SIST EN 14131:2003

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



SIST EN 14131:2003



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



EN 14131:2003 (E)2ContentspageForeword.31Scope.32Normative references.33Principle.34Reagents.45Apparatus.86Procedure.97Calculation.138Precision.149Test report.14Annex A (informative)
Optional enzyme treatment.16Annex B (informative)
Results of interlaboratory tests.19Bibliography.20SIST EN 14131:2003



EN 14131:2003 (E)3ForewordThis document (EN 14131:2003) 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 identicaltext or by endorsement, at the latest by December 2003, and conflicting national standards shall be withdrawnat the latest by December 2003.Annexes A and B are informative.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark,Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands,Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and the United Kingdom.1 ScopeThis European Standard specifies a microbiological method for the determination of the total folate content offoodstuffs 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 andadded folic acid (pteroylglutamic acid).2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications.These normative references are cited at the appropriate places in the text and the publications are listedhereafter. For dated references, subsequent amendments to or revisions of any of these publications apply tothis European Standard only when incorporated in it by amendment or revision. For undated references thelatest edition of the publication referred to applies (including amendments).EN ISO 3696, Water for analytical laboratory use — Specifications and test methods (ISO 3696:1987).3 Principletreatment may be used to further digest the food matrix. Naturally occurring folylpolyglutamates are[1] to folylmono- or folyldi-glutamates.Extracted folates are diluted with basal medium containing all required growth nutrients except folate. Thegrowth response of Lactobacillus casei, subsp. rhamnosus (ATCC 7469) to extracted folates is followedturbidimetrically and is compared to the growth response to calibrant solutions with known concentration.The method allows for the optional use of a semi-automated liquid-handling system and of a microplate or testtubes for incubation of the microorganism.SIST EN 14131:2003



EN 14131:2003 (E)44 Reagents4.1 GeneralDuring analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of atleast grade 1 as defined in EN ISO 3696. The water used for reagent preparation shall be glass distilled.4.2 Solvents and chemicals4.2.1 Glycerol, w(C3H8O3) = 80 %Mix 120 ml of glycerol with 30 ml of glass-distilled water.4.2.2 1-Octanol, C8H18O4.2.3 Toluene, C7H84.2.4 2-Mercaptoethanol, c(C2H6OS) = 0,1 mol/lAdd 70 µl of 2-mercaptoethanol to 10 ml of water.4.2.5 Sodium ascorbate, C6H7O6NaSodium ascorbate is used as a reagent in several solutions specified in this draft standard. Ascorbic acid mayequally well be used, but procedures for pH adjustment may need to be modified.4.2.6 Hydrochloric acid, c(HCl) = 1 mol/l4.2.7 Sodium hydroxide, w(NaOH) = 40 %Dissolve 400 g of sodium hydroxide in water and dilute to 1 l.4.2.8 Ammonium sulfate, H8N2O4S4.2.9 Sodium phosphate, monobasic, anhydrous, NaH2PO4The amounts of monobasic sodium phosphate used for buffer preparation (4.3) have been calculated for theanhydrous substance. The mono- or dihydrated substance may also be used, with the procedures adjustedaccordingly.4.2.10 2-(N-Cyclohexylamino)ethanesulfonic acid (CHES), C8H17NO3S4.2.11 N-(2-Hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), C8H18N2O4S4.2.12 Carbon powder, acid-washed4.2.13 Saline, sterileDissolve 9 g of sodium chloride in 1000 ml of water. Dispense 10-ml portions into 20 mm x 150-mm test tubes.Cap tubes and heat at + 121 °C for 15 min. Cool and store refrigerated.SIST EN 14131:2003



