【作者】 李颖畅；

【导师】 孟宪军；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2008， 博士

【摘要】 蓝莓是杜鹃花科（Ericaceae）越橘属（Vaccinium.spp）植物,在我国有丰富的蓝莓资源。研究表明,蓝莓果中含有丰富的花色苷类物质,具有消除眼睛疲劳,保护视力,以及抗氧化、抗衰老、抗溃疡、抗炎和抗癌等生理功能和药用功能。本文通过比较4个品种蓝莓花色苷含量的差异,筛选出花色苷含量比较高的“圣云”蓝莓作为试验材料,对其花色苷进行系统的研究,以明确其生物活性,为更好地开发和利用蓝莓资源提供理论支持。本文通过溶剂法和酶法研究蓝莓花色苷的提取工艺;研究AB-8型大孔树脂对花色苷静态和动态纯化条件,并采用聚酰胺和硅胶柱层析对花色苷进一步纯化,用硅胶薄层色谱对花色苷的组分成分进行分离。用反相高效液相色谱/质谱对蓝莓花色苷进行分离和组成成分分析;研究了蓝莓花色苷的稳定性,并对其进行微胶囊化和分子修饰;通过化学模拟试验对蓝莓花色苷的体外抗氧化作用进行了系统评价;通过动物试验研究花色苷对高脂血症大鼠血脂、抗氧化及动脉硬化的影响并探讨其作用机制。主要研究结论如下:1.溶剂法提取蓝莓花色苷最佳条件为:60%乙醇,料液比为1g:15mL,提取时间120min,pH3.0,提取温度40℃,提取2次即可。圣云蓝莓果中花色苷的含量约为328.91mg/100g鲜果。2.一定量的纤维素酶使蓝莓花色苷提取量提高,而果胶酶未能提高花色苷提取量,增加果胶酶用量,花色苷提取量下降。两种酶复合处理花色苷提取量低于相同用量纤维素酶的花色苷提取量,两种酶无协同作用。纤维素酶提取蓝莓花色苷的最佳条件为:酶用量5mg/g,料液比1g:8mL,酶解时间60min,pH5.0,酶解温度为45℃,提取2次即可,2次累计提取量与水提法相比,提高49.35%。3.通过对10种大孔树脂的静态吸附解吸研究,发现AB-8型大孔树脂是一种比较理想的树脂,吸附率大,解吸率高,较适合蓝莓果中花色苷类成分的纯化。AB-8型大孔树脂对花色苷的吸附属多分子层吸附,静态吸附解吸条件为:吸附平衡时间为4h,解吸平衡时间2h,吸附平衡最适浓度为750mg/L,pH3.0时吸附能力比较强,用pH3.0的60%乙醇溶液作为解吸液,低温有利于AB-8型树脂的吸附,而高温则有利于该树脂的解吸。动态吸附解吸条件为:吸附和解吸流速1mL/min,上样液浓度3.0mg/mL,用5倍柱床体积的60%酸性乙醇作为洗脱液。经大孔树脂纯化后的蓝莓花色苷为紫黑色粉末,再经硅胶、聚酰胺柱层析,其色价分别为79.20,85.30。以硅胶G作为层析介质,通过用不同的展开剂分离蓝莓花色苷,分离效果比较好的是正丁醇-乙酸-水（4:1:5）和水-乙醇-正丁醇-乙酸乙酯（12:8:24:36）为展开剂。4.蓝莓花色苷在酸性缓冲液和甲醇溶液中的可见区最大吸收波长分别520nm,535nm,通过高效液相色谱/质谱分析,“圣云”蓝莓含有17种花色苷单体,分别是芍药色素3-半乳糖苷、飞燕草色素3-半乳糖苷、矢车菊色素3-半乳糖苷、矢车菊色素3-葡萄糖苷、牵牛花色素3-半乳糖苷、牵牛花色素3-葡萄糖苷、矢车菊色素3-阿拉伯糖苷、芍药色素3-葡萄糖苷、锦葵色素3-半乳糖苷、锦葵色素3-葡萄糖苷、锦葵色素3-阿拉伯糖苷、牵牛花色素3-阿拉伯糖苷、飞燕草色素3-葡萄糖苷、芍药色素3-乙酰半乳糖苷、锦葵色素3-乙酰半乳糖苷、芍药色素3-乙酰葡萄糖苷、锦葵色素3-乙酰葡萄糖苷。通过制备型高效液相色谱制备2种花色苷单体,经紫外、红外、质谱、核磁共振分析鉴定,样品1主要为锦葵色素3-半乳糖苷,样品2主要为锦葵色素3-葡萄糖苷。5.蓝莓花色苷属于水溶性色素。该花色苷在pH小于3.0条件下比较稳定,在60℃以下热稳定性较好,在短时间内有一定的耐高温能力。对光比较敏感,微波对其稳定性无不良影响。该花色苷耐氧化还原性差,抗坏血酸使花色苷稳定性下降,加速花色苷溶液的褪色。蔗糖和葡萄糖对蓝莓花色苷稳定性均无不良影响,高浓度的蔗糖和葡萄糖对其有不同程度的护色效果。苯钾酸钠对蓝莓花色苷稳定性具有良好作用。K⁺不能使花色苷的吸光度增加,对花色苷的稳定性也无明显影响;Mg²⁺、Ca²⁺、Cu²⁺、Al³⁺具有增色作用,对花色苷的稳定性无显著影响:高浓度Na⁺、Zn²⁺、Mn²⁺具有增色作用,而且能够增强花色苷的稳定性:Fe²⁺、Fe³⁺、Pb²⁺对花色苷具有破坏作用,使花色苷的稳定性下降,含Fe³⁺、Pb²⁺的花色苷溶液出现浑浊,有沉淀生成。6.以阿拉伯胶为壁材微胶囊化蓝莓花色苷的工艺条件为:芯材与壁材比3:197,固形物含量为20%,进风温度为180℃,出风温度为70℃。以β-环糊精-麦芽糊精-阿拉伯胶为壁材微胶囊化蓝莓花色苷的工艺条件为:芯材与壁材比3:97,β-环糊精:麦芽糊精为1:2,阿拉伯胶占总固形物的百分比为7.5%,固形物含量为20%。以大豆分离蛋白-麦芽糊精为壁材对蓝莓花色苷进行微胶囊化的工艺条件:大豆分离蛋白与麦芽糊精比为3:7,芯材:壁材为4:96,固形物含量为15%。乙基纤维素作为壁材微胶囊化蓝莓花色苷的工艺条件为聚乙二醇含量15%,芯材与壁材比1:19,固形物含量4%。微胶囊产品在模拟胃液环境中释放率比较高,在模拟胃液环境中停留60min后,释放率均达到90%以上。蓝莓花色苷微胶囊化和酰化后,花色苷的光和热稳定性提高。7.蓝莓花色苷具有抗油脂和抗脂质体氧化能力,而且抗脂质体氧化能力强于抗坏血酸。蓝莓花色苷具有较强的还原能力:蓝莓花色苷具有清除羟基自由基、超氧阴离子自由基、DPPH自由基和H₂O₂能力。8.与高脂模型组相比,灌胃蓝莓花色苷能显著降低大鼠血清的总甘油三酯（TG）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）水平和载脂蛋白ApoB含量,显著提高高密度脂蛋白胆固醇（HDL-C）水平和载脂蛋白ApoAⅠ含量,动脉硬化指数显著下降。与高脂模型组相比,灌胃蓝莓花色苷可以有效提高大鼠血清和肝脏总抗氧化能力,抗氧化酶SOD和GSH-Px活性显著提高,脂质过氧化物MDA含量显著下降。结果表明:蓝莓花色苷能够降低高脂血症大鼠血脂水平,改善血脂代谢,抑制脂质过氧化,可以有效的预防动脉硬化（AS）的发生和发展。更多还原

【Abstract】 Blueberry is abundant and distributing over the country.Anthocyanins have been discussed in relation to a wide range of function characteristic such as vision improvement, antioxidant,antidecrepitude,antiulcer,antivirus and anticancer and so on.In this paper, Saintcloud blueberry with most anthocyanin content was selected after comparing anthocyanin content of 4 kinds of blueberry fruits.Anthocyanins were systematically studied from Saintcloud blueberry as material.It is very important to understand its bioactivities and use the blueberry resource.In this paper,anthocyanins were extracted from Saitcloud blueberry fruits.The optimum industrial process of extraction of blueberry