CEN/TC 302 - Milk and milk products - Methods of sampling and analysis

This document specifies a fluorimetric method for the determination of alkaline phosphatase (ALP) (EC 3.1.3.1) activity in raw and heat-treated whole milk, semi-skimmed milk, skimmed milk and flavoured milks.
This method is applicable to milk and milk-based drinks from cows, sheep and goats. It is also applicable to milk powder after reconstitution.
The instrument used for the determination of ALP can read activities up to 7 000 milliunits per litre (mU/l). If the activity is higher than 7 000 mU/l, it is diluted with ALP-free milk so as to obtain a measurement not higher than 7 000 mU/l.

This document specifies a fluorimetric method for the determination of alkaline phosphatase (ALP) (EC 3.1.3.1) activity in cheese.
This method is applicable to soft cheeses, semi-hard and hard cheeses provided that the mould is only on the surface of the cheese and not also in the inner part (e.g. blue veined cheeses). For large hard cheeses, specific conditions of sampling apply (see Clause 7).
The instrument used for the determination of ALP can read activities in the supernatant up to 7 000 milliunits per litre (mU/kg).

This document specifies a method for the determination of the moisture content of all types of dried milk and dried milk products.

This document specifies a method for the determination of the fat content of all types of cheese and processed cheese products containing lactose of below 5 % (mass fraction) of non-fat solids, and all types of caseins and caseinates.
The method is not applicable to fresh cheese types containing, for example, fruits, syrup or muesli. For such products, the Schmid-Bondzynski-Ratzlaff (SBR) principle is not applicable due to high concentrations of sugars. For these products, the method using the Weibull-Berntrop principle (see ISO 8262-3 | IDF 124-3) is appropriate.

This document gives guidance on the sample preparation of fresh cheese, (semi)soft cheese, (semi)hard cheese, processed cheese and whey cheese for physical and chemical analysis, including analysis by applying instrumental methods.
This document describes the (sub)sampling, and sample preparation steps carried out after sampling according to ISO 707 | IDF 50 and prior to method-specific sample preparations, e.g. as with analytical methods listed in References [2] to [22].
NOTE            Analysis on volatile substances, minor components or allergens can require additional precautionary measures in sample preparation in order to avoid loss of or contamination with one or more target analytes.

This document specifies a method for the determination of aflatoxin M1 content in milk and milk powder. The lowest level of validation is 0,08 µg/kg for whole milk powder, i.e. 0,008 µg/l for reconstituted liquid milk. The limit of detection (LOD) is 0,05 μg/kg for milk powder and 0,005 μg/kg for liquid milk. The limit of quantification (LOQ) is 0,1 μg/kg for milk powder and 0,01 μg/kg for liquid milk.
The method is also applicable to low-fat milk, skimmed milk, low-fat milk powder and skimmed milk powder.

This document gives guidelines for the establishment of a conversion relationship between the results of an alternative method and an anchor method, and its verification for the quantitative determination of the microbiological quality of milk.
NOTE     The conversion relationship can be used a) to convert results from an alternative method to the anchor basis or b) to convert results/limits, expressed on an anchor basis, to results in units of an alternative method.

This document specifies a method for the determination of inulin-type fructans (including oligofructose, fructooligosaccharides) in infant formula and adult nutritionals (both powder and liquid) containing 0,03 g/100 g to 5,0 g/100 g of fructans in the product as prepared ready for consumption.
The method has been validated in a multi laboratory study[1] with reconstituted standard reference material (SRM), infant/adult nutritional formula at a level of 0,204 g/100 g, adult nutritionals ready-to-feed (RTF) at levels of 1,28 g/100 g and 2,67 g/100 g, infant formula RTF at a level of 0,300 g/100 g, reconstituted follow-up formula at levels of 0,209 g/100 g to 0,275 g/100 g, reconstituted infant formula at levels from 0,030 8 g/100 g to 0,264 g/100 g. During the single laboratory validation study[2], spike-recovery experiments were performed up to 5 g/100 g in reconstituted infant formula powders (milk-based, partially hydrolysed milk-based and soy-based), adult nutritional RTF and reconstituted adult nutritional powders.

