1、BS EN ISO30024:2009ICS 65.120NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal feeding stuffs Determination ofphytase activity (ISO30024:2009)This British Standardwas published underthe authority of theStandards Policy andStrategy Committee on 30September 2
2、009 BSI 2009ISBN 978 0 580 62651 7Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 30024:2009National forewordThis British Standard is the UK implementation of EN ISO 30024:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuf
3、fs.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom
4、legal obligations.EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 30024July 2009ICS 65.120English VersionAnimal feeding stuffs - Determination of phytase activity (ISO30024:2009)Aliments des animaux - Dtermination de lactivitphytasique (ISO 30024:2009)Futtermittel - Bestimmung der Phytaseaktiv
5、itt (ISO30024:2009)This European Standard was approved by CEN on 17 June 2009.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 bibli
6、ographical 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 versions (English, French, German). A version in any other language made by translationunder the responsibility of
7、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, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland,
8、Italy, Latvia, Lithuania, Luxembourg, 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: Avenue Marnix 17, B-1000
9、Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 30024:2009: EBS EN ISO 30024:2009EN ISO 30024:2009 (E) 3 Foreword This document (EN ISO 30024:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding
10、 stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN, in collaboration with Technical Committee ISO/TC 34 “Food products“. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the lat
11、est by January 2010, and conflicting national standards shall be withdrawn at the latest by January 2010. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all
12、such patent rights. 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, Hungary, Icela
13、nd, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN ISO 30024:2009ISO 30024:2009(E) ISO 2009 All rights reserved iiiContents Page Foreword iv Introduction v 1 Scope . 1
14、2 Terms and definitions. 1 3 Principle. 1 4 Reagents 2 5 Apparatus 3 6 Sampling 4 7 Sample preparation 4 8 Procedure 4 8.1 Blank solutions . 4 8.2 Standards 4 8.3 Standard curve 5 8.4 Phytase level control 5 8.5 Feed test portions. 6 9 Calculations. 7 9.1 Formulation standard curve 7 9.2 Calculation
15、 phytase activity 8 9.3 Correction for phytic acid purity and water content . 9 9.4 Interference with blank values 9 10 Precision 9 10.1 Limit of detection and limit of quantification. 9 10.2 Interlaboratory test . 10 10.3 Repeatability 10 10.4 Reproducibility 10 11 Test report . 10 Annex A (informa
16、tive) Interlaboratory study results 11 Bibliography . 12 BS EN ISO 30024:2009ISO 30024:2009(E) iv ISO 2009 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing Internatio
17、nal 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 right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO
18、 also take part in the work. ISO collaborates closely with the International Electrotechnical 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 com
19、mittees is to prepare International Standards. Draft International Standards adopted by the technical 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
20、possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 30024 was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 327, Animal feeding stuff
21、s Methods of sampling and analysis, in collaboration with Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). BS EN ISO 30024:2009ISO 30024:2009(E) ISO 2009 All righ
22、ts reserved vIntroduction This International Standard has been developed to quantify phytase products in feed samples to enable the European Commission to control the phytase content of animal feed products. However, the method cannot be used to evaluate the in vivo efficacy of the phytase products.