anthocyanins were determined by solvent and enzyme method.Blueberry anthocyanins’ conditions of absorption and desorption which were static and dynamic were studied by AB-8 macroporous resin.Blueberry anthocyanins were purified by polyamide and silica-gel column chromatography.Blueberry anthocyanins were separated by thin-layer chromatography.Constitutes and structrue of blueberry anthocyanins was identified by RP-HPLC/MS.The stability of blueberry anthocyanins were tested.Microencapsulation and molecular modification of blueberry anthoeyanins were studied.The effect of blueberry anthocyanins on antioxidant was investigated by the method of chemical simulation in vitro.The effects of anthocyanions from blueberry fruits on hypolipidemic and antioxidative in hyperlipidemic rats were studied by animal tests.The main results were as follows:1.The optimum extraction technology parameters determined by ethanol were as follows: 60%ethanol,material-solvent ratio 1g:15mL,extraction time 120min,pH value 3.0, extraction temperature 40℃and extraction 2 times.Anthocyanins content was 328.91 mg/100g fresh fruits.2.The effect of cellulase,pectinase and their mixture on blueberry anthocyanins extraction was studied.The result showed that cellulose could enhance the extraction content of anthocyanins,but pectinse could not.In addition,the extraction content of anthocyanins by their mixture was lower than by cellulase.The optimum parameters of extraction technology by cellulase were enzymatic quantify 5mg/g,material-liquid ratio 1 g:8mL,enzymolysis time 60min,pH value 5.0,enzymolysis temperature 45℃and extraction 2 times.Blueberry anthocyanins content was more 49.53%by cellulase on extraction than water.3.Among 10 kinds of macroporous resins,AB-8 resin has better capabilities of absorption and desorption to purify crude blueberry anthocyanins.The conditions of static absorption and desorption of blueberry anthocyanins were that anthocyanins concentration in balance was 750mg/L,pH value of absorption was 3.0,the elution solvent was pH3.0 and 60%ethanol.The optimum conditions of dynamic absorption and desorption of blueberry anthocyanins were as follows:flow rate of absorption and desorption 1mL/min,concentration 3.0mg/mL.The elution solvent was 3BV（bed volume）60%ethanol.In those conditions,the anthocyanins produced by this technological process was purplish dark powder.Blueberry anthocyanins were purified by silica-gel and polyamide column chromatography after AB-8 resin purifying,the colour value was 79.20,85.30 respectively.Anthocyanins were separated by thin-layer chromatography.Separation was well done by butanol-acetic-water（4:1:5）and water-ethanol- butanol - acetic ester（12:8:24:36） as developing solvent.Two kinds of anthocyanins were isolated by preparative HPLC.Sample 1 was Malvidin3-arabinoside, Sample 2 was Malvidin3-glucoside by UV,IR,MS and NMR.4.The wavelength of absorption respectively was 520nm and 535nm in pH3.0 citric acid solvent and methanol.Saintcloud blueberry was found to contain 17 anthocyanins by HPLC/MS.The 17 of them were tentatively identified as Peonidin3-galactoside, Delphinidin3-galactosid,Cyanidin3-galactoside,Cyanidin3-glucoside,Petunidin3-galactoside ,Petunidin3-glucoside,Cyanidin3-arabinoside,Peonidin3-glucoside,Malvidin3-galactoside,Mal -vidin3-glucoside,Malvidin3-galactoside,Petunidin3-arabinoside,Delphinidin3-glucoside,Peon -idin3-（6"-acetyl）galactoside,Malvidin3-（6"-acetyl）galactoside,Peonidin3-（6"-acetyl） glucoside,Malvidin3-（6"-acetyl）glucoside.5.Blueberry anthocyanins are water-soluble pigments.The anthocyanins were stable under pH 3.0,below 60℃.It was heat-resistant to high temperature in a short time.The anthocyanins were sensitive to light and stable to microwave.It was found that oxidizer H₂O₂, reductive Na₂SO₃ and ascorbic acid could decrease the stability of anthocyanins and result in its more rapid discoloration.Sucrose and glucose had no harmful effect on the stability of blueberry anthocyanins,but had certain color maintenance at higher concentration.Sodium benzoate and K⁺ had no effect on the stability of anthocyanins.Mg²⁺,Ca²⁺,Cu²⁺and A1³⁺ had a certain effect of clout improvement,but no effect on the stability of anthocyanins.Na⁺,Zn²⁺ and Mn²⁺could not holy strengthen the colour at higher concentration,but enhance the stability of blueberry anthocyanins.Fe²⁺,Fe³⁺ and Pb²⁺ were harmful to the stability of blueberry anthocyanins while Fe³⁺ and Pb²⁺ could lead to precipitate in blueberry anthocyanins solution.6.The optimum technology parameters by using arabic gum as wall materials were as follows:the proportion of anthocyanins to wall materials was 3:197,emulsion concentration was 20%,inlet air and outlet air temperature were 180℃and 70℃respectively.The optimum technology parameters by using mixture ofβ-cyclodextrin,maltodextrinn,and arabic gum as wall materials were as follows:the ratio of core mateials to wall mateials 3:97,β-cyclodextfinto maltodextriun 1:2,arabic gum 7.5%,emulsion concentration 20%.The optimum technology parameters with mixture of soybean protein isolatidn（SPI） and maltodextrin showed that SPI and maltodextrin was in proportion of 3:7 as the materials,the proportion of core mateials to wall mateials was 4:96,the emulsion concentration was 15%（W/V）.The optimum technology parameters by using ethyl cellulose as wall materials were as follows:EPG concentration was 15%,the proportion of anthocyanins to wall materials was 1:19,emulsion concentration was 4%respectively.Anthocyanins of microencapsulation have higher releasing rate in vitro.Releasing rate could exceed 90%after 60min in gastric juice of simulation.The temperature and light stability of microencapsulation and anthocyanins of molecular modification could be improved.7.Blueberry anthocyanins could inhibit peroxidation of lard and lipid,and have stronger effect of lipid peroxidation than ascorbic acids.Blueberry anthocyanins have the reduction ability,and the positive correlation existed between reductive ability and concentration. Blueberry anthoeyanins could effectively scavenge hydroxyl free radicals,superoxide anions, DPPH and peroxide.8.The serum TG,TC,LDL-C concentration,ApoB content and atherosclerosis index in rats fed with anthocyanins from blueberry fruits（ABBF）were significantly lower than those in model group,while serum HDL-C concentration and ApoA I content were higher.Comparing with model group,T-AOC,SOD,GSH-Px were increased significantly in serum and liver by feeding with ABBF,while MDA content were decreased in serum and liver.ABBF could decrease blood lipids level,improve blood lipid metabolism,inhibit lipid peroxidation,inhibit formation and development of antherosclerosis.更多还原