High Lipase Activity Salinomycin Producing Strain Breeding And Fermentation Process Optimization

Salinomycin, produced by Streptomyces albus, is a polyether antibiotic specific for animals. As an anti-coccidia agent, Salinomycin is used world widely because of its high efficacy, broad spectrum and low residue. Its significant weight-gaining effect has been generally recognized by the poultry industry.Salinomycin was initially produced in China in early 90's. However the product is still lack of competitivity because of poor quality and high cost. Nonetheless it would be an effective way to improve the yield and reduce the cost through modification of the strain and optimization of the fermentation process.A spectrophotometric method was developed to determine the content of free fatty acid and soybean oil in fermentation broth. Free fatty acid could react with Cu²⁺ at a certain condition to form a compound, which has maximum absorption at 715 nm. The content of soybeen oil and the lipase activity can be calculated by determining the free fatty acid in broth and those derived from soybeen oil hydrolysis at different time during, fermentation.A highly efficient screening method for strains with high productivity was developed. Since a polyketide pathway is involved in the synthesis of salinomycin and the initiating building block is a three-carbon unit malony-CoA, plant or animal oil as carbon source in the medium would provide more three- and two-carbon units, thus facilitating the synthesis of salinomycin. Through comparing the lipase activity of different salinomycin producing strains, it was found that the lipase activity was co-related with the fermenting capacity of involved strains, with highly productive strains showing relatively high lipase activity. Therefore the possibility would be greatly enhanced to obtain strains with high productivity using lipase activity as an indicator during the primary screening. After several starting potential strains being purified, they were subjected to UV plus lithium chloride mutagenesis treatment and plated onto solid medium containing soybean oil. Colonies with high ratio of diameter of hydrolysis halo to that of colony were picked out and were re-screened by using shaking flasks. Finally a strain with a fermentation level of 13.5 percent higher than the starting strain was obtained.The obtained mutant strain was genetically stable, grew fast, had low demand for oxygen, and a very high lipase activity.The fermentation process was optimized from several points such as time and amount of inoculation, medium composition, control of pH to supplementation ofsoybean oil. Particularly the art of medium pH control and supplementation of soybean oil during fermentation was studied in detail. Fermentation level was increased from 20000 u/ml to 25000 u/ml after an appropriate amount of soybean oil was calculated according to the concentration of residual fatty acids and soybean oil in the medium. Fermentation level was also enhanced when fermentation period was prolonged with pH adjusted around 6.0 using sodium hydroxide during the fermentation.