23、 BS EN ISO 30024:2009BS EN ISO 30024:2009INTERNATIONAL STANDARD ISO 30024:2009(E) ISO 2009 All rights reserved 1Animal feeding stuffs Determination of phytase activity 1 Scope This International Standard specifies the determination of phytase activity in feed samples. The method does not distinguish
24、 between phytase added as a feed additive and endogenous phytase already present in the feed materials. The method cannot be used to evaluate or compare the in vivo efficacy of the phytase product. It is not a predictive method of the in vivo efficacy of phytases present on the market as they can de
25、velop different in vivo efficacy per unit of activity. The method is suitable and validated exclusively for the determination of phytase activity and exclusively in complete feeds. NOTE The harmonized method was developed on the basis of the presently existing phytase products E1600 (EC 3.1.3.8, 3-p
26、hytase), E1614 (EC 3.1.3.26, 4-phytase), and E1640 (EC 3.1.3.26, 4-phytase). Therefore, it might not necessarily be suitable as such for phytase products that are developed in the future. The harmonized method is thus a tool which is useful only to evaluate the total phytase activity in feed samples
27、 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 phytase unit U amount of enzyme that releases 1 mol of inorganic phosphate from phytate per minute under the reaction conditions specified in this International Standard 3 Principle Phytase re
28、leases phosphate from the substrate myo-inositol hexakisphosphate (phytate). The released inorganic phosphate is determined by forming a yellow complex with an acidic molybdate/vanadate reagent. The optical density (OD) of the yellow complex is measured at a wavelength of 415 nm and the inorganic ph
29、osphate released is quantified from a phosphate standard calibration curve. BS EN ISO 30024:2009ISO 30024:2009(E) 2 ISO 2009 All rights reserved4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized water or water of e
30、quivalent purity. WARNING This method requires the handling of hazardous substances. Observe local regulations for potentially hazardous chemicals to minimize risks to organizational, technical, and personal safety. 4.1 Ammonia solution, 25 % mass fraction; NH3. 4.2 Ammonium heptamolybdate tetrahydr
31、ate, (NH4)6Mo7O244H2O. 4.3 Ammonium monovanadate, NH4VO3. 4.4 Hydrochloric acid, 25 % mass fraction; HCl. 4.5 Nitric acid, 65 % mass fraction; HNO3. 4.6 Potassium dihydrogenphosphate, KH2PO4. 4.7 Phytate, phytic acid, dodecasodium salt, C6H6Na12O24P6xH2O, from rice, Sigma P01091). 4.8 Sodium acetate
32、 trihydrate, CH3COONa3H2O. 4.9 Polysorbate 202). 4.10 Dilute nitric acid. Dilute 1 volume nitric acid 65 % mass fraction (4.5) with 2 volumes water. Store at room temperature. The maximum storage time is indefinite. 4.11 Ammonium heptamolybdate reagent. Dissolve 100,0 g ammonium heptamolybdate tetra
33、hydrate (4.2) in approximately 800 ml water. Add 10 ml 25 % mass fraction ammonia solution (4.1) and make up with water to 1 000 ml. Store at room temperature in the dark. The maximum storage time is 2 months. 4.12 Ammonium vanadate reagent. Dissolve completely 2,35 g of ammonium monovanadate (4.3)
34、in approximately 400 ml water (50 C to 60 C). Add 20 ml dilute nitric acid (4.10) and make up with water to 1 000 ml. Store at room temperature in the dark. The maximum storage time is 2 months. 4.13 Molybdate/vanadate STOP reagent. Mix 1 volume ammonium vanadate reagent (4.12) with 1 volume ammoniu
35、m heptamolybdate reagent (4.11) and add 2 volumes dilute nitric acid (4.10). Mix and store at room temperature. The maximum storage time is 1 day. 4.14 Polysorbate 20, 10 % mass fraction. Dissolve 10,0 g of polysorbate 20 (4.9) with water and make up to 100 ml. Store at room temperature. The maximum