This document specifies the quantitative liquid chromatographic determination of specific sugars (galactose, glucose, fructose, sucrose, lactose and maltose) in various milk and milk products, applying arabinose as an internal standard.
The method is applicable to the following dairy matrices: milk, sweetened condensed milk, milk powder, cheese, whey powder, infant formula, milk dessert and yoghurt.
The method does not apply to dairy products containing soy or to the determination of the lactose content in low-lactose milk products at levels below 1 mg/g.
A high performance anion exchange chromatography method in combination with pulsed amperometric detection (HPAEC-PAD) method is applied[5][3][4]. With this method, thirteen different monosaccharides, disaccharides and trisaccharides can be separated: fucose, arabinose, galactose, glucose, fructose, sucrose, lactose, lactulose, maltose, melibiose, trehalose, isomaltulose and maltotriose.
The method is applicable to labelling for the six most important sugars that can be present by nature or by addition in milk and milk products. The method does not apply to sugar contents less than 0,1 %.

This document describes general workflows and protocols for the validation and the verification of qualitative screening tests for the detection of residues of veterinary drugs in liquid milk (raw, pasteurized, UHT and reconstituted milk powders and whey protein extracts) including biological methods. This guideline does not cover the validation of residue analysis by HPLC, UHPLC or LC-MS/MS.
This document is intended to be useful for manufacturers of screening test kits, laboratories validating screening methods or tests, competent authorities and dairies or end users of reagents or tests for the detection of veterinary drug residues in milk products. This document facilitates and improves the validation and verification of screening methods. The goals of this document are a harmonization in validation of methods or test kits in order for all stakeholders to have full trust in the result of residue screening and to limit the overlap and multiplication of validation work in different laboratories by sharing the validation results generated by an independent laboratory. Furthermore, a harmonized validation and verification procedure allows for comparison of the performance of different screening methods.
This document does not imply that all end users are bound to perform all verification work proposed.
The verification of the correct use of reagents/kits for the detection of antimicrobials is not part of the scope of this document.

This document specifies a method for the quantitative determination of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), phosphorus (P), potassium (K), sodium (Na), zinc (Zn), chromium (Cr), molybdenum (Mo) and selenium (Se) using inductively coupled plasma and mass spectrometry (ICP-MS).
The method is applicable for the determination of all 12 elements in infant formula and adult nutritional products. The method is also applicable for milk, milk powder, whey powder, butter and cheese excluding the determination of Cr, because all Cr results were below the quantification limit and reproducibility could not be determined in these matrices[1]. The present method is an extension of ISO 20649 | IDF 235 (AOAC 2011.19[2]) which was validated only for Cr, Mo and Se in infant formula and adult nutritional products.

ISO 20647:2015 specifies a method for the quantitative determination of total iodine in infant formula and adult nutritional formula.[1] The method is applicable to the measurement of total iodine in infant formula and adult nutritional formula from 0,5 µg/100g to 1 500 µg/100g reconstituted final product and for ready-to-feed products from 2,5 µg/100 g to 1 000 µg/100 g using ICP-MS.
Using various infant formula and adult nutritional products, the method was subjected to an interlaboratory study. Levels obtained ranged from 3,47 µg/100 g to 124 µg/100 g. For all precision data related to the interlaboratory study, see Table A.1 located in Annex A.

ISO 16958:2015 specifies a method for the quantification of individual and/or all fatty acids in the profile of milk, milk products, infant formula and adult nutritional formula, containing milk fat and/or vegetable oils, supplemented or not supplemented with oils rich in long chain polyunsaturated fatty acids (LC-PUFA). This also includes groups of fatty acids often labelled [i.e. trans fatty acids (TFA), saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega-3, omega-6 and omega-9 fatty acids] and/or individual fatty acids [i.e. linoleic acid (LA), α-linolenic acid (ALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)].
The determination is performed by direct transesterification in food matrices, without prior fat extraction, and consequently it is applicable to liquid samples or reconstituted powder samples with water having total fat ≥ 1,5 % m/m.
The fat extracted from products containing less than 1,5 % m/m fat can be analysed with the same method after a preliminary fat extraction using methods referenced in Clause 2. Dairy products, like soft or hard cheeses with acidity level ≤ 1 mmol/100 g of fat, can be analysed after a preliminary fat extraction using methods referenced in Clause 2. For products supplemented or enriched with PUFA with fish oil or algae origins, the evaporation of solvents should be performed at the lowest possible temperature (e.g. max. 40 °C) to recover these sensitive fatty acids.

Le présent document spécifie une méthode de détermination quantitative de la teneur en calcium (Ca), cuivre (Cu), fer (Fe), magnésium (Mg), manganèse (Mn), phosphore (P), potassium (K), sodium (Na) et zinc (Zn) par spectrométrie d'émission atomique avec plasma induit par haute fréquence (ICP-AES). La méthode est applicable au lait, au lait en poudre, au beurre, au fromage, au lactosérum, au lactosérum en poudre, aux formules infantiles et aux produits nutritionnels pour adultes dans les plages données dans le Tableau 1.

