacial acetic acid made up to 100 ml with water)\n4.5.2.\nDichloromethane\n4.5.3.\nAcetone\n4.6.\nProtein dissolving buffer\nDissolve\n5,75 g glycerol (87 % w/w)\n24,03 g urea\n250 mg dithiothreitol,\nin water and make up to 50 ml\nNote: Store in a refrigerator, maximum shelf-life one week.\n4.7.\nReagents for plasmin cleavage of caseins\n4.7.1.\nAmmonium carbonate buffer\nTitrate a 0,2 mol/l ammonium hydrogencarbonate solution (1,58 g/100 ml water) containing 0,05 mol/l ethylenediaminetetraacetic acid (EDTA, 1,46 g/100 ml with a 0,2 mol/l ammonium carbonate solution (1,92 g/100 ml water) containing 0,05 mol/l EDTA to pH 8.\n4.7.2.\nBovine plasmin (EC. 3.4.21.7), activity at least 5 U/ml\n4.7.3.\nε-Aminocaproic acid solution for enzyme inhibition\nDissolve 2,624 g ε-aminocaproic acid (6 amino-n-hexanoic acid) in 100 ml of 40 % (v/v) ethanol.\n4.8.\nStandards\n4.8.1.\nCertified reference standards of a mixture of renneted ewe's and goat's skimmed milk containing 0 % and 1 % of cow's milk are available from the Commission's Institute for Reference Materials and Measurements, B-2440 Geel, Belgium\n4.8.2.\nPreparation of laboratory interim-standards of buffalo's renneted milk containing 0 % and 1 % of cow's milk\nSkimmed milk is prepared by centrifuging of either buffalo or bovine raw bulk milk at 37 °C at 2 500 g for 20 minutes. After cooling the tube and contents rapidly to 6 to 8 °C, the upper fat layer is removed completely. For the preparation of the 1 % standard add 5,00 ml of bovine skimmed milk to a 495 ml of buffalo's skimmed milk in a 1 l beaker, adjust the pH to 6,4 by the addition of dilute lactic acid (10 % w/v). Adjust the temperature to 35 °C and add 100 μl of calf rennet (rennet activity 1: 10 000, c. 3 000 U/ml), stir for 1 minute and then leave the beaker covered with an aluminium foil at 35 °C for one hour to allow formation of the curd. After the curd has formed, the whole renneted milk is freeze-dried without prior homogenization or draining of the whey. After freeze-drying it is finely ground to produce a homogeneous powder. For the preparation of the 0 % standard, carry out the same procedure using genuine buffalo skimmed milk. The standards shall be stored at – 20 °C.\nNote: It is advisable to check the purity of the buffalo milk by isoelectric focusing of the plasmin-treated caseins before preparation of the standards.\nReagents for protein staining\n4.9.\nFixative\nDissolve 150 g trichloroacetic acid in water and make up to 1 000 ml.\n4.10.\nDestaining solution\nDilute 500 ml methanol and 200 ml glacial acetic acid to 2 000 ml with distilled water.\nNote: Prepare the destaining solution fresh every day; it can be prepared by mixing equal volumes of stock solutions of 50 % (v/v) methanol and 20 % (v/v) glacial acetic acid.\n4.11.\nStaining solutions\n4.11.1.\nStaining solution (stock solution 1)\nDissolve 3,0 g Coomassie Brilliant Blue G-250 (C.I. 42655) in 1 000 ml 90 % (v/v) methanol using a magnetic stirrer (approximately 45 minutes), filter through two medium-speed folded filters.\n4.11.2.\nStaining solution (stock solution 2)\nDissolve 5,0 g copper sulphate pentahydrate in 1 000 ml 20 % (v/v) acetic acid.\n4.11.3.\nStaining solution (working solution)\nMix together 125 ml of each of the stock solutions (4.11.1, 4.11.2) immediately prior to staining.\nNote: The staining solution should be prepared on the day that it is used.\n5.\nEQUIPMENT\n5.1.\nGlass plates (265 × 125 × 4 mm); rubber roller (width 15 cm); levelling table\n5.2.\nGel carrier sheet (265 × 125 mm)\n5.3.\nCovering sheet (280 × 125 mm). Stick on strip of adhesive tape (280 × 6 × 0,25 mm) to each long edge (see Figure 1)\n5.4.