1、November 2009DEUTSCHE NORM English price group 11No 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 65.120!$Zi“1557098www
2、.din.deDDIN EN ISO 30024Animal feeding stuffs Determination of phytase activity (ISO 30024:2009)English version of DIN EN ISO 30024:2009-11Futtermittel Bestimmung der Phytaseaktivitt (ISO 30024:2009)Englische Fassung DIN EN ISO 30024:2009-11www.beuth.deDocument comprises 18 pagesDIN EN ISO 30024:200
3、9-11 2 National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcommittee SC 10 “Animal feeding stuffs” (Secretariat: NEN, Netherlands) in collaboration with Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sa
4、mpling and analysis” (Secretariat: NEN, Netherlands). Based on the results of the European Standard. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee
5、 NA 057-03-03 AA Futtermittel. The DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 6497 EN ISO 6497 National Annex NA (informative) Bibliography DIN EN ISO 6497, Animal feeding stuffs Sampling parallel voting, ISO 30024:2009 has been adopted a
6、s aEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 30024 July 2009 ICS 65.120 English Version Animal feeding stuffs - Determination of phytase activity (ISO 30024:2009) Aliments des animaux - Dtermination de lactivit phytasique (ISO 30024:2009) Futtermittel - Bestimmung der Phytaseaktivitt
7、(ISO 30024:2009) 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 European Standard the status of a national standard without any alteration. Up-to-date lists and bibli
8、ographical 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 translation under the responsibility
9、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, Germany, Greece, Hungary, Iceland, Ire
10、land, 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 NORMUNG Management Centre: Avenue Marnix
11、17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 30024:2009: EContents 2 Page Foreword .3 Introduction.4 1 Scope5 2 Terms and definitions .5 3 Principle .5 4 Reagents 6 5 Apparatus.7 6 Sampling 8 7 Sampl
12、e preparation.8 8 Procedure.8 8.1 Blank solutions8 8.2 Standards .8 8.3 Standard curve 9 8.4 Phytase level control.9 8.5 Feed test portions .10 9 Calculations .11 9.1 Formulation standard curve.11 9.2 Calculation phytase activity .12 9.3 Correction for phytic acid purity and water content13 9.4 Inte
13、rference with blank values .13 10 Precision 13 10.1 Limit of detection and limit of quantification .13 10.2 Interlaboratory test14 10.3 Repeatability 14 10.4 Reproducibility 14 11 Test report14 Annex A (informative) Interlaboratory study results.15 Bibliography16 DIN EN ISO 30024:2009-11 EN ISO 3002
14、4:2009 (E) Foreword 3 This document (EN ISO 30024:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding 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 s
15、hall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest 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 d
16、ocument may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standar
17、d: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. EN
18、 ISO 30024:2009 (E) DIN EN ISO 30024:2009-11 Introduction 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 e
19、fficacy of the phytase products. 4 DIN EN ISO 30024:2009-11 EN ISO 30024:2009 (E) 1 Scope This International Standard specifies the determination of phytase activity in feed samples. The method does not distinguish between phytase added as a feed additive and endogenous phytase already present in th
20、e 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 develop different in vivo efficacy per unit of activity. The method is suitable and vali
21、dated 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-phytase), E1614 (EC 3.1.3.26, 4-phytase), and E1640 (EC 3.1.3.26, 4-phytase). Therefore
22、, 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. 2 Terms and definitions For the purposes of this document, the following terms and d
23、efinitions 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 releases phosphate from the substrate myo-inositol hexakisphosphate (phytate). The relea
24、sed 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 phosphate released is quantified from a phosphate standard calibration curve. 5 EN ISO 3
25、0024:2009 (E) DIN EN ISO 30024:2009-11 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity. WARNING This method requires the handling of hazardous substances. Observe local regula
26、tions 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 tetrahydrate, (NH4)6Mo7O244H2O. 4.3 Ammonium monovanadate, NH4VO3. 4.4 Hydrochloric acid, 25 % mass fraction; HCl.
