Cell culture and fermentation
Bacteria were cultured in liquid Gause media (LGM) with an adjusted pH of 7.3^{12 .} LGM media consists of soluble starch 20.0 g, KNO₃ 1.0 g, NaCl 0.5 g, MgSO₄ x 7 H₂O 0.5 g, K₂HPO₄ 0.5 g, FeSO₄ x 7 H₂O 10.0 mg, dissolved in 1000 mL distilled water. Culture supernatant for extraction was prepared by scaling up cultures. Initially, a 10 mL test tube of LGM was inoculated with a single loop inoculum. After 24 h incubation, the 10 mL starter culture was used to inoculate 90 mL of LGM. This culture was incubated at room temperature for 10 days. Supernatant was obtained by centrifuging the culture for 30 min at 6000 rpm. Crude supernatant was filtered through filter paper and extracted with a 1:1 methanol-ethyl acetate solvent mixture ¹³. The solvent was then evaporated using a rotary evaporator, yielding a paste that was lyophilized to form a powder.

Qualitative metabolites screening
Secondary metabolites content was determined by analyzing samples of the powdered extract. Testing was performed using a previously described qualitative approach¹⁴. Phenols, alkaloids, flavonoids, saponins, triterpenoids, anthraquinone glycosides, and tannins were prominent among identified compounds.

Infrared Spectroscopy
Infrared (IR) spectra of the crude extract were obtained using an Agilent Cary 630 spectrometer. Infrared spectra with wave numbers ranging from 400 to 4000 cm^-1 were obtained. Resolution was reported in two sizes per wave number.

In vitro antiplasmodial testing procedure
Antimalarial activity testing was performed in vitro¹⁵. This study used Plasmodium falciparum strain 3D7, which chloroquine-sensitive. One mg of sample extract was diluted in 100 mL of DMSO (stock solution, concentration 1.000 mg/mL). Serial dilution were made from the stock solution. In this study, synchronous parasites (ring stage) approximating 1% parasitemia were used. Fresh type O-positive human erythrocytes were used to maintain cultures, which were suspended at 4 percent hematocrit in complete medium¹⁶. Parasites were added to 96-well plates using a high-throughput liquid handler. Two microliters of various dilutions of extract were then added. Final extract concentrations were 100, 10, 1, 0.1, and 0.01 μg/mL. Plates were incubated under controlled gas condition (O₂ 5%, CO₂ 5%, N₂ 90%). Plates were incubated for 48 h at 37°C, then erythrocyte slides prepared using 20% Giemsa staining.

Data were analyzed by counting the number of infected erythrocytes per 1000 total erythrocytes microscopy. These data were used to calculate growth and inhibition percentages. The following formula was used to determine the growth percentage:

% growth = % parasitemia –D₀         …(1)

Where D₀ represents initial 1% parasitemia prior to incubation (%)
Inhibition is expressed as a percentage, and calculated using the formula below;

Percent inhibition =100 % – ((Xu/ Xk) x 100%         …(2)

Description
Xu = % growth in the presence of extract
Xk = % growth in the negative absence of extract

Extract concentration that inhibited parasite growth by up to 50% (EC₅₀), was calculated using the two formulae above. The inhibitory concentration (IC₅₀) was determined using probit analysis and inhibition percentage results.