【作者】 孙希云；

【导师】 孟宪军；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2011， 博士

【摘要】 蓝莓(Blueberry),又称越桔或蓝浆果,属杜鹃花科越桔属(Vaccinium)落叶灌木植物,因其具有较高的保健价值而风靡世界,是联合国粮农组织(FAO)推荐的五大健康水果之一。蓝莓中含有酚酸、花青苷及黄酮素等多种活性成分,国内外已有较深入研究。但对于蓝莓中含有的多糖还未见有详细报道,且有资料显示在蓝莓果渣中含有较多的多糖类物质(潘一峰,2005),具有非常大的潜在开发利用价值。本研究以蓝丰品种为原料,对蓝莓多糖进行了提取、纯化及分离鉴定,并对其体内抗肿瘤及免疫调节作用和部分体外活性进行了详细的研究,为今后蓝莓多糖的深入研究与开发利用奠定了理论基础。研究结果如下：(1)分别将水浸法、酶法、超声波法应用于蓝莓多糖的提取,并采用正交分析优化提取工艺,得到三种提取方法的最佳工艺条件。水浸法：料水比为1：60 g/mL,温度为90℃,时间为4 h,蓝莓多糖的得率为2.12%。酶法：酶解时间为100 min,酶解温度为40℃,酶添加量为0.6%,料水比为1：60 g/mL,蓝莓多糖的得率为2.32%。超声波辅助法：提取时间为40 mmin,提取温度为50℃,超声波功率为80W,料水比为1：60g/mL,蓝莓多糖的得率为2.34%。可以看出,超声波提取法成本低,效率高,而且提取量大,是提取蓝莓多糖较好的方法。(2)比较了Sevag法、三氯乙酸法、酶法与Sevag法结合脱蛋白的效果,结果表明酶法与Sevag法结合脱蛋白效率较高,其最佳处理条件为：木瓜蛋白酶用量为0.15%,pH 6.5,60℃恒温2h,离心得上清液,再用Sevag法脱蛋白4次基本上就可以将蛋白脱除干净,且此法对多糖的保留率较前两者都高,可达78.28%,是脱蛋白的较好方法。(3)采用双氧水进行脱色,综合考虑脱色率和最终样品的保留率,较好的选择应为：双氧水的添加量为40%,脱色pH为8.5,脱色温度为50℃,脱色时间为4 h,蓝莓多糖的脱色率为93.11%,多糖保留率为79.24%。(4)脱色脱蛋白后的多糖经过DEAE-52纤维素柱层析,分别用蒸馏水、0.1 mol/L NaCl、0.3 mol/L NaCl、0.5 mol/L NaCl洗脱,得到BBP1、BBP2、BBP3和BBP4四个组分,得率分别为11.77%、14.08%、34.65%和10.35%。将BBP3经Sephacryl S-300葡聚糖凝胶层析进一步分离纯化,经0.1 mol/L NaCl洗脱得到两个组分BBP3-1和BBP3-2。将BBP3-1经过紫外吸收光谱、Sephacryl S-300葡聚糖凝胶层析柱和HPLC检测验证其为单一多糖。(5)理化性质研究表明：蓝莓多糖BBP3-1为白色粉末固体,无特殊气味；易溶于热水,不溶于乙醇、乙醚、丙酮等有机溶剂；在水溶液中比旋光度[α]20D=+17。。鉴定反应表明：该多糖是不含有N元素,不含酚类、不含氨基酸或蛋白质的非淀粉类酸性多糖；BBP3-1重均分子量Mw为18643 Da,数均分子量Mn为9658 Da,峰位分子量Mp为3554 Da,分子量分布宽度(Mw/Mn)为3.186。蓝莓多糖(BBP)粘度特性研究表明：溶液浓度、温度、NaCl浓度、pH、热处理和剪切速率均对其黏度产生一定的影响。(6)气相色谱分析确定BBP3-1的单糖组成为：鼠李糖、半乳糖和葡萄糖,摩尔比为：1：1.5：2。红外光谱显示BBP3-1具有多糖的特征吸收峰,推测其是含有p-D-吡喃葡萄糖或半乳糖,不含甘露糖,含有糖醛酸的酸性多糖。核磁共振分析,可推测BBP3-1可能是以1,6键连接的吡喃葡萄糖链为主链,含有鼠李糖、半乳糖和葡萄糖的p构型多糖。(7)建立S180荷瘤小鼠模型,用400 mg/Kg体重.d、200 mg/Kg体重.d、100 mg/Kg体重.d三个剂量的蓝莓多糖溶液灌胃30d。结果表明,蓝莓多糖能显著抑制小鼠肿瘤的生长,且能提高荷瘤小鼠的胸腺指数、脾指数,显著增强荷瘤小鼠脾淋巴细胞增殖化能力,明显提高荷瘤小鼠腹腔巨噬细胞吞噬中性红的能力,显著促进荷瘤小鼠细胞因子TNF-a、IFN-γ、IL-2的分泌,明显提升荷瘤小鼠的NK细胞活性,说明蓝莓多糖可以通过免疫调节系统来抑制肿瘤细胞的生长,其中低剂量组100mg/Kg体重.d效果最好；对SRBC引起的小鼠迟发型过敏反应蓝莓多糖高剂量组400 mg/Kg体重.d有极显著的抑制作用。(8)蓝莓多糖体外给药对正常小鼠免疫功能有显著提升作用。实验表明,蓝莓多糖对脾淋巴细胞的转化能力有显著提升作用,但与ConA之间存在极明显的相互抑制作用；蓝莓多糖对巨噬细胞吞噬中性红的能力也有显著提升作用,且与ConA之间存在极明显的协同增效作用。(9)蓝莓多糖功能性质研究实验结果：抗氧化实验表明,蓝莓多糖对OH·和DPPH·自由基有较强的清除作用,且清除率随其浓度的增加而提高,但对02-·几乎没有作用效果；对豆油和猪油抗氧化作用表明蓝莓多糖具有一定的抗脂质过氧化作用,但作用不是很明显,与柠檬酸有显著协同增效作用。采用体外对α-淀粉酶的清除作用来评价蓝莓多糖的降血糖作用。试验结果表明,蓝莓多糖对α-淀粉酶的有一定的清除作用。通过对几种常见菌种的抑菌效果试验表明,蓝莓多糖没有明显的的抑菌作用。更多还原

【Abstract】 Blueberry,also known as bilberry or blue berry, belonging to the Vaccinium genus of Ericaceae, is a kind of deciduous shrub plant. It has fascinated the whole world for its higher value in health care and it is recommended by FAO as one of the most healthy fruits. Blueberry contains a variety of active ingredients, such as phenolic acid, anthocyanin and flavone etc., which have been studied intensively both home and abroad. But detailed reports on the polysaccharides from Blueberry are not seen yet and there is information shows that blueberry residue contains many polysaccharide substances (Pan Yifeng,2005), which has great potential for development and utilization. This paper, taking Lanfeng breed as raw material, extracted, purified, separated and indentified the polysaccharides from blueberry and carried out a detailed study on its anti-tumor and immunoregulation role in vivo and some activities in vitro. This study has laid a theoretical basis for future in-depth