EN 14131:2003 (E)54.2.14 Folic acid-free basal medium solution, double strengthFor each 100 ml needed, suspend the recommended amount of basal medium (Bacto Folic Acid CaseiMedium or equivalent1)) in 100 ml of glass-distilled water. Add 0,050 g of sodium ascorbate (4.2.5) and heat toboiling for 1 min to 2 min. Allow cooling to room temperature and adjust pH to 6,1 ± 0,1.4.2.15 Folic acid standard substanceFolic acid can be obtained from various suppliers and may contain up to 8 % water. The purity of the folic acidstandard may vary and it is therefore necessary to determine the concentration of the calibration solution byUV absorption measurement (see procedure for standardisation in 6.4.2).4.3 Buffers4.3.1 Phosphate buffer, pH 5,0 (c = 0,002 mol/l)Dissolve 0,24 g of sodium phosphate, monobasic (4.2.9) in 900 ml of water. Adjust pH to 5,0 ± 0,1 withsodium hydroxide (4.2.7) and dilute to 1000 ml with water.4.3.2 Phosphate buffer, pH 7,0 (c = 0,1 mol/l)Dissolve 12,0 g of sodium phosphate, monobasic (4.2.9) in 900 ml of water. Adjust pH to 7,0 ± 0,1 withsodium hydroxide (4.2.7) and dilute to 1000 ml with water.4.3.3 Phosphate buffer, pH 5,0 (c = 0,1 mol/l) with 2-mercaptoethanol (c = 10 mmol/l)Dissolve 12,0 g of sodium phosphate, monobasic (4.2.9) in 900 ml of water. Adjust pH to 5,0 ± 0,1, add0,70 ml of 2-mercaptoethanol (4.2.4) and dilute to 1000 ml with water.4.3.4 Phosphate buffer, pH 4,5 (c = 0,1 mol/l) with ascorbate (1 %)Dissolve 12,0 g of sodium phosphate, monobasic (4.2.9) and 10 g of sodium ascorbate (4.2.5) in 900 ml ofwater. Adjust pH to 4,5 ± 0,1 with sodium hydroxide (4.2.7) and dilute to 1000 ml with water. Prepare fresh onday of use.4.3.5 Phosphate buffer, pH 6,1 (c = 0,1 mol/l) with ascorbate (1 %)Dissolve 12,0 g of sodium phosphate, monobasic (4.2.9) and 10 g of sodium ascorbate (4.2.5) in 900 ml ofwater. Adjust pH to 6,1 ± 0,1 with sodium hydroxide (4.2.7) and dilute to 1000 ml with water. Prepare fresh onday of use.4.3.6 Phosphate buffer, pH 7,8 (c = 0,1 mol/l) with ascorbate (1 %)Dissolve 12,0 g of sodium phosphate, monobasic (4.2.9) and 10 g of sodium ascorbate (4.2.5) in 900 ml ofwater. Adjust pH to 7,8 ± 0,1 with sodium hydroxide (4.2.7) and dilute to 1000 ml with water. Prepare fresh onday of use.
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



EN 14131:2003 (E)64.3.7 CHES/HEPES buffer, pH 7,85 (c = 0,05 mol/l) with ascorbate and 2-mercaptoethanol (4.2.4) for thedialysis of rat blood plasma.Dissolve 23,8 g of HEPES (4.2.11), 20,7 g of CHES (4.2.10), 40 g of sodium ascorbate (4.2.5) and 1,4 ml of2-mercaptoethanol (4.2.4) in 1900 ml of water. Adjust pH to 7,85 ± 0,1 with sodium hydroxide (4.2.7) anddilute to 2000 ml with water. Add 4 g of acid-washed carbon powder (4.2.12). Prepare fresh on day of use.4.4 Enzymes4.4.1 Additional enzyme treatment (optional)Procedures for additional enzyme treatment are further discussed in Annex A.4.4.2 4.4.2.1 General0 from one of several sources. An4.4.2.2). A. Theappropriateness of the chosen enzyme preparation shall be checked with a suitable technique.NOTEA yeast powder, lyophilised pig’s liver (e.g. BCR CRM 487 [2]), or to a limited extent pteroyl-triglutamic acid canbe appropriate samples to use for the checking of the enzyme preparation.4.4.2.2 [3]Homogenise 250 g of fresh hog kidney at + 2 ºC in 750 ml of phosphate buffer with 2-mercaptoethanol (4.3.3).Centrifuge at + 2 ºC (18000 g, 20 min). Incubate supernatant at + 50 ºC for 2 h with gentle agitation andrepeat centrifugation. Fractionate the supernatant by precipitation with saturated ammonium sulfate (4.2.8).Collect the fraction precipitated between 50 % and 75 % saturation with ammonium sulfate. Suspendprecipitate in a minimal volume of phosphate buffer (4.3.1). Dialyse against the same buffer 2 x 24 h andcentrifuge (18000 g, 20 min). Transfer 1-ml aliquots to vials and lyophilise.4.5 Inoculum4.5.1 Test organismLyophilised culture of Lactobacillus casei subsp. rhamnosus (ATCC 7469)2).4.5.2 Culture mediumDilute 50 ml of double-strength folic acid-free basal medium solution (4.2.14) to 100 ml with glass-distilledwater. Add 0,5 ml of diluted folic acid stock solution (6.4.4), mix and sterile filter or heat at + 121 ºC for 15 minand rapidly cool to room temperature.4.5.3 Cryoprotected inoculumAseptically, add 1 ml of the prepared culture medium (4.5.2) to the lyophilised culture (4.5.1) and transfer0,15 ml of the resulting suspension to the culture medium according to 4.5.2. Incubate at + 37 ºC for 18 h.
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