This document specifies a protocol for the evaluation of instrumental alternative methods for total bacterial count in raw milk from animals of different species.
NOTE    The document is complementary to ISO 16140-2 and ISO 8196 | IDF 128 (all parts).

This document specifies a reference method for the determination of milk fat purity using gas chromatographic analysis of triglycerides. The method utilizes the differences in triglyceride fingerprint of milk fat from the individual triglyceride fingerprints of other fats and oils to determine samples which are outside the range normally observed for milk fat. This is achieved by using the defined triglyceride formulae based on the normalized weighted sum of individual triglyceride peaks which are sensitive to the integrity of the milk[6][7]. The integrity of the milk fat can be determined by comparing the result of these formulae with those previously observed for a range of pure milk fat samples[12]. Both vegetable fats and animal fats such as beef tallow and lard can be detected.
The method is applicable to bulk milk, or products made thereof, irrespective of the variation in common feeding practices, breed or lactation conditions. In particular, the method is applicable to fat extracted from milk products purporting to contain pure milk fat with unchanged composition, such as butter, cream, milk and milk powder.
Because a false-positive result can occur, the method does not apply to milk fat related to these circumstances:
a)    obtained from bovine milk other than cow's milk;
b)    obtained from single cows;
c)    obtained from cows whose diet contained a particularly high proportion of vegetable oils such as rapeseed, cotton or palm oil, etc.;
d)    obtained from cows suffering from serious underfeeding (strong energy deficit);
e)    obtained from colostrum;
f)     subjected to technological treatment such as removal of cholesterol or fractionation;
g)    obtained from skim milk, buttermilk or whey;
h)    obtained from cheeses showing increased lipolysis;
i)     extracted using the Gerber, Weibull?Berntrop or Schmid?Bondzynski?Ratzlaff methods, or that has been isolated using detergents (e.g. the Bureau of Dairy Industries method).
With the extraction methods specified in i), substantial quantities of partial glycerides or phospholipids can pass into the fat phase.
NOTE 1    In nature, butyric (n-butanoic) acid (C4) occurs exclusively in milk fat and enables quantitative estimations of low to moderate amounts of milk fat in vegetable and animal fats to be made. Due to the large variation of C4, for which the approximate content ranges from 3,1 % fat mass fraction to 3,8 % fat mass fraction, it is difficult to provide qualitative and quantitative information for foreign fat to pure milk fat ratios of up to 20 % mass fraction[11].
NOTE 2    In practice, quantitative results cannot be derived from the sterol content of vegetable fats, because they depend on production and processing conditions. Furthermore, the qualitative determination of foreign fat using sterols is ambiguous.
NOTE 3    Due to special feeding practices such as those related to c) and d), false-positive results have sometimes been reported for milk from certain Asian regions[15]. Moreover, grass-only diets such as mountain and, in particular, highland pasture feeding sometimes cause false-positive results, which can be substantiated by a content of conjugated linoleic acid (C18:2 c9t11) of ≥ 1,3 % fatty acid mass fraction[16][17]. Nevertheless, results conforming to the criteria of milk fat purity specified in this document are accepted, even if samples were undoubtedly produced under conditions reported in this note, including those described in h).
NOTE 4    In ca

ISO 9233-2 | IDF 140-2:2018 specifies a method for the determination of natamycin mass fraction in cheese, cheese rind and processed cheese of above 0,5 mg/kg and of the surface-area-related natamycin mass in cheese rind of above 0,03 mg/dm2.

ISO 9233-1 | IDF 140-1:2018 specifies a method for the determination in cheese rind of natamycin mass fraction of above 0,5 mg/kg and surface-area-related natamycin mass of above 0,03 mg/dm2.
NOTE       It is possible that the method is suitable for detecting migration of natamycin into the cheese.

ISO 8968-4|IDF 20-4:2016 specifies a method for the direct and indirect determination of the protein nitrogen content of liquid, whole or skimmed milk.

ISO 8968-1|IDF 20-1:2014 specifies a method for the determination of the nitrogen content and crude protein calculation of milk and milk products by the Kjeldahl principle, using traditional and block digestion methods. The methods are applicable to: liquid cow's (whole, partially skimmed or skimmed milk), goat's and sheep's whole milk; hard, semi-hard and processed cheese; dried milk and dried milk products (including milk-based infant formulae, milk protein concentrate, whey protein concentrate, casein and caseinate). The methods are not applicable to samples containing ammonium caseinate.

