Cs150fnx

Liquid medium, nutrient broth of 300 mL volume was used for the production of bacteriocins
¹⁰ . The Medium was sterilized in an autoclave at a pressure of 15 psi, at 121°C for 15 minutes. After one day at room temperature, the medium was inoculated with 5% of probiotic bacteria that had previously been incubated overnight at the optical density (OD) at 600nm ~ 0.1 (v/v) which was equivalent to 10
⁷ CFU/mL (OD measured with Thermo Scientific GENESYS 10S Uv-Visible)
^{11 (link)}. The inoculated media was then fermented in a shaking incubator at a speed of 150 rpm for ± 24 hours at 37 °C. After incubation, the fermented medium was cooled in a refrigerator at 5–10°C for ± one hour. The crude bacteriocin extract from the medium by centrifugation (Hitachi, Tokyo, Japan – CS150FNX) at 10,000 rpm for 10 min at 4°C. The supernatant was then separated by filtration through glasswool. The cell-free supernatant (extracted bacteriocins) produced, was tested for activity against pathogenic bacteria by using a disc diffusion method. Some of the supernatants was precipitated by the addition of 80% salt ammonium sulfate [(NH4) 2SO4], and then was tested for activity in the same manner, and finally was analyzed by using High-performance liquid chromatography (HPLC).

Gradients (2.0 ml) were prepared in a 7 × 20 mm centrifuge tube by successively layering with 0.4 ml of 25, 20, 15, 10, and 5% sucrose (W/V) in PBS. Lysates (postnuclear supernatant (PNS)) were prepared as described above. PNS (0.2 ml) were loaded on top of the gradients and centrifuged for 2 h at 55,000 r.p.m. at 4 °C (CS 150FNX, rotor: S55S, Hitachi, Tokyo, Japan). After centrifugation, eleven 0.2 ml fractions were collected from the top. Each fraction was analyzed by immunoblotting.

Bacteriocin was precipitated from the crude extract by the addition of 80% salt ammonium sulfate [(NH4) 2SO4] in a cold condition (temperatures of 5 °C to 10 °C) while stirring gently to achieve 80% saturation and was then left overnight. The precipitate was then separated from the filtrate by centrifugation (Hitachi – CS150FNX) in a cold state at 13,000 rpm for 10 minutes. After centrifugation, the precipitate was added with 0.05 M phosphate buffer solution at pH 7.0, and it was ready to be used for the bacteriocins activity test. The rest was put in microtubes and was stored in a freezer (-20 °C).

PMs were obtained as previously described [32 (link)]. Briefly, C. albicans CAF2-1 and KS028 cells were harvested (3 min, 6000 rpm) after 8, 14, or 24 h of culture (equal A₆₀₀ = 40 for all used strains) and lysis solution was added to the cells (1 M sorbitol, 0.1 M EDTA, 1% BME, 3 mg/mL zymolyase). After 30 min of incubation at 37 °C, cold H₂0_dd was added, and protoplasts were sonicated (5 s cycles, 2 min each) using an ultrasonic processor (Heilser UP50H). Next, lysates were centrifuged (10 min, 10,000 rpm, 4 °C), and the supernatant was then ultracentrifuged for 1 h, at 100,000 rpm, at 4 °C using micro ultracentrifuge CS150FNX (Hitachi; Tokyo, Japan). Pellets of PMs were resuspended in PBS, and mixture of CHCl₃–MeOH (1:2 v/v) was added. Finally, after continuous shaking for 24 h (4 °C), a phase containing CHCl₃ was added to glass vials and concentrated with nitrogen gas. The PM isolation was performed in 3 independent, biological replicates (n = 3).

The exosomes isolation was performed following standard protocol by ultracentrifugation.²⁵ Namely, LLC cells were cultured in 10 cm dishes, which were grown to 80% confluence, washed thrice with phosphate buffered saline (PBS), replaced with 10% exosome‐free serum and cultured for 24 hours. The supernatant was centrifuged at 200 g for 10 minutes to discard the pellet and then at 20 000 g for 20 minutes to discard the pellet. The obtained supernatant was centrifuged at 100 000 g for 70 minutes, and the obtained exosomes were further purified to remove the protein content in PBS implementing the same ultracentrifugation conditions. Ultracentrifugation was done using an ultracentrifuge (HITACHI; CS150FNX). Exosomes were observed using the transmission electron microscope HT7700 (HITACHI).

MB49 cells were washed twice with PBS when its confluence reach to 90%, following cultured with serum-free RPMI 1640 medium for 24 h. The culture supernatant was collected and centrifuged at 300g and 15,000g for 20 min respectively, which is to remove the suspended cells and cell debris. Then the collected supernatant was subjected to ultracentrifugation at 100,000g for 70 min at 4 °C (HITACHI ultracentrifuge, CS150FNX). The obtained exosome pellet was washed with PBS and concentrated after ultracentrifugation, which is to improve the purification of exosomes. The MB49-derived exosomes were verified by transmission electron microscopy (HT7800, HITACHI) and the expression of exosome specific markers CD63 and HSP90 by Western blot analysis.