1、 STD=BSI BS EN L2b32-ENGL 1777 = 1b211bb7 0785155 1111 BRITISH STANDARD Fruit and vegetable juices - Enzymatic determination of acetic acid (acetate) content - NAD spectrometric method The European Standard EN 126321999 has the status of a British Standard ICs 67.160.20 NO COPYING WITHOUT BSI PERMIS
2、SION EXCEPT AS PERMITTED BY COPYRIGHT LAW BS EN 12632:1999 STD-BSI BS EN LZb32-ENGL 1994 Lb24bb9 0785LSb 050 BS EN 12632:1999 National foreword This British Standard is the English language version of EN 12632: 1999. The UK participation in its preparation was entrusted to Technical Commiee AW/21, F
3、ruit and vegetable juices, which has the responsibility to: - aid enquirers to understand the te* - present to the responsible European committee any enquiries on the - monitor reiated intedonai and European developments and promulgate inteqretation, or proposals for change, and keep the UK interest
4、s informed them in the UK A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement internationa,l or European publications referred to in this document may be found in the BSI Standards Catalogue under t
5、he section entitled “Inkrnationai Standards Correspondence Index“, or by using the “Find“ facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contsact. Usem of British Standards axe responsible for their correct applica
6、tion. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 8, an inside back cover and a back cover. This British Standard, having Amendments issued s
7、ince publication been prepared under the direction of the Consumer products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 July 1999 Amd No. O BSI 07-1999 ISBN O 580 32212 2 Date STD*BSI BS EN 32b32-ENGL 3979 = 3b21ibb7 0785357
8、 T97 EUROPEAN STANDARD EN 12632 NORME EUR0PE”E EUROP- NOM February 1999 ICs 67.160.20 Descriptors: uit and vegetable juices, chemical adyss, determination of content, acetic acid, enzymac methos, spedmphotomehic ax p massconcentration; g acceleration due to gravity at the surface of the earth (9,81
9、d Coenzyme A; Adenosine-5-lii-phosphae; Adenosine-Mono-phosphate; Citrate synthase, Nicotjnamideaeninedinucleotide; -Nicotjnamideadeninedinucleotide, reduced form; Malate-dehydrogenase; 1 International Unit 0 of enzyme activity cataiyses the conversion of 1 wo1 of substance per minute at 25 “C under
10、 standard conditions. 4 Principle Acetic acid (acetate) is converted in the presence of the enzyme acetyloenzyme A-synthetase (ACS) with aden r is the radius of the centrifuge in centimetres, measured from the mid point (the centrifuge axis) to the bottom of the centrifuge tube when swung out; is th
11、e rotationai frequency per minute. n 6.7 Memm, membrane filter with a pore size 6.8 Centfifige tubes of 0,45 pl. 7 Procedure 7.1 Preparation of the test sample Dilute the fruit juice so that the acetic acid content is between 1 Fg and30 Fg per cuvette. This is equivalent to 10 mg to 300 mg acetic ac
12、id per litre sample (solution). If the concentration of acetic acid in the sample (solution) is less than 10 mg/l the sample volume to be pipeed into the cuvette can be increased up to 1,5 ml. If this occurs, the volume of water to be added must be reduced in order to obtain the same final volume in
13、 the cuvette. Use the (diluted) sample directly, even if it is slightly coloured The anaiysis by this method shall be on a volumetric basis, results being expressed per litre of sample. The anaysis of concentrated samples may also be carried out on a volumetric basis, after dilution to a known relat
14、ive density in this case, the relative density shall be indicaed Based on a weighed sample and taking the dilution factor for analysis into account, the results may also be expressed per kilogram of product. In products with a high viscosity andor a very high content of cells (for example pulp), det
15、ermination on the basis of a weighed test sample is the usual procedure. Mix cloudy samples well before dilution. Clarify cloudy samples containing low concentmtions of acetic acid by membrane ltration through a 0,45 pm filter (6.7) and centrifuge. O BSI 07-1999 STDDBSI BS EN L2b32-ENGL 1999 lb2Libb
16、9 0785LbL LiLB W 7.2 Test procedure 7.2.1 General The dekrmimtion shall normay be carried out at a constant temperature between 20 OC and 25 “C. A constant temperature in the range 25 OC to 37 OC may also be used, providing equivalent muits are obtained. The absorption maximum of NADH is at 340 nm.
