1、c - a 3 a C E E 3 5 c c 2 s $ E 2 5 C u1 E C o m I: c C 5 3 U u V 2 m il m E ? m V 5 Yi Yi s w r O z o Modified starch Determination of acetyl content by the enzymatic method (IS0 11213:1995) English version of DIN EN IS0 11 21 3 DIN - EN IS0 11213 This standard incorporates the English version of I
2、 so 1 1 2 1 3. ICs 67.180.20 Supersedes DIN 10387-1, Descriptors: Starch, acetyl content, testing. Modifizierte Strke; Bestimmung des Acetylgehaltes; enzymatisches Verfahren (IS0 11 213:1995) December 1986 edition. European Standard EN IS0 11 213: 1995 has the status of a DIN Standard. A comma is us
3、ed as the decimal marker. National foreword This standard has been published in accordance with a decision taken by CEN to adopt, without alteration, International Standard IS0 11 21 3 as a European Standard. The responsible German body involved in its preparation was the Normenausschu Lebensmittel
4、und landwirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Committee). DIN EN IS0 1666 is the standard corresponding to International Standard IS0 1666 and DIN IS0 3696 to IS0 3696 re- ferred to in clause 2. Amendments In comparison with DIN 10387-1, December 1986 edition, the
5、following amendments have been made. a) The titration method has been replaced by an enzymatic method. b) Data on precision have been included. Previous edition DIN 10387-1: 1986-12. Standard referred to (and not included in Normative references and Annex ZA) DIN EN IS0 1666 Starch; determination of
6、 moisture content; oven-drying method EN comprises 10 pages. Beuth Verlag GmbH, Berlin, has the exclusive right of sale for German Standards (DIN-NormenJ. DIN EN /SO 11 213 Engl. Price group 12.95 Sales No. 11 O8 EUROPEAN STANDARD NORME EUROPENNE EUROPAISCHE NORM EN IS0 11213 January 1995 ICs 67.1 8
7、0.20 Descriptors: Starch, acetyl content, testing. English version Modified starch Determination of acetyl content Enzymatic method (IS0 11213:1995) Amidon modifi; dosage de lactyle; mthode enzymatique (IS0 11 213: 1995) Modifizierte Strke; Bestimmung des Acetylgehaltes; enzymatisches Verfahren (IS0
8、 11 21 3 : 1995) This European Standard was approved by CEN on 1994-11-28 and is identical to the IS0 Standard as referred to. CEN members are bound to comply with the CENEENELEC Internal Regulations which stipu- late the conditions for giving this European Standard the status of a national standard
9、 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 European Standard exists in three official versions (English, French, German). A version in any other langu
10、age 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 national standards bodies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland,
11、 Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europben de Normalisatjon Europisches Komitee fr Normung Central Secretariat: rue de Stassari 36, B-1050 Brussels O 1995. Copyright reserved to all CEN m
12、embers. Ref. No. ENIS011213:1995E Page 2 EN ISO11213:1995 Foreword international Standard IS0 11 213: 1995 Modified starch; determination of acetyl content; enzymatic method, was prepared by ISOTTC 93 Starch (including derivatives and by-products) of the International Organization for Standardi- zat
13、ion, in collaboration with CEN/CS.lt was submitted for Formal Vote and approved by CEN as a European Standard. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, and conflicting national standards withdrawn, by July
14、1995 at the latest. In accordance with the CENKENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerla
15、nd and United Kingdom. Endorsement notice The text of the International Standard IS0 11 213:1995 was approved by CEN as a European Standard without any modifi- cation. NOTE: Normative references to international publications are listed in Annex ZA (normative). Page 3 ENIS011213:1995 1 Scope This Int
16、ernational 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- tent
17、s up to 2 YO (dm). 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 agr
18、eements 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 moistur
19、e content - Oven-drying methods. IS0 3696:1987, Water for analytical laboratory use - Specification 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 presenc
20、e of the enzyme acetyl-coA synthetase (ACS), acetate is converted with adenocine-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 mala
21、te 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 cont
22、ent 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 reagen
23、ts 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
24、are commercially avail- 4.1 Hydrochloric acid, 1 mol/l solution. 4.2 Sodium hydroxide, 5 mol/l solution. 4.3 Buffer solution. In about 70 mi 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 a
25、n endorsement by IS0 of the products named. Page 4 EN IS0 11 213: 1995 7.5 g of triethanolamine; 420 mg of L-malic acid; 5 Apparatus Usual laboratory apparatus and in particular the fol- lowing. 210 mg of magnesium chloride hexahydrate (MgCI,.GH,O). Add as much potassium hydroxide 5 mol/l solution a
26、s 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. 4.4 ATP-CoA-NAD solution. In 20 ml of water, dissolve the following reagents: 500 mg of crystallized disodium salt trihydrate of adenosine-5-triphosphate ATP-Na,H,.
