Study On Strain Breeding And Process For Production Of Hyaluronic Acid With Controllable Molecular Weight

Hyaluronic acid is a polysaccharide,which is formed by repeated unit of D-glucuronic acid and N-acetylglucosamine.As a kind of biopolymer material,HA has been widely used in the field of cosmetics,health food,medicine and others because of its excellent performance on bioactivity.The application of HA is closely related to its molecular weight.Generally,high molecular weight HA?Mr>1.0 MDa?is used in the field of cosmetics,and low molecular weight HA?Mr<0.1 MDa?is used in the field of food and medicine.HA can be produced by extraction from natural materials or microbial fermentation.In recent years,microbial fermentation has been the main method for production of HA.Therefore,studies on productivity and process optimization are carried forward for reducing production cost andIn the study,a strain of Streptococcus zooepidemicus?S.equi subsp.Zooepidimicus?as the original strain was irradiated by UV,microwave and ?-ray successively for breeding a high productive strain.And then,the medium composition and cultivation conditions were studied to improve the yield of HA.Afterwards,the methods of two-stage temperature and medium pH stabilization in fermentation were applied to improve the molecular weight of HA.Finally,degradation by selfenzyme in fermentation broth was studied to prepare the low molecular weight HA.The results achieved in the paper are as follows:Firstly,the strain S.equi S was irradiated by UV,microwave and ?-ray successively and lethality rate were calculated to determine the proper mutation dose.By screening,a HA high yield strain,designated as SXY36,was selected out,and the yield of HA reached 0.423 g/L,increased by 64.5% compared with that of the original strain S.Secondly,culture medium and cultivation conditions were optimized by the single-factor test and Response Surface Methodology.The result showed that the optimal culture medium was composed of 13.8 g/L glucose,11.2 g/L beef extracts,10.4 g/L peptone,4 g/L yeast extracts,2.5 g/L MgSO₄ and 4.5 g/L K₂HPO₄ with pH at 7.0.The proper cultivation condition were 37?,200 r/min with the loading volume of 80 mL in a 250 mL flask.After cultivation for 18 h,the yield of HA by strain SXY36 was increased to 0.692 g/L,63.6% improvement compared with the productivity of the original strain?0.423 g/L?.Thirdly,the effects of two-stage temperatures cultivation and medium pH stabilization on molecular weight were investigated.The yield of HA and the average molecular weight were increased to 0.731 g/L and 1.89 MDa when the cultivation was carried out at 38? for 12 h in the first stage,and then was turned 34? for 6 h in the second stage.Compared with the results in the original fermentation process?0.692 g/L and 0.923 MDa?,they were increased by 5.6% and 105.1% respectively.Fourthly,degradation by self-enzyme in fermentation broth was studied to prepare low molecular weight HA?LMWHA?.The results revealed that after centrifugation,the supernatant of fermentation broth was incubated at 38? with addition of 0.2 mol/L NaCl and adjustment of pH at 6.0,the molecular weight of HA was decreased to 0.095 MDa after incubation for 36 h,the content of HA was 0.706 g/L,not reduced significantly.