1、BRITISH STANDARD BS EN ISO 11213:1995 Modified starch Determination of acetylcontent Enzymatic method The European Standard EN11213:1995 has the status of a BritishStandardBSENISO11213:1995 This BritishStandard was published under the authority ofthe Standards Board andcomesinto effect on 15June1995
2、 BSI 01-2000 The following BSI references relate to the work on this standard: Committee reference AW/100 Draft for comment 93/507580 DC ISBN 0 580 24179 3 National foreword This BritishStandard has been prepared under the direction of the BSI Standards Board and is the English language version of E
3、N11213:1995 Modified starch Determination of acetyl content Enzymatic method, published by the European Committee for Standardization (CEN). It is identical with ISO11213:1995, published by the International Organization for Standardization (ISO), which has the same title. This BritishStandard has b
4、een produced to fulfil BSIs obligation to publish all approved European Standards but, because of the absence of interest in the UK in the subject concerned, there has been no UK participation in the preparation of EN ISO11213. Any queries relating to the EN should be directed to BSI quoting the ref
5、erence AW/100. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This docu
6、ment comprises a front cover, an inside front cover, pagesi andii, theEN ISO title page, pages2 to8, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front
7、cover. Cross-references Publication referred to Corresponding BritishStandard EN ISO 1666:1973 BS EN ISO1666:1994 Starch Determination of moisture Oven-drying method ISO 3696:1987 BS3978:1987 Specification for water for laboratory use Amendments issued since publication Amd. No. Date CommentsBSENISO
8、11213:1995 BSI 01-2000 i Contents Page National foreword Inside front cover Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents and materials 3 5 Apparatus 4 6 Preparation of the sample 4 7 Procedure 4 8 Expression of results 5 9 Precision 6 10 Test report 6 Annex A (informative)
9、Derivation of equation for calculation of acetyl content 7 Annex B (informative) Statistical results of the interlaboratory test 8 Annex C (informative) Bibliography 8 Annex ZA (normative) Normative references to international publicationswiththeir relevant European publications 8 Table 1 Analytical
10、 arrangement for enzymatic determination of acetic acid 6 List of references Inside back coverii blankEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11213 January 1995 ICS 67.180.20 Descriptors: Carbohydrates, starches, chemical analysis, determination of contents, acetyl group, enzymatic
11、method English version Modified starch Determination of acetyl content Enzymatic method (ISO 11213:1995) Amidons modifi Dosage de Lactyle Mthode enzymatique (ISO 11213:1995) Modifizierte Strke Bestimmung des Acetylgehaltes Enzymatisches Verfahren (ISO 11213:1995) This European Standard was approved
12、by CEN on1994-11-28. 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 concerning such national standards
13、 may be obtained on application to the Central Secretariat 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 its own language and notified to the
14、Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN Europ
15、ean Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1995 Copyright reserved to CEN members Ref. No. EN ISO 11213:1995 EENISO11213:1995 BSI 01-2000 2 Foreword The text of the International Standard IS
16、O11213:1995 has been prepared by Technical Committee ISO/TC93, Starch (including derivatives and by-products), in collaboration withCEN/CS. It has been submitted to Parallel Vote and has been approved on1994-11-28 as a European Standard. This European Standard shall be given the status ofa national
17、standard, either by publication of an identical text or by endorsement, at the latest by July1995, and conflicting national standards shallbe withdrawn at the latest by July1995. According to the CEN/CENELEC Internal Regulations, the following countries are bound to implement this European Standard:
18、 Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom.ENISO11213:1995 BSI 01-2000 3 1 Scope This International Standard specifies an enzymatic method for the determination of the ac
19、etyl content of modified starch, both granular and soluble in cold water. Total and free acetyl contents are determined and the bound acetyl content is calculated. The method is suitable for determining acetyl contents up to2%(m/m). 2 Normative references The following standards contain provisions w
