ISO TS 17193-2011 Milk - Determination of the lactoperoxidase activity - Photometric method (Reference method)《乳 氧化物酶活性的测定 光度法(参考方法)》.pdf

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1、 Access to Additional Content for ISO/TS 17193 First edition, Dated: 12/15/2011 (Click here to view the publication) This Page is not part of the original publication This page has been added by IHS as a convenience to the user in order to provide access to additional content as authorized by the Co

2、pyright holder of this document Click the link(s) below to access the content and use normal procedures for downloading or opening the files. ISO/TS 17193 Files Information contained in the above is the property of the Copyright holder and all Notice of Disclaimer an ISO Technical Specification (ISO

3、/TS) represents an agreement between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further three ye

4、ars, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn. Attention is drawn to the possibility that some of the

5、elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO TS 17193 IDF/RM 208 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy

6、 Federation (IDF). It is being published jointly by ISO and IDF. This corrected version of ISO/TS 17193IDF/RM 208:2011 incorporates the following corrections: a) in Clause 6, paragraph 1, “Technical Specification“ has replaced “International Standard“, b) in the Note to 8.1, “(1)“ has been inserted

7、after “chemical reaction“; c) in lines 2, 4, 5, and 6 of the variable definitions under Equation (2), an initial “2“ has been deleted, so that the affected values now correctly read, “0,05 ml“, “43,2 mol 1cm 1 “, “1,0 cm“, and “5“, respectively; d) in 10 c), “2011“ replaces “2012“. ISO/TS 17193:2011

8、(E) IDF/RM 208:2011(E) iv ISO and IDF 2011 All rights reservedForeword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country as well as regional dairy associations having sig

9、ned 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 of analysis and sampling for milk and milk products. The main task of S

10、tanding Committees is to prepare International Standards. Draft International Standards adopted by the Standing Committees are circulated to the National Committees for endorsement prior to publication as an International Standard. Publication as an International Standard requires approval by at lea

11、st 50 % of IDF National Committees casting a vote. In other circumstances, particularly when there is an urgent market requirement for such documents, a Standing Committee may decide to publish an other type of normative document which is called by IDF: Reviewed method. Such a method represents an a

12、greement between the members of a Standing Committee and is accepted for publication if it is approved by at least 50 % of the committee members casting a vote. A Reviewed method is equal to an ISO/PAS or ISO/TS and will, therefore, also be published jointly under ISO conditions. Attention is drawn

13、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/TS 17193 IDF/RM 208 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food

14、 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 Project Group on Photometric lactoperoxidase activity of the Standing committee on Analytical methods for processing aids and indicators under the aegis o

15、f its project leaders, Mr D. Tanzer (DE) and Mrs M. Nicolas (FR). This corrected version of ISO/TS 17193IDF/RM 208:2011 incorporates the following corrections: a) in Clause 6, paragraph 1, “Technical Specification“ has replaced “International Standard“, b) in the Note to 8.1, “(1)“ has been inserted

16、 after “chemical reaction“; c) in lines 2, 4, 5, and 6 of the variable definitions under Equation (2), an initial “2“ has been deleted, so that the affected values now correctly read, “0,05 ml“, “43,2 mol 1cm 1 “, “1,0 cm“, and “5“, respectively; d) in 10 c), “2011“ replaces “2012“. TECHNICAL SPECIF

17、ICATION ISO/TS 17193:2011(E) IDF/RM 208:2011(E) ISO and IDF 2011 All rights reserved 1 Milk Determination of the lactoperoxidase activity Photometric method (Reference method) 1 Scope This Technical Specification specifies a photometric method for the determination of the lactoperoxidase activity in

18、 milk in amounts exceeding 50 U/l. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 unit of lactoperoxidase activity amount of lactoperoxidase enzyme that catalyses the transformation of 1 mol of substrate per minute 3 Principle In the presenc

19、e of any lactoperoxidase enzyme derived from the sample, ABTS 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is catalytically converted to its radical cation (ABTS ) at pH 6,0 and 25 C. The amount of (ABTS ) liberated per minute is proportional to the lactoperoxidase activity and measured ph

20、otometrically at 420 nm. The liberation of the ABTS is based on the following chemical reaction: lactoperoxidase 22 2 2 ABTS H O 2H 2 ABTS 2H O (1) 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and doubly distilled water or water of equivalent purity. 4.1 B

21、uffer solution, pH 6,0. Dissolve 0,72 g of disodium hydrogenphosphate dihydrate (Na 2 HPO 4 2H 2 O) and 3,99 g of potassium dihydrogenphosphate (KH 2 PO 4 ) in a beaker in a volume of about 450 ml of water. Adjust the pH of the solution, if necessary, to 6,0 0,03 with a dilute sodium hydroxide, pota

22、ssium hydroxide or phosphoric acid solution. Transfer the solution to a 500 ml volumetric flask (5.6). Make up to the mark with water and mix. NOTE The buffer solution contains 8,09 mmol/l of disodium hydrogenphosphate dihydrate and 58,6 mmol/l of potassium dihydrogenphosphate. ISO/TS 17193:2011(E)

23、IDF/RM 208:2011(E) 2 ISO and IDF 2011 All rights reserved4.2 Hydrogen peroxide solution. WARNING Hydrogen peroxide is corrosive and should not be brought in contact with metals or readily flammable organic substances to prevent explosive mixtures forming. Pipette 0,1 ml of 30 % mass fraction H 2 O 2

24、into a 100 ml one-mark volumetric flask (5.6). Make up to the mark with water and mix. Prepare the hydrogen peroxide solution immediately before use (see 4.3). 4.3 ABTS reagent solution, 2 mmol/l solution of the diammonium salt of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) C 18 H 16 N 4 O

