1、BRITISH STANDARD BS 4401-18:1998 ISO 13965:1998 Methods of test for meat and meat products Part 18: Determination of starch and glucose contents (enzymatic method) ICS 67.120.10BS4401-18:1998 This British Standard, having been prepared under the directionof the Consumer Products and Services Sector
2、Committee, was published underthe authority of the Standards Committee and comesinto effect on 15 November 1998 BSI 06-1999 ISBN 0 580 30574 0 National foreword This British Standard reproduces verbatim ISO 13965:1998 and implements it as the UK national standard. The UK participation in its prepara
3、tion was entrusted to Technical Committee AW/6, Chemical analysis of meat and meat products, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the U
4、K interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publication
5、s referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions
6、 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 document comprises a front cover, an inside front cover, pages i and ii, the ISO title page, p
7、ages ii to iv, pages 1 to 8, 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 cover. Amendments issued since publication Amd. No. Date CommentsBS 4401
8、-18:1998 BSI 06-1999 i Contents Page National foreword Inside front cover Foreword iii Text of ISO 13965 1ii blankBS 4401-18:1998 ii BSI 06-1999 Contents Page Foreword iii 1 Scope 1 2 Normative reference 1 3 Definitions 1 4 Principle 1 5 Reagents 1 6 Apparatus 2 7 Sampling 3 8 Preparation of test sa
9、mple 3 9 Procedure 3 10 Calculation 5 11 Precision 6 12 Test report 7 Annex A (informative) Example of plotting and extrapolation ofabsorbance values 8 Annex B (informative) Bibliography Inside back cover Figure A.1 Example of plotting and extrapolation of absorbance values 8BS 4401-18:1998 BSI 06-1
10、999 iii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for w
11、hich 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 take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all
12、 matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard ISO
13、13965 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 6, Meat and meat products. Annex A and Annex B of this International Standard are for information only. Descriptors: Agricultural products, food products, animal products, meat, meat products, chemical a
14、nalysis, determination of content, starches, glucose, enzymatic methods.iv blankBS4401-18:1998 BSI 06-1999 1 1 Scope This International Standard specifies an enzymatic method for the determination of water-free starch content and glucose content of all kinds of meat and meat products, including poul
15、try. The method is suitable for the quantitative determination of starch and glucose contents down to levels of0,30 % (m/m). The method is not applicable for chemically modified starches or their derivatives. 2 Normative reference The following standard contains provisions which, through reference i
16、n this text, constitute provisions of this International Standard. At the time of publication, the edition indicated was valid. All standards are subject to revision, and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most rec
17、ent edition of the standard indicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 3696:1987, Water for analytical laboratory use Specification and test methods. 3 Definitions For the purposes of this International Standard, the following definitio
18、ns apply. 3.1 starch content of meat and meat products starch content determined in accordance with the procedure described in this International Standard, and expressed as a percentage by mass 3.2 glucose content of meat and meat products glucose content determined in accordance with the procedure
19、described in this International Standard, and expressed as a percentage by mass 4 Principle 4.1 Hydrolysis of the starch present in a test portion with the enzyme -amylase at pH = 5,0 for 15 min. Determination of the starch content using the following enzymatic reactions. 4.2 Hydrolysis of the solub
20、ilized starch to yield glucose using amyloglucosidase (AGS): For the determination of the glucose content, this step is omitted. 4.3 Phosphorylation of the glucose generated by means of adenosine 5-triphosphate (ATP) to yield glucose6-phosphate (G-6-P) using hexokinase (HK): 4.4 Oxidation of glucose
