【作者】 郭霞；

【导师】 孙敏；

【作者基本信息】 西南大学 ， 植物学， 2010， 博士

【摘要】 桑黄属于担子菌亚门（Basidiomycotina）、层菌纲（Hymenomycetes）、多孔菌目（Polyporales）、多层孔菌科（Hymenochaetacae）、针层孔菌属（Phellinus）,是一种多年生的珍稀药用真菌。桑黄主要含有多糖,其次还有黄酮、三萜和落叶松蕈酸等化合物,具有抗癌、增强免疫力、抗肝纤维化、抗脂质过氧化、抗肺炎、抗血管生成等功效。由于其生理复杂性、特殊性以及对外部生长环境的较高要求,其在自然界中形成子实体比较困难,尤其是需要多年才能形成可药用的子实体。加之,近几年人们对野生桑黄的大量采集,天然的桑黄资源已经非常稀少。人工栽培技术获得子实体,以及液体发酵法获得菌丝体,可以缓解供需矛盾获得有效药用成分。但桑黄人工栽培刚刚起步,并且子实体形成需要半年以上时间,而液体发酵法生产菌丝体,发酵周期短,不受季节气候影响,不受农药污染,具有广泛的应用前景。为了深入研究桑黄深层发酵过程中菌丝生长和胞外多糖合成的机理,为工业化大规模生产奠定基础。本文对桑黄菌丝发酵过程进行了优化,得到了大量生长良好,菌球松散的种子液和有利于桑黄菌丝体生长和胞外多糖合成的全合成培养基。筛选了对菌丝生长和胞外多糖合成有调控作用的碳源和植物激素,并从发酵动力学、菌丝体形态、氨基酸含量变化、多糖结构四个角度,分析了碳源和植物激素对桑黄胞外多糖合成代谢的调控机制。同时利用响应面优化了桑黄菌丝多糖提取工艺,并对胞外多糖结构进行了分析。最后以高脂饲料构建的高脂血症大鼠为实验对象,对发酵法生产的桑黄菌丝胞内、胞外多糖进行了降血脂活性分析。主要结果如下：1.通过桑黄种子液和全合成培养基筛选,优化了菌丝发酵过程种子液优化过程中,玻璃珠对增加菌球和菌丝数量,促进松散菌球形成具有显著作用。种龄是提高桑黄菌丝和胞外多糖产量的显著因素。优化后的种子培养条件为：3%葡萄糖、1.5%酵母粉上清液、0.1%MgSO₄、0.1%KH₂PO₄,加入一颗玻璃珠,10%接种量,96h种龄。优化后的种子液,菌球数量提高了62.5%、菌球直径和菌核直径分别缩小了39.1%和30.3%,得到了大量菌丝松散的种子液。通过正交试验优化了全合成培养基,其包括：4%葡萄糖、0.4%谷氨酸、0.4%（NH₄）₂SO₄.O.1％MgSO₄.O.1％KH2P04,初始pH为6.0。通过方差分析得到葡萄糖浓度、谷氨酸浓度、硫酸铵浓度和pH是影响菌丝生长的显著因素。桑黄菌丝发酵过程优化后,摇瓶发酵最大菌丝产量可达12.33±0.89 g.L^-1,最大胞外多糖产量可达1.21±0.08 g.L^-1。该全合成培养基成分简单,成本低廉,可有效促进桑黄菌丝生长和胞外多糖合成,同时便于发酵过程中各种成分含量的变化分析。2.研究了碳源（包括糖、植物油）和植物激素对桑黄胞外多糖合成的代谢调控通过单因素试验,研究了糖对菌丝生长和胞外多糖合成的影响,筛选出对胞外多糖合成有显著促进作用的乳糖。通过正交试验优化了乳糖添加时间和添加浓度对菌丝生长和胞外多糖的影响,得到最佳组合为发酵初始添加3%乳糖,菌丝产量和胞外多糖产量最大分别可达17.436士2.227 g.L^-1和0.963士0.391 g.L^-1。验证实验表明乳糖对菌丝体生长和胞外多糖合成有显著促进作用。通过单因素实验研究了植物油对菌丝生长和胞外多糖合成的影响,统计分析得出植物油对菌丝生长有显著促进作用,对胞外多糖合成有明显抑制作用。发酵初始摇瓶添加1%菜籽油,菌丝产量和胞外多糖产量最大可达34.633±1.6 g.L^-1和0.73±0.063 g.L^-1。通过168 h菌丝体形态分析,发现添加乳糖后菌丝体出现多处空胞,而添加菜籽油后菌丝体极少有空胞出现并且菌丝体粗壮。通过168 h发酵液氨基酸含量变化分析,结果表明初始添加3%乳糖的发酵液中,谷氨酸族含量降低了7%,天冬氨酸族含量增加了43%,芳香族氨基酸族含量降低了4%,丝氨酸族含量降低了76%,丙氨酸族含量增加了277%。初始添加1%菜籽油的发酵液,谷氨酸族含量增加了11%,芳香族氨基酸含量降低了7%,丝氨酸族含量降低了29%,丙氨酸族含量增加了342%,天冬氨酸族含量没有变化。通过油和乳糖氨基酸含量比较,推测天冬氨酸族含量与胞外多糖合成密切相关,丙氨酸族含量与菌丝体合成密切相关。从发酵动力学分析,分别添加油和乳糖后,菌体生长和胞外多糖部分偶联。通过单因素试验,筛选出对菌丝和胞外多糖合成有明显促进作用的萘乙酸。发酵初始添加5.0 mg.L^-1萘乙酸菌丝体最大产量为6.24士0.18 g.L^-1,最大胞外多糖产量为0.86士0.01 g.L^-1,与空白比较产量分别提高了15.98%和56.36%。通过发酵过程动力学分析,得出菌丝生长速率和胞外多糖合成速率同步,通过高效液相和红外光谱分析胞外多糖结构没有变化。因此,推测萘乙酸是通过提高菌丝体生物量,从而提高胞外多糖产量。3.应用响应面试验设计优化菌丝多糖提取工艺响应面试验优化了发酵法获得的湿菌丝多糖提取工艺,优化后的菌丝多糖提取条件为：提取温度70℃,料液比1∶6.2,提取时间1.5 h,胞内多糖得率为5.04%。比常规菌丝干粉提取法缩短了提取时间,降低了提取温度。4.色谱法分析桑黄胞外多糖结构通过高效液相色谱、红外色谱、气相色谱对发酵法获得的桑黄胞外多糖进行了结构的初步分析。结果表明：桑黄胞外多糖含糖量49.7%、蛋白质含量18.0%,由四部分多糖组成,分子量分别为6.4×10⁶g. mol^-1、3.3×10⁵g.mol^-1、2.7×10⁵ g. mol^-1、2.9 X103 g.mol^-1。气相色谱分析表明单糖组分主要含半乳糖。红外色谱分析其含有吡喃糖p型C-H变角振动的特征吸收峰。故推测胞外多糖主要是β-半乳聚糖。5.桑黄多糖降血脂活性分析通过高脂饲料饲养大鼠构建高脂血症动物模型,给予不同剂量的桑黄多糖进行干预,探讨发酵法生产的桑黄多糖降血脂活性。结果表明：低剂量和高剂量桑黄多糖均可显著降低高脂血症大鼠的血清总胆固醇、甘油三酯、低密度脂蛋白含量,与高脂血症大鼠相比,其最大降幅分别为19.7%、28.3%和32.9%；同时,多糖提高了高密度脂蛋白含量,最大增幅为12.6%。研究结果为桑黄开发为降血脂药物提供了理论参考。更多还原

