ENV 14166:2001

Foodstuffs - Determination of vitamin B6 by microbiological assay

This European Prestandard 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.

Lebensmittel - Mikrobiologische Bestimmung von Vitamin B6

Diese Europäische Vornorm 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 üblicher-weise 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.

Produits alimentaires - Dosage de la vitamine B6 par essai microbiologique

Živila - Določevanje vitamina B6 z mikrobiološko analizo

General Information

• Status: Withdrawn
• Publication Date: 20-Nov-2001
• Withdrawal Date: 26-May-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: 9960 - Withdrawal effective - Withdrawal
• Start Date: 27-May-2009
• Completion Date: 27-May-2009
• Ref Project: SIST ENV 14166:2002 - Foodstuffs - Determination of vitamin B6 by microbiological assay

Relations

• Replaced By: EN 14166:2009 - Foodstuffs - Determination of vitamin B6 by microbiological assay
• Effective Date: 22-Dec-2008

Standards Content (Sample)

SLOVENSKI STANDARD
SIST ENV 14166:2002
01-junij-2002
äLYLOD'RORþHYDQMHYLWDPLQD%]PLNURELRORãNRDQDOL]R
Foodstuffs - Determination of vitamin B6 by microbiological assay
Lebensmittel - Mikrobiologische Bestimmung von Vitamin B6
Produits alimentaires - Dosage de la vitamine B6 par essai microbiologique
Ta slovenski standard je istoveten z: ENV 14166:2001
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST ENV 14166:2002 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST ENV 14166:2002

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SIST ENV 14166:2002
EUROPEAN PRESTANDARD
ENV 14166
PRÉNORME EUROPÉENNE
EUROPÄISCHE VORNORM
November 2001
ICS 07.100.30
English version
Foodstuffs - Determination of vitamin B6 by microbiological
assay
Produits alimentaires - Détermination de la vitamine B6 par Lebensmittel - Mikrobiologische Bestimmung von Vitamin
essai microbiologique B6
This European Prestandard (ENV) was approved by CEN on 29 September 2001 as a prospective standard for provisional application.
The period of validity of this ENV is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the ENV can be converted into a European Standard.
CEN members are required to announce the existence of this ENV in the same way as for an EN and to make the ENV available promptly
at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the ENV) until the final
decision about the possible conversion of the ENV into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2001 CEN All rights of exploitation in any form and by any means reserved Ref. No. ENV 14166:2001 E
worldwide for CEN national Members.

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SIST ENV 14166:2002
ENV 14166:2001 (E)
Foreword
This European Prestandard has been prepared by Technical Committee CEN /TC 275, "Food analysis - Horizontal
methods", the secretariat of which is held by DIN.
Annexes A and B are informative.
This standard includes a Bibliography.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following coun-
tries are bound to announce this European Prestandard: Austria, Belgium, Czech Republic, Denmark, Finland,
France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden,
Switzerland and the United Kingdom.
1 Scope
This European Prestandard specifies a method for the determination of total vitamin B in foodstuffs by microbio-
6
logical assay (MBA). Vitamin B is determined as the mass fraction of pyridoxine, pyridoxal and pyridoxamine, in-
6
cluding their phosphorylated or glycosylated derivatives. It is usually expressed as milligram vitamin B per 100 g of
6
foodstuff. The method is applicable to samples that can be rendered homogeneous and do not contain high con-

centrations of antibiotics or other interfering substances.
2 Normative references
This European Prestandard 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 listed hereafter. For
dated references, subsequent amendments to or revisions of any of these publications apply to this European
Prestandard only when incorporated in it by amendment or revision. For undated references the latest edition of the
publication referred to applies (including amendments).
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 B vitamers from proteins and carbohydrates in the sample and hydrolyses the phosphates to the free
6

vitamers. The total Vitamin B content in the sample extract is then determined by comparing the growth response
6
of the assay test organism against growth obtained from appropriate standards, 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 as defined in EN ISO 3696:1995. The water used for reagent preparation shall be glass distilled. Once dis-
tilled, water shall be used within five days or discarded.
4.2 Chemicals and solutions
4.2.1 Sulfuric acid solution, substance concentration c (H S0 ) = 0,22 mol/l
2 4
4.2.2 Sodium hydroxide solution, c (NaOH) = 4 mol/l.
2

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SIST ENV 14166:2002
ENV 14166:2001 (E)
1)
4.2.3 Wort agar, (Difco 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)
4.2.4 Basal medium (Difco, Pyridoxine Y assay medium 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 pyri-
doxal. 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 auto-
clave 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, Sacchromyces 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 suit-
able 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 solution (4.2.8), centrifuging between washes. Re-suspend the cells in 50 ml of ster-
ile saline solution.
Using a sterile loop, transfer cells from this suspension onto three agar slopes (4.2.3) in a cross pattern and incu-
bate 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 fridge. 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.