21、 6-phosphate (G-6-P) by means of nicotinamide adenine dinucleotide phosphate (NADP) to gluconate 6-phosphate using glucose 6-phosphate dehydrogenase (G-6-PDH): 4.5 Spectrometric measurement of the amount of reduced nicotinamide dinucleotide phosphate (NADPH) at a wavelength of 340 nm. 5 Reagents Use
22、 only reagents of recognized analytical grade, unless otherwise specified. 5.1 Water, complying with at least grade 3 in accordance with ISO 3696.BS4401-18:1998 2 BSI 06-1999 5.2 -Amylase (EC 3.2.1.1), enzyme suspension. A liquid enzyme preparation of a heat-stable -amylase produced from Bacillus li
23、cheniformis (Termamyl 120L) 1) . 5.3 Sodium hydroxide solution, c(NaOH) = 5 mol/l. Dissolve 200 g of sodium hydroxide in water. Cool to room temperature, dilute to 1000 ml and mix. 5.4 Sodium hydroxide solution, c(NaOH) = 0,5 mol/l. Dissolve 20 g of sodium hydroxide in water. Cool to room temperatur
24、e, dilute to 1000 ml and mix. 5.5 Ammonium sulfate solution, c(NH 4 ) 2 SO 4 = 3,2 mol/l. Dissolve 422 g of ammonium sulfate in water. Dilute to 1000 ml and mix. 5.6 Acetate buffer, c(CH 3 CO 2 Na) = 0,1 mol/l, pH = 5,0. Dissolve 6,80 g of sodium acetate trihydrate (CH 3 CO 2 Na3H 2 O) in 400 ml of
25、water. Adjust the pH to 5,0 with hydrochloric acid or sodium hydroxide solution with a pH-meter (6.2). Dilute with water to 500 ml and mix. The solution is stable for at least 3 months at +4 C in the dark. 5.7 Citrate buffer, c(citrate) = 0,05 mol/l, pH = 4,6. Dissolve 440 mg of citric acid monohydr
26、ate (C 6 H 8 O 7 H 2 O) and 850mg of trisodium citrate dihydrate (C 6 H 5 Na 3 O 7 2H 2 O) in water. Dilute with water to 100 ml and mix. Check the pH with a pH-meter (6.2) and adjust if necessary with hydrochloric acid or sodium hydroxide solution. The solution is stable for at least 3 months at +4
27、 C in the dark. 5.8 Triethanolamine buffer, c(triethanolamine) = 0,75 mol/l, pH = 7,6. Dissolve 14,0 g of triethanolamine hydrochloride (C 6 H 15 NO 3 HCl) and 0,25 g of magnesium sulfate heptahydrate (MgSO 4 7H 2 O) in 80 ml of water. Set the pH at 7,6 with a pH-meter, using the sodium hydroxide so
28、lutions (5.3 and 5.4). Dilute with water to 100 ml and mix. The solution is stable for 4 weeks at +4 C in the dark. 5.9 Nicotinamide adenine dinucleotide phosphate solution, c(“-NADP-Na 2 ) = 12,7 10 3mol/l. Dissolve 100 mg of NADP disodium salt in 10,0 ml of water and mix. The solution is stable fo
29、r 4 weeks at +4 C in the dark. 5.10 Adenosine-5-triphosphate solution, c(59-ATP-Na 2 H 2 3H 2 O) . 81 10 3mol/l. Dissolve 500 mg of 59-ATP-Na 2 H 2 3H 2 O and 500 mg of anhydrous monosodium hydrogen carbonate (NaHCO 3 ) in 10,0 ml of water and mix. The solution is stable for 4 weeks at +4 C in the d
30、ark. 5.11 Amyloglucosidase (AGS; EC 3.2.1.3), enzyme suspension in ammonium sulfate solution (5.5), (AGS)= 10 mg/ml. The specific activity of the enzyme shall be 14 units per milligram. The suspension is stable for 1 year at +4 C in the dark. 5.12 Hexokinase (HK; EC 2.7.1.1)/glucose 6-phosphate dehy
31、drogenase (G-6-PDH; EC 1.1.1.49), enzyme suspension in ammonium sulfate solution (5.5), (HK) = 2 mg/ml and (G-6-PDH) = 1 mg/ml. The specific activity of both enzymes shall be 140 units per milligram. The suspension is stable for 1 year at +4 C in the dark. 6 Apparatus Usual laboratory apparatus and,
32、 in particular, the following. 6.1 Mechanical or electrical equipment, capable of homogenizing the laboratory sample. This includes a high-speed rotational cutter, or a mincer fitted with a plate with apertures not exceeding4,0 mm in diameter. 6.2 pH-meter. 1) Termamyl 120 L is an example of a suita
33、ble product available commercially from Novo, Denmark, and Tecator, Sweden. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product.BS4401-18:1998 BSI 06-1999 3 6.3 Fluted filter papers, glucose free, of diam
34、eter about 15 cm. 6.4 Pipettes, calibrated, for enzymatic analysis, or automatic micropipettes of equivalent quality with the following volumes: 20 4l, 50 4l, 100 4l and 200 4l. 6.5 Small plastics spatula, for mixing the contents of a cuvette (6.9). 6.6 Water bath, capable of being maintained at (60
35、 2) C. 6.7 Hot plate. 6.8 Spectrometer, capable of measuring at a wavelength of 340 nm. NOTEWhen a spectrometer fitted with a mercury vapour lamp is available, the readings can be carried out at 365 nm and 334 nm. The molecular absorption coefficient for NADPH is 3,51 lmmol 1 cm 1at 365 nm and 6,18
