1、August 2007DEUTSCHE NORM English price group 13No 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 67.100.01!$Hu;“1378224w
2、ww.din.deDDIN EN ISO 22160Milk and milk-based drinks Determination of alkaline phosphatase activity Enzymatic photo-activated system (EPAS) method (ISO 22160:2007)English version of DIN EN ISO 22160:2007-08Milch und flssige Milcherzeugnisse Bestimmung der Aktivitt der alkalischen Phosphatase Verfahr
3、en mit einem enzymatisch photoaktivierten System (EPAS) (ISO 22160:2007)Englische Fassung DIN EN ISO 22160:2007-08www.beuth.deDocument comprises 22 pages 10.07DIN EN ISO 22160:2007-08 2 National foreword This standard falls in the domain of Technical Committee CEN/TC 302 “Milk and milk products Meth
4、ods of sampling and analysis” (Secretariat: NEN, The Netherlands). This standard has been prepared by Subcommittee SC 5 “Agricultural food products” (Secretariat: NEN, The Netherlands) of Technical Committee ISO/TC 34 “Milk and milk products” (Secretariat: MSZT, Hungary) in collaboration with Techni
5、cal Committee CEN/TC 302. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Committee). Based on the results of parallel voting, ISO 22160 has been adopted as a European Standa
6、rd. The DIN Standards corresponding to the International Standards referred to in the EN are as follows: ISO 707 DIN EN ISO 707 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 ISO 11816-1 DIN EN ISO 11816-1 ISO 11816-2 DIN EN ISO 11816-2 National Annex NA (informative) Bibliography DIN EN ISO 70
7、7, Milk and milk products Guidance on sampling 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 for the determinati
8、on of repeatability and reproducibility of a standard measurement method DIN EN ISO 11816-1, Milk and milk products Determination of alkaline phosphatase activity Part 1: Fluorimetric method for milk and milk-based drinks DIN EN ISO 11816-2, Milk and milk products Determination of alkaline phosphata
9、se activity Part 2: Fluorometric method for cheese EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 22160 April 2007 ICS 67.100.10 Supersedes EN ISO 11816-1:2000 English Version Milk and milk-based drinks - Determination of alkaline phosphatase activity - Enzymatic photo-activated system (EP
10、AS) method (ISO 22160:2007) Lait et boissons base de lait - Dtermination de lactivit de la phosphatase alcaline - Mthode par un systme de photoactivation enzymatique (ISO 22160:2007) Milch und flssige Milcherzeugnisse - Bestimmung der Aktivitt der alkalischen Phosphatase - Verfahren mit einem enzyma
11、tisch photoaktivierten System (EPAS) (ISO 22160:2007) This European Standard was approved by CEN on 6 March 2007. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any a
12、lteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management 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
13、translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Ge
14、rmany, 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 STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMU
15、NG Management Centre: rue de Stassart, 36 B-1050 Brussels 2007 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 22160:2007: E2 EN ISO 22160:2007 (E) Contents Page Foreword3 1 Scope 5 2 Terms and definitions .5 3 Principle5 4 Rea
16、gents.6 5 Apparatus .7 6 Sampling.7 7 7.1 Test samples 8 7.2 Alkaline phosphatase-free milk8 8 Procedure (see Annex A) .8 8.1 Calibration 8 8.2 8.3 8.4 Determination 14 8.5 9 Calculation and expression of results 14 9.1 General. 14 9.2 Manual calculation 14 9.3 Expression of results . 15 10 Precisio
17、n 15 10.1 Interlaboratory test . 15 10.2 Repeatability 15 10.3 Reproducibility 16 11 Test report . 16 Annex A (informative) Procedure flowchart 17 Annex B (informative) Results of interlaboratory trials 18 Bibliography. 20 Calibration adjustment. 10 Heat-resistant microbial alkaline phosphatase cont
18、rol 14 Control tests and calibration check 13 Preparations.8 Foreword This document (EN ISO 22160:2007) has been prepared by Technical Committee ISO/TC 34 “Agricultural food products” in collaboration with Technical Committee CEN/TC 302 “Milk and milk products - Methods of sampling and analysis”, th
19、e 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 October 2007, and conflicting national standards shall be withdrawn at the latest by October 2007. This docum
20、ent supersedes EN ISO 11816-1:2000. 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, Germany, Greece
21、, 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 22160:2007 has been approved by CEN as EN ISO 22160:2007 without any modific
22、ations. 3 EN ISO 22160:2007 (E) 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 out the technical work.
