锁掷酵母联产油脂和胞外多糖的研究

发布时间：2018-02-15 06:40

本文关键词： 锁掷酵母油脂类胡萝卜素磷酸酯多糖　出处：《江南大学》2017年博士论文　论文类型：学位论文

【摘要】：上世纪70年代微生物油脂就开始进入大力研究阶段,但由于其生产成本较高限制了微生物油脂的大规模应用。为了克服成本过高的瓶颈,本文提出了一种新颖的、节约成本的方式联产微生物油脂和胞外多糖,为微生物油脂和磷酸酯胞外多糖的工业化应用奠定了基础。主要研究结果如下:(1)通过对锁掷酵母产油脂和胞外多糖的发酵条件优化,明确了该菌株微生物油脂和胞外多糖的产量都与生物量呈正相关。在摇瓶发酵条件下,葡萄糖浓度为126 g/L,玉米浆浓度为20 g/L,酵母膏浓度为10 g/L,硫酸铵浓度为2.5 g/L时,菌体干重、油脂产量和胞外多糖的产量最高,分别达到58 g/L、29 g/L和8 g/L。采用两阶段补料分批发酵(前期控制硫酸铵2.5-5 g/L促进菌体生长,后期控制无机氮源为零,促进油脂和胞外多糖的积累),菌体干重、油脂产量和胞外多糖的产量分别提高到72 g/L、39.6 g/L和12g/L。在发酵过程中随着胞内油脂的含量从20%升高到55%,类胡萝卜素的代谢向积累红酵母烯的方向移动。(2)对锁掷酵母破壁条件和油脂提取条件进行优化,确定了中试水平上的锁掷酵母超声波破壁工艺,然后对油脂中的主要营养成分进行分离纯化和定性定量分析。锁掷酵母油脂中的9种主要营养成分分别为角鲨烯、β-胡萝卜素、γ-胡萝卜素、麦角固醇酯、红酵母烯、甘油三酯、游离脂肪酸、麦角固醇和红酵母红素。油脂中麦角固醇总含量为3.2 g/kg油,其中16%以麦角固醇酯的形式存在。角鲨烯和类胡萝卜素的浓度分别为1.25g/kg油和508 mg/kg油。锁掷酵母油脂的脂肪酸组成主要为油酸(含量为73%)。锁掷酵母油脂不仅是一种高油酸油脂而且还含有丰富的脂溶性功能成分。(3)利用高效体积排阻色谱、原子力显微镜、红外光谱分析、甲基化分析和核磁分析测定了锁掷酵母胞外多糖的分子量、分子直径、溶液中的构象、单糖组成和结构单元。该多糖的平均分子量、水力学半径(Rh)和均方旋转半径(Rg)分别为1.3×106,45±2 nm和63±2 nm。结构参数Rg/Rh(1.4)和Mark-Houwink方程中的α值(0.638)都表明该多糖呈无规则卷曲结构,与原子力显微镜观察的结果一致。该多糖的单糖组成主要为半乳糖和葡萄糖,其比例接近2:1,不含有糖醛酸,多糖中含有2.1%的磷元素,通过其结构单元推测其可能为一种新型的磷酸二酯多糖。(4)多糖的稳态剪切特性符合Williamson model,不同浓度氯化钠对多糖的流变学特性影响较少,浓度较高的二价钙离子可以显著增强多糖的表观粘度。该多糖的触变性弱,多糖结构恢复快。从多糖的动态频率扫描发现,其主要表现为粘性流体。温度对多糖粘度的影响十分显著,随着温度的升高多糖粘度降低。静态剪切和动态震荡模式下,多糖的流变学特性都类似于呈柔顺的无规则卷曲结构的羧甲基纤维素钠并明显区别于呈弱凝胶特性的黄原胶。(5)锁掷酵母油脂和胞外多糖分别具有抗氧化和增强免疫的功能。实验结果表明红酵母烯的ABTS自由基的清除能力、抗脂质过氧化能力和缓解t-Booh引起的氧化损伤的能力均高于β-胡萝卜素,甚至略高于番茄红素。通过锁掷酵母胞外多糖对肿瘤细胞的抑制作用和免疫的调节功能发现,锁掷酵母多糖对肿瘤细胞没有直接的杀伤作用,但是能显著增强T/B淋巴细胞和巨噬细胞的增殖。锁掷酵母多糖可以被用于免疫增强药物的开发。
[Abstract]:In 70s the microbial oil began to enter the vigorous research stage, but due to its high production cost limits the large-scale application of microbial oils. In order to overcome the bottleneck of the high cost, this paper proposes a novel, cost-effective way of production of microbial oils and extracellular polysaccharide, which laid the foundation for the industrial application of microbial polysaccharides grease and extracellular phosphate. The main results are as follows: (1) the fermentation conditions for producing yeast grease and lock throw exopolysaccharide optimization, clear yield of polysaccharides of the strain and extracellular microbial lipids are associated with the biomass was positively. In shake flask fermentation conditions, the concentration of glucose was 126 g/L. Corn slurry concentration is 20 g/L, yeast extract concentration was 10 g/L, the concentration of ammonium sulfate was 2.5 g/L, the cell dry weight, cell yield and fat polysaccharide yield was the highest, reached 58 g/L, 29 g/L and 8 g/L. by two Fed batch fermentation stage (pre control ammonium sulfate 2.5-5 g/L promote cell growth, late control inorganic nitrogen source is zero, the accumulation of polysaccharides promote lipid and extracellular), cell dry weight, yield and yield of polysaccharide and fat cells were increased to 72 g/L, 39.6 g/L and 12g/L. in the fermentation process with the content intracellular fat increased from 20% to 55%, the metabolism of carotenoids accumulation of red yeast to move ene direction. (2) the lock breaking conditions and throw yeast oil extraction conditions were optimized to determine the test level of the lock on the throwing yeast by ultrasonic technology, and analyses and qualitative and quantitative analyses of separation and