After centrifugation, P. freudenreichii cells were washed and resuspended in 20 mM phosphate buffer (pH 7.0), then disrupted in a boiling water for 30 min to release intracellular vitamin B 12 and heme [22] . After removing cell debris by centrifugation at 10,000 × g for 10 min, the supernatant was used to analyze the concentration of vitamin B 12 and heme. HPLC analysis of vitamin B 12 was performed with a Hypersil GOLD C18 column (5 μm × 4.6 mm × 250 mm; Dikma) at 254 nm with the same HPLC system as described above [20] . The mobile phase consisted of 0.02 M sodium acetate buffer solution (pH 3.5, adjusted with acetic acid), and acetonitrile (gradient of 5%-24%, 0-30 min). The elution was carried out at 25 °C with a ow-rate of 1.0 mL/min. Commercially available vitamin B 12 was used as an external standard.
The concentration of heme was measured by colorimetry [23] . Commercially available hemin was used as external standard. First, hemin standard solutions (1-10 µg/mL) were prepared. Pyridine-NaOH (2 mL, 33% pyridine and 0.1 mol/L NaOH) solution was added to 1 mL of hemin standard solution. After mixing, 3 mg sodium sul te were added and reacted for 30 min to reduce the heme iron. A linear standard calibration curve was obtained using absorbance at 557 nm. When measuring a fermentation sample, there was no need to add sodium sul te.
The concentration of heme was measured by colorimetry [23] . Commercially available hemin was used as external standard. First, hemin standard solutions (1-10 µg/mL) were prepared. Pyridine-NaOH (2 mL, 33% pyridine and 0.1 mol/L NaOH) solution was added to 1 mL of hemin standard solution. After mixing, 3 mg sodium sul te were added and reacted for 30 min to reduce the heme iron. A linear standard calibration curve was obtained using absorbance at 557 nm. When measuring a fermentation sample, there was no need to add sodium sul te.