【作者】 郭守东；

【导师】 毛文君；

【作者基本信息】 中国海洋大学 ， 药物化学， 2010， 博士

【摘要】 海洋和南极特殊环境微生物产生的胞外多糖,由于其新颖的结构特征、独特的化学性质、丰富的生物活性以及潜在的商业价值,成为了近几年新的研究热点。本文以十六种海洋和极地来源的微生物产生的胞外多糖为研究对象,对其产率、理化性质、单糖组成以及抗氧化活性进行分析,从中筛选出三种分别由海洋迟钝型爱德华氏细菌(Edwardsiella tarda)、南极树粉孢属真菌(Oidiodendron truncatum)以及海南蕾二岐灯芯柳珊瑚(Dichotella gemmacea)共附生真菌赭曲霉(Aspergillus ochraceus)产生的胞外多糖,进行分离纯化、结构研究以及抗氧化活性评价。研究结果如下：1.十六种微生物胞外多糖的产率为0.03-0.45 g/L,糖含量为57.20%-94.71%,蛋白含量为3.41%-15.50%,葡萄糖醛酸的含量为0.75%-6.03%,葡萄糖胺的含量为0.3%-12.4%,所有胞外多糖中硫酸根的含量均低于检测限。两株南极真菌产生的胞外多糖的单糖组成以甘露糖、葡萄糖和半乳糖为主；海洋微生物来源的十四种胞外多糖的单糖组成有十种以甘露糖为主,两种以葡萄糖和半乳糖为主,两种(FU-FANG和QD08-1)以葡萄糖占绝对优势。结果表明,不同环境来源的微生物产生的胞外多糖,在理化性质、单糖组成和DPPH自由基清除能力等方面均具有较大差异。最终选取了抗氧化活性较强的三种胞外多糖作进一步的研究,这三种胞外多糖分别由海洋迟钝型爱德华氏细菌(Edwardsiella tarda)、南极树粉孢属真菌(Oidiodendron truncatum)以及海南蕾二岐灯芯柳珊瑚(Dichotella gemmacea)共附生真菌赭曲霉(Aspergillus ochraceus)产生。2.海洋迟钝型爱德华氏细菌产生的胞外多糖经Q-Sepharose F.F.阴离子交换柱层析和Sephadex G150凝胶柱层析分离纯化获得两种分子量分别为29.0 kDa和70.0kDa的胞外多糖,ETW1和ETW2。经HPLC、GC、甲基化、GC-MS、IR及NMR分析表明这两种胞外多糖均是由[β-D-Manp(1→]、[→2)-α-D-Manp(1→]、[→3)-α-D-Manp(1→]、[→2,3)-α-D-Manp(1→]和[→3,6)-α-D-Manp(1→]糖基以约2：1：2：1：1的摩尔比组成的；二者均是以[→3)-α-D-Manp(1→]为主链的七糖重复单元组成的甘露聚糖,并且[β-D-Manp(1→]和[→2)-α-D-Manp(1→]以Manp(β1→)和Manp(β1→2)Manp(α1→)的形式分别连接在主链的C-6位和C-2位上；ETW1约由26个该类型的七糖重复单元组成,而ETW2由大约62个该类型的七糖重复单元组成。这是首次从新型迟钝型爱德华氏细菌产生的胞外多糖中获得结构新颖尚未见报道的甘露聚糖。3.南极树粉孢属真菌胞外多糖经Q-Sepharose F.F.阴离子交换柱层析和Sephadex G100凝胶柱层析分离纯化获得五种胞外多糖AFW1、AFW2、AFS1、AFS2和AFS3,分子量依次为60.0 kDa、7.5 kDa、64.5 kDa、19.0 kDa和9.0 kDa。其中AFS1、AFS2和AFS3中含有少量蛋白,蛋白含量依次为1.8%、1.9%和5.1%。经HPLC、GC、GC-MS、IR及NMR分析表明AFW2是由[→6)-α-D-Glcp(1→]糖基组成的线性葡聚糖；AFS2的主链也是[→6)-α-D-Glcp(1→]糖基,但平均每六个糖基中有一个糖基在C-3位存在[α-D-Glcp(1→]的分支；AFS3的糖链则是由[→2)-α-D-Glcp(1→]和[→6)-α-D-Glcp(1→]:糖基以1：6的摩尔比组成；AFW1是由[→6)-α-D-Glcp(1→]、[→4,6)-α-D-Glcp(1→]、[→2,4,6)-α-D-Manp(1→]和[Galp (1→]糖基以1：1：1：3的摩尔比组成；AFS1是由[→6)-α-D-Glcp(1→]、[→4,6)-(1]α-D-Glcp(1→]、[→2,4,6)-α-D-Manp(1→]、[→2)-α-D-Manp(1→]和[Galp(1→]糖基以约1：1：1：1：3的摩尔比组成,其中[α-D-Galp(1→]和[β-D-Galp(1→]连接于AFW1和AFS1糖链的非还原末端。其中,AFW2属于具有广泛应用价值的Dextran系列的线性葡聚糖。4.蕾二岐灯芯柳珊瑚共附生真菌赭曲霉的胞外多糖经Q-Sepharose F.F.阴离子交换柱层析和Saphacryl S-400凝胶柱层析分离纯化获得两种分子量分别为29.0kDa和7.4 kDa的半乳甘露聚糖,AW1和AW2。采用可控酸降解的方法制备寡糖,并结合Bio-Gel P4凝胶柱层析获得八个寡糖组分；经GC、ESI-MS、ESI-CID-MS/MS、GC-MS和NMR分析表明,所得寡糖的聚合度为2-9,且以罕见的[→5)-β-D-Galf(1→]糖基的形式存在。AW1和AW2的GC-MS和NMR的分析结果表明,AW1和AW2中的甘露糖以[a-D-Manp(1→]、[β-D-Manp(1→]、[→2)-a-D-Manp(1→]和[→2,6)-a-D-Manp(1→]糖基的形式存在,而半乳糖以[β-D-Galf(1→]和[→5)-β-D-Galf(1→]糖基的形式存在；经可控酸降解后AW1和AW2中的[→2)-α-D-Manp(1→]糖基的摩尔比显著上升而[→2,6)-α-D-Manp(1→]和[→5)-β-D-Galf(1→]糖基的摩尔比显著下降,说明不同聚合度的呋喃型半乳寡糖以[→5)-β-D-Galf(1→]糖基的形式连接于以[→2)-α-D-Manp(1→]为主链的核心甘露聚糖的C-6位上。该研究首次从赭曲霉(Aspergillus ochraceus)产生的胞外多糖中获得了两种结构新颖尚未见报道的半乳甘露聚糖,同时提供了一个从海洋微生物获取罕见的[→5)-β-D-Galf(1→]寡糖的新途径,也为“海洋糖库”提供了新型的特征寡糖。该研究对海洋共附生真菌胞外多糖的研究具有重要的参考价值。5.通过DPPH自由基清除能力、羟基自由基清除能力、超氧阴离子自由基清除能力、抗脂质过氧化能力以及BSA氧化损伤的SDS-PAGE分析表明,纯化后的七种胞外多糖都表现出较强的清除自由基和抑制脂质过氧化的能力,并在一定程度上能对BSA的氧化损伤起到保护作用。七种胞外多糖的抗氧化活性顺序为：AFS3＞AFS2＞AFS1＞ETW1＞AFW2＞ETW2＞AFW1。其中AFS3清除DPPH自由基和超氧阴离子自由基的EC50值分别为0.46mg/mL和0.55mg/mL。体内抗氧化实验表明,AFS3可以提高D-半乳糖诱导的衰老模型小鼠体内抗氧化酶SOD.GPx和CAT的活力,有效降低脂质过氧化产物MDA的水平,在一定程度上达到对抗氧化应激的功效；AFS3也可能通过提高小鼠的免疫功能或通过体外抗氧化的作用模式而达到清除ROS的功效。胞外多糖抗氧化活性的差异与其分子量有一定的相关性,推测20-30 kDa左右的多糖抗氧化效果较佳；另外,多糖复合物中的蛋白也与其抗氧化活性相关。更多还原

