【作者】 孙利芹；

【导师】 王长海；

【作者基本信息】 大连理工大学 ， 生物化工， 2009， 博士

【摘要】 紫球藻（Porphyrodium cruentum）是红藻门中唯一的单细胞类群,在生长过程中能合成一类细胞外多糖（Extracellular polysaccharide;EPS）,其产量很大程度上受到培养条件的影响。国内外关于紫球藻的培养和活性成分的研究取得了一定进展,但有关紫球藻多糖分子修饰,尤其是在紫球藻多糖结构与其生物活性之间的关系研究方面尚不多见。本文采用生态调控和工艺培养条件优化等手段,有效地提高了紫球藻多糖的产量,为紫球藻多糖的大量制备奠定了良好的基础。在紫球藻多糖理化性质研究和结构分析的基础上,利用对多糖的降解和分子修饰等手段获得了多种不同分子质量和不同硫酸基含量的多糖,并对这些不同性质多糖的抗氧化、抗肿瘤和免疫调节活性以及生物活性与分子结构之间的关系进行了详细的研究,取得了以下研究结果:1、提高紫球藻多糖产量的培养条件（1）通过考察无机盐、金属离子及植物生长激素等对紫球藻多糖合成的影响,获得了有利于提高多糖产量的培养基配方（OCMⅡ）:NaHCO₃ 3.5g,KH₂PO₄20mg,NaNO₃2g,Na₂SO₄20mg,FeCl₂·6H₂O 10μM,MnCl₂·4H₂O 0.2μm,V_B1 0.9 mg,V_B12 2.7μg,无菌海水1000mL。采用OCMⅡ培养基、15d培养,紫球藻多糖的最大产量达到了672 mg/L,分别比2/f和Koch对照培养基的多糖产量提高了1.79和1.62倍。（2）在自行研制的平板式光生物反应器中,考察了光照密度、光照时间、光质和反应器光径对紫球藻多糖合成的影响。在光通量密度为80μE·m^-2s^-1、光暗循环周期为18:6、自然光照射条件下,多糖的产量达到0.96g/L。在光径为30mm的反应器中多糖产量高达1.13g/L,而在光径为50mm的反应器中多糖的面积产率最高达到5.80 g/m²/d的水平。（3）采用均匀设计法对紫球藻半连续培养的工艺参数进行优化。在NaN03浓度、更新速率和更新周期分别为3.5 g/L、27%和2.91d的优化条件下,经过23d的培养,紫球藻多糖的总采收量达到29.4 g,平均多糖产量为1.96g/L,分别是批式培养的2.35和1.57倍。2、不同性质紫球藻多糖的制备及其结构性质分析（1）利用DE52离子交换柱层析及Sephacryl S-400凝胶过滤柱层析获得了纯化的多糖。该多糖的平均分子量为2918kDa,硫酸基含量14.63%,糖醛酸含量7.8%;多糖溶液为典型的假塑性流体特征,其粘度不受温度、加热时间等的影响。GC分析表明该多糖由木糖、葡萄糖和半乳糖组成,为一种杂多糖,其摩尔比为:2.96:1.25:3.06;化学分析和光谱分析结果表明紫球藻多糖糖链连接方式以p-（1→3）为主,存在少量1→4及1→6糖苷键;Gal在支链或链末端有较大量的存在,Xyl和Glc在主链或靠近主链区域有特定分布。综合上述分析,推断EPS可能的重复单元结构如下:或者,（2）采用密闭微波降解技术分别制备了相对分子量分别为6553 Da、60.66 kDa、和256.26 kDa的小分子量多糖;采用超声波辅助过氧化氢降解技术分别制备了203.6kDa、606.6 kDa、802.6 kDa、903.3 kDa和1002 kDa的小分子量多糖。通过控制不同的衍生温度制备了硫酸根含量分别为31.0%、31.5%、47.5%和42.4%的硫酸酯多糖衍生物。降解后的小分子量多糖的理化性质没有明显的变化,衍生化后紫球藻多糖的水溶性明显增加。3、紫球藻多糖的生物学活性（1）紫球藻多糖的抗氧化活性所制备的各类紫球藻多糖均表现出很强的清除羟自由基（·OH）、超氧阴离子（O₂^-·）自由基和1,1-二苯基苦基苯肼（DPPH·）自由基的活性,并且清除作用均与其浓度正相关;对由Fe²⁺/抗坏血酸诱发的小鼠肝匀浆氧化损伤和由H₂O₂诱发的小鼠红细胞氧化溶血也具有浓度依赖性的抑制作用。多糖分子量对其抗氧化活性有显著的影响,降解前原糖样的抗氧化活性未能检测出,但是降解后随着多糖分子量的减小其抗氧化活性显著增加,尤其是分子量为6553Da的多糖的抗氧化效果最好。经过硫酸化修饰后,多糖的抗氧化活性明显增加,对-OH和O₂^-·的半数抑制浓度分别为3.92 mg/mL和0.4mg/mL;随着硫酸基含量的增加,衍生化多糖对三种自由基的清除效果明显增强,但是差异不十分显著。（2）紫球藻多糖的抗肿瘤作用SRB法测定紫球藻体外抗肿瘤活性,结果表明高分子量的紫球藻多糖没有体外抑瘤效果,分子量为6.55 kDa和256 kDa的多糖对肺腺癌癌细胞（A549）和喉表皮样癌癌细胞（Hep-2）和肝癌细胞株（SMMC7721）表现出一定的抑制作用。荷S180实体瘤小鼠灌胃给药的结果显示,高、中、低三个剂量组（200、100、50mg/kg/d）的紫球藻多糖均可显著抑制S180实体瘤的生长,其抑瘤率分别为53.3%、47.5%和40.5%,表明该多糖具有明显的抗肿瘤作用。并且紫球藻多糖能显著地提高荷瘤小鼠的脾指数和胸腺指数,明显促进脾淋巴细胞的增殖。在剂量为200μg/mL时,脾淋巴细胞的增殖指数相对于模型组为2.41,相对于CTX阳性对照组为2.15。（3）紫球藻多糖的体外免疫活性在25-200μg/mL浓度范围内,紫球藻多糖能显著提高小鼠腹腔巨噬细胞释放NO能力,增强小鼠腹腔巨噬细胞吞噬中性红的能力,促进小鼠腹腔巨噬细胞和脾淋巴细胞的增殖。分子量与硫酸基含量对免疫调节功能有显著影响。分子量为6.55 kDa的多糖的免疫增强活性最强;除了硫酸基含量为32%的多糖对腹腔巨噬细胞的增殖促进作用最强外,其余都是随着硫酸根含量的增高,免疫促进作用增强,硫酸根含量高于40%的多糖的免疫增强活性较好。更多还原

【Abstract】 The Porphyridiun cruentum （P.cruentum） are the only unicellular marine microalga in Rhodophyta, which can synthesize and secrete extracellular polysaccharides （EPS） into culture liquid when cells are grown in steady-state culture. The yield of EPS usually correlates with the culture condition of microalga. There have been made some progress about cultivation and bioactive compounds of P. cruentum. However, the researches about the molecular modification of P. cruentum polysaccharide, especially the relationship between structure and biological activities are still rare. In present work, the yield of EPS was improved effectively by ecology control and optimization of technical condition. Based on analysis of physicochemical properties and chemical structure of polysaccharide, different molecular and different sulfate content derivatives of polysaccharide extracted from P. cruentum were prepard. Thereafter, the relationship between different properties polysaccharide and the antioxidant activity, anti-tumor activity and immunomodulation activity