\nElectrofocusing chamber with cooling plate (e.g. 265 × 125 mm) and suitable power supply (≥ 2,5 kV) or automatic electrophoresis device\n5.5.\nCirculation cryostat, thermostatically controlled at 12 ± 0,5 °C\n5.6.\nCentrifuge, adjustable to 3 000 g\n5.7.\nElectrode strips (≥ 265 mm long)\n5.8.\nPlastic dropping bottles for the anode and cathode solutions\n5.9.\nSample applicators (10 × 5 mm, viscose or low protein-adsorption filter paper)\n5.10.\nStainless steel or glass staining and destaining dishes (e.g. 280 × 150 mm instrument trays)\n5.12.\nAdjustable rod homogenizer (10 mm shaft diameter), rpm range 8 000 to 20 000\n5.13.\nMagnetic stirrer\n5.14.\nUltrasonic bath\n5.15.\nFilm welder\n5.16.\n25 μl micropipettes\n5.17.\nVacuum concentrator or freeze-dryer\n5.18.\nThermostatically controlled water bath adjustable to 35 and 40 ± 1 °C with shaker\n5.19.\nDensitometer equipment reading at λ = 634 nm\n6.\nPROCEDURE\n6.1.\nSample preparation\n6.1.1.\nIsolation of caseins\nWeigh the amount equivalent to 5 g dry mass of cheese or the reference standards into a 100 ml centrifuge tube, add 60 ml distilled water and homogenize with a rod homogenizer (8 000 to 10 000 rpm). Adjust to pH 4,6 with dil. acetic acid (4.5.1) and centrifuge (5 minutes, 3 000 g). Decant the fat and whey, homogenize the residue at 20 000 rpm in 40 ml distilled water adjusted to pH 4,5 with dil. acetic acid (4.5.1), add 20 ml dichloromethane (4.5.2), homogenize again and centrifuge (5 minutes, 3 000 g). Remove the casein layer that lies between the aqueous and organic phases (see Figure 2) with a spatula and decant off both phases. Rehomogenise the casein in 40 ml distilled water (see above) and 20 ml dichloromethane (4.5.2) and centrifuge. Repeat this procedure until both extraction phases are colourless (two to three times). Homogenize the protein residue with 50 ml acetone (4.5.3) and filter through a medium-speed folded filter paper. Wash the residue on the filter with two separate 25 ml portions of acetone each time and allow to dry in the air or a stream of nitrogen, then pulverize finely in a mortar.\nNote: Dry casein isolates should be kept at –20 °C.\n6.1.2.\nPlasmin cleavage of β-caseins to intensify γ-caseins\nDisperse 25 mg of isolated caseins (6.1.1) in 0,5 ml ammonium carbonate buffer (4.7.1) and homogenize for 20 minutes by e.g. using ultrasonic treatment. Heat to 40 °C and add 10 μl plasmin (4.7.2), mix and incubate for one hour at 40 °C with continuous shaking. To inhibit the enzyme add 20 μl ε-aminoproic acid solution (4.7.3), then add 200 mg of solid urea and 2 mg of dithiothreitol.\nNote: To obtain more symmetry in the focused casein bands it is advisable to freeze-dry the solution after adding the ε-aminocaproic acid and then dissolving the residues in 0,5 ml protein dissolving buffer (4.6).\n6.2.\nPreparation of the urea containing polyacrylamide gels\nWith the aid of a few drops of water roll the gel carrier sheet (5.2) onto a glass plate (5.1), removing any extraneous water with paper towel or tissue. Roll the cover sheet (5.3) with spacers (0,25 mm) onto another glass plate in the same way. Lay the plate horizontally on a levelling table.\nAdd 10 μl Temed (4.1.3.1) to the prepared and de-aerated gel solution (4.1.2), stir and add 10 μl PER-solution (4.1.3.2), mix thoroughly and immediately pour out evenly onto the centre of the cover sheet. Place one edge of the gel carrier plate (sheet side down) on the cover sheet plate and lower it slowly so that a gel film forms between the sheets and spreads out regularly and free of bubbles (Figure 3). Carefully lower the gel carrier plate completely using a thin spatula and place three more glass plates on top of it to act as weights. After polymerization is complete (about 60 minutes) remove the gel polymerized onto the gel carrier sheet along with the cover sheet by tipping the glass plates. Clean the reverse of the carrier sheet carefully to remove gel residues and urea. Weld the gel sandwich into a film tube and store in a refrigerator (maximum six weeks).