Biomedical Engineering Reference
In-Depth Information
Fig. 4
Schematic workfl ow for scale-up fermentation and bioassay-guided fractionation of selected extracts
process described earlier, these new extracts were arranged in
96-well plates called “refers” for re-fermentation. These plates
were tested again to confi rm the activity and to know if the micro-
organism produces the same active metabolite at 100 mL growth
condition. Active extracts identifi ed were cross-checked with those
listed in Natural Products Dictionary (distributed by CRC Press
Taylor and Francis Group:
http://www.crcpress.com/
), and the
ones already known were excluded from further analysis, a process
known as dereplication. Semi-preparative HPLC was made with
the active extracts into 80 fractions. This new plate (called HPF)
was tested again in the assay to detect the active fraction/s.
Active extracts with clear chemical profi les were cultured in a
larger scale using a 1 L fl ask. Different fractionation processes were
made by following the peak activity and the process end with the
purifi cation and the elucidation of the molecular structure of the
active compound using liquid chromatography-mass spectrometry
(LC-MS) and NMR (Fig.
4
).
3.3 Extraction
of Microbial Culture
Broths and Fractiona-
tion of Extracts
1. Extract liquid culture broths (1 L) by addition of an equal volume
of acetone (1 L) under continuous shaking at 220 rpm for 1 h.
2. Separate biomass by centrifugation, and concentrate the super-
natant to 1 L under a stream of nitrogen.
