1、BRITISH STANDARD BS ISO 11814:2002 Dried milk Assessment of heat treatment intensity Method using high-performance liquid chromatography ICS 67.100.10 BS ISO 11814:2002 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Policy and Strategy Co
2、mmittee, was published under the authority of the Standards Policy and Strategy Committee on 13 December 2002 BSI 13 December 2002 ISBN 0 580 40948 1 National foreword This British Standard reproduces verbatim ISO 11814:2002 and implements it as the UK national standard. The UK participation in its
3、preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international publications referre
4、d to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisio
5、ns of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or
6、 proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to v, a blank page, pages 1 to 9 and a back co
7、ver. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date Comments Reference numbers ISO 11814:2002(E) IDF 162:2002(E)INTERNATIONAL STANDARD ISO 11814 IDF 162 First edition 2002-11-15 Dried milk Assessment of
8、 heat treatment intensity Method using high- performance liquid chromatography Lait sec valuation de lintensit du traitement thermique Mthode par chromatographie en phase liquide haute performance BSISO11814:2002BSISO11814:2002iiIS:41811 O2002(E) ID:261 F2002(E) I SO dna ID 2002 F All irhgts seredev
9、r iiiForeword 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 whic
10、h 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 ma
11、tters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circ
12、ulated 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 International Standard ISO 11814|IDF 162 may be the subject of patent right
13、s. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO 11814|IDF 162 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AO
14、AC International. It is being published jointly by ISO and IDF and separately by AOAC International. Annex A of this International Standard is for information only. BSISO11814:2002iiiIS:41811 O2002(E) ID:261 F2002(E) vi I SO dna ID 2002 F All irhgts seredevrForeword IDF (the International Dairy Fede
15、ration) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of sta
16、ndard methods 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 National Committee
17、s casting a vote. International Standard ISO 11814|IDF 162 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and
18、 separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team Characterization of milk and milk products according to heat treatment, of the Standing Committee on Minor components and characterization of physical properties, under the aegis of its project leader,
19、Mr M.A.J.S. van Boekel (NL). BSISO11814:2002ivIS:41811 O2002(E) ID:261 F2002(E) I SO dna ID 2002 F All irhgts seredevr vIntroduction The limits for (extra) low-heat skim milk powder can be influenced by the origin of the milk used for production, as well as the heat treatment applied during processi
20、ng of the powder or of the reference sample (see 4.4). Therefore in this International Standard no limits are given for the relative ratio, r (see 9.1) and the relative sample ratio, r t(see 9.3). It is advisable to have r and r tstandard limits for extra-low-heat powder which depend on the applicat
21、ion requirements. The standard limits should be based on the national legislation of the country concerned. BSISO11814:2002vNITERNATNOIAL STANDARD IS:41811 O2002(E) ID:261 F2002(E)I SO dna ID 2002 F All irhgts seredevr 1Dried milk Assessment of heat treatment intensity Method using high-performance
22、liquid chromatography 1 Scope This International Standard specifies a high-performance liquid chromatographic (HPLC) method for an assessment of the heat treatment intensity to be applied during the processing of milk powder, in order to differentiate extra-low-heat skim milk powder from low-heat sk
23、im milk powder. 2 Term and definition For the purposes of this International Standard, the following term and definition applies. 2.1 extra-low-heat skim milk powder skim milk powder with a minimum content of denatured whey proteins, as determined by the procedure specified in this International Sta
24、ndard 3 Principle A test portion of milk powder is dissolved in water. Casein and denatured whey proteins are precipitated iso- electrically at pH 4,6. The undenatured whey proteins present in the filtrate are determined by high-performance liquid chromatography. The results obtained are interpreted
25、. 4 Reagents Use only reagents of recognized analytical grade and distilled or demineralized water or water of at least equivalent purity. 4.1 Hydrochloric acid, c(HCl) 1 mol/l. Dilute 80 ml of concentrated hydrochloric acid 37 % (mass fraction) to 1 000 ml with water and mix. 4.2 Eluent, pH 6,0 Dis
26、solve 1,74 g of dipotassium hydrogen phosphate (K 2 HPO 4 ), 12,37 g of potassium dihydrogen phosphate (KH 2 PO 4 ) and 21,41 g of sodium sulfate (Na 2 SO 4 ) in about 700 ml of water. Adjust the pH to 6,0 with phosphoric acid solution (85 %) or potassium hydroxide solution (10 mol/l), if necessary.
