SIST EN 14166:2009

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Mikrobiologische Bestimmung von Vitamin B6Produits alimentaires - Détermination de la vitamine B6 par essai microbiologiqueFoodstuffs - Determination of vitamin B6 by microbiological assay07.100.30Mikrobiologija živilFood microbiologyICS:Ta slovenski standard je istoveten z:EN 14166:2009SIST EN 14166:2009en,fr,de01-december-2009SIST EN 14166:2009SLOVENSKI
STANDARDSIST ENV 14166:20021DGRPHãþD



SIST EN 14166:2009



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14166May 2009ICS 07.100.30Supersedes ENV 14166:2001
English VersionFoodstuffs - Determination of vitamin B6 by microbiologicalassayProduits alimentaires - Détermination de 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 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 Management 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 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, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14166:2009: ESIST EN 14166:2009



EN 14166:2009 (E) 2 Contents Page Foreword .31Scope .42Normative references .43Principle .44Reagents .45Apparatus .76Procedure .77Calculation .98Criteria for acceptance of data . 109Precision . 1110Test Report . 11Annex A (informative)
Vitamin B6 calibration lines obtained by MBA using the test organism Saccharomyces uvarum . 12Annex B (informative)
Precision data . 13Bibliography . 14 SIST EN 14166:2009



EN 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, 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. 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, 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, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. SIST EN 14166:2009



EN 14166:2009 (E) 4 1 Scope This European Standard specifies a method for the determination of total vitamin B6 in foodstuffs by microbiological assay (MBA). Vitamin B6 is determined as the mass fraction of pyridoxine, pyridoxal and pyridoxamine, including 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 concentrations 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 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) 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 B6 vitamers from proteins and carbohydrates in the sample and hydrolyses the phosphates 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 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 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) = 4 mol/l 4.2.3 Wort agar, (Difco1)
or suitable alternative) Dissolve the agar in glass distilled water according to the manufacturer’s 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.
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. SIST EN 14166:2009



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 that the manufacturer’s 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 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. 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 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, Saccharomyces 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-walled 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 suspension 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 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 should 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 Working 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 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 third 10 ml portion of inoculum rinse. This is used for the assay inoculum. SIST EN 14166:2009



EN 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, ρ(C8H11NO3) ≈ 200 µg/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 mark with glass distilled water and mix. This solution is stable for two weeks if kept refrigerated. 4.6 Concentration test
Dilute 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 µg/ml of the stock solution according to equation (1): ερVMA××=w (1) where:
A is the absorbance value of the solution at 290 nm; 0 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, (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 calibration 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 distilled water. Prepare on day of use. 4.8.3 Pyridoxine calibration solution 5, (C8H11NO3)
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