\nNote: The cover sheet with the spacers can be re-used. The polyacrylamide gel can be cut to smaller sizes, recommended when there are few samples or if an automatic electrophoresis device is used (two gels, size 4,5 × 5 cm).\n6.3.\nlsoelectric focusing\nSet the cooling thermostat to 12 °C. Wipe off the reverse of the gel carrier sheet with kerosene, then drip a few drops of kerosene (4.2) onto the centre of the cooling block. Then roll the gel sandwich, carrier side down, onto it, taking care to avoid bubbles. Wipe off any excess kerosene and remove the cover sheet. Soak the electrode strips with the electrode solutions (4.3, 4.4), cut to gel length and place in the positions provided (distance of electrodes 9,5 cm).\nConditions for isoelectric focusing:\n6.3.1.\nGel size 265 × 125 × 0,25 mm\nSample application: After pre-focusing (step 1), pipette 18 μl of the sample and standard solutions onto the sample applicators (10 × 5 mm), place them on the gel at 1 mm intervals from each other and 5 mm longitudinally from the anode and press lightly. Carry out focusing using the above conditions, carefully removing the sample applicators after the 60 minutes of sample focusing.\nStep\nTime(min.)\nVoltage(V)\nCurrent(mA)\nPower(W)\nVolt-hours(Vh)\n1.\nPre-focusing\n30\nmaximum\n2 500\nmaximum\n15\nconstant 4\nc. 300\n2.\nSample focusing\n60\nmaximum\n2 500\nmaximum\n15\nconstant 4\nc. 1 000\n3.\nFinal focusing\n60\nmaximum\n2 500\nmaximum\n5\nmaximum\n20\nc. 3 000\n40\nmaximum\n2 500\nmaximum\n6\nmaximum\n20\nc. 3 000\n30\nmaximum\n2 500\nmaximum\n7\nmaximum\n25\nc. 3 000\nNote: If thickness or width of the gels are changed, the values for current and power have to be suitably adjusted (e.g. double the values for electric current and power if a 265 × 125 × 0,5 mm gel is used).\n6.3.2.\nExample of a voltage programme for an automatic electrophoresis device (2 gels of 5,0 × 4,5 cm), electrodes without strips applied directly to the gel\nStep\nVoltage\nCurrent\nPower\nTemp.\nVolt-hours\n1.\nPre-focusing\n1 000 V\n10,0 mA\n3,5 W\n8 °C\n85 Vh\n2.\nSample focusing\n250 V\n5,0 mA\n2,5 W\n8 °C\n30 Vh\n3.\nFocusing\n1 200 V\n10,0 mA\n3,5 W\n8 °C\n80 Vh\n4.\nFocusing\n1 500 V\n5,0 mA\n7,0 W\n8 °C\n570 Vh\nPlace sample applicator in step 2 at 0 Vh.\nRemove sample applicator in step 2 at 30 Vh.\n6.4.\nProtein staining\n6.4.1.\nProtein fixation\nRemove the electrode strips immediately after turning off the power and put the gel immediately into a staining/destaining dish filled with 200 ml fixative (4.9); leave for 15 minutes, shaking continuously.\n6.4.2.\nWashing and staining the gel plate\nThoroughly drain off the fixative and wash the gel plate twice for 30 seconds each time with 100 ml destaining solution (4.10). Pour off the destaining solution and fill the dish with 250 ml staining solution (4.11.3); allow to stain for 45 minutes with gentle shaking.\n6.4.3.\nDestaining the gel plate\nPour off the staining solution, wash the gel plate twice using a 100 ml destaining solution (4.10) each time, then shake with 200 ml destaining solution for 15 minutes and repeat the destaining step at least two or three times until the background is clear and uncoloured. Then rinse the gel plate with distilled water (2 × 2 minutes) and dry in the air (2 to 3 hours) or with a hairdryer (10 to 15 minutes).