Study On Breeding Of Oxygen Tolerant Lactobacillus Delbrueckii Strains By Protoplast Technique

The protoplast technique has become an effective mean of industrial microbial breeding, which including protoplast transformation breeding, protoplast fusion breeding, protoplast mutation breeding, protoplast regeneration breeding and other kinds of protoplast breeding. In this article, to obtain oxygen-tolerant performance improved Lactobacillus delbrueckii subsp. bulgaricus strains, the protoplast transformation and the single inactivated protoplast fusion technique were adopted, and the results were as follows:The starting strain L. delbrueckii FQ is Gram-positive bacteria, the cell wall is compact. Therefore, suitable concentration of lysozyme and mutanolysin were combined in the process of protoplast preparation. Enzyme concentration, enzymo lysis time and enzymo lysis method could affect the protoplast formation and regeneration, L. delbrueckii FQ strain was treated by different concentration of mutanolysin and lysozyme with ultrasound at37℃, protoplast formation and regeneration rate increased gradually along with the increase of different concentration of mutanolysin. When the final concentration of enzyme was10μg/mL mutanolysin and1mg/mL lysozyme, and enzymolysis time was120minutes, the product of protoplast formation and regeneration rate got maximum, the protoplast formation and regeneration rate was99.99%and4.93%respectively. Enzyme concentration was too high and the protoplast regeneration rate decreased on the contrary. The starting strain was treated with optimal enzyme concentration and ultrasound for30minutes,60minutes,90minutes,120minutes at37℃, results showed that protoplast formation rate was99.99%, which didn’t increase with increase of enzymolysis time; and when the L. delbrueckii FQ strain was treated with ultrasound for90minutes at37℃, maximal regeneration rate was6.36%. Therefore, the optimal enzymolysis condition of L. delbrueckii FQ strain:concentration of enzyme was lOμg/mL mutanolysin and lmg/mL lysozyme, and enzymolysis time was90minutes with ultrasound at37℃. The composition of regeneration medium was the key factor that affected the protoplast regeneration, the double regeneration medium Ⅱ (RM Ⅱ) was the optimal protoplast regeneration medium of the six media for the L. delbrueckii FQ strain, the regeneration rate could reach to10.59%, and the sequence of regeneration rate:RM Ⅱ> RM I> RM IV> RM Ⅲ, and the protoplast could not regenerate in RM V and RM VI medium. The plasmid pFL010of Lactococcus lactis FC strain and plasmid pNZ8148of its control strain Lactococcus lactis FL, was respectively induced to the starting strain L. delbrueckii FQ to obtain the oxygen-tolerant performance improved L. delbrueckii strains by protoplast transformation technique. Results of experiment showed that the optimal enzymo lysis condition of L. delbrueckii FQ strain was10μg/mL mutanolysin and lmg/mL lysozyme, and treated with ultrasound for90minutes at37℃. The optimal transformation condition was400g/L PEG6000which induced plasmids for5minutes at20℃, pH6.5. After5generations of the continuous passage, two transformants was obtained, named by QC and QL respectively. Both of the cell density of transformant QC and QL were higher than that of L. delbrueckii FQ strain in the mode of static culture, results showed that oxygen-tolerant performance of transformants were improved certainly. But the cell density of transformant QC and QL in the mode of static culture were higher than that in the mode of shaking culture, which suggested that high concentration of oxygen had negative effect on the growth of the transformant QC and QL.Single inactivated protoplast fusion technique was adopted in the fusion of L. delbrueckii FQ strain and inactivated protoplast of L. lactis FC strain and its control strain L. lactis FL. The condition of protoplast formation, regeneration and influence factors of fusion process were examined in this paper. Results of experiment showed that the optimal enzymo lysis condition of L. delbrueckii FQ strain was10μg/mL mutanolysin and lmg/mL lysozyme, and treated with ultrasound for90minutes at37℃. With0.5mg/mL glycine and50mg/mL lysozyme, the maximal protoplast formation rate of L. lactis FC strain was99.96%for8hours at37℃. And the inactivated rate of L. lactis FC strain protoplast was99.99%for120minutes at65℃. The results of fusion factors indicated that the optimal fusion condition was400g/L PEG6000which induced protoplast for10minutes at20℃, pH6.5, and the highest fusion rate reached to1.178×10-5. Compared to the single inactivated fusion rate2.74x10"7of the reference, the fusion rate was improved about43times. After5generations of the continuous passage, a chloramphenicol-tolerant fusant was obtained, named by QC10. The cell density of the fusant could reach to6.533in the mode of shaking culture, which demonstrated the oxygen-tolerant performance of the fusant was significantly improved.With1mg/mL glycine and10mg/mL lysozyme, the maximal protoplast formation rate of L. lactis FL strain was99.97%for90minutes at37℃. And the inactivated rate of L. lactis FL strain protoplast was96.89%for120minutes at65℃. The results of fusion factors indicated that the optimal fusion condition was400g/L PEG6000which induced protoplast for5minutes at20℃, pH6.5, and the highest fusion rate reached to2.72×10-6. Compared to the single inactivated fusion rate2.74×10-7of the reference, the fusion rate was improved about10times. After5generations of the continuous passage, a chloramphenicol-tolerant fusant was obtained, named by QL10. The cell density of the fusant QL10could reach to1.396in the mode of static culture, the cell density was improved0.25times contrast to the parent strain L. delbrueckii FQ.