27、 Dilute with water to 1 000 ml and mix. Filter the eluent through a 0,45 m membrane filter prior to use. 4.3 Flushing solvent Mix 100 ml of acetonitrile (CH 3 CN) with 900 ml of water. Filter the mixture through a 0,45 m membrane filter prior to use. BSISO11814:20021IS:41811 O2002(E) ID:261 F2002(E)
28、 2 I SO dna ID 2002 F All irhgts seredevrOther flushing solvents may be used, provided that they inhibit the growth of bacteria and do not affect the separation performance of the column. 4.4 Reference sample Use extra-low-heat skim milk powder, with a minimum content of denatured whey proteins. 5 A
29、pparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, capable of weighing to the nearest 1 mg, with readability to 0,1 mg. 5.2 Beakers, of capacity 50 ml. 5.3 Graduated cylinder, of capacity 50 ml. 5.4 Graduated pipettes, capable of delivering 2 ml. 5.5 Magne
30、tic stirrer. 5.6 Filter paper, medium grade, of diameter about 15 cm. 5.7 Filter funnels, of diameter about 7 cm. 5.8 Conical flasks, of capacity 50 ml. 5.9 HPLC equipment, consisting of the following. 5.9.1 Magnetic stirrer, provided with a heater for keeping the eluent at 85 C 1 C. 5.9.2 Pump, cap
31、able of delivering a flow of 1,0 ml/min. 5.9.3 Injector, hand or automatic, with a 20 l injection capacity. 5.9.4 Zorbax Bio column, series GF-250 column, length 25 cm and 0,94 cm internal diameter, or an equivalent column in combination with a precolumn, length 3 cm and 0,3 cm internal diameter, pa
32、cked with protein I-125 (Millipore Waters) or an equivalent packing material. 1)NOTE Typical retention times obtained by the procedure specified in this International Standard and using the GF-250 column are (see Figures A.1 and A.2 for examples): immunoglobulin fraction (Ig): 8,2 min bovine serum a
33、lbumin fraction (BSA): 8,8 min -lactoglobulin A and B fraction ( -Lg): 9,7 min -lactalbumin ( -La): 10,6 min 5.9.5 Thermostatic column oven, capable of maintaining a temperature of 30 C 1 C. 5.9.6 UV-detector, capable of operating at 280 nm. 1) Zorbax Bio column and Millipore Waters packing material
34、 are examples of suitable products available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF of these products. BSISO11814:20022IS:41811 O2002(E) ID:261 F2002(E) I SO dna ID 2002 F All irhgts ser
35、edevr 35.9.7 Integrator, capable of measuring peak area. Chose the integration control parameters in such a way that a) the baseline is allocated at the beginning and at the end of the chromatogram (see Figure A.2), b) the peak areas of the whey proteins are measured by the perpendicular drop method
36、 (see Figure A.2), and c) any late peaks from the last sample do not interfere with the integration of the next sample. 6 Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707. It is important that the laboratory recei
37、ve a sample which is truly representative and has not damaged or changed during transport or storage. Store the sample in such a way that deterioration and change in composition are prevented. 7 Preparation of test sample Transfer the test sample to a container of capacity about twice the volume of
38、the sample, provided with an airtight lid. Close the container immediately. Mix the milk powder thoroughly by repeatedly shaking and inverting the container. 8 Procedure 8.1 Test portion Weigh, to the nearest 1 mg, 2 g of the prepared test sample (clause 7) into a beaker (5.2). 8.2 Test solution 8.2
39、.1 Add 40 ml of water preheated to 40 C. Dissolve the test portion by stirring for 30 min using a magnetic stirrer (5.5). 8.2.2 Adjust the test solution to pH 4,6 by a dropwise addition of the hydrochloric acid (4.1) using a graduated pipette (5.4). Stir during the pH adjustment with the aid of the
40、magnetic stirrer (5.5). Allow the mixture stand for 15 min at room temperature. Check the pH of the mixture and, if necessary, re-adjust the pH to 4,6. 8.2.3 Filter the mixture through a filter paper (5.6) into a conical flask (5.8), discarding the first fraction of the filtrate. 8.3 Reference solut
41、ion Weigh, to the nearest 1 mg, 2 g of the reference sample (4.4) into a beaker (5.2) and proceed as in 8.2. 8.4 Determination by HPLC 8.4.1 During HPLC analysis, maintain the eluent reservoir at a temperature of 85 C in order to keep the eluent degassed and to prevent bacterial growth. BSISO11814:2
42、0023IS:41811 O2002(E) ID:261 F2002(E) 4 I SO dna ID 2002 F All irhgts seredevr8.4.2 Prior to use, condition the column by repeatedly injecting 20 l of the standard sample solution until constant peak areas and retention times are obtained. Typical retention times obtained by the procedure specified
43、in this International Standard are given in 5.9.4 (see also Figure A.2). 8.4.3 Inject 20 l of the reference solution (8.3) and the test solution (8.2.3) respectively into the HPLC equipment operating at a flow rate of 1,0 ml/min of eluent (4.2). 8.4.4 The integrator (5.9.7) automatically calculates
44、the peak area of the whey proteins. The baseline location shall be checked in every chromatogram. For the test solution and the reference solution, equal baseline sets shall be obtained. The analysis or the integration shall be repeated if the baseline is improperly located. During the reintegration
45、 procedure, the use of forced baseline-set times is allowed only if no baseline drift in the chromatograms of the test solution or the reference solution is observed. 8.4.5 In the case of a series of analyses, perform repeated injection of the reference solution after every five test solutions. If a
46、 slight drift is noticed for the results of the reference solution, correct the peak area of the individual whey proteins of the test solution. If a large drift is noticed, check that the apparatus is functioning properly and/or recondition the column (see 8.4.2) and repeat the analyses. When the co
47、lumn is not being used for more than 1 day after finishing the analyses, wash it with the flushing solvent (4.3) at a flow rate of 0,2 ml/min for at least 3 h. Never store the column in the eluent. 9 Calculation and expression of results 9.1 Calculation of the relative ratio Calculate the relative r
48、atio, r, of the sum of the peak areas of immunoglobulin (Ig) and bovine serum albumin (BSA) by using the following equation: it bt 1 ir br 100 AA rf AA + = +where A itis the numerical value of the peak area of the immunoglobulin (Ig) fraction of the test sample solution, determined as in 8.4.4; A bt
49、is the numerical value of the peak area of the bovine serum albumin (BSA) fraction of the test sample solution; A iris the mean numerical value of the peak area of the immunoglobulin (Ig) fraction of the reference sample solutions used before and after the test sample solution; A bris the mean numerical value of the peak area of the bovine serum albumin (BSA) fraction of the reference sample solutions used before and after the test