ISO 5536|IDF 23:2009 specifies a method for the determination of the water content of milk fat products by the Karl Fischer method.
The method is applicable to butteroil (anhydrous butteroil, anhydrous butterfat, anhydrous milk fat) with a water content not exceeding 1,0 % mass fraction.

ISO 12779/IDF 227:2011 specifies a method for the determination of the water content of lactose by Karl Fischer (KF) titration.

2013-05-15 LVO: Text arrived in ISO/CS (see notification of 2013-05-13 in dataservice).

2011-10-03 - KA - French version only

ISO 1211|IDF 1:2010 specifies the reference method for the determination of the fat content of milk of good physicochemical quality.
The method is applicable to raw cow milk, raw sheep milk, raw goat milk, reduced fat milk, skimmed milk, chemically preserved milk, and processed liquid milk.
It is not applicable when greater accuracy is required for skimmed milk, e.g. to establish the operating efficiency of cream separators.

ISO 5764|IDF 108:2009 specifies a reference method for the determination of the freezing point of raw bovine milk, heat-treated whole, reduced fat and skimmed bovine milk, as well as raw ovine and caprine milk, by using a thermistor cryoscope.
The freezing point can be used to estimate the proportion of extraneous water in milk. Calculation of the amount of extraneous water is subject to daily and seasonal variations, and is not within the scope of ISO 5764|IDF 108:2009.
Results obtained from samples with a titratable acidity exceeding 20 ml of 0,1 mol/l sodium hydroxide solution per 10 g of non-fat solids are not representative of the original milk.

ISO - Taking over of an ISO Technical Corrigendum

ISO 8381¦IDF 123:2008 specifies the reference method for the determination of the fat content of milk-based infant foods.
The method is applicable to liquid, concentrated and dried milk-based infant foods with no, or not more than a mass fraction of 5 % (dry matter) of such added matter as starch, dextrin, vegetables, fruit and meat.
The method is not applicable to products which do not dissolve completely in ammonia owing to the presence of starch or dextrin at mass fractions of more than a few percent, or to the presence of hard lumps. The method is also not applicable to products which contain free fatty acids in significant quantities. The results obtained for these products will be too low.

ISO 1854¦IDF 59:2008 specifies the reference method for the determination of fat content of whey cheese.
The method is not applicable to products which do not dissolve completely in ammonia solution or which contain free fatty acids in significant quantities.

ISO 1737|IDF 13:2008 specifies the reference method for the determination of the fat content of all types of evaporated milk and sweetened condensed milk (liquid sweetened and unsweetened concentrated milk).

ISO 1736|IDF 9:2008 specifies the reference method for the determination of the fat content of dried milk and dried milk products. The method is applicable to dried milk with a fat content of 40 % mass fraction or more, dried whole, dried partially skimmed, and dried skimmed milk, dried whey, dried buttermilk and dried butter serum.
The method is not applicable when the powder contains hard lumps which do not dissolve in ammonia solution or free fatty acids in significant quantities.

ISO 2450|IDF 16:2008 specifies the reference method for the determination of the fat content of raw, processed and sour cream in which no appreciable separation or breakdown of fat, due to lipolysis, has occurred.
The method is not applicable to sour creams with starch or other thickening agents.

ISO 7208|IDF 22:2008 specifies the reference method for the determination of the fat content of liquid skimmed milk, whey and buttermilk. It is a particularly accurate gravimetric method especially for the purpose of establishing the operating efficiency of cream separators.
ISO 7208|IDF 22:2008 also specifies the reference method for establishing correction tables for procedures with skimmed milk butyrometers.

ISO 7328¦IDF 116:2008 specifies the reference method for the determination of the fat content of most milk-based edible ices and ice mixes.
The method is also applicable to concentrated and dried ice mixes.
The method is not applicable to some milk-based edible ices and ice mixes, in which the level of emulsifier, stabilizer or thickening agent or of egg yolk or of fruits, or of combinations of these constituents makes the Roese-Gottlieb method unsuitable.

ISO 20541|IDF 197:2008 specifies a method for the determination of the nitrate content of milk and milk products by molecular-absorption spectrometry after Griess reaction (preceded by enzymatic reduction).
The method is, in particular, applicable to whole, partly skimmed, skimmed and dried milk, hard, semi-hard and soft cheeses, processed cheese, whey cheese, caseins, caseinates, dried whey and milk protein concentrates.
The method can be used at contents corresponding to a measured concentration in the sample solution (with blank subtracted) of more than 0,2 mg/l.

