The extraction of the PGRs was carried out adopting the
methodology of [21; 24]. The broth cultures were
centrifuged at 7,700 x g for 30 min. 100 mL of
supernatant was collected in a separatory funnel. The
pH of the supernatant was adjusted to 2.8 with 1N HCl
and extracted twice 300 mL with ethyl acetate. The
ethyl acetate and the aqueous fraction were separated.
The aqueous fraction was used for further extraction.
The pH of aqueous fraction was adjusted 7.0 with 1 N
NaOH and extracted with water saturated n-butanol.
The n-butanol and aqueous fractions were separated.
The above ethyl acetate and n-butanol fractions were
evaporated to dryness at 40-450 C. The residue thus
obtained was dissolved in absolute 2 mL of methanol
and further filtered through membrane filter (0.45