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中国精品科技期刊2020
冯瑞雪, 史瑛, 姬中伟, 钱肖华, 徐岳正, 毛健, 许正宏. 黄酒多肽的多级分离纯化及其对小鼠巨噬细胞免疫调节的影响[J]. 食品工业科技, 2020, 41(13): 289-295. DOI: 10.13386/j.issn1002-0306.2020.13.046
引用本文: 冯瑞雪, 史瑛, 姬中伟, 钱肖华, 徐岳正, 毛健, 许正宏. 黄酒多肽的多级分离纯化及其对小鼠巨噬细胞免疫调节的影响[J]. 食品工业科技, 2020, 41(13): 289-295. DOI: 10.13386/j.issn1002-0306.2020.13.046
FENG Rui-xue, SHI Ying, JI Zhong-wei, QIAN Xiao-hua, XU Yue-zheng, MAO Jian, XU Zheng-hong. Multistep Separation and Purification of Huangjiu Peptides and its Effects on the Immune Regulation of Macrophage in Mice[J]. Science and Technology of Food Industry, 2020, 41(13): 289-295. DOI: 10.13386/j.issn1002-0306.2020.13.046
Citation: FENG Rui-xue, SHI Ying, JI Zhong-wei, QIAN Xiao-hua, XU Yue-zheng, MAO Jian, XU Zheng-hong. Multistep Separation and Purification of Huangjiu Peptides and its Effects on the Immune Regulation of Macrophage in Mice[J]. Science and Technology of Food Industry, 2020, 41(13): 289-295. DOI: 10.13386/j.issn1002-0306.2020.13.046

黄酒多肽的多级分离纯化及其对小鼠巨噬细胞免疫调节的影响

Multistep Separation and Purification of Huangjiu Peptides and its Effects on the Immune Regulation of Macrophage in Mice

  • 摘要: 本研究以黄酒为原料,通过大孔树脂吸附、凝胶色谱等方法多级分离纯化黄酒中活性肽组分,并探究各级分离肽组分对小鼠巨噬细胞RAW264.7免疫调节作用的影响。通过脂多糖LPS(1 μg/mL)刺激RAW264.7细胞建立体外炎症模型,以不同浓度的各级分离纯化的黄酒多肽样品进行干预,对NO释放抑制率最高的黄酒多肽组分进行结构分析。结果表明,可通过大孔树脂初步分离纯化黄酒活性多肽。对3种不同型号大孔树脂的吸附解析性能进行比较,最终选择DA201-C大孔吸附树脂富集黄酒多肽,收集洗脱液旋蒸冻干后获得黄酒多肽SJ组分。采用MTT法检测细胞活力,黄酒多肽SJ作用RAW264.7细胞的安全范围为≤2.5 mg/mL。采用Griess Reagent法检测细胞培养液中一氧化氮(NO)含量,与LPS模型组相比,黄酒多肽SJ浓度为0.3、0.6、1.2、2.5 mg/mL时能明显抑制NO释放(抑制率分别为20.85%、36.42%、68.58%、95.01%)。黄酒多肽SJ经Sephadex G-15凝胶色谱柱分离得到3个组分即G1、G2、G3,分别收集这三组洗脱峰并测定其对RAW264.7细胞NO分泌量的影响。研究发现G1组分活性高于其他2种组分,G1、G2、G3的NO释放半抑制浓度分别是0.68、0.86、0.75 mg/mL。对G1组分进行氨基酸组成分析得知其疏水氨基酸占比32.63%,必需氨基酸占比28.46%。研究结果证明黄酒多肽能显著抑制LPS诱导的RAW264.7分泌NO,对LPS刺激的RAW264.7细胞炎症具有保护作用,从而促使巨噬细胞发挥免疫作用。

     

    Abstract: In this study, the active peptide components in huangjiu were purified by macroporous resin adsorption, gel filtration chromatography and other methods. The effects of different peptide components on immune regulation of mouse macrophages were investigated.In vitro cell validation model was created by stimulating RAW264.7 cells with lipopolysaccharide (1 μg/mL), different concentrations of purified huangjiu peptide samples were used as intervention to determine their immune regulatory activity, the structure of the peptides in huangjiu with the highest inhibition rate of NO release was analyzed. The results showed that the active peptides could be separated and purified by macroporous resin. The adsorption and desorption capabilities of three different types of macroporous resin were compared, and DA201-C macroporous resin was finally selected to enrich the huangjiu peptide, and the eluent was collected to obtain the huangjiu peptide SJ after rotary evaporation and freeze-drying. The RAW264.7 cells were treated with different concentration of huangjiu peptides, cell viability was determined with MTT methods. The safe range of huangjiu peptide SJ was less than 2.5 mg/mL.The Griess Reagent method was used for the detection of nitric oxide (NO) content in cell culture solution. Compared with no intervention group (LPS model group), the huangjiu peptides SJ in the concentration of 0.3, 0.6, 1.2, 2.5 mg/mL could significantly inhibit the release of NO (the inhibition rates were 20.85%, 36.42%, 68.58% and 95.01%, respectively).The huangjiu peptide SJ were further separated by gel filtration on Sephadex G-15 into 3 peaks, namely G1, G2 and G3.Elution peaks of these three groups were collected.The study found that immune regulatory activity of G1 component was higher than the other two components, and the semi-inhibited concentration of NO release of G1, G2 and G3 were 0.68, 0.86 and 0.75 mg/mL, respectively. The amino acid composition analysis of G1 component showed that the proportion of hydrophobic amino acid was 32.63% and the proportion of essential amino acid was 28.46%. The results showed that huangjiu peptide could significantly inhibit LPS-induced RAW264.7 secretion of NO, and had a protective effect on LPS-stimulated RAW264.7 cell inflammation, thus promoting macrophages to play an immune role.

     

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