【作者】 杨强；

【导师】 李新华；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2013， 博士

【摘要】 银杏(Ginkgo biloba L)又称白果树、公孙树,属银杏科银杏属裸子植物,主产中国(梁立兴,1988)。其叶、果和外种皮等皆具有药用价值,被称为“全身都是宝的活化石”(张丽娇,等.2009)。银杏果是银杏树的种子,因其中种皮为白色的硬壳,故俗称白果。近年来,国内外对银杏果的研究主要集中在银杏酸、银杏酚、银杏蛋白及银杏油上,对银杏果多糖的报道很少。本研究以银杏果为原料,对银杏果中淀粉的理化性质和糊化特性进行了研究,对银杏果中非淀粉多糖的提取、分离纯化、结构鉴定、降血脂以及抗氧化活性进行了详细研究,以期为银杏果的研究和开发奠定理论基础。主要结论如下：(1)从银杏果中提取银杏果淀粉,对其特征性状进行了分析,结果表明：银杏果淀粉中的直链淀粉含量较高,约为32.1%；银杏果淀粉颗粒多呈多面球形和卵圆形,分子大小分布较为均匀,淀粉颗粒表面较为光滑；银杏果淀粉的透明度、溶解度、膨胀度比玉米淀粉高,比马铃薯淀粉低；凝沉性、冻融稳定性比玉米淀粉强,比马铃薯淀粉差；银杏果淀粉的粘度介于马铃薯淀粉和玉米淀粉之间；与马铃薯淀粉和玉米淀粉相比,银杏果淀粉的热稳定性差、抗老化能力弱；随着淀粉乳浓度的增加,银杏果淀粉糊的粘度增大,热稳定性下降；随着转数的增加,银杏果淀粉糊的粘度下降、热稳定性和抗老化性增强、糊化时间缩短、糊化温度降低；食盐的添加降低了银杏果淀粉糊的粘度、提高了热稳定性、增强了抗老化能力、出峰时间延长、糊化温度升高；蔗糖的添加提高了银杏果淀粉糊的粘度,延长糊化时间；柠檬酸的添加降低了银杏果淀粉糊的粘度和热稳定性,增强了银杏果淀粉糊的抗老化能力。(2)分别将热水浸提法、微波法应用于银杏果非淀粉多糖的提取,并采用正交分析优化提取工艺,得到两种提取方法的最佳工艺条件。热水浸提法：料液比为25mL/g、温度为90℃、时间为5h,在此条件下银杏果多糖的提取率为2.76%。微波法：微波辅助浸提法提取银杏白果多糖的最佳工艺条件为：料液比为40mL/g、微波功率为700W、萃取温度为50℃、萃取时间为5min,在此条件下银杏果多糖的提取率为4.87%。可以看出,微波提取法成本低,效率高,但微波法有可能会对多糖结构造成一定的影响。(3)研究了银杏果非淀粉多糖的脱蛋白、脱色工艺,并对工艺条件进行了优化。脱蛋白工艺：通过对Sevage法、三氯乙酸法、盐酸法和酶+Sevage法四种脱蛋白方法的比较,选取了木瓜蛋白酶联合Sevage法,在单因素试验的基础上进行正交试验,得出了木瓜蛋白酶联合Sevage法脱蛋白的最佳条件：酶用量O.1g,酶解温度40℃,酶解时间1.5h、溶液pH为7,在此条件下的蛋白脱除率为71.2%,多糖损失率15.3%。脱色工艺：采用大孔树脂法对银杏果多糖进行脱色,通过静态试验筛选出了脱色1号、D900和DA-201C三种树脂进行正交试验,通过正交试验和动态吸附试验对脱色条件进行优化,结果表明：脱色1号是银杏果多糖脱色的理想树脂。色素脱除的优化条件为：pH值为4.5,温度为25℃,上柱速度为1.5mL/min,上样浓度为选择4mg/mL,柱容量为2BV的条件下,多糖的脱色率为82.37%,多糖保留率为79.12%,蛋白去除率为88.39%。(4)脱色脱蛋白后的银杏果多糖(命名为GBSP)经过DEAE-52纤维素柱层析,分别用蒸馏水、0.lmol/L NaCl、0.3mol/L NaCl、0.5mol/L NaCl洗脱,主要得到蒸馏水洗脱组分GBSP1和0.1mol/L NaC洗脱组分GBSP2两个组分,得率分别为29.7%和37.9%。将GBSP1经SephadexG-200葡聚糖凝胶层析进一步分离纯化,经蒸馏水洗脱得到两个组分GBSP1-1和GBSP1-2,纯度分别为79.48%、100%。将纯化所得银杏果多糖GBSP1-2,经过紫外吸收光谱、SephadexG-200葡聚糖凝胶层析柱和HPLC检测验证为较纯的多糖,纯度可达100%。(5)银杏果多糖GBSP1-2理化性质表明：GBSP1-2中不含淀粉、蛋白质、还原糖等；为淡黄色粉末固体,无特殊气味；易溶于热水,不溶于乙醇、乙醚、丙酮等有机溶剂。液相色谱分析法分析银杏果多糖GBSP1-2的单糖组成为：甘露糖、半乳糖醛酸、葡萄糖和半乳糖。银杏果多糖GBSP1-2红外光谱显示,银杏果多糖GBSP1-2具有多糖的特征吸收峰,可推测多糖GBSP1-2是含有β-吡喃糖苷键的酸性多糖。(6)银杏果非淀粉多糖降血脂功能作用的试验结果表明：银杏果多糖能显著降低经高脂饲料喂养后小鼠的体重,能使肝指数下降、肾指数升高；银杏果多糖能显著降低高脂血症小鼠血清中TC、TG和LDL-C的含量、提高HDL-C的含量,且高剂量组和阳性对照组各项指标之间均无明显差异,说明高剂量的银杏果多糖具有降血脂功能,对高脂血症的发生有一定的预防作用；银杏果多糖能够显著降低动脉粥样硬化指数(AI),说明其对冠心病和动脉粥样硬化有一定的预防作用。(7)银杏果非淀粉多糖体外抗氧化活性验结果表明,银杏果多糖对DPPH自由基、超氧自由基和羟基自由基的清除效果显著,且随质量浓度的增大而增大,但清除效果不如抗氧化剂Vc。(8)银杏果非淀粉多糖体内抗氧化活性试验对高脂血症模型小鼠体内血清和肝脏中蛋白质、SOD、GSH-Px、CAT和MDA的变化进行了研究。结果显示,高脂血症可使小鼠血清和肝脏中蛋白质、SOD、GSH-Px和CAT的含量降低、MDA的含量升高,而银杏果多糖则能显著提高血清和肝脏中蛋白质、SOD、GSH-Px和CAT的含量、降低血清和肝脏中MDA的含量,具有很好的抗氧化作用。更多还原

【Abstract】 Ginkgo{Ginkgo biloba L), also known as white nut,maidenhair tree, is a gymnosperm Ginkgo Ginkgo and is mainly produced in China (Liang Lixing,1988). Leaves, nuts and testa mused has medicinal value, known as the "body is a treasure living fossil"(Zhang Li Jiao, etc.,2009). Ginkgo ginkgo is seeds of tree, due to which the seed coat of white hard shell and it is commonly known as ginkgo. In recent years, research of ginkgo mainly is in ginkgo acid, ginkgo phenol, the ginkgo protein and oil ginkgo but rarely the polysaccharide reports of ginkgo nut. In this study, ginkgo nut as raw material, physical and chemical properties of the ginkgo starch polysaccharides and pasting properties were studied, and the non-starch polysaccharides ginkgo extract, purification, structural identification, lipid-lowering effect and antioxidantdetailed study of the theoretical