23、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 International Standard requi
24、res 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. ISO 22160IDF 209 was pre
25、pared 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 Heat treatment, of the Standing Committee on Minor
26、 components a decrease in background will cause a corresponding increase to the mean value of each calibrator). In this case, change the luminometer background to this new background setting according to the following equations. a) Use Equation (3) when calibrator A1is out of range: ()1m A132 55ANu
27、u (3) where NA1is the numerical value of the adjustment to bring the mean value of calibrator A1into range; A1mis the mean value of calibrator A1 (calculated in 8.1.11); 32 is the lower limit value for calibrator A1; 55 is the upper limit value for calibrator A1. b) Use Equation (4) when calibrator
28、A2is out of range: ( )2m A245 110ANuu (4) where NA2is the numerical value of the adjustment to bring thecalibrator A2mean value into range; A2mis the mean value of calibrator A2; 45 is the lower limit value for calibrator A2; 110 is the upper limit value for calibrator A2. 10 EN ISO 22160:2007 (E) c
29、) Use Equation (5) when either calibrator B1or B2is out of range: ( )mB145 205BNuu (5) where NBis the numerical value of the adjustment to bring the mean value of calibrator B1or B2into range; Bmis the mean value of calibrator B1or B2 (calculated in 8.1.12); 145 is the lower limit value for calibrat
30、or B1or B2; 205 is the upper limit value for calibrator B1or B2. After determining NABfrom Equation (3), (4) or (5), verify whether the other calibrator, X calibrator B1or B2if Equation (3) or (4) was used, or calibrator A1or A2if Equation (5) was used, will stay in range, using Equations (6) and (7
31、): 1pro 2x xR XRuu and (6) pro m ABXXN= (7) where Rx1is the lower limit of the mean of other calibrator X; Rx2is the upper limit of the mean other calibrator X; Xprois the projected mean of calibrator X after NABadjustment; Xmis the mean value of other calibrator X (calculated in 8.1.11 or 8.1.12);
32、NABis the numerical value of the adjustment for calibrator A1, A2, B1or B2 calculated from Equation (3), (4) or (5). If the calibrator, X, is in the specified range, then adjust the luminometer background value with the NABvalue to bring the calibrators into range according to Equation (8): g1 g ABB
33、BN=+ (8) where Bg1is the new luminometer background value; Bgis the current luminometer background value. 8.2.1.2 In cases where the mean values of one calibrator are significantly outside the specified ranges (greater than 10), divide the obtained mean value of the calibrator concerned by its targe
34、t values (A1= 44 mU/l or A2= 88 mU/l, and B1and B2= 175 mU/l). Average the obtained two ratios for calibrators A and B to determine a mean correction ratio as show in Table 1. 11 EN ISO 22160:2007 (E) Table 1 Calibrator ratio Calibrator Target value Mean value Ratio (mean/target) A144 A1mRA1= A1m/ 4
35、4 A288 A2mRA2= A2m/ 88 B1or B2175 B1m or B2mRB= B1mor B2m/ 175 Mean ratio (Rm) Rm=(RA1or RA2)+ RB/2 Multiply the obtained mean ratio for calibrators A and B by the luminometer correction using Equation (9): r1 r mCCR= (9) where Cr1is the new luminometer correction; Cris the calculated luminometer co
36、rrection (see 8.1.7); Rmis the mean ratio of calibrators A and B (see Table 1); Change the thus-obtained new luminometer correction to its new value, Cr1. 8.2.1.3 In cases where the mean value is significantly less (less than 10 units) than the target value and the other calibrator mean value is sig
37、nificantly greater (more than 10 units) than the target value, then determine the difference between the mean value of calibrator A (A1= 44 mU/l or A2= 88 mU/l). Add this value to the background value of the luminometer; if the value is less than the target value, the difference is a negative number
