1、8069:2007)g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58and lactatesThe European Standard EN ISO 8069:2007 has the status of a British StandardICS
2、 67.100.10Dried milk Determination of content of lactic acid BRITISH STANDARDBS EN ISO 8069:2007Incorporating amendment no. 1 (renumbers BS ISO 8069:2005 as BS EN ISO BS EN ISO 8069:2007This British Standard was published under the authority of the Standards Policy and Strategy Committee on 10 Janua
3、ry 2006 BSI 2007ISBN 978 0 580 59611 7Amendments issued since publicationAmd. No. Date Comments17261 31 August 2007 Renumbers BS ISO 8069:2005 as BS EN ISO 8069:2007contract. Users are responsible for its correct application. Compliance with a British Standard cannot confer immunity from legal oblig
4、ations.National forewordThis British Standard is the UK implementation of EN ISO 8069:2007. It is identical with ISO 8069:2005. It supersedes BS 1743-8:1987 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk produc
5、ts. A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 8069June 2007ICS 67.100.10English VersionDried milk - Determinati
6、on of content of lactic acid and lactates(ISO 8069:2005)Lait sec - Determination de la teneur en acide lactique et enlactates (ISO 8069:2005)Milchpulver - Bestimmung des Gehalts an Milchsure undLactaten (ISO 8069:2005)This European Standard was approved by CEN on 19 May 2007.CEN members are bound to
7、 comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Mana
8、gement Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as th
9、eofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia,
10、Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2007 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN
11、national Members.Ref. No. EN ISO 8069:2007: EForeword The text of ISO 8069:2005 has been prepared by Technical Committee ISO/TC 34 “Agricultural food products” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 8069:2007 by Technical Committee CEN/TC 302 “M
12、ilk and milk products - Methods of sampling and analysis“, the secretariat of which is held by NEN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by December 2007, and conflicting national standard
13、s shall be withdrawn at the latest by December 2007. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France
14、, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of ISO 8069:2005 has been approved by CEN as EN ISO 8069:2007 with
15、out any modifications. EN ISO 8069:2007Reference numbersISO 8069:2005(E)IDF 69:2005(E)INTERNATIONAL STANDARD ISO8069IDF69Second edition2005-09-15Dried milk Determination of content of lactic acid and lactates Lait sec Determination de la teneur en acide lactique et en lactates EN ISO 8069:2007ii iii
16、Contents Page Foreword iv 1 Scope . 1 2 Terms and definitions. 1 3 Principle. 1 4 Reagents 1 5 Apparatus 3 6 Sampling 3 7 Preparation 4 7.1 Preparation of test sample. 4 7.2 Test portion . 4 7.3 Blank test. 4 7.4 Preparation of solution and deproteination. 4 8 Procedure 4 8.1 Test to check the activ
17、ity of reagents. 4 8.2 Determination 5 9 Calculation and expression of results 6 9.1 Calculation. 6 9.2 Expression of results . 7 10 Precision 7 10.1 Interlaboratory test . 7 10.2 Repeatability 7 10.3 Reproducibility 8 11 Test report . 8 Annex A (normative) Good laboratory practice (GLP) rules for t
18、he performance of enzymatic analyses. 9 Bibliography . 13 EN ISO 8069:2007iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO
19、 technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates cl
20、osely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Dr
21、aft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this doc
22、ument may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 8069IDF 69 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is bein
23、g published jointly by ISO and IDF. This edition of ISO 8069IDF 69 cancels and replaces ISO 8069:1986, which has been technically revised. EN ISO 8069:2007vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member countr
24、y. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams
25、and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject
26、 of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 8069IDF 69 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by ID
27、F and ISO. All work was carried out by the Joint ISO-IDF Action Team on Lactose and lactate determination, of the Standing Committee on Main components of milk, under the aegis of its project leader, Mr J Romero (US). This edition of ISO 8069IDF 69 cancels and replaces IDF 69:1987, which has been te
28、chnically revised. EN ISO 8069:2007blank1Dried milk Determination of content of lactic acid and lactates 1 Scope This International Standard specifies an enzymatic method for the determination of the lactic acid and lactates content of all types of dried milk. 2 Terms and definitions For the purpose
29、s of this document, the following terms and definitions apply. 2.1 lactic acid and lactates content mass of substances determined by the procedure specified in this International Standard NOTE It is expressed as milligrams of lactic acid per 100 g of non-fat solids. 3 Principle A test portion of dri
30、ed milk is dissolved in warm water. The fat and proteins are precipitated then filtered. The filtrate is treated with the following enzymes and biochemical substances, added simultaneously, but acting in sequence: a) L-lactate dehydrogenase (L-LDH) and D-lactate dehydrogenase (D-LDH), in the presenc
