采用Isoptericola halotolerans CGMCC 5336褐藻胶裂解酶制备褐藻寡糖,通过液质联用技术(liquid chromatograph-mass spectrometer, LC-MS)对其组成进行初步分析,后经过凝胶色谱柱对其进行分离、纯化,得到4种组分,分别命名为Fr1、Fr2、Fr3、Fr4。通过LC-MS分析各组分纯度并确定分子量,使用G-25凝胶柱进行脱盐。通过RT-PCR和Western Blot技术,研究所获得的4个组分对低密度脂蛋白受体(low-density-lipoprotein receptor,LDLR)基因和蛋白水平的调节作用以及对前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)蛋白水平的调节作用。结果表明,Fr1中含有2种聚合度的寡糖,为五糖和六糖;Fr2、Fr3和Fr4均为聚合度均一的寡糖,分别为四糖、三糖和二糖。4个组分中,Fr1、Fr3、Fr4均能显著上调LDLR的蛋白水平,其中Fr3和Fr4的活性尤为显著。Fr3能下调PCSK9的蛋白水平,推测Fr3对LDLR蛋白表达的上调作用可能与PCSK9有关。而Fr4对LDLR的上调作用与PCSK9无关,可能受固醇反应原件结合蛋白-2(SREBP-2)的调节。结果表明,Fr3和Fr4可开发作为食品添加剂用于降低血浆胆固醇。
Alginate oligosaccharides were prepared by hydrolyzing alginate with algae lyase from Isoptericola halotolerans CGMCC 5336. The components were analyzed by liquid chromatography-mass spectrometry and purified by gel chromatography. Four main components, named Fr1, Fr2, Fr3, and Fr4, were purified and identified. Their effects on the transcription and translation of low-density-lipoprotein receptor (LDLR) gene and pro-protein convertase subtilisin/kexin type 9 (PCSK9) were investigated. The results showed that Fr1 contained two oligosaccharides, pento- and hexo-oligosaccharides; Fr2, Fr3 and Fr4 were tetra-, tri- and di-oligosaccharides. Among them, Fr1, Fr3, and Fr4 significantly up-regulated the level of LDLR while Fr3 down-regulated the level of PCSK9, indicating that the up-regulated LDLR by Fr3 might be related to PCSK9. The up-regulation of LDLR by Fr4 was not related to PCSK9, however, might be regulated by the sterol response binding protein-2 (SREBP-2). These indicated that Fr3 and Fr4 may be further developed as new food additives for lowering the plasma cholesterol level.
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