1、STD.BSI BS EN 12138-ENGL 1778 9 1b2Libb7 Ob BRITISH STANDARD Fruit and vegetable juices - Enzymatic determination of D-malic acid content - NAD spectrometric method The European Standard EN 121% : 1997 has the status of a British Standard ICs 67.160.20 NO COPYING WTHOUT BSI PERMISSION EXCEPT AS PERM
2、ITTED BY COPYRIGHT LAW I581 2T8 D BS EN 12138 : 1998 - STD.BS1 BS EN 12138-ENGL 1978 D 1b24bb b73582 134 D AmdNo. Date BS EN 12138 : 1998 Text affected National foreword This British Standard is the English language version of EN 12138 : 1997. The UK pasticipation in its preparation was entrusted to
3、 Technical Committee AWE1, Fruit and vegetable juices, which has the responsibility to: - aid enquirers to understand the text; - present to the responsible internationallEuropean committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; - monitor re
4、lated intemational and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secrem. IS0 5725 : 1986, to which informative reference is made in the text, has been superseded by IS0 57251 : 1994, IS0 57252 : 1994,
5、IS0 5725-3 : 1994, IS0 57254 : 1994 and IS0 57256 : 1994 which are identical with BS IS0 5725 Accuracl/ (trueness and precision) of measurement methods and results, BS IS0 57251 : 1994 Generalpnnciples and definitions, BS IS0 57252 : 1994 Basic method for the determination of repeatability and repmd
6、ucibity of a stard mm.surernnt method, BS IS0 57253 : 1994 IntemLediate measures of the precision of a stam 6.2.2 bottle 2 with approximately 210 mg of lyophilized NAD; 6.2.3 three bottles with lyophilized D-MDH, approximately 8 KJ each 5.3 calcium hydroxide; 6.4 ethanol approximately 98 ?$ 6.6 pota
7、ssium hydroxide. 6 Apparatus Usual laboratory appasatus and, in particdar, the following: 6.1 Emyrne test pipettes, graduated along the stem only, with long ungraduated delivery tip. 6.2 Pipettes, with an equivalent accuracy to that of 6.1, e.g. positive displacement capillary pipettes. 6.3 Cuvettes
8、, made of quartz, glas or plastics, of 10 mm optical path length, and which do not have a sigruficant absorption at wavelengths of 334 nm, 340 nm or 365 nm. 6.4 Spectd-line photometer, with a mercury lamp and filters for measuring at wavelengths of 365 nm or 334 nm. 6.5 Spectrometer, with variable w
9、avelength for measuring at 340 nm (aiteniative to 6.4). 6.6 VolumetmCks, of 50 ml capaciw. 6.7 Membrane filter, of pore size 0,2 km. ) Boehringer Mannheim is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not
10、constitute an endorsement by CEN of this product. O BSI 1998 Page 4 EN i2138 : 1997 7 Procedure 7.1 Sample preparation 7.1.1 Normal preparation Normally products shall not be pretreated, however dilution may be necessary and their analysis by this method shall be on a volumetric basis, results being
11、 expressed per litre of sample. The analysis of concentrated products may also be carried out on a volumetric basis, transfer quantitatively into a 50 ml volumetric flask (6.6) and diiute to the mark Clarify solution (as given 7.1.1) and use colourless clear solution in enzyme assay. 7.2 Test proced
12、ure 7.2.1 General The determination shall be carried out between 20 “C and 25 “C or a constant temperatun? in the range of 25 “C to 37 “C providing equivalent results are obtained. The absorption maximum of NADH is at a wavelength of 340 nm. When using a variable wavelength spectrometer (6.6), measu
13、re at the absorption maximum only. When using a mercury vapour lamp, spectd-ljne photometer (6.4), measure at a wavelength of 334 run or 365 nm. Do not use singlemark transfer pipettes for pipetting the solutions. Solutions of enzyme, coenzyme and 7.2.3 !lest sample solution Pipette into the cuvette
14、 1,OO mi of the HEPES buffer (6.2.1), OJO ml of the NAD solution (6.2.2), 1,70 mi of water and 0,lO mi of sample solution. Mix and after 6 min, read the absorbance (AlSmple) against air (with no cuvette in the lightpath). 7.2.4 Enzyme reaction and quantif2cation Start the reaction by the addition of
15、 0,05 ml of D-MDH solution (6.2.3) to each of the solutions given in 7.2.2 and 7.2.3. Mix and wait until the reaction has stopped (approximately 20 min) and read the absorbances (A2) of the solutions against air. if the reaction has not stopped after 20 min, continue to read the absorbances at5min i
16、ntervais until it increase at a constant rate and extrapolate this back to the time when the final enzyme solution D-MDH was added (This process allows for side reactions and, the so called creep reaction, which is described in more detail in annex B). 8 Calculation According to the reaction on whic
17、h the determination is based, there is a hear proportionality between the amount of NADH formed (and therefore the absorbance difference) and the concentration of D-mdC acid, as given in the following equaon: The cdcuiation of the concentration of a substance in dilute solution by absorptiometric me
18、asurement is based on the Beer-Lambert law. The D-Wc acid content of the sample (in g4) is calculated from the following equation: AA = (A2 - AilSmpie - (A2 - A1)Biank (1) where M is the molecular mass of D-malic acid V is the total volume of the test solution within the cuvette (in mi); v is sample
19、 volume (i mi); 6 is the lightpath of the cuvette (in cm); E is the extinction coefficient of NADH ; at 340 nm = 6,3 (l-mmol-l-cm-l); 365 nm = 3,4 (.mmoi- 1-cm-1); (134,09 gmol-1) buffer may be added from suitable automatic pipettes. Enzyme test pipettes (6.1) or their equivalent (6.2) shail be used
20、 for pipettjng the sample solution. It is recommended to include D-malic acid as a standard solution in this assay. 334 nm = 6,18 (l.mmo1- km-l). If the volumes given in 7.2 are adhered to, the caiculaion becomes, in g/i of malic acid: 3,956 P (D-malic acid) = 7 AA decimal. O BSI 1998 STD-BSI BS EN
21、LZL38-ENGL 1798 Lb24bb7 b73587 7Lb m Page 5 EN 12138 : 1997 9 Precision Details of the interlaboratory test on the precision of the method are summarized in Annex C. The values derived from the interlaboratory test may not be applicable to anaiyte concentration ranges and matsices other than given i
22、n Annex C. 9.1 Repeatability The absolute difference between two single test results found on identical test material by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatabiity limit T in not more than 5 % of the cases. The value is : T = 2,03 +
23、0,044. p(pm - a reference to this European Standard - the date and type of sampiing procedure - the date of receipt; - the date of test; - the test resuits and units in which they have been expressed; - whether the repeatability of the method has been verified - any particular points observed in the
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