不同分子质量糖基化核桃肽的制备及功能特性分析

Preparation and Functional Properties of Glycosylated Walnut Peptides with Different Molecular Masses

以核桃粕制备的核桃蛋白为原料,通过酶解、超滤制备3种不同分子质量的核桃肽,分别与5种糖类(葡萄糖、蔗糖、乳糖、麦芽糊精、葡聚糖)进行糖基化改性,以乳化性质和抗脂质氧化能力为指标,研究核桃肽糖基化改性最优工艺条件。利用凝胶电泳、傅里叶变换红外光谱及圆二色谱表征核桃肽的糖基化共价接枝反应产物,研究其理化特性,同时基于激光共聚焦显微镜明确糖基化改性产物的乳状液稳性。结果表明:分子质量大于30 kDa核桃肽与麦芽糊精的糖基化反应最佳工艺条件为核桃肽-麦芽糊精比例1∶2、反应时间8 h、反应温度80℃、肽质量浓度15 mg/mL,十二烷基硫酸钠-聚丙烯酰氨凝胶电泳实验结果证实了分子质量大于30 kDa核桃肽与麦芽糊精通过糖基化反应生成共价复合物,傅里叶变换红外光谱分析表明糖分子以共价键的形式接入到核桃肽分子中;圆二色谱表明肽结构发生改变,α-螺旋从4.85%增加至6.43%、反平行从30.16%增加至35.91%、平行从3.28%增加至4.33%、β-转角从23.21%降至20.77%、无规卷曲从33.59%降至31.22%。糖基化反应改性除Fe^(2+)螯合能力没有改善,其溶解性、乳化性、乳化稳定性、吸水性、吸油性、表面疏水性、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除力和抗脂质氧化能力均提高(乳化性为(83.24±1.64)m^(2)/g、乳化稳定性为(218.49±3.55)min、吸水性为(3.92±0.36)g/g、吸油性为(3.17±0.24)g/g、表面疏水性为251.05±6.91、DPPH自由基清除率为(86.74±2.14)%、抗脂质氧化能力为(62.17±3.51)%);激光共聚焦结果显示核桃肽糖基化改性后对核桃油的包裹能力增强,对提高乳液稳定性具有一定的潜在利用价值。核桃肽的糖基化改性为核桃粕副产物加工利用提供新思路,同时为糖基化改性在食品开发中的应用提供一定的参考。

Three walnut peptides with different molecular masses were prepared from walnut meal protein by sequential enzymatic hydrolysis and ultrafiltration,and separately glycosylated with five sugars(glucose,sucrose,lactose,maltodextrin and dextran).The reaction conditions were optimized based on emulsifying properties and anti-lipid oxidation capacity.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),Fourier transform infrared(FTIR)spectroscopy and circular dichroism(CD)spectroscopy were used to characterize the glycosylated peptides,their physicochemical properties were studied,and their emulsion stability was determined by laser confocal microscopy(LCM).The results showed that the optimal conditions for the glycosylation reaction between walnut peptide with molecular mass greater than 30 kDa and maltodextrin were as follows:ratio of walnut peptide to maltodextrin 1:2,reaction time 8 h,reaction temperature 80℃,and peptide concentration 15 mg/mL.SDS-PAGE confirmed the formation of a covalent complex between the walnut peptide and maltodextrin through glycosylation reaction.FTIR spectroscopy showed that sugar molecules were connected to walnut peptide molecules by covalent bonds.CD spectroscopy showed that the peptide’s structure changed.The proportion of α-helix increased from 4.85% to 6.43%,the proportion of antiparallelism increased from 30.16% to 35.91%,the proportion of parallelism increased from 3.28% to 4.33%,the proportion of β-turn decreased from 23.21% to 20.77%,and the proportion of random coil decreased from 33.59% to 31.22%.The glycosylation modification did not improve the Fe^(2+)chelating capacity,but increased the solubility,emulsifying capacity to(83.24±1.64)m^(2)/g,emulsion stability to(218.49±3.55)min,water absorption capacity to(3.92±0.36)g/g,oil absorption capacity to(3.17±0.24)g/g,surface hydrophobicity to 251.05±6.91,1,1-diphenyl-2-picrylhydrazyl(DPPH)radical-scavenging capacity to(86.74±2.14)%,and anti-lipid oxidation capacity to(62.17±3.51)%.The results of LCM showed that the encapsulation capacity of walnut peptides for walnut oil was enhanced after glycosylation,suggesting a potential application for improving the stability of emulsions.The glycosylation modification of walnut peptides provides a new idea for the processing and utilization of walnut meal by-products and provides a reference for the application of glycosylation modification in food development.