17、When using a variable wavelength spectrophotometer (6.6), measure at the absorption maximum oniy When using a mercury vapour lamp, spectd-line photometer, measure at a wavelength of 334 nm or 366 nm. Do not use smglemark transfer pipettes for pipetting the solutions. Solutions of enzyme, coenzyme an
18、d buffer may be added from suitable automatic pipettes. Enzyme test pipettes (6.1) or their equivaent (6.2) shaii be used for pipetting the sample solution. Include a standard solution of acetic acid in each analytical run. 7.2.2 Blank test solution Also see the pipetting scheme given in 7.2.3. pipe
19、tte into Cuveaes 1,OO mi of the bufer solution (6.16), 0,lO ml of the NAD/CoA solution (6.16), OJO ml of the ATP solution (6.17) and 2,oO ml of water. Pipette into cuvettes Buffer solution (6.16) NADKoA solution (6.16) ATP solution (6.17) Sample solution (7.1) Water Page 5 EN 12632:1999 Mix and afte
20、r 3 min read the absorbanceA0. Add 0,02 ml of the MDWCSsolution (6.18), mix and after 2 min read the absorbance Al. Add 0,Ol ml of the ACS suspension (6.19), mix and after 10 min to 15 min read the absorbance A2. 7.2.3 Acetic aid test solution Pipe into cuvees 1,OO mi of the buffer solution (6.16),
21、0,lO ml of the NADXoA solution (6.16), 0,lO ml of the ATP solution (6.17), 0,lO ml of the sample solution (7.1) and 1,90 mi of water. Mix and affer3min read the absorbanceA0. Add 0,OZ ml of the MDWCSsolution (6.18), mix and aRer 2 min read the absorbance Al. Add 0,Ol ml of the ACS suspension (6.19),
22、 mix and after the Won has stopped (10 min to 15 min) read the absorbanceA2. Check for completion of the reaction by reading the finai absorbances at 2 min intervais for 10 min. If necessary (a constantly increasing vaiue) correct for the “creep“ reaction by exraplating the absorbance back to the ti
23、me of the addition of MDWCS (see annex C). Table 1 o, 10 OJO Sample ml 1,oo OJO 0,lO 0,lO 1,m I Mix and read the absorbances Ao of the solutions after approximately 3 min then add MDWCS solution (6.18) I0,02 I0,02 Mix and read the absorbances Al of the solutions after approximately 2 min. Start the
24、reaction by adition of I ACS suspension (6.19) I0,Ol I0,Ol Mix and after completion of reaction (approx. 15 min.) read absorbances A2 of blank and sample. If the Won has not stopped after 15 min, continue to read the absorbances at 2 min intervals una the absorbances increase constantly for 10 min.
