为充分利用柚子资源并减少环境污染,以金柚柚皮为原料,采用改良Osborne法分级提取柚皮蛋白,通过扫描电镜、红外光谱、氨基酸组成分析对蛋白的结构进行初步鉴定,并使用MTT法和ELISA检测法分别检测小鼠RAW 264.7巨噬细胞的存活率、NO含量以及IL-6和TNF-α的表达水平。实验发现,柚皮清蛋白、球蛋白、醇溶蛋白和谷蛋白的提取率分别为(15.47±0.17)%、(4.20±0.09)%、(0.18±0.02)%和(0.04±0.01)%,扫描电镜观察到柚皮清蛋白为片状,球蛋白为多孔状,醇溶蛋白为条状相连的片状结构,3种蛋白只在N—H、—OH和—CH的官能团处发生构象变化,柚皮蛋白中的甘氨酸和丙氨酸的含量最为丰富,含量分别为125.49和50.48 g/100 g。在免疫活性方面,清蛋白在800 μg/mL时刺激RAW 264.7 细胞产生NO的能力以及促进细胞产生TNF-α和IL-6的能力最强,浓度分别为95.46 μmol/L,9 528.05和1 769.33 pg/mL,实验数据表明柚皮清蛋白的免疫调节能力最强,具有保健食品开发和医学用药开发的潜力。
In order to make full use of pomelo resources and reduce environmental pollution, pomelo peel was used as the raw material and an improved Osborne method was used to extract proteins in stages. The structure of the protein was identified by scanning electron microscope, infrared spectrum and amino acid composition analysis. And the survival rate of mouse RAW 264.7 macrophages, NO content, and the expression level of IL-6 and TNF-α were detected by MTT, and ELISA detection, respectively. It was found that the extraction rates of albumin, globulin, gliadin, and glutenin were (15.47±0.17)%, (4.20±0.09)%, (0.18±0.02)% and (0.04±0.01)%, respectively. The results of scanning electron microscope showed that the albumin was in the form of a sheet and the globulin was in a porous form, while the gliadin was in a strip-like structure. Moreover, the conformations of the three proteins only changed at the functional groups of N—H, —OH, and —CH. Furthermore, the content of glycine and alanine were the most abundant which reached 125.49 g/100 g and 50.48 g/100 g respectively. Albumin had the strongest ability to stimulate RAW 264.7 cells to produce NO and TNF-α and IL-6 at 800 μg/mL, at 95.46 μmol/L, 9 528.05 pg/mL and 1 769.33 pg/mL, respectively. The experimental data showed that pomelo peel albumin had the strongest immune activity, suggesting its potential as a health food development and medical drug.
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