basis for the research and development of the ginkgo. The main conclusions are as follows:(1) Ginkgo starch polysaccharide extracted from ginkgo nut and its characteristic traits were analyzed, the results showed that:ginkgo starch amylose content is higher, and at around32.1%; Ginkgo nut starch granules is mostly multi-faceted spherical and oval, which molecular size distribution is more uniform and smooth surface of starch granules. Transparency, solubility, expansibility, retrogradation and freeze-thaw stability of ginkgo starch are better than corn but worse than potato; Viscosity of ginkgo starch is between corn and potato, close to potato, twice as much as corn. Compare with potato and corn, the thermostability and anti-aged of ginkgo starch are worse; With the increasing of the concentration of starch milk, the starch viscosity is strengthened and thermostability is weakened. With the increasing of the speed, the starch thermostability and anti-aged are strengthened and viscosity is weakened, the time of gelatinization is shorter and the temperature is lower. The addition of sodium chloride decreases the viscosity and improves the anti-aging and thermostability, makes the peak time later and the pasting temperature higher. The addition of sucrose improves the viscosity, makes the pasting time later. The addition of citric acid decreases the viscosity and thermostability and strengthens the anti-aging.(2) The hot water extraction and the microwave method respectively applied to the extraction of ginkgo non-starch polysaccharides and orthogonal optimize the extraction process, to get the optimum conditions of the two extraction methods. The hot water extraction:solid-liquid ratio to25mL/g, a temperature of90℃and for5h, and2.76%of the the polysaccharides extraction rate under this condition ginkgo nut.Microwave method:Microwave-assisted Extraction of Ginkgo biloba ginkgo polysaccharide optimum conditions:the solid-liquid ratio of40mL/g, microwave power to700W, extraction temperature of50℃and extraction time of5min under this condition ginkgo nut polysaccharide extraction rate of4.87%. As it can be seen, the microwave extraction is low of cost, high efficiency, but the microwave method might have affected in polysaccharide structure.(3) Non-starch polysaccharides of ginkgo nut of the deproteinized, bleaching process, and the process conditions were optimized.The deproteinized process:Comparison of four methods of removing protein of the Sevage law, trichloroacetic acid, hydrochloric acid and enzymes+Sevage law, and selected Papain Stock Sevage method on the basis of single factor test orthogonal test of which was out the papain Stock Sevage of law deproteinized the best conditions:enzyme dosage0.1g, reaction temperature40℃, the hydrolysis time1.5h and the pH of the solution is7Under these conditions, and the protein removal rate of71.2%, and polysaccharideloss rate of15.3%.Bleaching process:decolorization of ginkgo nut polysaccharide by macroporous resin method, and screened out by static tests Decolorization No.1, the D900and DA-201C of three resins orthogonal test, the bleaching conditions by orthogonal test and dynamic assayoptimization, and results show that:Decolorization No.1is a ideal resin of ginkgo nut polysaccharide decolorization. The pigment removal optimal conditions:the pH of the solution is4.5, a temperature of25℃, the speed of the column was1.5mL/min, the concentration of the sample of selection is4mg/mL and the column capacity is2BV conditions under polysaccharide decolorization rate of82.37%, polysaccharides retention rate of79.12%, and protein removal rate of88.39%.