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1、 Reference number ISO 15914:2004(E) ISO 2004 INTERNATIONAL STANDARD ISO 15914 First edition 2004-02-01 Animal feeding stuffs Enzymatic determination of total starch content Aliments des animaux Dtermination enzymatique de la teneur totale en amidon Copyright International Organization for Standardiz
2、ation Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:45:46 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15914:2004(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with
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6、may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Te
7、l. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2004 All rights reserved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 0
8、4/20/2007 03:45:46 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15914:2004(E) ISO 2004 All rights reserved iii Contents Page Forewordiv 1 Scope1 2 Normative references .1 3 Terms and definitions.1 4 Principle.2 5 Reagents2 6 Apparatus.4 7 Preparation of test sample.5
9、 8 Procedure.5 9 Calculation and expression of results7 10 Precision8 11 Test report9 Annex A (informative) Notes on procedure .10 Annex B (informative) Results of interlaboratory test11 Bibliography .12 Copyright International Organization for Standardization Provided by IHS under license with ISO
10、Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:45:46 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15914:2004(E) iv ISO 2004 All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation o
11、f 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 right to be represented on that committee. In
12、ternational 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. International Standards are drafted in accordance with
13、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 an International Standard requires approval
14、 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 identifying any or all such patent rights. ISO 15914 was prepared by Technical Committee
15、ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:45:46 MDTNo reproduction or networking permitted without l
16、icense from IHS -,-,- INTERNATIONAL STANDARD ISO 15914:2004(E) ISO 2004 All rights reserved 1 Animal feeding stuffs Enzymatic determination of total starch content 1 Scope This international Standard specifies a method for the enzymatic determination of the total starch content of animal feeding stu
17、ffs and raw materials for animal feeding stuffs. The method is also applicable to the determination of the purity of starch. It is important that in the sample matrix no components are present which contribute to the measured extinction at 340 nm. The analytical range of the method is 40 g/kg to 1 0
18、00 g/kg starch. The standard procedure is applicable to the range 200 g/kg to 1000 g/kg. For the lower range, 40 g/kg to 200 g/kg, another dilution procedure for the standard glucose solution and samples can be used. 2 Normative references The following referenced documents are indispensable for the
19、 application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO 6498:1998, Animal feedi
20、ng stuffs Preparation of test samples 3 Terms and definitions For the purpose of this document, the following terms and definitions apply. 3.1 starch natural vegetable polymer consisting of long linear unbranched chains of 1,4-D-glucose units (amylose) and/or long -1,6-branched chains of -1,4-linked
21、 glucose units (amylopectin) 3.2 starch content mass fraction of starch and its high molecular mass breakdown products, insoluble in 40 % ethanol, and determined in accordance with the method of this International Standard NOTE The starch content is expressed in grams per kilogram. Copyright Interna
22、tional Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:45:46 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15914:2004(E) 2 ISO 2004 All rights reserved 4 Principle The
23、 milled test sample is extracted with 40 % ethanol to remove the soluble sugars. Sample disintegration and starch solubilization is achieved by dispersing the extracted test portion in aqueous DMSO (90 % volume fraction) at 100 C, followed by addition of concentrated hydrochloric acid at 60 C. The s
24、olubilized and dissolved starch is quantitatively converted into glucose by the enzyme amyloglucosidase. The glucose quantification is carried out by the well-known hexokinase method (see 1, 2). 5 Reagents Use only reagents of recognized analytical grade. 5.1 Water, complying with at least grade 3 i
25、n accordance with ISO 3696:1987. 5.2 Ethanol (C2H5OH), 40 % (volume fraction) Take 417 ml of ethanol (96 % volume fraction) and dilute with water to 1 000 ml 5.3 Hydrochloric acid, c(HCl) = 12 mol/l. 5.4 Aqueous sodium hydroxide, c(NaOH) = 4 mol/l. Dissolve in a beaker 40 g of NaOH in about 50 ml wa
26、ter. After cooling, transfer quantitatively to a 250 ml volumetric flask and dilute to the mark with water. WARNING Heat develops. Wear safety glasses. 5.5 Acetic acid solution, c(CH3COOH) = 2 mol/l. Add to a 500 ml volumetric flask about 200 ml water, followed by 59 ml of glacial acetic acid. Dilut
27、e to the mark with water. 5.6 Sodium acetate solution, c(CH3COONa) = 2 mol/l. Dissolve, in a 500 ml volumetric flask, 82,0 g of sodium acetate in water and dilute to the mark with water. 5.7 Sodium acetate buffer, c(CH3COONa/H) = 2 mol/l, pH = 4,8. Mix 41 ml of acetic acid solution (5.5) with 59 ml
28、of sodium acetate solution (5.6). Check the pH with a pH-meter and, if necessary, adjust to obtain the correct pH with the acetic acid or the sodium acetate solution. Prepare a fresh buffer solution daily. 5.8 Aqueous dimethylsulfoxide, (DMSO) = 90 % (volume fraction). Mix pure DMSO and water in a v
