Only 143 metabolites were detected following this secondary extraction, and the levels were reduced by an average of 81% (median reduction of 93%) compared to the level in the initial extract (Additional file 2), indicating near complete extraction using this method.
The factorial experimental design showed that microcrystalline cellulose PH 102 and corn starch were the best absorbents, and that drying in a fluidized bed was the best method, offering advantages in terms of physicochemical properties such as particle size, density, voluminosity, flowability and hygroscopicity, compared to the initial extract and to the other formulations.
Individual libraries (n = 9) were subsequently pooled according to their initial extract number (C11 3, C21 3 and C31 3) to form three final libraries labelled C1, C2 and C3, respectively.
We estimated the bacterial load of leaf litter samples by counting the number of colonies (Colony-Forming Unit) per initial gram of dry leaf matter (40 ml of initial extract corresponded to 200 mg of dry leaf litter; 200 µl plated on agar corresponded to 1 mg of dry leaf litter).
cPurification fold= Specific activity at given step/Specific activity of initial extract.
The negative control wells contained cells without virus or the initial extract dilution.
The presence of the redox agent dithiothreitol (DTT) in the initial extract prevents the formation of disulfide bridges.
For LC MS MS analysis the initial extract (3.2 mL for the API2000 and 0.48 mL for the API3000) was transferred to a disposable glass tube.
The dsDNA concentration of the initial extract of feed streams spanned a wide range (3 600 mg/L) and both the shake flask and extractions ranked combinations similarly.
The same initial extract (i.e. without any clean-up) can be used for LC MS MS analysis, after changing the solvent to methanol water.
Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract.
