1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58acid content Enzymatic methodICS 67.100.30Cheese and processed cheese products Determination of cit
2、ric DRAFT FOR DEVELOPMENTDD ISO/TS 2963:2006DD ISO/TS 2963:2006This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 29 September 2006 BSI 2006ISBN 0 580 49110 2an international Standard, to extend the life of the Technical Specification or to
3、 withdraw it. Comments should be sent to the Secretary of the responsible BSI Technical Committee at British Standards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products.A list of
4、 organizations represented on AW/5 can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Amendments issued since publicationAmd. No. Date Commentsthat UK experience can be
5、 reported to the international organization responsible for its conversion to an international standard. A review of this publication will be initiated not later than 3 years after its publication by the international organization so that a decision can be taken on its status. Notification of the st
6、art of the review period will be made in an announcement in the appropriate issue of Update Standards.According to the replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into National forewordThis Draft for Development was pu
7、blished by BSI. It is the UK implementation of ISO/TS 2963:2006. It supersedes BS 770-7:1997 which is withdrawn.This publication is not to be regarded as a British Standard. It is being issued in the Draft for Development series of publications and is of a provisional nature. It should be applied on
8、 this provisional basis, so that information and experience of its practical application can be obtained.Comments arising from the use of this Draft for Development are requested so Reference numbersISO/TS 2963:2006(E)IDF/RM 34:2006(E)TECHNICAL SPECIFICATION ISO/TS2963IDF/RM34First edition2006-05-01
9、Cheese and processed cheese products Determination of citric acid content Enzymatic method Fromages et fromages fondus Dtermination de la teneur en acide citrique Mthode enzymatique DD ISO/TS 2963:2006ii iiiContents Page Foreword iv Foreword. v Introduction . vi 1 Scope . 1 2 Terms and definitions.
10、1 3 Principle. 1 4 Reagents 1 5 Apparatus 3 6 Sampling 3 7 Preparation of test sample. 3 8 Procedure 4 8.1 Check tests 4 8.2 Test portion . 4 8.3 Reagent blank test 4 8.4 Deproteination. 5 8.5 Determination 5 9 Calculation and expression of results 6 9.1 Calculation. 6 9.2 Expression of results . 7
11、10 Precision 7 10.1 Interlaboratory test . 7 10.2 Repeatability 7 10.3 Reproducibility 7 11 Test report . 7 Annex A (normative) Good Laboratory Practice (GLP) rules for the performance of enzymatic analyses. 8 Bibliography . 12 DD ISO/TS 2963:2006iv Foreword ISO (the International Organization for S
12、tandardization) 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 interested in a subject for which a technical committee has been established has the ri
13、ght 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 Commission (IEC) on all matters of electrotechnical standardization. Internationa
14、l 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 technical committees are circulated to the member bodies for voting. Publication as
15、an International Standard requires approval by at least 75 % of the member bodies casting a vote. In other circumstances, particularly when there is an urgent market requirement for such documents, a technical committee may decide to publish other types of normative document: an ISO Publicly Availab
16、le Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the
17、members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further three years, revised to become an International S
18、tandard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn. Attention is drawn to the possibility that some of the elements of this document may be the subj
19、ect of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO/TS 2963IDF/RM 34 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 joi
20、ntly by ISO and IDF. This edition of ISO/TS 2963IDF/RM 34 cancels and replaces ISO 2963:1997, which has been technically revised. DD ISO/TS 2963:2006vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Eve
21、ry 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. The main task of technical committees is to prepare Internationa
22、l Standards. Draft International Standards adopted by the Action 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. In other circumstances, part
23、icularly when there is an urgent market requirement for such documents, a Standing Committee may decide to publish an other type of normative document which is called by IDF: Reviewed method. Such a method represents an agreement between the members of a Standing Committee and is accepted for public
24、ation if it is approved by at least 50 % of the committee members casting a vote. A Reviewed method is equal to an ISO/PAS or ISO/TS and will, therefore, also be published jointly under ISO conditions. Attention is drawn to the possibility that some of the elements of this document may be the subjec
25、t of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO/TS 2963IDF/RM 34 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 jointl
26、y 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 C. Hughes (NZ). This edition of ISO/TS 2963IDF/RM 34 cancels and replaces IDF 34C:1992, wh
27、ich has been technically revised. DD ISO/TS 2963:2006vi Introduction The method described in ISO 2963:1997 and IDF 34C:1992 did not fulfil the requirements for a fully validated International Standard. No new interlaboratory tests could be organized with the method according to ISO 5725-1 and ISO 57
28、25-2 due to a lack of participants, hence the publication of this revision as a Technical Specification/Reviewed method. Reliable results with enzymatic methods will only be obtained if the good laboratory practice (GLP) rules for such analyses are applied strictly. These GLP rules are given in Anne
29、x A. DD ISO/TS 2963:20061Cheese and processed cheese products Determination of citric acid content Enzymatic method 1 Scope This Technical Specification specifies an enzymatic method for the determination of the citric acid content of cheese and processed cheese products. 2 Terms and definitions For
30、 the purposes of this document, the following terms and definitions apply. 2.1 citric acid content mass fraction of substances determined by the procedure described in this Technical Specification NOTE The citric acid content is expressed as mass fraction in percent. 3 Principle An extract of the sa
31、mple is treated with the following enzymes and biochemical substances: a) citrate lyase (CL) to convert citric acid to oxalacetate and acetate; b) malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), in the presence of reduced nicotinamide adenine dinucleotide (NADH), to catalyse the reductio
32、n of oxalacetate and its decarboxylation product pyruvate to L-malate and L-lactate, respectively, with the subsequent conversion of NADH to its oxidized form (NAD+). The decrease in concentration of NADH is determined by measurement of the absorbance of the test solution at 340 nm. The citric acid
33、content is proportional to the decrease in NADH concentration. 4 Reagents Use only reagents of recognized analytical grade and distilled water or water of at least equivalent purity, unless otherwise specified. Take note of the production and expiry dates given by the manufacturer of the reagents. 4
34、.1 Enzymes If an enzyme suspension is applied with other than the prescribed activity, the volume of the suspension stated in the pipetting scheme (8.5.1) shall be increased or decreased proportionally. The reagents described in 4.7 to 4.10 inclusive may be obtained commercially as a test combinatio
35、n. 4.2 Trichloroacetic acid solution (CCl3COOH). Dissolve 200,0 g of trichloroacetic acid in water. Dilute to 1 000 ml with water and mix. DD ISO/TS 2963:20062 4.3 Sodium hydroxide solution I, c(NaOH) = 5,0 mol/l. Dissolve 200,0 g of sodium hydroxide in water. Dilute to 1 000 ml with water and mix.
