Strain Screening And Optimization Of Fermentation Medium For Higher Production Of Hyaluronidase

Hyaluronic acid(HA), anonbranchedPolysaccharide, is composedofregularly alternatingunitsof N-acetylglucosamineandD-glucuronic acidlinkedbyβ-(1�'4)andβ-(1�'3) glycosidic bonds. Theaveragemolecularweight(Mw)rangeofnaturalHAwithin105～107D,andthemolarratioo ftthetwo monosaccharide inthemolecularstrueture is 1:1.HA is widely applied for cosmetic industry, medical domain andhealthfood, because of its good viscoelasticity, plasticity,extraordinary water-holding function,penetrabilityand highly biological absorbency. The biological activity and using effect of HA are connected with its molecular weight. The molecular weight (Mr) ofLMWHA is with (1～8)×104Da, and Mr of o-HA is below 1×104Da. The micromolecule polysaccharose can resistance to tumor, promote wound healing, promote bone and angiogenesis, regulate immune function, having potential medical application prospect. They has different biological activities from high molecular weight HA and even have opposite biological activities. The biological activity of LMWHA and o-HA has attracted widespread attention.Enzymatic procession of HA by hyaluronidase is gentle、pollution free、 process controlled and high yeld. Besides, hyaluronidase has important biological values in medical domain.Based on this, this paper systematically studied the screening of hyaluronidase-secreting strains, fermentation optimization. The main results are summarized as follows.1. Screening and identification of hyaluronidase-producing bacterial strains.A marine strain TF18 with high hyaluronidase production was isolated from sedimentary bacteria in the air.16S rDNA gene sequence was sequenced,and its morphological characteristics, culturalcharacteristics, physiological and biochemical characteristics, we could prove that the strain TF18 belonged to Bacillus. But it was identified as a new bacteriathat was different from the other bacteria strains of Bacillusthrough normal screening and analysis of 16S rDNA.In the process of strain selection, the method of transparent circle of the plate, make the screening results more intuitive and accurate, and effectively improve the screening efficiency. Simultaneous enzymatic activity was measured by ultraviolet spectrophotometry (UV) method, and the method was used to replace the turbidity method (《Chinese Pharmacopoeia 2010 Edition》). This method can greatly reduce the workload of the enzyme activity assay, improve the accuracy, and reduce the error due to the operation, reagent, environment and other factors.2. Optimization of fermentation conditions of Bacillus sp.TF18In this study,TF18(Bacillus sp.TF18)was used as the starting strain, and the seed culture conditions were optimized:the best carbon source was glucose, the best nitrogen source was yeast powder and peptone, shaking speed was 150r/min, the temperature was 36℃. Using single factor test to get the best proportion of fermentation medium:glucose40g/L, yeast 7g/L, peptone 9g/L, MgSO4·7H2O 2.5g/L, KH2PO4·5H2O 2.2g/L.