Lysozyme

TKGs (approximately 14 mm in diameter) growing in renewed milk for 24 h were sampled and washed thoroughly with sterile water to remove any adhered kefir and attached microbes prior to DNA extraction. Approximately 40 mg of biomass from each grain (n = 3) was taken aseptically, pestled with TissueRuptor (Tiangen, Beijing, China) and then suspended in 600 μl of 1.2 M sorbitol buffer. After addition of 50 U lyticase (Tiangen, Beijing, China), the samples were allowed to react for 30 min at 30 °C. The samples were centrifuged at 1500×g for 10 min, and the deposits were resuspended in buffer (20 mM Tris-HCl, 2 mM sodium EDTA, 1.2% Triton×-100) with 20 mg/ml lysozyme (Tiangen, Beijing, China) for 50 min at 37 °C. Subsequently, total DNA was extracted according to the instructions of the Marine Animals DNA Kit (Tiangen, Beijing, China) with a modification involving the addition of 50 μl of (20 mg/ml) Proteinase K solution (Tiangen, Beijing, China).

All aqueous humor sample collections were performed using a standard sterilization procedure. Undiluted aqueous humor samples (200 μL from one eye) were obtained from the patients via anterior chamber paracentesis. The samples were flash-frozen and kept at 80°C. 1.5 mL microcentrifuge tube with 200 μL sample and 250 μL 0.5 mm glass bead (BioSpec Products, OK, United States) were attached to a horizontal platform on a vortex mixer (Vortex-Genie2 vortex mixer, Scientific Industries, United States) and agitated vigorously at 2,800–3,200 rpm for 30 min. Then 7.2 μL lysozyme (Tiangen Biotech, Beijing, China) was added for wall-breaking reaction. 0.3 mL sample was separated into a new 1.5 mL microcentrifuge tube and DNA was extracted using the TIANamp Micro DNA Kit (DP316, Tiangen Biotech, Beijing, China) according to the manufacturer’s recommendation.