【作者】 陈耀华；

【导师】 陈健； 钱列生；

【作者基本信息】 华南理工大学 ， 轻工技术与工程， 2013， 硕士

【摘要】 金黄喇叭菌是一种药食兼用的真菌，它分布广泛、味美可口且营养丰富。目前对金黄喇叭菌的研究主要集中在金黄喇叭菌的营养成分及其抗菌活性等方面。但对金黄喇叭菌多糖的研究甚少，本文主要研究了金黄喇叭菌多糖的提取、分离、纯化、结构鉴定和生物活性等。主要研究结果如下：金黄喇叭菌干品中的粗蛋白、总糖、粗脂肪、粗纤维、灰分、水分含量分别为：22.82%、49.58%、4.36%、4.58%、9.18%、6.98%，且富含多种矿物质元素和人体必须氨基酸。进行金黄喇叭菌多糖水提工艺优化研究。通过单因素试验及正交实验分析表明水提工艺对多糖得率的影响因素依次为：提取次数﹥提取时间﹥提取温度﹥液料比，金黄喇叭菌多糖的水提取工艺的最适条件为：提取温度为90℃，液料比为30:1，提取时间为3h，提取次数为2次，金黄喇叭菌多糖的得率为10.9%。金黄喇叭菌多糖的分离纯化：多糖提取液经过脱色、醇沉、脱蛋白及再醇沉后得到金黄喇叭菌粗多糖CAB，经DEAE-52离子交换柱层析、Sepharose CL-6B琼脂糖凝胶柱层析、超滤离心管分离得到单一组分CABⅠ、CABⅡ-2和CABⅢ，并经过高效凝胶渗透色谱测定其重均分子量分别为16271Da、19513Da、56004183Da。CABⅠ的初级结构分析：红外光谱显示CABⅠ在波数3421cm^-1处有很强的O-H伸缩振动，在波数2925cm^-1处有C-H的伸缩振动，1417cm^-1处是多糖羧基中C-O的伸缩振动吸收峰，1151¹043cm^-1表明多糖结构中有吡喃环，867cm^-1、800cm-1处的吸收峰表明有甘露糖的存在。利用气相色谱进行单糖组成研究，结果表明CABⅠ含岩藻糖、甘露糖、葡萄糖和半乳糖，其摩尔比为1︰2.11︰3.13︰6.45。高碘酸氧化和Smith降解显示CABⅠ中含1→6、1→、1→2以及1→3型糖苷键，岩藻糖和甘露糖可能完全含不可氧化的1→3型糖苷键，葡萄糖几乎完全被氧化主要含1→6、1→、1→2型糖苷键，半乳糖完全被氧化含1→6、1→、1→2型糖苷键。核磁共振结果分析表明CABⅠ含有α、β两种糖苷构型，部分C-2、C-3和C-6被取代，C-4没有被取代。多糖组分CABⅠ、CABⅡ和CABⅢ的生物活性研究结果表明，金黄喇叭菌多糖对DPPH·自由基有清除活性并且有较好的还原力，表明其具有一定的抗氧化活性；以豚鼠血清为补体的模型进行抗补体实验，表明金黄喇叭菌多糖具有良好的抗补体活性；采用MTT法检测了金黄喇叭菌多糖在体外对人体肝癌细胞Hep G2和乳腺癌细胞MCF-7增殖的抑制作用，结果发现CABⅠ、CABⅡ和CABⅢ对两种癌细胞均有一定程度的抑制作用；金黄喇叭菌多糖在三种生物活性中整体上都呈剂量依赖性，且单一多糖表现出较好的生物活性。更多还原

【Abstract】 Craterellus aureus Berk. et Curt. is an edibable and medicinal fungi, which is widelydistributed, delicious and nutritious. At present, the research of Craterellus aureus Berk. etCurt. focuses on the nutrients, antibacterial activity and so on. But there are few studies onCraterellus aureus Berk. et Curt. polysaccharide. This paper mainly studies the extraction,separation, purification, primary structure and biological activity of Craterellus aureus Berk.et Curt. polysaccharide. The main results are as follows:The content of crude protein, total polysaccharide, crude fat, crude fiber, ash and waterin the dry sample were:22.82%,49.58%,4.36%,4.58%,9.18%and6.98%. Craterellusaureus Berk. et Curt. was rich in minerals and essential amino-acid.The optimized water extracting technology of Craterellus aureus Berk. et Curt.polysaccharide was investigated. The single factor experiment and orthogonal experimentshowed that the influence factors of polysaccharide yield were: extraction times> extractiontime> extraction temperature> liquid-solid ratio. The optimum water conditions were asfollows: extraction temperature90℃, liquid-solid ratio30:1（mL/g）, extraction time3h,extraction times2times, the yield could come to10.9%in these conditions.The separation and purification of Craterellus aureus Berk. et Curt.polysaccharide（CAB）: CAB was obtained after polysaccharide extracted in the optimumconditions, with decolored, ethanol subsided, deproteinized and ethanol subsided. CAB wasseparated and purified by DEAE-52ion-exchange column chromatography, Sepharose CL-6Bgel column chromatography and100KD ultrafiltration centrifugal tube. Then three purifiedpolysaccharides were obtained which is named CAB Ⅰ, CABⅡ-2, CAB Ⅲ. The weightaverage relative molecular mass of CABⅠ, CABⅡ-2and CAB Ⅲ were16271Da,19513Daand56004183Da which were detected by GPC.The structure of CAB Ⅰw as analysed. The infrared spectra showed CABⅠ contained thestretching vibration of O-H at3412cm^-1, the stretching vibration of C-H at2925cm^-1, thestretching vibration of a group of carboxylic at1417cm^-1. CAB Ⅰ contained pyranoid ring andmannose because of the absorption peak at1151¹043cm^-1and867cm^-1,800cm^-1.Monosaccharide composition analysis of CABⅠby GC demonstrated CAB Ⅰwas consistedof fucose, mannose, glucose and galactoside with the mole ratios was1:2.11:3.13:6.45.Periodate oxidation and Smith degradation showed CABⅠ contained1→6,1→,1→2and1→3type glycosidic bond. Fucose and mannose couldn’t be oxidized and should be1→3typeglycosidic bond. Large amounts of glucose could be oxidized, the glycosidic bond mainly was 1→6,1→or1→2. All galactoside could be oxidized, the glycosidic bond was1→6,1→or1→2. NMR result analysis proved that CABⅠcontained α, β-configuration, and partsubstitutional bond was C-2, C-3and C-6, non substitutional bond was C-4.The study results of biological activity of CABⅠ, CABⅡand CABⅢ showedCraterellus aureus Berk. et Curt. polysaccharides had ability of scavenging DPPH· freeradicals and good ability of reducing power. Both of them indicated they had antioxidantactivity. Anticomplement research chose guinea pig blood serum as the complement, andresult showed Craterellus aureus Berk. et Curt. polysaccharides owned good anticomplementactivity. Antitumor activity of Craterellus aureus Berk. et Curt. polysaccharides wasdetermined on two kinds of cancer cells, human hepatoma cells Hep G2and human breastcancer cells MCF-7by MTT assay. The results showed that CABⅠ, CABⅡand CABⅢ havecertain inhibition to this two kinds of tumor.In the three biological activities Craterellusaureus Berk. et Curt. polysaccharides was dose-dependent on the whole and the purifiedpolysaccharide showed good biological activity.更多还原