Determination of vitamin B6 by microbiological assay

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Golden standard

EN 14166:2009 Foodstuffs - Determination of vitamin B6 by microbiological assay


Method indicator

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The European Standard EN 14166:2009 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, pyridoxamine including their phosphorylated or glycosylated derivatives. The method can be used to determine vitamin B6 from samples that can be homogenised and do not contain high concentrations of interfering substances like antibiotics.


Sample pretreatments include acid hydrolysis where vitamin B6 vitamers are liberated from proteins and carbohydrates, and phosphates are hydrolysed to free vitamers. Method determines the total vitamin B6 content in the sample, and the determination is done by comparing the growth of the test organism in a sample to the growth of the test organism in a pyridoxine hydrochloride standard.

Key steps

Preparation of the sample solution

  • The sample must be homogenised properly. 
  • Sulfuric acid solution is added and the solution is autoclaved (121 °C, 5 h)
  • After autoclaving, sodium hydroxide solution is added and the pH is adjusted to 4,5.
  • The sample solution is filtered.

Microbiological assay

  • Different vitamin B6 vitamers show varying growth responses to the assay organism (Saccharomyces uvarum ATCC 9080).
    • The dose response of pyridoxine is the highest.
  • Usually pyridoxine (standard substance: pyridoxine hydrochloride) is used as a calibrant.
  • Test tubes or microtitre-plate may be used.
  • EN 14166:2009 describes different assay formats.
  • The test tubes are sterilised to prevent contamination and after that inoculated.
  • The test tubes are incubated in a water bath
    • 30 °C, 18-24 h
  •  The growth of the test organism is determined by measuring the optical density in the visible region of the spectrum.

Calibration and calculation

  • There are duplicates for all standards.
    • Usually pyridoxine hyrdochloride is used as a standard.
  • Uninoculated blank test tubes must be clear and inoculated blank test tubes may show only very small amount of turbidity.
  • Absorbances of the tubes are plotted against the concentrations of standard in the corresponding assay tubes.
  • The calibration line is drawn.
  • The amount of tested analyte is determined by comparing the absorbance of the test sample to the calibration line.
  • The mass fraction of vitamin B6 in mg/100 g is calculated.
  • The result of vitamin B6  in mg/100 g of the sample is calculated as a mean value of all dilution levels.
    • The pyridoxine hydrochloride is usually used for calibration, and in that case the result is expressed as the microbiological activity referred to the pyridoxine hydrochloride.


  • Laboratory glassware should be washed with detergents that will not stimulate or inhibit the growth of the test organism.
  • Usually pyridoxine is used as a calibrant but this may cause an underestimate when sample contains significant amount of pyridoxal and/or pyridoxamine.
  • Minimum criteria for acceptance of data:
    • inoculated blank solutions should not exceed the maximum turbidity set
    • the calibration solution with the highest concentration level must exceed the minimum turbidity set
    • variation between replicates and different dilution levels should not exceed the amount of variation accepted
    • suitable reference control samples should be analysed
  • Transfers of test organism onto agar maintenance medium should be done regularly to ensure the maintenance of the organism.

Criteria for analytical performance and analytical quality control

Certified Reference Materials/Standard Reference Material

EuroFIR assistance to this method/guidelines