【Abstract】 The exopolysaccharides(EPSs) produced by marine and Antarctic derived microorganisms of special environments become a new research front due to their novel structural characteristics, particular chemical properties, various bioactivities as well as commercial opportunities in recent years. In this paper, the yield, chemical properties, monosaccharide component and antioxidant activity of the EPSs obtained from 16 marine and Antarctic microorganisms were analyzed, based on the analysis results the EPSs produced by marine bacterium Edwardsiella tarda, Antarctic fungi Oidiodendron truncatum and Dichotella gemmacea epiphytic fungi Aspergillus ochraceus were further purified, their structural characteristics and antioxidant activities were investigated. The research results are as follows:1. The yields of the EPSs produced by 16 marine and Antarctic microorganisms were 0.03-0.45g/L, and their content of sugar, protein, GlcA and GlcN were 57.20%-94.71%,3.41%-15.50%,0.75%-6.03% and 0.3%-12.4%, respectively, while their sulfate content were all below the detection limit. The two EPSs obtained from Antarctic fungi were mainly composed of Man, Glc and Gal; Ten EPSs produced by marine microorganisms were mainly contained Man with various contents of Glc and Gal; two EPSs mainly consisted of Glc and Gal, while the rest two contained majority of Glc. The results showed that the chemical properties, monosaccharide component and DPPH radicals scavenging abilities of the EPSs obtained from microorganisms varying with their living environments. Finally, the EPSs with better antioxidant activities produced by marine bacterium E. tarda, Antarctic fungi O. truncatum and epiphytic fungi A. ochraceus were screened out for further investigation. 2. Two purified EPSs, ETW1 and ETW2 with molecular weights of 29.0 kDa and 70.0 kDa, respectively, were isolated from the crude EPS of E. tarda using Q-Sepharose F.F. anion exchange chromatography and Sephadex G150 gel permeation chromatography. The results indicated that ETW1 and ETW2 had a similar structure with [β-D-Manp(1→], [→2)-α-D-Manp(1→],[→3)-α-D-Manp(1→], [→2,3)-α-D-Manp(1→] and [→3,6)-α-D-Manp(1→] in a molar ratio of 2:1:2:1:1. Both of them had a heptasaccharide repeating unit with a [→3)-α-D-Manp(1→] backbone chain, which were substituted by Man(β1→2)Man(α1→) and Man(β1→) at the C-2 and C-6 positions, respectively. ETW1 and ETW2 were composed of 26 and 62 the repeating units, respectively. The two mannans with novel structures separated from the fermentation broth of the unusual E. tarda are firstly reported in this paper.3. Five purified EPSs AFW1, AFW2, AFS1, AFS2 and AFS3 were isolated from the crude EPS of O. truncatum, their molecular weights were 60.0 kDa,7.5 kDa,64.5 kDa,19.0 kDa and 9.0 kDa, respectively. The protein content of AFS1, AFS2 and AFS3 were 1.8%,1.9% and 5.1%, respectively. The results indicated that AFW2 was a linear glucan with [→6)-α-D-Glcp(1→] glycosyls as its backbone chain; AFS2 also had a [→6)-α-D-Glcp(1→] glycosyl backbone