Study On The Separation, Purification, Property Of Pigment Produced By Oligotrophic Bacterium AT52 (Alteromonas Stellipolaris) From Antarctic Seas

Oligotrophic bacteria AT52(Alteromonas stellipolaris) collected from deep sea of the Antarctica, possessed special genetic, physiological and biochemical properties for the uniquely environment of extreme cold, dry and strong radiation in Antarctic, can exocytosis a bright brown pigment. In order to widely applied to numerous fields about foodstuff, cosmetic and pharmacy in the future, the fermentation condition、extraction、physicochemical activity and structural analysis for the brown pigment from oligotrophic bacteria AT52(Alteromonas stellipolaris) were studied. The results in detail were stated as follows:The optimal fermentation conditions were selected by orthogonal experiment and mono-factor design. And the optimal culture conditions were as follows:through selecting improved Zobell 2216E medium:(peptone0.4%、yeast extract0.075%、seawater, added to 5μmol/L Mg2+ 5%), initial pH 7.7、volume 70mL of 250 mL Erlenmeyer flask、shaker rotate speed 150 rpm/min、medium concentration 100%、inoculum 8mL、culture temperature 11℃and fermentation culture time 10 days. Under this optimal condition, the fermentation liquor could reach the biomass could reach 0.978g/L and an absorbance (475nm) of 0.596 at the end of fermentation.On the foundation of the single-factor tests, the pigment extraction by oligotrophic bacteria AT52(Alteromonas stellipolaris) produced was designed and its result was analyzed using Design-Expert 7.0 software. The relation model of pigment absorbance with different operating variables is established. Normal probability plot of residual for the pigment extraction and response surface for desirability function of the pigment extraction with different factors are given in this paper. The results indicated that the extraction temperature has extremely remarkable influence on AT52 brown pigment extraction rate, with methanol density and pH having less influence compared to extraction temperature. According to derivation of the quadratic regress equation:Y=-0.3193+0.02327X-0.28475Y+0.024127Z+0.0002XY-0.00006XZ-0.00005YZ-0.0001572 5X2+0.0261Y2-0.00012975Z2,the optimized operation condition for pigment extraction is obtained, which is the material-to-solution ratio 1:20, the extraction time 25hours, the methanol density 62.8%, pH 4.5, the extraction temperature 60.1℃, the absorbance and crude yield of brown pigments can reach 0.650 and 17.2mg/mL.And the result is obvious increased 14.12 percent compared with the single factor extraction experiment. The paper also proposed an effective and availability method to purification of brown pigment obtained by oligotrophic bacteria AT52. According to color reaction, TLC, HPLC, it is showed that the natural pigment inCLuding DOPA and at least three types of main ingredients were found in the pigment after the purification of centrifugalization and filtration membrane, organic solvent, acid-base complex solvent, silica gel column chromatography. Based on the spectrum of UV and IR, they showed that the brown pigment with functions of hydroxyenzene, carboxyl and indole was a kind of indole-framework eumelanin.At last, the paper studied the pigment stability. The crude pigments are easy to dissolve in water and methanol, while the pigments after purification are easy to dissolve in methanol only. The maximum absorption wavelength is 214nm. The pigment was stable in pH>4.0, heat, and illumination (natural light or ultraviolet radiation) conditions, unstable in NaCLO, and strong against H2O2 and HNO3, Na2SO3 and ascorbic acid were protective to the pigment. Fe3+, Fe2+, Pb2+, but Mg2+ solution was protective to it. Other metals had weak effects on the pigment. The result of pigment antioxidation test demonstrated that the brown pigment has good antioxidation and hacteriostasis effect. And the results showed that it has high activity on H2O2, DNA oxidative damage,hydroxyl radical and superoxide anion, while lower in DPPH·scavenging. The results of antibacterial activities examined showed that the brown pigment had highly significant bacteriostatic activities on mold.