1、BS ISO 9231:2008 ICS 67.100.01 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BRITISH STANDARD Milk and milk products Determination of the benzoic and sorbic acid contentsThis British Standard was published under the authority of the Standards Policy and Strategy Committee on
2、 31 2008 BSI 2008 ISBN 978 0 580 56534 2 Amendments/corrigenda issued since publication Date Comments BS ISO 9231:2008 National foreword This British Standard is the UK implementation of ISO 9231:2008. The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analys
3、is of milk and milk products. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Stand
4、ard cannot confer immunity from legal obligations. DecemberBS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008Reference numbers ISO 9231:2008(E) IDF 139:2008(E) ISO and IDF 2008INTERNATIONAL STANDARD ISO 9231 IDF 139 First edition 2008-07-15 Milk and milk products Determination of the benzoic and sor
5、bic acid contents Lait et produits laitiers Dtermination de la teneur en acide benzoque et en acide sorbique BS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008 ISO 9231:2008(E) IDF 139:2008(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, th
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8、as been taken to ensure that the file is suitable for use by ISO member bodies and IDF national committees. In the unlikely event that a problem relating to it is found, please inform the ISO Central Secretariat at the address given below. COPYRIGHT PROTECTED DOCUMENT ISO and IDF 2008 All rights res
9、erved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO or IDF at the respective address below. ISO copyright office Interna
10、tional Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published
11、in Switzerland ii ISO and IDF 2008 All rights reservedBS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008 ISO 9231:2008(E) IDF 139:2008(E) ISO and IDF 2008 All rights reserved iii Contents Page Foreword iv Foreword. v 1 Scope. 1 2 Normative references. 1 3 Terms and definitions. 1 4 Principle. 1 5 Re
12、agents 1 6 Apparatus 3 7 Sampling 3 8 Preparation of test sample. 3 8.1 Yogurt and other fermented milks 3 8.2 Other milk products 4 9 Procedure 4 9.1 Precipitation of fats and proteins and clarification. 4 9.2 High-performance liquid chromatography (HPLC) . 4 10 Calculation and expression of result
13、s 5 10.1 Calculation. 5 10.2 Expression of results. 5 11 Precision 5 11.1 Interlaboratory testing 5 11.2 Repeatability 5 11.3 Reproducibility 5 12 Test report. 5 Annex A (informative) Interlaboratory trials 7 Bibliography . 9 BS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008 ISO 9231:2008(E) IDF 13
14、9:2008(E) iv ISO and IDF 2008 All rights reservedForeword 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
15、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 take part in the work. ISO collaborates closely with the International
16、 Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards
17、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. Attention is drawn to the possibility that some of the elements of this document may be the subject of
18、patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 9231 IDF 139 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO
19、 and IDF. BS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008 ISO 9231:2008(E) IDF 139:2008(E) ISO and IDF 2008 All rights reserved v Foreword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in
20、 every member country as well as regional dairy associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods
21、of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the IDF National Committees cast
22、ing a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 9231 IDF 139 was prepared by the International Dairy Federation (IDF) and Technical C
23、ommittee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by the Joint ISO-IDF Action Team Food additives and vitamins of the Standing Committee on Analytical methods for additives and contaminants under the a
24、egis of its project leader, Dr. M. Carl (DE). ISO 9231 IDF 139:2008 cancels and replaces IDF 139:1987, which has been technically revised. BS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008BS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008 INTERNATIONAL STANDARD ISO 9231:2008(E) IDF 139:2008(E) ISO
25、and IDF 2008 All rights reserved 1 Milk and milk products Determination of the benzoic and sorbic acid contents 1 Scope This International Standard specifies a method for the determination of the benzoic and sorbic acid contents in milk and milk products. The method is applicable to milk, dried milk
26、, yogurt and other fermented milks, and cheese and processed cheese, and is suitable for measuring the contents of both compounds at levels of more than 5 mg/kg. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, on
27、ly the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 1042, Laboratory glassware One-mark volumetric flasks 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1
28、benzoic and sorbic acid contents mass fractions of benzoic acid and sorbic acid determined by the procedure specified in this International Standard NOTE The benzoic acid and sorbic acid contents are expressed in milligrams per kilogram of product. 4 Principle Fats and proteins are removed from a sl
29、ightly alkaline solution of the product by Carrez precipitation. Following dilution of the resultant solution with methanol, the supernatant liquid is filtered. The benzoic acid and sorbic acid are separated by high-performance liquid chromatography (HPLC) on a reversed-phase C 18column, measuring t
30、he absorbance at 227 nm and 250 nm. 5 Reagents Unless otherwise specified, use only reagents of recognized analytical grade and distilled water or demineralized water or water of equivalent purity. 5.1 Methanol (CH 3 OH). BS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008 ISO 9231:2008(E) IDF 139:20
