【摘要】：濱海白首烏中蛋白質(zhì)和多糖含量豐富,是其具有生物理活性的重要物質(zhì)基礎(chǔ),具有較好的研究和開發(fā)的價值。本研究分別提取濱海白首烏中這兩種重要物質(zhì),對其功能性進行全面的分析,以期為其在食品工業(yè)中的應(yīng)用提供理論依據(jù)。本研究對白首烏中的蛋白質(zhì)進行提取,評價了其理化性質(zhì)和加工性質(zhì);然后采用響應(yīng)面法優(yōu)化白首烏多糖的提取條件,并對所得粗多糖進行純化并分析了白首烏多糖的理化性質(zhì)和體外抗氧化性。具體研究內(nèi)容及實驗結(jié)果如下:首先本研究比較不同溶劑提取法對白首烏可溶性蛋白得率的影響,通過電泳法分析了蛋白的提取效果,同時對白首烏可溶性蛋白進行初步表征;采用液相色譜串聯(lián)質(zhì)譜法對提取得到的白首烏蛋白進行鑒定:大豆分離蛋白作為對照,對白首烏蛋白的加工特性進行分析。結(jié)果表明:PBS法提取白首烏蛋白優(yōu)于傳統(tǒng)的堿液提取和Tris-HCl法提取;白首烏蛋白的分子量主要分布在348 kDa左右,另一條相對遷移率較低、分子量在700 kDa附近的條帶推測為其聚集體,白首烏蛋白分子主要由一種亞基(分子量為28 kDa)構(gòu)成:白首烏蛋白具有良好的加工性能,特別是持油性、乳化性及乳化穩(wěn)定性和起泡性,均好于大豆分離蛋白,白首烏蛋白的溶解性與大豆分離蛋白的溶解性隨pH變化趨勢基本一致。實驗選擇對料液比、提取溫度和提取時間3個因素進行單因素試驗。根據(jù)單因素試驗結(jié)果設(shè)計中心組合試驗,以多糖得率為響應(yīng)值,采用響應(yīng)面分析法獲得白首烏多糖的最優(yōu)工藝參數(shù)。結(jié)果表明,白首烏多糖的最優(yōu)提取工藝為料液比1:17、提取溫度為91℃、提取時間為3 h。在此條件下,白首烏多糖得率為1.58%,與模型的預期值1.61%基本相符。白首烏多糖提取液經(jīng)過酶法淀粉、Sevge法去蛋白、95%的乙醇沉淀后,再經(jīng)過透析和凍干后得到不含有核酸和蛋白質(zhì)粗多糖。經(jīng)過DEAE-Sepharo Fast Flow陰離子交換色譜和葡聚糖G-150凝膠色譜柱色譜對白首烏多糖進行了分級分離,最終得到一個純化組分。通過HPGPC測定其相對分子質(zhì)量為830.93 KDa。采用PMP柱前衍生化法分析白首烏多糖的單糖組成,結(jié)果表明白首烏多糖主要是由半乳糖醛酸、半乳糖、阿拉伯糖、鼠李糖、葡萄糖醛酸和甘露糖組成,其摩爾比為甘露糖:鼠李糖:葡萄糖醛酸:半乳糖醛酸:半乳糖:阿拉伯糖=0.058:0.280:0.071:1.455:0.918:0.469。采用4種抗氧化活性測定的方法,對濱海白首烏抗氧化清除自由基能力進行分析和研究,結(jié)果表明:白首烏多糖具有一定的清除DPPH自由基、超氧自由基、ABTS+·自由基能力,并且隨著多糖的濃度增加,清除率逐漸增高。清除DPPH自由基、超氧自由基、ABTS+·自由基能力的IC_(50)分別為0.5543mg/mL、0.5881mg/mL和0.1232 mg/mL,其中清除ABTS+·自由基能力較強,與VC相接近;白首烏多糖的ORAC值為109.94μmol Trolox/g,表明白首烏多糖具有較強的抗氧化能力。最后,通過建立TNF-α誘導人臍靜脈血管內(nèi)皮細胞產(chǎn)生氧化應(yīng)激反應(yīng)模型,來研究白首烏多糖的抗氧化性。結(jié)果發(fā)現(xiàn),白首烏多糖可顯著提高由TNF-α誘導的人臍靜脈血管內(nèi)皮細胞的活力和SOD酶活性,顯著抑制細胞中活性氧(ROS)含量,這表明白首烏多糖能夠減少血管內(nèi)皮細胞受到氧化損壞。
[Abstract]:The content of protein and polysaccharide in the coastal white fleece-flower root is rich, it is the important material foundation of its physical activity, and has good value for research and development. In this study, the two important substances in the coastal white fleece-flower root are respectively extracted, and the function of the two important substances is comprehensively analyzed, so as to provide a theoretical basis for the application of the two important substances in the food industry. In this study, the extraction of protein from Radix Cynanchi Auriculati was carried out, and its physical and chemical properties and processing properties were evaluated. Then, the extraction condition of the polysaccharide was optimized by the response surface method, and the obtained crude polysaccharide was purified and the physical and chemical properties and the in vitro oxidation resistance of the polysaccharide were analyzed. The contents and experimental results are as follows: First, the effect of different solvent extraction method on the soluble protein of Radix Polygoni Multiflori is compared, and the effect of protein extraction is analyzed by electrophoresis, and the soluble protein of Radix Cynanchi Auriculati is initially characterized. The obtained white fleece-flower root protein was identified by liquid chromatography-tandem mass spectrometry: as a control, the processing characteristics of the white fleece-flower root protein were analyzed. The results showed that the extraction of the white fleece-flower root protein by the method of PBS was better than that of the conventional alkaline solution extraction and the Tris-HCl method, the molecular weight of the white fleece-flower root protein was mainly about 348 kDa, the other relative mobility was low, and the band with the molecular weight near 700 kDa was presumed to be an aggregate, The white fleece-flower root protein is mainly composed of a subunit (molecular weight of 28 kDa), the white fleece-flower root protein has good processing performance, in particular to oil-holding, emulsifying and emulsifying stability and foaming property, The solubility of the white fleece-flower root protein and the solubility of the soybean protein isolate are basically consistent with the change of pH. The single factor test was carried out for three factors, such as the ratio of liquid to liquid, the extraction temperature and the extraction time. Based on the results of single factor test, the optimal process parameters of the polysaccharide of Cynanchum multiflorum were obtained by using the response surface analysis method. The results showed that the optimum extraction process of the polysaccharide was 1: 17, the extraction temperature was 91 鈩，

本文編號：2358993