Alireza Fayazi - biology department, science and research branch, islamic azad university, Tehran
Malihe Keramati - Nano-Biotechnology Department, Pasteur Institute of Iran, Tehran

Background and Aim : Hyaluronic acid (HA) as a biodegradable, biocompatible, non-immunogenic linear polysaccharide has been used in many applications such as pharmaceutical, clinical and cosmetics. Currently, commercial HA for human use is mainly produced by fermentation, specifically using Streptococcus sp. In present study we improved HA production by mutant strain of Streptococcus equisimilis as one of the best strains producing hyaluronic acid capsule.Two of the most important factors in medium culture are pH and temperature.The most frequently used optimization strategy is “one-at-a-time”strategy. This approach is not only time consuming, but also ignores the combined interactions between physiochemical parameters, so in this study we optimized this two factors in medium culture conditions by response surface method. RSM combines statistical experimental designs and empirical model developing by regression with a purpose of process or product optimization.Methods : In first step we used the Minitab۱۷ software to design of experiment in RSM by Central composite design(CCD) method. Maximum and minimum values were also considered according to recent studies. Minimum level for temperature was determined ۳۰°C and maximum level ۳۷°C, as well as minimum level for pH was determined ۵.۵ and maximum level ۸.۰. Finally, experiment was carried out with ۱۴ run, ۲ block and ۶ center points. In the next step the primary seed of Streptococcus equisimilis were cultured in Todd Hewitt Broth for ۱۶ h. The production medium containing Na۲HPO۴·۱۲H۲O, KH۲PO۴·۵H۲O, MgSO۴·۷H۲O, yeast extract, and glucose was inoculated by ۱۰% of seed at OD۶۰۰nm ۱, and incubation for ۶ h of HA production (in ۱۸۰ RPM, ۳۷°C) . Then, HA was precipitated by A special method. The amount of proteins and nucleic acids was assayed at OD۲۸۰nm and OD۲۶۰nm. The HA concentration was determined by complexometery method using carbazole assay at ۵۵۰ nm. Finally, HA values was evaluated by the MINITAB ۱۷ software.Results : Finding showed that the yield of HA extracted from Streptococcus equisimilis in medium culture with pH:۸.۰ and temperature ۳۷°C is ۳.۲۷ mg/ml and its yield was more than other values of pH and temperature.Conclusion : This suggested that we optimized temperature and pH in medium components with central composite design (CCD) method for high production of HA.

Hyaluronic acid, Streptococcus equisimilis, Carbazole, response surface methodology, central composite design