2.2 GC-MS
To identify the hydrophilic components of yeast extract, non-targeted
GC-MS analyses were performed after trimethylsilylation according to a
previous report.[15] Each 5.0 g/L yeast extract
sample (E1, E2, E3, E4, M1, M2, M3, and M4) and mixed samples (E1-E4,
E2-E4, E3-E4, E4-M1, E4-M2, E4-M3, E1-M3, E2-M3, E3-M3, M3-M1, M3-M2,
and M3-M4) were prepared and autoclaved at 121°C for 20 min. The sample
(100 µL) was combined with 20 mg/mL ribitol (60 µL). Then, 900 µL of
water, methanol, and chloroform at a ratio of 1:2.5:1, respectively,
were added. After extraction with thorough mixing, the tubes were
centrifuged at 4°C for 5 min with 16,000×g . The top water phases
(600 µL) were transferred into new tubes, dried partly by a centrifuge
evaporator, and freeze-dried by a lyophilizer. Methoxyamine chloride (20
mg/mL in pyridine) was added to the lyophilized samples and incubated at
30°C for 90 min. After the incubation,N -methyl-N -(trimethylsilyl)trifluoroacetamide was added
and the mixture was incubated at 37°C for 30 min. The samples were then
introduced into the GC-MS system.
The Agilent GC-MS system, 7980B and 5977A MSD, was used with a HP-5 ms
UI column (30 m × ϕ 0.25 mm × firm thickness 0.25 µm). The instrument
conditions were set as described previously.[15]Peaks were obtained from total ion chromatograms using the decombolution
program in MassHunter software (Agilent Technology, CA, USA). The peak
area was normalized by the internal standard (ribitol) peak. Peak
annotation was performed with support from the NIST14 database.