Study On Improvement Strain Screening And Fermentation Conditions Of A Novel Bacteriocin-like Producing Strain

A novel bacteriocin-like producing strain was screened and isolated from mature alcohol distillery grain and yellow water samples. A new way was used to isolate the bacterium producing antibiotic substance: culture bacterium in Ep tubes then isolate them by paper-disk test and cylinder-plate method. The strain was identified as Bacillus subtilis by physiological-biochemical testing and morphologic characteristics. It was named LL18-4.In the procedure of selecting bacteriocin-like high-producing strain, gentamycin sulfate and rifampicin were added in the culture medium as selection pressure. This new method can obviate low-producing strain and increasing the successful rate of obtaining high-producing strain, it is better than traditional screening approach. Experiment showed that using Bacillus subtilis LL18-4(with the yield of bacteriocin-like being 164u/ml) as parent strain, after UV mutation, the mutant LL-Ⅰ -E65 resistant to 0.3g/L of gentamycin sulfate was obtained, the mutant produced 217u/ml bacteriocin-like and increased by 32% compared with the parent strain. Subsequently, the mutant LL- Ⅰ -E65 was used as parent strain, after DES mutation, the mutant LL- Ⅱ -337 was resistant to 0.5g/L of rifampicin was obtained, the mutant produced 288u/ml bacteriocin-like and increased by 33% compared with the parent strain and 76% compared with the firstparent strain Bacillus subtilis LL18-4. The high-producing strain LL-II -337 was stable in bacteriocin-like production after continuous subcultures for seven generations.Fermentation conditions for bacteriocin-like producing have been established: 1% glucose as carbon source, 2% beef extract as nitrogen source, 1% extract liquid of the fresh distillery grain as growth factor, initial pH6.5, 100ml culture medium in 250ml conical flask, seeds age for 24h, inoculation 1% and culture temperature 37°C for 96h. The bacteriostasis activity can reach 419u/ml.There was no activity appears when the supernatant was treated with proteinase K. This kind of bacteriocin-like substance has very high heat stability when it was under acid condition.The plasmid was isolated from the cell of LL- II -337. Theexperiment of plasmid confirmed that the plasmid has some relation tothe ability of its bacteriostasis substance producing.