【Abstract】 Phellinus igniarius is a precious medicinal mushroom belonging to the subphylum Basidomycotina, class Hymenochaetacae, order Polyporales, family Hymenochaetaceae, genus Phellinus. It contains many kinds of compounds such as polysaccharides, flavonoids, triterpenoids, larch mushroom acids and so on. These compounds are able to prevent and cure many diseases including anti-cancer, anti-liver fibrosis, anti-pneumonia, anti-angiogenesis and enhancing immunity. Because of the physiological complexity, particularity and high requirement of growing environment, it is very difficult to cultivate fruit body with high quantity of bioactive compounds, In addition, the amounts of natural P. igniarius were decreased sharply due to the mass collection in recent years. It is a promising approach by artificial cultivation and fungi fermentation to address the contradiction between supply and demand. Using this method, polysaccharides from P. igniarius can be produced in large scale and used in medical care causes.In order to illuminate the mechanism of mycelia growth and biosynthesis of extracellular polysaccharides （EPS） by P. igniarius, the optimization of fermentation process was carried out. The seed culture with a large amount of loose pellets and a chemical defined media for mycelial growth and EPS synthesis were obtained. Fermentation kinetics, mycelial morphology, contents of amino acids and the characterization of EPS were used to analyze the metabolic regulation of EPS biosynthesis. Response surface methodology was used for optimizing extraction conditions of mycelia and the preliminary characterization of EPS was analyzed. The anti-hyperlipemia activity of intracellular polysaccharides （IPS） and EPS was analyzed by adult rats. The main results were as follows: 1. The optimization of fermentation process including seed culture and a chemical defined media were carried out. Glass beads and seed age affected the amount of pellets formation. The optimal seed medium was including 3% glucose,1.5% yeast source,0.1% MgSO₄ and 0.1% KH₂PO₄. Inoculation volume and inoculation time were 10% and 96 h, respectively. During the seed culture, a glass bead was used to increase the amount of pellets. After optimization of seed culture, the amounts of pellets were increased 62.5%. The diameter of pellets and core pellets decreased 39.1% and 30.3%, compared with control. A chemically defined medium for mycelial growth and EPS production by submerged culture of P. igniarius was investigated. The mainly defined medium compositions were optimized by orthogonal matrix method. The optimal defined medium （per liter） included 40.0 g glucose,4.0 g glutamic acid,4.0 g （NH₄）₂SO₄,1.0 g MgSO₄ 1.0 g KH₂PO₄ and initial pH value was 6.0. Under the optimal medium, the maximal mycelial biomass and EPS production were 12.33±0.89 g. L"1 and 1.21±0.08 g. L^-1 at 192 hour in shake flask.2. Effects of different kinds of carbon sources and phytohormones on EPS biosynthesis were studied. Our results showed that lactose was able to significantly increase the production of mycelia and EPS. Plant oil was benefit to mycelia growth. Especially, rapeseed oil significantly increased the production of mycelia, whereas it inhibited EPS synthesis. Adding 3% lactose in initial medium was benefical for EPS biosynthesis, and