1、September 2007DEUTSCHE NORM English price group 12No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.100.10!$I0o“13813
2、76www.din.deDDIN EN ISO 8069Dried milk Determination of content of lactic acid and lactates (ISO 8069:2005)English version of DIN EN ISO 8069:2007-09Milchpulver Bestimmung des Gehalts an Milchsure und Lactaten (ISO 8069:2005)Englische Fassung DIN EN ISO 8069:2007-09www.beuth.deDocument comprises 19
3、pages11.07DIN EN ISO 8069:2007-09 2 National foreword This standard falls in the domain of Technical Committee CEN/TC 302 “Milk and milk products Methods of sampling and analysis” (Secretariat: NEN, The Netherlands). This standard has been prepared by Subcommittee SC 5 “Milk and milk products” (Secr
4、etariat: NEN, The Netherlands) of Technical Committee ISO/TC 34 “Food products” (Secretariat: MSZT, Hungary) in collabora-tion with Technical Committee CEN/TC 302. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und land-wirtschaftliche Produkte (Food and
5、 Agricultural Products Standards Committee), Technical Committee Milch und Milchprodukte Probenahme- und Untersuchungsverfahren. Based on the results of the unique acceptance procedure, ISO 8069:2005 has been adopted as a European Standard. The DIN Standards corresponding to the International Standa
6、rds referred to in the EN are as follows: ISO 707 DIN EN ISO 707 ISO 1736 DIN EN ISO 1736 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN EN ISO 707, Milk and milk products Guidance on sampling DIN EN ISO 1736, Dried milk and dried milk products D
7、etermination of fat content Gravimetric method (Reference method) DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method
8、for the determination of repeatability and reproducibility of a standard measurement method EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 8069June 2007ICS 67.100.10English VersionDried milk - Determination of content of lactic acid and lactates(ISO 8069:2005)Lait sec - Determination de la te
9、neur 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 comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for givi
10、ng 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 Management Centre or to any CEN member.This European Standard exists in three official versio
11、ns (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 theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bul
12、garia, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZ
13、ATIONCOMIT 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 national Members.Ref. No. EN ISO 8069:2007: EContents Page 1 2 3 4 5 6 7 7.1 7.2 7.3 7.4
14、8 8.1 8.2 9 9.1 9.2 10 10.1 10.2 10.3 11 Annex A (normative) Good laboratory practice (GLP) rules for the performance of enzymatic EN ISO 8069:2007 (E)2Foreword 3 Scope . 5 Terms and definitions. 5 Principle. 5 Reagents 5 Apparatus 7 Sampling 7 Preparation 8 Test portion . 8 Preparation of solution
15、and deproteination. 8 Preparation of test sample. 8 Blank test. 8 Procedure 8 Test to check the activity of reagents. 8 Determination 9 Expression of results . 11 Precision 11 Interlaboratory test . 11 Reproducibility 12 Test report . 12 Calculation. 10 Bibliography . 17 Calculation and expression o
16、f results 10 Repeatability 11 analyses. 13 Foreword. 4 EN ISO 8069:2007 (E) 3 Foreword 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
17、Technical Committee CEN/TC 302 “Milk 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, an
18、d conflicting national standards 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, De
19、nmark, Estonia, Finland, France, 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
20、by CEN as EN ISO 8069:2007 without any modifications. Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying ou
21、t 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 and Standing Committees are circulated to the National Committees for voting. Publication as an Intern
22、ational 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 of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. IS
23、O 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 IDF and ISO. All work was carried out by the Joint ISO-IDF Action Team on Lactose and lactate determinat
24、ion, 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 technically revised. EN ISO 8069:2007 (E)41 Scope This International Standard specifies an enzymatic met
25、hod for the determination of the lactic acid and lactates content of all types of dried milk. 2 Terms and definitions For the purposes 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 In
26、ternational Standard NOTE It is expressed as milligrams of lactic acid per 100 g of non-fat solids. 3 Principle A test portion of dried 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,
27、 added simultaneously, but acting in sequence: a) L-lactate dehydrogenase (L-LDH) and D-lactate dehydrogenase (D-LDH), in the presence 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
28、the presence of L-glutamate, to transform pyruvate into L-alanine and to convert L-glutamate to -ketoglutarate. The amount of NADH produced 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 reag
29、ents recognized analytical grade. The water used in the preparation of the enzyme solutions shall be of at least doubly glass-distilled 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
30、) = 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 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 8069:2007 (E)54.3 Sodiu
31、m hydroxide solutions 4.3.1 Sodium hydroxide solution I, c(NaOH) = 10 mol/l. Dissolve 400 g of sodium hydroxide in water. Dilute with water 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
32、. 4.4 Glycerol solution (C3H8O3), with a volume fraction of 50 % glycerol. 4.5 Ammonium sulfate solution, c(NH4)2SO4 = 3,2 mol/l. Dissolve 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
33、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 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 sol
34、ution (NAD). Dissolve 350 mg of nicotinamide adenine dinucleotide (C21H27N7O14P2) in 10 ml of water. This solution may be kept for 4 weeks 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
35、), from hog muscle suspension. Dissolve 10 mg of L-lactate dehydrogenase suspension in 1 ml of glycerol solution (4.4). The pH of the obtained 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.
36、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 +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. Di
37、ssolve 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 specific 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. Th
38、e 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, keep 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
39、sulfate solution (4.5). The pH of the obtained suspension should be about 7. The specific activity of the glutamate pyruvate transaminase (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 8069:2007 (E)6Add 1,0 ml of ammonium sulfate
40、 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 acceleration 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
41、 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. 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 Di
42、ssolve 50 mg of lithium D-lactate (C3H5O3Li) in water. Dilute with water to 500 ml and mix. 5 Apparatus Usual laboratory equipment and, in particular, 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.
43、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, 0,05 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 diame
44、ter 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 Plastic 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
45、 optical path length 1 cm. 5.12 ParafilmTM1). 6 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method
46、 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 endo
47、rsement by ISO or IDF of this product. EN ISO 8069:2007 (E)77 Preparation 7.1 Preparation of test sample Transfer the test sample to a container with capacity about twice the volume of the sample and provided with an airtight lid. Close the container immediately. Mix the sample thoroughly by repeate
48、dly shaking and inverting the container. During preparation, avoid exposure of the test sample to the atmosphere in order to minimize adsorption of water. 7.2 Test portion Weigh, to the nearest 1 mg, 1,0 g of the test sample in a 50 ml glass beaker (5.2). 7.3 Blank test Carry out a blank test by pro
49、ceeding as specified in 7.4 and 8.2, using all reagents but omitting the test portion. 7.4 Preparation of solution and deproteination 7.4.1 Dissolve the test portion (7.2) in about 20 ml of water preheated to between 40 C and 50 C, while stirring with the glass rod (5.9) or suitable means. Transfer the contents of the glass beaker quantitatively to a 100 ml one-mark volumetric flask (5.4) by rinsing the beaker with water. Cool the contents of the flask to about 20 C. 7.4.2 Add to the solution (7.4.1), in the following