20、hich, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this International Standard are encouraged to investigate the possibili
21、ty of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 1666:1973, Starch Determination of moisture content Oven-drying methods. ISO 3696:1987, Water for analytical laboratory use Specificatio
22、n and test methods. 3 Principle The total acetyl content is determined by heating the sample with dilute hydrochloric acid which hydrolyses the acetyl fraction and solubilizes the starch. In the presence of the enzyme acetyl-CoA synthetase (ACS), acetate is converted with adenosine-5-triphosphate (A
23、TP) and coenzyme A (CoA) to acetyl-Co-A. The latter then reacts with oxaloacetate to form citrate in the presence of citrate synthase (CS). The oxaloacetate required for the reaction is formed from malate and nicotinamide adenine dinucleotide (NAD) in the presence of malate-dehydrogenase (MDH). In t
24、his reaction, the NAD is reduced to NADH and the formation of NADH can be determined by measuring the increase in absorbance at a specified wavelength. (Seereference1 in Annex C.) The free acetyl content is determined by making a suspension of the modified starch in water, filtering, and determining
25、 the acetyl content of the filtrate as already described. The bound acetyl content is calculated by subtracting the free acetyl content from total acetyl content. 4 Reagents and materials The reagents used shall be of recognized analytical grade, unless otherwise specified. The water used shall comp
26、ly with the specifications of ISO3696, grade2. The enzymes used shall be of a quality equivalent to the relevant enzymes of Boehringer Mannheim 1) . NOTE 1Suitable test kits which are commercially available can be used. 4.1 Hydrochloric acid,1mol/l solution. 4.2 Sodium hydroxide,5mol/l solution. 4.3
27、 Buffer solution In about70ml of water, dissolve the following reagents: 7,5g of triethanolamine; 420mg of L-malic acid; 210mg of magnesium chloride hexahydrate (MgCl 2 .6H 2 O). Add as much potassium hydroxide5mol/l solution as necessary in order to obtain a pH of8,4. The volume required is about8m
28、l. The solution is stable for1 year when stored at+4 C. 4.4 ATP-CoA-NAD solution In20ml of water, dissolve the following reagents: 500mg of crystallized disodium salt trihydrate of adenosine-5-triphosphate ATP-Na 2 H 2 .3H 2 O; 98%(m/m); 500mg of anhydrous sodium hydrogen carbonate; 50mg of lyophili
29、zed trilithium salt of coenzyme A about85%(m/m) CoA; 250mg of lyophilized free acid monohydrate of nicotinamide adenine dinucleotide -NAD.H 2 O98%(m/m). The solution is stable for1 week when stored at+4 C. 4.5 MDH-CS suspension Disperse about1100 U (international units) of malate-dehydrogenase (MDH
30、from pig heart; EC1.1.1.37) and about270U of citrate synthase (CS from pig heart; EC4.1.37) in0,4ml of ammonium sulfate solution, c(NH 4 ) 2 SO 4 =3,2mol/l. The solution is stable for1 year when stored at+4 C. NOTE 2One international unit (1 U) catalyses14mol/min at25 C from the relevant substrate.
31、1) This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of the products named.ENISO11213:1995 4 BSI 01-2000 4.6 ACS solution Dissolve 20mg of lyophilizate containing5mg of acetyl-coenzyme A synthetase (ACS from yeast; EC6
32、.2.1.1; . 16U) in0,4ml of water. The solution is stable for5d when stored at+4 C. 5 Apparatus Usual laboratory apparatus and in particular the following. 5.1 Conical flasks, of capacity250ml, equipped with screw caps. 5.2 Boiling water bath, equipped with a shaker. 5.3 Volumetric flasks, of capacity
33、200ml. 5.4 Micropipettes or syringes 5.5 Molecular absorption spectrometer, suitable for operation at340nm. 5.6 Cuvettes, of quartz glass or other materials transparent at340nm, with a thickness of10mm 0,1mm. 5.7 Sieve, with an aperture of8004m. 5.8 Blade mill 5.9 Water bath, capable of being thermo
34、statically controlled between20 C and25 C. 6 Preparation of the sample Sieve through a8004m sieve(5.7). If material does not pass through the sieve, grind the sample with a blade mill(5.8) so that it will completely pass through the8004m sieve. Homogenize the sample. 7 Procedure 7.1 Hydrolysis of ac
35、etyl groups 7.1.1 Dispersion of granular starch Weigh, to the nearest1mg, approximately1g of the prepared sample and place it in a conical flask(5.1). Add50ml of hydrochloric acid(4.1) while agitating to ensure good dispersion. Continue as described in7.1.3. 7.1.2 Dispersion of pregelatinized starch
