【作者】 张森；

【导师】 袁其朋；

【作者基本信息】 北京化工大学 ， 微生物与生化药学， 2012， 硕士

【摘要】 本文以红法夫酵母为研究对象，从诱变、培养、优化和添加物影响几个方面对红法夫酵母生产虾青素进行了系统的研究。常温常压等离子体诱变（ARTP）是一种新型诱变方法，具有操作容易、上手简单、自身安全和对环境无污染等优点。目前利用其诱变红法夫酵母的文章还没有，故我们将野生型酵母菌株AS2.1557作为原始菌株，利用ARTP进行诱变。确定3×10^-4β-紫罗酮作为筛选剂，经筛选得到突变菌株A7，并经传代培养检测遗传稳定性较好。用YM培养基作为发酵培养基培养，诱变菌株虾青素的产量约为1.67mg/L，较原始菌株提高了40%，虾青素含量为224.6μg/g，较出发菌株提高了42%。通过单因素试验考察了不同碳源、氮源等培养基对红法夫酵母生长和虾青素积累的影响，挑选出三个重要因素，利用响应面法（RSM）和BBD优化确定了诱变菌株的最佳培养基：蔗糖36.64、硫酸铵3.1、酵母提取物1.79、 CaCl₂0.1、 KH₂PO₄2、MgSO₄0.5、NaCl₂0.1（单位g/L）。然后对发酵条件进行了单因素试验，简单确定了合适的培养条件，10%接种量，50mL装液量，培养温度20°C，摇床转速200rpm，pH=5.5。揺瓶培养细胞干重和虾青素产量为7.8g/L和3.6mg/L。另外本文还研究了诱变菌株在发酵罐中的生长情况并绘制了红法夫酵母的各种动力学曲线，为以后的动力学研究和发酵罐的扩大培养奠定基础。本文研究了添加物对原始菌株和A7的影响及作用机制。首先我们研究了添加物对原始菌株影响，48h添加烟酰胺在20mg/L时虾青素产量达到最大值1.8mg/L比空白增加了15%，烟酸浓度为9mg/L时其虾青素产量达到最大值量比空白增加了6%。氧载体双氧水浓度20μL/L时达到最大值2.47mg/L比空白增加了大约20%。豆油浓度为10ml/L达到最大值为2.38mg/L比空白增加了大约37.6%，效果显著。亚甲基蓝浓度为0.5g/L的时候，虾青素的产量达到最大值1.962mg/L比空白增加了20%。不饱和脂肪酸影响微小，亚麻酸浓度为20μl/L时，虾青素产量增加到2.69mg/L比空白增加了13%左右。另外本文研究了添加物对诱变菌株A7的影响。异戊烯醇和香叶醇是甲羟戊酸途经的前体类似物，其中异戊烯醇浓度50.9mg/L时，虾青素产量达到最大值4.07mg/L比空白增加了10%。吲哚乙酸和乙烯利和水杨酸都对红法夫酵母有影响，尤其是吲哚乙酸（IAA）浓度1mg/L，虾青素产量达到4.4mg/L比空白增加了40%。更多还原

【Abstract】 Phaffia rhodozyma was chosen as the producer of astaxanthin. Theeffect of mutagenesis, optimization of the medium and additives onastaxanthin yield was studied.Atmospheric and Room Temperature Plasma （ARTP） is a newmutagenesis method, which has the advantage of simple and safeoperation and no pollution to the environment. At present, there are notrelatively reports about Phaffia rhodozyma with ARTP. An astaxanthinhyper-producing mutant A7was obtained by ARTP method of originalstrain AS2.1557and3×10^-4beta-ionone screening. A7strain has geneticstability through continuous passage culture. Astaxanthin production andastaxanthin content of mutant A7are reached1.67mg/L and224.6μg/gby YM medium, which increased40%and42%compared with those ofAS2.1557.Effects of different carbon sources, nitiogen sources, growth factorson astaxanthin biosynthesis by mutant A7were studied through the singlefactor experiment. We chose three significant factors and these factorsand their interaction were studied by response surface methodology （RSM）. A optimized medium was obtained, which was composed of36.64g/L sucrose,3.1g/L （NH₄）₂SO₄,1.79g/L yeast extract,0.1g/LCaCl₂,2g/L KH₂PO₄,0.5g/L MgSO₄and0.1g/L NaCl₂. The effect ofinoculum size, agitation speeds, temperature, liquid volume and initial pHvalue on the astaxanthin production by X. dendrorhous was studied. Theoptimum conditions were10%,200rpm,20°C,50ml and5.5,respectively. In this medium, the biomass of Phaffia rhodozyma,astaxanthin yield were8g/L and3.6mg/L. In addition, we also studiedthe growth of mutant strains in the fermentation tank and drew a varietyof kinetic curves of the Phaffia rhodozyma. We could continue to do theresearch of the subsequent kinetics and fermenter culture in furtute.We studied the influence of additives on the growth and astaxanthinproduction by original strains, firstly. When20mg/L nicotinamide wasadded, astaxanthin production was1.8mg/L. A15%was observdcompared with the control and the addition of9mg/L nicotinic acid onlyslightly improved astaxanthin production. The effect of oxygen carrier onthe astaxanthin production was studied. Among them, the stimulation ofH₂O₂and soya bean oil was remarkable. When20μL/L H2O2was added,astaxanthin production was2.47mg/L. A15%was observd comparedwith the control. The addition of10ml/L soya bean oil improved37.6%.Among unsaturated fatty acids, only linolenic acid improved13%astaxanthin production, the others almost had no positive effect. We also studied the influence of additives on the growth and astaxanthinproduction by A7strains. When50.9mg/L Isoamyl enolate was added,astaxanthin production was4.07mg/L inereased10%than the control.The astaxanthin production was4.4mg/L improved40%with theaddition of1mg/L indole-3-acetic acid.更多还原