Extraction And Isolation Of Alfalfa Ice Structuring Proteins And Its Antifreeze Protection Forangle Yeast

Ice structuring proteins(ISPs)is kinds of polypeptide,with thermal hysteresis activity,restrain crystallization or re-crystallization,which is generated by emergency reaction to resist the cold environmental stimulus.In this study,ISPs were extracted,separated and purified,from the raw materials of Alfalfa,to detection of the antifreeze activity using ice combined with phosphate buffer solution.Additionally,the freezing preservation of Alfalfa ISPs was deeply characterized in angel yeast.Furthermore,extraction conditions for Alfalfa ISPs were optimized by calculation of extraction efficiency,single-factor experiment and response surface methodology.The results revealed that the extraction yield of Alfalfa ISPs was up to 2.87%,when the extraction was carried out in 61 mmol/L phosphate buffer solution at pH 8.0 with a solid/liquid ratio of 1:20 and 46 ice pieces were added in per 100 milliliters of crude extracts.And thermal hysteresis activity value of Alfalfa ISPs was 0.54 in the extraction condition.Additionally,the observed ice crystal morphology suggested that the Alfalfa ISPs are able to significantly inhibit the growth and modify the conformations of ice crystals.Amino acid analysis showed that Alfalfa ISPs contain 17 kinds of amino acids which control the proportion of Hydrophilic/hydrophobic amino acids are 7 to 4.Moreover,DEAE-52 of anion exchange column chromatography together with Polyacrylamide gel electrophoresis(SDS-PAGE)was employed to separate and purify the Alfalfa ISPs.There are many important elements were optimized,such as optimum concentration and pH value of buffer solution,or loading quantity of sample were measured at 20mmol/L,pH 7.5 and 40mg/mL,respectively.Ultimately,we obtained two components of Alfalfa ISPs which protein molecular weight is 34kDa and 52kDa.The results suggested that their thermal hysteresis activity values are all of 0.41,and both of them are able to patently modify the conformations and inhibit growth of ice crystals.Dyeing appraisal of SHIFF showed that the 34 kDa is composed of glycoprotein,while the 52 kDa is not.Additionally,the secondary structures have been defined of the two Alfalfa ISPs using infrared spectroscopy(IR).The preliminary results revealed that α-helix,β-sheet,β-turn and random coil is 13.19%,60.21%,12.21%,19.16%and 16.87%,39.41%,26.69%,17.03%,respectively,which is different in 34 kDa and 52 kDa of Alfalfa ISPs.In addition,we further characterized the functional role of Alfalfa ISPs in freezing preservation of angel yeast.The results showed considerable survival rate and glutathione release quantity in Alfalfa ISPs added,comparing with in control,under different conditions which include freezing time,cryogenic temperature and freeze-thaw cycles.Collectively,the results indicated that Alfalfa ISPs would be effectively to raise the antifreeze protection in angel yeast.