Study on Fermentation Strategies of Arachidonic Acid by Mortierella alpina

花生四烯酸（Arachidonicacid，ARA）属于ω-6系列多不饱和脂肪酸，因具有促进婴幼儿脑部发育、提高机体免疫力等多种重要的生理功能而被广泛应用于医药、食品等各种领域。传统的ARA主要来源于动物油等，难以满足市场需求，而利用微生物产ARA具有不受产地、原料、条件等限制的优点，成为当前研究的热点之一。 本文首先对目前用来生产ARA最常用的微生物高山被孢霉（Mortierellaalpina，M.alpina）的培养条件进行了研究，通过摇瓶实验考察了碳源、碳氮比、初始pH值、接种量和微量元素等对ARA的影响。结果发现在培养基中使用葡萄糖为碳源，碳氮比维持在40:1至50:1之间，初始p...Arachidonic acid (ARA) is one of omega-6 polyunsaturated fatty acids (PUFAS), which has been widely applied in medicine, food industry and other fields because of its various physiological functions such as promoting the brain development of infants and increasing immunity of human body. Traditional ARA mainly comes from animal oils, which is difficult to meet the demand of market Therefore, the p...学位：工学硕士院系专业：化学化工学院化学工程与生物工程系_化学工程学号：2062009115127

Domestication of the high-sugar-tolerant Mortierella alpina on arachidonic acid(ARA) production

通讯作者：Tel: 86-592-2186038; : [email protected][中文摘要]【目的】提高花生四烯酸(Arachidonic acid,ARA)产量,克服ARA产生菌高山被孢霉(Mortierella alpina)在长期的保存及使用过程中易受到外界条件影响发生退化,从而导致菌种耗糖量降低、影响菌种摄入营养的能力和不利于工业化生产的缺点。【方法】首先采用固体培养基驯化,将菌种逐级涂布于梯度高糖PDA平板(含糖量分别为2%、5%、7%、10%和15%)培养,挑选经固体驯化后能耐受10%高糖浓度平板的菌种,转接到两种含不同氮源的梯度高糖(含糖量分别为3%、4%、5%和6%)液体培养基中进行驯化,最后对驯化后的菌种进行2 L发酵罐放大实验。【结果】当培养基中以酵母粉为氮源时,驯化后菌体的最高耗糖量由3 g/(L.d)提高到12 g/(L.d);当培养基中以玉米浆为氮源时,驯化后菌体的最高耗糖量由7 g/(L.d)提高到12 g/(L.d)。摇瓶驯化实验结果表明以玉米浆为氮源驯化的菌种发酵效果较好,发酵罐实验结果显示菌体生物量为50 g/L,总油脂为18 g/L,目的产物ARA产量为8 g/L。相比未驯化之前的发酵结果,生物量和总油脂含量提高了近3倍,ARA产量提高了近4倍。【结论】经过高糖驯化, 菌种的耗糖能力得到提高, 生物量、总油脂及ARA 的产量也都有所增加, 从而可以使菌种在保存和使用过程中不易退化,保持稳定。[英文摘要][Objective] In order to improve arachidonia acid(ARA) production,and prevent the degeneration of ARA-producing strain Mortierella alpina in long-term culture preservation and cultivation,which could lead to low consumption rate of substrates like carbon source.[Methods] Mortierella alpina strain was first domesticated in high-sugar PDA plate with gra-dient sugar content(2%、5%、7%、10% and 15%).The strain which grew better in the solid medium containing 10% sugar was then selected and transferred to two liquid high-sugar media with different nitrogen sources to domesticate. The gradient sugar contents were 3%、4%、5% and 6%, respectivlely. Finally, the domesticated strain was used to produce ARA in a 2 L bioreactor. [Results] The experimental results show that the consumption of sugar increased from 3 g/(L·d) up to 12 g/(L·d) in the medium with yeast extract, and from 7 g/(L·d) up to 12 g/(L·d) in the medium with corn steep liquor. After cultivation for 144 h in a 2 L bioreactor,the yield of ARA reached 8 g/L, which was 4 times higher than that obtained with the original strain. [Conclusion] The domestication of the high-sugar-tolerant Mortierella alpina increased the consumption of sugar, biomass, total fatty acid and ARA production. The domesticated strain could maintain the high sugar resistance in the preserving process and turn to be more stable.福建省科技厅重大专项项目(No.2010NZ0001-4);; 国家自然科学基金项目(No.31071488