research and utilization of blueberry polysaccharides and its study results are as follows:(1) Immersion method, enzymatic method and ultrasonic wave method were used separately in extracting polysaccharides from blueberry and the extraction process was optimized by orthogonal test to acquire the optimal process conditions of each method. Immersion method:ratio of material to water 1:60 g/mL,Temperature 90℃, time 4 hours, the yield of blueberry polysaccharide is 2.12%. Enzymatic method:enzymolysis time 100 mins, temperature 40℃, enzyme additive amount 0.6%, ratio of material to water 1:60 g/mL, and the yield of polysaccharide is 2.32%. Ultrasonic-assisted extraction method:extraction time 40mins, temperature 50℃, power of ultrasonic wave 80 W, ratio of material to water 1:60 g/mL, the yield of polysaccharide is 2.34%. The results indicated that the ultrasonic-assisted method is characterized by low cost, high efficiency and the more extractive quantities. Therefore, the ultrasonic-assisted method has already been suggested as a way of extractiving polysaccharides from blueberry.(2) Effect of de-protein was compared between Sevag method, TDA method and Enzyme method combined with Sevag, the results showed Enzyme method combined with Sevag has higher de-protein efficiency. Its optimal treatment conditions are:papain usage is 0.15%, pH 6.5,60℃constant temperature for 2 hours, supernatant was acquired by centrifugation, then de-protein 4 times by Sevag method to basically remove protein thoroughly. And this method showed better retention ratio for polysaccharide than the first two methods, blueberry polysaccharide retention ratio can reach 78.28%, indicating that it is a better method.(3) Hydrogen peroxide was used for decoloration. Considering the decloration rate and the retention rate of the final sample, the better choice is the hydrogen peroxide additive amount is 40%, pH 8.5, temperature 50℃, treatmeat time 4 h.The resultant decoloration rate and retention rate of blueberry polysaccharide are 93.11% and 79.24% respectively.(4) After decoloration and de-protein, blueberry polysaccharide went thorough DEAE-52 cellulose colum chromatography and then washed out by distilled water, 0.1mol/L NaCl、0.3 mol/L NaCl、0.5 mol/L NaCL in order, the four components of BBP1、BBP2、BBP3 and BBP4 were acquired at 11.77%、14.08%、34.65% and 10.35% yield respectively. BBP3 was further purified by going through Sephacryl S-300 chromatography and washing out by 0.1 mol/L NaCl to get BBP3-1 and BBP3-2 components. BBP3-1 was tested and verified as single polysaccharide through ultraviolet absorption spectrometry, Sephacryl S-300 chromatography and HPLC analysis.(5) Physicochemical properties research shows:BBP3-1 is white powder solid, no special odor, easy soluble in hot water and insoluble to organic solvents, like ethanol, ether and acetone etc. Its specific rotation in water solution is [α]20D=+17°. This polysaccharide was tested and verified as a kind of none-starch acid polysaccharide which does not contain N element, phenol, amino acids or protein. The weight-average molecular weight (Mw) of BBP3-1 is 18643 Da, the number-average molecular weight (Mn) of it is 9658 Da and the peak position molecular weigh (Mp) is 3554 Da and the distribution width of molecular weight (Mw/Mn) is 3.186. Viscosity character of blueberry polysaccharide research indicates: solution concentration, temperature, NaCl concentration, pH, heat treatment and shearing rate all have some certain effects on the viscosity of BBP.