中甸牦牛乳酪蛋白源生物活性肽的鉴定及抗氧化活性

Identification and Antioxidant Activity of Bioactive Peptides Derived from Zhongdian Yak Milk Casein

以中甸牦牛乳酪蛋白为研究对象,将其进行酶解超滤后,分别对分子质量>3 kDa和<3 kDa酶解组分的促细胞增殖活性和抗氧化活性进行测定,并采用液相色谱-串联质谱、生物信息学及分子对接分析中甸牦牛乳酪蛋白源的生物活性肽。结果表明:酪蛋白酶解超滤后,分子质量>3 kDa和<3 kDa酶解组分均能保证RAW264.7细胞的增殖率在80%以上,2种酶解液均具有1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和2,2′-联氮-双-(3-乙基苯并噻唑啉-6-磺酸)(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),ABTS)阳离子自由基清除能力,分子质量<3 kDa酶解组分的促细胞增殖、DPPH自由基、ABTS阳离子自由基清除能力优于>3 kDa酶解组分;基于液相色谱-串联质谱技术从中甸牦牛酪蛋白分子质量<3 kDa的酶解组分中共鉴定到895条肽段,其中96条肽段为已知活性的肽段,主要为抗氧化肽(39.6%)、血管紧张素转化酶抑制肽(30.2%)等,说明中甸牦牛乳酪蛋白是生物活性肽的重要来源;基于肽段的活性和疏水性从预测的20条潜在抗氧化肽中筛选到抗氧化肽GYF、RPW,通过分子对接发现GYF、RPW与ABTS阳离子自由基、DPPH自由基的结合能均为负数,具备结合潜力,且主要通过形成氢键和疏水相互作用发挥抗氧化活性。

The enzymatic hydrolysate of Zhongdian yak milk casein was fractionated by ultrafiltration into two fractions:molecular mass>3 kDa and<3 kDa.The cell proliferation activity and antioxidant activity of the two fractions were determined.The bioactive peptides derived from yak milk casein were analyzed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),bioinformatics and molecular docking.The results showed that the proliferation rate of RAW264.7 cells was above 80%in the presence of either the molecular mass>3 kDa or<3 kDa fraction.Both fractions had the ability to scavenge 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical and 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid)(ABTS)cation radical.The cell proliferation and free radical scavenging activity of the molecular mass<3 kDa fraction were better than those of the>3 kDa fraction.A total of 895 peptides were identified from the molecular mass<3 kDa fraction,of which 96 were known bioactive peptides,mainly antioxidant peptides(39.6%)and angiotensinconverting enzyme inhibitory peptides(30.2%),indicating that Zhongdian yak casein is an important source of bioactive peptides.Based on their activity and hydrophobicity,antioxidant peptides GYF and RPW were selected from the predicted 20 potential antioxidant peptides.The results of molecular docking showed that the binding energies of GYF and RPW with ABTS cation and DPPH radicals were all negative,indicating that they had binding potential and exerted antioxidant activity mainly by forming hydrogen bonds and hydrophobic interactions.