Abstract:To obtain peptides with high antifreeze activity，the degreased Antarctic krill was studied with alkaline protease as the optimal protease. The amino acid composition of its hydrolysate was analyzed. Antifreeze peptides from defatted Antarctic krill （AKAPs） were isolated，purified，and identified using ultrafiltration，Sephadex G-25 gel filtration chromatography，and liquid chromatography-tandem mass spectrometry （LC-MS/MS）.The changes in the antifreeze activity were determined by measuring the enzyme activity of hydrogen peroxide before and after a freeze-thawing cycle. The antifreeze activity of biosynthesized AKAPs was further verified by determining the cryoprotective ability against Lactobacillus rhamnosus. The results showed that AKAPs were enriched in proline （20.67%），alanine （9.53%），and glycine （9.24%）. After isolation and purification of AKAPs，the fraction containing more small molecular peptides had strong antifreeze activity. The antifreeze activity was significantly improved （10.12%）. The hexapeptide DINFRY （DY） and the nonapeptide LGDGMSLST （LT） were identified by LC-MS/MS. DY and LT were biosynthesized and intercalated to obtain the composite peptide DY+LT. The DY，LT，and DY+LT treatment groups after freeze-thawing cycle were significantly higher than the saline treatment group（P＜0.05） in the relative survival rates of L. rhamnosus，with better cryoprotection than commercial cryoprotectants. It was shown that the amino acid sequences of AKAPs are Asp-Ile-Asn-Phe-Arg-Tyr，Leu-Gly-Asp-Gly-Met-Ser-Leu-Ser-Thr.