1、BRITISH STANDARD CEN EN*ISO*LL213 95 3404589 0095473 585 H Modified starch - Determination of acetyl content - Enzymatic method The European Standard EN 11213 : 1995 has the status of a British Standard BS EN IS0 11213 : 1995 CEN EN*ISO*LL213 95 3404589 0095474 411 comes into effect on Amd. No. Date
2、 15 June 1995 BS EN IS0 11213 : 1995 Txt affected O BSI 1995 The following BSI references relate to the work on this standard: Committee reference AW/lOO Draft for comment 93507580 DC ISBN O 580 24179 3 CEN EN*ISO*11213 95 B 3404589 0095475 358 BS EN IS0 11213 : 1995 Contents National foreword Page
3、i Foreword Method 1 2 3 4 5 6 7 8 9 10 Scope Normative references Principle Reagents and materiais Apparatus Preparation of the sample Procedure Expression of results Precision Test report 2 3 3 3 3 4 4 4 5 7 7 Annexes A (informative) Derivation of equation for calculation of acetyl content 8 B (inf
4、ormative) Statistical results of the interlaboratory test 9 C (informative) Bibliography 10 ZA (normative) Normative references to international publications with their relevant European publications 10 ble 1 Analytical arrangement for enzymatic determination of acetic acid 6 i CEN EN*ISO*LL2L3 95 W
5、 3404589 0095476 294 BS EN IS0 11213: 1995 National foreword This British Standard has been prepared under the direction of the BSI Standards Board and is the English language version of EN 11213 : 1995 Modified starch - Determination of acetyl content - Enzymatic method, published by the European C
6、ommittee for Standardization (CEN). It is identical with IS0 112 13 : 1995, published by the International Organization for Standardization (ISO), which has the same title. This British Standard has been produced to fulfil BSIs obligation to publish all approved European Standards but, because of th
7、e absence of interest in the UK in the subject concerned, there has been no UK participation in the preparation of EN IS0 11213. Any queries relating to the EN should be directed to BSI quoting the reference AW/lOO. Cross-references Publication referred to Corresponding British Standard EN IS0 1666
8、: 1973 BS EN IS0 1666 : 1994 Starch - Determination of moisture - Oven-drying method IS0 3696 : 1987 BS 3978 : 1987 Specification for water for laboratory use Compliance with a British Standard does not of itself confer immunity from legal obligations. ii CEN EN*ISO*l1213 95 3404589 O095477 120 = EU
9、ROPEAN STANDARD EN IS0 11213 NORME EUROPENNE EUROPISCHE NORM January 1995 ICs 67.180.20 Descriptors: Carbohydrates, starches, chemical analysis, determination of contents, acetyl group, enzymatic method English version Modified starch - Determination of acetyl content - Enzymatic method (IS0 11213 :
10、 1995) Amidons modifi - Dosage de Lactyle - Mthode enzymatique (IS0 11213 : 1995) Modifizierte Strke - Bestimmung des Acetylgehaltes - Enzymatisches Verfahren (IS0 11213 : 1995) This European Standard was approved by CEN on 1994-11-28. CEN members are bound to comply with the CENXENELEC Internal Reg
11、ulations 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 Central Secretariat or to any CEN member. This
12、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 Central Secretariat has the same status as the official versions. CEN members are the
13、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 European Committee for Standardization Comit Europen de Normalisation Europisches Komitee f
14、r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels O 1995 Copyright reserved to CEN members Ref. No. EN IS0 11213 : 1995 E CEN EN*ISO*L1213 95 I 3404589 0095478 Ob7 = Page 2 EN IS0 11213 : 1995 Foreword The text of the International Standard IS0 11213 : 1995 has been prepared by Rchn
15、ical Committee ISO/TC 93, Starch (including derivatives and by-products), in collaboration with CEN/CS. It has been submitted to Parallel Vote and has been approved on 1994-11-28 as a European Standard. This European Standard shall be given the status of a national standard, either by publication of
16、 an identical text or by endorsement, at the latest by July 1995, and conflicting national standards shall be withdrawn at the latest by July 1995. According to the CENXENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finl
17、and, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom. CEN EN*ISO*LL2L3 95 W 3404589 0095479 TT3 Page 3 EN IS0 11213 : 1995 Modified starch - Determination of acetyl content - Enzymatic method 1 Scope This Interna
18、tional Standard specifies an enzymatic method for the determination of the acetyl 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 con- tents up
19、 to 2 % (ndnz). 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publi- cation. the editions indicated were valid. All standards are subject to revision, and parties to agreem
20、ents based on this International Standard are encouraged to investigate the possibility of applying the most re- cent editions of the standards indicated below. Members of IEC and IS0 maintain registers of cur- rently valid International Standards. IS0 1666: 1973, Starch - Determination of moisture