EN 14131:2003 (E)7Heat 150 ml of glycerol (4.2.1) at + 121 ºC for 15 min and cool in ice bath. Cool incubated bacterial culture inice bath and add 100 ml sterilised and cooled glycerol. Mix gently. Dispense 2-ml aliquots into sterile vials.Store at – 20 ºC for up to three months or at – 70 ºC for up to six months.NOTEIt is essential to maintain aseptic conditions throughout the whole process.4.5.4 Working inoculum4.5.4.1 Working inoculum for tube culturesDilute 2 ml cryoprotected inoculum (4.5.3) to 50 ml with sterile saline (4.2.13). Vortex mix.4.5.4.2 Working inoculum for microplate cultures (optional)Add 5 ml of sterile saline (4.2.13) to 2 ml cryoprotected inoculum (4.5.3). Vortex mix.4.5.5 Inoculated folic acid-free basal medium solution, for microplate assay (optional).Add 1 µl working inoculum (4.5.4.2) per 1 ml folic acid-free basal medium solution (4.2.14). Mix thoroughly.5 ApparatusUsual laboratory apparatus, glassware and, in particular, the following:5.1 Centrifuge, cooled, for preparation of hog kidney conjugase (4.4.2.2), suitable for 18 000 g.5.2 Heating device, Autoclave or equivalent5.3 Incubator,
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



EN 14131:2003 (E)86 Procedure6.1 GeneralFolates are sensitive to UV light and oxidation. Perform all operations away from natural and strongfluorescent light. Use amber glassware where possible.Carry out the determination in duplicate as single determinations on two different occasions.6.2 Preparation of test sampleThe test sample shall be homogeneous. Coarse material shall be rendered homogeneous using anappropriate mill and/or blender. Care shall be taken to avoid exposure to high temperatures during thisprocess. Homogenised test samples shall be stored in air-tight containers, free from exposure to light.6.3 Preparation of sample solution6.3.1 Generalamylase treatment to hydrolyse proteins and carbohydrates, thus facilitating folate extraction. The samplelpolyglutamates tomay be used provided that sufficient enzyme activity is ensured. Procedures for additional enzyme treatmentolase from other sources are further described in Annex A.6.3.2 ExtractionAccurately weigh a suitable amount of the test sample (corresponding to 2 g to 2,5 g dry weight) into a 100-mlvolumetric flask. Add 20 ml of phosphate buffer with ascorbate pH 6,1 (4.3.5) and mix thoroughly. Add 30 mlof water and 0,1 ml to 1 ml of 1-octanol (4.2.2). Cover flask and heat at 100 °C to 121 °C for 15 min. Cool, anddilute to 100 ml with phosphate buffer with ascorbate pH 6,1 (4.3.5).6.3.3
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



EN 14131:2003 (E)9VbMAwhere:Ais the absorbance value of the solution at 282 nm;Mis the molar mass of folic acid, i.e. 441,4 g mol-1;is the molar absorption coefficient of folic acid [4] at 282 nm, i.e. 27600 l mol-1 cm-1;bis the absorption path length in cm;Vis the dilution factor (if any).6.4.3 Folic acid standard solution (1 µg/ml)Add 5 ml of stock solution (6.4.1) to 475 ml with water and adjust pH to 7,5 with sodium hydroxide (4.2.7).Dilute to 500 ml with water. Prepare fresh on day of use. Further dilutions may be prepared to suit the assayformat used.6.4.4 Diluted folic acid standard solution (100 ng/ml) for use in inoculum.Add 10 ml of standard solution (6.4.3) to approximately 60 ml of water and adjust pH to 7,5 wi
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