36、 Add50ml of hydrochloric acid(4.1) to a conical flask(5.1). Introduce a magnetic stirrer and start agitation. Slowly and carefully add about1g of the prepared sample. Ensure a good, lump-free dispersion. Determine the mass of the test portion by weighing by difference to the nearest1mg. 7.1.3 Hydrol
37、ysis and filtration Seal the conical flask tightly with the screw cap and place it in the boiling water bath(5.2) with the shaker operating for30min. Remove the flask and cool to about20 C 5 C by immersing it in an ice bath. Open the flask when its content is fully cooled, add10ml of sodium hydroxid
38、e solution(4.2), mix, and wash the contents quantitatively into a200ml volumetric flask(5.3). Place the volumetric flask in the water bath(5.9) for temperature equilibration between20 C and25 C. Control the temperature and make up to the mark with distilled water. Filter through a suitable filter pa
39、per. Reject the first20ml to30ml of filtrate and directly use the remaining solution as the test solution in the enzymatic determination, as described in7.4. 7.2 Free acetate 7.2.1 Dispersion of granular starch Disperse10g of the prepared sample, while stirring, in100ml of distilled water in a conic
40、al flask(5.1). Continue as described in7.2.3. 7.2.2 Dispersion of pregelatinized starch Add about100ml of distilled water to a conical flask(5.1). Introduce a magnetic stirrer and start agitation. Slowly and carefully add about2g of the prepared sample. Ensure a good, lump-free dispersion. Determine
41、 the mass of the test portion by weighing by difference to the nearest1mg. 7.2.3 Dissolution and filtration Seal the conical flask and agitate for30min. Transfer the contents of the conical flask quantitatively to a200ml volumetric flask(5.3). Place the volumetric flask in the water bath(5.9) for te
42、mperature equilibration between20 C and25 C. Control the temperature and make up to the mark with distilled water. Filter through a suitable filter paper. Reject the first20ml to30ml of filtrate and directly use the remaining solution as the test solution in the enzymatic determination, as described
43、 in7.4. 7.3 Check test To check the method, the assay can be performed on a reference material such as pure anhydrous sodium acetate acetyl content=52,4%(m/m). For this, weigh to the nearest0,1mg, about100mg of anhydrous sodium acetate. Then transfer to a1000ml volumetric flask. Place the volumetric
44、 flask in the water bath(5.9) for temperature equilibration between20 C and25 C. Control the temperature and make up to the mark with distilled water. Continue as described in7.4.ENISO11213:1995 BSI 01-2000 5 7.4 Enzymatic determination of acetic acid Carry out the enzymatic determination of acetic
45、acid according to the following analytical arrangement and conditions: wavelength:340nm; temperature: 20 C to25 C. Read the absorbances against a cuvette(5.6) filled with water. NOTE 3Measurements can also be made at the following wavelengths with the corresponding molar absorption coefficients (x)
46、used in the calculations: Hg, 365nm: x =3,4lmmol 1 cm 1 ; Hg, 334nm: x =6,18lmmol 1 cm 1 ; Pipette into cuvettes(5.6) the volumes of reagents indicated in the analytical arrangement in Table 1. 8 Expression of results 8.1 Absorbance difference Calculate the absorbance difference using the equation:
47、8.2 Total acetyl content Calculate the total acetyl content using the equation If an aliquot of solution with sample other than the0,50ml specified in Table 1 is taken, the equation has to be adjusted accordingly. When the check test(7.3) is effected, a dilution factor of5 has to be included because
48、 the test solution is made up to1000ml and not200ml as in the procedure for the test sample (see7.1 and7.2). NOTE 4A full explanation of the derivation of the equation is given in Annex A. 8.3 Free acetyl content Calculate the free acetyl content using the equation 8.4 Bound acetyl content Calculate
49、 the bound acetyl content using the equation where %A is the numerical value of the absorbance difference; A 0 , A 1 and A 2 are the numerical values of absorbances measured according to the analytical arrangement of Table 1; s is an index designating the solution with sample; b is an index designating the blank. where w a is the numerical value of the total acetyl content, in percentage by mass, of the test sample; %A is the numerical value of the absorbance difference calculated according to8.1; x i
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