Accelerated green process of tetramethylpyrazine production from glucose and diammonium phosphate

Background: 2,3,5,6-Tetramethylpyrazine (TMP) is a popular food flavour additive. This biologically active ingredient has additional potential dietotherapy functions, such as in cardiovascular and cerebrovascular health. Previous production methods from renewable materials suffer from low yields and efficiency which seriously hampers the scaling up of the laboratory processes. Results: We have found a novel temperature-controlled two-step preparation method of TMP with highly elevated production yield and efficiency. In the first step, 30.1 g L-1 of acetoin, the precursor of TMP, was biosynthesized in 29 h from 100 g L-1 of glucose by Bacillus subtilis CICC 10211 in a fermentor controlled at 37 degrees C, pH 7.0 +/- 0.2, 500 rpm of stirring, and 1.0 vvm of airflow. In the following step, the fermentation broth, supplemented with 67.7 g L-1 of diammonium phosphate, was transferred into another reactor controlled at 95 degrees C. After 2.5 h of spontaneous reaction, 8.34 g L-1 of TMP was obtained. A temporary by-product, which was identified as 2-(1-hydroxyethyl)-2,4,5-trimethyl-3-oxazoline, was formed but then decomposed rapidly under higher temperature. The novel method received consistent results when it was tested using Serratia marcescens CICC 10187 and Paenibacillus polymyxa CICC 23617, respectively. Crystallized TMP with high purity (99.9%) from the cooled reaction mixture was harvested by filtration. Conclusions: The novel method can be expanded for other acetoin-producing strains and it gives the highest product titre and production efficiency so far devised for TMP production from renewable materials. The complex components of the reaction mixture have no effect on the purity of the product obtained from the crystallization process.