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1、DRAFT FOR DEVELOPMENT DD ENV 14166:2001 Foodstuffs Determination of vitamin B6 by microbiological assay ICS 07.100.30; 67.050 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:28:15 GMT+00:0
2、0 2006, Uncontrolled Copy, (c) BSI DD ENV 14166:2001 This Draft for Development, having been prepared under the direction of the Consumer Products and Services Sector Policy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 19 December 2001 B
3、SI 19 December 2001 ISBN 0 580 37677 X National foreword This Draft for Development is the official English language version of ENV 14166:2001. This publication is not to be regarded as a British Standard. It is being issued in the Draft for Development series of publications and is of a provisional
4、 nature in order to assess the method. It should be applied on this provisional basis, so that information and experience of its practical application may be obtained. Comments arising from the use of this Draft for Development are requested so that UK experience can be reported to the European orga
5、nization responsible for its conversion into a European Standard. A review of this publication will be initiated 2 years after its publication by the European organization so that a decision can be taken on its status at the end of its three-year life. The commencement of the review period will be n
6、otified by an announcement in Update Standards. According to the replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into a European Standard, to extend the life of the prestandard or to withdraw it. Comments should be sent in
7、 writing to the Secretary of BSI Technical Committee AW/-/3, Food analysis Horizontal methods, at 389 Chiswick High Road, London W4 4AL, giving the document reference and clause number and proposing, where possible, an appropriate revision of the text. A list of organizations represented on this com
8、mittee can be obtained on request to its secretary. Cross-references The British Standards which implement international 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
9、using the “Find” facility of the BSI Standards Electronic Catalogue. Summary of pages This document comprises a front cover, an inside front cover, the ENV title page, pages 2 to 13 and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amend
10、ments issued since publication Amd. No. DateComments Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:28:15 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EUROPEAN PRESTANDARD PRNORME EUROPENNE EUROPISCHE VORNORM ENV 14166 November 2001 ICS 07.100.30 English versi
11、on Foodstuffs - Determination of vitamin B6 by microbiological assay Produits alimentaires - Dtermination de la vitamine B6 par essai microbiologique Lebensmittel - Mikrobiologische Bestimmung von Vitamin B6 This European Prestandard (ENV) was approved by CEN on 29 September 2001 as a prospective st
12、andard for provisional application. The period of validity of this ENV is limited initially to three years. After two years the members of CEN will be requested to submit their comments, particularly on the question whether the ENV can be converted into a European Standard. CEN members are required
13、to announce the existence of this ENV in the same way as for an EN and to make the ENV available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the ENV) until the final decision about the possible conversion of the
14、 ENV into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARD
15、IZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2001 CENAll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. ENV 14166:2001 E Licensed Copy: London South Bank Univers
16、ity, London South Bank University, Tue Dec 12 05:28:15 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ENV 14166:2001 (E) 2 Foreword This European Prestandard has been prepared by Technical Committee CEN /TC 275, “Food analysis - Horizontal methods“, the secretariat of which is held by DIN. Annexes A and
17、 B are informative. This standard includes a Bibliography. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following coun- tries are bound to announce this European Prestandard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece
18、, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. 1 Scope This European Prestandard specifies a method for the determination of total vitamin B6 in foodstuffs by microbio- logical assay (MBA). Vitamin B6 is determined as the mass
