1、September 2015Translation by DIN-Sprachendienst.English price group 10No part of this translation 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).I
2、CS 67.060!%F08“2351321www.din.deDDIN EN ISO 6647-1Rice Determination of amylose content Part 1: Reference method (ISO 6647-1:2015);English version EN ISO 6647-1:2015,English translation of DIN EN ISO 6647-1:2015-09Reis Bestimmung des Amylosegehalts Teil 1: Referenzverfahren (ISO 6647-1:2015);Englisc
3、he Fassung EN ISO 6647-1:2015,Englische bersetzung von DIN EN ISO 6647-1:2015-09Riz Dtermination de la teneur en amylose Partie 1: Mthode de rfrence (ISO 6647-1:2015);Version anglaise EN ISO 6647-1:2015,Traduction anglaise de DIN EN ISO 6647-1:2015-09SupersedesDIN EN ISO 6647-1:2007-11www.beuth.deIn
4、 case of doubt, the German-language original shall be considered authoritative.Document comprises 14 pages08.15 DIN EN ISO 6647-1:2015-09 2 A comma is used as the decimal marker. National foreword This document (EN ISO 6647-1:2015) has been prepared by Technical Committee ISO/TC 34 “Food products” (
5、Secretariat: AFNOR, France/ABNT, Brazil), Subcommittee SC 4 “Cereals and pulses” (Secretariat: SAC, China) in collaboration with Technical Committee CEN/TC 338 “Cereal and cereal products” (Secretariat: AFNOR, France). The responsible German body involved in its preparation was the DIN-Normenausschu
6、ss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-05-06 AA Getreide und Getreideerzeugnisse. DIN EN ISO 6647 consists of the following parts, under the general title Rice Determination of amylose content: Part 1: Refer
7、ence method Part 2: Routine methods The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 5725-1 DIN EN ISO 5725-1 ISO 5725-2 DIN EN ISO 5725-2 ISO 6647-2 DIN EN ISO 6647-2 ISO 24333 DIN EN ISO 24333 Amendments This standard differs from DIN
8、EN ISO 6647-1:2007-11 as follows: a) the method has been revised and brought in line with the state of the art; b) calibration values for standards are determined by means of size exclusion chromatography, SEC. Previous editions DIN EN ISO 6647-1: 2007-11 DIN EN ISO 6647-1:2015-09 3 National Annex N
9、A (informative) Bibliography 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 determination of repeatabilit
10、y and reproducibility of a standard measurement method DIN EN ISO 6647-2, Rice Determination of amylose content Part 2: Routine methods DIN EN ISO 24333, Cereals and cereal products Sampling DIN EN ISO 6647-1:2015-09 4 This page is intentionally blank EN ISO 6647-1May 2015 ICS 67.060 Supersedes EN I
11、SO 6647-1:2007English Version Rice - Determination of amylose content - Part 1: Reference method (ISO 6647-1:2015) Riz - Dtermination de la teneur en amylose - Partie 1:Mthode de rfrence (ISO 6647-1:2015)Reis - Bestimmung des Amylosegehalts - Teil 1: Referenzverfahren (ISO 6647-1:2015) This European
12、 Standard was approved by CEN on 16 April 2015. 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 bibliographical references concern
13、ing such national standards may be obtained on application to the CEN-CENELEC 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 of a CEN member into
14、its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Ger
15、many, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of expl
16、oitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 6647-1:2015 EEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGDIN EN ISO 6647-1:2015-09 EN ISO 6647-1:201
17、5 (E) 2Contents Page Foreword . 3 1 Scope . 4 2 Normative references. 4 3 Terms and definitions 4 4 Principle 4 5 Reagents . 4 6 Apparatus 5 7 Sampling . 5 8 Procedure 6 8.1 Preparation of test samples 6 8.2 Test portion and preparation of the solutions 6 8.3 Debranching to obtain linear chains of s
18、tarch 6 8.4 Blank test 6 8.5 Operating conditions of SEC 6 8.6 Calculation of amylose values 6 9 Expression of results . 7 10 Precision . 7 10.1 Interlaboratory test . 7 10.2 Repeatability . 7 10.3 Reproducibility . 7 11 Test report . 7 Annex A (informative) Results of an interlaboratory test 8 Bibl
19、iography 10 Foreword This document (EN ISO 6647-1:2015) has been prepared by Technical Committee ISO/TC 34 Food products in collaboration with Technical Committee CEN/TC 338 “Cereal and cereal products” the secretariat of which is held by AFNOR. This European Standard shall be given the status of a
