Isolation of LAB
Eighteen samples of pickles (pickled Chinese cabbage) were purchased from different parts of China. Each sample (1 g) was blended with 9 mL of pure water and diluted sample was plated onto MRS medium (Difco, USA), and incubated at 37 °C until single colonies grew. Each single colony was preliminarily screened for LAB by gram staining, H2O2 test and microscopic evaluation (100 × oil). All samples were stored at -80 °C.
Measurement of inosine and guanosine levels
HPLC was used to detect inosine and guanosine levels simultaneously. 33.7 mg inosine and 35.7 mg guanosine were dissolved in 100 mL K3PO4 solution (100 mmol/L, pH 7.0) and 20 uL of each solution was injected into a HPLC device (LC-20A, Shinadzu Corporation, China) equipped with a column 5C18-AR-II (4.6 × 250 mm, Cosmosil, China). The final flow phase (0.1 umol/L NaHPO4, 0.187 mol/L H3PO4) was performed at a flow rate of 1 mL/min, a wavelength of 254 nm, a retention time of 40 mins and a column temperature of 30 °C.
To assess their ability to degrade inosine and guanosine, 1% LAB was inoculated in 10 mL of liquid MRS and cultured for 48 h at 37 °C in oxygen free conditions. MRS solution (2 mL) was then centrifuged at 4000×g for 5 mins at 4 °C. The sediment was rinsed with pure water and resuspended in 750 uL of inosine-guanosine solution at 37 °C for 60 min with shaking (120 rpm). The solutions were analyzed by HPLC as described above. The rate of inosine and guanosine degradation by LAB strains was assessed according to the following equation: (v = [(0.9C-X) 0.9C] × 100%, X: the remaining area of ionisine and guanosine on the chromatogram V = speed of degradation (g/L/min).
Acid tolerance assay
Candidate LAB strains (1%) were inoculated in liquid MRS (Difco, USA), and cultured for 24 h at 37 °C under oxygen free conditions in anaerobic chest with AnaeroPack (Beijing BY Tech, China). Liquid MRS (5%) was then injected into 10 ml MRS and cultured at 37 °C. ODs were assessed at hourly intervals from 0 to 4 h using a spectrophotometer (Nanodrop, Gene Limited, China). Briefly, the number of bacteria in the bacterial suspension per unit volume was measured at OD600. A standard curve was established with the corresponding OD value and bacteria number (OD600 = 1, 2 × 109 cfu/ml).
Bile tolerance assay
Candidate LAB strains (1%) were inoculated in liquid MRS and cultured for 24 h at 37 °C under oxygen free conditions. Liquid MRS (5%) was then injected into 10 ml of MRS (pH 5.6, 0.1 g/100 mL) and cultured at 37 °C. ODs were assessed at hourly intervals from 0 to 4 h using a spectrophotometer.
Pepsin and trypsin tolerance assay
Candidate LAB strains (1%) were inoculated in liquid MRS and cultured for 24 h at 37 °C under oxygen free conditions. Liquid MRS (5%) was injected into 10 ml MRS of pH 3, pepsin (0.3 g/100 mL) and of pH 8, trypsin (0.1 g/100 mL), respectively, and cultured at 37 °C. ODs were assessed at hourly intervals from 0 to 4 h.
Sequencing
16S rDNA sequencing was performed following previously described method [19]. Briefly, DNA was extracted using the CTAB method and DNA concentrations were quantified using a NanoDrop and agarose gel electrophoresis. Illumina Miseq sequencing was used to perform multiple splicing. VSEARCH (v2.4.2) software was used to classify all high-quality sequences with 97% similarity for OTU classification and beta diversity analysis. Finally, an evolutionary tree was built and compared to the Silva database (v123). The microbial communities of the four samples were compared and analyzed according to OTU classification. Since there were multiple OTUs corresponding to the same genus or species, Heatmap was drawn and the TOP30 species in richness ranking were provided.
Animal model
Animal experiments were carried out at Guangzhou Medical University (Guangzhou, China) following NIH guidelines for the Care and Use of Laboratory Animal and the protocols were approved by Animal Use and Care of Committee of Guangzhou Medical University (Approval No. GMU00327). Male SD rats (42 days old) were provided by Animal Center of the Traditional Chinese Medicine University of Guangzhou, China (SCXK 2013–0034). The rats were housed in a specific pathogen-free (SPF) laboratory at a constant temperature (22 ± 1 °C) and 40–50% humidity, with a 12 h light/dark cycle and free access to chow and water. The rats were randomly divided into three groups (n = 10): (1) control; (2) hyperuricemia model; (3) S12 strain treatment. All groups (excluding controls) were administered with yeast extract (20 g/kg body weight) by gavage combined with oxygen oxazine acid potassium (350 mg/100 g) by intraperitoneal injection for 20 days. On the 10th day, S12 strain group was administered 1.0 ml of S12 (1.5 × 109 cfu/ml) by gavage for 10 days. On days 0, 10 and 20, blood was collected and centrifuged at 4000 r/min for 5 mins at 4 °C to obtain serum. Serum uric acid (UA), urea nitrogen (BUN) and creatinine (Cr) levels were then determined using Hitachi 747 automatic analyzer following standard protocols. At the end of experiments, the rats were sacrificed after being intraperitoneally anesthetized with sodium pentobarbital (65 mg/kg) according to the guidelines for euthanasia in the Guide for Care and Use of Laboratory Animals. The kidney tissues were dissected and fixed in 40 g/L paraformaldehyde, wax-embedded and cut into serial sections. The sections were then stained with hematoxylin and eosin (HE), and observed under microscope.
Statistical analysis
Statistical analysis was performed using SPSS software and GraphPad Prism 5. Data are presented as the mean ± SD. P-value < 0.05 was considered statistically significant.