1、BRITISH STANDARD Fruit and vegetable juices - Determination of free amino acids content - Liquid chromatographic method The European Standard EN 12742:1999 has the status of a British Standard ICs 67.160.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW BS EN 12742: 1999 STD-
2、BSI BS EN 327YZ-ENGL 3999 P lbZLlbb9 0783770 257 = 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 June 1999 Amd. No. O BSI O81999 ISBN O 580 32215 7 BS EN 12742:1999 Da
3、te Comments National foreword This British Standard is the English language version of EN 127421999. The UK participation in its preparation was entsusted to Technical Committee AW/21, Fniit and vegetable juices, which has the responsibility to: - aid enquirers to understand the text; - present to t
4、he responsible European committee any enquiries on the - monitor related intedonal and European developments and promulgate interpretation, or proposals for change, and keep the UK interests informed; them in the UK. A list of organizations represented on this committee can be obtained on request to
5、 its secretaqy. Cross-references The British Standards which implement intedonal or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Sta
6、ndards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of p
7、ages This document comprises a front cover, an inside front cover, the EN tite page, pages 2 to 10, an inside back cover and a back cover. ICs 67.160.20 Descriptors: fruit and vegetable juices, chemical analysis, determination of content, amino acids, chromatographic analysis Enghsh version Frwt and
8、 vegetable juices - Determination of the fh amino acids content - Liquid chromatographic method Jus de iuits et de lgumes - Dosage des teneurs en acides amins libres - Mthode par chromatographie en phase liquide Frucht- und Gemusesafte - Bestimmung der Gehalte an hien Aminosauren - Fligchrohisches V
9、ei.Eahren This European Standard was approved by CEN on 17 January 1999. CEN members are bound to comply with the CENKENELEC internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Uptodate lists and bibiographic
10、al references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by lnmslation under the responsibility of a CEN me
11、mber into its own language and notified to the Centml Secretariat has the same status as the official versions. CEN members are the national standards bodies of Awtxia, Belgium, Czech Republic, Denmark, Finland, France, Germanx Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portug
12、al, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standar-on Comit Europen de Normalisation Europisches Komitee fur Normung Central Secretariat: rue de Stassart 36, 5-1060 Brussels O 1999 GEN All rights of exploitation in any form and by any means reserved worldwide for C
13、EN national Members. Ref. No. EN 12742:1999 E Foreword This European Standard has been prepared by lchnicai Committee CENITC 174, Fruit and vegetable juices - Methods of anaiysis, the Secretariat of which is held by AFNOR. This European Standard shall be given the status of a nationai standard, eith
14、er by publication of an identicai text or by endorsement, at the latest by August 1999, and conflicting national standards shall be withdrawn at the latest by August 1999. According to the CENKENELEC Intemal Regulations, the national standards opmimtions of the following countries are bound to imple
15、ment this European Standard: AustSia, Belgium, Czech Republic, Denmark, Finland, France, Gem Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Foreword 1 Scope 2 Normative references 3 Symbols 4 Principle 5 Reagent
16、s 6 APPW 7 Procedure 8 Calculation 9 Precision 10 Test report Annex A (ionnative) Bibliography Annex B (informative) Statktical results of the inter-laboratory test Annex C (informative) Details of typical conditions and reagents used in the analysis of free amino acids in fruit and vegetable juices
17、 page 2 3 3 3 3 3 3 4 4 5 5 6 6 9 O BSI 061999 STD.BS1 BS EN 32742-ENGL 1999 Lb24bb7 0783773 Tbb 1 scope This European Standard specifies a chromatographic procedure for the determination of the free amino acid content in fruit and vegetable juices and related products. 2 Normative references “his E
18、uropean Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publicati
19、ons apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN IS0 3696: 1995, Wa,ter for analytical Qbomtory use - Spec p mass concentration; g acceleration due to gravity at the surface
20、 of the earth (9,i m/s2). 4 Principle Amino acids are separated on a cation exchange chromatography column by stepwise elution with a range of lithium citrate buffers of different molarities and pH values. After a colour reaction, with ninhydrin, the amino acids are quantified photometrically at a w
