EN 14166:2009

Foodstuffs - Determination of vitamin B6 by microbiological assay

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.

Lebensmittel - Mikrobiologische Bestimmung von Vitamin B6

Diese Europäische Norm legt ein mikrobiologisches Verfahren (microbiological assay - MBA) zur Bestimmung des Gesamtgehaltes an Vitamin B6 in Lebensmitteln fest. Vitamin B6 wird als Massenanteil von Pyridoxin, Pyridoxal und Pyridoxamin einschließlich deren phosphorylierter oder glykosylierter Derivate bestimmt. Dieser wird üblicherweise als Milligramm Vitamin B6 je 100 g Lebensmittel angegeben. Das Verfahren ist auf Proben anwendbar, die homogenisiert werden können und keine hohen Konzentrationen an Antibiotika oder anderen störenden Substanzen enthalten.
Dieses Verfahren wurde in einem Ringversuch an angereicherten und nicht angereicherten Proben validiert, wie Vollkornmehr, Milchpulver, Mischgemüse und Schweineleber, jeweils bei Gehalten von 0,5 mg/100 g bis 1,9 mg/100 g. Weitere Information zu Validierungsdaten, siehe Anhang B.

Produits alimentaires - Détermination de la vitamine B6 par essai microbiologique

La présente Norme européenne spécifie une méthode de dosage de la vitamine B6 totale présente dans les
produits alimentaires par essai microbiologique. La vitamine B6 est dosée comme étant la fraction massique
de la pyridoxine, du pyridoxal et de la pyridoxamine, y compris leurs dérivés phosphorylés ou glycosylés. Elle
est généralement exprimée en milligrammes de vitamine B6 par 100 g de produits alimentaires. La méthode
est applicable aux échantillons qui peuvent être homogénéisés et qui ne contiennent pas d’antibiotiques ou
autres substances interférentes en concentrations importantes.
La méthode a été validée par un essai interlaboratoire sur des échantillons enrichis et non enrichis tels que
farine complète, lait en poudre, assortiment de légumes et foies de porc, à des concentrations allant de
0,5 mg/100 g à 1,9 mg/100 g. Pour de plus amples informations sur les données de validation, voir
l’Annexe B.

Živila - Določevanje vitamina B6 z mikrobiološko analizo

General Information

• Status: Published
• Publication Date: 26-May-2009
• Withdrawal Date: 29-Nov-2009
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 9 - Vitamins and Carotenoids
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 28-Oct-2020
• Completion Date: 23-Sep-2025
• Ref Project: SIST EN 14166:2009 - Foodstuffs - Determination of vitamin B6 by microbiological assay

Relations

• Replaces: ENV 14166:2001 - Foodstuffs - Determination of vitamin B6 by microbiological assay
• Effective Date: 22-Dec-2008

Overview

EN 14166:2009 - Foodstuffs: Determination of vitamin B6 by microbiological assay (MBA) is a European Standard published by CEN (approved 23 April 2009). The method specifies a validated microbiological assay for determination of total vitamin B6 (pyridoxine, pyridoxal, pyridoxamine and their phosphorylated/glycosylated derivatives) in foodstuffs. Results are normally reported as milligrams vitamin B6 per 100 g of food. The procedure is applicable to homogeneous samples that do not contain high concentrations of antibiotics or other interfering substances and was validated in an inter‑laboratory study on fortified and non‑fortified matrices (wholemeal flour, milk powder, mixed vegetables, pig’s liver) at 0.5–1.9 mg/100 g.

Key Topics and Requirements

• Analytical principle: Acid hydrolysis to liberate vitamers and dephosphorylate derivatives, followed by a microbiological growth response assay (MBA) using the test organism Saccharomyces uvarum. Growth is compared to a pyridoxine hydrochloride calibration series.
• Scope of measurement: Total vitamin B6 expressed as mass fraction (mg/100 g) covering pyridoxine, pyridoxal, pyridoxamine and related derivatives.
• Sample preparation: Homogenisation, acid hydrolysis (autoclave), neutralisation and pH adjustment to 4.5 ± 0.1, filtration and dilution to fit calibration range.
• Reagents & standards: Defined reagents (sulfuric acid, NaOH, basal media), pyridoxine hydrochloride standard and calibration solutions (example concentrations 5, 10, 20 ng/ml), and grade‑1 water per EN ISO 3696.
• Test organism & maintenance: Use and weekly maintenance of Saccharomyces uvarum (ATCC 9080) and preparation of working inoculum.
• Assay formats: Tube and microtitre‑plate formats described; example volumes and calibration layouts provided. Incubation at 30 °C (≈18–24 h) and optical density measurement in the visible region (≈550–600 nm).
• Validation & quality: Annex A provides calibration behaviour; Annex B contains inter‑laboratory precision data. Criteria for acceptance of data and reporting requirements are specified.

Applications and Users

• Who uses it: Food testing laboratories, national food control authorities, food manufacturers, nutritional labs, and research institutions performing quantitative vitamin B6 analysis in foods.
• Why use it: Standardized MBA enables reliable determination of total vitamin B6 across diverse food matrices where chromatographic separation may not be required or feasible, supports compliance, nutritional labelling and research.
• Limitations: Not suitable for samples with high antibiotic content or strong assay inhibitors; differences in response between vitamers (pyridoxine vs pyridoxamine/pyridoxal) can affect results - pyridoxine is commonly used as calibrant.

Related Standards

• EN ISO 3696:1995 - Water for analytical laboratory use (referenced for reagent water quality).
• Supersedes: ENV 14166:2001.
• Prepared by CEN/TC 275 (Food analysis - Horizontal methods); see Annexes A and B in EN 14166:2009 for calibration lines and precision data.

Standards Content (Sample)

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

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

Vitamin B6 calibration lines obtained by MBA using the test organism Saccharomyces uvarum . 12Annex B (informative)
Precision data . 13Bibliography . 14 SIST EN 14166:2009

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

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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