Zircoprep mini

Three representative chicken cecum samples were selected at each time point, to exclude the samples with maximum and minimum bacterial counts, and subjected to 16S rRNA sequencing analysis. Aliquots of the BPW suspensions (1 ml) were centrifuged at 21,500 × g for 10 min at 4°C. The pellets were then resuspended in 400 μl of homogenization solution containing 2 μl of proteinase K (Promega, Madison, WI, USA). After incubation at 37°C for 10 min, the samples were vortexed for 5 min with Zirconia beads (ZircoPrep Mini; Nippon Genetics, Tokyo, Japan) on a Disruptor Genie instrument (Scientific Industries, Bohemia, NY, USA). After centrifugation at 11,000 × g for 5 min, 100 μl of each supernatant were transferred into 300 μl of lysis buffer (Promega). DNA extraction was then carried out using a Maxwell Blood DNA kit in a Maxwell RSC instrument (Promega). The concentration and quality of the extracted DNA were measured on a Tape Station 4150 system (Agilent Technologies, Santa Clara, CA, USA), and the samples were stored at −80°C until use.

S. mutans ATCC25175, S. oralis ATCC6249, and P. gingivalis ATCC33277, which are indigenous to the oral cavity, were inoculated with the same broth containing SPE at various dilutions. Briefly, inoculated samples were incubated aerobically or anaerobically at 37 °C for 24 h. Bacterial cells were harvested through centrifugation at 5000× g for 10 min under 4 °C chamber conditions and washed twice with Phosphate buffered saline (PBS). Cells (approximately 1.0 × 10⁸) were suspended in 150 μL solution containing 50 mM Tris-HCl buffer (pH 7.6), 1 mM EDTA, and 0.5% triton-X 100. Subsequently, the suspension was placed in ZircoPrep Mini (Nippon Genetics Co. Ltd., Tokyo, Japan) and agitated for 10 min in a Cell disruptor (µT-12, TAITEC, Tokyo, Japan). After sample centrifugation, supernatant was used.
Superoxide Dismutase Assay Kit (Cayman Chemical Company Inc., Ann Arbor, MI, USA) was used to establish SOD amounts in tested bacterial cells, whereas Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) was used to establish bacterial hydrogen peroxide amounts following our earlier work [49 (link)]. Both kits were performed according to the manufacturer’s recommendation.