21、avelength of 570 nm (or 440 nm for proline). 6 Reagents 6.1 General Use only reagents of recognized anaiytical grade and only water in accordance with at least grade 1 of EN IS0 3696: 1995. Page 3 EN 12742:lM 6.2 Buffer solutions for amino acid elution Prepare the buffer solutions in accordance with
22、 the manufacturers instructions. Ready-prepared lithium ci- buffer of different pH values and lithium ion molarities can also be used. The molarities and pH values of buffer solutions can differ in accordance with the equipment manufacturers instructions (e.g. the length and chneter of the analytica
23、l column and type of resin). Therefore it is impossible to give exact details for the prepamtion of the various buffer solutions. The description of the buffer solutions detailed in annex C are therefore only for use as a guide for the user of this standard. 6.3 Lithium hydroxide solution c(Li0H) =
24、0,2 molll to 0,4 mom An example for the preparation of this solution for column regeneration is given in annex C. 6.4 Colour reagent Ninhydrin in a solution of Zmethoxyethanol and sodium acetate buffer. The sability of this mixture is very variable and ranges itom a few days to a few weeks. An examp
25、le for the preparation of the solution used in the colour development stage of this process is given in annex C. 6.6 Lithium citrate buffer (pH = 2,OO to 2,20) for sample dilution An example for the preparation of this dilution buffer is given in annex C. 6.6 Standard amino acids Weigh out individua
26、l portions of amino acids so that, after appropriate dilution, the absolute levels in the analyser faIl withim the linear range for each amino acid It is useful to use a standard solution of simii concentsation to that expected in the sample. The use of certified reference materials1) is recommended
27、 for this procedure. An example for the preparation of the amino acid test solution is given in annex C. 6 Apparatus Usual laboratory apparatus and, in particular, the following: 6.1 Amino acid analyser, with one of the following column dimensions: 9mm X 500mm, 6mm x 200mm, 4,6mm X 200mm or270mm,4mm
28、 X 200mm,4mm X 1OOmm or 3,2 mm X 140 mm The columns can be made of glass or steel. The various columns, described above, give significant differences in peak resolution and analysis times. 1 These materiais can be supplied by the Institute for reference materiais and measurement, C.C.R Retieseweg, 2
29、400 Geel, Belgium. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. O BSI W1999 Page 4 EN 127429999 6.2 Chromatographic resins
30、(cation exchanger) The resins are only available under trade names and are specifically adapted for the various commercial analysers, e.g. Durrum2) DCGa, Biotronik2) BTC 3118, Biotmnikz) BTC 2710, LKB ltro ac?) II, LKB also available for a range of these resins to remove traces of ammoNum saits from
31、 the buffer solutions and to help prolong the effective life of the analytical columns (examples are: Dumm2) G3, Biotron) BTGF, LKB Ultropac2) i). 6.3 Centrifuge, capable of producing a centrifugd force of 4 O00 g at the base of the cenmge tube (6.4). NOTE The rotational speed required to give corre
32、ct centrifugal acceleration can be calculated from the following equation: Ultropac2) 8,6300/7300 Beckman f: 1. Pre-columns are a = ii, x r x (n/i000)* (1) where a is the centrifugal acceleration; r is the radius of the centrifuge in centimetres, measwed from the mid point (the centrifuge axis) to t
33、he bottom of the centrifuge tube when swung out; n is the rotational frequency per minute. 6.4 Centrifuge tubes 6.6 Membrane filter, non sterie hydrophilic syringe lter(s) with a pore size of O, T = 0,013 KUTIOV; where: S, = 0,0026 + 0,0132 X c KUIIOM. 9.2 Reproducibility The absolute difference bet
34、ween single test results on identical test material reported by two laboratories will exceed the reproducibility value R in not more than 5 % of the cases. The values are: - for amino acids present at low concentrations c c 0,l mom. R = 0,019 mo* - for amino acids present at high concentrations c 0,
35、l mom R = 2,a sR; where: SR = 0,007 + 0,0473 X c IIUIIOM. 10 Wst Report The test report shall contain the foliowing dah - ali information necessary for the identification of the sample (kmd of sample, origin of sample, designaon); - a reference to this European Standard; - the date and type of sampl
36、uig procedure (if hown); - the date of receipt; -thedateoftest; - test resuis and units in which they have been expressed; - whether the repeatability has been verified - any particular points observed in the course of the test; - any operations not specified in the method or regarded as optional, w
37、hich might have affected the resuits. O BSI 06-1-1999 Page 6 EN 12742:1999 Annex A (informative) Bibliography Determination of repeatability and reproducibility for a standan test method by inter-ubomny tests. Moore, S. and Steh, W.H. Chromatography of amino acids on suifonakd poiystyrene resins. J.
