1、Ref. No. DIN 10483-2 : 2002-08English price group 06 Sales No. 010610.03DEUTSCHE NORM August 200210483-2Continued on pages 2 and 3. No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e.V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,ha
2、s the exclusive right of sale for German Standards (DIN-Normen).Determining the lactoperoxidase activity in milkby reflectance spectroscopyTranslation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.ICS 67.100.10Bestimmung der Laktop
3、eroxidase-Aktivitt in Milch Teil 2: Reflektometrisches VerfahrenForewordThis standard has been prepared by Technical Committee Chemische und physikalische Milchuntersuchungof the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural Prod-ucts Standards Committee)
4、.1 ScopeThe method specified in this standard serves to determine the lactoperoxidase activity in raw milk, milk forretail and reconstituted milk by reflectance spectroscopy using test strips. On the basis of the reproducibilitystandard deviation in the low-activity range, the limit of detection can
5、 be put at 6 units per litre.2 Normative referencesThis standard incorporates, by dated or undated reference, provisions from other publications. These nor-mative references are cited at the appropriate places in the text, and the titles of the publications are listedbelow. For dated references, sub
6、sequent amendments to or revisions of any of these publications apply tothis standard only when incorporated in it by amendment or revision. For undated references, the latestedition of the publication referred to applies.DIN 10483-1 Determining the lactoperoxidase activity in milk by photometry (re
7、ference method)DIN 12690 Class A and Class B one-mark bulb pipettes for laboratory useDIN EN ISO 707 Milk and milk products Guidance on sampling (ISO 707 : 1997)ISO 5725-1 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 1: Gen-eral principles and definitionsISO 5725-
8、2 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 2: Basicmethod for the determination of repeatability and reproducibility of a standard measure-ment method3 ConceptSee DIN 10483-1.4 PrincipleA test strip containing 3,3,5,5-tetramethylbenzidine (TMB) as substrate is
9、 immersed in milk diluted withwater. In the presence of hydrogen peroxide, the catalytic action of the lactoperoxidase converts thesubstrate into a coloured compound, the reaction being as follows:Page 2DIN 10483-2 : 2002-082TMB + H2O2+ 2H+ idaselactoperox2TMB+ 2 H2O (1)The amount of oxidized substr
10、ate liberated is proportional to the lactoperoxidase activity and can be measuredreflectometrically at a suitable wavelength (e.g. in the 650 nm to 660 nm range).NOTE: The hue may also be established visually using a colour index.5 Reagents5.1 GeneralAnalytical grade reagents shall be used and the w
11、ater used shall be distilled or at least of equivalent purity.The following reagents shall be used.5.2 Test strips, consisting of an elongated plastic base layer to which absorbent material (e.g. paper) con-taining the TMB substrate has been attached in a suitable way (e.g. by gluing).NOTE: Test str
12、ips with TMB as substrate are commercially available.1)5.3 Hydrogen peroxide solution, prepared by pipetting 1 ml of 30 % (m/m) hydrogen peroxide into a 100 mlvolumetric flask and making up to the mark with water.CAUTION. Hydrogen peroxide is caustic and contact with metals or readily flammable orga
13、nic sub-stances shall be avoided since explosive mixtures may be produced.5.4 Peroxidase-free UHT2) milk, for use as a diluent in preparing calibration samples as specified in theappendix of DIN 10483-1, August 2002 edition.6 ApparatusIn addition to standard laboratory equipment, the following shall
14、 be used.6.1 Reflectometer, for measuring diffuse reflection at a suitable wavelength (e.g. in the 650 nm to 660 nmrange), equipped with a device for analysing test strips.1)6.2 Pipettes, of nominal capacities 5 ml and 20 ml (e.g. pipettes as in DIN 12690).7 SamplingSee DIN EN ISO 707.8 ProcedureNOT
15、E: The calibration specified in subclause 8.2 will generally not be necessary in the case of commerciallyavailable test systems and, in addition, their use simplifies the evaluation as in clause 9.8.1 Sample dilutionMilk samples that have been refrigerated shall be heated to ambient temperature and
16、carefully mixed.Depending on the lactoperoxidase content of the milk and the sensitivity of the reflectometer, it will be necessaryto dilute the milk sample with water in a suitable ratio by volume of not less than 1:5 (e.g. 5 ml of sample and20 ml of water).UHT milk samples shall be diluted only in