27、 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 trihydrate, CH3COONa3H2O. 4.9 Polysorbate 202). 4.10 Dilute nitric acid. Dilute 1 volume nitric acid 65
28、% 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 tetrahydrate (4.2) in approximately 800 ml water. Add 10 ml 25 % mass fraction ammonia solution (4.1) and make
29、 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) in approximately 400 ml water (50 C to 60 C). Add 20 ml dilute nitric acid (4.10) and make up with water
30、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 ammonium heptamolybdate reagent (4.11) and add 2 volumes dilute nitric acid (4.10). Mix and store at room temper
31、ature. 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 storage time is 6 months. 4.15 Acetate buffer, pH 5,5; 0,25 mol/l. Dissolve 34,0 g of sodium acetate tri
32、hydrate (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 storage time is 2 weeks. 4.16 Acetate buffer with 0,01 % mass fraction polysorbate 20, pH 5,5; 0,25 mol/l
33、. 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 polysorbate 20 (4.14) and make up to 1 000 ml with water. Store at room temperature. The maximum storage time is
34、 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 ISO of this product. 2) Tween 20 is an example of a suitable product available commercially. This informa
35、tion is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. 6 DIN EN ISO 30024:2009-11 EN ISO 30024:2009 (E) 4.17 Acetate buffer with 0,01 % mass fraction polysorbate 20, pH 5,5; 0,50 mol/l. Dissolve 68,0 g of sodium acetat
36、e 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) and make up to 1 000 ml with water. Store at room temperature. The maximum storage time is 2 weeks. 4.18 Phytate substrate s
37、olution, 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 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
38、,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 potassium dihydrogenphosphate (4.6) at 105 C for 2 h and store it in a dessicator. Weigh approximately 682 mg of dried potass
39、ium 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 (4.16). Calculate the exact concentration of the phosphate stock standard solution. Store at room temperature. The maximum st
40、orage 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 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
41、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 controlled (with inserts for 2 ml tubes). 5.2 pH-meter, capable of being read to at least two places of decimals. 5.3 Magnetic
42、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 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 Electron
43、ic 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 2 ml. 7 EN ISO 30024:2009 (E) DIN EN ISO 30024:2009-11 6 Sampling A representative sample should have been sent to the labo
44、ratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling procedure is given in ISO 6497 1. 7 Sample preparation Perform two weighings for each sample. Weigh two portions of pell
45、ets 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
46、(5.12) and centrifuge (5.8) for 3 min at 11 000g to 20 000g. Inhomogeneity in the sample can lead to high coefficients of variation (CVs). For feed samples showing CVs 15 %, such inhomogeneity can derive from inhomogeneous particle size distribution in products or inhomogeneous feed preparation. If
47、feed samples show high CVs, grind the feed samples using an Ultra centrifugal mill3)with a sieve of nominal size of openings 1 mm. Grind 150 g feed and extract as described in this clause. 8 Procedure 8.1 Blank solutions Inorganic phosphate in the sample contributes to colour formation. Therefore, b
48、lanks are included for each sample. For calculation of phytase activity, subtract blank values from the test values. 8.2 Standards 8.2.1 Phosphate standard solutions The phosphate stock standard solution (4.19) is diluted with 0,25 mol/l acetate buffer containing 0,01 % mass fraction polysorbate 20
49、(4.16) according to Table 1. Table 1 Dilution steps to obtain standard colorimetric solutions for the phosphate curve Standard solution Volumes phosphate stock standard solution (4.19) Volumes 0,25 mol/l acetate buffer with 0,01 % mass fraction polysorbate 20 (4.16) Dilution factor Concentrationmol/mlaA 1 1 2 25 B 1 3 4 12,5 C 1 7 8 6,25 D 15 16 3,125 aCalculate the exact concentrations (4.19). 3) Example of a suitable product available commercially. This information is given for the convenience of users of this International Standard an