ISO 707|IDF 50:2008 gives guidance on methods of sampling milk and milk products for microbiological, chemical, physical and sensory analysis, except for (semi)automated sampling.

ISO 13366-1|IDF 148-1:2008 specifies a microscopic method (reference method) for the counting of somatic cells in both raw and chemically preserved milk.
ISO 13366-1|IDF 148-1:2008 is applicable for the counting of somatic cells in cows' milk, provided that the eventually mentioned prerequisites are met.
This method is suitable for preparing standard test samples and determining reference method values that are required for calibrating mechanized and automated cell-counting methods.

ISO 8968-3|IDF 20-3:2004 specifies a method for the determination of the nitrogen content of liquid, whole or skimmed milk.
It concerns a semi-micro rapid routine method following the block-digestion principle.

ISO 8069|IDF 69:2005 specifies an enzymatic method for the determination of the lactic acid and lactates content of all types of dried milk.

ISO 22160|IDF 209:2007 specifies a method for the determination of the alkaline phosphatase activity in pasteurized whole milk, semi-skimmed milk, skimmed milk, cream and flavoured milks using a chemiluminescent (EPAS) method.
The method is applicable to milk and milk-based drinks from cows, sheep, buffalo and goats.
The method is also suitable for any liquid-based sample if diluted in such a way that the diluted alkaline phosphatase activity has less than 7 000 milliunits per litre.

ISO 14637|IDF 195:2004 specifies an enzymatic method for the determination of the urea content of milk by measurement of the difference in pH.

CMC - Superseding information

ISO 13366-2|IDF 148-2:2006 gives guidance on the operating conditions for counting somatic cells, in both raw and chemically preserved milk, using fluoro-opto-electronic somatic cell counters in which either a rotating disc technique or flow cytometry is applied in the counting section.
The guidance is applicable to the counting of somatic cells in raw cow milk. The guidance is also applicable to raw milk of other species, such as goat, sheep and buffalo, if the specified prerequisites are met.

ISO 5534|IDF 4:2004 specifies the reference method for the determination of the total solids content of cheese and processed cheese.

ISO 13969|IDF 183:2003 gives guidance for a standardized description of microbial inhibitor tests for milk and milk products. It is intended to give a framework and basis for the evaluation/validation of microbial inhibitor tests, allowing the comparison of data obtained from different tests and experimental studies.

ISO 14673-3|IDF 189-3:2004 specifies a routine method for the determination of the nitrate and nitrite contents of milk and milk products by cadmium reduction and flow injection analysis (FIA). The method is applicable to hard, semi-hard and soft cheeses of various ages, and processed cheese. The detection limits of the method are 0,5 mg of nitrate ions per kilogram and 1,0 mg of nitrite ions per kilogram, respectively.
The method is also applicable to whey powder, milk powder and milk-based infant food.

ISO 14673-2|IDF 189-2:2004 specifies a routine method for the determination of the nitrate and nitrite contents of milk and milk products by segmented flow analysis. The method is applicable to milk, cheese, and liquid and dried milk products and infant foods.

ISO 14673-1|IDF 189-1:2004 specifies a method for the determination of the nitrate and nitrite contents of milk and milk products by cadmium reduction and spectrometry. The method is applicable to: whole and partly skimmed and skimmed dried milk; hard, semi-hard and soft cheeses; processed cheese; whey cheese, caseins and caseinates and dried whey.
The method may be performed using automatic equipment, in particular by segmented flow analysis (SFA) or flow injection analysis (FIA), thus reducing cadmium contamination in laboratory work places and waste water.

ISO 17189|IDF 194:2003 specifies a method for the determination of the fat content of butter, edible oil emulsions and spreadable fats (margarine, vegetable oil spreads, dairy spreads and blended spreads).

ISO 18330|IDF 188:2003 gives guidelines for the standardized description of immunoassays or receptor assays for the detection of antimicrobial residues in milk and milk products.
It is intended to provide a framework and basis for the evaluation/validation of tests based on the binding of an antimicrobial compound to its specific antibody or to other types of detecting molecules.
In addition to immunoassays [e.g. enzyme-immunoassay (EIA) and radio-immunoassay (RIA)], there are several quantitative, semi-quantitative and qualitative test formats based on the binding of antimicrobial compounds to microbial receptors or to receptor proteins. Enzymatic assays and particle-based assays based on receptor proteins are referred to as receptor assays in ISO 18330|IDF 188:2003.