the maximal production of dry cell weight （DCW） and EPS were 17.436±2.227 g. L^-1 and 0.963±0.391 g. L^-1, respectively, while adding 1% rapeseed oil in initial medium was helpful for mycelia growth, the maximal production of DCW and EPS were 34.633±1.6 g. L^-1 and 0.73±0.063 g. L^-respectively. Morphology analysis revealed that some of mycelia cultured in lactose were hollow, but mycelia cultured in rapeseed oil were robust. Effect of phytohormones including indole-3-acetic acid （IAA）, indoIe-3-butyric acid （IBA） and 1-naphthalentacetic acid （NAA） on mycelial growth of medicinal mushroom Phellinus linteus were investigated. The production of DCW and EPS with addition of5.0mg. L^-1 NAA was 6.24±0.18 g. L^-1 at 168 h and 0.86±0.01 g. L^-1 at 192 h, which were enhanced by 15.98% and 56.36% compared to the control, respectively. However, the molecular weight and infrared spectrum of EPS were coincident with the control. Results indicated that NAA at the proper concentration was beneficial in stimulating mycelial growth and EPS biosynthesis, whereas it could not alter the molecular structure of EPS. The fermentation dynamic analysis showed thet specific growth rate was accord with specific production rate. These results indicated that the positive effect of NAA on EPS biosynthesis maybe cause by enhancing the mycelial growth.3. The methods of high performance liquid chromatography, infrared spectrometry and gas chromatography were used to analysis the preliminary character of EPS. The results showed that it contained 49.7% sugar,18.0% protein. It contained four parts of polysaccharide, molecular weight (g. mol^-1) were 6.4×10⁶,3.3xl0⁵,2.7×10⁵ and 2.9xlO³, respectively. The structure of monosaccharide was mainly galactose according to gas chromatogphy. The structure of polysaccharide could be composed ofβ-galactan.4. Response surface methodology was applied to optimize the extraction conditions of IPS of P. igniarius. The optimal conditions were extraction temperature 70℃, extraction time 1.5 h and the ratio of mycelia to water 1:6.2. Under the optimal condition, the maximal yield of crude IPS from mycelia was 50.39±0.41 mg. g^-1. Compared with the extraction of dry power mycelia, the optimal extraction process was favor to save extraction time and decrease extraction temperature.5. The EPS and IPS extracted from mycelia can obviously reduce the level of the blood cholesterol （TC）, serum triglyceride （TG）, and serum low-density lipoprotein （LDL）, and increase the level of high-density lipoprotein （HDL） of the hyperlipemia rats. Polysaccharide could decrease the level of TC, TG and LDL remarkably with decreasing rate of 19.7%,28.3% and 32.9%. Polysaccharide could increase the levels of HDL with the highest rates of 12.6%. Polysaccharides showed a significant effect of antihyperlipidaemia activity. The results indicated that polysaccharides from submerged culture of medicinal fungus have favorable potency to develop anti-hyperlipermia drugs.更多还原