31、e of nicotinamide adenine dinucleotide (NAD), to oxidize lactate to pyruvate and to convert NAD to its reduced form NADH; b) glutamate pyruvate transaminase (GPT), in the presence of L-glutamate, to transform pyruvate into L-alanine and to convert L-glutamate to -ketoglutarate. The amount of NADH pr
32、oduced is determined by spectrophotometric measurement at a wavelength of 340 nm, and is proportional to the lactic acid and lactates content. 4 Reagents Use only reagents recognized analytical grade. The water used in the preparation of the enzyme solutions shall be of at least doubly glass-distill
33、ed purity and the water used for other purposes shall be glass-distilled or of at least equivalent purity. 4.1 Potassium hexacyanoferrate(II) solution, c(K4Fe(CN)63H2O) = 35,9 g/l. Dissolve 35,9 g of potassium hexacyanoferrate(II) trihydrate in water. Dilute with water to 1 000 ml and mix. 4.2 Zinc
34、sulfate solution, c(ZnSO47H2O) = 71,8 g/l. Dissolve 71,8 g of zinc sulfate heptahydrate in water. Dilute with water to 1 000 ml and mix. EN ISO 8069:20072 4.3 Sodium hydroxide solutions 4.3.1 Sodium hydroxide solution I, c(NaOH) = 10 mol/l. Dissolve 400 g of sodium hydroxide in water. Dilute with wa
35、ter to 1 000 ml and mix. 4.3.2 Sodium hydroxide solution II, c(NaOH) = 0,1 mol/l. Dissolve 4,0 g of sodium hydroxide in water. Dilute with water to 1 000 ml and mix. 4.4 Glycerol solution (C3H8O3), with a volume fraction of 50 % glycerol. 4.5 Ammonium sulfate solution, c(NH4)2SO4 = 3,2 mol/l. Dissol
36、ve 422,84 g of ammonium sulfate in water. Dilute with water to 1 000 ml and mix. 4.6 Buffer solution, pH 10. Dissolve 7,92 g of glycylglycine (C4H8N2O3) and 1,47 g of L-glutamic acid (C5H9NO4) in about 80 ml of water. Adjust the pH to 10,0 0,1 at 20 C with sodium hydroxide solution I (4.3.1). Dilute
37、 with water to 100 ml and mix. This solution may be kept for 3 months if stored in a refrigerator at between 0 C and +5 C. 4.7 Nicotinamide adenine dinucleotide solution (NAD). Dissolve 350 mg of nicotinamide adenine dinucleotide (C21H27N7O14P2) in 10 ml of water. This solution may be kept for 4 wee
38、ks if stored in a refrigerator at between 0 C and +5 C. When the solution is being used, keep the vessel immersed in crushed ice. 4.8 L-Lactate dehydrogenase (L-LDH), from hog muscle suspension. Dissolve 10 mg of L-lactate dehydrogenase suspension in 1 ml of glycerol solution (4.4). The pH of the ob
39、tained suspension should be about 7. The specific activity of the L-lactate dehydrogenase (L-LDH, EC 1.1.1.27) suspension shall be at least 5 500 units/ml at 25 C. If not, prepare another L-LDH suspension. The L-LDH suspension may be kept for 12 months if stored in a refrigerator at between 0 C and
40、+5 C. When the suspension is being used, keep the vessel immersed in crushed ice. 4.9 D-Lactate dehydrogenase (D-LDH), from Lactobacillus leichmannii suspension. Dissolve 5 mg of D-LDH suspension in 1 ml of ammonium sulfate solution (4.5). The pH of the obtained suspension should be about 6. The spe
41、cific activity of the D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) suspension shall be at least 1 500 units/ml at 25 C. If not, prepare another D-LDH suspension. The D-LDH suspension may be kept for 12 months if stored in a refrigerator at between 0 C and +5 C. When the suspension is being used, kee
42、p the vessel immersed in crushed ice. 4.10 Glutamate pyruvate transaminase (GPT), from pig heart suspension. Dissolve 20 mg of GPT suspension in 1,0 ml of ammonium sulfate solution (4.5). The pH of the obtained suspension should be about 7. The specific activity of the glutamate pyruvate transaminas
43、e (GPT, EC 2.6.1.2) suspension shall be at least 1 600 units/mI at 25 C. If not, prepare another GPT suspension. EN ISO 8069:20073Add 1,0 ml of ammonium sulfate solution (4.5) to the 1 ml suspension with 20 mg of GPT and mix. Centrifuge this 2,0 ml suspension containing 10 mg of GPT/ml at a radial a
44、cceleration of 4 000 g for 10 min. Transfer 1,0 ml of the clear supernatant liquid and discard the remaining solution and pellet. The suspension may be kept for 12 months if stored in a refrigerator at between 0 C and +5 C. When the suspension is being used, keep the vessel immersed in crushed ice.
45、4.11 Lithium L-lactate solution Dissolve 50 mg of lithium L-lactate (C3H5O3Li) in water. Dilute with water to 500 ml and mix. 4.12 Lithium D-lactate solution Dissolve 50 mg of lithium D-lactate (C3H5O3Li) in water. Dilute with water to 500 ml and mix. 5 Apparatus Usual laboratory equipment and, in p
46、articular, the following. 5.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg. 5.2 Glass beaker, of capacity 50 ml. 5.3 Graduated cylinder, of capacity 50 ml. 5.4 One-mark volumetric flasks, of capacity 100 ml. 5.5 Pipettes, capable of delivering 0,02 ml, 0,
47、05 ml, 0,2 ml, 1,0 ml and 2,0 ml. 5.6 Graduated pipettes, capable of delivering 5 ml and 10 ml, graduated in 0,1 ml divisions. 5.7 Glass filter funnel, of diameter about 7 cm. 5.8 Filter paper, medium fast grade, of diameter about 15 cm, free from lactic acid and lactates. 5.9 Glass rod. 5.10 Plasti
48、c paddles, capable of mixing the sample-enzyme mixture in the spectrometric cell. 5.11 Spectrophotometer, capable of measuring at 340 nm, equipped with cells of optical path length 1 cm. 5.12 ParafilmTM1). 6 Sampling A representative sample should have been sent to the laboratory. It should not have
49、 been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707IDF 50. Store the sample in such a way that deterioration and change in composition are prevented. 1) ParafilmTMis an example of a product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF of this product. EN ISO 8069:20074 7 Preparation 7.1 Preparation of test