(6) The constituents of the monosaccharide BBP3-1 were determined by gas chromatography (GC) as:rhamnose, galactose and glucose at molar ratio of 1:1.5:2. Infrared spectrum(IR) displayed that the BBP3-1 has the characteristic absorption peak of polysaccharides and therefore, it was inferred that it is a kind of acid polysaccharide with uronic acid. It containsβ-D-glucopyranose or galactose, but does not contains mannose. Through nuclear magnetic resonance(NMR) analysis, it may infer that the BBP3-1 is aβ-polysaccharide of which the 1-6-glucopyranose chain as its main chain and it contains rhamnose, galactose and glucose.(7) S180 tumor-bearing mice model was established through lavage with 30d blueberry polysaccharide solution at 400 mg/Kg weight.d、200 mg/Kg weight.d、100 mg/Kg weight.d separately. The results indicated that the blueberry polysaccharide can significantly inhibit the mouse tumor’s growth and lift up the thymus index, spleen index of the tumor-bearing mice, notably enhance the proliferation capacity of the tumor-bearing mice’s spleen lymphocyte, markedly strengthen the cytophagy of the macrophage inside the tumor-bearing mice’s abdominal cavity on neutral red, strikingly encourage the secretion of TNF-α、IFN-γ、IL-2, sharply increase the viability of NK cell of the tumor-bearing mice, indicating that the blueberry polysaccharide can inhibit tumor’s growth through immune regulatory system. The lowest dosage of 100mg/Kg.d showed best effect. The result of delayed hypersensitivity induced by SRBC showed that blueberry polysaccharide in high dosage of 400 mg/Kg体重.dinhibits delayed hypersensitivity significantly.(8) Drug delivery of blueberry polysaccharide in vitro can also significantly boost the immunologic function of the normal mice. The study showed that blueberry polysaccharide significantly promotes the transformation capacity of the spleen lymphocyte, but there exists a mutual inhibitory action between it and ConA. Blueberry polysaccharide can also significantly strengthen the cytophagy of the macrophage on neutral red, also there exists a significant synergy between it and ConA.(9) Some functional properties of blueberry polysaccharide were studied. Anti-oxidation test in vitro showed the blueberry polysaccharide has strong clearing effect on OH·and DPPH·radicals and its clearing rate increases with its concentration increasing, but it has hardly any effect on O2·-. The test of blueberry polysaccharide on soybean oil and lard oil’s anti-oxidation showed it has some effect on anti-lipid peroxidation, but its effect is not significant. It also found it has synergy effect with citric acid. By using blueberry polysaccharide’s clearing action in vitro on a-amylase, the effect of lowering blood-glucose was evaluated. The test showed that it has some certain effect on clearingα-amylase. Through the inhibitory effect test on several common strains, it showed that blueberry polysaccharide do not have significant inhibitory effect.更多还原