\nNote 1: Carry out fixing, washing, staining and destaining at 20 °C. Do not use elevated temperatures.\nNote 2: If more sensitive silver staining (e.g. Silver Staining Kit, Protein, Pharmacia Biotech, Code No 17-1150-01) is preferred, plasmin-treated casein samples have to be diluted to 5 mg/ml.\n7.\nEVALUATION\nEvaluation is performed by comparing the protein patterns of the unknown sample with reference standards on the same gel. Detection of cow's milk in cheeses from ewe's milk, goat's milk and buffalo milk and mixtures of ewe's, goat's and buffalo milk is done via the γ3- and γ2-caseins, whose isoelectric points range between pH 6,5 and pH 7,5 (Figures 4 a, b, Figure 5). The detection limit is less than 0,5 %.\n7.1.\nVisual estimation\nFor visual evaluation of the amount of bovine milk it is advisable to adjust the concentrations of samples and standards to obtain the same level of intensity of the ovine, caprine and/or buffalo γ2- and γ3-caseins (see ‘γ2 E,G,B’ and ‘γ3 E,G,B’ in Figures 4 a, b and Figure 5). After which the amount of bovine milk (less than, equal to or greater than 1 %) in the unknown sample can be judged directly by comparing the intensity of the bovine γ3- and γ2-caseins (see ‘γ3 C’ and ‘γ2 C’ in Figures 4 a, b and Figure 5) to those of the 0 % and 1 % reference standards (ewe, goat) or, laboratory interim-standards (buffalo).\n7.2.\nDensitometric estimation\nIf available, apply densitometry (5.19) for the determination of the peak area ratio of bovine to ovine, caprine and/or buffalo γ2- and γ3-caseins (see Figure 5). Compare this value to γ2- and γ3-casein peak area ratio of the 1 % reference standard (ewe, goat) or laboratory interim-standard (buffalo) analysed on the same gel.\nNote: The method is operating satisfactorily, if there is a clear positive signal for both bovine γ2- and γ3-caseins in the 1 % reference standard but not in the 0 % reference standard. If not, optimize the procedure following the details of the method precisely.\nA sample is judged as being positive, if both bovine γ2- and γ3-caseins or the corresponding peak area ratios are equal to or greater than the level of the 1 % reference standard.\n8.\nREFERENCES\nAddeo F., Moio L., Chianese L., Stingo C., Resmini P., Berner I, Krause I., Di Luccia A., Bocca A.: Use of plasmin to increase the sensitivity of the detection of bovine milk in ovine and/or caprine cheese by gel isoelectric focusing of γ2-caseins. Milchwissenschaft 45, 708-711 (1990).\nAddeo F., Nicolai M.A., Chianese L., Moio L., Spagna Musso S., Bocca A., Del Giovine L.: A control method to detect bovine milk in ewe and water buffalo cheese using immunoblotting. Milchwissenschaft 50, 83-85 (1995).\nKrause I., Berner I, Klostermeyer H.: Sensitive detection of cow milk in ewe and goat milk and cheese by carrier ampholyte — and carrier ampholyte/immobilized pH gradient — isoelectric focusing of γ-caseins using plasmin as signal amplifier. in: Electrophoresis-Forum 89 (B. J. Radola, ed.) pp 389-393, Bode-Verlag, München (1989).\nKrause Ι., Belitz H.-D., Kaiser K.-P.: Nachweis von Kuhmilch in Schaf and Ziegenmilch bzw. -käse durch isoelektrische Fokussierung in harnstoffhaltigen Polyacrylamidgelen. Z. Lebensm. Unters. Forsch. 174, 195-199 (1982).\nRadola B.J.: Ultrathin-layer isoelectric focusing in 50-100 μm polyacrylamide gels on silanised glass plates or polyester films. Electrophoresis 1, 43-56 (1980).\nFigure 1\nSchematic drawing of the covering sheet\nFigure 2\nCasein layer floating between aqueous and organic phases after centrifugation\nFigure 3\nFlapping technique for casting of ultrathin polyacrylamide gels\na = spacer tape (0,25 mm); b = covering sheet (5.3); c, e = glass plates (5.1); d = gel solution (4.1.2); f = gel carrier sheet (5.2)\nFigure 4a\nIsoelectric focusing of plasmin-treated caseins from ewe's and goat's milk cheese containing different amounts of cow's milk.