36、lmmol 1 cm 1 at 334 nm. 6.9 Cuvettes, made of quartz or glass, with lid, or disposable cuvettes for single use, made of polymethacrylate, of 10 mm optical path length. 6.10 Analytical balance, capable of weighing to the nearest 0,1 mg. 7 Sampling Sampling is not part of the method specified in this
37、International Standard. A recommended sampling method is given in ISO 3100-1 1. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage. Start from a representative sample of at least 200 g. Store the sample i
38、n such a way that deterioration and change in composition are prevented. 8 Preparation of test sample Homogenize the laboratory sample with the appropriate equipment (6.1). Take care that the temperature of the sample material does not rise above 25 C. If a mincer is used, pass the sample at least t
39、wice through the equipment. Fill a suitable airtight container with the prepared sample. Close the container and store in such a way that deterioration and change in composition are prevented. Analyse the sample as soon as practicable, but always within 24 h after homogenization. 9 Procedure NOTEMus
40、cle glycogen, which occurs normally in, for example, sausages, does not interfere with the determination. Maltose interferes because this disaccharide is hydrolysed by amyloglucosidase into glucose. Maltose (and glucose) can, however, be extracted from the sample with alcohol. 9.1 Test portion If th
41、e test sample contains maltose, make sure that the water content does not exceed 20 % (m/m). If necessary, dry the test sample. Weigh about 400 mg of the prepared test sample (see clause 8) to the nearest 0,1 mg into a centrifuge tube. Proceed in accordance with 9.2. If the test sample does not cont
42、ain maltose, weigh between 100 mg and 1,0 g (m) of the prepared test sample to the nearest 0,1 mg into a 100 ml conical flask. Add 30 ml of acetate buffer (5.6) and proceed in accordance with 9.3. 9.2 Extraction of maltose (and glucose) Wash the sample three times with 10 ml of 40 % (V/V) ethanol an
43、d centrifuge after each washing. Filter the supernatant. Combine the precipitate in the centrifuge tube with that on the filter and transfer to a100ml conical flask using 4 5 ml of acetate buffer (5.6). Add 10 ml of acetate buffer (5.6). 9.3 Preparation of extract Pipette (6.4) 50 4l of -amylase sus
44、pension (5.2) into the conical flask containing the sample. Cover the conical flask with aluminium foil and boil on a hot plate (6.7) for 15 min. Shake the flask at intervals.BS4401-18:1998 4 BSI 06-1999 Then keep the conical flask at 60 C in the water bath (6.6) for 15 min. Quantitatively transfer
45、the contents of the conical flask to a 100 ml volumetric flask. Rinse the conical flask with warm water and add the washings to the volumetric flask. Allow to cool to room temperature and dilute to the mark with water. Filter (6.3) at least 10 ml of the sample extract, discarding the first few milli
46、litres, and immediately proceed in accordance with 9.4. Keep samples containing fat for 1 h in the refrigerator before filtration. 9.4 Determination 9.4.1 Prepare a reagent blank solution as follows. Pipette (6.4) 200 4l of citrate buffer (5.7) and 100 4l of water into a cuvette (6.9) containing a s
47、patula(6.5). 9.4.2 Prepare a test solution for the glucose determination as follows. Pipette (6.4) 200 4l of citrate buffer(5.7) and 100 4l (V 2 ) of the sample extract (9.3) into a cuvette (6.9) containing a spatula(6.5). 9.4.3 Prepare a test solution for the starch determination as follows. Pipett
48、e (6.4) 200 4l of citrate buffer(5.7) and 100 4l (V 2 ) of the sample extract (9.3) into a cuvette (6.9) containing a spatula (6.5). If the concentration of starch in the sample solution exceeds 0,4 g/l, dilute it before analysis. 9.4.4 Pipette (6.4) 20 4l of AGS suspension (5.11) into the cuvette c
49、ontaining the reagent blank solution(9.4.1) and into the cuvette containing the test solution for the starch determination (9.4.3). Pipette 20 4l of water into the cuvette containing the test solution for the glucose determination (9.4.2). Mix the contents of the cuvettes by swirling or by moving the spatula up and down. Close the cuvettes with the lid or otherwise (e.g.paraffin sheet). Keep the cuvettes in the water bath (6.6) for 15 min at (60 2) C. The above pipette procedure is schematically presented b
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