19、 fraction of pyridoxine, pyridoxal and pyridoxamine, in- cluding their phosphorylated or glycosylated derivatives. It is usually expressed as milligram vitamin B6 per 100 g of foodstuff. The method is applicable to samples that can be rendered homogeneous and do not contain high con- centrations of
20、antibiotics or other interfering substances. 2 Normative references This European Prestandard 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 dat
21、ed references, subsequent amendments to or revisions of any of these publications apply to this European Prestandard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies (including amendments). EN ISO 3696:1995, Water
22、 for analytical laboratory use Specification and test methods (ISO 3696:1987). 3 Principle Pyridoxine, pyridoxal and/or pyridoxamine are extracted from foodstuffs by acid hydrolysis. The hydrolysis step liberates the B6 vitamers from proteins and carbohydrates in the sample and hydrolyses the phosph
23、ates to the free vitamers. The total Vitamin B6 content in the sample extract is then determined by comparing the growth response of the assay test organism against growth obtained from appropriate standards, see 1. 4 Reagents 4.1 General During analysis, unless otherwise stated, use only reagents o
24、f recognised analytical grade and water of at least grade 1 as defined in EN ISO 3696:1995. The water used for reagent preparation shall be glass distilled. Once dis- tilled, water shall be used within five days or discarded. 4.2 Chemicals and solutions 4.2.1 Sulfuric acid solution, substance concen
25、tration c (H2S04 ) = 0,22 mol/l 4.2.2 Sodium hydroxide solution, c (NaOH) = 4 mol/l. Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:28:15 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ENV 14166:2001 (E) 3 4.2.3 Wort agar, (Difco1) or suitable alternative) Disso
26、lve the agar in glass distilled water according to the manufacturers instructions. Heat to boil. Dispense 5 ml aliquots into glass bottles, cap and autoclave at 121 C for 15 min. Cool at an angle for slopes to form. Store in a refrigerator for up to three months. 4.2.4 Basal medium (Difco, Pyridoxin
27、e Y assay medium1) or suitable alternative) The concentration of the assay medium should be chosen, depending upon the assay format used, to ensure that the manufacturers recommended concentration is obtained in the final assay volume. 4.2.5 Liquid culture medium Dilute basal medium (4.2.4) with an
28、equal volume of water containing 2,0 ng/ml pyridoxine, pyridoxamine and pyri- doxal. Add 10 ml portions to screw-topped tubes and autoclave at 121C for 5 min and cool rapidly. Store in refrigerator for up to one month. 4.2.6 Inoculum rinse Dilute basal medium (4.2.4) with an equal volume of water. A
29、dd 10 ml portions to screw-topped tubes and auto- clave at 121 C for 5 min and cool rapidly. Store in refrigerator for up to one month. 4.2.7 Sodium chloride, mass fraction w (NaCl) ? 98,0 % 4.2.8 Sterile saline solution Dissolve 0,9 g of sodium chloride (4.2.7) in 100 ml of glass distilled water. A
30、utoclave at 121 C for 15 min. 4.2.9 Hydrochloric acid, c (HCl) = 0,1 mol/l 4.2.10 Sodium hydroxide solution, c (NaOH) = 0,1 mol/l 4.3 Test organism, Sacchromyces Uvarum ATCC 9080. (Freeze-dried yeast) 4.3.1 Test organism maintenance (stock culture) The test organism is maintained by weekly transfers
31、 onto agar maintenance medium (4.2.3) using the following procedure: Prepare 50 ml portions of Pyridoxine basal medium (4.2.4) and place in a 100 ml thick-walled glass bottle or suit- able flask. Add 2 ml of Pyridoxine calibration solution 20 (4.8.1), cap and autoclave at 121 C for 5 min. Cool as ra
32、pidly as possible in cold water to below 30 C. Aseptically, add 1 ml of autoclaved medium to the freeze dried culture (4.3) and add 0,5 ml of the resultant suspension to the remaining medium using a sterile pipette. Incubate at 30 C for 16 h. After incubation, the organism should show thick growth.
33、Transfer the medium to suitable sterile, centrifuge tubes and centrifuge at 2000 g for 5 min. Discard the supernatant and wash the cell residue with two 50 ml portions of sterile saline solution (4.2.8), centrifuging between washes. Re-suspend the cells in 50 ml of ster- ile saline solution. Using a
34、 sterile loop, transfer cells from this suspension onto three agar slopes (4.2.3) in a cross pattern and incu- bate for 16 h to 20 h at 30 C. After incubation, the cross should show visible growth and there should be no growth in the surrounding areas. Store the organism in a fridge. The organism sh
35、ould be transferred to fresh agar slopes on a weekly basis. It is essential to maintain aseptic conditions during preparation and transfer of solutions. 1)This information is given for the convenience of users of this Prestandard and does not constitute an endorsement by CEN of the product named. Eq