20、national standard, either by publication of an identical text or by endorsement, at the latest by November 2015, and conflicting national standards shall be withdrawn at the latest by November 2015. Attention is drawn to the possibility that some of the elements of this document may be the subject o
21、f patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 6647-1:2007. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this
22、 European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia
23、, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 6647-1:2015 has been approved by CEN as EN ISO 6647-1:2015 without any modification. “”DIN EN ISO 6647-1:2015-09EN ISO 6647-1:2015 (E)31 ScopeThis part of ISO 6647 specifies a reference method f
24、or determining calibration values for standards that will be used to make a standard curve for the quantification of amylose content in milled, non-parboiled rice in the range of amylose content from 0 % to 30 %.2 Normative referencesNo normative references cited in this document.3 Terms and definit
25、ionsFor the purposes of this document, the following terms and definitions apply.3.1amylosemolecules consisting of linear chains containing more than 200 linked glucose units3.2amylopectinmolecules consisting of branched chains ranging from 6 to 100 linked glucose units3.3waxy ricewaxy rice contains
26、 no chains of length consistent with being amylose4 PrincipleThe linear chains of starch are separated on the basis of hydrodynamic volume and molecular weight by size exclusion chromatograph.2Flour is gelatinised in a solution of sodium hydroxide and the molecules of starch in the solution are debr
27、anched with isoamylase,12The linear chains are separated by size exclusion chromatography (SEC), and the proportion of amylose chains is calculated by the area under the amylose peak relative to the full detector response.5 ReagentsAll the reagents used shall be of recognized analytical quality and
28、the water used shall be distilled, or demineralised water, or water of equivalent purity.5.1 Ethanol, 95 % (v/v).5.2 Sodium hydroxide, 0,25 mol/l solution.5.3 Glacial acetic acid.DIN EN ISO 6647-1:2015-09 EN ISO 6647-1:2015 (E) 45.4 Sodium acetate buffer, 0,2 mol/l solution, brought to pH4 with glac
29、ial acetic acid. Mix 10 ml buffer with additional 360 l glacial acetic acid.5.5 lsoamylase.5.6 Ion exchange mixed bed resin, for example, AG 501-X8 and Bio-Rex MSZ 501(D)1).5.7 Ammonium acetate eluant, 0,05 mol/l solution, pH 4,75.6 ApparatusThe usual laboratory apparatus and, in particular, the fol
30、lowing:6.1 Stirrer and stirring magnets.6.2 Hot plate.6.3 Microcentrifuge.6.4 Microcentrifuge tubes, 2,0 ml capacity.6.5 Scintillation vials, 20 ml capacity.6.6 Vortex mixer.6.7 Water baths, capable of holding 50 C and of reaching boiling point.6.8 Analytical balance, capable of weighing to the near
31、est 0,000 1 g.6.9 Pipettes and micropipettes, capacity of 0,2 ml, 1 ml, 5 ml, and 10 ml.6.10 Syringe, capable of delivering 25 l.6.11 Size Exclusion Chromatograph (SEC) fitted with a refractive index (RI) detector.6.12 SEC column, suitable for separating chains of MW 1 620 000.6.13 Grinder, capable
32、of reducing uncooked milled rice to flour which will pass through a 150 m to 180 m (80 to 100 Mesh) sieve. A cyclone mill with 0,5 mm screen is recommended.6.14 Sieve, size 150 m to 180 m (80 to 100 Mesh).7 SamplingIt is important that the laboratory receives a sample which is truly representative a
33、nd has not been damaged or changed during transport or storage.Sampling is not part of the method specified in this part of ISO 6647. A recommended sampling method is given in ISO 24333.1) AG 501-X8 and Bio-Rex MSZ 501 (D) are examples of suitable products available commercially. This information is
34、 given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.DIN EN ISO 6647-1:2015-09EN ISO 6647-1:2015 (E)58 Procedure8.1 Preparation of test samplesThe calibration is carried out using 300 g of flour of each of the five standards from specific
35、 varieties2), each standard carrying one of the five alleles of the Waxy gene, which is the gene responsible for amylose synthesis.In the grinder (6.13), grind at least 10 g of milled rice of each sample to very fine flour which will pass through the sieve (6.14).8.2 Test portion and preparation of
