1、September 2009DEUTSCHE NORM English price group 10No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 07.100.30!$YqZ“15478
2、55www.din.deDDIN EN 14166Foodstuffs Determination of vitamin B6 by microbiological assayEnglish version of DIN EN 14166:2009-09Lebensmittel Mikrobiologische Bestimmung von Vitamin B6Englische Fassung DIN EN 14166:2009-09SupersedesDIN V ENV 14166:2002-02www.beuth.deDocument comprises pages16DIN EN 14
3、166:2009-09 National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was done by Working Group WG 9 “Vitamins and Carotenoids”. The responsible German body involved in its preparation was the
4、Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-13 AA Vitamine und Carotinoide. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN I
5、SO 3696 Amendments This standard differs from DIN V ENV 14166:2002-02 as follows: a) Pyridoxine hydrochloride is now used as the only standard substance for the preparation of stock and calibration solutions. b) The standard has been revised in form and substance and has been brought in line with th
6、e current rules of presentation. Previous editions DIN V ENV 14166: 2002-02 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods 2 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14166May 2009ICS 07.100.30 Supersedes ENV 1416
7、6:2001 English VersionFoodstuffs - Determination of vitamin B6 by microbiologicalassayProduits alimentaires - Dtermination de la vitamine paressai microbiologiqueLebensmittel - Mikrobiologische Bestimmung von Vitamin B6This European Standard was approved by CEN on 23 April 2009.CEN members are bound
8、 to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN M
9、anagement Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as
10、 theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovaki
11、a, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN
12、 national Members.Ref. No. EN 14166:2009: EB6EN 14166:2009 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Procedure .77 Calculation 98 Criteria for acceptance of data 109 Precision 1110 Test Report 11Annex A (informative) Vitamin B6 calibrati
13、on lines obtained by MBA using the test organism Saccharomyces uvarum . 12Annex B (informative) Precision data . 13Bibliography . 14DIN EN 14166:2009-09 EN 14166:2009 (E) 3 Foreword This document (EN 14166:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
14、, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November 2009, and conflicting national standards shall be withdrawn at the latest by November 2009. Atte
15、ntion is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes ENV 14166:2001. According to the CEN/CENELEC Internal Regulations
16、, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands
17、, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 14166:2009-09 EN 14166:2009 (E) 4 1 Scope This European Standard specifies a method for the determination of total vitamin B6in foodstuffs by microbiological assay (MBA). Vitamin B6is d
18、etermined as the mass fraction of pyridoxine, pyridoxal and pyridoxamine, including their phosphorylated or glycosylated derivatives. It is usually expressed as milligram vitamin B6per 100 g of foodstuff. The method is applicable to samples that can be rendered homogeneous and do not contain high co
19、ncentrations of antibiotics or other interfering substances. This method has been validated in an inter-laboratory test on fortified and non-fortified samples such as wholemeal flour, milk powder, mixed vegetables and pigs liver at levels from 0,5 mg/100 g to 1,9 mg/100 g. For further information on
20、 the validation data, see Annex B. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments)
21、applies. EN ISO 3696:1995, Water 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 vitamin B6vitamers from proteins and carbohydrates i
22、n the sample and hydrolyses the phosphates to the free vitamers. The total vitamin B6content in the sample extract is then determined by comparing the growth response of the assay test organism against growth obtained from a pyridoxine hydrochloride standard, see 1. 4 Reagents 4.1 General During ana
23、lysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at least grade 1 according to EN ISO 3696:1995. The water used for reagent preparation shall be glass distilled. Once distilled, water shall be used within five days or discarded. 4.2 Chemicals and solution
24、s 4.2.1 Sulfuric acid solution, substance concentration c(H2SO4 ) = 0,22 mol/l 4.2.2 Sodium hydroxide solution, c(NaOH) = 4 mol/l 4.2.3 Wort agar, (Difco1)or suitable alternative) Dissolve the agar in glass distilled water according to the manufacturers instructions. Heat to boil. Dispense 5 ml aliq
25、uots 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. 1) This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named.
