Sample preparation for the metabolomics investigation of poly-gamma-glutamate-producing Bacillus licheniformis by GC–MS

• Perchloric acid is more suitable than methanol as a quencher for γ-PGA sample.
• Cold methanol method could extract more intracellular metabolites.
• Metabolite could cover the relational pathway of γ-PGA synthesis.
• This method could be used in other high viscous samples, like xanthan gum.

Metabolomics aims to analyze global intracellular metabolites of organisms. To study the intracellular metabolites of poly-γ-glutamate-producing Bacillus licheniformis, the quenching and extraction conditions were assessed and optimized. It indicated that perchloric acid was an appropriate quenching solution for B. licheniformis by measuring livability of cells, leakage of ATP, energy charge and intracellular metabolites. 0.85% NaCl was an appropriate washing solvent for a sample because it did not lead to serious leakage and would not affect operation of GC–MS. Among the four different extraction methods (cold pure methanol, PM; hot ethanol/water (75:25 v/v), HE; cold methanol/water (50:50 v/v), MW; and cold chloroform/methanol/water (50:25:25 v/v/v), CMW), HE was superior to others on the basis of the energy charge and intracellular metabolites, which could effectively inhibit enzymatic activities and extract more intracellular metabolites. The method could obtain some metabolites which were involved in the most important synthesis pathways of poly-γ-glutamate, including glycolysis, citric acid cycle and glutamate metabolism. It is the first evaluation of a sample preparation of poly-γ-glutamate-producing bacteria, which might be used as one model for the preparation of polymer-producing samples for metabolomics analysis.