Evaluation of carob extract as a carbon source in hyaluronic acid production and optimization of production conditions

Studies, starting from isolation of hyaluronic acid from animal sources reveal that it is a very important biopolymer for human health. This polysaccharide, which is a straight chain natural glycosaminoglycan polymer, can be produced by biotechnological methods by using the natural producer species Group A and Group C Streptococcus. This biopolymer, which has high bioavailability due to its high water holding capacity, viscoelasticity and its ability to reach high molecular weights, can be widely used in the treatment of orthopedic ailments, eye diseases and eye surgery, filling materials used in aesthetic surgery, skin renewal and wrinkle removal, cancer cells detection and treatment, drug transport systems and cosmetics industry either directly or as an auxiliary material. Improving the fermentation conditions and lowering the production costs are vey important for the biotechnological production of hyaluronic acid, having high utilization rate. In this respect, this thesis was designed to demonstrate the producibility of this polymer, which has a high added value, by using the carob grown in our country as a carbon source. In this study, media of hyaluronic acid production by Streptococcus equi subsp. zooepidemicus ATCC 3992, which include carob extract as a carbon source, was optimized. Firstly, with the literature review, carob extract medium components were determined with 4 different nitrogen sources as casein, yeast extract, beef extract and peptone and 5 different mineral sources such as MgSO4.7H2O, KH2PO4, NaCl, K2HPO4 and FeSO4. In order to perform media optimization, the Plackett-Burman design, which includes the inoculation rate in addition to these components, was created. The 15 different trial fermentations offered by the design were performed in incubator at 37°C, 150 rpm shaking speed, 7.0 initial pH. HA quantities detected after fermentation were used as response surfaces and medium optimization was performed with the Plackett-Burman design. The amount of hyaluronic acid was determined as 2.60 g/L at the end of the fermentation which was carried out in the incubator with using optimum medium. In addition, as a result of analysis of variance (ANOVA); The model, KH2PO4, NaCl and FeSO4 were found statistically significant (P<0.05). Accordingly, hyaluronic acid production was carried out in bioreactor using by the optimum medium composition. The effect of fermentation parameters on the molecular weight of the produced hyaluronic acid was investigated using the Box-Behnken response surface design. The experimental design was created with the independent variables, which were selected as the amount of aeration, agitation speed and initial biomass density, and fermentations were carried out. In addition, molecular weight of hyaluronic acid were determined by molecular separation (size exclusion, SEC) chromatography using preperative HPLC. The model and the amount of aeration were found to be statistically significant as a result of bioreactor experiments (P <0.05). As a result of the thesis, 2.60 g/L hyaluronic acid was produced in optimum carob fruit extract medium with shaking incubator. In addition, the average molecular weight of the hyaluronic acid produced in the bioreactor was found to between 500 kDa and 1750 kDa. Thus, it has been proved that carob fruit can be used in the production of hyaluronic acid, a product with high added value, and the optimum medium composition and fermentation conditions of this raw material have been determined.