purification the main nutritional ingredients in the grease. Lock 9 main nutritional components in the oil were throwing yeast squalene, beta carotene, gamma carotene, ergosterol esters, red yeast squalene, triglycerides, free fatty acids, ergot solid alcohol and torularhodin oil. The fat of ergosterol in total content was 3.2 g/kg oil, of which 16% is in the form of ester. Ergosterol concentration of squalene and carotenoids were 1.25g/kg oil and 508 mg/kg oil. The fatty acid composition of a yeast oil lock mainly oleic acid (was 73%). A lock of yeast oil is not only a kind of high oleic acid oil it is also rich in fat soluble components. (3) using high performance size exclusion chromatography, atomic force microscopy, infrared spectroscopy, molecular polysaccharide lock throw yeast extracellular volume, determined by methylation analysis and NMR analysis of molecular diameter, conformation in solution, monosaccharide composition and average molecular structure unit. The polysaccharide content, hydrodynamic radius (Rh) and mean square radius (Rg) were 1.3 and 63 * 106,45 + 2 nm + 2 nm. Rg/Rh (1.4) and the structural parameters of the Mark-Houwink equation in the alpha value (0.638) showed that the polysaccharide was random coil structure, with the atom Force microscopy results. The monosaccharide composition of polysaccharides mainly for galactose and glucose, the ratio is closer to 2:1, not containing uronic acid, phosphorus polysaccharide containing 2.1%, through the structural unit of speculation that it may be a new type of phosphate ester two polysaccharide. (4) the steady shear properties sugar with Williamson model, different concentration of sodium chloride on the rheological characteristics of polysaccharide is less affected, the price two higher calcium ion concentration can significantly increase the apparent viscosity of the polysaccharide. The polysaccharide weak thixotropy, polysaccharide structure and quick recovery. From the dynamic frequency of polysaccharide scan found that the main performance for the viscous fluid. The influence of temperature on the viscosity of the polysaccharide is very significant, with the increase of polysaccharide viscosity in the lower temperature. The static and dynamic shear turbulence model, random coil structure of sodium carboxymethyl cellulose and rheological properties of polysaccharides are similar to a smooth Xanthan gum is significantly different from weak gel properties. (5) a lock of yeast fat and extracellular polysaccharide respectively with antioxidant and enhance immune function. The experimental results show that the ABTS free radical scavenging capacity of red yeast squalene, anti lipid peroxidation and alleviate t-Booh induced oxidative damage ability was higher than that of beta carotene, or even slightly higher than lycopene. Through the lock throw yeast extracellular polysaccharide on tumor cell inhibition and immune regulation function, yeast polysaccharide did not lock a direct killing effect on tumor cells, but significantly increased the T/B lymph cells and macrophage proliferation. A lock of yeast polysaccharide can be used for immune enhancing drug development.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ929

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