1、 Reference numbers ISO 8069:2005(E) IDF 69:2005(E) ISO and IDF 2005INTERNATIONAL STANDARD ISO 8069 IDF 69 Second edition 2005-09-15 Dried milk Determination of content of lactic acid and lactates Lait sec Determination de la teneur en acide lactique et en lactates ISO 8069:2005(E) IDF 69:2005(E) PDF
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6、er ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightis
7、o.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2005 All rights reservedISO 8069:2005(E) IDF 69:2005(E) ISO and IDF 2005 All rights reserved iii Contents Page Foreword iv 1 Scope . 1 2 Terms and definitions. 1 3 Principle. 1 4 Reagents 1 5 App
8、aratus 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 activity of reagents. 4 8.2 Determination 5 9 Calculation and expression of results 6 9.1 Calculation.
9、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 the performance of enzymatic analyses. 9 Bibliography . 13 ISO 8069:2005(E) IDF 69:2005(E) iv ISO a
10、nd IDF 2005 All rights reservedForeword 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 technical committees. Each member body intere
11、sted 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 closely with the International Electrotechnical
12、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. Draft International Standards adopted by the tec
13、hnical 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 document may be the subject of patent rights. ISO
14、 shall not be held responsible for identifying any or all such patent rights. ISO 8069 IDF 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 being published jointly by ISO and IDF. This edit
15、ion of ISO 8069 IDF 69 cancels and replaces ISO 8069:1986, which has been technically revised. ISO 8069:2005(E) IDF 69:2005(E) ISO and IDF 2005 All rights reserved v Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every mem
16、ber country. 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 Act
17、ion Teams 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 t
18、he subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 8069 IDF 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 jo
19、intly by IDF 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 8069 IDF 69 cancels and replaces IDF 69:1987, which
20、 has been technically revised. INTERNATIONAL STANDARD ISO 8069:2005(E) IDF 69:2005(E) ISO and IDF 2005 All rights reserved 1 Dried 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
21、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 International Standard NOTE It is expressed as mill
22、igrams 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, added simultaneously, but acting in sequence: a)
23、 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 the presence of L-glutamate, to transform pyruvat
24、e 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 reagents recognized analytical grade. The water used
25、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(K 4 Fe(CN) 6 3H 2 O) = 35,9 g/l. Dissolve 35,9 g of potassium
26、hexacyanoferrate(II) trihydrate in water. Dilute with water to 1 000 ml and mix. 4.2 Zinc sulfate solution, c(ZnSO 4 7H 2 O) = 71,8 g/l. Dissolve 71,8 g of zinc sulfate heptahydrate in water. Dilute with water to 1 000 ml and mix. ISO 8069:2005(E) IDF 69:2005(E) 2 ISO and IDF 2005 All rights reserve
27、d4.3 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 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
28、ml and mix. 4.4 Glycerol solution (C 3 H 8 O 3 ), with a volume fraction of 50 % glycerol. 4.5 Ammonium sulfate solution, c(NH 4 ) 2 SO 4 = 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 (
29、C 4 H 8 N 2 O 3 ) and 1,47 g of L-glutamic acid (C 5 H 9 NO 4 ) 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 Ni
30、cotinamide adenine dinucleotide solution (NAD). Dissolve 350 mg of nicotinamide adenine dinucleotide (C 21 H 27 N 7 O 14 P 2 ) 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 cr
31、ushed 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 obtained suspension should be about 7. The specific activity of the L-lactate dehydrogenase (L-LDH, EC 1.1.1.27) suspensi
32、on 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 +5 C. When the suspension is being used, keep the vessel immersed in crushed ice. 4.9 D-Lactate dehydrogenase (D-LDH),
33、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 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.
34、 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, keep the vessel immersed in crushed ice. 4.10 Glutamate pyruvate transaminase (GPT), from pig heart suspension. Dissolve 2
35、0 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 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. ISO 80
36、69:2005(E) IDF 69:2005(E) ISO and IDF 2005 All rights reserved 3 Add 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 acceleration of 4 000 g for 10 min. Transfer 1,0 ml of the clear s
37、upernatant 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. 4.11 Lithium L-lactate solution Dissolve 50 mg of lithium L-lacta
38、te (C 3 H 5 O 3 Li) in water. Dilute with water to 500 ml and mix. 4.12 Lithium D-lactate solution Dissolve 50 mg of lithium D-lactate (C 3 H 5 O 3 Li) in water. Dilute with water to 500 ml and mix. 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, cap
39、able 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, 0,05 ml, 0,2 ml, 1,0 ml and 2,0 ml. 5.6 Graduated pipet
40、tes, 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 Plastic paddles, capable of mixing the sample-enzyme mixtur
41、e in the spectrometric cell. 5.11 Spectrophotometer, capable of measuring at 340 nm, equipped with cells of optical path length 1 cm. 5.12 Parafilm TM1) . 6 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storag
42、e. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707 IDF 50. Store the sample in such a way that deterioration and change in composition are prevented. 1) Parafilm TMis an example of a product available commercially. This i
43、nformation is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF of this product. ISO 8069:2005(E) IDF 69:2005(E) 4 ISO and IDF 2005 All rights reserved7 Preparation 7.1 Preparation of test sample Transfer the test sample to a conta
44、iner with capacity about twice the volume of the sample and provided with an airtight lid. Close the container immediately. Mix the sample thoroughly by repeatedly shaking and inverting the container. During preparation, avoid exposure of the test sample to the atmosphere in order to minimize adsorp
45、tion 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 proceeding 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
46、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 conte
47、nts of the flask to about 20 C. 7.4.2 Add to the solution (7.4.1), in the following order, 5,0 ml of potassium hexacyanoferrate(II) solution (4.1), 5,0 ml of zinc sulfate solution (4.2) and 10,0 ml of sodium hydroxide solution II (4.3.2), swirling thoroughly after each addition. Dilute with water to
48、 the 100 ml mark. Mix thoroughly and allow the mixture to stand at room temperature for 30 min. 7.4.3 Filter through a filter paper (5.8), discarding the first fraction of the filtrate. Use of a centrifuge is a suitable alternative to filtration. 8 Procedure CAUTION Avoid contamination, especially w
49、ith perspiration. 8.1 Test to check the activity of reagents 8.1.1 Whenever a new batch of reagents (4.6 to 4.10 inclusive) is prepared, or when such reagents have been kept in a refrigerator without being used for more than 2 weeks, or when restarting analytical work after a period of analytical inactivity, or whenever other conditions may justify it, perform the following test for the recovery of lactates. 8.1.2 Pipette 10 ml of lithium L-lactate solution (4.11) into each of two 100 ml one-mark volumetric f
