EN 14131:2003

Foodstuffs - Determination of folate by microbiological assay

This European Standard specifies a microbiological method for the determination of the total folate content of foodstuffs by turbidimetric detection of the growth of the microorganism Lactobacillus casei, subsp. rhamnosus (ATCC 7469).
The method allows for the determination of folates in foodstuffs, including naturally occurring folates and added folic acid (pteroylglutamic acid).

Lebensmittel - Mikrobiologische Bestimmung von Folat

Diese Europäische Norm legt ein mikrobiologisches Verfahren zur Bestimmung des Gesamtgehaltes an Folat in Lebensmitteln fest, wobei das Wachstums des Mikroorganismus Lactobacillus casei, subsp. rhamnosus (ATCC 7469) durch Trübungsmessung bestimmt wird.
Dieses Verfahren erlaubt die Bestimmung von Folat einschließlich natürlich vorkommender Folate und hinzugefügter Folsäure (Pteroylglutaminsäure) in

Produits alimentaires - Détermination des folates par essai microbiologique

La présente Norme européenne spécifie une méthode microbiologique permettant de déterminer la teneur totale en folates des produits alimentaires par mesure turbidimétrique de la croissance du microorganisme Lactobacillus casei, subsp. rhamnosus (ATCC 7469).
Cette méthode permet la détermination des folates dans les produits alimentaires, y compris les folates naturels et l'acide folique ajouté (acide ptéroylglutamique).

Živila - Določevanje folata z mikrobiološko metodo

General Information

• Status: Published
• Publication Date: 03-Jun-2003
• 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: 17-Sep-2024
• Completion Date: 14-Apr-2025
• Ref Project: SIST EN 14131:2003 - Foodstuffs - Determination of folate by microbiological assay

Overview - EN 14131:2003 (CEN)

EN 14131:2003 is a European Standard (CEN) that specifies a validated microbiological method for the determination of total folate in foodstuffs. The assay measures naturally occurring folates and added folic acid (pteroylglutamic acid) by turbidimetric detection of the growth response of the microorganism Lactobacillus casei subsp. rhamnosus (ATCC 7469). The standard (approved April 2003) is intended for routine food analysis laboratories and research users needing a standardized protocol for folate quantification.

Key topics and technical requirements

• Method principle: Extraction of folates from homogenized food samples, optional enzymatic deconjugation of folylpolyglutamates, dilution into a folate-free basal medium, and turbidimetric monitoring of L. casei growth compared to calibrated folic acid standards.
• Microorganism: Uses lyophilised Lactobacillus casei subsp. rhamnosus (ATCC 7469) as the folate-responsive test organism.
• Sample extraction: Typical sample mass corresponds to 2–2.5 g dry weight; extraction in phosphate buffer with ascorbate (pH 6.1) and heat treatment (100–121 °C, 15 min) to release folates.
• Optional enzyme treatment: Annex A describes additional enzyme steps to convert folylpolyglutamates to mono/di-glutamates when required.
• Calibration and standards: Folic acid stock standard (example: 100 µg/ml) is standardized by UV absorbance; working standards (e.g., 1 µg/ml and diluted 100 ng/ml) are used to generate calibration curves.
• Assay formats: Supports test-tube assays or microplate (96-well) formats with turbidimetric or nephelometric readout; incubation typically at 37 °C.
• Quality & reporting: Method includes procedures for duplicate determinations, precision assessment (interlaboratory results in Annex B), and required test report elements.
• Reagents & controls: Details on buffers (phosphate buffers at multiple pH values with ascorbate), enzyme preparations, inoculum preparation, and cryoprotected stocks are specified. EN ISO 3696 (water for analytical use) is a normative reference.

Practical applications and users

Who uses EN 14131:2003:

• Food testing laboratories performing folate analysis for nutritional labeling and compliance.
• Food manufacturers and ingredient suppliers (cereals, beverages, dairy, fortified foods) verifying added folic acid and natural folate content.
• Regulatory agencies and public health laboratories conducting surveillance of dietary folate.
• Research groups studying folate bioavailability, food composition databases, and fortification programs.

Benefits:

• Standardized, reproducible microbiological assay for total folate.
• Applicable to a wide range of foodstuffs, including fortified products.
• Adaptable to semi-automated and microplate workflows for higher throughput.

Related standards

• EN ISO 3696 - Water for analytical laboratory use (referenced for reagent-grade water).
• CEN/TC 275 - Technical committee for food analysis horizontal methods (development context).

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Mikrobiologische Bestimmung von FolatProduits alimentaires - Détermination des folates par essai microbiologiqueFoodstuffs - Determination of folate by microbiological assay07.100.30Mikrobiologija živilFood microbiologyICS:Ta slovenski standard je istoveten z:EN 14131:2003SIST EN 14131:2003en01-november-2003SIST EN 14131:2003SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14131June 2003ICS 07.100.30English versionFoodstuffs - Determination of folate by microbiological assayProduits alimentaires - Détermination des folates par essaimicrobiologiqueLebensmittel - Mikrobiologische Bestimmung von FolatThis European Standard was approved by CEN on 21 April 2003.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 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 Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14131:2003 ESIST EN 14131:2003

Optional enzyme treatment.16Annex B (informative)
Results of interlaboratory tests.19Bibliography.20SIST EN 14131:2003

1)Bacto Folic Acid Casei Medium is the trade name of a product supplied by Difco. This information is given for theconvenience 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 14131:2003

2)Distributors include National Collection of Industrial and Marine Bacteria Ltd (Aberdeen, UK) and Culture Collection,University of Göteborg (Gothenburg, Sweden). This information is given for the convenience of users of this EuropeanStandard and does not constitute an endorsement by CEN of the distributors named.SIST EN 14131:2003

or water-bath for incubation at (+37 ±0,2) ºC5.4 Spectrometer/nephelometer,
for measurement of turbidity of folate extracts after incubation in testtubes5.5 Test tube racks with cover,
to hold test tubes during heating and incubation5.6 Dialysis tube (optional),
with molecular mass cut-off of 12000 to 14000, for the preparation of ratplasma conjugase (Annex A).5.7 Liquid-handling system (optional),
for automated assay-tube reading5.8 Microplate reader (optional),
for measurement of turbidity after incubation in microplates5.9 Flat-bottomed 96-well microplates, sterile (optional)SIST EN 14131:2003

ml water and store in ice.Carefully mix in a 10-ml volumetric flask: 1 ml of sample extract (6.3.2), 3,5 ml of phosphate buffer withascorbate pH 4,5 (4.3.4) and 0,5 ml of reconstituted
37 °C for 3 h.Inactivate enzyme by heating at 100 °C for 3 min. Cool, and add to volume with phosphate buffer withascorbate pH 4,5 (4.3.4). Centrifuge (1000 g, 10 min) to remove precipitated protein. Store supernatant at – 18 °C for subsequent analysis.6.4 Calibration6.4.1 Folic acid stock solution (100 µg/ml)Accurately weigh 50 mg of folic acid (4.2.15) that has been dried to constant weight and dissolve in phosphatebuffer (4.3.2) in a 500-ml volumetric flask. Dilute to volume with phosphate buffer. Top with toluene to keepsurface covered.6.4.2 Concentration test of folic acid stock solutionTransfer 10 ml of stock solution (6.4.1) to a 100-ml volumetric flask and dilute to volume with phosphate bufferpH 7,0 (4.3.2). Measure the absorbance of the solution at 282 nm using phosphate buffer as a blank.Calculate the folic acid concentration () in gram per litre according equation (1):SIST EN 14131:2003
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