26、 Equivalent products may be used if they can be shown to lead to the same results. DIN EN 14166:2009-09 EN 14166:2009 (E) 5 4.2.4 Basal medium (Difco Pyridoxine Y Medium1)or suitable alternative) The concentration of the assay medium should be chosen, depending upon the assay format used, to ensure
27、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 equal volume of water containing 2,0 ng/ml pyridoxine, pyridoxamine and pyridoxal. Add 10 ml portions to screw-topped tubes and autoclave at 121 C f
28、or 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. Add 10 ml portions to screw-topped tubes and autoclave at 121 C for 5 min and cool rapidly. Store in refrigerator for up to one month. 4.2.7 Sodium ch
29、loride, 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. Autoclave 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, Sacchar
30、omyces uvarum ATCC 9080 (freeze-dried yeast) 4.3.1 Test organism maintenance (stock culture) The test organism is maintained by weekly transfers 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-w
31、alled glass bottle or suitable flask. Add 2 ml of pyridoxine calibration solution 20 (4.8.1), cap and autoclave at 121 C for 5 min. Cool as rapidly 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 su
32、spension to the remaining medium using a sterile pipette. Incubate at 30 C for 16 h. After incubation, the organism should show thick growth. 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
33、 portions of sterile saline (4.2.8), centrifuging between washes. Re-suspend the cells in 50 ml of sterile saline solution. Using a sterile loop, transfer cells from this suspension onto three agar slopes (4.2.3) in a cross pattern and incubate for 16 h to 20 h at 30 C. After incubation, the cross s
34、hould show visible growth and there should be no growth in the surrounding areas. Store the organism in a refrigerator. The organism should be transferred to fresh agar slopes on a weekly basis. It is essential to maintain aseptic conditions during preparation and transfer of solutions. 4.3.2 Workin
35、g inoculum On the day before required, transfer cells 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 supern
36、atant. Cells can be washed with 2 x 10 ml inoculum rinse (4.2.6) discarding the supernatant each time. Re-suspend cells in a third 10 ml portion of inoculum rinse. This is used for the assay inoculum. DIN EN 14166:2009-09 EN 14166:2009 (E) 6 4.4 Standard substance 4.4.1 Pyridoxine hydrochloride, w(C
37、8H11NO3 . HCl) 98 % 4.5 Stock solution 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 volumetric flask. Dilute to the
38、mark with glass distilled water and mix. This solution is stable for two weeks if kept refrigerated. 4.6 Concentration testDilute 2 ml of the stock solution (4.5.1) to 20 ml with 0,1 mol/l HCl. Measure the absorbance value at 290 nm against 0,1 mol/l HCl solution (pH 1). Calculate the mass concentra
39、tion, , in g/ml of the stock solution according to equation (1): VMA =w(1) where: A is the absorbance value of the solution at 290 nm; is the molar extinction coefficient in a hydrochloric acid solution of c(HCl) = 0,1 mol/l at max= 290 nm (here: 8 400 mmol-1cm-1, see 2); Mw is the molar mass of the
40、 standard substance, in gram per mol; V is the dilution factor, i.e. 10. 4.7 Intermediate pyridoxine 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. 4.8 Calibration solutions 4.8.1 Pyridoxine ca
41、libration solution 20, (C8H11NO3) = 20 ng/ml Dilute 5 ml intermediate calibration solution (4.7) to 100 ml with glass distilled water. Prepare on day of use. 4.8.2 Pyridoxine calibration solution 10, (C8H11NO3) = 10 ng/ml Dilute 25 ml of pyridoxine calibration solution 20 (4.8.1) to 50 ml with glass
42、 distilled water. Prepare on day of use. 4.8.3 Pyridoxine calibration solution 5, (C8H11NO3) = 5 ng/ml Dilute 25 ml of pyridoxine calibration solution 20 (4.8.1) to 100 ml with glass distilled water. Prepare on day of use. NOTE Alternative concentrations may be prepared to suit the assay format to b
43、e used, see 6.3.1 and 6.3.2. DIN EN 14166:2009-09 EN 14166:2009 (E) 7 5 Apparatus 5.1 General Usual laboratory equipment and glassware. All glassware shall be washed with detergents that will not stimulate or depress the growth of the assay test organism. Glassware shall be thoroughly washed and rin
44、sed with glass distilled water. If glass test tubes are used for the assay format, they should be thoroughly washed and then heated at a minimum temperature of 160 C overnight before use. The following items are also required. 5.2 Autoclave, or similar heating device 5.3 UV spectrometer 5.4 Incubato
45、r or water-bath, capable of maintaining a constant and defined temperature and with a shaking facility. 5.5 Sterile pipettes and/or syringes 5.6 Autoclavablebottles or flasks 5.7 Sterile bottles or tubes 6 Procedure 6.1 Preparation of the test sample The test sample shall be homogeneous. Coarse mate
46、rial shall be rendered homogeneous using an appropriate mill and/or blender. Pre-cooling of the sample may be necessary to prevent exposure to high temperatures. 6.2 Preparation of the test sample solution Weigh an appropriate amount of sample (between 0,5 g and 10 g corresponds to about 2 g of vita
47、min B6) into a screw top glass bottle or conical flask, to the nearest 1 mg. Add 150 ml of sulfuric acid solution (4.2.1) and swirl the contents to mix.Cap the bottle (or flask) and autoclave at 121 C for 5 h. Cool to room temperature in cold water, add 15 ml of sodium hydroxide solution (4.2.2) and
48、 cool again. Adjust the pH of the sample solution to 4,5 0,1 using sodium hydroxide solution (4.2.10) and a pH meter. Transfer the solution to a 200 ml volumetric flask and dilute to the mark with glass distilled water. Mix thoroughly by inversion and filter through a fine porosity filter paper. Dil
49、ute with glass distilled water to an analyte concentration suitable for the assay calibration range used. A reagent blank should be run with every batch of samples. 6.3 Determination of vitamin B6by microbiological assay 6.3.1 Assay configuration - Standards The growth response of the different vitamin B6vitamers to the assay organism varies (see Annex A). Pyridoxal shows a slightly lower dose response than pyridoxine and pyridoxamine shows an even lower dose response (e.g. pyri