36、 storage time is 6 months. 4.15 Acetate buffer, pH 5,5; 0,25 mol/l. Dissolve 34,0 g of sodium acetate trihydrate (4.8) in approximately 900 ml water. Adjust the pH with 25 % mass fraction hydrochloric acid (4.4) to 5,50 0,02 and make up to 1 000 ml with water. Store at room temperature. The maximum
37、storage time is 2 weeks. 4.16 Acetate buffer with 0,01 % mass fraction polysorbate 20, pH 5,5; 0,25 mol/l. Dissolve 34,0 g of sodium acetate trihydrate (4.8) in approximately 900 ml water. Adjust the pH with 25 % mass fraction hydrochloric acid (4.4) to 5,50 0,02. Add 1 ml 10 % mass fraction polysor
38、bate 20 (4.14) and make up to 1 000 ml with water. Store at room temperature. The maximum storage time is 2 weeks. 1) Example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by
39、ISO of this product. 2) Tween 20 is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. BS EN ISO 30024:2009ISO 30024:2009(E) ISO 2009 All rights
40、reserved 34.17 Acetate buffer with 0,01 % mass fraction polysorbate 20, pH 5,5; 0,50 mol/l. Dissolve 68,0 g of sodium acetate trihydrate (4.8) in approximately 900 ml water. Adjust the pH with 25 % mass fraction hydrochloric acid (4.4) to 5,50 0,02. Add 1 ml 10 % mass fraction polysorbate 20 (4.14)
41、and make up to 1 000 ml with water. Store at room temperature. The maximum storage time is 2 weeks. 4.18 Phytate substrate solution, 7,5 mmol/l (5 mmol/l end-concentration in the reaction). Dissolve 2,00 g of dodecasodium phytate (4.7) whose inorganic phosphorus content is u 0,1 % mass fraction (see
42、 9.3) in approximately 200 ml acetate buffer (4.15). Adjust the pH with 25 % mass fraction hydrochloric acid (4.4) to 5,50 0,02 and make up with acetate buffer (4.15) to 250 ml. The maximum storage time is 2 weeks at 4 C. 4.19 Phosphate stock standard solution, 50 mmol/l. Dry approximately 10 g of p
43、otassium dihydrogenphosphate (4.6) at 105 C for 2 h and store it in a dessicator. Weigh approximately 682 mg of dried potassium dihydrogenphosphate, transfer it quantitatively to a 100 ml volumetric flask and make up to 100 ml with 0,25 mol/l acetate buffer with 0,01 % mass fraction polysorbate 20 (
44、4.16). Calculate the exact concentration of the phosphate stock standard solution. Store at room temperature. The maximum storage time is 2 weeks. 4.20 Phytase stock standard solution. Weigh 100,0 mg to 300,0 mg of a certified phytase standard, transfer it quantitatively to a 100 ml volumetric flask
45、 and dissolve it in 100 ml 0,25 mol/l acetate buffer with 0,01 % mass fraction polysorbate 20 (4.16). Stir it for 15 min to 45 min. Store at room temperature. The maximum storage time is 1 day. 5 Apparatus Usual laboratory apparatus, in particular, the following. 5.1 Water bath, thermostatically con
46、trolled (with inserts for 2 ml tubes). 5.2 pH-meter, capable of being read to at least two places of decimals. 5.3 Magnetic stirrers (W 20 W power). 5.4 Egg-shaped stirring bars (40 mm 20 mm). 5.5 Analytical balance, capable of being read to at least 0,1 mg. 5.6 Balance, capable of being read to at
47、least 0,01 g. 5.7 Vortex mixer. 5.8 Centrifuge for microcentrifuge tubes (5.12), capable of 11 000g to 20 000g. 5.9 Electronic dispenser. 5.10 Pipettes (electronic and manual), in the range 10 l to 2 000 l. 5.11 Spectrophotometer double beam or microplate reader. 5.12 Microcentrifuge tubes, capacity
48、 2 ml. BS EN ISO 30024:2009ISO 30024:2009(E) 4 ISO 2009 All rights reserved6 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.
49、A recommended sampling procedure is given in ISO 6497 1. 7 Sample preparation Perform two weighings for each sample. Weigh two portions of pellets or mash, of about 50 g each, into 500 ml conical flasks. Add 500 ml water and 0,5 ml of 10 % mass fraction polysorbate 20 (4.14) to the feed and mix vigorously for 45 min on a magnetic stirrer (5.3) with egg-shaped stirring bars (5.4). Transfer 2 ml of the feed extract to a microcentrifuge tube (5.12) and centrifuge (5.8) for 3 min at 11 000g to 20 000g. Inhomogeneity in the sa