chain with an average branch of [α-D-Glcp(1→] at the C-3 position of every six [→6)-α-D-Glcp(1→] glycosyls; AFS3 had a linear structure with [→6)-α-D-Glcp(1→] and [→2)-a-D-Glc(1→] in a molar ratio of 6:1; AFW1 was composed of [Galp(1→], [→4,6)-α-D-Glcp(1→], [→2,4,6)-a-D-Manp(1→] and [→6)-α-D-Glcp(1→] glycosyls in a molar ratio of 3: 1:1:1; AFS1 consisted of [Galp(1→], [→6)-α-D-Glcp(1→], [→4,6)-a-D-Glcp(1→], [→2,4,6)-a-D-Manp(1→], and [→2)-α-D-Manp(1→] in a molar ratio of 3:1:1:1:1. The branches of AFW1 and AFS1 were majority of [α-D-Galp(1→] and [β-D-Galp(1→] glycosyls. Among them, AFW2 belongs to the dextran series linear glucan with wide applications.4. Two novel galactomannans, AW1 and AW2 with molecular weights of 29.0 kDa and 7.4 kDa, respectively, were obtained from the crude EPS of A. ochraceus. Both of them were subjected to depolymerization by controllable acid hydrolysis with optimized conditions, and nine oligosaccharides were obtained by Bio-Gel P4 chromatography. The results indicated that galacto-oligosaccharides with consecutive degrees of polymerization from one to nine were in the uncommon form of [→5)-β-D-Galf(1→], the mannopyranosyls in AW1 and AW2 were in the form of [a-D-Manp(1→], [β-D-Manp(1→], [→2)-α-D-Manp(1→],[→2,6)-a-D-Manp(1→], while the galactofuranosyls were in the form of [β-D-Galf(1→] and [→5)-β-D-Galf(1→]; After depolymerization the molar ratio of [→2)-α-D-Manp(1→] glycosyls increased observably in AW1 and AW2, while [-+2,6)-a-D-Manp(1→] and [→5)-β-D-Galf(1→] glycosyls decreased remarkably, all of which suggested that the galacto-oligosaccharides should be linked to the C-6 positions of the mannan core, whose backbone chain was [→2)-α-D-Manp(1→] glycosyls. This study provides a new way to obtain uncommon [→5)-β-D-Galf(1→] linked oligosaccharides from marine microorganisms; and supplies novel characteristics of galacto-oligosaccharide for the "marine carbohydrate database".5. All of the seven purified EPSs exhibited various degrees of antioxidant activities in vitro, which were evaluated by DPPH radicals, hydroxyl radicals and superoxide radicals scavenging abilities, lipid peroxidation inhibition and BSA oxidation damage SDS-PAGE assays. Their antioxidant activities were in the order of AFS3>AFS2> AFS1>ETW1>AFW2>ETW2>AFW1. The EC50 values of AFS3 on DPPH radicals, superoxide radicals were 0.46 mg/mL and 0.55 mg/mL, respectively. AFS3 also showed antioxidant activity in D-Gal induced aging mice by improving the activities of antioxidant enzymes (SOD, GPx and CAT), it restored the high levels of lipid peroxidation product MDA and inhibited the "oxidative stress" in D-Gal induced aging mice. In addition, its antioxidant activity may also exerted by enhancing the immune function of the aging mice or scavenging the ROS directly by the tested models in vitro. The molecular weights of EPSs may be related to their antioxidant activities, and 20-30 kDa seems to be good for their antioxidant effects; while the proteins bounded to the EPSs may also play roles in their antioxidant activities.更多还原