\n% CM = percentage of cow's milk, C = cow, E = ewe, G = goat\nUpper half of the IEF gel is shown.\nFigure 4b\nIsoelectric focusing of plasmin treated caseins from cheese made from mixtures of ewe's, goat's and buffalo milk containing different amounts of cow's milk.\n% CM = percentage of cow's milk; 1 + = sample containing 1 % of cow's milk and spiked with pure bovine casein at the middle of the track. C = cow, E = ewe, G = goat, B = buffalo.\nTotal separation distance of the IEF gel is shown.\nFigure 5\nSuperposition of densitograms of standards (STD) and cheese samples made from a mixture of ewe's and goat's milk after isoelectric focusing.\na,b = standards containing 0 and 1 % of cow's milk; c-g = cheese samples containing 0, 1, 2, 3 and 7 % of cow's milk. C = cow, E = ewe, G = goat.\nUpper half of the IEF gel was scanned at λ = 634 nm.\nANNEX IX\nEvaluation of the analyses\n1.\nQuality assurance\nAnalyses shall be performed by laboratories designated in accordance with Article 12 of Regulation (EC) No 882/2004 (**) or designated by the competent authorities of the Member State.\n2.\nSampling and disputes over the results of analysis\n1.\nSampling shall be carried out in accordance with the relevant regulation for the product under consideration. If no sampling provisions are expressly provided for, then the provisions laid down in ISO 707, Milk and milk products – Guidance on sampling, shall be used.\n2.\nLaboratory reports of the results of the analysis shall contain sufficient information to allow an evaluation of the results to be carried out in accordance with the Appendix.\n3.\nDuplicate samples shall be taken for analyses required under Union rules.\n4.\nIf a dispute arises over the results, the paying agency shall have the necessary analysis on the product in question carried out again, and the cost shall be met by the losing party.\nThe above mentioned analysis shall be carried out provided that sealed duplicate samples of the product are available and have been stored appropriately with the competent authority. The manufacturer shall send a request to the paying agency to conduct the analysis within 7 working days following the notification of the results of the first analysis. The analysis shall be carried out by the paying agency within 21 working days following receipt of the request.\n5.\nThe appeal result shall be the definitive one.\n6.\nIf the manufacturer can prove, within five working days of sampling, that the sampling procedure was not carried out correctly, sampling shall be repeated where possible. If sampling cannot be repeated, the consignment shall be accepted.\nAppendix\nEvaluation of compliance of a consignment with the legal limit\n1.\nPrinciple\nWhere public intervention and private storage legislation lay down detailed sampling procedures then those procedures shall be followed. In all other cases a sample of at least 3 sample units taken randomly from the consignment submitted to control shall be used. A composite sample may be prepared. The result obtained shall be compared with the legal limits by calculation of a 95 % confidence interval as 2 x standard deviation, where the relevant standard deviation depends on whether (1) the method is validated through international collaboration with values for σr and σR or (2) in the case of in-house validation, an internal reproducibility has been calculated. This confidence interval will then equate to the measurement uncertainty of the result.\n2.