25、 6 S 4 (NH 4 ) 2 . Weigh 55 mg of the di-ammonium salt of ABTS into a 50 ml volumetric flask (5.6). Add about 30 ml of buffer solution (4.1) to dissolve the salt. Then add 1,0 ml of hydrogen peroxide solution (4.2). Make up to the 50 ml mark with the buffer solution and mix. Prepare a fresh reagent

26、solution every day. 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg. 5.2 pH-meter, capable of measuring 0,1 unit of pH, with a readability of 0,01 unit. 5.3 Spectrophotometer, capa

27、ble of measuring absorbance at 420 nm, with a temperature-controlled cuvette holder capable of maintaining a temperature of 25 C 0,5 C. 5.4 Plastic cuvette, of pathlength 1,0 cm, provided with a lid. NOTE The use of glass or quartz cuvettes can give results that are too low due to adsorption losses.

28、 5.5 Air-displacement pipettes or piston-operated pipettes, ISO 8655-2 5 , of nominal capacities 0,05 ml, 0,1 ml, 1,0 ml, and 2,0 ml. 5.6 One-mark volumetric flasks, of nominal capacities 5 ml, 10 ml, 25 ml, 50 ml, 100 ml and 500 ml, ISO 1042 2 , class A. 5.7 Water bath or dry bath, capable of maint

29、aining a temperature of 25 C 0,5 C. 6 Sampling Sampling is not part of the method specified in this Technical Specification. A recommended sampling method is given in ISO 707 IDF 50 1 . It is important the laboratory receive a truly representative sample which has not been damaged or changed during

30、transport or storage. ISO/TS 17193:2011(E) IDF/RM 208:2011(E) ISO and IDF 2011 All rights reserved 37 Procedure 7.1 Preparation of test sample dilution Warm refrigerated test samples to room temperature and mix carefully. It is usually not necessary to reheat the test sample for proper mixing. In no

31、 case shall the temperature exceed 35 C. Depending on the lactoperoxidase content of the sample, mix the test sample with water in a suitable ratio by volume of at least 1 5. To calculate the lactoperoxidase activity, the differential absorbance, A, found has to be in the range 0,02 min 1to 0,5 min

32、1 . Depending on the measurement range, the dilutions with water specified in Table 1 are applicable. Table 1 Test sample dilution Measurement range U/l Dilution 50 to 1 200 1 5 100 to 2 500 1 10 250 to 6 000 1 25 For dilution of UHT milk samples, a ratio of 1 5 (by volume) shall be used. 7.2 Determ

33、ination Prior to the photometric determination, adjust the photometer (5.3) to its zero level at a wavelength of 420 nm with air as reference (without a cuvette in the beam path). Pipette 2 ml of ABTS reagent solution (4.3) into a cuvette (5.4). Cover the cuvette and place it in the water bath or th

34、e dry bath (5.7) set at 25 C for the time necessary to attain this temperature into the cuvette. Then add 0,05 ml of the test sample diluted as specified in 7.1. Immediately cover the cuvette again and mix its contents by carefully inverting. Place the cuvette quickly in the cuvette holder of the ph

35、otometer (5.3) preheated at 25 C. Within 15 s after adding the diluted test sample to the reagent solution in the cuvette, measure the absorbance A 1at a wavelength of 420 nm using air as reference. Record the absorbance A 2after precisely 2 min. IMPORTANT For a reliable measurement, make sure that

36、the interval between adding the diluted test sample to the reagent and reading A 1is 15 s. If using a 0,05 ml piston-operated pipette, ensure that the tip is rinsed beforehand with the sample dilution. ISO/TS 17193:2011(E) IDF/RM 208:2011(E) 4 ISO and IDF 2011 All rights reserved8 Calculation and ex

37、pression of results 8.1 Calculation NOTE In the chemical reaction (1) underlying this determination, there is a linear relation between the change in absorbance per minute and the lactoperoxidase activity. The activity of an enzyme in a dilute solution determined by absorption photometry conforms to

38、 the BeerLambert law. Calculate the lactoperoxidase activity, a l , expressed as units per litre (U/l) of the sample using Equation (2): l 1 2 1000 2 VAf a dV (2) where V 1is the total volume, in millilitres, of the test solution (7.2) (V 1 2,05 ml); V 2is the volume, in millilitres, of the sample d

39、ilution (7.1) (V 2 0,05 ml); A is the differential absorbance, (A 2 A 1 )/min; is the absorptivity of the oxidized ABTS at 420 nm ( 43,2 mol 1cm 1 ); d is the pathlength, in centimetres, of the cuvette (d 1,0 cm); f is the dilution factor (f 5 for UHT milk); 1 000 is the factor for conversion to uni

40、ts per litre U/l; 2 is the stoichiometric coefficient. 8.2 Expression of results Express the results to the nearest whole number. 9 Precision At the time of publication, no interlaboratory study fulfilling the requirements of ISO 5725-1 3and ISO 5725-2 4could be organized. Consequently this method i

41、s published as a Technical Specification and not as an International Standard. 10 Test report The test report shall contain at least the following information: a) all information necessary for the complete identification of the sample; b) the sampling method used, if known; c) the test method used,

42、with reference to this Technical Specification (ISO/TS 17193 IDF/RM 208:2011); d) all operating details not specified in this Technical Specification, or regarded as optional, together with details of any incidents which may have influenced the test result(s); e) the test result(s) obtained; f) if the repeatability has been checked, the final quoted result obtained.