29、olume ratio of 9:1. 5.9 Clarifying solutions, according to Carrez, prepared as follows. 5.9.1 Potassium hexacyanoferrate(II) solution, cK4Fe(CN)6 = 0,25 mol/l. In a 1 litre volumetric flask, dissolve 106 g of potassium hexacyanoferrate(II) trihydrate K4Fe(CN)63H2O in water. Dilute to the mark with w
30、ater. 5.9.2 Zinc acetate, solution in 0,5 mol/l acetic acid, cZn(CH3CO2)2 = 1 mol/l. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:45:46 MDTNo reproduction or networking per
31、mitted without license from IHS -,-,- ISO 15914:2004(E) ISO 2004 All rights reserved 3 In a 1 litre volumetric flask, dissolve 219,5 g of zinc acetate dihydrate Zn(CH3CO2)22H2O and 30 g of glacial acetic acid in water. Dilute to the mark with water. 5.10 Iodine solution in potassium iodide In a 1 li
32、tre volumetric flask, dissolve 12,7 g of iodine (I2) and 24,0 g of potassium iodide (KI) in water. Dilute to the mark with water. Dilute this solution 10-fold before use. 5.11 Standard glucose solution 5.11.1 Samples containing 200 g/kg to 1 000 g/kg starch Prepare three independent standard glucose
33、 solutions (c = 0,0194 mol/l). In each 100 ml volumetric flask dissolve 350 mg anhydrous glucose (C6H12O6) to the nearest mg in water. Dilute to the mark with water. 5.11.2 Samples containing 40 g/kg to 200 g/kg starch Prepare three independent standard glucose solutions (c = 0,0039 mol/l). In each
34、500 ml volumetric flask, dissolve 350 mg 1 mg of anhydrous glucose (C6H12O6) in water. Dilute to the mark with water. Prepare fresh standard glucose solutions daily. 5.12 Amyloglucosidase solution, 160 U/ml AMG. Dissolve, in a mixture of 9 ml of water and 1 ml of sodium acetate buffer (5.7), 267 mg
35、of amyloglucosidase (AMG) EC 3.2.1.3 (Aspergillus niger; Roche Diagnostics, No. 1 202 367, 6 U/mg).1) With respect to storage and the use of the enzymes, follow carefully the recommendations of the enzyme supplier. When another enzyme is used, the activity should be estimated as given in Note 1. The
36、 mass of the enzyme should be adapted to the activity found. NOTE 1 Different enzyme suppliers use different definitions for the units of activities of enzymes. In this International Standard the following definition for the unit of activity for AMG has been used: 1 unit of amyloglucosidase will rel
37、ease 1 mol of glucose from glycogen in 1 min at 25 C and pH = 4,75. NOTE 2 10 ml of enzyme solutions is enough for about 75 determinations. 5.13 D-Glucose UV test set, for quantifying glucose enzymaticly, with the hexokinase method (R-Biopharm, No. 7162512), according to the manufacturer, the unused
38、 kits may be stored for 1 year at 4 C), as given in 5.13.1 to 5.13.3. 5.13.1 Buffer/substrate solution (Bottle 1) Dissolve the content of Bottle 1 in 45 ml of freshly prepared distilled water. Store this reagent in a cool (4 C) and dark place for not longer than for 4 weeks. Use the required amount
39、of this solution at ambient temperature. 5.13.2 Enzyme solution (Bottle 2) This solution is ready for use. 1) Roche Diagnostics No. 1 202 367 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
40、 constitute an endorsement by ISO of this product. 2) R-Biopharm AG No. 716251 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 products. Copyright I
41、nternational Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/20/2007 03:45:46 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 15914:2004(E) 4 ISO 2004 All rights reserved 5.13.3 Co
42、louring solution Mix 22,5 ml of buffer/substrate solution (5.13.1) with 95 ml of water and 0,45 ml of enzyme solution (5.13.2) and homogenize. This amount of reagent is enough for a series of about 40 measurements. If more or less measurements are be carried out, the mixing volumes may be adapted. O
43、nly use fresh prepared colouring solutions at ambient temperature. 6 Apparatus Usual laboratory apparatus and, in particular, the following. 6.1 Analytical balance, capable of weighing to the nearest 0,1 mg. 6.2 Centrifuge, with an acceleration of (180 10) g and of 3 000 g, suitable for screw cap tu
44、bes of 12 ml (the value of g is given for the bottom of the tube). 6.3 Water bath, adjusted to (100 2) C. 6.4 Water bath, adjusted to (60 1) C. 6.5 pH-meter, calibrated, with a combined glass electrode, capable of measuring the pH to the nearest 0,01 pH unit. 6.6 Microlitre pipettes, calibrated and
45、adjustable from 40 l to 200 l, 200 l to 1 000 l, and 1 ml to 5 ml, and a dispenser, adjustable from 1 ml to 5 ml. 6.7 Spectrometer, with a flow cuvette, capable of measuring at 340 nm. 6.8 Rotary shaker, speed 50 r/min, for screw-cap-closed centrifuge tubes (6.11). 6.9 Dispensor/diluter (e.g. Hook E
46、0gs is the numerical value of the absorbance of the standard glucose solution; E0wb is the numerical value of the mean of the absorbance of the water blanks. The average corrected absorbance value of the water blanks is (by definition) equal to zero. Using linear regression analyses, calculate the c
47、alibration graph for the corrected absorbance values against the glucose content (in grams per litre) of the undiluted standard glucose solutions. Calculate the corrected absorbance value of each of the sample solutions using the equation: () 1s0s0sb EEE= where E1s is the numerical value of the corr
48、ected absorbance of the sample solution; E0s is the numerical value of the absorbance of the sample solution; E0sb is the numerical value of the mean of the absorbance of the sample blanks. Using the calibration graph (9.1), calculate the glucose content (g) of the undiluted sample solutions (8.5), in grams per litre. See Annex A. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/2