(4) Bleaching the polysaccharide after the deproteinized after DEAE-52cellulose column chromatography, and respectively with distilled water,0.1mol/L NaCl,0.3mol/L NaCl,0.5mol/L NaCl elution primarily and then to get GBSP1and GBSP2, two a component, and yield29.7%and37.9%, respectively. GBSP1by the SephadexG-200dextran gel chromatography are further isolated and purified and then distilled water afforded two components GBSP1-1, and GBSP1-2, and purity of79.48%and100%, respectively. Purified ginkgo nut polysaccharide of GBSP1-2after UV absorption spectrum, Sephadex G-200Sephadex chromatography and HPLC validation pure polysaccharide, up to100%purity.(5) Physical and chemical properties show that:the polysaccharide GBSP1-2of ginkgo nut does not contain starch, protein, reducing sugar and so on; solid as a pale yellow powder, no special smell; soluble in water, insoluble in alcohol, ether, acetone and other organic solvents. By high performance liquid chromatography analysis the the ginkgo polysaccharide GBSP1-2single sugar composition:mannose, galactose, glucuronic acid, glucose and galactose. The infrared spectrum of the polysaccharide GBSP1-2of the Ginkgo nut show that ginkgo nut the polysaccharide GBSP1-2is a polysaccharide characteristic absorption peak and speculated that the polysaccharide GBSP1-2is the acidic polysaccharide containing beta-pyran-glycosidic bond.(6) The ginkgo nut NSP lipid-lowering function of the test results show that:ginkgo nut polysaccharides can significantly reduce the weight by the high fat diet mice, liver index can decrease,renal index increased; ginkgo nut polysaccharides can significantly reduced the TC, TG and LDL-C, increased HDL-C of mice, and no significant differences between the high-dose group and positive control group the indicators, indicating that high doses of ginkgo nut polysaccharide with lipid-lowering function for the occurrence of high-hyperlipidemia some preventive effect; ginkgo nut polysaccharides can significantly reduce the atherosclerosis index (AI) and have some preventive effect on coronary heart disease and atherosclerosis hardening.(7) Ginkgo non-starch polysaccharides in vitro antioxidant activity of experimental results show that ginkgo nut polysaccharides on DPPH radical, superoxide and hydroxyl radicals scavenging effect was significant, and increases with the increase of the mass concentration, but Cleareffective as antioxidants Vc.(8) Non-starch polysaccharides of ginkgo nut antioxidant activity in vivo experiment use hyperlipidemia model mice and research the serum and liver protein, SOD, GSH-Px, CAT and MDA changes. The results showed that hyperlipidemia mice can in serum and liver protein, SOD, GSH-Px and CAT decreased, MDA content increased but ginkgo nut polysaccharides can significantly improve the protein in serum and liver SOD GSH-Px and CAT content, decreased serum and liver MDA content, and with good antioxidant.更多还原