沙棘籽粕蛋白肽的稳定性及分离纯化

Stability and Separation of Peptides from Seabuckthorn Seed Protein

以沙棘籽粕蛋白为原料,酶解制备具有抑制胰脂肪酶活性的多肽,通过超滤、大孔树脂分离、超高效液相色谱-串联质谱和分子对接对沙棘籽粕蛋白肽(seabuckthorn seed peptides from alcalase hydrolysates,SSPA)进行分离纯化、结构鉴定和筛选。以猪胰脂肪酶(porcine pancreatic lipase,PPL)抑制率为指标,稳定性分析表明,SSPA具有热稳定性和pH值稳定性,适量Na^(+)和Mg^(2+)可显著提高SSPA的PPL抑制活性,SSPA在短期贮藏过程中不会因暴露于空气而影响PPL抑制活性。超高效液相色谱-串联质谱鉴定得到31种多肽,结合分子对接筛选得到6个肽段,小于3 kDa组分中上述寡肽含量分别为VR(2.90%)、FR(7.40%)、RDR(1.10%)、APYR(1.50%)、NLLHR(1.40%)和EEAASLR(1.10%)。固相合成测定其IC_(50)值分别为VR(371.07μg/mL)、FR(243.07μg/mL)、RDR(250.50μg/mL)、APYR(350.41μg/mL)、NLLHR(220.70μg/mL)、EEAASLR(510.55μg/mL)。分子对接表明以上多肽通过氢键、π-π堆积与PPL结合。Pearson相关性分析表明,分子对接结合能与SSPA的PPL抑制率之间存在显著正相关(R^(2)=0.865,P<0.05)。

In this study,peptides with inhibitory activity against porcine pancreatic lipase(PPL)from the alcalase hydrolysate of seabuckthorn seed meal protein were purified by ultrafiltration and macroporous resin separation,and their structures were investigated by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)and molecular docking.The peptides had good thermal and pH stability.The PPL inhibitory activity was significantly improved by adding appropriate amounts of Na^(+) and Mg^(2+) but not influenced by short-term exposure to the air.A total of 31 peptides were identified by UPLC/MS-MS,and six peptides were selected by molecular docking,whose contents in the ultrafiltration fraction with molecular mass less than 3 kDa were as follows:VR(2.90%),FR(7.40%),RDR(1.10%),APYR(1.50%),NLLHR(1.40%)and EEAASLR(1.10%),respectively.The half-maximal inhibitory concentration(IC_(50))values of VR,FR,RDR,APYR,NLLHR and EEAASLR prepared by solid phase synthesis were 371.07,243.07,250.50,350.41,220.70,and 510.55μg/mL,respectively.The results of molecular docking showed that each of these peptides could combine with PPL by hydrogen bonding andπ-πstacking interactions.Pearson correlation analysis showed that there was a positive correlation between molecular binding energy and the PPL inhibitory activity of the peptides from seabuckthorn seed protein(R^(2)=0.865,P<0.05).