36、4.4 Sodium hydroxide solution II, c(NaOH) = 1,0 mol/l. Dissolve 40,0 g of sodium hydroxide in water. Dilute to 1 000 ml with water and mix. 4.5 Sodium hydroxide solution III, c(NaOH) = 0,1 mol/l. Dissolve 4,0 g of sodium hydroxide in water. Dilute to 1 000 ml with water and mix. 4.6 Zinc chloride so
37、lution, c(ZnCl2) = 800 mg/l. Dissolve 800 mg of zinc chloride in water. Dilute to 1 000 ml with water and mix. 4.7 Buffer solution, pH 7,8. Dissolve 71,3 g of glycylglycine in about 700 ml of water. Adjust to pH 7,8 with sodium hydroxide solution I (4.3). Add 100 ml of zinc chloride solution (4.6).
38、Dilute to 1 000 ml with water and mix. The buffer solution may be kept for 4 weeks if stored in a refrigerator at between 0 C and +5 C. 4.8 Reduced nicotinamide adenine dinucleotide solution Dissolve 50 mg of reduced nicotinamide adenine dinucleotide disodium salt (C21H27N7O14P2Na2) and 100 mg of so
39、dium hydrogen carbonate (NaHCO3) in 10 ml of water. The reduced nicotinamide adenine dinucleotide solution may be kept for 4 weeks if stored in a refrigerator at between 0 C and +5 C. 4.9 Malate dehydrogenase/lactate dehydrogenase suspension Mix suitable amounts of malate dehydrogenase (MDH from pig
40、 heart; suspension in ammonium sulfate solution, 3,2 mol/l, at pH 6,0 0,2; EC 1.1.1.37)1)and lactate dehydrogenase (LDH from rabbit muscle; suspension in ammonium sulfate solution, 3,2 mol/l, at pH 7 0,2; EC 1.1.1.27). Dilute with ammonium sulfate solution (3,2 mol/l) so as to obtain a final suspens
41、ion containing about 600 units2)of MDH/ml and 1 400 units2)of LDH/ml. The malate dehydrogenase/lactate dehydrogenase suspension may be kept for 1 year if stored in a refrigerator at between 0 C and +5 C. 4.10 Citrate lyase solution Dissolve a suitable amount of citrate lyase lyophilisate (CL) from A
42、erobacter aerogenes; EC 4.1.3.6 in ice-cold water so as to obtain a solution containing 40 units/ml 2). The citrate lyase solution may be kept for 1 week if stored at between 0 C and +5 C, and for 4 weeks if stored at 20 C. 4.11 Citric acid standard solution. Dissolve 1,600 g of citric acid monohydr
43、ate (C6H8O7H2O) in water. Dilute to 1 000 ml with water and mix. 1) The EC number refers to the Enzyme Classification number as given in Reference 4. 2) This unit (often called International or Standard Unit) is defined as the amount of enzyme which will catalyse the transformation of 1 mol of subst
44、rate per minute under standard conditions. DD ISO/TS 2963:200635 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 pH meter. 5.3 Glass beakers, of capacity 50 ml. 5.4 Macerator, e
45、quipped with a suitable beaker. 5.5 One-mark volumetric flasks, of capacity 100 ml. 5.6 Pipettes, capable of delivering 0,02 ml, 1 ml, 2 ml, 5 ml, 25 ml and 40 ml, respectively. 5.7 Graduated pipettes, capable of delivering 10 ml, graduated in 0,1 ml divisions. 5.8 Measuring cylinder, of capacity 50
46、 ml. 5.9 Filter funnel, of diameter approximately 7 cm. 5.10 Filter paper, medium grade, of diameter approximately 15 cm. 5.11 Spectrometer, suitable for measuring at a wavelength of 340 nm, equipped with cells of optical path length 1 cm. 5.12 Plastic paddles, suitable for mixing the sample-enzyme
47、mixture in the spectrometer cell. 5.13 Water bath, capable of maintaining a temperature of 20 C to 25 C, with rack suitable for holding the spectrometer cell (5.11) during the incubation period (optional; see 8.5.1). Incubation of the cells in the water bath is only necessary if the room temperature
48、 is below 20 C. 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 Technical Specification. A recommended sampling method is given in ISO 707|IDF 50.
49、7 Preparation of test sample Prepare a homogeneous sample taking care to avoid loss of moisture, using the following procedure. a) In the case of cheese, remove the rind or mouldy surface layer of the cheese in such a way as to provide a sample representative of the cheese as it is usually consumed. Grind or grate the sample by using an appropriate device, mix the ground or grated mass quickly and, if possible, grind or grate a second time and again mix thoroughly by intensive stirring and kneading. b) In
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