36、the solutionsWeigh triplicate samples of 50 mg 0,5 mg of each test sample into glass scintillation vials of known weight (6.5). To this, carefully add a small magnetic stirrer (6.1), as well as 0,5 ml of ethanol (5.1), using a pipette (6.9), washing down any of the test portion adhering to the side
37、of the vial. Shake slightly in order to wet all of the sample. Pipette 2,0 ml of sodium hydroxide solution (5.2) and mix. Disperse starch completely by heating the mixture to a gentle boil on a hot plate (6.2) for about 10 min, ensuring mixture does not bubble over. When the solution becomes clear (
38、about 10 min of boiling gently), remove it from the hot plate. Weigh each vial and adjust the weight to 4 g with water that has been heated to 60 C to 70 C.8.3 Debranching to obtain linear chains of starchTransfer 800 l of each gelatinised solution to a microcentrifuge tube (6.4) by using the approp
39、riate pipette (6.9). Add 200 l of the sodium acetate buffer (5.4). Add 2,5 activity units (U) isoamylase using the syringe (6.10). Mix well using a vortex mixer (6.6). Incubate for 2 h in a water bath (6.7) at 50 C, agitating every half hour, then boil for 5 min to denature the isoamylase. Transfer
40、supernatant to a clean microcentrifuge tube and add 0,1 g ion exchange resin (5.6). Incubate at 50 C for 30 min, then centrifuge. Carefully transfer supernatant to a clean microcentrifuge tube. The sample is now ready to be injected into an SEC (6.11) and should be injected the day of preparation.8.
41、4 Blank testCarry out a blank test in parallel with the determination, by the same procedure, using the same quantities of all the reagents as in 8.2 and 8.3, but using 800 l of the sodium hydroxide solution (5.2) that does not contain gelatinised starch.8.5 Operating conditions of SECThe SEC (6.11)
42、 should comprise a separation module, a refractive index detector, software, and a suitable column (6.12). If an autosampler is used, it is preferable that it contains a sample heater to maintain the debranched starch samples at 40 C. Ensure that the SEC system and needle wash are primed, the inject
43、or is purged, the seals are washed, and the column is equilibrated according to the operating instructions of the SEC system and column provider.For the analysis of starch, eluant (5.7) should be flowing at 0,5 ml/min through an appropriate size exclusion column (6.12). The column (6.12) is maintain
44、ed at 60 C. Once the column is calibrated and the baseline of the RI (6.11) is stable, samples can be injected. Each sample and blank is run for 40 min.8.6 Calculation of amylose valuesOnce the SEC runs are completed, determine the peak for amylose chains based on the peak missing from the sample th
45、at is waxy and which does not contain amylose. After fixing baseline issues and normalizing 2) Standard rice flours are available from the International Rice Research Institute, DAPO 7777, Metro Manila, Philippines. This information is given for the convenience of users of this International Standar
46、d and does not constitute an endorsement by ISO. Equivalent products may be used if they can be shown to lead to the same results.DIN EN ISO 6647-1:2015-09 EN ISO 6647-1:2015 (E) 6the detector response, calculate the area under each peak associated with starch, and then the area under the peak assoc
47、iated with amylose chains. The percent amylose follows the following formula:A=SaSt100% (1)whereA is the percent amylose;Sa is the area under the amylose peak;St is the total area under the starch peaks.9 Expression of resultsThe amylose peak is identified by comparing with the SEC trace of a waxy v
48、ariety because these varieties do not contain amylose. The amylose content is calculated for each of the three replicates for each of the five samples by taking the arithmetic mean of the three replicates as the amylose value. Any replicate that is significantly different from the others should be repeated completely from 8.1.10 Precision10.1 Interlaboratory testDetails of an international interlaboratory test on the precision of the method are summarized in Annex A. The values derived from this test ma
