0.45 μm membrane filter

To determine the antimicrobial activity of LAB cultured in MRS or juice, spot‐on‐the‐lawn assays (Lee & Chang, 2016) or paper disk assays (Yang & Chang, 2010) were used. Briefly, LAB strain was cultivated in MRS broth at 30℃ for 24 hr or in juice at 15℃ for 0–5 days. The culture was then centrifuged (10,100 g, 15 min, 4℃) and the supernatant sterilized by passing through a 0.45 μm membrane filter (Sartorius) and used as culture filtrate or juice filtrate.
The prepared culture filtrate or juice filtrate was used for the antimicrobial activity assay. Plates were prepared by adding fungi (6.0 log spore per 20 ml MEA or PDA) to 1.5% bacto agar (Duchefa) for antifungal assays or by spreading the bacteria (6.0 log CFU/mL) onto MRS, LB, TSB, or NB + 2% NaCl agar for antibacterial assays (Lee & Chang, 2016; Yang & Chang, 2010). For the paper disk assay, paper disks (diameter 8 mm; Advantec, Tokyo, Japan) on MRS plates were spotted with 100 µl of sample. The plates were then incubated at 30℃ for 24 hr and examined for inhibition zones. For the spot‐on‐the‐lawn assay, 10 µl of sample was spotted onto the sensitive mold and bacterial plates. Antimicrobial activity, which was defined as the reciprocal of the highest dilution at which microbial growth was inhibited, was expressed as arbitrary units (AU) per milliliter.

HPLC-based fingerprinting was accomplished using two-dimensional HPLC (Agilent HP1200, Agilent Technologies, San Jose, CA, USA). Ephedrine (Sigma-Aldrich, St. Louis, MO, USA), as a principal component of Es, was used as a standard. Briefly, 100 μg of water extract of Es, 10 μg of Ephedrine and 200 mg of rat fecal samples from the end of microbiota preparation experiment were dissolved in 1 mL 50% of methanol separately. After sonication and centrifuging, the supernatant of samples were filtrated by 0.45 μm membrane filter (Sartorius AG, Goettingen, Germany) and prepared for HPLC analysis. The Agilent HPLC system consisted of a quaternary pump, on-line solvent degasser, Agilent sample manager/column heater module, and Agilent UV detector. An Agilent Eclipse XDB-C18 (5 mm, 4.6 mm × 250 mm) column was used and the compounds were eluted with solvents composed of acetonitrile: water: phosphoric acid (40:60:0.1 v/v/v) containing sodium lauryl sulfate at a concentration of 5 g/L as an ion-pairing agent. Peak identity of Ephedrine in the samples was confirmed by comparison of spectrum and retention time. HP1200 ChemStation software (Agilent Technologies, Santa Clara, CA, USA) was applied and all chromatograms were obtained using a wavelength of 215 nm.

S. pneumoniae was diluted with half saline until the turbidity reached 0.5 using the McFarland standard, inoculated in 19 volumes of Todd-Hewitt broth (Difco) and then incubated on an orbital shaker (150 rpm) at 37 °C in a 5 % CO₂ incubator for 24 h. The culture supernatants were centrifuged, passed through a 0.45 μm membrane filter (Sartorius, Göttingen, Germany) and stored at −80 °C until use. Todd-Hewitt broth incubated without S. pneumoniae was used as a control.