21、content - Oven-drying methods. IS0 3696:1987, Water for analytical laboratory use - Specification and rest 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
22、of the enzyme acetyl-coA synthetase (ACS), acetate is converted with adenosine-5-triphosphate (ATP) 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
23、 and nicotinamide adenine dinucleotide (NAD) in the presence of malate-dehydrogenase (MDH). In this 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. (See reference l in annex C.) The free acetyl conten
24、t is determined by making a suspension of the modified starch in water, filtering, and determining the acetyl content of the filtrate as already described. The bound acetyl content is calcu- lated by subtracting the free acetyl content from total acetyl content. 4 Reagents and materials The reagents
25、 used shall be of recognized analytical grade, unless otherwise specified. The water used shall comply with the specifications of IS0 3696, grade 2. The enzymes used shall be of a quality equivalent to the relevant enzymes of Boehringer Mannheim”. NOTE 1 able can be used. Suitable test kits which ar
26、e commercially avail- 4.1 Hydrochloric acid, 1 moll1 solution. 4.2 Sodium hydroxide, 5 mol/l solution. 4.3 Buffer solution. In about 70 ml of water, dissolve the following re- agents: 1 ) This information is given for the convenience of users of this International Standard and does not constitute an
27、 endorsement by IS0 of the products named. - CEN EN*ISO*LL2L3 95 I 3404589 0095480 715 m Page 4 EN IS0 11213 : 1995 7,5 g of triethanolamine; 5 Apparatus 420 mg of L-malic acid; 210 mg of magnesium chloride hexahydrate Usual laboratory apparatus and in particular the fol- lowing. 5.1 Conical flasks,
28、 of capacity 250 ml, equipped with screw caps. 5.2 Boiling water bath, equipped with a shaker. (MgCI,.GH,O). Add as much potassium hydroxide 5 moi/l solution as necessary in order to obtain a pH of 8,4. The volume required is about 8 ml. The solution is stable for 1 year when stored at + 4 “C. 5.3 V
29、olumetric flasks, of capacity 200 ml 5.4 Micropipettes or syringes. 4.4 ATP-CoA-NAD solution. 5.5 Molecular absorption spectrometer, suitable for operation at 340 nm. In 20 ml of water, dissolve the following reagents: 5.6 Cuvettes, of quartz glass or other materials 500 mg of crystallized disodium
30、salt trihydrate of transparent at 340 nm, with a thickness of adenosine-5-triphosphate ATP-Na2H,.3H,O; 10 mm 5.7 Sieve, with an aperture of 800 pm. 500 mg of anhydrous sodium hydrogen carbonate; 5.8 Blade mili. 5.9 Water bath, capable of being thermostatically controlled between 20 “C and 25 “C. 50
31、mg of lyophilized trilithium salt of coenzyme A about 85 % (dm) COA; 250 mg of lyophilized free acid monohydrate of nicotinamide adenine dinucleotide B-NAD.H,O 98 YO (dm). 6 Preparation of the sample The solution is stable for 1 week when stored at + 4 “C. Sieve through a 800 pm sieve (5.7). If mate
32、rial does not pass through the sieve, grind the sample with a blade mill (5.8) so that it will completely pass through the 800 pm sieve. Homogenize the sample. 4.5 MDH-CS suspension. 7 Procedure Disperse about 1 100 U (international units) of malate-dehydrogenase IMDH from pig heart; 7.1 Hydrolysis
33、of acetyl groups EC 1.1.1.37) and about 270 U of citrate synthase (CS from pig heart; EC 4.1.371 in 0.4 ml of ammonium 7.1.1 Dispersion of granular starch sulfate solution, c(NH,),SO, = 3,2 mol/l. Weigh, to the nearest 1 mg, approximately 1 g of the The solution is Stable for 1 Year when stored at p
34、repared sample and place it in a conical flask (5.11. + 4 OC. Add 50 ml of hydrochloric acid (4.1) while agitating to NOTE 2 One international unit (1 U) catalyses ensure good dispersion. Continue as described in 1 pmoi/min at 25 “C from the relevant substrate. 7.1.3. 7.1.2 Dispersion of pregelatini
35、zed starch 4.6 ACS solution. Add 50 ml of hydrochloric acid (4.1) to a conical flask Dissolve 20 mg of lyophilizate containing 5 mg of acetylcoenzyme A synthetase (ACS from yeast; EC 6.2.1.1 ; % 16 U) in 0,4 ml of water. The solution is stable for 5 d when stored at + 4 OC. (5.1). Introduce a magnet
36、ic stirrer and start agitation. Slowly and carefully add about 1 g of the prepared sample. Ensure a good, lump-free dispersion. Deter- mine the mass of the test portion by weighing by difference to the nearest 1 mg. CEN EN*ISO*11213 95 3404587 0075481 651 m 7.1.3 Hydrolysis and filtration Seal the c
37、onical flask tightly with the screw cap and place it in the boiling water bath (5.2) with the shaker operating for 30 min. Remove the flask and cool to about 20 “C f 5 “C by immersing it in an ice bath. Open the flask when its content is fully cooled, add 1 O ml of sodium hydroxide solution (4.2), m
38、ix, and wash the contents quantitat- ively into a 200 ml volumetric flask (5.3). Place the volumetric flask in the water bath (5.9) for tempera- ture equilibration between 20 “C and 25 “C. Control the temperature and make up to the mark with dis- tilled water. Filter through a suitable filter paper.