27、3H20; 98 Yo (dm); 5.1 with 5.2 5.3 5.4 5.5 Conical flasks, of capacity 250 ml, equipped screw caps. Boiling water bath, equipped with a shaker. Volumetric flasks, of capacity 200 ml. Micropipettes or syringes. Molecular absorption spectrometer, suitable for operation at 340 nm. 5.6 Cuvettes, of quar
28、tz glass or other materials transparent at 340 nm, with a thickness of 10 mm -r 0,l mm. 5.7 Sieve, with an aperture of 800 pm. 500 mg of anhydrous sodium hydrogen carbonate: 5.8 Blade mill. 50 mg of lyophilized trilithium salt of coenzyme A about 85 Yo (dm) COA; 250 mg of lyophilized free acid monoh
29、ydrate of nicotinamide adenine dinucleotide p-NAD.H,O 98 YO (dm). The solution is stable for 1 week when stored at + 4 “C. 4.5 MDH-CS suspension. Disperse about 1 100 U (international units) of malate-dehydrogenase (MDH from pig heart; EC 1 .I .I ,371 and about 270 U of citrate synthase (CS from pig
30、 heart; EC 4.1.37) in 0.4 ml of ammonium sulfate solution, c(NH EC 6.2.1 .I; l 16 U) in 0,4 ml of water. The solution is stable for 5 d when stored at + 4 OC. 5.9 Water bath, capable of being thermostatically controlled between 20 “C and 25 “C. 6 Preparation of the sample Sieve through a 800 pm siev
31、e (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 the 800 pm sieve. Homogenize the sample. 7 Procedure 7.1 Hydrolysis of acetyl groups 7.1.1 Dispersion of granular starch Weigh, to the nearest 1 mg, approximately 1
32、g of the prepared sample and place it in a conical flask (5.1). Add 50 ml of hydrochloric acid (4.1) while agitating to ensure good dispersion. Continue as described in 7.1.3. 7.1.2 Dispersion of pregelatinized starch Add 50 ml of hydrochloric acid (4.1) to a conical flask (5.1). Introduce a magneti
33、c stirrer and start agitation. Slowly and carefully add about 1 g of the prepared sample. Ensure a good, lurnp-free dispersion. Deter- mine the mass of the test portion by weighing by difference to the nearest 1 mg. Page 5 EN IS0 11 213:1995 7.1.3 Hydrolysis and filtration Seal the conical flask tig
34、htly 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 It 5 “C by immersing it in an ice bath. Open the flask when its content is fully cooled, add 10 ml of sodium hydroxide solution (4.21, mix, and wash the
35、 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. Re- ject the fi
36、rst 20 ml to 30 mi 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 a conical flask
37、(5.11. 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 dispersion. Deter- mine
38、 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 the water bath (5
39、.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 the enzymatic de
40、termination, 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 % (dm). For this, weigh to the nearest 0,l mg, about 100 mg of anhydrous sodium acetate. Then transfer to a 1 O00 ml vo
41、lumetric 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 acetic acid Carry out the enzymatic determina
42、tion of acetic acid according to the following analytical arrangement and conditions: - wavelength: 340 nm; - temperature: 20 “C to 25 OC. Read the absorbances against a cuvette (5.6) filled with water. NOTE 3 Measurements can also be made at the follow- ing wavelengths with the corresponding molar
43、absorption coefficients (x) used in the calculations: - Hg, 365 nm: x = 3.4 bmmol- 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 Absorbance difference Calculate the absorban
44、ce difference using the equation: where AA is the numerical value of the absorbance difference; 4, A, and A2 are the numerical values of absorbances measured according to the analytical arrangement of table 1 ; is an index designating the solution with sample; s b is an index designating the blank.
45、Page 6 ENIS011213:1995 Reagent and action Table 1 - Analytical arrangement for enzymatic determination of acetic acid I I Solution with sample Blank 0,20 ml 2,OO ml Buffer solution (4.3) I I,OO mi I 1.00 mt 0.20 ml 1,50 ml ATP-CoA-NAD solution (4.4) Double-distilled water Test solution - 0,50 ml ACS
46、 solution (4.6) MDH-CS suspension (4.5) I O,OI mi I O,OI mi 0,02 ml 0,02 ml Mix the contents of each cuvette, wait until the reaction has stopped (about 10 min to 15 min) and read the absorbance (A2). If the reaction has not stopped after about 15 min, continue to read the absorbance at 2 min interv
47、als until the absorbance increases constantly for 2 min. NOTE - The volume of solution with sample can be changed to accommodate very high or very low acetate levels; the final total volume must always be 3.23 ml. 8.2 Total acetyl content If an aliquot of solution with sample other than the 0,50 ml
48、specified in table 1 is taken, the equation has to be adjusted accordingly. When the check test (7.3) is effected, a dilution factor of 5 has to be in- cluded because the test solution is made up to 1 O00 ml and not 200 ml as in the procedure for the test sample (see 7.1 and 7.2). NOTE 4 A full expl
49、anation of the derivation of the Calculate the total acetyl content using the equation 556 x AA 1 O0 w= a xxm, 100 - wm where wa AA n m1 Wm is the numerical value of the total acetyl content, in percentage by mass, of the test sample; is the numerical value of the absorbance difference calculated according to 8.1; is the numerical value of the molar ab- sorption coefficient of NADH at 340 nm, in litres per millimole centimetre (x = 6,30 I-mrnol- cm- ); is the numerical value of the mass, in grams, of the test portion (see 7.1.1 or 7.1 21; is the numerical val