38、 BWZ. Chem. (1951), 192,663-681. Determirdon of free amino acids: No. 57,1989. Analyses Coiiecion/inte&onai Federation of Fruit Juice Producers. Loose-leaf edition, as of 1989. Zug: Swiss Fruit Union. IS0 57251986, Preczsh Of test methods - Annex B (informative) Statistical results of the inter-labo
39、ratory test In accordance with IS0 57251986, the following parcuneters have been defined in an interiaboratory test (for literature pertaining to the method see annex A). The test was conducted by the Max von Pettenkofer-Institute of the Federai Health Office, Food Chemistry Department, Berlin (FRG)
40、. Year of interlaboratory test 1987 Number of samples 2 O ES1 06-1999 STD-BSI BS EN L2742-ENGL L777 = lb24bb9 0783777 bOL Methionine Iso-Leucine Leucine mosine Phenyl alanine Amino butyric acid Ethanolamine Ammonia (f = 46) (f= 55) 0,028 0,00136 0,004 0,00749 0,065 0,00519 0,015 0,00693 (f= 47) (f =
41、 56) (f= 47) (f=5r) (f=47) (f=57) (f = 39) (f = 47) 0,059 0,00511 0,014 0,00990 0,12 0,00556 0,016 0,01172 0,25 0,00652 0,02 0,02196 (f = 43) (f= 52) 3963 0,04450 0,13 0,2028 (f = 40) (f = 49) 0,38 0,01945 0,06 0,04947 (f= 33) = 40) Very important deviations from mean value. Page 7 EN 12742A999 Orni
42、thine usine Histidine Arginine Asparagine Glutamine limit .- 0,048 0,00433 0,012 0,00692 0,02 0,27 0,00971 0,03 0,03360 o, 10 OJO 0,00520 0,015 0,01258 0104 4,24 0,06826 o, 19 0,1915 O,% (f = 32) (f= 39) (f = 50) (f= 60) (f=47) (f = 57) (f= 52) cf=43) (f= 52) 2,m 0,04439 O, 13 0,1585 0,45 Very impor
43、tant deviations from mean value. R (movi) Fi 0,171 4 0,06 . O BSI 06-1999 STD-BSI BS EN L2742-ENGL L779 Lb24bb7 0783778 548 W Page 8 EN 12742d999 Aspartic Acid lble B.2 - Apple juice Mean Value 5 (movi) 0,77 Repeatability standard deviation s, (movi I Threonine Repeatability Reproducibility Reproduc
44、ibility limit standard limit deviation T (mou) (movi R (movi) 0,01239 (f=w 0,00449 U=) (f = 39) 0,00493 0104 0,04190 o, 12 (f = 52) 0,013 0,00644 0,018 (f = 52) (f= 47) 0,014 0,01087 0,03 Proline Serine I OJO 0,20 Glutamic acid Alanine 0,27 Valine 0,01009 (f= 35) (f = 35) (f = 46) 0,00563 0,00390 I
45、0,047 0103 0,03614 OJO (f= 42) (f = 42) (f= 55) 0,016 0,02083 0,06 0,011 0,00551 0,016 I Methionine I de), and 5 ml of hydrochloric acid c(HCI = 10 movi. Following a similar procedure to that detailed above for buffer A, prepare and adjust the solutions pH to 2,20 and make up to volume. Then add the
46、 preservative to the buffer as detailed above for buffer A. C.6 Preparation of amino acid test solution C.6.1 General Values for the concentrations in mmoii of the amino acids and other materials are given in the brackets and these should be suitable for most purposes. C.6.2 Standard solution A Cont
47、ains monia (e = 2,0), ethanolamine (e = 1,O) and the foliowing amino acids: asparticacid (1,5) threonine (1,O) serine glutamic acid (1,5) proline (6,O) glycine (1,O) alanine (1,5) valine (1,O) methionine (1,O) &leucine (170) leucine (1,O) tyrosine (1,O) phenylalanine (1,O) gamma-amino-%-butyric acid
48、 (1,5) ornithine (170) me (1,O) histidine (1,O) arginine (1,O) C.6.3 Standad solution B Contains the foilowing amino acids: asparagine (3,O) and giutamine (1,O). C.6.4 Standard solution C Contains 1 mmovi of either nor-leucine or nor-valine which is used as an intrnai standard. The aqueous solutions
49、 will keep for a few months if For calibration of the instrument, mix 1 mi of each of the three sandard solutions A, B and C and make up to 10 mi with the dilution buffer (see 6.6). stored at - 18 “C. O BSI O-1999 BSI 389 Chiswick High Road London w4 4AL4 STDOBSI BS EN 12742-ENGL 1777 m lb24bb7 0781781 032 BS EN 12 742: 1999 BSI - British Standards Institution BSI is the independent nationai body responsible for preparing British Standards. It presents the K view on standards in Europe and at the intemational level. It is incorporated by Roya
