丝瓜多糖的提取纯化及其抗氧化活性的研究

发布时间：2018-03-04 18:27

本文选题：丝瓜　切入点：多糖　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：丝瓜,是一种常见的可供食用的植物,其化学成分复杂,含有多种活性成分。近年来,植物多糖的研究成为众人关注的焦点,主要是由于植物多糖是一种天然的具有良好生物活性的物质,可用于治疗多种疾病及提高人类的身心健康。然而对于丝瓜多糖的研究还处于初级阶段,研究丝瓜多糖的性质及结构为日后丝瓜多糖产品的研发提供了理论依据。本文主要对丝瓜多糖的提取条件、分离纯化方法、理化性质、抗氧化活性及结构进行了研究,为扩大丝瓜的使用范围,研究丝瓜功能性食品提供参考依据。试验内容与结果如下:(1)采用超声波-酶复合法对丝瓜多糖进行提取,利用响应面法优化丝瓜多糖提取工艺参数。以丝瓜多糖提取率为指标,对影响多糖提取率的四个因素:纤维素酶添加量、液料比、超声温度、超声时间,进行工艺参数优化。在提取过程中,因素对丝瓜多糖提取率的影响大小顺序为:液料比纤维素酶添加量超声温度超声时间;提取工艺的优化条件为:纤维素酶添加量5.6%,液料比33:1,超声温度72℃,超声时间30min,该条件下得到的丝瓜多糖提取率为16.74%,说明该方法能较好的用于丝瓜多糖提取条件的优化。将得到的粗多糖命名为LCP。(2)以丝瓜粗多糖LCP为原料,采用Sevage法脱蛋白,利用正交试验优化脱蛋白条件。结果表明在V氯仿:V正丁醇比为3:1,VLCP:VSevage比为5:1,振荡时间为20min的条件下脱蛋白效果最好,测得脱蛋白率为26.73%,多糖损失率为10.26%。(3)采用大孔树脂法对脱蛋白后的丝瓜粗多糖LCP经行了脱色试验。采用静态吸附筛选出了大孔树脂D301进行动态吸附试验,最后结果表明上样浓度3mg/mL,上样量为10mL,上样流速为2.5mL/min的条件下脱色效果最佳,得到的脱色率为74.05%,保留率为68.92%。(4)以脱蛋白脱色后的丝瓜粗多糖LCP为原料,采用DEAE-52纤维素柱层析,分别以蒸馏水、O.1mol/LNaCl溶液、0.2mol/LNaCl溶液、0.5mol/LNaCl溶液进行洗脱得到LCPⅠ、LCPⅡ、LCPⅢ、LCPⅣ四个组分,收集多糖含量最高的组分LCPⅡ,多糖含量为63.53%。将LCPⅡ利用Sephadex G-75凝胶柱层析纯化,以蒸馏水为洗脱液,得到单一的洗脱峰,收集浓缩、透析、冻干后得到LCPⅡ-1。经紫外光谱扫描图分析可知,LCPⅡ-1中几乎不含蛋白质、多肽、核酸等杂质。(5)将丝瓜多糖LCPⅡ-1经红外光谱扫描可知,LCPⅡ-1具有多糖特征吸收峰,存在毗喃环结构的单体。LCP Ⅱ-1的相对分子质量为16766。(6)分离纯化获得丝瓜多糖LCPⅡ-1为白色絮状固体,易溶于水,不溶于乙醇、乙醚、丙酮、乙酸乙酯等有机溶剂。经化学反应试验表明,该样品中含有多糖组分,不含有蛋白质、淀粉类物质。体外抗氧化活性测定结果显示,LCPⅡ-1对清除羟基自由基的能力较强,丝瓜多糖浓度为1.0mg/mL时,清除率最高为97.65%,和Vc相近;LCPⅡ-1对清除DPPH自由基的能力与Vc相比能力较弱,多糖浓度为10mg/mL时,清除率达到最高为39.62%;清除超氧阴离子自由基能力不及Vc,但随浓度增大呈上升趋势,当浓度为5mg/mL时,其清除率达到45.58%;LCPⅡ-1的总还原能力远不及Vc的还原能力,但在测量范围内呈上升趋势,具有一定的抗氧化能力。
[Abstract]:Luffa is a common edible plants, the complex chemical composition, contains many active ingredients. In recent years, the research of plant polysaccharides become the focus of attention, mainly because the plant polysaccharide is a natural substance with good biological activity, can be used for the treatment of various diseases and improve human the physical and mental health. However, for the study of Luffa polysaccharide is still at the primary stage, provides a theoretical basis for the research on the nature and structure of Luffa polysaccharide in the days after the gourd polysaccharide products. In this paper the extraction conditions of sponge gourd polysaccharide, methods of separation and purification, physicochemical properties, antioxidant activity and structure were studied, for to expand the scope of the use of sponge gourd, to provide reference for the research of Luffa functional food. The test contents and results are as follows: (1) the gourd polysaccharides were extracted by ultrasonic enzymatic method, optimization of Luffa using response surface method The extraction parameters in Luffa polysaccharide. The extraction rate of polysaccharide as the index, the four factors affecting the extraction rate of polysaccharides: the amount of cellulase, solid-liquid ratio, ultrasonic temperature, ultrasonic time, optimize the process parameters. In the extraction process, factors of Luffa polysaccharide extraction effect of the size of the order: liquid feed amount of ultrasonic temperature of ultrasonic time than cellulase; optimization of extraction conditions for cellulase addition 5.6%, solid-liquid