High tetramethylpyrazine production by the endophytic bacterial Bacillus subtilis isolated from the traditional medicinal plant Ligusticum chuanxiong Hort.

Tetramethylpyrazine (TMP) with significant protective effects on cardiovascular is the active ingredient of traditional Chinese medicine Rhizoma Chuanxiong (RC). However, many studies have reported the low content of TMP in RC. The endophytes of medicinal plants have the biosynthetic potential to produce the same or similar active metabolites as the host, while few reports were conducted to explore the endophytic bacteria of Ligusticum chuanxiong Hort. and its productive capacity for the important ingredient TMP. The present paper focuses on the isolation and identification of TMP producing endophytic bacteria from RC. In this study, the endophytic bacteria were isolated from the rhizome of Ligusticum chuanxiong Hort. (Umbelliferae). Yeast extract peptone glucose medium (YP) was used for fermentation medium (37 °C, 220 rpm agitation, 144 h). GC and GC/MS were performed to determine and verify the product, the fermentation characteristics were investigated. Morphological observation, physiological and biochemical indexes combining with 16S rRNA sequence analysis were carried out to identify the endophytic bacteria. As a result, five strains of endophytic Bacillus subtilis were firstly isolated and identified from RC, named as LB3, LB3-2-1, LB6-2, LB4, LB5 respectively. All five strains of endophytic B. subtilis produced TMP, while LB5 had the highest production of 10.69 g/L at the 144 h fermentation. This work demonstrates the fact that the endophytic B. subtilis of RC can produce a high level of TMP, indicating the endophytic B. subtilis might play a role in the accumulation of TMP during the growth period of RC.