The Preparation And The Physicochemical Properties Of The Antifreeze Protein From Antarctic Krill

Antarctic krill(Euphausia superba)is a small marine zooplankton living in the Antarctic ocean.It possesses an estimated biomass of 6.5–10 millions.This animal contains the largest amount of protein(over 65 % of dry weight)among all the organisms,known as the largest renewable animal protein granary for human beings.Antarctic krill can withstand a low water temperature of-1.9 °C,which may be due to its body containing some proteins possessing antifreeze activity.Antifreeze proteins(AFPs)are a kind of special proteins which can reduce the freezing point,modify the ice crystal,and inhibit the growth and recrystallization of ice crystals.The main features are thermal hysteresis activity(THA)and ice recrystallization inhibition activity(RI).Because of its excellent performance,it is widely used in food,biological,clinical medicine and chemical and other fields.Firstly to carry out the researches of the key factors and optimal conditions of extraction of Antarctic Krill AFPs by specific affinity adsorption.On the basis of single factor experiment,sample concentration,adsorption temperature,adsorption time and adsorption number were optimized by response surface methodology with the AFPs extraction yield and THA as response value.The results indicated that the optimal extraction process was sample concentration of 1.0 mg/mL,adsorption temperature of-1.70 °C,adsorption time of 11 h,the adsorption number of 2.Under the optimal conditions,the model optimum solution would be 29.34 % and 0.0805 °C.The relative errors were 2.66 % and 1.86 % between actual and model value.Secondly to analyze the influence of DSC on the THA evaluation of Antarctic krill AFPs.The THA of the purified Antarctic krill AFP was determined by DSC.The effect of sample cooling rate,sample concentration,ice crystal content,and buffer system on the determination of THA by DSC was studied.The results showed that the molecular weight of AFP was about 76 kDa and the THA was 1.76 °C.The cooling rate should be 1.0 °C/min,the concentration of sample should be 1.0 mg/mL,and the content of ice crystal should be between 10 % and 15 %.The choice of buffer system has a great influence on THA.The presence of ions and pH may play an important role for THA.Therefore,optimized DSC can be used for the evaluation of the THA of Antarctic krill AFP.Thirdly,some physicochemical properties of Antarctic krill AFPs were studied.The results showed that AFPs were non-thermally stable AFPs with a denaturation temperature of 30.3 oC by DSC.The pH value of AFPs sample solution of Antarctic krill had a great influence on its THA.And the maximum THA was observed in the vicinity of pH value of 7.7.The agarose gel experiment showed that the AFPs of Antarctic krill could inhibit the crystallization and recrystallization of ice crystals obviously,and it had a high application value in the food industry.The AFPs of Antarctic krill were gradually adsorbed to the ice crystals by the specific affinity adsorption,but there was a certain proportion of Antarctic krill AFPs in the solution part.Finally,for the preliminary study on the structure of AFPs of Antarctic krill,the amino acid composition,UV spectrum,circular dichroism spectrum(CD)and Fourier transform infrared spectroscopy(FR-TI)were determined.The types and proportions of amino acids in AFPs of Antarctic krill were determined.There were two absorption peaks in the ultraviolet wavelength range,the strong UV absorption peak near 220 nm caused by the peptide bond and the weak absorption peak near 280 nm caused by the aromatic amino acid.And it can be seen that the AFPs of the Antarctic krill have ?-helix,?-sheet and random coil structure by CD.The secondary structure is roughly as follows: the ratio of ?-helix is 24.18 %~24.96 %,the proportion of ?-sheet is 31.90 %~33.17 %,and the proportion of ?-angle is 36.65 % ~ 38.07 %,the ratio of random curl to 5.22 % to 5.85%.by FR-TI.Conclusion: Antarctic krill AFPs were prepared and purified by specific affinity adsorption based on the material of Antarctic krill protein.The antifreeze activity of Antarctic krill AFPs was evaluated by DSC based on THA.The physicochemical properties of AFPs were preliminarily analyzed,which provided basic information for the exploration of antifreeze mechanism of Antarctic krill AFPs.It provided a new idea for clarifying the biological mechanism of Antarctic krill adaptation to the Antarctic low temperature environment.