38、 and the calculation should result in a decrease in background. a) Calculate the new background value Bg2 for calibrator A1, by using Equation (10): ()g2 g 1m44BBA=+ (10) where Bgis the calculated luminometer background value (see 8.1.8) Bg2is the new luminometer background value; A1mis the mean val
39、ue of calibrator A1(see 8.1.11); 44 is the target value of calibrator A1. b) Calculate the new background value Bg3 for calibrator A2, by using Equation (11): ( )g3 g 2m88BBA=+ (11) where Bgis the calculated luminometer background value (see 8.1.8) Bg3is the new luminometer background value; A2mis t
40、he mean value of calibrator A2(see 8.1.11); 88 is the target value of calibrator A2. 12 EN ISO 22160:2007 (E) c) Adjust the luminometer background setting as calculated. Reassay calibrator B and determine the mean value Bm(1 or 2). Divide the obtained mean by the target value (B1and B2= 175 mU/l). M
41、ultiply this ratio by the luminometer correction according to Equation (12): m(1 or 2)r2 r175BCC= (12) where Cris the calculated luminometer correction (see 8.1.7) Cr2is the new luminometer correction; Bm(1or2)is the mean value of calibrator B1 or B2measured in the reassay; 175 is the target value o
42、f calibrator B1or B2. Adjust the luminometer correction to the new value. 8.2.2 Repeat steps 8.1.11 to 8.1.13. The mean readings of the luminometer shall be between 32 mU/l and 55 mU/l for working calibrator A1, and between 45 mU/l and 110 mU/l for working calibrator A2. Repeat the determination if
43、any one value is greater or less than 40 % of the mean value. The mean value for working calibrator B (either B1or B2) shall be between 145 mU/l and 205 mU/l. Repeat the determination if any one value varies by more than 30 % of the mean value. If both calibrator mean values are in range, follow ste
44、p 8.3.3. If either calibrator mean value is still out of range, follow step 8.1.1. 8.3 Control tests and calibration check 8.3.1 Negative control test Perform a negative control test according to the determination procedure (8.4) without adding the test portion (8.4.2). Perform a negative control te
45、st daily to verify the luminometer calibration and reagent performance. The system is verified (negative result) when the obtained alkaline phosphatase activity levels are less than 5 mU/l in the appropriately calibrated luminometer channel. 8.3.2 Positive control test Rehydrate the positive control
46、 (4.4) with previously tested milk (dairy drink) showing a mean alkaline phosphatase activity of less than 5 mU/l (less than 15 mU/l with flavoured-milk products or cream products) or the prepared negative sample (7.2). Perform a positive control test daily to verify the luminometer calibration and
47、reagent performance. If the value determined exceeds 585 mU/l or is less than 300 mU/l, repeat with another rehydrated positive control. If the thus-obtained value is still out of range, recalibrate the luminometer (see 8.1). 8.3.3 Calibration check 8.3.3.1 After performing the calibration steps in
48、8.1 and 8.2, proceed with the negative control test (8.3.1) and triplicate determination of the positive control (8.3.2) to verify the calibration result. 8.3.3.2 The negative control shall give a value of less than 5 mU/l and the positive control (N = 3) mean value test shall be in the range 380 mU
49、/l to 510 mU/l. If the negative control value is out of range, use a new substrate (4.1) and repeat the test. If the positive control value is out of range, or any individual value exceeds 585 mU/l or is less than 300 mU/l, check the temperature of the stopping solution (4.2) and repeat the test with another rehydrated positive control. If the thus-obtained value is still out of range on retests, recalibrate (see 8.1.1). 13 EN ISO 22160:2007 (E) NOTE Triplicate determination of the positive control is used for verification of calibr