\nThe method is validated through international collaboration\nIn this case, the repeatability standard deviation σr and the reproducibility standard deviation σR have been established and the laboratory can demonstrate compliance with the performance characteristics of the validated method.\nCalculate the arithmetic mean x – of the n repeated measurements.\nCalculate the expanded uncertainty (k = 2) of x – as\nU = 2 σ 2 R − n − 1 n σ 2 r\nIf the final result x of measurement is calculated using a formula of the form x = y 1 + y 2, x = y 1 – y 2, x = y 1 · y 2 or x = y 1/y 2 the usual procedures for combining standard deviations in such cases shall be followed.\nThe consignment is judged to be not in compliance with the upper legal limit UL if\nx – − U > UL ;\notherwise it is judged to be in compliance with UL.\nThe consignment is judged to be not in compliance with the lower legal limit LL if\nx – + U < LL ;\notherwise it is judged to be in compliance with LL.\n3.\nIn-house validation with calculation of internal reproducibility standard deviation\nIn cases where methods not specified in this Regulation are used and precision measures have not been established, an in-house validation shall be carried out. Internal repeatability standard deviation σir and the internal reproducibility standard deviation σi R shall be used instead of σr and σ R , resp., in the formulae for the computation of the expanded uncertainty U.\nThe rules to be followed to determine compliance with the legal limit are as set out under point 1. However, if the consignment is judged to be non-compliant with the legal limit, the measurements shall be repeated with the method specified in this Regulation and the result evaluated in accordance to point 1.\n(*)\nThe produce Ampholine® pH 3,5-9,5 (Pharmacia) and Resolyte® pH 5-7 and pH 6-8 (BDH, Merck) have proved particularly suitable for obtaining the required separation of γ-caseins.\n(**)\nRegulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules (OJ L 165, 30.4.2004, p. 1).’",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "footnote",
      "label": "f1",
      "text": "The method to be applied shall be approved by the paying agency."
    },
    {
      "type": "footnote",
      "label": "f2",
      "text": "Scorched particles' analyses may be conducted systematically. However, such analyses shall always be conducted if no sensory checks are performed."
    },
    {
      "type": "footnote",
      "label": "f3",
      "text": "The method to be applied shall be approved by the paying agency (one or both methods)."
    },
    {
      "type": "footnote",
      "label": "f4",
      "text": "The method to be applied shall be approved by the paying agency."
    },
    {
      "type": "footnote",
      "label": "f5",
      "text": "Sensory checks shall be performed where deemed necessary after risk based analysis approved by the paying agency."
    },
    {
      "type": "footnote",
      "label": "f6",
      "text": "The method to be applied shall be approved by the paying agency."
    },
    {
      "type": "footnote",
      "label": "f7",
      "text": "Sample application: After pre-focusing (step 1), pipette 18 μl of the sample and standard solutions onto the sample applicators (10 × 5 mm), place them on the gel at 1 mm intervals from each other and 5 mm longitudinally from the anode and press lightly. Carry out focusing using the above conditions, carefully removing the sample applicators after the 60 minutes of sample focusing."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eur/2018/150/annex",
    "retrieved_at": "2026-11-01T14:44:17.618495+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}