1、Ref. No. DIN 10483-1 : 2002-08English price group 07 Sales No. 010710.03DEUTSCHE NORM August 200210483-1Continued on pages 2 to 4. 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,has
2、 the exclusive right of sale for German Standards (DIN-Normen).Determining the lactoperoxidase activity in milkby photometry (reference method)Translation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.67.100.10Bestimmung der Laktop
3、eroxidase-Aktivitt in Milch Teil 1: Photometrisches Verfahren (Referenzverfahren)In keeping with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the decimal marker.ForewordThis standard has been prepared by Techn
4、ical Committee Chemische und physikalische Milchuntersuchungof the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural Prod-ucts Standards Committee).1 ScopeThis standard specifies a photometric reference method of determining lactoperoxidase activity exceeding
5、50 units per litre in raw milk, milk for retail and reconstituted milk. It is also suitable for calibrating the methoddescribed in DIN 10483-2.2 Normative referencesThis standard incorporates, by dated or undated reference, provisions from other publications. These nor-mative references are cited at
6、 the appropriate places in the text, and the titles of the publications are listedbelow. For dated references, subsequent 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
7、the publication referred to applies.DIN 10483-2 Determining the lactoperoxidase activity in milk by the reflectometric methodDIN 12650-2 Piston-operated pipettes for laboratory useDIN 12690 Class A and Class B one-mark bulb pipettes for laboratory useDIN EN ISO 707 Milk and milk products Guidance on
8、 sampling (ISO 707 : 1997)DIN EN ISO 1042 Laboratory glassware One-mark volumetric flasks (ISO 1042 : 1998)ISO 5725-1 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 1: Gen-eral principles and definitionsISO 5725-2 : 1994 Accuracy (trueness and precision) of measurem
9、ent methods and results Part 2: Basicmethod for the determination of repeatability and reproducibility of a standard measure-ment methodPage 2DIN 10483-1 : 2002-083 ConceptLactoperoxidase activityThe enzyme activity determined by the method described in this standard.NOTE: Lactoperoxidase activity i
10、s reported in units per litre, one unit being the quantity of enzyme that converts1 mol of substrate per minute under standardized conditions (in this case at a temperature of 25 C and apH value of 6).4 PrincipleAfter the milk has been diluted with water, the conversion of 2,2-azinobis(3-ethylbenzot
11、hiazoline-6-sulfonicacid) (ABTS) by lactoperoxidase per unit time is determined photometrically, the reaction being as follows:2 ABTS + H2O2+ 2 H+ idaselactoperox2 ABTS+ 2 H2O (1)The number of ABTS free-radical cations (ABTS+) liberated per unit time is proportional to the lactoperoxidaseactivity an
12、d can be determined photometrically at a wavelength of 420 nm.5 Reagents5.1 GeneralAnalytical grade reagents shall be used and the water used shall be double-distilled or at least of equivalentpurity.The following reagents shall be used.5.2 Buffer solution, prepared by dissolving 0,72 g (8,09 mmol/l
13、) of disodium hydrogenphosphate dihydrateand 3,99 g (58,6 mmol/l) of potassium dihydrogenphosphate together in water in a 500 ml volumetric flask andmaking up to the mark with water.The pH value of the buffer solution shall not differ by more than t 0,03 from 6. Otherwise, dissolve the buffersalts i
14、n a smaller volume of water (about 450 ml) and, if necessary, adjust the pH value to 6 using a dilute acidor hydroxide solution. After adjustment, the solution shall be transferred to a 500 ml volumetric flask and madeup to the mark with water.5.3 Hydrogen peroxide solution, prepared by pipetting 0,
15、1 ml of 30 % (m/m) hydrogen peroxide into a100 ml volumetric flask and making up to the mark with water. The solution shall be used immediately (seesubclause 5.4).CAUTION. Hydrogen peroxide is caustic and contact with metals or readily flammable organic sub-stances shall be avoided since explosive m
16、ixtures may be produced.5.4 2 mmol/l reagent solution, prepared by weighing 55 mg of diammonium 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid), C18H16N4O6S4(NH4)2, into a 50 ml volumetric flask, dissolving it in about 30 mlof buffer solution, adding 1 ml (about 0,2 mmol/l) of the hydrogen per
17、oxide solution and making up to the markwith the buffer solution.Prepare a fresh reagent solution every day.6 ApparatusIn addition to standard laboratory equipment, the following shall be used.6.1 Spectrophotometer, for measuring absorbance at 420 nm, equipped with a temperature-controlled cu-vette
18、holder.6.2 Plastic cuvettes, with lid, having a path length of 1 cm.NOTE: As a result of adsorption, glass or quartz cuvettes may give results that are slightly low.6.3 Pipettes, of nominal capacities 5 ml and 20 ml (e.g. DIN 12690 pipettes).6.4 Piston-operated pipettes, of nominal capacities 0,05 m
19、l, 0,1 ml, 1 ml and 2 ml (e.g. piston-operatedpipettes as in DIN 12650-2).6.5 Volumetric flasks, of nominal capacities 50 ml, 100 ml and 500 ml (e.g. volumetric flasks as inEN ISO 1042).6.6 Water bath, capable of being maintained at (25 t 0,5) C, for adjusting the temperature of the reagentsolution
20、or of the cuvette holder.lactoperoxidasePage 3DIN 10483-1 : 2002-087 SamplingSee DIN EN ISO 707.8 Procedure8.1 Sample dilutionMilk samples that have been refrigerated shall be heated to ambient temperature and carefully mixed.The differential absorbance per minute, DE/min, found experimentally and u
