1、BS EN 14166:2009ICS 07.100.30NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determinationof vitamin B6 bymicrobiological assayThis British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 30 June2009. BSI 2009I
2、SBN 978 0 580 58611 8Amendments/corrigenda issued since publicationDate CommentsBS EN 14166:2009National forewordThis British Standard is the UK implementation of EN 14166:2009. Itsupersedes DD ENV 14166:2001 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommit
3、tee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a
4、British Standard cannot confer immunityfrom legal obligations.BS EN 14166:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14166May 2009ICS 07.100.30 Supersedes ENV 14166:2001 English VersionFoodstuffs - Determination of vitamin B6 by microbiologicalassayProduits alimentaires - Dtermination de
5、la vitamine B6 paressai microbiologiqueLebensmittel - Mikrobiologische Bestimmung von VitaminB6This European Standard was approved by CEN on 23 April 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the sta
6、tus 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 Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A
7、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 theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, D
8、enmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISAT
9、IONEUROPISCHES 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 national Members.Ref. No. EN 14166:2009: EBS EN 14166:2009EN 14166:2009 (E) 2 Contents Page Foreword 31 Scope 42 Normative
10、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 calibration lines obtained by MBA using the test organism Saccharomyces uvarum . 12Annex B (informative) Precision data
11、 . 13Bibliography . 14BS EN 14166:2009EN 14166:2009 (E) 3 Foreword This document (EN 14166:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard,
12、 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. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. C
13、EN 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, the national standards organizations of the following countries are bound to implement this European Standard: Au
14、stria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 1
15、4166:2009EN 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 determined as the mass fraction of pyridoxine, pyridoxal and pyridoxamine, including their phosphorylated or glycosylated
16、 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 concentrations of antibiotics or other interfering substances. This method has been validated in an inter-laboratory test
17、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 the validation data, see Annex B. 2 Normative references The following referenced documents are indispensable for the a
18、pplication of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principl
19、e Pyridoxine, pyridoxal and/or pyridoxamine are extracted from foodstuffs by acid hydrolysis. The hydrolysis step liberates the vitamin B6vitamers from proteins and carbohydrates in the sample and hydrolyses the phosphates to the free vitamers. The total vitamin B6content in the sample extract is th
20、en 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 analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at least grade 1 according
21、 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 solutions 4.2.1 Sulfuric acid solution, substance concentration c(H2SO4 ) = 0,22 mol/l 4.2.2 Sodium hydroxide solution, c(NaOH)
22、= 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 aliquots into glass bottles, cap and autoclave at 121 C for 15 min. Cool at an angle for slopes to form. Store in a refriger
23、ator 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. Equivalent products may be used if they can be shown to lead to the same results. BS EN 14166:2009EN 14166:2009 (E) 5 4
24、.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 that the manufacturers recommended concentration is obtained in the final assay volume. 4.2.5 Liquid culture medium Dilute ba
25、sal 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 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 a
26、n 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 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 gla
27、ss 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, Saccharomyces uvarum ATCC 9080 (freeze-dried yeast) 4.3.1 Test organism maintenance (stock culture) The test organism is maintained
28、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-walled glass bottle or suitable flask. Add 2 ml of pyridoxine calibration solution 20 (4.8.1), cap and autoclave at 121 C for
29、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 suspension to the remaining medium using a sterile pipette. Incubate at 30 C for 16 h. After incubation, the organism should sh
30、ow 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 portions of sterile saline (4.2.8), centrifuging between washes. Re-suspend the cells in 50 ml of sterile saline solution. U
31、sing 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 should show visible growth and there should be no growth in the surrounding areas. Store the organism in a refrigerator. The o
32、rganism 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 Working inoculum On the day before required, transfer cells from the stock culture (4.3.1) into two tubes of the liquid culture med
33、ium (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. Cells can be washed with 2 x 10 ml inoculum rinse (4.2.6) discarding the supernatant each time. Re-suspend cells in a
34、third 10 ml portion of inoculum rinse. This is used for the assay inoculum. BS EN 14166:2009EN 14166:2009 (E) 6 4.4 Standard substance 4.4.1 Pyridoxine hydrochloride, w(C8H11NO3 . HCl) 98 % 4.5 Stock solution 4.5.1 Pyridoxine stock solution, (C8H11NO3) 200 g/ml Accurately weigh 121,5 mg to the neare
35、st 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 mark with glass distilled water and mix. This solution is stable for two weeks if kept refrigerated. 4.6 Concentration testDilute
36、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 concentration, , 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
37、 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 standard substance, in gram per mol; V is the dilution factor, i.e. 10. 4.7 Intermediate pyridoxine calibration solution, (C8H11N
38、O3) 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 calibration solution 20, (C8H11NO3) = 20 ng/ml Dilute 5 ml intermediate calibration solution (4.7) to 100 ml with glass distilled wa
39、ter. 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 distilled water. Prepare on day of use. 4.8.3 Pyridoxine calibration solution 5, (C8H11NO3) = 5 ng/ml Dilute 25 ml of pyridoxine
40、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 be used, see 6.3.1 and 6.3.2. BS EN 14166:2009EN 14166:2009 (E) 7 5 Apparatus 5.1 General Usual laboratory equipment and glassware.
41、 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 rinsed with glass distilled water. If glass test tubes are used for the assay format, they should be thoroughly washed and then heated at
42、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 Incubator or water-bath, capable of maintaining a constant and defined temperature and with a shaking facility. 5.5 Sterile pipettes and/or syr
43、inges 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 material shall be rendered homogeneous using an appropriate mill and/or blender. Pre-cooling of the sample may be necessary to prevent expo
44、sure 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 vitamin 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
45、 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 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 solu
46、tion 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. Dilute with glass distilled water to an analyte concentration suitable for the assay calibration range used. A reagent blank should be run
47、 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 pyridoxam
48、ine shows an even lower dose response (e.g. pyridoxine for plant based foods, pyridoxal for dairy based foods, pyridoxal or pyridoxamine for meats). It is common practice to use pyridoxine as calibrant but this may cause an underestimate where significant levels of pyridoxamine are present. BS EN 14
49、166:2009EN 14166:2009 (E) 8 Microbiological assays may be performed in test tubes or in microtitre-plate format.The volumes of standards, sample extracts, and assay media used will vary between laboratories depending upon assay format.Two assay formats, which use low and high volumes of test sample extract respectively, are provided in Tables 1 and 2below. Depending on the assay format (see Tables 1 and 2),pipette the appropriate amount of the calibration solutions (4.8.1 to 4.8.3)