were investigated. The results were summarized as follows:1 The culture conditions for improvement of EPS production（1） In order to probe the effect of nutrient salts on polysaccharide yield of P. cruentum, several important inorganic compounds such as nitrogen, phosphor, sulfate, metal ion and plant growth hormone were investigated. The optimal cultural medium （OCMⅡ） for increasing EPS yield were obtained with NaHCO₃ 3.5 g, KH₂PO₄ 20 mg, NaNO₃ 2 g, Na₂SO₄ 20 mg, FeCl₂·6H_O 10μM, and MnCl₂·4H₂O 0.2μM, V_B1 0.9 mg, V_B12 2.7μg, sterile seawater 1000 mL. The maximal EPS production achieved as 672 mg/mL after 15 d cultivation in OCMⅡmedium, which increased 1.79 times and 1.62 times compared with that cultivated in 2/f and Koch medium.（2） In order to investigate light quality on yield of EPS, the P. cruentum were cultivated in flat plate photobioreactors （FPPBR） developed by ourselves and different light illumination intensity, light/dark cycle, light spectrum and light pathway of reactor were designed. The optimum light illumination conditions for maximal EPS yield were photon flux density 80μE·m^-2s^-1, light/dark cycle 18:6, natural light or yellow light, and the maximal yield of polysaccharide was 0.96 g/L. The maximum EPS yield was observed as 1.13 g/L in FPPBR with light-pathway of 30 mm, but the maximal areal output rate of EPS achieved as 5.8 g /rn²/d in FPPBR with light-pathway of 50 mm. （3） To probe the effects of renewal regime on the production of polysaccharides, P. cruentum were cultured semicontinuously in flat plate photobioreactor. Uniform design was used to optimize renewal conditions. Quadratic mathematic models related to output rate, total recovery yield of biomass and polysaccharides were set up to clarify the influence of individual factors and their interactions. According to the mathematic models, the optimal semi-continuous condition for total yield of EPS was NaNO₃ 3.5 g/L, renewal rate 27%, renewal period 2.91 d. With the optimal renewal regime, the maximal total recovery yields and average production of EPS achieved at 29.4 g and 1.96 g/L, respectively, which was 2.35 and 1.57 times higher than that of batch cultivation.2 Preparation of different EPSs and analysis of structure and properties（1） The EPSs were isolated and purified by DE52 hydronium exchange chromatography column and Sephacryl S-400 gel chromatography column, then the homogeneous water-solubility polysaccharides were obtained. Analysis of physicochemical properties showed that the apparent molecular weight （MW） of EPS was about 2918 kDa, sulfate content was 14.63%, glucurouic acid content was 7.8%. Rheological behavior showed that the aqueous solution of EPS was typical pseudoplastic fluid, and the viscosity of polysaccharide could not change with temperature and heating time. GC analysis showed that the monosaccharide components of EPS were mainly composed of with xylose, glucose and galactose, and molar ratio of each sugar is 2.96: 1.25: 3.06. Results of chemical and spectral analysis showed that the main linkage wasβ-（1→3）, and there were a few of 1→4 and 1→6 linkages. A large of galactoses exsited at the end or branch of sugar chain, but xylose and glucose were distributed specially close to main chain. So it can be concluded that the possible repeating unit of EPS could be shown as: （2） The molecular weights of degraded polysaccharides prepared by hermetical microwave were 6553 Da, 60.66 kDa and 56.26 kDa, respectively. Oxidative degradation with H₂O₂ under ultrasonic