ISO/TC 34/SC 5 - Milk and milk products
Standardization of methods of analysis and sampling for milk and milk products, covering the dairy chain from primary production to consumption
Lait et produits laitiers
Normalisation des méthodes d'analyse et d'échantillonnage du lait et des produits laitiers, couvrant la filière laitière, de la production primaire à la consommation
General Information
This document specifies a protocol for the evaluation and validation of alternative quantitative methods of milk analysis. This document is also applicable for the validation of new alternative methods where, due to a limited number of operational instruments, the execution of an interlaboratory study and ISO 8196-1 | IDF 128-1 is not feasible. The protocol is applicable to milk parameters such as, for example, fat, protein, lactose, urea and somatic cells in milk. It can also be extended to other parameters. This document also establishes the general principles of a procedure for granting international approvals for the performance of the alternative methods. These principles are based on the validation protocol defined in this document.
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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.
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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.
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This document specifies a method for the determination of melamine and cyanuric acid with liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). The method has been validated in an interlaboratory study via the analysis of spiked samples of milk-based infant formula, soy-based infant formula, milk powder, whole milk, soy drink and milk chocolate ranging from 0,71 mg/kg to 1,43 mg/kg for melamine and 0,57 mg/kg to 1,45 mg/kg for cyanuric acid. The limits of quantification (LOQ) for melamine and cyanuric acid in food are 0,05 mg/kg and 0,25 mg/kg, respectively. The upper limit of the working range is up to 10 mg/kg for melamine and up to 25 mg/kg for cyanuric acid.
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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.
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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.
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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.
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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 %.
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This document gives guidelines for the use of near infrared (NIR) spectrometry in the analysis of milk and milk products in liquid, semi-solid or solid form. Depending on the sample form and application, different instrument setups for transmittance, diffuse reflectance or transflectance can be applied.
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This document specifies a liquid chromatography tandem mass spectrometry (LC?MS/MS) method for the quantification of the inhibitory substance, nitrofurazone, in milk and milk products. The method has been validated for measuring trace levels of intact nitrofurazone to levels down to 1 ng/g in fluid milk and powdered dairy products on a whole product (i.e. powder) basis. While the method is expected to apply to other dairy matrices, additional validation will be required to demonstrate this.
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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.
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This document specifies a method for the determination of the whey to casein protein ratio, ranging from 20:80 to 80:20 in cow milk-based infant formula powders. This method does not apply to the analysis of infant formulas containing hydrolysed protein or proteins from other sources (e.g. plants or milk from other mammals).
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This document gives guidelines for using infrared spectrometry in in-line and on-line applications for dairy processing. These applications are distinct to those covered in ISO 21543 | IDF 201. It is applicable, but not limited, to: — the determination of protein, fat and total solids in liquid milk and milk products using mid and near infrared spectrometry; — the determination of protein, fat and moisture in solid or semi-solid products, such as milk powder, and butter and liquid dairy streams using near infrared spectrometry.
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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).
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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 cases where a positive result is suspected to be caused by circumstances related to c) or d), another analytical method, such as fatty acid or sterol analysis, can be applied to confirm the finding. Due to similar or increased limitations (e.g. as described in NOTE 1 and NOTE 2), a negative result obtained by another method is not appropriate to contrastingly confirm milk fat purity.
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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.
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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.
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This document specifies a method for the determination of chloride in milk, milk products, infant formula and adult nutritionals by potentiometry[1][2][3][4] with an analytical range of 0,35 mg chloride/100 g to 711,6 mg chloride/100 g product, or ready-to-feed products.
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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.
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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.
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ISO 19662 | IDF 238:2018 specifies a method, the acido-butyrometric or "Gerber", for determining the fat content of milk. It is applicable to whole milk and partially skimmed milk. It is also applicable to milk containing authorized preservatives (potassium dichromate, bronopol). It does not apply to formalin milk, nor to milks that have undergone a homogenisation treatment.
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ISO 196660 | IDF 237:2018 specifies an acidobutyrometric method for determining the fat content of cream. The reference method remains the gravimetric method (by ammoniacal ether extraction) described in ISO 2450 | IDF 16. This method is applicable to cream having a fat content between 20 % and 50 % inclusive: - intended for manufacturing butter; - sweet, unmatured and non-inoculated; - raw or having undergone a heat treatment; - non-homogenized; - with or without preservatives (2-bromo-2-nitropropane, 1,3 diol or bronopol).
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ISO/TS 19046-2 | IDF/RM 233-2:2017 specifies a method for the determination of propionic acid level in cheese, using ion exchange chromatography.
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ISO/TS 19046-1| IDF/RM 233-1:2017 specifies a method for the determination of propionic acid level in cheese, using gas chromatography.
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ISO 11816-2:2016|IDF 155-2:2016 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 can read activities in the supernatant up to 7 000 milliunits per litre (mU/l).
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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.
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ISO 27105:2016 specifies a method for the quantitative determination of hen's egg white lysozyme content in milk and cheese. The method is suitable for measuring low levels of hen's egg white lysozyme with a quantification limit of 10 mg/kg.