25、O BSI 07-1999 Page 6 EN I26321999 Correct for a possible “creep” reaction by extrapolating the absorbance to the time of the addition of ACS (see annex C). Determhe the absorbance differences for blank and sample (Al - Ao) and (A2 - Ao). With preceding indicator reactions there is not a linear propo
26、rtionality between the measured absorbance Merence and the acetic acid concentration. The following fornula serves to calculate the macetic acid: 8 Calculation The acetic acid content, expressed in milligrams per litre, is calculated using the following equation: where VI M F V2 6 E is the finai vol
27、ume in miliihq is the molecular weight of acetic acid is the diluton factor of the sample solution; is the sample volume in millilitres, is the iight path of the cuvette in centimetres; is the absorption coefficient of NADH ak 340 = 6,3 (l X mol- X m- ); (f3),05 ou; 365 nm= 3,4 (l x moi-1 x cm-1); 3
28、34 nm= 6,lS (l X m01-l X a-). If the volumes given in 7.2.2 and 7.2.3 are not altered, then the acetic acid content in milligrams per litre of sample is calculated hm the following quatiom 3323 x ,E - a reference to this European Standard; - the date and type of samphg procedure (if known); - the da
29、te of receipt; - the date of test; - test resuits and units in which they have been exprd, - whether the repeatability has been verified, - any particuiar points observed in the course of the test; - any operations not specified in the method or regarded as optional, which might have affected the re
30、sults. O BSI 07-1999 STDoBSI BS EN 12b32-ENGL 1779 1b29bbS 0785Lb3 290 Page 7 EN 126321999 Sample Number of laboratories retained after eliminating outliers Number of outliers (laboratories) Number of accepted results Mean value (z) (md) Repeatability standard deviation (Sr) (ma) Repeatability imit
31、(r) (ma) Reproducibility standar deviation (SR) (mg/i) Reproducibility relative standard deviation (RSDR) (%) Reproducibility limit (R) (mgIl) Repeatability reiative standard deviation (RSDr) (%) Annex A (informative) Bibliography Determination of repeatability and reproducibility for a standard tes
32、t method by inter-labomm tests. Determination of acetic acid Enzymatic method, No. 66,1996, Analyses Collection international Federation of Wt Juice producers. Loose-leaf eition, as of 1996. Zug: Swiss Fruit Union. Determination of acetic acid Enzymatic method, Methods of biochemical analysis and fo
33、od analysis using single reagents. Boehnnger Mannheim. IS0 57251986, precision Of test mthods - A B C D 19 16 21 20 3 6 77 65 a3 80 252 325 249 215 7365 8,28 5,30 5,11 22 23 15 14 18,69 23,62 11,69 15,55 7,4 793 477 7,2 53 66 33 44 1 - 370 216 291 274 Annex B (informative) Statistical results of the
34、 inter-labratory tests in accordance with Is0 57251986, the following parameters have been define in an inter-laboratory test. (For literature pertauung to the method see annex A). The test was conducted by the Max von Pettenkofer institute of the Federal Health Office, Food Chemistty Department, Be
35、rlin, ERG Year of inter-iaboWry test 1991/1 199m Number of laboratories 22 21 Number of sample 2 2 Sample types: A Tomatojuice; B Cherry nectar; C Apple juice; D Orangejuice. O BSI 07-1999 Page 8 EN 12632:1999 Annex C (informative) Wormation on how to treat “creep” lX?actions “Creep” reactions are g
36、eneraiiy due to side reactions of the enzyme, presence of other enzymes in the sample matrix, or interaction of one or more components of the matrix with a reagent of the enzymic reaction. In the normal reaction the absorbance comes to a constant value after a certain time, typically 10 min to 20 mi
37、n, which depends on the speed of the particular enzymic reaction. However, when side reacons occur the absorbance does not reach a constant value but steadily increaw with time and this type of process is commonly died a “creep” reaction. If this problem occurs the absorbance of the solution should
38、be measured at regular intervais (2min to 5min) after the time required for the standard solution to reach its final absorbance. When the absorbance is increasing at a constant rate (Wdt = constant) take 5 to 6 readings and then extrapolate, either graphically or mathematically, to the absorbance th
39、e solution would have had at the point when the final enzyme was added (To). The extrapolated absorbance difference at this point (Af - A,) is used in the calculation of the substrate concentration. Absorbance Af P dNdt = constant Main reaction 1 I I 1 I l I 1 I I I l O O TO (addition of final enzym
40、e) Figure C.1- Creep reaction Time O BSI 07-1999 BS EN 12632: 1999 BSI 389 Chiswick High Road London w4 4AIJ BSI - British Standards Institution BSI is the independent national body responsible for preparing British Standards. It presents the UK view on standads in Europe and at the international le
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