Research On Enhancing Activity Of Soybean-Sourced Antifreeze Protein By Enzymolysis

Antifreezing proteins(AFP) can effectively lower the freezing point of organic fluids, thereby preventing the growth of macroscopic ice crystals.According to this, AFP has great market future and obvious financial effect in food storage, ice-slurry system and cryopreservation of cells and organs in medicine. Soybean meal is inrich of protein, Nevertheless, the concentration of AFP in organism is very low, which limit its application prospect.it is important to extract AFP from soybean meal and enhance the antifreeze activity by enzymolysis.Soybean meal as raw material, petroleum ether as degrease solvent to defat,then drying, Extracting antifreeze protein with “alkali-solution and acid-isolation” method, Enhancing the antifreeze activity with Enzymolysis. Using differential scanning calorimetry(DSC) to measure thermal hysteresis activity and microscopic observation to wacth the ice crystal size。Through single factor and orthogonal experiments,extraction of protein from soybean meal and its antifreeze activity were studied by using DSC and microscopic observation.the order of five factors effected of the thermal hysteresis activity of antifreezing proteins of soybean meal are as follows: the alkaline solution p H> reaction temperature> material/solvent rati> the acidic solution p H> time. the results showed that: under this conditions:alkaline solution p H 9.00, material-liquidr ratio 1∶20(w/v), time 60 min, temperature 45 ℃, acid-isolation p H4.50, then the thermal hysteresis activity reach to 0.54 ℃. The molecular weight of the major contents of antifreezing protein is about 37.99 k Da,81.62 k D and 91.92 k Da by SDS-PAGE. At the same time, the soybean meal exhibit some frost resistance.Enzymolysis may enhance the antifreeze activity,thereby, the crude extracted proteins as substrate, enzymolysis conditions: time, enzyme concentration, substrate concentration, temperature, p H are studied for getting the highest active peptide products. The result show that the influence of reaction temperture are insignificant,The studies found the order of the other four factors effected of the thermal hysteresis activity of antifreezing proteins of soybean meal from strong to weak as follows: substrate concentration > enzyme concentration> time> p H. when hydrolysis time at 4.0 h,substrate concentration of 3%,enzyme concentration8000u/g, substrate p H=10.00, reaction temperture 55 ℃,the thermal hysteresis is 0.834 ℃. Bovine serum albumin as control, comparing observed crystals exhibit some frost resistance. The molecular weight distribution is in the range of 7.32 k Da-14.88 k Da,while the DH is 46.9%.