Screening High-yield Bacteria Lactobacillus Plantarum Carring CLA By Low Energy Ions Implantation

This test takes a CLA-producing lactobacillus plantarum as original strain, and studies the CLA-producing conditions of such strain, then mutagenesis selective development is conducted to the original strain with 10 kev nitrogen ion for several times, hoping to get a high-productivity CLA strain, at the same time, in order to compare the differences between original strain and mutation strain in molecule, it further studies the damages of ion implantation to genetic material of microorganisms through microorganisms DNA and protein variation detection with RAPD analysis technology and SDS-PAGE. The major research outcome is as follows:1. N+ implanting is made to lactobacillus plantarum original strain ANCLA01, when the implanting volume is 10 kev, dosaze is 0 (comparative) -120×2.6×10¹³ ions/cm², and mutagenesis is conducted to original strain, survival curve is primarily in conformity with"Saddle-shaped"curve as reported in literature. The best implanted energy is achieved when implanting volume is 60×2.6×10¹³ N⁺/cm². After repeated mutagenesis, the CLA productivity of mutation high-yielding strain ANCLA02 rises from 43.44μg/mL of original strain to 106.67μg/mL, After the mutant strain is cultivated for 5 generations, the CLA productivity tends to be stable.2. Through preliminary studies on conditions of original strain to produce CLA, it acquired the most suitable conditions for lactobacillus plantarum to produce CLA: take linoleic acid as inducer, and volume of addition is 0.1%, inoculum size is 4%, PH value is 6.5, it is fermented 24 hours at 37℃.3. The original strain and mutant strain are tested in whole-cell protein electrophoresis, and protein expression difference between the original strain and mutant strain is studied. The original strain and mutant strain are tested through SDS-PAGE method and the outcome indicates that there is no difference between the original strain and mutant strain in whole-cell protein expression.4. In order to compare differences at themolecular level between the original strain and the mutant strain, 30 pieces of random primers were designed to clarify the genetic background by randomly amplified polymorphic DNA (RAPD). By RAPD analysis, the DNA mutant strain is changged, of 2 primers whose amplification products showed polymorphic characterwere selected from 30 primers, which can be used to discern the mutant strain. The primer rate of polymorphism amplification production is 6.67%, and the polymorphismis stable with fine repeatability.