1、BSI Standards PublicationBS EN ISO 6647-1:2015Rice Determination ofamylose contentPart 1: Reference methodBS EN ISO 6647-1:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO6647-1:2015. It supersedes BS EN ISO 6647-1:2007 which iswithdrawn.The UK participa
2、tion in its preparation was entrusted to TechnicalCommittee AW/4, Cereals and pulses.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its
3、correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 74415 0ICS 67.060Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strateg
4、y Committee on 31 May 2015.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 6647-1 May 2015 ICS 67.060 Supersedes EN ISO 6647-1:2007English Version Rice - Determination of amylose content - Part 1: Reference method (ISO 6647-1:2015) Riz -
5、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 Standard was approved by CEN on 16 April 2015. CEN members are bound to comply with the CEN/CENELEC Internal Regulati
6、ons 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 concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
7、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 its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN m
8、embers are the national standards bodies of 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, Por
9、tugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any
10、form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 6647-1:2015 EBS EN ISO 6647-1:2015EN ISO 6647-1:2015 (E) 3 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/T
11、C 338 “Cereal and cereal products” the secretariat of which is held by AFNOR. 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 November 2015, and conflicting national standards shall be withdrawn a
12、t the latest by November 2015. 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 such patent rights. This document supersedes EN ISO 6647-1:2007. According
13、to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Gree
14、ce, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, 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 wit
15、hout any modification. BS EN ISO 6647-1:2015ISO 6647-1:2015(E)Foreword iv1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 15 Reagents 16 Apparatus . 27 Sampling 28 Procedure. 38.1 Preparation of test samples . 38.2 Test portion and preparation of the solutions . 38.3 Debranc
16、hing to obtain linear chains of starch . 38.4 Blank test . 38.5 Operating conditions of SEC 38.6 Calculation of amylose values 39 Expression of results 410 Precision . 410.1 Interlaboratory test. 410.2 Repeatability . 410.3 Reproducibility . 411 Test report . 4Annex A (informative) Results of an int
17、erlaboratory test 5Bibliography 7 ISO 2015 All rights reserved iiiContents PageBS EN ISO 6647-1:2015ISO 6647-1:2015(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Stand
18、ards 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, also ta
19、ke part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In pa
20、rticular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of the elements of
21、 this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see
22、www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to
23、the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 4, Cereals and pulses.This second edition cancels and replaces the first edition (ISO 6647-1
24、:2007), of which it constitutes a minor revision.ISO 6647 consists of the following parts, under the general title Rice Determination of amylose content: Part 1: Reference method Part 2: Routine methodsiv ISO 2015 All rights reservedBS EN ISO 6647-1:2015Rice Determination of amylose content Part 1:
25、Reference method1 ScopeThis part of ISO 6647 specifies a reference method for 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 r
26、eferencesNo normative references cited in this document.3 Terms and definitionsFor 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 chai
27、ns ranging from 6 to 100 linked glucose units3.3waxy ricewaxy rice contains 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 soluti
28、on of sodium hydroxide and the molecules of starch in the solution are debranched 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 R
29、eagentsAll the reagents used shall be of recognized analytical quality and 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.INTERNATIONAL STANDARD ISO 6647-1:2015(E) ISO
30、2015 All rights reserved 1BS EN ISO 6647-1:2015ISO 6647-1:2015(E)5.4 Sodium acetate buffer, 0,2 mol/l solution, brought to pH4 with glacial 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
31、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 following: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 Vor
32、tex mixer.6.7 Water baths, capable of holding 50 C and of reaching boiling point.6.8 Analytical balance, capable of weighing to the nearest 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 (
33、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 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 Sie
34、ve, size 150 m to 180 m (80 to 100 Mesh).7 SamplingIt is important that the laboratory receives a sample which is truly representative and 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
35、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 given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.2 ISO 2015 All rights reservedBS EN ISO 6647-1:20
36、15ISO 6647-1:2015(E)8 Procedure8.1 Preparation of test samplesThe calibration is carried out using 300 g of flour of each of the five standards from specific varieties2), each standard carrying one of the five alleles of the Waxy gene, which is the gene responsible for amylose synthesis.In the grind
37、er (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 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, carefu
38、lly 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 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 compl
39、etely 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 (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
40、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 appropriate 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 usi
41、ng 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 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 tra
42、nsfer 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.8.4 Blank testCarry out a blank test in parallel with the determination, by the same procedure, using the same quantities of all the reagents as
43、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) should comprise a separation module, a refractive index detector, software, and a suitable column (6.12). If an autosampler is used, it is pre
44、ferable 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 injector is purged, the seals are washed, and the column is equilibrated according to the operating instructions of the SEC system and column provide
45、r.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 maintained 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 fo
46、r 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 that is waxy and which does not contain amylose. After fixing baseline issues and normalizing 2) Standard rice flours are available from the Inte
47、rnational Rice Research Institute, DAPO 7777, Metro Manila, Philippines. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO. Equivalent products may be used if they can be shown to lead to the same results. ISO 2015 All
48、 rights reserved 3BS EN ISO 6647-1:2015ISO 6647-1:2015(E)the detector response, calculate the area under each peak associated with starch, and then the area under the peak associated with amylose chains. The percent amylose follows the following formula:A=SaSt100% (1)whereA is the percent amylose;Sa
49、 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 variety 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 Interlabor
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