Breeding Of Lactobacillus With Oxalate-Degrading Capacity By Protoplast Fusion

Almost 80% of kidney stones are comprised of calcium oxalate. Level of oxalate in urine is the most significant risk factor in the formation of kidney stones. Some studies have reported that bacterial such as Bifidobacterium lactis with oxalate-degrading capacity can efficiently reduce the oxalate absorption and the urinary oxalate level, and it can be used to prevent and treat kidney stone diseases. While B. lactis is poorly oxygen-tolerant, and the application of transgenic technology hinders it from being as safety microbial ecological agents. The aim of this work was to breeding of strains with oxalate-degrading capacity and oxygen-tolerant characteristic by protoplast fusion of B. lactis and Lactobacilhis acidophilus strains. The related results are as follows:The optimized condition of preparation for protoplasts from B. lactis and L. acidophilus was as follows:the best lysozyme concentration were 6mg/mL and 12mg/mL, and the duration of enzyme treatment were 25min and 30min. The protoplasts formation rate reached 98.3%,86.5%, the regeneration rate were 42.2%,46.5%, respectively. The fusion rate reached 7.6% for about 7 min with the PEG60O0 used as fusion agents in such conditions:50% PEG6000,30℃,0.02mol/L CaCl2,0.5 mol/L MgCl2. Some fusants were screened in the medium containing oxalate during aerobic growth, and six fusants with almost same oxalate-degrading capacity were obtained for further investigation.The result of 16S rDNA sequence analysis showed that L. acidophilus was the main parent of fusant. Further PCR test indicated that the OXC gene was successfully and stably integrated on the chromosome DNA of fusant. The stability test found that the level of oxalate-degrading of the fusants were stable. Simulation experiment in vitro gastrointestinal showed that three strains have good acid tolerance and bile salt resistant characteristic.To improve the density of the fusant SZY2-1, the orthogonal experiment was used to optimize the culture medium and fermentation conditions. The optimized medium contained 2.5% sucrose,3% soybean tryptone,0.05% magnesium sulfate,0.35% dipotassium hydrogen phosphate,0.5% sodium acetate,0.025% manganous sulfate,0.01% ferric chloride,0.2% dibasic ammonium citrate; the living bacteria number reached 3.9×1010 cfu/mL after 15h cultivation under the optimized condition in which pH, inoculation amount, culture temperature were 6.0,4%,37℃, respectively.