Heto lyolab 3000

The parts of baobab fruit used in this study were pulp, seeds, fibrous filaments, and shell (Figure 2). They were obtained from one batch of fruits and were supplied by ARWA Foodtech AB (Lund, Sweden).
The seeds were frozen, freeze-dried by a Heto LyoLab 3000 (Thermo scientific Heto, Denmark), and then ground using a GRINDOMIX 2000 (Retsch Italia, Verdere Scientific S.r.l, Bergamo, Italy). Ground seeds were defatted with n-hexane (20 mL per g seeds), and defatted seeds were used for dietary-fiber analysis. Seeds were also extracted to obtain oil [41 (link)]. Each sample (5 g) was weighed in triplicate and placed in a Falcon tube with 20 mL of a chloroform:methanol (2:1) solution. The mixture was shaken on a tilting laboratory shaker for 20 min and then centrifuged for 10 min. The supernatant was removed and placed into a separating funnel; the extraction was repeated four times. Thereafter, 40 mL of a 0.5% sodium chloride solution was added to the separating funnel, and a vigorous shaking for 1 min was applied. After 5 min, the chloroform phase was collected in a Falcon tube, 15 g of sodium anhydrous sulfate was added, and the mixture was filtered through a filter paper. The lipid extract was obtained by evaporation of the chloroform using a Rotavapor. From 5 g of dry sample, we obtained 0.79 g of oil with a total extraction yield of 16%.

Surface sterilized naïve and heat-killed pathogen challenged termite workers (24 g each) were suspended, separately, in 120 ml of 20 mM Tris-HCl, 20 mM NaCl (pH = 7.5) buffer and homogenized as previously described [12 ]. The crude CFE was quickly frozen in liquid nitrogen and lyophilized (Heto Lyolab 3000, Thermo Fisher Scientific, Pittsburgh, PA) at -57°C overnight before being dissolved in MQ water to achieve the final concentration of approximately 20 mg/ml protein concentration as determined by the Bradford assay (Bio-Rad, Hercules, CA) [21 (link)].
Additionally, a sample of each crude CFE (15 ml) was sequentially size fractionated with Microsep^™ Advance Centrifugal Devices (Pall Corporation, Port Washington, NY) with the molecular weight cut-offs (MWCO) of 100K, 30K, 10K, and 3K. This separated the CFE into five fractions containing proteins with approximate molecular weight of >300 kDa, 90–180 kDa, 30–90 kDa, 10–20 kDa, and <10 kDa, respectively. The fractionated solutions were lyophilized and dissolved in MQ water to achieve the final protein concentration of approximately 20 mg/ml to match that of the crude extract. All samples were stored at -80°C until the antibacterial assays.