wave were used to degrade EPS from 2918 kDa to 203.6 kDa, 606.6 kDa, 802.6 kDa, 903.3 kDa and 1002 kDa. The EPS derivatives with sulfate content of 31.0%, 31.5%、47.5% and 42.4% were prepared by controlling different reaction temperature. The physicochemical properties before and after degradation had no obvious changes, and the water-solubility of derivate EPS increased obviously.3 Biological activities of EPS（1） The antioxidant properties of EPS were evaluated by determining the scavenging ability of free radicals, inhibitory effects on Iipid peroxidation in mouse liver homogenates and hemolysis of mouse erythrocytes. The results showed that all degraded polysaccharide fragments had strong scavenging effects on hydroxyl-radical （·OH）, Superoxide-radical （O₂^-·） and 1, 1-Diphenyl-2-picrylhydrazyl （DPPH·）. Besides, the scavenging activities of EPS improved with an increase of samples concentration. All degraded polysaccharides clearly inhibited Iipid peroxidation induced by Fe²⁺/ascorbic acid in mouse liver homogenates, and markedly inhibited H₂O₂-induced hemolysis of mouse red blood cells （RBCs） in a dose-dependent manner.MW of EPS had significant effect on antioxidant ability. High-molecular-weight EPS before degradation had no obvious antioxidant activity, but the antioxidant activities increased with the decrease of MW. The 6.55 kDa fragment had stronger antioxidant activity than the others. The antioxidant activity of sulfate derivatives of EPS improved distinctly, and the IC₅₀ values of scavenging·OH and O₂^-·were 3.92 mg/mL and 0.4mg/mL,respectively. With an increase of sulfate content, the scavenging effects of polysaccharide derivatives increased, but the difference between them was not obvious significance.（2） The inhibitory effects of EPS on tumor cells in vitro were determined by method of SRB. The results showed that high-MW EPSs had no anti-tumor activity. The 6.55 kDa-fragment and 256 kDa-fragment had some inhibitory effects on lung adenocarinoma cells （A549）, laryngeal epidermal cancer cells （Hep-2） and hepatoma cells （SMMC7721）, but the effects were not obvious.The effects of EPS on Sarcoma 180 （S180） tumor growth were observed after different doses of EPS were administrated by gastric perfusion to S180-bearing mice. The growth of S180 tumor was significantly inhibited by EPS, and the growth inhibitory rate were 53.3%、47.5% and 40.5%, repectively at the dose of 200, 100 and 50 mg/kg/d. The effects of EPS on immune system in vivo indicated that EPS could increase remarkably spleen and thymus index in S180-bearing mice, and could also stimulate significantly spleen lymphocyte. The pro- liferation indexes of lymphocyte were 2.41 and 2.15 compared with model groups and positive control （CTX） groups, respectively.（3） The immunomodulating assay in vitro showed EPS could improve production of NO of mouse macrophage, boost up neutral red uptake, stimulate mouse macrophage and spleen lymphocyte proliferation at the dose range of 25-200μg/mL in a dosage-dependent way. The immunomodulating ability between different molecular weight EPS had significant differences,and 6.55 kda-fragment had strongest activity. Sulfate content of EPS derivatives can also affect the immunomodulation activities significantly. With an increase of sulfate content, the immuno-accelerating effects enhanced, so the derivative with sulfate content of 48% had the strongest activity. However, the best effect on macrophage proliferation was observed at fragment with sulfate content of 32%.更多还原