36、uivalent products may be used if they can be shown to lead to the same results. Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:28:15 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ENV 14166:2001 (E) 4 4.3.2 Working inoculum On the day before required, transfer c
37、ells from the stock culture (4.3.1) into two tubes of the liquid culture medium (4.2.5) keeping the transfers as sterile as possible. Incubate for 16 h to 20 h at 30 ?C. Under aseptic conditions, centrifuge culture at 2000 g for 2 min and decant supernatant. Wash cells with 2 x 10 ml inoculum rinse
38、(4.2.6) dis- carding the supernatant each time. Re-suspend cells in a third 10 ml portion of inoculum rinse. This is used for the assay inoculum. 4.4 Standard substances 4.4.1 Pyridoxine hydrochloride, w (C8H11NO3.HCl) ? 98 % 4.4.2 Pyridoxal hydrochloride, w (C8H9NO3.HCl) ? 98 % 4.4.3 Pyridoxamine d
39、ihydrochloride, w (C8H12N2O2.2HCl) ? 98 % 4.5 Stock solutions 4.5.1 Pyridoxine stock solution, ? (C8H11NO3) ? 200 g/ml Accurately weigh 121,5 mg to the nearest 0,1 mg pyridoxine hydrochloride (4.4.1) in a small beaker. Dissolve in glass distilled water, then transfer quantitatively to a 500 ml volum
40、etric flask. Dilute to the mark with glass distilled water and mix. This solution is stable for two weeks if kept refrigerated. 4.5.2 Pyridoxal stock solution, ? (C8H9NO3) ? 200 g/ml Prepare as for Pyridoxine using 121,8 mg to the nearest 0,1 mg pyridoxal hydrochloride (4.4.2). This solution is stab
41、le for two weeks if kept refrigerated. 4.5.3 Pyridoxamine stock solution, ? (C8H12N2O2) ? 200 g/ml Prepare as for Pyridoxine using 143,4 mg to the nearest 0,1 mg pyridoxamine dihydrochloride (4.4.3). This solution is stable for two weeks if kept refrigerated. 4.6 Concentration test Depending on the
42、standard substance used, dilute 2 ml of the appropriate stock solution (4.5.1 to 4.5.3) to 20 ml with 0,1 mol/l HCl. Measure the absorbance at the wavelengths shown in Table 1, against 0,1 mol/l HCl solution (pH 1). Table 1 Concentration test parameters 2 StandardSolvent? ?maxMolar Extinction Coeff.
43、 ? ? PyridoxineHCl0, 1 mol/l HCl2908400 PyridoxalHCl0,1mol/l HCl2889000 Pyridoxamine2 HCl0,1 mol/l HCl2928200 Calculate the mass concentration ?, in g/ml of the stock solution according to equation (1): ? ? VMA? ? w (1) where: Ais the absorbance value of the solution at the relevant wavelength; ?is
44、the appropriate molar extinction coefficient from Table 1; Licensed Copy: London South Bank University, London South Bank University, Tue Dec 12 05:28:15 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ENV 14166:2001 (E) 5 Mwis the molecular weight of the standard substance, in gram per mol; Vis the dilu
45、tion factor, i.e. 10. 4.7 Intermediate calibration solutions, ? ? 400 ng/ml. 4.7.1 Pyridoxine intermediate calibration solution, ? (C8H11NO3) ? 400 ng/ml. Dilute 2 ml of Pyridoxine stock solution (4.5.1) to 1000 ml with glass distilled water. Prepare on day of use. 4.7.2 Pyridoxal intermediate calib
46、ration solution, ? (C8H9NO3) ? 400 ng/ml. Dilute 2 ml of Pyridoxal stock solution (4.5.2) to 1000 ml with glass distilled water. Prepare on day of use. 4.7.3 Pyridoxamine intermediate calibration solution, ? (C8H12N2O2) ? 400 ng/ml. Dilute 2 ml of Pyridoxamine stock solution (4.5.3) to 1000 ml with
47、glass distilled water. Prepare on day of use. 4.8 Calibration solutions 4.8.1 Pyridoxine calibration solution 20, ? (C8H11NO3) = 20 ng/ml Dilute 5 ml intermediate calibration solution (4.7.1) to 100 ml with glass distilled water. Prepare on day of use. 4.8.2 Pyridoxine calibration solution 10, ? (C8
48、H11NO3) = 10 ng/ml Dilute 25 ml of 20 ng/ml calibration solution (4.8.1) to 50 ml with glass distilled water. Prepare on day of use. 4.8.3 Pyridoxine calibration solution 5, ? (C8H11NO3) = 5 ng/ml Dilute 25 ml of 20 ng/ml calibration solution (4.8.1) to 100 ml with glass distilled water. Prepare on
49、day of use. 4.8.4 Pyridoxal calibration solution 20, ? (C8H9NO3) = 20 ng/ml Dilute 5 ml intermediate calibration solution (4.7.2) to 100 ml with glass distilled water. Prepare on day of use. 4.8.5 Pyridoxal calibration solution 10, ? (C8H9NO3) = 10 ng/ml Dilute 25 ml of 20 ng/ml calibration solution (4.8.4) to 50 ml with glass distilled water. Prepare on day of use. 4.8.6 Pyridoxal calibration solution 5, ? (C8H9NO3) = 5 ng/ml Dilute 25 ml of 20 ng/ml calibration solution (4.8.4) to 100 ml with glass distilled water. Prepare on day of use. 4.8.7 Pyr