Mutation Breeding And Fermentation Conditions Optimization Of Hyaluronic Acid Producing Bacteria

Hyaluronic acid (HA) is a high molecular weight polysaccharide. It is extensively applied areas including ophthalmology, orthopedics and cosmetic industry. The biopolymer is widespread in nature, having been identified in vertebrate soft tissues and the capsule of group A and C streptococci. The aim of this thesis is to study the production of HA by Streptococcus Zooepidemicus.The accuracy and precision of Bitter-Muir method and CTAB turbidimetric method were examined to assay the concentration of hyaluronic acid in fermentation broth. The presence of constituents in fermentation broth has a considerable interference for Bitter-Muir method, but slight for CTAB turbidimetric method. The CTAB turbidimetric method is simple and rapid, which can be directly used to analyze HA fermentation broth without pretreatment.In this study, the original strain was Streptococcus equi subsp. Zooepidemicus ATCC 39920. It was mutagenized with ion beam, Nd:YAG laser and NTG. After times mutation, a high HA production strain LZ3200 was obtained.The HA yield of LZ3200 was 0.44g/L, 51.7% higher than that of original strain.The flask shaking fermentation techniques conditions of LZ3200 were discussed. The results showed that glucose was suitable carbon source, and brain heart infusion was suitable nitrogen source. The proper temperature, pH, shake speed and volume of shake flask were 37℃, 7.0, 200r/min and 25/250mL respectively. After a 30-hour fermentation, the final HA concentration was 0.452g/L.The flask shaking medium composition of LZ3200 was optimized using genetic algorithms. The optimization task of 6 constituents, each of which has 64 concentration levels, was finally finished within 40 cases. The optimized culture medium is composed of glucose 47.0 g/L, yeast extract 13.6 g/L, peptone 7.8 g/L, beef extract 2.2 g/L, KH₂PO₄ 1.55 g/L, MgSO₄ 2.55 g/L. The concentration of hyaluronic acid with optimized culture medium is 0.494 g/L, 9.29% higher than that of initial culture medium.