ratio 33:1, ultrasonic temperature 72, ultrasonic time 30min, obtained the conditions of Luffa polysaccharide extraction rate is 16.74%, which shows that the method can optimize the extraction conditions of polysaccharide for Luffa better. The obtained crude polysaccharide named LCP. (2) in Luffa LCP polysaccharide as raw material, removal of protein by Sevage method, using orthogonal test conditions. The results show that the removal of protein in V chloroform: n-butanol V ratio of 3:1, VLCP: and VSevage ratio is 5:1, oscillation time Under the condition of 20min protein removal effect is the best, the measured protein removal rate was 26.73%, the polysaccharide loss rate was 10.26%. (3) by macroporous resin method for protein removal after the Luffa LCP crude polysaccharide were studied by static adsorption and decolorization test. Selected D301 macroporous resin by dynamic adsorption test, finally the results showed that the concentration of sample 3mg/mL, sample volume is 10mL, the optimum sample flow rate of decolorization effect under the condition of 2.5mL/min, the decolorization rate was 74.05%, the retention rate of 68.92%. (4) to remove protein after decolorization of Luffa LCP polysaccharide as raw material, using DEAE-52 cellulose chromatography respectively with distilled water. O.1mol/LNaCl solution, 0.2mol/LNaCl solution, 0.5mol/LNaCl solution was eluted with LCP I and LCP II, LCP III, LCP IV four components, collect the highest polysaccharide content components of LCP II, the polysaccharide content was 63.53%. LCP II by Sephadex G-75 gel column chromatography, distilled water As the eluent, elution peak, single collect concentration, dialysis and freeze-dried to obtain LCP II -1. by UV scan analysis, LCP II -1 almost no protein, polypeptide, nucleic acids and other impurities. (5) the gourd polysaccharide LCP II -1 by infrared spectroscopy scanning showed that LCP II -1 has the characteristic absorption peaks of polysaccharide monomer.LCP II and furan ring structure of the relative molecular weight of -1 was 16766. (6) purified from Luffa polysaccharide LCP II -1 white flocculent solids, soluble in water, insoluble in ethanol, ether, acetone, ethyl acetate and other organic solvents by. Reaction test showed that the samples containing polysaccharides, not contain protein, starch material. The determination results of antioxidant activity in vitro showed that LCP II -1 hydroxyl radical scavenging ability, gourd polysaccharide concentration is 1.0mg/mL, the clearance rate of up to 97.65%, and similar to Vc; scavenging DPPH free LCP II -1 Radical ability compared with Vc capacity is weak, the polysaccharide concentration is 10mg/mL, the clearance rate reached 39.62%; super anion free radical scavenging ability than Vc, but with the increase of concentration increased when the concentration of 5mg/mL, the removal rate reached 45.58%; LCP II -1 total reduction ability than the reduction ability of Vc, but the upward trend in the measurement range, has certain antioxidant capacity.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ281

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