一种富集高温栅藻Desmodesmus sp. F51的新型生物絮凝剂

【背景】海科贝特氏菌(Cobetia marina)可产生大量具有絮凝活性的胞外产物，可视为一种新型的生物絮凝剂。高温栅藻(Desmodesmus sp. F51)是一种具有较高叶黄素含量的微藻，被认为是一种新兴的叶黄素来源，但利用该生物絮凝剂高效富集高温栅藻的相关研究迄今尚未见报道。【目的】以高温栅藻为对象，研究该新型生物絮凝剂的絮凝效率，并对絮凝机理进行初步探讨。【方法】探索在不同生长阶段微藻培养液添加生物絮凝剂、添加量、絮凝时间、pH对絮凝效率的影响，分析生物絮凝剂的功能基团，并测定在不同pH条件下添加生物絮凝剂前后高温栅藻的Zeta电位变化，以及在显微镜下分析藻细胞在添加生物絮凝剂前后的形态。【结果】在高温栅藻生长至稳定期，pH 8.0添加2 mL生物絮凝剂，絮凝15 min絮凝效果最佳，达82.1%。傅里叶红外图谱显示了多糖及酰胺结构的特征吸收峰，由此推测生物絮凝剂主要是多糖的混合物，含有少量蛋白质。根据Bradford法测定絮凝剂中蛋白含量约为0.4%(质量比)，通过苯酚-硫酸法测定总糖质量分数约为34.5%(质量比)，与FTIR谱分析结果基本相符。生物絮凝剂在pH 4.0～11.0保持60%以上的絮凝效率，说明无论是酸性或是碱性条件下絮凝效率都较高，结合Zeta电位的分析表明，推测生物絮凝剂对高温栅藻的絮凝机理中占主导地位的可能是吸附架桥作用。【结论】该研究对微藻生物絮凝具有重要的理论和实践意义。厦门市科技计划项目(3502Z20173018)福建省自然科学基金(2017J01077)国家自然科学基金项目 (31871779

Study on Protoplast Transformation of Mortierella alpina and Its Homologous Expression of Elongase Gene

为了有效提高高山被孢霉(MOrTIErEllA AlPInA)产花生四烯酸的能力,采用聚乙二醇介导的原生质体转化法将重组Pd4质粒(含高山被孢霉合成花生四烯酸的延长酶基因glElO)转入高山被孢霉中,对其转化条件和同源表达进行了研究.结果表明,原生质体最佳转化条件为:以山梨醇作为稳定剂,50MMOl/l的CA2+和聚乙二醇4000作为转化介质,于25℃转化15MIn后温育,原生质体转化率最高为1.8μg-1.该转化子的摇瓶实验结果显示重组菌中花生四烯酸的产量比原始菌株的产量最高提高了近30%.该研究结果为高山被孢霉的性状改进及其他真菌的的遗传转化提供参考.In order to improve the production of Arachidonic acid(ARA),the protoplasts from Mortierella alpina were transformed with recombinant pD4 plasmid carrying the GLELO gene(elongase gene) in PEG system to study its homologous expression.The results showed that the best transformation system for preparing protoplasts sorbitol as osmotic stabilizer with 50 mmol/L Ca2+ and PEG4000 as the transformant medium,which was incubated at 25 ℃ for 15 min.After regenerating,the highest transformation ratio of 1.8 μg-1 was got.The results in shaking culture showed that ARA yield was almost improved by 30% in the recombinant Mortierella alpina than the original strain.It′s suggested that it will be useful for studying on the improvements of traits in Mortierella alpina and the genetic transformation in other fungi.国家自然科学基金项目(31071488);福建省科技厅重大专项(2010NZ0001-4

Determination of the number of ψ(3686) events taken at BESIII

The number of ψ(3686) events collected by the BESIII detector during the 2021 run period is determined to be (2259.3±11.1)×106 by counting inclusive ψ(3686) hadronic events. The uncertainty is systematic and the statistical uncertainty is negligible. Meanwhile, the numbers of ψ(3686) events collected during the 2009 and 2012 run periods are updated to be (107.7±0.6)×106 and (345.4±2.6)×106, respectively. Both numbers are consistent with the previous measurements within one standard deviation. The total number of ψ(3686) events in the three data samples is (2712.4±14.3)×10^