Membrane filter

¹³C-labeling analysis was performed by the protocol reported before with a slight modification [59 (link)]. 15 ml cells of the later exponential phase at the OD₆₀₀ of 1.3 ± 0.1 were pre-cultured in minimal medium with ¹²C-methanol and 5 g/l ¹²C-labeled β-alanine, and then were rapidly passed through the membrane filter (0.22 μm, 47 mm, Sartorius). The filter was immediately removed and placed on an agar plate with ¹²C-methanol and ¹²C-labeled β-alanine for 30 min at 30 °C, then transferred to another agar plate with the same concentration of ¹²C methanol and ¹³C-labeled β-alanine for different times (i.e. 2, 5 and 12 h). Then the filter was immediately transferred to a 50 ml tube with liquid nitrogen for quenching. The sample was stored at − 80 °C freezer until it was ready for subsequent extractions. The metabolites were extracted and analyzed as the described above.

The processes of YFV production and purification are similar to those described by Pato and coworkers [10 (link)]. The working virus seed of YF 17D-213 infects the Vero cells (WHO-approved cell line) and undergoes bioreactor culturing. The supernatant of the culture medium containing the virus was treated with β-propriolactone for 24 h at room temperature to inactivate virus, and then concentrated 100 times by using a molecular weight cutoff of 300 kDa membrane filter (Sartorius, Germany), followed by downstream chromatography purification. Chromatography was performed using Akta pilot, operated with the UNICORN software (https://www.cytivalifesciences.com/en/us/shop/chromatography/software/unicorn-7-p-05649) (Cytiva, Marlborough, MA, USA). Ion exchange (Q Sepharose Fast Flow) and Capto Core700 (Cytiva, USA) resin prepacked in an XK50 Column were used for capturing and polishing, respectively, and the entire purification process. The purified sample was subsequently filtered using 0.45 μm filter units (Sartorius, Goettingen, Germany).

Conjugation was performed by membrane filter technique as described previously (22 ). Here, cefotaxime resistant STEC 1050 and 1152 strains were selected as donor strains, while plasmid-free nalidixic acid resistant mutant of E. coli ATCC 25922.1 (Nal
^r) selected as recipient strain (spontaneous Nal
^rcolonies were generated by gradient plate technique). Conjugation was carried out by mixing 2 ml of donor and 3 ml of recipient cells in a sterile Petri -plate, the mixture was passed through a conjugation assembly. The membrane filter (Sartorius, Germany) was then removed and placed on Tryptic Soy agar (TSA) plate at 37°C for 24 h. Mating was disrupted by vigorous vortexing of the membrane in 5 ml D/W and serially diluted (10
⁻²– 10
⁻⁸) with 5 ml normal saline. 200 μl of each dilution was spread on TSA medium selective for transconjugants (containing 25 μg of cefotaxime per ml and 100 μg of nalidixic acid per ml) and recipient cells (containing 100 μg of nalidixic acid per ml). Colonies that grew on selective agar plates were considered transconjugants. The frequency of conjugation was then calculated as number transconjugants colonies divided by number of recipient multiply dilution factor. Simultaneously, control for donor and recipient strains were carried out to check the presence of any spontaneously developed mutants.

Thin film hydration technique or TFH was used for the synthesis of niosome formulations^{51 (link),52} . At the first, stock solutions of each niosome component at concantration of 50 mg/mL in choloroform was prepared. Then, Span 60, Tween 60, ergosterol and Carum or TQ at molar ratios of 30:30:30:10 were add to a RB flasks (50 mL). Rotary evaporator was used for cholorophorm evaporation (Eppendorf, Germany) at reduced pressure, 60 °C for 60 minutes. Resulted lipid film was hydrated with 5 mL of phosphate-buffered saline (PBS) 7.4 solution at 60 °C for 45 minutes. The obtained milky solution containing MLV niosomes was further sonicated for 10 min by Ultrasonic Bath Sonicator (Tecno-Gaz Ultrasonic system; Tecna S.p.A, Bologna, Italy) to produce SUVs that have a more homogenoius sizes. For further homogeneous formulation, filtration by membrane filter was performed (0.22 uM, Sartorius AG, Göttingen, Germany).

The P. freudenreichii KCTC 1063 strain (Korean Collection for Type Cultures, Jeongeup-si, Korea) was grown in RCM at 37 °C for 36–40 h in an anaerobic chamber with a BD GasPak™ EZ container system (Becton Dickinson, Sparks, MD, USA). After 3 activation periods, live P. freudenreichii (LPF) was harvested by centrifugation at 3,000 × g for 5 min at 4 °C, washed twice with phosphate-buffered saline (PBS), and diluted to a concentration of 10⁸ colony-forming units (CFUs)/mL with PBS. The supernatant of the P. freudenreichii culture (SPFC) was adjusted to a pH 7.0 ± 0.1 using a small amount of 5 M NaOH and filtered with a membrane filter (0.22 μm pore size) (Sartorius, Goettingen, Germany). The concentration of propionate in SPFC was 32.7 mM and the chromatogram is shown in Supplementary Fig. S4. For an experimental control, RCM was incubated and processed in the same manner as SPFC to exclude the possible effects of any factors derived from RCM on the LS 174T cells.