31、08(E) 2 ISO and IDF 2008 All rights reserved5.2 Precipitating reagents, as follows: 5.2.1 Potassium hexacyanoferrate(II) solution Dissolve 10,6 g of potassium hexacyanoferrate(II) trihydrate (K 4 Fe(CN) 6 .3H 2 O) in water in a 100 ml one-mark volumetric flask (6.3). Dilute to the mark with water an
32、d mix. NOTE The 100 ml solution is sufficient for 40 runs. 5.2.2 Zinc acetate solution Dissolve 21,9 g of zinc acetate dihydrate (CH 3 COO) 2 Zn.2H 2 O and 32 ml of acetic acid (CH 3 COOH) in water in a 100 ml one-mark volumetric flask (6.3). Dilute to the mark with water and mix. If the zinc acetat
33、e dihydrate does not dissolve completely, heat the 100 ml flask and its contents in a water bath (6.2) maintained at 70 C while swirling. When the zinc acetate dihydrate has dissolved completely, cool the solution thus obtained back to room temperature. Dilute to the mark with water and mix again. N
34、OTE The 100 ml solution is sufficient for 40 runs. 5.3 Phosphate buffer solution, pH 6,7. Dissolve 2,5 g of potassium dihydrogen phosphate (KH 2 PO 4 ) and 2,5 g of potassium hydrogen phosphate trihydrate (K 2 HPO 4 .3H 2 O) in 1 l of water and mix. Filter the solution thus obtained through the solv
35、ent filtration system (6.8). 5.4 Mobile phase, for HPLC. Mix 10 volumes of methanol (5.1) with 90 volumes of phosphate buffer solution (5.3). Remove any dissolved gas by applying a slight vacuum. 5.5 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. Dissolve 4,0 g of sodium hydroxide pellets in water
36、in a 1 000 ml one-mark volumetric flask (6.3). Dilute to the mark with water and mix. 5.6 Sulfuric acid, c(H 2 SO 4 ) = 0,5 mol/l. Pour cautiously 15 ml of concentrated sulfuric acid, with a mass fraction of at least 95 % to 98 %, into 250 ml of water in a 500 ml one-mark volumetric flask (6.3) and
37、allow to cool. Dilute to the mark with water and mix. 5.7 Sorbic acid and benzoic acid standard solutions, as follows: 5.7.1 Stock standard solution Dissolve 50 mg of sorbic acid and 50 mg of benzoic acid in methanol (5.1) in a 100 ml one-mark volumetric flask (6.3). Dilute with methanol (5.1) to th
38、e mark and mix. The stock standard solution is stable for at least three weeks if stored in a refrigerator at between 4 C and 7 C. 5.7.2 Working standard solution Mix 500 ml of methanol (5.1) with 500 ml of water to obtain an aqueous-methanol solution with a volume fraction of 50 %. On the day of us
39、e, pipette 5 ml of stock standard solution (5.7.1) into a 250 ml one-mark volumetric flask (6.3). Dilute with the 50 % aqueous-methanol solution to the mark and mix. The resulting working standard solution contains 10 g/ml of both the sorbic and benzoic acid. BS ISO 9231:2008 BS ISO 9231:2008 BS ISO
40、 9231:2008 ISO 9231:2008(E) IDF 139:2008(E) ISO and IDF 2008 All rights reserved 3 6 Apparatus Usual laboratory equipment and, in particular, the following: 6.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg. 6.2 Water bath, capable of maintaining a tempera
41、ture of 70 C 2 C. 6.3 One-mark volumetric flasks, of capacities 100 ml, 250 ml, 500 ml and 1 000 ml, meeting the requirements for class A as specified in ISO 1042. 6.4 Liquid chromatograph, equipped with a pump capable of generating pressures of up to 4,37 MPa (6 000 psi), an injector, a dual-wavele
42、ngth or diode-array UV detector, and a recorder or integrator. The dual-wavelength detector shall have a 1 cm light path flow-through optical cell and shall be capable of measuring absorbance at 227 nm (for benzoic acid) and 250 nm (for sorbic acid). 6.5 HPLC column, made of stainless steel, of leng
43、th 250 mm, of internal diameter 4 mm, containing a reversed-phase, octadecyl (ODC) treated silica adsorbent, i.e. Micro-Bondapak C 18 1)or similar. 6.6 Syringe for HPLC. 6.7 Sample clarification kit, for membrane filtration of sample extracts, with filters of pore size 0,45 m for aqueous solutions.
44、6.8 Solvent filtration system, for membrane filtration of solvents, with filters of pore size 0,45 m for aqueous solutions. 6.9 Ultrasonic bath. 6.10 pH-meter. 7 Sampling A representative sample should have sent to the laboratory. It should not have been damaged or changed during transport or storag
45、e. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707 IDF 501 . Store the test sample in such a way that deterioration and change in composition are prevented. 8 Preparation of test sample 8.1 Yogurt and other fermented milk
46、s Prior to starting the procedure, homogenize the sample by warming it gently to 40 C while stirring. Weigh, to the nearest 0,1 g, 20 g of the homogenized sample into a 100 ml one-mark volumetric flask (6.3). 1) Micro-Bondapak C 18 is the name of a product available commercially. This information is
47、 given for the convenience of the users of this International Standard but does not constitute an endorsement by either ISO or IDF of the product named. BS ISO 9231:2008 BS ISO 9231:2008 BS ISO 9231:2008 ISO 9231:2008(E) IDF 139:2008(E) 4 ISO and IDF 2008 All rights reserved8.2 Other milk products W
48、eigh, to the nearest 0,1 g, 3 g of sample into a 20 ml glass beaker. Disperse the test sample completely in 10 ml water added in small portions while stirring with a glass rod. Transfer the solution quantitatively to a 100 ml one-mark volumetric flask (6.3), rinsing the beaker twice with 5 ml of wat
49、er. 9 Procedure 9.1 Precipitation of fats and proteins and clarification Add 25 ml of sodium hydroxide solution (5.5) to the test sample (see 8.1 or 8.2) and mix. Either place the flask and its contents in an ultrasonic bath (6.9) for 15 min or place the flask and its contents in a water bath (6.2) maintained at 70 C and heat for 15 min, subsequently allowing the solution to cool to room temperature. Adjust the pH to 8 1 by adding sulfuric acid solution (5.6) while mixing. Then add 2 ml of potassium hexacyanofe