17、 a ratio by volume of 1:5.8.2 CalibrationThe photometric method described in Part 1 of this standard is a reference method for calibrating the methoddescribed here for determining lactoperoxidase activity.At least six calibration samples (series of milk dilutions), the lactoperoxidase activity of wh
18、ich has been deter-mined photometrically and lies within the scope of the method dealt with here, shall be used. Although the scopeof the photometric method does not extend to below 50 U/l, lower values are theoretically obtainable when usingreflectance spectrometry if evaluation is as in clause 9.1
19、) The reagents and test strips specified in subclauses 5.2 and 5.3 and the reflectometer specified in subclause6.1 are available as Reflectoquantperoxidase milk test kits and as an RQflexreflectometer, respectively,from Merck Eurolab GmbH, Darmstadt, Germany. They are examples of suitable commercial
20、 products and arementioned solely as guidance for the user of this standard without implying approval of the products by DIN.Other products may be used if they are shown to give identical results.2) UHT = Ultra high temperature.lactoperoxidasePage 3DIN 10483-2 : 2002-08To prepare suitable calibratio
21、n samples, high-activity milk shall first be diluted with a suitable volume of peroxi-dase-free UHT milk. After further dilution with water, the lactoperoxidase activity of the calibration samples shallbe determined by photometry as described in DIN 10483-1 and their remission determined as describe
22、d insubclause 8.3.Recalibration shall be performed for every fresh batch of test strips.8.3 AnalysisBring 5 ml of the milk sample, which has previously been diluted as specified in subclause 8.1, to a temperatureof (23 t 1) C, add 0,25 ml of hydrogen peroxide solution and then immerse the test strip
23、 in the sample for abouttwo seconds until it is completely wetted. Shake off excess liquid from the strip or drain it using an absorbentcloth. Determine the relative remission at a suitable wavelength after precisely three minutes, ensuring that thetemperature is (23 t 1) C throughout this period.NO
24、TE: A deviation of 1 C from the temperature will result in an error of about 5 %. To avoid fairly high errors,the test strip may be placed in an incubator capable of being maintained at (23 t 1) C.9 EvaluationThe remission values of the calibration samples of known lactoperoxidase activity determine
25、d as specified insubclause 8.3 shall be used to calculate a linear regression curve by using semi-log paper to plot the remissionas a function of the lactoperoxidase activity.10 Precision10.1 GeneralThe precision data were determined in 2000 on heat-treated milk in an interlaboratory test involving
26、24 labora-tories and carried out in accordance with ISO 5725-1 and ISO 5725-2.Any laboratory using this standard is obliged to use its quality assurance system to ensure compliance with theprecision data set out in this clause and to confirm such compliance (e. g. by participating in interlaboratory
27、quality assurance or laboratory performance tests or using reference material, preferably as part of a routineinternal laboratory statistical control scheme).10.2 Repeatability limit(same operator, same equipment)The absolute difference between two successive results obtained under repeatability con
28、ditions will not exceedthe repeatability limit, r, in more than 5 % of cases on average.A value of 3 U/l was determined for a mean of 10 U/l and of 50 U/l for a mean of 365 U/l. If these limits areexceeded, both results shall be regarded as unreliable.10.3 Reproducibility limit(different operators,
29、different equipment)The absolute difference between two individual results obtained under reproducibility conditions will not exceedthe reproducibility limit, R, in more than 5 % of cases on average.A value of 6 U/l was determined for a mean of 10 U/l and of 155 U/l for a mean of 365 U/l. If these l
30、imits areexceeded, both results shall be regarded as unreliable.11 Test reportThe test report shall refer to this method and include the following details:a) type, origin and description of sample;b) sampling method and date of sampling;c) lactoperoxidase activity, expressed in milliunits per millilitre (mU/ml) or units per litre (U/l);d) date of testing;e) the reason for any deviation from this standard.BibliographySonntag, O. Trockenchemie Analytik mit trgergebundenen Reagenzien (Dry-state chemistry Analyses in-volving support-bound reagents). G. Thieme Verlag.
