1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58alkaline phosphatase activity Enzymatic photo-activated system (EPAS) methodThe European Standard E
2、N ISO 22160:2007 has the status of a British StandardICS 67.100.10Milk and milk-based drinks Determination of BRITISH STANDARDBS EN ISO 22160:2007BS EN ISO 22160:2007This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 May 2007 BSI 2007ISBN 978
3、 0 580 50651 2Amendments issued since publicationAmd. No. Date Commentscontract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations. National forewordThis British Standard was published by BSI. It is the UK implementatio
4、n of EN ISO 22160:2007.The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products.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
5、 necessary provisions of a EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 22160April 2007ICS 67.100.10 English VersionMilk and milk-based drinks - Determination of alkalinephosphatase activity - Enzymatic photo-activated system(EPAS) method (ISO 22160:2007)Lait et boissons base de lait - Dter
6、mination de lactivitde la phosphatase alcaline - Mthode par un systme dephotoactivation enzymatique (ISO 22160:2007)Milch und flssige Milcherzeugnisse - Bestimmung derAktivitt der alkalischen Phosphatase - Verfahren miteinem enzymatisch photoaktivierten System (EPAS) (ISO22160:2007)This European Sta
7、ndard 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 EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning suc
8、h nationalstandards 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 translationunder the responsibility of a CEN member into its own language a
9、nd notified to the CEN Management Centre has the same status as theofficial 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
10、, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, 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 ex
11、ploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 22160:2007: EForeword 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 mi
12、lk 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 October 2007, and conflicting national standards shall be
13、withdrawn at the latest by October 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, Germany, G
14、reece, 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 mo
15、difications. EN ISO 22160:2007Reference numbersISO 22160:2007(E)IDF 209:2007(E)INTERNATIONAL STANDARD ISO22160IDF209First edition2007-04-01Milk and milk-based drinks Determination of alkaline phosphatase activity Enzymatic photo-activated system (EPAS) method Lait et boissons base de lait Dterminati
16、on de lactivit de la phosphatase alcaline Mthode par un systme de photoactivation enzymatique EN ISO 22160:2007ii iiiContents Page Foreword iv 1 Scope . 1 2 Terms and definitions. 1 3 Principle. 1 4 Reagents 2 5 Apparatus 3 6 Sampling 3 7 Preparations 4 7.1 Test samples . 4 7.2 Alkaline phosphatase-
17、free milk. 4 8 Procedure (see Annex A) 4 8.1 Calibration . 4 8.2 Calibration adjustment. 6 8.3 Control tests and calibration check 9 8.4 Determination 10 8.5 Heat-resistant microbial alkaline phosphatase control 10 9 Calculation and expression of results 10 9.1 General. 10 9.2 Manual calculation 10
18、9.3 Expression of results . 11 10 Precision 11 10.1 Interlaboratory test . 11 10.2 Repeatability 11 10.3 Reproducibility 12 11 Test report . 12 Annex A (informative) Procedure flowchart 13 Annex B (informative) Results of interlaboratory trials 14 Bibliography . 16 EN ISO 22160:2007iv Foreword ISO (
19、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 interested in a subject for which a technical commi
20、ttee 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 Commission (IEC) on all matters of electrotec
21、hnical 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 technical committees are circulated to the membe
22、r 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 shall not be held responsible for identifyin
23、g any or all such patent rights. ISO 22160IDF 209 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. EN ISO 22160:2007vForeword IDF (the International Dai
24、ry 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. IDF collaborates with ISO in the development of standard methods of
25、 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 requires approval by at least 50 % of the IDF National Committees castin
26、g 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 prepared by the International Dairy Federation (IDF) and Technical Com
27、mittee 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 components a decrease in background will cause a corresponding inc
28、rease 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 u (3) where NA1is the numerical value of the adjustment to bring th
29、e 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 A2is out of range: ( )2m A245 110ANuu (4) where NA2is the numerical
30、 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. EN ISO 22160:20077c) Use Equation (5) when either calibrator B1or B2is out of range: ( )mB145
31、 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 calibrator B1or B2; 205 is the upper limit value for calibrator B1or B2. After det
32、ermining 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): 1pro 2x xR XRuu and (6) pro m ABXXN= (7) where Rx1is the lower limit of
33、 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); NABis the numerical value of the adjustment for calibrator A1, A2, B1or B2
34、 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 ABBBN=+ (8) where Bg1is the new luminometer background value; Bgis the curren
35、t 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 target values (A1= 44 mU/l or A2= 88 mU/l, and B1and B2= 175 mU/l). Average the
36、 obtained two ratios for calibrators A and B to determine a mean correction ratio as show in Table 1. EN ISO 22160:20078 Table 1 Calibrator ratio Calibrator Target value Mean value Ratio (mean/target) A144 A1mRA1= A1m/ 44 A288 A2mRA2= A2m/ 88 B1or B2175 B1m or B2mRB= B1mor B2m/ 175 Mean ratio (Rm) R
37、m=(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 correction (see 8.1.7); Rmis the mean ratio of calibrators A and B (see Table 1);
38、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 significantly greater (more than 10 units) than the target value, then determine th
39、e 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 and the calculation should result in a decrease in background. a) Calculate the
40、 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 value of calibrator A1(see 8.1.11); 44 is the target value of calibrator A1. b) Cal
41、culate 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 the mean value of calibrator A2(see 8.1.11); 88 is the target value of calibrator
42、 A2. EN ISO 22160:20079c) 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). Multiply this ratio by the luminometer correction according to Equation (12): m(1 or 2)r
43、2 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 of calibrator B1or B2. Adjust the luminometer correction to the new value. 8.2.2 Repeat
44、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 any one value is greater or less than 40 % of the mean value. The mean value for workin
45、g 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 step 8.3.3. If either calibrator mean value is still out of range, follow step 8.1.1. 8.3
46、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 test daily to verify the luminometer calibration and reagent performance. The system is v
47、erified (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 (4.4) with previously tested milk (dairy drink) showing a mean alkaline phosphatase ac
48、tivity 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 reagent performance. If the value determined exceeds 585 mU/l or is less than 300 mU/l,
49、 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 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 r