1) This information is given for the convenience of users of this Prestandard 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.
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SIST ENV 14166:2002
ENV 14166:2001 (E)
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. Wash cells with 2 x 10 ml inoculum rinse (4.2.6) dis-
carding the supernatant each time. Re-suspend cells in a third 10 ml portion of inoculum rinse. This is used for the
assay inoculum.
4.4 Standard substances
4.4.1 Pyridoxine hydrochloride, w (C H NO HCl)
98 %
8 11 3.
4.4.2 Pyridoxal hydrochloride, w (C H NO .HCl)
98 %
8 9 3
4.4.3 Pyridoxamine dihydrochloride, w (C H N O .2HCl)
98 %
8 12 2 2
4.5 Stock solutions
4.5.1 Pyridoxine stock solution,
(C H NO )  200 μg/ml
8 11 3
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.
4.5.2 Pyridoxal stock solution,
(C H NO )  200 μg/ml
8 9 3
Prepare as for Pyridoxine using 121,8 mg to the nearest 0,1 mg pyridoxal hydrochloride (4.4.2). This solution is
stable for two weeks if kept refrigerated.

4.5.3 Pyridoxamine stock solution,
(C H N O )  200 μg/ml
8 12 2 2
Prepare as for Pyridoxine using 143,4 mg to the nearest 0,1 mg pyridoxamine dihydrochloride (4.4.3). This solution
is stable for two weeks if kept refrigerated.
4.6 Concentration test
Depending on the standard substance used, dilute 2 ml of the appropriate stock solution (4.5.1 to 4.5.3) to 20 ml
with 0,1 mol/l HCl. Measure the absorbance at the wavelengths shown in Table 1, against 0,1 mol/l HCl solution
(pH ~ 1).
Table 1 — Concentration test parameters [2]
Standard Solvent
 Molar Extinction Coeff. 
max
Pyridoxine·HCl 0, 1 mol/l HCl 290 8400
Pyridoxal·HCl 0,1mol/l HCl 288 9000
Pyridoxamine·2 HCl 0,1 mol/l HCl 292 8200
Calculate the mass concentration
, in μg/ml of the stock solution according to equation (1):
A  M V
w

 (1)

where:
A is the absorbance value of the solution at the relevant wavelength;
 is the appropriate molar extinction coefficient from Table 1;
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SIST ENV 14166:2002
ENV 14166:2001 (E)
M is the molecular weight of the standard substance, in gram per mol;
w
V is the dilution factor, i.e. 10.

4.7 Intermediate calibration solutions,
 400 ng/ml.
4.7.1 Pyridoxine intermediate calibration solution,
(C H NO )  400 ng/ml.
8 11 3
Dilute 2 ml of Pyridoxine stock solution (4.5.1) to 1000 ml with glass distilled water. Prepare on day of use.
4.7.2 Pyridoxal intermediate calibration solution,
(C H NO )  400 ng/ml.
8 9 3
Dilute 2 ml of Pyridoxal stock solution (4.5.2) to 1000 ml with glass distilled water. Prepare on day of use.
4.7.3 Pyridoxamine intermediate calibration solution,
(C H N O )  400 ng/ml.
8 12 2 2
Dilute 2 ml of Pyridoxamine stock solution (4.5.3) to 1000 ml with glass distilled water. Prepare on day of use.
4.8 Calibration solutions
4.8.1 Pyridoxine calibration solution 20,
(C H NO ) = 20 ng/ml
8 11 3
Dilute 5 ml intermediate calibration solution (4.7.1) to 100 ml with glass distilled water. Prepare on day of use.
4.8.2 Pyridoxine calibration solution 10,
(C H NO ) = 10 ng/ml
8 11 3
Dilute 25 ml of 20 ng/ml calibration solution (4.8.1) to 50 ml with glass distilled water. Prepare on day of use.
4.8.3 Pyridoxine calibration solution 5,
(C H NO ) = 5 ng/ml
8 11 3
Dilute 25 ml of 20 ng/ml calibration solution (4.8.1) to 100 ml with glass distilled water. Prepare on day of use.
4.8.4 Pyridoxal calibration solution 20,
(C H NO ) = 20 ng/ml
8 9 3
Dilute 5 ml intermediate calibration solution (4.7.2) to 100 ml with glass distilled water. Prepare on day of use.
4.8.5 Pyridoxal calibration solution 10,
(C H NO ) = 10 ng/ml
8 9 3
Dilute 25 ml of 20 ng/ml calibration solution (4.8.4) to 50 ml with glass distilled water. Prepare on day of use.
4.8.6 Pyridoxal calibration solution 5,
(C H NO ) = 5 ng/ml
8 9 3
Dilute 25 ml of 20 ng/ml calibration solution (4.8.4) to 100 ml with glass distilled water. Prepare on day of use.

4.8.7 Pyridoxamine calibration solution 40,
(C H N O ) = 40 ng/ml
8 12 2 2
Dilute 10 ml intermediate calibration solution (4.7.3) to 100 ml with glass distilled water. Prepare on day of use.

4.8.8 Pyridoxamine calibration solution 20,
(C H N O ) = 20 ng/ml
8 12 2 2

Dilute 25 ml of 40 ng/ml calibration solution (4.8.7) to 50 ml with glass distilled water. Prepare on day of use.

4.8.9 Pyridoxamine calibration solution 10,
(C H N O ) = 10 ng/ml
8 12 2 2
Dilute 25 ml of 40 ng/ml calibration solution (4.8.7) 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.
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SIST ENV 14166:2002
ENV 14166:2001 (E)
5 Apparatus
5.1 General
Usual laboratory equipment and glassware. All glassware shall be washed with detergents that will not st
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