21、sed to calculate the lactoperoxidaseactivity, should be 0,02 to 0,5. Depending on the lactoperoxidase content of the milk, it will therefore benecessary to dilute the milk sample with water in a suitable ratio by volume of not less than 1:5 (e.g. 5 ml ofsample and 20 ml of water). Table 1 indicates
22、the dilutions suitable for different measurement ranges.Table 1DilutionMeasurement range,in U/l1:5 150 to 12001:10 100 to 25001:25 250 to 6000UHT*) milk samples shall be diluted only in a ratio by volume of 1:5.8.2 AnalysisPrior to the photometric determination, zero the photometer at a wavelength o
23、f 420 nm with air as a reference(i.e. without a cuvette in the beam path).Pipette 2 ml of the reagent solution into a cuvette, place the cuvette in the photometer cuvette holder and preheatto (25 t 0,5) C. Then add 0,05 ml of the milk sample diluted as described in subclause 8.1, having rinsed theti
24、p of the 0,05 ml piston-operated pipette with diluted sample solution beforehand. Seal the cuvette immedi-ately, mix its contents carefully by inverting it and return it immediately to the photometer cuvette holder.Determine the absorbance at a wavelength of 420 nm, E1, with air in the reference pat
25、h. As a guide, the time lapsebetween adding the diluted milk sample and reading off E1shall be 15 seconds. After precisely two minutes,record the absorbance, E2.9 CalculationIn the reaction on which the analysis is based, the change in absorbance per unit time is in direct proportion tothe lactopero
26、xidase activity. The calculation of the activity of an enzyme in dilute solution by absorption pho-tometry is based on the Lambert-Beer formula.Calculate the lactoperoxidase activity, aLPO, in milliunits per millilitre (mU/ml) or units per litre (U/l) of the sample,using the following equation:aLPO=
27、 2000121VdFEV(2)whereV1is the total volume of test solution, in ml (here, V1= 2,05 ml);V2is the volume of diluted sample, in ml (here, V2= 0,05 ml);DE is the differential absorbance, (E2 E1)/min;e is the absorptivity of the oxidized ABTS at 420 nm (43,2 cm2/mol);d is the path length of the cuvette,
28、in cm (here, d = 1 cm);F is a dilution factor (5 for UHT milk);1000 is the factor for converting to mU/ml or U/l;2 is the stoichiometric coefficient.Report the calculated lactoperoxidase activity in U/l to one decimal place.*) UHT = Ultra high temperature.Page 4DIN 10483-1 : 2002-0810 Precision10.1
29、GeneralThe precision data were determined in 2000 on heat-treated milk in an interlaboratory test involving eightlaboratories 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 thepr
30、ecision data set out in this clause and to confirm such compliance (e.g. by participating in interlaboratoryquality assurance or laboratory performance tests or by using reference materials, preferably as part of a routineinternal laboratory statistical control scheme).10.2 Repeatability limit(same
31、operator, same equipment)The absolute difference between two successive results obtained under repeatability conditions will not exceedthe repeatability limit, r, in more than 5 % of cases on average.A value of 30 U/l was determined for a mean of 400 U/l. If this limit is exceeded, both results shou
32、ld be regardedas unreliable.10.3 Reproducibility limit(different operators, 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 65 U/l was det
33、ermined for a mean of 400 U/l. If this limit is exceeded, both results should be regardedas 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 activ
34、ity, in mU/ml or U/l;d) date of testing;e) the reason for any deviation from this standard.AppendixCalibration of reflectometric methodThe photometric method described in this part of DIN 10483 is a reference method for calibrating the rapidreflectometric method described in Part 2.At least six cali
35、bration samples comprising a series of milk dilutions that have lactoperoxidase activities withinthe measurement range of the photometric method (see subclause 8.1) will be needed. The range of the pho-tometric method does not extend below 50 U/l, but lower lactoperoxidase activity values can theore
36、tically beused to calibrate the reflectometer if evaluated as in clause 9 using linear regression.To prepare suitable samples, a high-activity milk shall first be diluted with a suitable volume of peroxidase-freeUHT milk and then mixed with water in a suitable ratio by volume of not less than 1:5. T
37、he subsequent procedureis then as described in subclause 8.3 of DIN 10483-2, August 2002 edition.A suitable dilution series containing at least ten different dilution stages can be prepared by mixing raw milk withperoxidase-free UHT milk in a ratio by volume of 1:3 to 1:50 and then additionally dilu
38、ting the mixtures obtainedwith water. Because the lactoperoxidase activity in raw milk varies naturally, it may be necessary to alter theamount of peroxidase-free milk added to obtain samples having a lactoperoxidase activity suitable for themeasurement range of the photometric method.BibliographyWe
39、rner, W., Rey, H.-G. and Wielinger, H. ber die Eigenschaften eines neuen Chromogens fr die Blutzucker-bestimmung nach der GOD/POD-Methode (The properties of a new chromogen for blood sugar determinationby the GOD/POD method), Z. Anal. Chem., 1970: 252, 224228.Gallati, H. Peroxidase aus Meerrettich:
40、Kinetische Studien sowie Optimierung der Aktivittsbestimmung mit denSubstraten H2O2und 2,2-Azino-di-(3-ethyl-benzothiazolinsulfonsure-(6) (ABTS) (Peroxidase from horserad-ish: Kinetic studies and optimization of activity determination with the substrates H2O2and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), J. Clin. Chem. Clin. Biochem., 1979: 17, 17.