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ISO 19344:2015 specifies a standardized method for the quantification of active and/or total lactic acid bacteria and probiotic strains in starter cultures used in dairy products by means of flow cytometry. The method is also applicable to probiotics used in dairy products and to fermented milk products such as yogurts containing primarily lactic acid bacteria. This International Standard does not apply to taxonomical differentiation of bacteria. Due to its non-specificity, the method may quantify other bacteria than those within the scope of this International Standard, when present in the sample. This may lead to overestimation of the counts. The minimum bacterial cell concentration in the sample before applying this standardized method depends on the dilution rates used in the individual protocols. Typically 106 cells per gram or ml are considered within the minimum range.
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ISO 20649:2015 specifies a method for the quantitative determination of chromium, selenium and molybdenum in infant formula and adult nutritional formula.[1] The method has been validated in an interlaboratory study on seven different matrices.
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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.
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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.
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ISO/TS 17758|IDF/RM 87:2014 specifies a method for the determination of the dispersibility in water of instant dried milk. The method is applicable to instant dried skimmed milk manufactured by either the "straight-through" or the "re-wet" process and also to instant dried whole milk.
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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.
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ISO 11816-1|IDF 155-1:2013 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 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 alkaline phosphatase-free milk so as to obtain a measurement not higher than 7 000 mU/l.
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ISO 9622|IDF 141:2013 gives guidelines for the quantitative compositional analysis of milk and liquid milk products, such as raw milk, processed milk, cream and whey, by measurement of the absorption of mid-infrared radiation. Additional built-in instrument features, such as a conductivity sensor, can improve the performance in the determination of compositional parameters and allow for the estimation of other parameters. The guidelines specified are applicable to the analysis of cow's milk. The guidelines are also applicable to the analysis of milk of other species (goat, ewe, buffalo, etc.) and derived liquid milk products, provided adequate calibrations are generated for each application and adequate control procedures are in place.
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ISO/TS 18083|IDF/RM 51:2013 specifies the calculation of the approximate content of phosphorus from added phosphate salts and phosphorus containing pH-regulating agents in processed cheese products. The method is applicable to processed cheese products derived mainly from cheese varieties with a P/N ratio of 0,12 ± 0,02 and containing no ingredients in quantities substantially affecting the P/N ratio of the cheese raw material.
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ISO/TS 22113|IDF/RM 204:2012 specifies a routine method for determining the titratable acidity of milk fat. The method is applicable to milk fat obtained from: a) raw milk; b) heat-treated milk; c) milk reconstituted from milk powder; d) cream with any fat content, provided the product is diluted so as to obtain a mass fraction of between 4 % and 6 % fat. The method is not applicable to fermented milk or milk that has undergone bacterial or enzymatic damage.
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ISO 15163:2012 specifies a reference method for the determination of the amounts of chymosin and bovine pepsin present in a test sample of calf rennet and adult bovine rennet. In addition, it can be used for mixtures of calf/bovine rennet with fermentation-produced bovine chymosin (FPC).
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ISO 15174:2012 specifies a method for comparison of the total milk-clotting activity of a microbial coagulant sample with the milk-clotting activity of an international microbial coagulant reference standard on a standard milk substrate prepared with a calcium chloride solution of concentration 0,5 g/l (pH ~6,5).
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ISO/TS 11869|IDF/RM 150:2012 specifies a potentiometric method for the determination of the titratable acidity of natural yoghurt, flavoured yoghurt, fruit yoghurt, drinking yoghurt, fresh cheese with or without fruit, buttermilk with or without fruit, and other fermented milk products.
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ISO/TS 17193¦IDF/RM 208:2011 specifies a photometric method for the determination of the lactoperoxidase activity in milk in amounts exceeding 50 U/l.
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ISO 13082:2011 specifies a method for the determination of the lipase activity. It is intended for the preparation of pregastric lipase and rennet paste, both of animal origin.
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ISO 12779/IDF 227:2011 specifies a method for the determination of the water content of lactose by Karl Fischer (KF) titration.
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ISO 27871|IDF 224:2011 specifies a method for determining the nitrogenous fractions in cheeses and processed cheese from cow milk.
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ISO 6731|IDF 21:2010 specifies the reference method for the determination of the total solids content of milk, cream and evaporated milk.
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ISO 6734|IDF 15:2010 specifies the reference method for the determination of the total solids content of sweetened condensed milk.
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ISO/TS 15495|IDF/RM 230 gives guidance for the quantitative determination of melamine and cyanuric acid content in milk, powdered milk products, and infant formulae by electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS).
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ISO 10932|IDF 223:2010 specifies a method for determining the minimal inhibitory concentration (MIC) of a series of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB). ISO 10932|IDF 223:2010 recommends the broth microdilution method as the standard method.
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ISO 26462|IDF 214:2010 specifies an enzymatic method for the determination of the lactose content of milk and reconstituted milk by measurement of the difference in pH (differential pH measurement).
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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.
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