39、 Re- ject the first 20 ml to 30 ml of filtrate and directly use the remaining solution as the test solution in the enzymatic determination, as described in 7.4. 7.2 Free acetate 7.2.1 Dispersion of granular starch Disperse 10 g of the prepared sample, while stirring, in 100 ml of distilled water in
40、a conical flask (5.1). Continue as described in 7.2.3. 7.2.2 Dispersion of pregelatinized starch Add about 100 ml of distilled water to a conical flask (5.1). Introduce a magnetic stirrer and start agitation. Slowly and carefully add about 2 g of the prepared sample. Ensure a good, lump-free dispers
41、ion. Deter- mine the mass of the test portion by weighing by difference to the nearest 1 mg. 7.2.3 Dissolution and filtration Seal the conical flask and agitate for 30 min. Transfer the contents of the conical flask quantitat- ively to a 200 ml volumetric flask (5.3). Place the volumetric flask in t
42、he water bath (5.9) for tempera- ture equilibration between 20 “C and 25 “C. Control the temperature and make up to the mark with dis- tilled water. Filter through a suitable filter paper. Re- ject the first 20 ml to 30 ml of filtrate and directly use the remaining solution as the test solution in t
43、he enzymatic determination, as described in 7.4. 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 YO (dm). For this, weigh to the nearest 0,l mg, about 100 mg of Page 5 EN IS0 11213 : 1995 anhydrous sod
44、ium acetate. Then transfer to a 1 O00 ml volumetric flask. Place the volumetric flask in the water bath (5.9) for temperature equilibration between 20 “C and 25 OC. Control the temperature and make up to the mark with distilled water. Con- tinue as described in 7.4. 7.4 Enzymatic determination of ac
45、etic acid Carry out the enzymatic determination of acetic acid according to the following analytical arrangement and conditions: - wavelength: 340 nm; - temperature: 20 “C to 25 “C. Read the absorbances against a cuvette (5.6) filled with water. NOTE 3 Measurements can also be made at the follow- in
46、g wavelengths with the corresponding molar absorption coefficients (x) used in the calculations: - Hg, 365 nm: x = 3,4 Immol-cm-; - Hg. 334 nm: x = 6,18 I.mmol-cm-. Pipette into cuvettes (5.6) the volumes of reagents indicated in the analytical arrangement in table 1. 8 Expression of results 8.1 Abs
47、orbance difference Calculate the absorbance difference using the equation: L J where AA Ao, Al S b is the numerical value of the absorbance difference; and A, are the numerical values of absorbances measured according to the analytical arrangement of table 1 ; is an index designating the solution wi
48、th sample; is an index designating the blank. CEN EN*ISO*LL2L3 95 I 3404589 0095482 598 1,00 ml 0,20 ml 2.00 ml - prtge 6 EN IS0 11213 : 1995 1.00 ml 0.20 ml 1.50 ml 0.50 ml Table 1 - Analytical arrangement for enzymatic determination of acetic acid MDH-CS suspension (4.5) 0,Ol ml 0.01 ml Reagent an
49、d action 1 Solution with I sample I Blank Buffer solution (4.3) ATP-CoA-NAD solution (4.4) Doubledistilled water Test solution bx the contents of each cuvette and read the absorbance (Ao). To each cuvette add Mix the contents of each cuvette and read the absorbance (A,) after about 3 min. Start the reaction by adding to each cuvette 1 0.02 ml I 0.02 mi I I ACS solution (4.6) I Mix the contents of each cuvette, wait until the reaction has stopped (about 1 O min to 15 min) and read the absorbance (A2). If the reaction has not st
